in vitro maturation, in vitro fertilization and

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In Vitro Maturation, in Vitro Fertilization andDevelopment of Bovine Oocytes.Li ZhangLouisiana State University and Agricultural & Mechanical College

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300 North Zeeb Road, Ann Arbor Ml 48106-1346 USA 313/761-4700 800/521-0600

IN VITRO MATURATION, IN VITRO FERTILIZATION AND DEVELOPMENTOF BOVINE OOCYTES

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partial fulfillment of the

requirements for the degree of Doctor of Philosophy

in

The Department of Animal Science

by Li Zhang

B.S., Beijing Normal University, 1982 M.S., Chinese Academy of Agricultural Sciences, 1986

December, 1995

UMI Number: 9618336

UMI Microform 9618336 Copyright 1996, by UMI Company. All rights reserved.

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UMI300 North Zeeb Road Ann Arbor, MI 48103

ACKNOWLEDGEMENTS

The people who supported and contributed to this project will be always

remembered. First of all, I like to give my heartfelt thanks to my major professor, Dr.

Robert A. Godke, for his invaluable guidance and encouragement through this project.

His trust and support since the beginning of my research work in his laboratory greatly

encouraged me to make my "dream” come true, and he has proved to be far more of

a role model than he might anticipate.

Special thanks goes to Dr. Patricia J. Wozniak for her statistical guidance and

unlimited availability. The statistical analysis methods that I have learned from her

enabled me to complete much more comprehensive and interesting experiments. I would

like to thank Dr. William Hansel for his suggestions on this project. 1 am so lucky

having him in my dissertation advisory committee and so impressed by his keen insight,

his incredible memory and his kindness. Many thanks to Dr. Joseph D. Roussel for

always being there, willing to discuss ideas and give help. I would like to acknowledge

my appreciation to Dr. John W. Lynn for his interest to this project, allowing me access

to the confocal microscope in his laboratory and spending time to educate me about his

area of expertise.

I would also like to send my thanks to Dr. Don P. Wolf in Oregon Regional

Primate Research Center, Dr. Thomas D. Bunch and Dr. Shiquan Wang in Utah State

University, Dr. Xiangzhong Yang in Cornell University, Dr. Danie M. Barry in

University of Stellenbosch in South Africa for their unwavering support though my

graduate study. I am particularly grateful to Richard S. Denniston, for without him, I

would not have had the material, the supply and the instruments around which this

project based. My thanks and gratitude also go to my fellow graduate students Griff

Blakewood, Mike Myers, Leyi Li and Nancy Vitatle for their friendship and help.

Finally, special thanks is owed to my parents, Jingshan Zhang and Qingmei Liang

for supporting my educational development from childhood, to my brother Jianzhong and

my sister Lingling for their loving friendship and commitment to see me finish this

process, and to my husband Daxun Zhang and my son Tingting Zhang for their

continuous encouragement, cooperation and love. Without their help I would not have

been able to complete this project.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS .............................................................................................. ii

LIST OF TABLES ....................................................................................................... vi

LIST OF FIG URES.................................................................................................... viii

ABSTRACT .................................................................................................................. ix

CHAPTER1. INTRODUCTION...............................................................................................1

2. LITERATURE REVIEW: IN VITRO FERTILIZATION AND DEVELOPMENT OF BOVINE O O C Y TES.................................................. 6

Oocyte In Vitro Maturation ........................................................................ 6Sperm Capacitation ................................................................................. 15In Vitro Fertilization ................................................................................. 21In Vitro Development of IVF-Derived E m bryos.....................................23Recent Progress in Bovine In Vitro Fertilization.....................................31

3. A SIMPLE METHOD FOR IN VITRO MATURATION,IN VITRO FERTILIZATION AND CO-CULTURE OFBOVINE O O CY TES.......................................................................................35

Introduction.......................................................... 35Materials .....................................................................................................37Procedure.....................................................................................................39Results and Discussion .............................................................................. 43

4. EFFECT OF TEMPERATURE AND C02 LEVELS ON IN VITRO MATURATION AND FERTILIZATION OF BOVINE O O C Y T ES....................................................................................................... 53

Introduction..................................................................................................53Materials and Methods .............................................................................. 55R esults.......................................................................................................... 61Discussion.....................................................................................................64

5. CUMULUS CELL FUNCTION DURING BOVINE OOCYTES MATURATION, FERTILIZATION AND EMBRYO DEVELOPMENT IN V IT R O .........................................................................73

Introduction..................................................................................................73Materials and Methods .............................................................................. 74R esults.......................................................................................................... 80Discussion.....................................................................................................87

6. EVALUATING BOVINE OVIDUCT CELLS USED IN COMBINATION WITH BOVINE CUMULUS CELLS TO CO-CULTURE IVF-DERIVED BOVINE EMBRYOSIN VITRO .......................................................................................................... 94

Introduction.................................................................................................. 94Materials and Methods ...............................................................................95R esults.......................................................................................................... 98Discussion................................................................................................. 100

7. EFFECT OF GROWTH FACTORS AND GROWTH MODULATORS ON BOVINE OOCYTEIVM /IV F/IVC.............................................................................................. 106

Introduction.............................................................................................. 106Materials and Methods ......................................................................... 108R esults...................................................................................................... 119Discussion................................................................................................. 129

8. BIRTH OF LIVE CALVES AFTER TRANSFER OF FROZEN- THAWED BOVINE EMBRYOS FERTILIZED IN V IT R O ................. 140

Introduction.............................................................................................. 140Material and Method ............................................................................. 141R esults...................................................................................................... 143Discussion................................................................................................. 146

9. CONCLUSION ........................................................................................... 151

LITERATURE C IT E D ................................................................................................. 158

APPENDIXESA. BRACKETT-OLIPHANT M E D IU M ...................... 186

B. PREPARATION OF Ca++ IONOPHORE A23187 FORSPERM CAPACITATION........................................................................ 188

C. COMPOSOTION OF CZB MEDIUM ..................................................... 189

VITA ............................................................................................................................. 190

v

LIST OF TABLES

1. Results after IVM, IVF and cumulus cell co-culture of bovineoocytes during a 30-day t r i a l .............................................................................. 45

2. Experimental design for Experiment 4.1 56

3. Experimental design for Experiment 4.2 .............................................................. 58

4. Ovary holding temperature on oocyte maturation,fertilization and subsequent embryo development............................................. 62

5. Incubation temperature and C 02 level on bovine oocyte fertilization.................63

6. Experimental design for Experiment 5.1 76

7. Experimental design for Experiment 5.2 ..............................................................77

8. Effects of cumulus cell removal at various times and co-cultureon oocyte maturation, fertilization and embryo developm ent..........................81

9. Effects of cumulus cell removal at various times on the developmentof IVF-derived bovine embryos in the presence of co-culture ....................... 83

10. Effects of cumulus cell removal at various times on the developmentof IVF-derived bovine embryos in the absence of co-cu ltu re ..........................84

11. Effect of cumulus cells on fertilization and embryo development...................... 86

12. Main effect of partial and complete cumulus cell rem oval..................................88

13. Experimental design for Experiment 6 ...................................................................97

14. Effect of cell types and culture media on in vitro developmentof IVF-derived bovine embryos ................................................................... 101

15. Experimental design for Experiment 7.1 I l l

16. Experimental design for Experiment 7.2 ........................................................ 112

17. Experimental design for Experiment 7 .3 a ........................................................ 114

18. Experimental design for Experiment 7 .3 b ........................................................ 115

vi

19. Experimental design for Experiment 7 .3 c ....................................................... 117

20. Bovine oocytes matured in vitro with and without insulin ............................ 121

21. Effect of growth factors on IVF-derived bovine embryos ............................ 123

22. Arachidonic acid and PDGF on bovine oocyte in vitromaturation, fertilization and development..................................................... 124

23. Adding AA at different time intervals on bovine oocytesIVF and development .................................................................................... 126

24. Dose response of AA on the development of IVF-derivedbovine em b ry o s............................................................................................... 128

25. Results of culturing and transferring frozen-thawed in vitrofertilized bovine e m b ry o s .............................................................................. 145

26. Development of frozen-thawed in vitro fertilized bovineembryos after co-culture with cumulus c e l ls ............................................... 148

vii

LIST OF FIGURES

1. Bovine cumulus oocyte complexes used for producingIVF-derived e m b ry o ............................................................................................ 47

2. IVF-derived em bryos................................................................................................ 48

3. Somatic cell monolayers used for co-culture of IVF-derivedbovine em b ry o s.................................................................................................... 99

4. A blastocyst and a hatched blastocyst developed from insulin treatmentgroup after Hoechst 33342 staining of the nuclei......................................... 120

5. IVF-derived embryos produced from this s tu d y .............................................. 144

6. First frozen-thawed IVF-derived embryo resulting in live birth .................. 147

vii i

ABSTRACT

To improve the efficiency of the culture system for bovine in vitro fertilization

(IVF), several factors affecting the success of in vitro maturation (IVM), IVF and in vitro

culture (IVC) of bovine oocytes were studied. A simple and efficient procedure has been

developed for IVM/IVF/IVC of bovine follicular oocytes, which consistently produced

45 to 50% morulae and blastocysts during the treatment of over 6,000 oocytes. Ovary

holding temperatures of 0°C, 18°C and 25°C were found to affect the rate of maturation,

fertilization and cleavage (PC0.05). When ovaries were held at 25°C for 3 h, 20% of

the oocytes developed to blastocysts and hatched in vitro, indicating that this temperature

is suitable for holding ovaries during transportation. The highest cleavage rate (79%)

was obtained when oocytes were matured and fertilized both at 39 °C in 5% COj.

Fertilization rate was impaired with a lowered incubation temperature (37°C) and C 02

level (2.5%). Cumulus cells were shown to be important and necessary for IVM and

acquisition of competence for full embryonic development. Cumulus cell removal before

IVM, before IVF or 7 h after IVF reduced the rates of maturation, fertilization and

development at all embryo stages evaluated (P < .05). Cumulus cell removal at 20 h after

fertilization resulted in a rate of development similar to control embryos (P > .05). Co

culture enhanced the development of IVF-derived embryos. More embryos developed

to blastocysts and hatched when co-cultured with bovine cumulus cells (BCC), bovine

oviduct epithelial cells (BOEC) and BCC plus BOEC cells than in medium alone

(P C .05). A simple serum-free medium (CZB) supported more IVF-derived early

embryos to the morula stage than a complex serum-containing medium (TCM-199).

However, fewer embryos hatched in CZB medium than in serum-containing TCM-199

(P < .05). Among growth factors evaluated, arachidonic acid (AA) had a significant

stimulatory effect on embryo development (P < .05). The optimal AA concentration was

50 ng/ml; a high concentration (500 ng/ml) was toxic. Bovine IVM/IVF/IVC embryos

were successfully cryopreserved, thawed and transferred into recipient animals. Nine

clinical pregnancies were established with the birth of five normal calves. This success

indicates that viable bovine embryos can be produced in the laboratory.

x

CHAPTER 1

INTRODUCTION

In vitro fertilization (IVF) is a controlled process by which oocytes and sperm

cells interact with each other under culture condition to produce embryos. In most

mammals, fertilization occurs in the ampullar region of the oviduct. The resulting zygote

continues to develop as it passes along the oviduct and enters the uterus, and remains free

within the lumen of the female reproductive tract until implantation. In vitro fertilization

avoids the influence of the female reproductive tract and allows gamete interaction to

occur in culture, which is a potentially useful tool to investigate the physiological

requirements and mechanisms for mammalian fertilization and early embryo

development.

For many years, efforts have been made to fertilize mammalian oocytes and

develop early embryos in vitro. Some of the most extensive studies were conducted in

1951 by Austin and Chang. Their two independent studies clearly demonstrated that

sperm cells must undergo a process called capacitation, a series of changes that normally

occur in the female reproductive tract, to achieve the capacity to penetrate an oocyte

(Austin, 1951; Chang, 1951). In 1959, Chang reported the first birth of mammalian live

young after in vitro fertilization in the rabbit. In his study, in v/vo-matured oocytes

(ovulated) and in v/vo-capacitated sperm cells were used to produce transferable embryos.

Thereafter studies on the physiological, endocrinological and biochemical aspects

of oocyte maturation and sperm capacitation, as well as early embryo development

1

extended our knowledge for developing an in vitro system for mammalian fertilization.

Successful in vitro fertilizations producing early pregnancies and live births have been

reported in many species, including the mouse (Whittingham, 1968), rat (Toyoda and

Chang, 1974), hamster (Yanagimachi and Chang, 1964), cat (Pope et al., 1994), pig

(Cheng et al., 1986; Mattioli et al., 1989), cow (Brackett et al., 1982a), sheep (Cheng

et al., 1986; Crozet et al., 1987), goat (Hanada, 1985a), horse (Palmer et al., 1990),

buffalo (Suzuki et al., 1992), monkey (Kuehl and Dukelow, 1979; Bavister et al., 1983)

and human (Steptoe and Edwards, 1978). As research continued, in vitro culture systems

became more effective; follicular oocytes could be matured in vitro, ejaculated sperm

were successfully capacitated in vitro and preimplantation embryos were routinely

produced in the laboratory for research purposes, or for embryo transfer to obtain live

young. The most success in applying this IVF technology has been in humans. Today,

in vitro fertilization has become an effective and widely used method of helping infertile

couples conceive children.

Studies of bovine in vitro fertilization were begun in 1970’s. Early studies

achieved fertilization by inseminating follicular oocytes or ovulated oocytes in the oviduct

of a recipient animal or surrogate female (Sreenan, 1970; Hunter et al., 1972; Trounson

et al., 1977). The first successful in vitro fertilization in cattle was reported by Iritani

and Niwa (1977). In their study, 21 of 103 oocytes were fertilized in vitro with in vivo

capacitated sperm cells. However, none of the oocytes were fertilized with in vitro

capacitated sperm. Brackett et al. (1978) reported that fertilization occurred in 14 of 25

follicular or ovulated bovine oocytes using sperm cells capacitated in vitro with a high

ionic strength solution. This study demonstrated that bovine sperm capacitation can be

induced in vitro. This was the first a repeatable protocol reported for in vitro

fertilization in cattle. In 1982, this same group (Brackett et al., 1982a) reported the first

calf bom as a result of in vitro fertilization of an ovulated oocyte and surgical embryo

transfer. Subsequently, Lambert et al. (1983) reported the birth of six calves after

transfer of in vitro fertilized embryos produced from laparoscopically recovered oocytes

from preovulatory follicles, and cultured in rabbit oviducts.

Later, Hanada et al. (1986), Critser et al. (1986) and Xu et al. (1987) reported

live young bom after successful in vitro fertilization of in vitro matured (IVM) bovine

oocytes. In those studies, surrogate animal oviducts were used for early embryo

development. The first IVF calves produced by nonsurgical transfer of IVM, IVF and

in vitro cultured (IVC) bovine embryos were reported by Lu et al. in 1987, and soon

after by Goto et al. (1988). Co-culture systems were used in their studies and IVF-

derived embryos were developed to morula or blastocyst stages in vitro before being

transferred nonsurgically to recipient females. Pregnancies and live births have also been

reported after transfer of frozen-thawed IVM/IVF/IVC embryos (Fukuda et al., 1990;

Kajihara et al., 1992; Zhang et al., 1993). The use of frozen-thawed IVF-derived

embryos eliminates the need for synchronizing recipient animals, giving more flexibility

to the reproductive management system.

Among farm animals, cattle have become the best studied species for in vitro

fertilization. Cattle have been used as a model animal to study follicular growth, oocyte

maturation, sperm capacitation, sperm-oocyte interaction and fertilization, and nutrient

and growth factor requirements for early embryo development. Cattle have also been

used to study maternal-embryonic genomic transition, pregnancy recognition and in

bioengineering studies including embryo sexing, animal cloning and the production of

transgenic animals. One advantage of using the cow as a model to study in vitro

fertilization is the ready availability of both sperm cells and oocytes. Follicular oocytes

can be easily obtained from the ovaries of slaughterhouse animals, and fresh semen can

be collected from bulls or cryopreserved for IVF. Within resent years, thousands of

articles have been published on bovine IVF and embryo related studies. Several novel

protocols for bovine IVF have now been reported (see review by Gordon, 1994).

Several laboratories now are able to produce IVM/IVF/IVC bovine embryos for research

purposes and for commercial transplantation. However, the efficiency of current

IVM/IVF/IVC systems is still low, and the mechanisms underlying in vitro fertilization

and early mammalian development are far from being fully understood.

The objective of this study was to improve the efficiency of IVM/IVF/IVC of

bovine oocytes by studying several factors that may affect the success rates. A simple

procedure described in Chapter 3 was developed to produce a large number of

IVM/IVF/IVC bovine embryos from follicular oocytes obtained from abattoir ovaries.

Based on this procedure, a series of factors were studied including: 1) the ovary holding

temperature during transportation from a local abattoir to the laboratory and the

incubation temperature and C 02 level during oocyte maturation and fertilization in vitro

(Chapter 4); 2) the effect of cumulus cells on IVM/IVF/IVC (Chapter 5); 3) the effects

of different culture media and "helper" cells on in vitro development of IVF-derived

bovine embryos (Chapter 6) and, 4) the effects of exogenous growth factors on the

development rates of IVM/IVF/IVC bovine oocytes (Chapter 7). Finally, IVM/IVF/IVC

bovine embryos were cryopreserved and thawed approximately one year after storage in

liquid nitrogen, and transferred non-surgically to recipient females to verify the viability

of these in vitro produced bovine embryos by producing live young (Chapter 8).

CHAPTER 2

LITERATURE REVIEW:IN VITRO FERTILIZATION AND DEVELOPMENT OF BOVINE OOCYTES

Oocyte In Vitro Maturation

One of the prerequisites for successful fertilization is the completion of oocyte

maturation. In mammalian oogenesis, the meiotic process is initiated during fetal life and

arrested shortly before or after birth in the prophase diplotene of the first meiotic division

(germinal vesicle or GV stage). By the time a female reaches puberty, a group of

follicles (a cohort) start to develop under the influence of gonadotrophins, and one or

more growing follicles become dominant follicles (Graafian follicles). Oocyte(s) within

dominant follicle(s) resume the meiotic maturation process after a preovulatory

luteinizing hormone (LH) surge, GV breakdown (GVBD), release of the first polar body,

and are again arrest at metaphase of the second meiotic division (M-II). At this time,

oocyte maturation (which also includes the changes in the ooplasm known as cytoplasmic

maturation) is completed and ovulation takes place.

The phenomenon that mammalian oocytes can undergo spontaneous meiotic

resumption when isolated from their follicles was first demonstrated by Pincus and

Enzmann (1935). They reported that rabbit oocytes can be matured in vitro even in a

hormone-free medium. This phenomenon was later verified in many other mammalian

species, including the cow (Chang, 1955a; Edwards, 1965). In 1970’s, the studies on

bovine oocyte in vitro maturation showed that nuclear maturation can be achieved after

in vitro culture of follicular oocytes, and that these oocytes could be fertilized in the

6

oviduct of inseminated cows (Sreenan, 1970; Hunter et al., 1972; Shea et al., 1976;

Trounson et al., 1977).

Iritani and Niwa (1977) cultured bovine follicular oocytes in modified Kreb’s

Ringer bicarbonate medium for 24 hours, and inseminated these oocytes with in vivo

capacitated sperm. They observed sperm penetration and pronuclei formation in 20%

of the inseminated oocytes. The first birth of calves after in vivo fertilization of in vitro

matured oocytes was reported by Newcomb et al. (1978). In their study, oocytes

aspirated from small follicles were cultured in Ham’s F-10 medium supplemented with

20% estrous cow serum (ECS), 1 fig/ml estradiol 17-/3 (E J, 1 lU/ml human chorionic

gonadotropin (hCG) for 22 to 24 hours resulting in 72% of cultured oocytes developing

to the M-II stage; 23% of oocytes recovered a week later from inseminated recipient

cows were fertilized and developed to > 8-cell stage. This study further verified the

viability of in v/rro-matured bovine oocytes.

The selection of follicular oocytes has been shown to be an important factor on

the success of in vitro maturation and fertilization. Bovine oocytes are usually aspirated

from follicles of the abattoir ovaries using a needle attached to a syringe, or the ovaries

of live animals using a needle attached to vacuum pump and guided with ultrasonography

or laparoscopy.

Fukui and Sakuma (1980) reported that ovarian activity (presence corpora lutea

and/or corpus albicans) and the size of the follicles ( £ 5 mm, 6 to 10 mm and 11 to 20

mm) had no effect on oocyte in vitro maturation. However, the absence of cumulus cells

significantly decreased oocyte maturation rate. They also found that the proportion of

oocytes without cumulus cells was increased approximately 30% in the oocytes collected

from large follicles compared with that of the smaller follicles. Shioya et al. (1988)

reported significantly lower maturation rates, lower fertilization and cleavage rates, and

high degeneration rates from naked oocytes compared with cumulus enclosed oocytes

after IVM/IVF. Similar findings were also reported by other research groups (Leibfried

et al., 1979, Suss et al., 1988; Sirard and Bilodeau, 1990). These findings suggest that

the presence of cumulus cells surrounding oocytes is the best indicator of the ability of

oocyte to complete meiosis maturation in vitro. The oocytes without cumulus cells (most

from large follicles) might have been collected from atretic follicles, and thus

degenerating at the time of collection (de Loos et al., 1991).

Suss et al. (1988) and Sirard et al. (1989) studied the time sequence of nuclear

progression of bovine oocytes during in vitro maturation. They reported that during in

vitro culture, oocytes that have compacted cumulus cells had a nuclear configuration of

GV from 0 to 6.6 hours, GVBD at 6.6 to 8.0 hours, metaphase I (M-I) at 10.3 to 15.4

hours, anaphase I (A-I) at 15.4 to 16.6 hours, telophase I (T-I) at 16.6 to 18.0 hours,

and M-II at 18.0 to 24.0 hours. This time sequence paralleled the time sequence

reported for in vivo matured oocytes (Hafez and Ishibashi, 1964; Kruip et al., 1983;

Hunter, 1980; Hyttel et al., 1986), and by the end of the in vitro culture period (24

hours), »80% of cultured oocytes had reached M-II stage.

Although studies have shown that nuclear maturation can be completed during in

vitro culture, cytoplasmic maturation was of primary concern. Thibault and Gerard

(1973) reported that in vitro cultured rabbit oocytes could be penetrated by sperm, but

that the sperm head would not enlarge to form normal male pronuclei. Trounson et al.

(1977) found 49% of in vitro- matured oocytes fertilized after transfer to the oviduct of

an inseminated cow, few had developed to morulae or blastocysts by 96 hours later. In

contrast, 39% of in vivo-matured oocytes developed to blastocysts under the same

condition and 13 of 16 blastocysts developed to normal fetuses after embryo transfer.

Unfortunately, there is no distinct indicator for cytoplasm maturation in oocytes.

Therefore, fertilizability and developmental competence of oocytes are often used to

indicate the cytoplasm maturity.

Efforts have been made to improve oocyte cytoplasmic maturation by using

different culture media, protein supplements, hormones, growth factors, follicular fluid

and co-culture. The selection of the medium for culture of bovine follicular oocytes was

based on the results of early embryo culture studies. Although some studies have shown

that phosphate-buffered medium (PBS) could support bovine and ovine embryo

development in vitro (Moore and Spry, 1972; Trounson et al, 1976), no penetration was

detected after insemination when this medium was used for an oocyte maturation culture

(Baker and Polge, 1976).

Bicarbonate buffered media are generally used for in vitro maturation of bovine

oocytes and in vitro fertilization. Eng et al. (1986) reported that bicarbonate ions are

required for normal polar body formation. Bicarbonate buffered medium (TCM-199E;

Earle’s salts) was used for pig oocyte maturation culture resulting in twice the percentage

of polar body formation as the same medium with phosphate buffers (TCM-199H;

Hank’s salts). When Hank’s based medium was supplemented with bicarbonate buffer,

10polar body formation was restored to the level in Earle’s buffered medium. Several

other studies also reported the beneficial effect of bicarbonate buffering systems on

oocyte maturation and early embryo development over that of other media (Wales et al.,

1969; Quinn and Wales, 1973; Kane, 1975; Rajamahendran et al., 1985).

Both simple defined and complex media have been used for oocyte maturation in

previous studies. Iritani and Niwa (1977) used Kreb’s Ringer medium and Hunter et al.

(1972) used Tyrode’s medium to in vitro mature bovine oocytes. Normal fertilization

was observed after insemination with in vivo-capacitated sperm cells or after the transfer

to oviducts of inseminated cows. Newcomb et al. (1978) used a complex medium

(Ham’s F-10) for oocyte maturation culture and obtained the first birth of calves from

IVM oocytes. The same medium has also been used to produce IVM/IVF calves and

IVM/IVF/IVC calves (Xu et al., 1987, 1992b).

The most commonly used culture medium for bovine oocyte maturation is Tissue

Culture Medium-199 (TCM-199). Many laboratories have reported good maturation and

fertilization rates, moderate morula and blastocyst formation rates and the establishment

of pregnancies after embryo transfer using TCM-199 as culture medium (Lu et al.,

1987a; Goto et al., 1988; Brackett et al., 1989; Fukuda et al., 1990; Zhang et al.,

1992b). Bavister et al. (1992) compared seven different culture media for oocyte

maturation in vitro and found that the medium used for in vitro maturation can strongly

affect the developmental capacity of in v/'/ro-produced bovine embryos. In five of seven

media (TCM-199, SFRE, MEMa, M EM a+, RPMI:MEMa) tested in their study, the

proportion of oocytes that cleaved (76 to 82%), developed to morulae (25 to 32%) and

11blastocysts (12 to 19%) were similar. However, cleavage and development to morulae

and blastocysts were significantly reduced when Waymouth and Ham’s F-12 (50 and

35%, 14 and 3%, 3 and 1%, respectively) were used for in vitro maturation of bovine

oocytes.

Protein supplements were considered as an essential component in culture media

for oocyte in vitro maturation. Blood serum and serum albumin are major proteins added

to the culture media. It has been reported that the addition of serum to the medium

significantly increased the percentage of oocyte maturation over that of medium alone

(Sanbuissho and Threlfall, 1990). Both Hunter et al. (1972) and Iritani and Niwa (1977)

used bovine serum albumin (BSA) as a protein supplement and obtained in vitro

maturation and in vitro fertilization. Newcomb et al. (1978) added estrous cow serum

(ECS) to culture medium for oocyte in vitro maturation and established pregnancy after

transfer of in v/Vro-matured oocytes to inseminated cows.

Shea et al. (1976) reported in vitro maturation and subsequent fertilization using

fetal bovine serum (FBS) as a protein supplement. Leibfried et al. (1986) compared the

effect of FBS and BSA on bovine IVM/IVF and found that FBS was superior to BSA in

promoting in vitro maturation and fertilization of bovine oocytes. Unfortunately, the

effective components in serum are undefined. Eppig et al. (1992) reported that although

approximately 70% of mouse oocytes underwent maturation in both serum and BSA

containing media, fertilization and development to blastocysts were significantly reduced

when oocytes were matured in BSA-containing medium compared with serum- containing

medium. Using a chymotrypsin digestion test, they found that the zona pellucida of

12

oocytes matured in BSA-containing medium were four times more resistant than the zona

pellucida of oocytes matured in vivo or in FBS-containing medium, which could be one

explanation for reduced fertilization rates. While the hardening of the zona pellucidae

of oocytes matured in BSA-containing medium could be prevented by addition of fetuin

(a glycoprotein component of FBS), the developmental competence of these oocytes was

still not as good as those matured in serum-containing medium. This study suggests that

serum growth factors may function during oocyte growth and maturation to promote

events leading to successful fertilization and subsequently embryogenesis.

Recently, several research groups reported successful in vitro maturation and

fertilization of bovine oocytes in serum-free and even protein-free medium. Saeki et al.

(1991) cultured bovine oocytes in TCM-199 supplemented with .3% polyvinyl

pyrrolidone (PVP) and obtained 93% maturation rate, which was not significantly

different from that of TCM-199 with 10% FBS (88%). Oocytes that matured in protein-

free medium could also be fertilized (60 to 94%) and developed to blastocysts (27%) in

vitro in protein-free medium (Saeki et al., 1993).

Moor and Trounson (1977) reported that the developmental capacity of in vitro-

matured sheep oocytes was affected by both hormones and follicular factors. In their

study, oocytes removed from follicles resumed meiosis in culture, but did not undergo

normal development after transfer to inseminated animals. When oocytes were in vitro

matured within follicles, more oocytes that were cultured in hormone-containing medium

[follicle stimulating hormone (FSH), LH, estradiol-17/3 (Ej)] developed to blastocysts

after transplantation than those cultured in hormone-free medium. Newcomb et al.

13

(1978) obtained a live birth from in vitro matured and in vivo fertilized bovine oocytes

using Ham’s F-10 supplemented with estrous cow serum (ECS), gonadotropins and E2

as the oocyte maturation medium. Many research groups thereafter studied the effect of

serum, gonadotropins and steroid hormones on in vitro maturation of bovine oocytes, and

the results remain controversial.

Younis et al. (1989) compared different sera and hormone additions during bovine

oocyte maturation on subsequent in vitro maturation and in vitro fertilization rates. They

found that more oocytes matured when using cow sera (Day-0, Day-1, Day-10 and Day-

20) compared with FBS, and more fertilized oocytes developed to 4- to 8-cell stage when

Day-20 cow serum was used during in vitro maturation. The addition of hormones (Ej,

Ej + LH, Ej + FSH) improved IVM results and, when LH or FSH was added with Ej,

the development to 4- to 8-cell stage was markedly enhanced.

Brackett et al. (1989) reported that oocytes matured with high LH resulted in

embryos of excellent viability. Pregnancies were established after embryo transfer.

Their further study (Zuelke and Brackett, 1993) indicated that LH acts via the cumulus

cells to increase glutamine metabolism within oocytes and, thus, enhance oocyte quality

during the maturation process. Sanbuissho and Threlfall (1990) and Saeki et al. (1991)

reported improved pronucleus formation, fertilization, cleavage and blastocyst formation

when hormones (LH, FSH, Ej) were presented in in vitro maturation medium. The

maturation rates, however, did not differ among treatment groups.

In contrast, Fukui and Ono (1989) reported neither the addition of serum (FBS

or ECS) nor hormones affected in vitro maturation, cleavage and blastocyst formation in

cattle. Kruip et al. (1988) reported a negative effect of high concentrations of Ej on

bovine oocyte spindle formation and polar body extrusion. Yang et al. (1993) combined

different oocyte maturation and sperm capacitation procedures and found that when Ca++

ionophore was used for sperm capacitation, no differences were detected in cleavage and

blastocyst formation between hormones and non-hormone maturation groups. However,

when heparin was used for sperm capacitation, more cleaved embryos developed to

blastocysts in the hormone-treated group than the hormone-free group. Their findings

suggested that the difference in oocyte developmental capability could be due to the age

of oocyte at the time of fertilization. Since it has been reported that capacitation of

bovine sperm by heparin in vitro normally takes »4 .5 hours (Parrish et al., 1989) and

the hormone (FSH, LH and E2 ) addition delayed the course of bovine oocyte maturation

(Suss et al., 1988). Therefore, when 22 to 24 hours was used as the maturation

incubation interval and in vitro insemination took place at the same time, better embryo

development rates resulted from those oocytes matured with hormones and inseminated

with heparin-capacitated sperm. Oocytes that matured without hormones could be over

matured at the time of penetration by heparin-capacitated sperm, and had impaired

developmental capability.

Recently, Boediono et al. (1994) compared superovulated cow serum (SCS) with

FBS on bovine oocyte maturation, fertilization and development to blastocyst stage in

vitro, and reported a significantly higher percentage of embryos developing to the

blastocyst stage in the presence of SCS than FBS. However, the results of SCS analysis

showed that the improved development was not related to the level of LH and

15

progesterone in the sera. The serum that had a low concentration of glucose, fatty acids

and cholesterol supported cleavage and blastocyst development in vitro.

Sperm Capacitation

The sperm released from the male reproductive tract of most mammals are not

immediately fertile. They have to undergo a physiological change that normally occurs

in the female reproductive tract, termed "capacitation" (Austin, 1951; Chang, 1951). At

the site of fertilization, fertilizing sperm pass through the cumulus cell layers of the

oocyte, bind to the zona pellucida, complete the acrosome reaction (AR), penetrate the

zona pellucida and fuse with the plasma membrane of the oocyte (Wassarman, 1987;

Saling, 1991). It has been reported that only capacitated sperm are able to enter the

cumulus complex, while uncapacitated and acrosome-reacted sperm are excluded from

this outer extracellular matrix of the oocyte (Cummins and Yanagimachi, 1986).

Unlike the acrosome reaction, which is clearly defined as the multiple fusions

between the outer acrosomal membrane and the overlying sperm plasma membrane and

the release of acrosome hydrolases, capacitation is a process with little detectable

morphological change in the sperm membrane. Capacitated sperm could be reversed to

uncapacitated state by introducing seminal plasma, which is called "decapacitation"

(Chang, 1957). It has been reported that the decapacitation factor(s)-treated capacitated

sperm cannot pass through the corona radiata (Farooqui, 1983). However, the

decapacitation factor(s) can be removed by centrifugation (Bedford and Chang, 1962).

It is believed that capacitation involves a modification or removal of a substance coating

the sperm surface, which leads to a decrease in membrane negative surface charge, an

16

efflux of membrane cholesterol and an influx of calcium between the plasma and outer

acrosome membranes (Langlais and Robert, 1985).

Changes in sperm flagellar and associate swimming trajectories have been

reported for capacitated sperm (Yanagimachi, 1969; Yanagimachi, 1981). This

movement has been termed "hyperactivation". It is thought that the vigorous thrusting

of the flagellum may mechanically aid sperm passage through the oocyte investment

(Yanagimachi, 1981). In vitro studies demonstrated that altered motility patterns were

calcium-dependent and related to fertilizing ability of sperm cells in several different

species (Fraser, 1977; Yanagimachi, 1972; Cummins, 1982; Bird et al., 1989).

Therefore, the hyperactivity is often used as an indicator of the sperm capacitation

process. It has been proposed that hyperactive movement may result from an elevated

intracellular calcium level during capacitation, which is controlled by the calcium pump

in the mitochondria (Bradley et al., 1979).

The discovery of sperm capacitation greatly stimulated the study of in vitro

fertilization. Early studies used the female uterus to capacitate sperm and obtained in

vitro fertilization in rabbits (Chang, 1955b; Bedford, 1969). Barros and Austin (1967)

demonstrated that the tubal fluid collected from the oviducts of hamster was capable of

inducing sperm capacitation and acrosome reaction. Yanagimachi (1969) used follicular

fluid to capacitate hamster sperm in vitro. Gwatkin et al. (1972) reported that cumulus

cells surrounding the oocytes could induce sperm capacitation.

The factors which induce sperm capacitation in the female genital tract do not

appear to be species-specific. It was reported that rabbit sperm could be capacitated in

17

the uteri of rats and dogs (Hamner and Sojka, 1967) and hamster sperm could be

capacitated with bovine follicular fluid (Gwatkin and Andersen, 1969).

One hypothesis of sperm capacitation proposes that neuraminic acid and steroid

sulfate that coat the sperm plasma membrane during sperm maturation in the epididymis

are removed by the enzymes (neuraminidase, steroid sulfatase and arylsulfatase) in the

female reproductive tract, that causes destablization of the sperm membrane (Farooqui,

1983). However, the exact molecular basis of capacitation still remains to be

determined.

Successful in vitro capacitation of sperm in simple, chemically defined culture

media were reported in several species in early 1970s (Toyoda, 1971; Yanagimachi,

1972; Yanagimachi et al., 1976). These studies demonstrated that mammalian sperm

capacitation could occur in the absence of the female reproductive tract or female tract

secretion. However, there appears to be a species difference in the requirement of

physicochemical conditions, such as the time of exposure and the type of energy sources,

for in vitro sperm capacitation (Chang and Hunter, 1975; Rogers and Yanagimachi,

1975). Although the reason for the differences remain unknown, it is likely due to the

chemical characteristics of the sperm plasma membrane (Yanagimachi, 1977).

The early attempts at bovine in vitro fertilization with in vitro-capacitated sperm

were relatively unsuccessful (Sreenan, 1970; Baker and Polge, 1976; Iritani and Niwa,

1977). Failed fertilization with in v/'rro-capacitated sperm suggested that the seminal

plasma component(s) coating the bovine sperm was very stable, and could not be

18

removed by simple washing and incubating in culture medium like those for mouse and

human sperm capacitation.

Brackett et al. (1978) used a high ionic strength solution (380 mOsm) to induce

bovine sperm capacitation for in vitro insemination resulting in normal fertilization and

subsequently 2- to 4-cell development in vitro. In 1982, the same group reported the

first live young birth from bovine IVF using the high ionic strength medium treated

sperm (Brackett et al., 1982a). The latter study along with those of others (Oliphant and

Brackett, 1973; Koehler, 1976; Kinsey and Koehler, 1978) suggested that the treatment

of sperm with hypertonic medium resulted in the loss or removal of antigens over the

acrosomal region of sperm head.

Iritani et al. (1984) reported fertilization of in v/fro-matured bovine oocytes with

sperm capacitated in a chemically defined isotonic medium. In their study, sperm were

collected 1 day before insemination, kept for 14 to 18 hours at 20°C in a test tube,

preincubated in m-KRB solution for 8 hours, and 58% of IVM oocytes were fertilized

after insemination. A similar method using long incubation times has also been used to

capacitate bovine sperm by Wheeler and Seidel (1987).

First and Parrish (1988) developed a method using heparin for inducing bovine

sperm capacitation. Their group systematically studied bovine tubal fluid and found that

estrual cow oviduct fluid contains a capacitating factor. The same factor, however, was

not found to be present at the luteal phase of the estrous cycle. They further found the

active factor was heat stable, unaffected by protease digestion, precipitated by ethanol,

and inactivated by nitrous acid, a reagent that degrades heparin-like glycosaminoglycans.

19

Their in vitro study demonstrated that heparin was effective in inducing bovine

sperm capacitation in a dose and time dependent manner. Bovine sperm were capacitated

with heparin by 4 hours of incubation, with the maximal effect occurring with 7.5 pg/ml

heparin (Parrish et al., 1988). They found that heparin must bind to capacitating sperm

and once capacitation has occurred the heparin could be displaced from sperm without

causing decapacitation. They also noted that calcium was required for heparin to

capacitate sperm, and when sperm were incubated with heparin an uptake of 45Ca++

occurred. Today, heparin treatment is a well accepted method for inducing bovine sperm

capacitation. Pregnancies and live birth have been reported using heparin capacitated

sperm for in vitro fertilization from several different research groups (Critser et al.,

1986; Lu et al., 1987a; Xu et al., 1987).

Caffeine is another reagent that has been used to induce bovine sperm

capacitation. It has been reported that caffeine was able to increase motility, fertilizing

capability and the ability of the sperm to penetrate the cervical mucus in human

(Schoenfeld et al., 1973; Atiken et al., 1983), mouse (Frazer, 1979), and rhesus monkey

(Boatman and Bavister, 1984), It is believed that caffeine inhibited intracellular

phosphodiesterase activity, which caused the accumulation of cyclic adenosine mono

phosphate (cAMP) (Cai and Marik, 1989). The elevated cAMP levels likely involved

control of the calcium ion flux across the sperm membrane, which in turn increased both

motility and velocity of normal sperm (Barkay et al., 1984).

Goto et al. (1988) reported live birth from nonsurgical transfer of IVM/IVF and

in v/'/ro-cultured (IVC) bovine embryos, using caffeine as a sperm stimulating or

20

activating agent during capacitation. In this study, frozen-thawed epididymal sperm were

preincubated for 2 to 3 hours in Brackett-Oliphant (B-O) medium containing BSA and

2.5 mM caffeine before insemination.

Calcium fluxes through the sperm plasma membrane is a universal event that has

been reported during sperm capacitation and the acrosome reaction (Dan, 1956, 1967;

Yanagimachi and Usui, 1974). In Ca++-free media, sperm do not undergo the acrosome

reaction (AR), even if they are exposed to an AR-inducing stimuli (Yanagimachi, 1975).

Didion and Graves (1989) studied the role of Ca++ on bovine sperm and reported that

these sperm could survive in a Ca++-free medium, but both sperm binding and

penetration of the zona pellucida were Ca++ dependent. Since calcium influx has been

found consistently during sperm capacitation, efforts have been made to capacitate sperm

by inducing Ca++ fluxes. One agent that has been used successfully to induce bovine

sperm capacitation and AR is the divalent cation ionophore A23187. Addition of calcium

ionophore A23187 to a sperm-medium suspension in the presence of calcium were shown

to induce sperm capacitation and AR in mouse (Suarez et al., 1987), pig (Smith et al.,

1983), human (Atiken et al., 1984), sheep (Shams-Borhan and Harrison, 1981) and

bovine (Byrd, 1981; Takahashi and Hanada, 1984; Bird et al., 1989).

Talbot et al. (1976) proposed that calcium ionophore A23187 can form a

lipophilic complex with calcium ion and facilitates its transport across the sperm plasma

membrane, resulting in sperm capacitation and acrosome reaction. Shams-Borhan and

Harrison (1981) have reported that an ionophore-induced sperm acrosome reaction in the

ram was similar to natural acrosome reactions in three features: 1) the induction is

21

calcium-dependent; 2) no morphological differences were found in sperm; and 3) sperm

plasma membrane modification was found only in the acrosomal region. Their study also

found that higher concentration of ionophore had a detrimental effect on sperm motility.

By adding serum albumin shortly after the addition of the ionophore, however, motility

could be preserved, while the acrosome reaction occurred as usual.

Hanada (1985) reported fertilization and cleavage development of in virro-matured

caprine oocytes inseminated with ionophore treated buck sperm. Using .1 or .2 /*M

calcium ionophore A23187 for 30 or 60 seconds, they obtained a fertilization rate of

78.1%, which was significantly greater than that of the no ionophore group (29.4%).

Fukuda et al. (1990) inseminated oocytes with calcium ionophore A23187 treated bovine

sperm and reported birth of normal calves after transfer IVM/IVF/IVC embryos

In Vitro Fertilization

In vitro fertilization of bovine oocytes can be obtained by co-incubating matured

oocytes and capacitated sperm in a simple chemically-defined medium, termed defined

medium (DM), Brackett-Oliphant medium, or Tyrode’s albumin lactate pyruvate (TALP)

medium. This medium is a modified Tyrode’s medium developed by Brackett and

Oliphant (1975). The insemination medium is basically the same as the sperm

capacitation medium, except without the capacitating agent and with added bovine serum

albumin (BSA). The medium contains high bicarbonate content and is adjusted to a pH

of 7.8. Incubation of the sperm and oocytes is usually completed under 5% CO; in air

(First and Parrish, 1987) or 5% C 02, 5% 0 2 and 90% N2 (Younis et al., 1989). It has

been established that the pH and the bicarbonate ion play an important role during sperm

22

capacitation, sperm penetration and male pronuclear formation (Ijaz and Hunter, 1989;

Boatman and Robbins, 1991b).

Co-incubations are usually conducted in 50 to 100 fil microdroplets with 5 to 25

oocytes/drop in petri dishes covered with mineral oil. The sperm concentration for in

vitro fertilization ranges from 1 to 20 x 10s motile sperm/ml of medium (Brackett et al.,

1982a; Goto et al., 1989), and oocytes are usually co-incubated with sperm for 6 to 24

hours (Parrish et al., 1986; Goto et al., 1989), washed several times to remove extra

sperm cells, and then transferred into embryo development culture medium. It is

important to remove oocytes from the sperm suspension as soon after penetration as

possible because excess dead sperm in the culture system could be toxic to the early stage

embryos and, therefore, decrease the viability of cultured embryos (Brackett, 1985).

Sperm penetration takes *»6 hours from the time of insemination in cattle (Xu and

Greve, 1988). It should be noted that this also depends on the method used for both

oocyte maturation and sperm capacitation (Yang et al., 1993).

The incubation temperature has been shown to be critical to the success of in vitro

fertilization in farm animals. Early studies using 37°C for bovine IVF resulted in lower

fertilization rates than that of in vivo fertilization (see review by Wright and Bondioli,

1981). Lenz et al. (1983) first suggested that in vitro maturation and fertilization of

bovine oocyte are temperature-dependent processes. When bovine oocytes were in vitro

matured and fertilized at 39°C, which was the highest fertilization rate of the

temperatures evaluated in their study, 58% of oocytes were penetrated by sperm.

23

Another factor that has been reported to effect the outcome of IVF is the source

of the semen. Sperm from different bulls result in different fertilization rates in vitro

(Hanada, 1985b; Iritani et al., 1986). This phenomena has also been reported in rabbit

(Brackett, 1982b), sheep (Fukui et al., 1988) and goat (Zhang et al., unpublished). In

our laboratory, we also found the variation among different ejaculations from the same

buck (Zhang et al., unpublished). Therefore, it is important to screen the semen sample

individually prior to the onset IVF. Parrish et al. (1986) obtained an acceptable

fertilization rate using frozen-thawed bovine semen to inseminate IVM oocytes.

Pregnancies resulted with the use of frozen-thawed semen from the same group (Critser

et al., 1986). These findings allowed IVF laboratories to decrease the variability often

noted from one semen batch to another. Semen from the same bull and same ejaculation

can be cryopreserved separately in many straws and tested before its use in IVF.

In Vitro Development of IVF-Derived Embryos

The methodology for the in vitro development of farm animal IVF embryos is

based on the early embryo culture studies in which the embryos were collected from

superovulated females. The earliest attempts to culture bovine embryos in vitro were

reported by Pincus (1951), Brock and Rowson (1952) and Sreenan et al. (1968), but

limited development was observed from these early stage embryos. Tervit et al. (1972)

reported successful culture of early stage bovine embryos to the morula and blastocyst

stages using synthetic oviduct fluid (SOF) medium. Three of five 8-cell bovine embryos

developed to blastocysts after being cultured for 6 days, however, all the 1-cell embryos

were retarded by completion of the culture interval. Shea et al. (1974) cultured early

24

stage bovine embryos in BMOC-3 and SOF medium supplemented with HEPES buffer

and found that the embryos at 8- to 12-cell stages developed at a higher rate than

embryos at an earlier stage of development. When Kanagewa (1979) cultured bovine

embryos in BMOC-3 medium, 82% of ^8-cell stage embryos developed into expanded

blastocyst after 2 to 4 days of culture. There was no development, however, from 2- to

3-cell stage embryos. These initial findings suggested that there is a 8- to 16-cell stage

in vitro developmental block in bovine embryos.

The in vitro developmental block was first described in mouse embryos

(Whittingham and Biggers, 1967, 1968). It has been found that mouse embryos from

outbred strains do not generally develop into blastocysts when cultured from the 1-cell

stage in chemically defined media, but arrest development at the 2-cell stage (2-cell

block). However, embryos collected from the same mice at the 2-cell stage or 1-cell

stage embryos from inbred strains can develop into normal blastocysts in the same

culture medium (Whittingham and Biggers, 1967, 1968; Biggers, 1971; Whittingham,

1974). Studies have shown that the "2-cell block" could be prevented by injecting

cytoplasm from embryos of a non-blocking strain (Muggleton-Harris et al., 1982), and

"2-cell blocked" mouse embryos could be rescued by transferring to oviducts placed in

an organ culture system (Whittingham and Biggers, 1967). These findings suggested that

the in vitro developmental block is due to a culture-induced cytoplasmic defect, which

does not permit normal development of those mouse embryos beyond 2-cell stage.

In vitro developmental block has also been found in other laboratory and farm

animals, including hamster at 2- to 4-cell stage (Whittingham and Bavister, 1974), rat at

4- to 8-cell stage (Bavister, 1988), sheep at 8- to 16-cell stage (Wintenberger et al.,

1953), goat at 8- to 16-cell stage (Betteridge, 1977), cow at 8- to 16-cell stage (Thibault,

1966; Camous et al., 1984) and pig at 4- to 8-cell stage (Davis and Day, 1978). These

in vitro developmental blocks were found to be related to the maternal-embryonic genome

transition in early stage embryos. In many mammalian animals, the earliest stages of

embryogenesis are dependent upon stored product of maternal genome (Davison, 1976),

Both maternal RNA and protein are accumulated within the oocyte that are sufficient for

the progression of the embryo development through the first cleavage division in the

mouse (Golbus et al., 1973), the second cleavage division in the rabbit (Van Blerkom and

Manes, 1974) and the pig (Schoenbeck et al., 1993) and the third cleavage division in the

sheep (Crosby et al., 1988) and the cow (Frei et al., 1986). Subsequent embryonic

development is dependent upon the activation of the embryonic genome and the synthesis

of embryonic RNA and protein. During this transition, the metabolic requirements of

embryo development in some species or strains appear to be difficult to satisfy in vitro.

Efforts have been made to overcome the 8- to 16-cell block during in vitro

culture of early stage bovine embryos. Wright et al. (1976b) tested different culture

media [minimum essential medium (MEM), TCM-199, BMOC-3, SOF and Whitten's

medium] and protein supplements (FBS and BSA). Blastocyst expansion and hatching

was achieved from 2- to 8-cell stage embryos when cultured in Ham's F-10 medium

supplemented with 10% FBS. No development past early blastocyst stage was achieved

when BSA was used as a protein supplement. Although the success rate was low (two

of fourteen 1- to 2-cell embryos developed to expanded blastocysts and one hatched), this

26

was the first report that described complete hatching in vitro of bovine embryos cultured

from 1- to 2-cell stage.

Since there was no efficient in vitro culture system available for bovine embryos

prior to the 1980s, early stage IVF-derived embryos were either transferred surgically

to the oviduct of recipient cows or transferred into sheep or rabbit oviducts, flushed out

5 to 7 days later to recover morula and blastocyst stage embryos and then transferred

nonsurgically into recipient cows.

Brackett et al. (1982a) reported the first IVF live birth in cattle after transfer of

an IVF-derived embryo to a recipient cow. In this study, oocytes were recovered

surgically from the ovaries prior to ovulation or from oviducts shortly after ovulation and

co-incubated with the sperm cells that had been treated with high ionic strength medium

(380 mOsm/kg) to induce capacitation. A 4-cell stage IVF-derived embryo was

transferred surgically into the oviduct of a recipient cow resulting a normal pregnancy

and live birth.

Critser et al. (1986) transferred IVM/IVF 1-cell stage bovine embryos into the

oviducts of sheep for cleavage and blastocyst development. Recovered blastocysts were

then nonsurgically transferred into recipient cows. Pregnancies and normal development

were reported from this study. Lu et al. (1987a) and Xu et al. (1987) used the same

approach for developing early stage IVF-derived bovine embryos and reported live birth

after nonsurgical embryo transfer. Although the successes of producing live young from

IVF-derived embryos were encouraging, these procedures were relatively complicated

and time consuming.

27

Kuzan and Wright (1982) were among the first to use a somatic cell co-culture

system to develop bovine embryos to the blastocyst stage in vitro. They cultured morula

stage embryos with bovine uterine fibroblast cells (BUFC) and bovine testicular fibroblast

cells (BTFC) in MEM medium supplemented with 10% FBS, and found that more

embryos developed to blastocysts and hatched blastocysts when co-cultured with BUFC

than cultured in MEM alone. The beneficial effect of BUFC on the in vitro development

of bovine morula stage embryos have also reported by other research groups. Voelkel

et al. (1985) co-cultured bisected bovine morula stage embryos on a monolayer o f BUFC

for 72 hours. The embryo viability was significantly improved compared with those

demi-embryos cultured with Ham’s F-10 medium alone. Wiemer et al. (1987) developed

a fetal bovine uterine fibroblasts cell (FBUFC) co-culture system to in vitro develop

bovine morula stage embryos, and reported enhanced development over that of culturing

in medium alone.

Camous et al. (1984) used bovine trophoblastic vesicles to co-culture 1- to 8-cell

stage bovine embryos, and reported that 46% of co-cultured embryos developed to

morula stage, which was significantly greater than that of embryos cultured in medium

alone (18%). Hey man et al. (1987) used trophoblastic vesicle conditioned-medium to

culture 1- to 2-cell stage bovine embryos, and 38.8% (14/36) of the embryos developed

to the 16-cell stage compared with 41.8% (23/55) of embryos developed to the same

stage when directly co-cultured with trophoblastic vesicles. These findings suggested that

trophoblast vesicles may produce embryotrophic factor(s) and release the factor(s) into

28

the culture medium, and this factor(s) is able to enhance the in vitro development of

bovine embryos beyond the block stage.

Lu et al. (1987b) used a co-culture system to develop IVM/IVF-derived embryos,

and reported the first live birth after nonsurgical transfer of IVM/IVF/IVC embryos. In

their study, two IVF-derived embryos were co-cultured with bovine oviduct epithelial

cells (BOEC) for 6 days before recipient transfer resulting in the birth of twin calves.

Goto et al. (1988) also reported live birth from nonsurgical transfer of IVF-derived

embryos co-cultured with bovine cumulus cells (BCC).

Within a short period of time, co-culture has become a routine method to in vitro

develop IVF-derived embryos of the farm animals (see review by Thibodeaux and

Godke, 1993). Various types of somatic cells have been found to benefit early stage

bovine embryo development, by overcoming 8- to 16-cell in vitro developmental block

and promoting morula and blastocyst formation.

The cells that have been used for co-culture bovine IVF-derived embryos were

from both reproductive and non-reproductive origins. Among reproductive origin cells,

BOEC (Lu et al., 1988; Fukui, 1989; Aoyagi et al., 1990; Berg and Brem, 1990; Saeki

et al., 1991; Nagao et al., 1991) and BCC (Kajihara et al., 1987; Zhang et al., 1990a;

Fukuda et al., 1990; Younis and Brackett, 1990; Iwasaki et al., 1990) are the most

extensively used "helper" cells with proved embryo viability by producing live birth after

embryo transfer. BOEC monolayers are usually prepared from the abattoir oviducts 48

hours prior to embryo co-culture (Thibodeaux et al., 1992). The BCC monolayers are

usually obtained from the cumulus cells that attached to the bottom of the culture dish

29

during oocyte in vitro maturation incubation and maintained for embryo co-culture

(Zhang et al., 1992b).

BOEC cell lines and bovine trophoblast cell lines have also been shown to support

bovine IVF embryo development in co-culture. Scodras et al. (1991) co-cultured IVF

zygotes with BOEC, BOEC cell line and trophoblast cell line, the percentages of the

cultured zygotes that developed to the compacted morula and blastocyst stages (9.7 and

20.9%, 19.4 and 25.8%, 10.1 and 27.6%, respectively) were significantly greater than

that of zygotes cultured in medium alone (2 .9 and 2.9%). Their results showed that cell

lines can support the development of IVF zygotes to morula and blastocyst stages.

Another interesting finding from this study was that more compacted morulae resulted

from BOEC cell line than freshly prepared BOEC monolayers.

Broussard et al. (1994) used frozen-thawed cumulus cells for co-culture IVF-

derived bovine embryos. Cleaved IVF embryos were cultured in medium alone as a

control, co-cultured with fresh cumulus cells or co-cultured with frozen-thawed cumulus

cells. On day 5 of culture the percentage of embryos that developed to blastocyst stage

were 1.5, 20.6 and 23.0%, respectively, and on day 6 of culture the percentage of

embryos that developed to blastocysts increased to 3.1, 31.2 and 30.4% respectively.

This study demonstrated an improvement of embryo development in co-culture to days

5 and day 6 compared with the control group. However, no differences were found

among co-culture groups, which suggested that frozen-thawed cumulus cells can enhance

embryo development similar to fresh cells during in vitro culture.

30

Several kinds of non-reproductive origin cells and cell lines have also been used

for support IVF embryo development in vitro. Kim et al. (1991) reported that bovine

fetal spleen (BFS) cells, chick embryo fibroblasts (CEF) cells were able to support the

development of early stage IVF-derived bovine embryos to hatched blastocysts. Scodras

et al. (1991) used a bovine kidney epithelial cell line (MDBK) and a fetal murine

fibroblasts cell line (STO) to co-culture bovine IVF zygotes. Both resulted in improved

embryo development over that of medium alone control group.

Voelkel and Hu (1992) used a buffalo rat liver (BRL) cell line to co-culture IVF

bovine embryos. When early stage embryos were co-cultured for 6 days, 29% of the

embryos developed beyond compacted morula stage. Those embryos were subsequently

frozen and thawed. After thawing, 53% were viable. Twenty frozen-thawed embryos

were transferred into recipient cows resulting in 6 pregnancies (30%). This study further

proved the viability of the bovine IVF embryos that developed on a commercially

available cell lines by producing normal pregnancies.

Myers et al. (1994) compared the effect of BOEC, BRL cell line, MDBK cell line

and a African green monkey kidney (Vero) cell line on the development of IVF-derived

bovine 2- to 4-cell stage embryos. Both monolayer co-culture and conditioned medium

culture were evaluated. These results showed that co-culture improved development to

the blastocyst stage compared with control medium alone, and the highest development

was observed after co-culture with BOEC. Conditioned medium also enhanced

development to morulae and blastocysts compared with the control medium; however,

no differences were detected among different type of cells.

31

The supportive effect of somatic cell co-culture on the development of IVF-

derived bovine embryos is obvious, however, the mechanism of this effect remains

unclear. It has been proposed that the "helper" cells may produce factors that promote

embryonic cell proliferation and differentiation and/or metabolize embryotoxic substances

from the culture medium (Kane et al., 1992).

Recent Progress in Bovine In Vitro Fertilization

The successes of bovine oocyte maturation, sperm capacitation, fertilization and

early stage embryo development in vitro has greatly stimulated the study of early

mammalian development, as the oocytes, zygotes and embryos can be routinely produced

in the laboratory using slaughterhouse ovaries. These successes have markedly increased

our understanding of the physiologically requirement and regulation of mammalian early

development. However, the efficiency of current IVM/IVF and in vitro culture systems

used to produce viable morula and blastocyst stage embryos from abattoir ovaries is still

low, with an estimated average 4.6 blastocysts per animal (Lu and Polge, 1992).

The blastocysts produced in vitro had fewer cells, a lower proportion of inner cell

mass (Iwasaki et al., 1990), as well as lower post-thaw viability (Rorie et al., 1990;

Leibo and Loskutoff, 1993) compared with in vivo produced embryos. Recent studies

of the metabolism and energy substrate requirements of mouse, rat and hamster embryo

development have shown that glucose and phosphate (that normally present in body fluids

and contained in the majority of culture medium) can actually be toxic to early stage

embryos and/or cause in vitro developmental block. In contrast, the addition of glutamine

to the culture medium enabled these embryos to overcome the in vitro developmental

block (Schini and Bavister, 1988; Chatot et al., 1989; Seshagiri and Bavister, 1991;

Miyoshi et a l., 1994). Seshagiri and Bavister (1989) reported that phosphate is required

for inhibition of development by glucose of hamster 8-cell embryos in vitro. A 2- to 3-

fold decrease in oxygen consumption was observed in the embryos cultured with glucose

and phosphate (Seshagiri and Bavister, 1991). They proposed that there is a "Crabtree

effect" (enhanced glycolysis inhibits respiratory activity and oxidative metabolism) on

early stage embryonic cell development, which indicates that the metabolic properties of

these embryonic cells differ markedly from those of most somatic cells and resemble

some cancer cells. Although glucose seems not to block early bovine embryo

development (Pinyopummintr and Bavister, 1991), these findings suggest that there is a

potential impact of imbalanced culture medium to cultured embryos.

Kane et al. (1992) suggested that the oocytes and early stage embryos may have

enough maternal origin nutrients to support their development before hatching. The

reduced developmental competence of in vitro produced and cultured embryos using

current culture systems may be explained by the fact that the embryo is swimming in an

ocean of fluid into which it is leaking its endogenous nutrients. This is supported by:

1) there is little or no change in protein content of prehatched embryo from 1-cell to

morula stage in some of the common mammalian species (Brinster, 1967; Schiffner and

Spielmann, 1976; Wright et al., 1981) and 2) the studies on increasing embryo density

and reducing the medium volume showed an improvement in embryo ability to overcome

the in vitro developmental block and enhancement of morula and blastocyst formation

(Paria and Dey, 1990; Nieder and Caprino, 1991; Lane and Gardner, 1992). All of

33

these findings suggest that the current culture systems are nutrient unbalanced systems

for early embryos, and the direction of further studies on the requirement of early

mammalian development in vitro should be focused on both the addition of nutrient

components and the balance of the nutrient components in the culture medium.

The presence of autocrine and paracrine effects of growth factors has also been

proposed. The observations that increased embryo density and decreased culture medium

volume could enhance embryo development (Paria and Dey, 1990; Nieder and Caprino,

1991; Lane and Gardner, 1992) suggests that certain factors of embryonic origin

participated in an autocrine regulation of embryo development. In contrast, the impacted

development of in vitro cultured embryos compared with in vivo produced embryos

(Bowman and McLaren, 1970; Iwasaki et al., 1990) indicates that the complement of

early stage embryo development requires additional paracrine factors that originate from

the reproductive tract.

More progress may be made in the future if growth factors that are involved in

early mammalian embryonic development are identified. The expression of growth factor

genes (Rappolee et al., 1988), the presence of growth factor receptors (Rappolee et al.,

1990; Adamson, 1990), and the growth promoting effect of exogenous growth factors on

oocyte and embryo development (Paria and Dey, 1990; Harvey and Kaye, 1992; Das et

al., 1991; Yoshida and Kanagawa, 1984; Harper and Brackett, 1993) are now being

reported. Using molecular biology techniques, several growth factor ligand and receptor

gene expression patterns have been characterized by in vitro produced embryos of mouse,

sheep and cattle (Rappolee et al., 1988; Watson et al., 1992; 1994). Transcripts

34

encoding growth factors and growth factor receptors have also been detected in oviductal

epithelial cell monolayer cultures, which may explain the beneficial effect of co-culture

on early embryo development (Watson et al., 1994). More information is needed to

support the hypothesis that growth factors may play an important role in regulation early

embryo development.

Our knowledge of early development of mammalian embryos is far from

complete. A better understanding of gamete interaction and embryo development from

further studies should result in the discovery of culture systems that are more reliable and

are more efficiently produce bovine embryos in vitro.

CHAPTER 3

A SIMPLE METHOD FOR IN VITRO MATURATION, IN VITRO FERTILIZATION AND CO-CULTURE

OF BOVINE OOCYTES

Introduction

The first calf resulting from in vitro fertilization (IVF) was reported by Brackett

et al. in 1982. This feat was duplicated by members of the same research group, with

several IVF calves including a set of dizygotic IVF twins, reported in 1984 (Brackett et

al., 1984). Since the mid-1980s, IVF technologies have sparked interest (Lu et al.,

1987a) and bovine IVF research has thus markedly increased in both the scientific

community and in the commercial livestock sector. Key IVF methodologies for bovine

oocytes have been reported from different laboratories for early stage in v/vo-matured

follicular oocytes (Brackett et al., 1982a; Sirard and Lambert, 1986) and from in vitro-

matured oocytes harvested from abattoir ovaries (Parrish et al., 1986; Lu et al., 1987a;

Xu et al., 1987; Lu et al., 1988; Sirard et al., 1988). Efforts have been made with in

vitro culture of fertilized bovine oocytes to overcome 8- to 16-cell in vitro developmental

block (Thibault, 1966; Eyestone and First, 1986) to produce IVF embryos at later

morphologic stage for nonsurgical transfer to recipient females (Xu et al., 1987; Lu et

al., 1988; Sirard et al., 1988). A major barrier remaining in the bovine IVF procedure

is to develop an efficient in vitro culture system for the IVF-derived embryos (Lu et al.,

1987a; Fukui et al., 1988; Goto et al., 1988).

35

36

Transplant pregnancies have resulted from the transfer of IVF-derived bovine

embryos using the oviducts of the rabbit (Sirard and Lambert, 1986; Sirard et al., 1985),

the sheep (Critser et al., 1986; Lu et al., 1987a; Parrish et al., 1986) and the cow (Lu

et al., 1987a; Parrish et al., 1986) as intermediate incubators for developing embryos.

More recently, early stage IVF-derived bovine embryos have been cultured to the morula

and blastocyst stage in vitro using bovine oviduct epithelial cell (Fukui and Ono, 1988;

Lu et al., 1988) and the chick embryo amnion (Blakewood and Godke, 1990).

Previously, Motlik and Fulka (1981) reported that good success occurs with IVF of

rabbit oocytes when incubated with rabbit granulosa cells. More recently, Baird et al.

(1990) reported improved in vitro embryo development resulted when early stage mouse

embryos were co-cultured with granulosa cells harvested from hamsters. Success has

also been reported when IVF-derived bovine embryos were in vitro cultured with bovine

cumulus cells (Goto et al., 1988); however, improvements are needed to increase the

efficiency of this procedure. Unfortunately, the basic in vitro culture procedures

presently in laboratory use are labor intensive, time consuming and generally lack

efficiency.

An effort has been made to modify bovine in vitro maturation (IVM)-IVF

procedures and to combine this approach with a simple, efficient procedure for co-

culturing the IVF-derived embryos with the animal’s own follicular cumulus cells. This

procedure for bovine IVF and co-culture is currently underway in this station and has

been used for producing bovine embryos for embryo transfer, freezing, and

micromanipulation.

Materials

A. Equipment

1. Laminar-flow hood, Nuaire1

2. Temperature-controlled incubator with 5% COj, 50°C

3. Temperature-controlled water bath, 50°C

4. Stereomicroscope (10 to 40X), Zeiss2

5. Inverted microscope with an UV light attachment (10 to 100X), Nikon*

6. Centrifuge, table top, 1000 rpm

7. pH Meter, Microelectrodes

8. Osmometer

9. Hemacytometer, standard, with a 10-jtl pipette

B. Chemicals

1. TCM-199 (25 mM HEPES, Earle’s salts), no. 380-2340AG, GIBCO4

2. Dulbecco’s phosphate-buffered saline (PBS) (pH 7.2, 270 mOsm), no.

1300EB4

3. Heat-treated fetal bovine serum (FBS), no. 240-6000AG*

4. Brackett-Oliphant medium (B-O medium) (Appendix Table 1)

5. Calcium ionophore (A23187), no. C-7522, Sigma3

6. Bovine serum albumen (Fraction V), no. A-43783

1 Nuaire, Minneapolis, MN2 Zeiss, Thomwood, NY3 Nikon, New York, NY4 GIBCO, Grand Island, NY3 Sigma Chemical, St. Louis, MO

7. Polyvinyl alcohol (PVA), no. P-81365

8. Penicillin-G, potassium, 100 IU/ml, Squibb-Marsam6

9. Streptomycin sulfate, 100 jig/ml, Eli Lilly7

10. Hoechst stain, no. 33342, no. B-22615

11. 70% Ethanol solution

12. Caffeine-sodium benzoate5

13. Mineral oil, medical grade

14. Distilled, deionized water, Milli-Q, 1200 ml

C. Supplies

1. Sterile conical centrifuge tubes, 15 ml, Coming®

2. Sterile culture dishes, four-well multidishes, Nunclon9

3. Plastic petri dish, 100 mm, Falcon10

4. Plastic petri dish, 35 mm10

5. Sterile plastic syringe, 5 ml, Becton-Dickenson11

6. Sterile needles, 20 gauge11

7. Sterile Pasteur glass pipettes

8. Acrodisc syringe filter, 0.22 /un, no. 4192, Gelman12

6 Squibb-Marsam, Cherry Hill, NJ7 Eli Lilly & Co., Indianapolis, IN8 Coming, New York, NY9 Nunclon, Denmark

10 Falcon, New York, NY11 Becton Dickenson & Co., Lincoln Park, NJ12 Gelman Sciences, Ann Arbor, MI

9. Sterile glass beakers, 500 ml

10. Adjustable pipettes, 2 tolO /*1, 10 to 100 /tl, 100 to 1,000 /tl

Procedure

Ovary collection and oocyte in vitro maturation

1. Transport the ovaries obtained from heifers and mature cows at an abattoir

to the laboratory at temperatures S25°C within 3 to 5 hours (h) in sterile

Dulbecco’s PBS with antibiotics (100 IU/ml of penicillin-G and 100 /rg/ml

of streptomycin sulfate).

2. Wipe the bench surface and rinse hands with 70% ethanol.

3. In the laboratory, rinse the ovaries 3 times in Dulbecco’s PBS with penicillin

and streptomycin. As a precautionary measure, latex rubber gloves are used

when handling the ovaries.

4. Gently dry each ovary with a sterile paper towel.

5. Aspirate 3- to 8-mm diameter follicles at room temperature (22° to 25 °C)

with a 20-gauge needle attached to 5-ml plastic syringe. Place follicular fluid

aspirated from follicles in 15-ml conical centrifuge tubes.

6. After a sedimentation interval (5 min after the last follicle contents are added

to the centrifuge tube), aspirate the top layer of follicular fluid leaving 1 to

2 ml of fluid in the bottom of the centrifuge tube.

7. Add 8 ml of PBS with .1% PVA and antibiotics (washing medium) to

centrifuge tube, mix and allow oocytes to settle by gravity and then discard

40supernatant. Wash the oocytes three times using this procedure, allowing the

oocytes to settle via gravity between each washing.

8. After the third washing, resuspend the oocytes in 8 ml of washing medium

and place contents into a 100-mm sterile plastic petri dish for oocyte

evaluation. Rinse the centrifuge tube twice with the washing medium to

recover any remaining oocytes.

9. Transfer oocytes with intact cumulus cells to a 35-mm petri dish and wash

twice in washing medium under a laminar flow hood. We recommend not

using fluorescent light during this procedure because it may be harmful to

oocytes during maturation (Hirao and Yanagimachi, 1978a).

10. Wash cumulus-intact oocytes (20 to 25/group) with .5 ml of room temperature

TCM-199 with 5% FBS. Place the oocytes in .5 ml of TCM-199 with 5%

FBS (22° to 25 °C) in each culture well of a four-well culture plate. Cover

each of these culture wells with .5 ml room temperature-equilibrated mineral

oil.

11. Incubate the four-well culture plates at 39 °C under 5% C 02 in air for 22 to

24 h.

12. To verify oocyte maturation, cumulus-intact oocytes that did not fertilize after

the maturation procedure were exposed to 2% aceto-orcein stain and nuclear

material evaluated for metaphase-II morphology. These oocytes were then

carefully evaluated for extrusion of the first polar body.

41B. Sperm Cell Capacitation

1. Frozen semen from a group of progeny-test dairy and beef bulls (n=5) was

evaluated for oocyte IVF capacity and semen from one ejaculate from two of

these IVF-compatible bulls and was individually processed and stored in LN2

for the subsequent IVF procedure.

2. During the IVF procedure, 2 units (e.g, .25 ml plastic semen straw) of semen

(from the same bull or two different bulls) were thawed in 25 °C water bath

for 1 min. Thawing procedure may vary depending on specific

recommendations of the organization packaging the frozen semen. In each

case, it is recommended to carefully follow the thawing procedure prescribed

by the processor.

3. Underneath a sterile hood, the semen straw contents were transferred into a

15-ml conical centrifuge tube.

4. The sperm cells were washed twice with 5 ml of B-O medium (Appendix A)

supplemented with 10 mM caffeine sodium benzoate, each time centrifuging

at 800 rpm for 6 min.

5. The sperm cell pellet resulting from centrifugation is then resuspended in that

volume of B-0 medium (usually 2 ml) to adjust the sperm cell concentration

to 3 x 106 motile sperm cells/ml.

6. A 20-pM Ca++ ionophore solution was prepared by adding 2.5 ml of Milli-Q

water to 50 pi of a 1 mM stock solution of Ca++ ionophore A23187

(Appendix B).

42

7. The 20-/tM Ca++ ionophore solution (10 /tl) was added to 2 ml of the

resuspended sperm cells (3 x 10s motile sperm cells/ml) for 1 min. The final

concentration for sperm cell capacitation medium will then be . 1 /*M of Ca++

ionophore.

C. In vitro Fertilization

1. After oocyte maturation for 24 h, 20 to 25 cumulus intact oocytes were

washed once with B-O medium supplemented with 20 mg/ml of bovine serum

albumin (BSA) and then transferred immediately into 50-/il microdrops of the

B-O medium with BSA into a 35-mm petri dish.

2. The oocytes were then covered with a 2.5-ml layer of mineral oil (22° to

25°C).

3. A aliquots (50-Atl) of capacitated sperm suspension were added to each droplet

to give a concentration of 1.5 x 106 motile sperm/ml of B-O medium with

BSA.

4. Capacitated sperm cells and cumulus-intact oocytes within the 100 /il

fertilization droplets were then incubated at 39° C with 5% C 02 in an

atmosphere of humidified air for 5 h.

D. Embryo Development During In Vitro Co-culture

1. After 5 h of incubation with sperm cells, the oocytes with attached cumulus

cells were washed once with .5 ml of fresh TCM-199 supplemented with 5%

FBS.

43

2. The oocytes were covered with a layer mineral oil and then incubated in an

atmosphere of 5% C 02 and air, at 39°C for an additional 43 h.

3. After 48 h of incubation (insemination interval), cumulus cell attached to the

embryos were removed by gently teasing with a 30-gauge needle. The

cumulus cells were then allowed to remain in the culture wells with the

developing embryos.

4. One half of the TCM-199 culture medium was removed at this time and

replaced with fresh TCM-199 medium with 5, 10 or 20% FBS to activate the

cumulus cell culture system.

3. The culture medium was replaced with fresh medium at 48-h intervals during

co-culture, To maximize in vitro development, fresh cumulus cells was added

to the culture system at the time the culture medium was replaced. The

embryos were examined for development using an inverted microscope (40-

100X) at the time the medium was changed.

6. On days 7 and 8 after IVF morulae and blastocysts derived from this

procedure were either transferred to recipient females or frozen in liquid

nitrogen. Some embryos were stained with the nuclear stain Hoechst stain

no. 33342 to aid in assessing cell number during and following co-culture.

Results and Discussion

The experimental results obtained using this modified approach to the in vitro

production of bovine morulae and blastocysts are encouraging. Embryos derived from

in vitro maturation and in vitro fertilization progressed rapidly through the 8- to 16-cell

44in vitro developmental block stage when the embiyos were co-cultured with endogenous

cumulus cells. The actual result from one recent 30-day trial using the procedure

described herein on dairy and beef cows/heifers are presented in Table 1. These results

are similar to and, in most cases, better than those reported to date for IVM and IVF-

derived embryos from bovine oocytes obtained at slaughter (Fukui and Ono, 1988; Iritani

et al., 1984; Lu et al., 1988). This approach to in vitro culture makes the IVF procedure

less time consuming, more efficient, and less costly when compared with the other in

vivo and in vitro culture methods presently in use at commercial cattle embryo transplant

stations.

This approach simplifies many of the procedures reported in the scientific

literature that have subsequently become a part of many bovine oocyte in vitro maturation

and in vitro fertilization protocols used today. After processing over 6,000 bovine

oocytes using this modified procedure in our laboratory, it now seems that the need for

adding gonadotropin [e.g., luteinizing hormone (LH) and follicle stimulating hormone

(FSH)] and estradiol-17/3 to the maturation medium, the need for oocyte movement

(rocking action) during the maturation procedure, the need to conduct in vitro maturation

and fertilization procedures in a heated room (>30°C), and the need for "swim up" and

extended capacitation incubation procedures are certainly lessened or possibly eliminated

from the standard procedure with the simplified method described herein. With this

procedure we normally expect a 90 to 95% maturation rate, an 85 to 90% fertilization

rate, a 65 to 75% cleavage rate and 42 to 50% of the IVF-derived embryos to reach the

morula stage of development from a random group of bovine oocytes harvested from

45

Table 1. Results after IVM, IVF and cumulus cell co-culture of bovineoocyte during a 30-day trial

No. of No. Mean Morphological development*

oocytes exposed CCE No. No. No. No. No. No.

selected11 to IVMC score*1 MATR FERT CLEV MORL EXBLST HBLST3,212 3,081 3.81 2,927 2,650 2,101 1,350 493 293

% 96% — 95% 86% 68% 44% 16% 9.5%

*MATR=:Matured; FERT= fertilized; CLEV= cleaved; MORL= morulae;EXBLST=expanded blastocyst; HBLST=hatched blastocyst.

bOocytes with tightly compacted cumulus cell layers.

°Final evaluation and selection for in vitro maturation procedure.

dCumuIus complex expansion score: l=poor to 4 = excellent.

46

ovaries of healthy, well-nourished, cyclic cows at the time of slaughter (Figures 1 and

2).

The simplified procedure outlined does not call for the addition of supplemental

LH, FSH, or estradiol-170 to the maturation medium as previously been proposed

(Staigmiller and Moor, 1984), and used routinely by others in bovine (Aoyagi et al.,

1988; Shioya et al., 1988), ovine (Fukui et al., 1988), and porcine (Mattioli et al., 1988;

Nagoi et al., 1988) oocyte maturation procedures. The approach described herein does

include 5% heat-treated FBS in the maturation medium, which could make available to

the oocytes small amounts of these hormones during the maturation and culture process.

The value of FBS to in vitro maturation and fertilization of bovine oocytes, however, has

not been clearly defined. In a recent experiment at this laboratory (unpublished data),

400 bovine oocytes were placed in the TCM-199 with 5% FBS or placed in TCM-199

without FBS. Maturation rates (95.4 vs. 96.5) were similar but significantly increased

fertilization and cleavage rates, and a greater percentage of the IVF-derived embryos

reaching the blastocyst stage in vitro were found in the system containing FBS compared

with the same maturation system without FBS. It is interesting to note that in this study

80% of the oocytes were fertilized and 67% of the oocytes cleaved after oocyte

maturation without either supplemental hormones or FBS. Further observations in our

laboratory suggest that parthenogenic activation and cleavage (2-cell stage) occurs in up

to S3% of the bovine IVM oocytes not exposed to sperm cells.

The simplified procedure uses caffeine (Kajihara et al., 1987; Niwa and Ohgoda,

1988) and Ca++ ionophore A23187 to aid in capacitation of the frozen-thawed bovine

47

Figure I. Bovine cumulus oocyte complexes (COC) used for producing IVF-derived embryos in this study. Immature cumulus-intact oocytes aspirated from 2 to 8 mm follicles (a). Oocytes that matured for 24 h in TCM-199 with 5% FBS (b). Magnification: X100.

48

Figure 2. IVF-derived embryos produced from this study. Eight- to 16-cell stage embryos developed 3 days post-insemination with cumulus cell co-culture. Magnification: X200 (a). Expanded blastocysts and hatched blastocysts developed 10 days postinsemination with cumulus cell co-culture. Magnification: X100 (b).

49

sperm cells similar to that previously reported (Aoyagi et al., 1988; Hanada, 198S;

Sugawara et al., 1985). With this method the sperm cells are exposed to the ionophore

for only 1 min. This approach was modified from that of an earlier experiment with in

vitro capacitation of mammalian sperm cells (Brackett and Oliphant, 1975), and is much

less time consuming than the longer, more commonly used methodologies using heparin

to enhance capacitation (Leibfried-Rutledge et al., 1987; Parrish et al., 1986).

In a recent experiment in this laboratory (Zhang et al., 1990), the effect of three

different ovary holding temperatures during transport from the slaughterhouse to the

laboratory were evaluated. When bovine ovaries were cooled on ice at 0°C to 2°C

during transport, lower rates of fertilization and embryo development resulted (P < .05)

compared with transport temperatures of 18°C or 25°C. This has recently been

confirmed by another laboratory making similar temperature comparisons on bovine

ovaries (Gordon and Lu,1990). The reason for this is unclear. When IVM-IVF

procedures were conducted at room temperature (22 to 25 °C) and at 30 to 32 °C in other

preliminary experiments in this laboratory, there was no difference in fertilization,

cleavage and subsequent embryo development rates. These findings indicate that cooling

the bovine ovary before processing may be more detrimental than once expected;

however, allowing cumulus-intact oocytes to cool to room temperature during laboratory

processing may not be as critical to the overall IVM-IVF procedure as once thought

(Lenz et al., 1983).

The results of this study verify the reports of others (Aoyagi et al., 1988;

Leibfried-Rutledge et al., 1987; Parrish et al., 1986) in that sperm from different bulls

50

within the same breed differ greatly in their ability to fertilize ova during the IVF

procedure. We have also noted that frozen semen from the same collection gives more

repeatable results during bovine IVF than that of different ejaculates from the same bull

or from different bulls.

In a recent study in this laboratory (Zhang et al., 1991), cumulus-intact oocytes

(n=170) from heifers were compared with cumulus-intact oocytes (n = 170) collected

from mature cows using the same IVM-IVF procedure. The results from this study

showed that oocytes harvested from mature cows gave similar maturation, fertilization

and early cleavage rates; however, the percentage of the cleaving embryos reaching the

expanded blastocyst and the hatched blastocysts stages in vitro was greater (P< .05) for

mature cows than of heifers. These observations on parity of the females are in

agreement with results of other recent IVF studies (Kajihara et al., 1987, 1991).

Possibly, the time interval needed for oocyte maturation is different between mature cows

and young heifers (Kajihara,Y, personal communication).

Using the animal’s own follicular granulosa/cumulus cells to aid in embryo

development to morula-stage embryos has consistently given the better results than of

other in vitro culture and co-culture systems presently in use in our laboratory. The

number of blastomeres per cultured embryo at the morula stage was similar to the

number stained and counted for fresh embryos collected from donor cows on the same

day post-fertilization. After a basic IVM-IVF procedure with cumulus cell co-culture in

our laboratory, 16 good quality morulae were individually transferred to day 7 and 8

recipient cattle, resulting in eight transplant pregnancies (50%) past 90 days and the birth

51

of seven live, healthy IVF offspring. The current consensus is that development of IVF-

derived embryos to the blastocyst stage in vitro suggests that normal fertilization has

occurred under laboratory conditions (Staigmiller and Moor, 1984).

Despite the use of various chemically defined media, very little success has

resulted with in vitro embryo culture procedures. This has been overcome to some

degree by using various cell types in co-culture systems. The use of endogenous

cumulus cells and increasing levels of FBS in the culture system certainly has merit and

should not be overlooked as a culture system for IVF embryos. The cumulus cell co

culture system is easy to maintain, efficient, and produces results similar to and in most

cases better than those reported for other culture or co-culture systems. This approach

is cost efficient because the cumulus cells are a by-product of the IVM-IVF procedure.

One drawback with this co-culture system becomes evident when one cultures

IVF-derived oocytes to the expanded blastocyst and hatching stages in vitro. When only

homologous cumulus cells supplemented with FBS are used in the culture system, half

or less of the embryos develop from the morula to the expanded blastocyst stage of

development. The reason for this reduced development is unclear. Apparently, other

components (growth factors) are needed in the in vitro system that are not present or not

available to the embryos in sufficient concentrations during extended incubation with

bovine cumulus cells. Furthermore, one can not rule out the possibility of increasing

levels of toxic metabolic by-products in the co-culture system hindering further

embryonic development at this stage of incubation.

52

To maximize in vitro development of IVF-derived embryos, we suggest adding

fresh cumulus cells to the culture system at 48-h intervals during incubation, when the

culture medium is exchanged. This may make enough of the unidentified growth

factor(s) available to increase the number of embryos developing to the blastocyst stage.

Possibly, a multilayered cell culture system of more than one type of cell (e.g., oviductal

and cumulus cells) could also be used to maintain the development of these IVF-derived

morular to expanded or hatched blastocyst stage (Rodriguez et al., 1991).

CHAPTER 4

EFFECT OF TEMPERATURE AND C02 LEVELS ON IN VITRO MATURATION AND FERTILIZATION OF BOVINE OOCYTES

Introduction

In vitro maturation (IVM), fertilization (IVF) and culture (IVC) of farm animals

oocytes can provide a large supply of early stage embryos for basic embryo research,

and will allow scientists to study the details of the maturation and fertilization process.

At present, although the procedure for IVM/IVF/IVC of bovine oocytes has been

established (Brackett et al., 1977; Iritani and Niwa, 1977; Brackett et al., 1982a; Parrish

et al., 1986) and calves have been bom after nonsurgical transfer of fresh IVM/IVF/IVC

embryos (Lu et al., 1988; Goto et al., 1988; Xu, et al., 1992a; Reichenbach et al., 1992)

or frozen-thawed IVM/IVF/IVC embryos (Fukuda et al., 1990; Zhang et al., 1993) from

abattoir ovaries, the efficiency of the in vitro system is relatively low. Since the current

IVF procedure is simply an effort to mimic the reproductive tract of the female animal,

the physiological factors, such as in vitro temperature and C Q environment on

maintaining oocyte viability, maturation, fertilization and early development are not fully

understood.

Cooling intact abattoir ovaries has been reported to be detrimental to oocyte

viability (Yang et al., 1990; Smedt et al., 1992). Apparently, the sensitivity of the

oocyte to cooling varies among different species (Smith and Tunney, 1978). However,

a negative effect on later development of IVF-derived embryos has also been reported

53

54

after bovine ovaries were held at a temperature close to body temperature (37°C) (Yang

et al., 1990; Sekin et al., 1992).

Presently, the recommended incubation conditions for bovine IVF is 39 °C in 5%

C 02 in air or 5% C 02, 5% Oa and 90% N2 (First and Parrish, 1987; Younis et al.,

1989). This incubation temperature is thought to be based on internal core body

temperature of the cow (Anderson, 1970). An early research study, using 37°C for

bovine IVF, strongly suggested that this temperature lowered the success rate of in vitro

fertilization (see review by Wright and Bondioli, 1981). Later, Brackett et al. (1982)

obtained the first bovine IVF offspring following fertilization incubation at 38°C. Lenz

et al. (1983) subsequently reported that bovine in vitro fertilization was a temperature

dependent process and that higher fertilization rates should result with increased

temperatures. Grinsted et al. (1980) reported that in vivo follicle temperatures were

lower compared with the internal core body temperature of the rabbit by 2.8°C, and that

the temperatures were similar for both large and small follicles. The physiological

significance of a temperature gradient on follicle growth and oocyte maturation in vivo

is presently unclear.

A bicarbonate buffering system has been shown to be beneficial for oocyte

maturation and embryo development in mammalian species (Wales et al., 1969; Quinn

and Wales, 1974; Kane, 1975; Wright et al., 1976a). Eng et al. (1986) showed that the

appropriate level of C 02 in the gas phase is required not only for maintaining the

appropriate medium pH, but is also intimately involved in in vitro oocyte and embryo

55

metabolism. However, C 02 levels have not been investigated extensively as a variable

that may influence oocyte maturation and in vitro fertilization.

The objectives of this study were: 1) to evaluate the effect of different ovary

holding temperatures during transportation on the developmental potential of bovine

oocytes and 2) to evaluate the effect of incubation temperature and CQj levels on bovine

oocytes in in vitro maturation and fertilization.

Materials and Methods

Experimental design

Experiment 4.1. Ovary holding temperature and bovine IVM/IVF/IVC

The first experiment was designed to evaluate ovary holding temperatures on

oocyte maturation, fertilization and subsequent embryo development. In this experiment,

three holding temperatures were evaluated. Prior to animal slaughter, three thermos

bottles were filled with water at either 25°C, 18°C or 0°C (with ice). Three 50-ml

sterile plastic vials containing physiological saline were placed into each thermos.

Ovaries from six immature slaughterhouse beef heifers (■*6 month old) were

immediately placed randomly in one of three 50-ml vials, and placed into the thermos

bottles. The bottles were then transported to the St. Gabriel Physiology Laboratory. The

elapsed time from ovary collection to oocyte aspiration was 3 h. The ovaries were then

subjected to a standard bovine IVF procedure, as outlined in Chapter 3. The

experimental outline is shown in Table 2.

56

Table 2. Experimental design for Experiment 4.1

Ovary temperature No. of ovaries* No. of oocytes

0°C 4 25

18°C 4 25

25 °C 4 25

•Ovaries randomly placed across treatment temperatures.

57

Experiment 4.2. Incubation temperature, C 02 levels and bovine oocyte fertilization

The second experiment was designed to evaluate incubation temperatures and C 02

levels on oocyte maturation and fertilization. Cumulus oocyte complexes (COC)

harvested from mixed-breed beef heifers were randomly assigned to one of four treatment

groups as follows: 1) oocytes matured at 37CC and fertilized at 37°C in 2.5 % CO* in air,

2) oocytes matured at 37°C but fertilized at 39°C in 2.5% CO2 in air, 3) oocytes

matured at 39°C and fertilized at 39°C in 2.5% CQ2 in air and 4) oocytes matured at

39°C and fertilized at 39°C in 5% C 02 in air (standard IVF procedure, as in Chapter 3).

The experiment treatments and number of oocytes assigned per treatment is shown in

Table 3. After 24 h of maturation, cultured oocytes were inseminated with ionophore

A23187-treated sperm cells. The cleavage rate was evaluated 48 h after insemination and

used as an indication of successful in vitro fertilization. All cleaved embryos were

subsequently cultured at 39°C in 5% C 02 in humidified air and randomly assigned to

another experiment (data not shown here).

Ovary transportation and oocyte collection

Ovaries for these experiment were harvested from cyclic, mixed breed beef

heifers at a local abattoir immediately after slaughter and transported at 25°C (or other

temperatures as described in specific experiments) to the St. Gabriel Physiology

Laboratory in physiological saline (.9% NaCl). In the laboratory, ovaries were rinsed

three times in saline containing antibiotics (100 IU penicillin-G, 100 ftg streptomycin

sulfate/ml) and dried with a sterile paper towel. Oocytes were collected by aspiration

of 2- to 8-mm antral follicles using an 18-gauge needle attached to a 6-ml sterile, plastic

58


Culture condition* No. of oocytes

37°C/37°C (2.5% COj) 600

37°C/39°C (2.5% CO^ 600

39°C/39°C (2.5% COj) 600

39°C/39°C (5% COj) 600

♦Culture condition means the incubation temperature and C 02 level in each treatment group: maturation temperature/ fertilization temperature (C02 level).

59

syringe. Follicular fluid was placed in 50-ml conical centrifuge tubes. After a

sedimentation interval (3 to 5 min.), supernatant was discarded and 10 ml of Dulbecco’s

phosphate-buffered saline (PBS, Gibco, Grand Island, NY) with .1% Polyvinyl alcohol

(PVA, Sigma, St. Louis, MO) was added to the centrifuge tube. The oocyte-PBS

suspension was placed into a 100-mm sterile plastic petri dish for oocyte evaluation.

Oocytes with intact cumulus and homogeneous cytoplasm were used in this study. Ovary

washing and oocyte collecting were performed at room temperature (23 to 25°C).

In vitro maturation and fertilization

The bovine IVF procedure used was previously developed in this laboratory

(Chapter 3). The standard procedure was changed only for temperatures and CO2 levels

as designated by the experimental design. Briefly, selected oocytes were washed two

times in PBS with .1% PVA, two times in Tissue Culture Medium-199 (TCM-199; 25

mM HEPES, Earle’s salts, Gibco) supplemented with 5% fetal bovine serum (FBS,

Hyclone, Logan, UT) and placed in four-well culture plates (Nunclon, Denmark) (20-25

oocytes/well) containing .5 ml of TCM-199 with 5% FBS in each well. Oocytes were

incubated 22 h for maturation at 39° C in 5% CO2 in an atmosphere of humidified air.

Approximately 1 to 2 h prior to insemination, two straws of frozen semen from

a pre-selected Holstein dairy bull of known fertility were thawed in a 25 °C water bath

for 1 min. The sperm cells were washed twice in Brackett-Oliphant (B-O) medium

(Brackett and Oliphant, 1975) supplemented with 10 mM caffeine (Niwa and Ohgoda,

1988) and adjusted to a concentration of 3 x 10® motile sperm cells/ml. Sperm cells were

then exposed to .1 /xM of calcium ionophore A23187 for 1 min to aid in capacitation

60

(Hanada, 1985; Zhang et al., 1992b). Approximately 50 pd of a sperm suspension was

quickly added to each 50 /*1 fertilization droplet containing oocytes. The fertilization

medium was B-O medium supplemented with 20 mg/ml bovine serum albumin (BSA,

Fraction-V, Sigma). Oocytes and sperm cells were co-incubated at 39°C in 5% COi in

humidified air.

After 6 h of incubation, oocytes with attached cumulus cells were washed with

TCM-199 supplemented with 5% FBS and transferred into four-well culture plates

containing .5 ml TCM-199 with 5% FBS. Oocytes were cultured for an additional 43

h to evaluate the cleavage rate. The cleaved embryos were subsequently cultured on a

cumulus cell monolayer (Goto et al., 1988; Zhang et al., 1992b) at 39°C in 5% C 02 in

humidified air until the embryos reached the hatched blastocyst stage. The culture

medium (TCM-199 with 5% FBS) was replaced with fresh medium at 48-h intervals and

the embryo development recorded immediately after medium change. All uncleaved

oocytes (in Experiment 4.1) were fixed at 48 h post-insemination in ethanol:acetic acid

(3:1) for 24 h and stained with 2% aceto-orcein to examine the arrested stages (GV, M-I,

M-II and PN).

Statistical analysis

The difference in the proportion of oocytes that matured, fertilized and developed

to blastocyst and hatched blastocyst stage among treatment groups were tested using Chi-

square analysis for both Experiment 4.1 and Experiment 4.2. A P-value of < .05 was

defined as statistically significant.

Results

Experiment 4.1

In this experiment, the effect of three ovary holding temperatures (0°C, 18°C and

25 °C) on oocyte maturation, fertilization and subsequent embryo development was

evaluated. As shown in Table 4, oocyte maturation was impaired when ovaries were

held at 0°C or 18°C for 3 h during transportation compared with 25°C (20%, 58% and

100%, respectively; PC .05). Fertilization rates in 0°C, 18°C and 25°C group were

10 %, 54 % and 95 %, respectively, which was also significantly different among treatment

groups (PC.05). There was no cleavage when ovaries were hold at 0°C during

transportation although 10% of the oocytes had decondensed sperm heads or showed

evidence of two pronuclei. Significantly more oocytes cleaved in the 25 °C group (85%)

than in the 18°C group (25%) (PC.05). The proportions of the oocytes developing to

the morula and blastocyst stages from the 25°C treatment group were also greater than

those from the 18°C treatment group (50% vs. 8% and 20% vs. 4%, respectively;

PC .05). All blastocysts developed from the 25°C group hatched in vitro; however, none

of blastocysts hatched from the 18°C treatment group.

Experiment 4.2

The effect of the combination of two incubation temperatures (37 °C and 39 °C)

during maturation and fertilization and two C 02 levels (2.5% and 5%) on oocyte

fertilization was evaluated in this experiment. The result is shown in Table 5. Only 49

(8%) of the 600 oocytes cleaved when maturation and fertilization temperature were both

37°C and the C 02 level was 2.5% (37°C/37°C and 2.5% COj). The cleavage rate

62

Table 4. Ovary holding temperature on oocyte maturation, fertilization and subsequent embryo development in Experiment 4.1*

OvaryTEMP

No. ofoocytes

MATR%

FERT%

CLEAV%

MORL%

BLST%

HBLST%

0°C 20 201 10* 0* - - -

18°C 24 58* 54b 25b 8* 4* 0*

25 °C 20 100b 95e 85* 50b 20b 20b

*TEMP=temperature; MATR=matured; FERT=fertilized; CLEAV= cleaved; MORL= morula; BLST=blastocyst; HBLST=hatched blastocyst.

*bcValues with different superscripts within columns are different (P<.05).

63

Table 5. Incubation temperature and C 02 level on bovine oocyte fertilization

Culture condition* No. of oocytes No. cleaved (%)

37°C/37°C (2.5% CO,) 600 49 (8 .1 /

37°C/39°C (2.5% CO,) 550 152 (27.6)b

39°C/39°C (2.5% COj) 600 148 (24.7)b

39°C/39°C (5% C02) 610 480 (78.7)e

♦Culture condition means incubation temperatures and COz levels in each treatment groups: maturation temperature/fertilization temperature (CO, level).

*,b cValues with different superscripts within columns are different (P < .05).

64

(27.6%) was significantly increased when oocytes were matured at 37°C, but fertilized

at 39°C in 2.5% CO2 (37°C/39°C and 2.5% CO2 ). When oocytes were matured at 39°C

and fertilized at 39°C in 2.5% COz in air (39°C/39°C and 2.5% COz) the cleavage rate

was 24.7%, which was significantly greater than 37°C/37°C and 2.5% COz(P<.05),

but not different from 37°C/39°C and 2.5% C 02 (P> .05). The greatest cleavage rate

(78.7%) was obtained when oocytes were matured at 39°C, fertilized at 39°C and the

C 02 level was raised to 5% (39°C/39°C, 5% COz).

Discussion

From previous observations made in our laboratory, it was suggested that ovary

holding temperature is a possible source of variability in the success of in vitro

fertilization of bovine oocytes. To test if ovary holding temperature during transportation

of slaughterhouse ovaries to the embryo laboratory was detrimental, a comparison of

three holding temperatures (25°C, 18°C and 0°C) was performed. The result showed

a significant effect of ovary holding temperatures on oocyte maturation, fertilization and

subsequent development. Temperatures below 25°C (18°C and 0°C) decreased oocyte

maturation and embryo development rates, and this detrimental effect was greater at 0°C

than at 18°C.

These results are in agreement with those of Yang et al. (1990), who reported

the detrimental effect of holding ovaries at 4°C on cleavage and blastocyst formation

compared with those transported at 25°C. However, in that experiment, they did not test

any temperatures between 25°C and 4°C that could easily occur during ovary

65

transportation, if the temperature was not well controlled. Our results showed that

oocyte maturation and embryo development were also impaired when ovaries were held

at 18°C for 3 h compared with 25°C. This finding suggests that bovine oocytes are

more sensitive to low temperature than mouse oocytes. Smith and Tenney (1978) have

reported that the in vitro maturation of mouse oocytes was not affected by cooling murine

ovaries on ice as long as 4 h before maturation incubation. The different sensitivity

between these two species may be due to the difference in plasma membrane structure,

organelle structure and/or composition of the cytoplasm (Pickering et al., 1990).

One of the detrimental effects of cooling oocytes that has been reported recently

is disruption and disassembly of microtubules of the second meiotic spindle. Park and

Ruffing (1992) have demonstrated that more than 50% of bovine IVM oocytes had

reduced or absent meiotic spindles when exposed to 25 °C for 1 min. Pickering et al.

(1990) also reported that cooling human oocytes to room temperature for 10 min caused

spindle disorganization in 50% of the oocytes examined, and less than one half of those

oocytes with disorganized spindles were able to reassemble the spindle to the normal

appearance following 4 h of recovery at 37°C. In contrast, our results showed no visible

detrimental effect on oocyte maturation, fertilization and subsequent development when

ovaries were held at 25°C for 3 h. The possible explanation may be that the cooling

damage to the oocyte is developmental stage dependent. In the present study, ovaries

containing germinal vesicle (GV) stage oocytes were cooled rather than metaphase- II

(M-II) oocytes that were used in the study by Pickering et al. (1990) and Park and

Ruffing (1992). GV-stage bovine oocytes may be able to tolerate room temperature

66

without being impaired for later development. M-Il oocytes, however, are likely to be

more sensitive to low temperature because of the presence of the meiotic spindle in which

the microtubules are easily disturbed by cooling. If this is the case, the survival rates

after cooling or freezing of GV-stage bovine oocytes should be better than that of M-n

stage oocytes.

Another possible cause of cooling damage to the oocyte is premature cortical

granule reaction, which causes reduced in vitro fertilization rates in mouse (Johnson et

al., 1988). This may be true in present study since only 10% of the oocytes from

ovaries held at 0°C were considered to be fertilized. This was less likely to occur in the

18°C treatment group because in vitro fertilization across matured oocytes appeared to

be normal. Therefore, the reduced oocyte maturation and embryo development rates

when ovaries were held at 18°C may due to some unknown factor(s), such as the

changes in oocyte cytoplasm and/or membrane structure and function (Quinn, 1989).

The second experiment evaluated the effect of different combinations of incubation

temperatures (37°C vs. 39°C) and C 02 levels (2.5% vs. 5%) on in vitro fertilization of

in vitro matured oocytes. A large number of oocytes were used in this experiment, and

some interesting findings were generated. First, the results showed that the incubation

temperature during fertilization had a significant effect on cleavage rate when the same

maturation temperature and the same C 02 level were used. The fertilization rates were

8.1% for maturation/fertilization at 37°C/37°C and 2.5%COi and 27.6% for 37°C/39°C

and 2.5%C02 (P < .0001). Secondly, when oocytes were matured at 37°C but fertilized

at 39°C under 2.5%C02, the cleavage rate (27.6%) was as good as that of oocytes

matured at 39°C and fertilized at 39°C under 2.5%C02 (24.7%) (P> .05), which

suggested that 37 °C may be a suitable incubation temperature for bovine oocyte in vitro

maturation. This findings are in partial agreement with those of Lenz et al. (1983), who

reported that 39 °C was significantly better than 37 °C for both in vitro maturation and

in vitro fertilization of bovine oocytes. Although the recommended incubation

temperatures for bovine oocyte in vitro maturation and fertilization, at present, are both

39°C, which is similar to the core body temperature of the cow, the temperature of the

antral follicles of female rabbit has been reported to be *>3°C lower than the internal

core body temperature of the doe. Similar temperatures were found from both large and

small follicles (Grinsted et al., 1980).

Jn vitro studies have also given evidence of a beneficial effect of 37® C on oocyte

maturation in cattle compared with 39°C. Katska and S mo rag (1985) reported no

significant differences were found for the percentages of oocytes reaching metaphase-II

after 20 h of culture at 37, 38 and 39°C. However, the results of the fluorescein

diacetate (FDA) viability test showed that the fluorescein accumulation in the oocytes

decreased proportionally to increased culture temperature, which indicates that the oocyte

viability was decreased as culture temperature increased. Thus, the relatively low

temperature in the follicles of the female (the site of oocyte maturation in vivo in most

mammalian species), as well as in vitro maturation incubation may have a positive effect

on oogenesis that is similar to the effect of lower temperature on spermatogenesis in the

male testes (Fisch et al., 1990).

68In contrast, fertilization apparently requires a higher temperature than oocyte

maturation. The elevated temperature during in vitro fertilization seems to be able to

stimulate the metabolic processes of both male and female gametes. It has been reported

that the sperm capacitation and acrosome reaction are temperature dependent (Mahi and

Yanagimachi, 1973). The percentage of bovine sperm cells undergoing acrosome

reaction increased as the incubation temperatures were increased (Lenz et al., 1983).

The sperm-oocyte fusion and post-fusion events (Hirao and Yanagimachi, 1978), as well

as, the cleavage and metabolic rates of preimplantation embryos (Lavy et al., 1988; Ryan

et al., 1992) have been shown to be temperature dependent. It is also well known in

humans that the basal body temperature raises .5 to .8°C at the time of ovulation, which

indicates that fertilization and subsequent embryo development occurs at a higher relative

temperature in vivo. This stimulatory effect of elevated temperature on in vitro

fertilization and subsequent embryo development, however, has been found only within

a narrow temperature range. Further elevation of incubation temperature caused

decreased sperm viability, percent oocyte maturation, fertilization (Lenz et al., 1983) and

an increased embryo degeneration rate (Lavy et al., 1988; Ryan et al., 1992).

Oviductal fluid is rich in bicarbonate and has been shown beneficial for in vitro

fertilization (Brackett and Mastroianni, 1974; Maas et al., 1977). Bicarbonate-buffered

culture medium with 5% C 02in air (First and Parrish, 1987) or 5% C 02, 5% 0 2 and

90% N2 (Younis et al., 1989) as the gas phase are commonly used for bovine in vitro

maturation and fertilization. Since bicarbonate ion and C 02 can not be separated in

69

current culture conditions, changes in the C 02 level in gas phase will change the

concentration of bicarbonate ion as well as pH of the culture medium.

Recently, contradictory findings have been reported on the effects of the COa

level on embryo development in vitro. McKierana and Bavister (1990) reported that

significantly more 2-cell hamster embryos developed to the blastocyst stage with

increased C 02 concentration from 5 to 7.5% or 10%. However, Wang et al. (1992)

reported that a significantly greater blastocyst yield was obtained under 5% CO, than

10% C 02 when culturing 2- to 8-cell IVF-derived bovine embryos. In the present study,

we evaluated the effects of bicarbonate and C 02 on bovine in vitro fertilization by

comparing maturation and fertilization incubation under 5% C 02 with 2.5% CQj in air.

More oocytes cleaved under 5% C 02 than 2.5% CC^ (78.7% vs. 24.7%; P < .0001) after

in vitro maturation and fertilization at 39°C. This finding further verifies that the

bicarbonate ion plays an important role during in vitro fertilization of bovine oocytes.

The pH of the medium was also measured under each gas phase and a .2 pH difference

(pH 7.1 under 5% C 02 vs. pH 7.3 under 2.5% CO2 ) was found. Since media at pH 7.2

to 7.4 are routinely used in mammalian embryo culture (Brinster, 1965), this stimulatory

effect of CO2 was unlikely due to a change in pH of the medium.

One possible explanation for this phenomena is that the higher level of CO* in the

gas phase during in vitro fertilization may accelerate sperm capacitation and/or acrosome

reaction. Boatman and Robbins (1991b) showed that bicarbonate and C 02 affect the

fertilizing ability of the hamster sperm primarily by stimulating both motility and an

earlier stage of capacitation of hamster sperm cells. When bicarbonate and COj were

70

continuously present, both progressive and hyperactivated motility (an indication of

capacitation) were stimulated in a dose-dependent manner. It was also found that

hyperactivation and zona penetration of the sperm were highly correlated. Capacitation

is a series of changes in sperm cells that normally occur in the female reproductive tract,

and only capacitated sperm are thought to be able to penetrate zona-intact oocytes

(Chang, 1951; Austin, 1952).

Another possibility is that bicarbonate ion concentration and C 02 levels may affect

oocyte in vitro maturation. Eng et al. (1986) reported that bicarbonate ion was required

for normal polar body formation. They used bicarbonate buffered medium (TCM-199E;

Earle’s salts) for pig oocyte maturation and noted twice the percentage of polar body

formation as the same medium based on a phosphate buffer system (TCM-199H; Hank’s

salts). When the Hank’s salt based medium was supplemented with a bicarbonate buffer

system, polar body formation was restored to the level in Earle’s salt based medium.

In the present study, oocyte maturation status was not evaluated. However, the low level

of C 02 (2.5%) may have impaired oocyte maturation and, therefore, decreased the

cleavage rate.

The low cleavage rate obtained by decreasing C 02 level in this study may also

reflect an interruption of metabolic events causing the failure of completion of

fertilization and first cell division. Fixation of bicarbonate/C02 has been found in the

preimplantation embryos of mouse (Biggers et al., 1967; Wales et al., 1969), rabbit

(Kane, 1975; Quinn and Wales, 1974), and monkey (Kuehl and Dukelow, 1979). Quinn

and Wales (1971, 1973) suggested that CO2 fixation by embryos may related to the

71

normal enzyme activities that are required during embryogenesis. They observed a

reduced incorporation of low molecular weight precursors into macromolecules when

embryos were cultured in phosphate-buffered medium. Thus, a low level of COa

concentration in the gas phase 'may be necessary to ensure that there are enough

bicarbonate/C02 can be incorporated into oocytes to complete the process of fertilization

and normal cell division.

In summary, the present study has demonstrated that bovine oocytes are sensitive

to low temperature. Cooling the ovary to 18°C significantly decreased oocyte maturation

and subsequent embryo development rates. Furthermore, when ovaries were maintained

on ice (0°C) for 3 h prior to processing, no cleavage occurred after in vitro insemination.

It appears that a holding temperature at 25°C is more suitable for bovine ovary

transportation than either lower or higher temperatures. Yang et al. (1990) and Sekin

et al. (1992) have also reported detrimental effects of higher ovary holding temperatures

(37 °C and 39 °C) on IVM/IVF of bovine oocytes. Our results also showed that

incubation temperature and C 02 level are very important factors on in vitro maturation

and fertilization of bovine oocyte. Best cleavage rates resulted when oocytes were both

matured and fertilized at 39°C in 5% C 02. In contrast, maturation at 37°C did not

decrease fertilization rate. Further study is necessary to evaluate the developmental

potential of those IVF-derived bovine embryos when oocytes are matured at 37°C,

fertilized and cultured for in vitro development at 39°C, since this approach may more

closely mimic the physiological body conditions found in the cow.

72

This study clearly showed that the small changes in environmental conditions can

have a big impact on oocyte in vitro maturation, fertilization and subsequent embryo

cleavage rates. Therefore, proper handling of ovaries and control of culture conditions

are essential in all embryo laboratories. In our laboratory, by employing the optimal

ovary holding temperature (25 °C) and in vitro culture conditions (39°C, 5% COJ, 65

to 75% cleavage rates and 45 to 50% morulae were consistently produced with large

numbers of oocytes (Zhang et al., 1992b; Zhang et al., 1995). Additional studies on the

basic physiological conditions, as well as metabolic requirements during fertilization are

needed to generate a better understanding of early embryo development. This will allow

us to develop a more efficient in vitro system to produce bovine embryos for research

and commercial animal production.

CHAPTER 5

CUMULUS CELL FUNCTION DURING BOVINE OOCYTE MATURATION, FERTILIZATION AND EMBRYO DEVELOPMENT IN VITRO

Introduction

The mammalian oocyte has a close relationship to the somatic cells during its

growth and developmental phase. The cumulus-oocyte complexes (COC) are maintained

by delicate cell-to-cell connections among the cumulus cells and with the oocytes (Moor

et al., 1980; Eppig, 1982). The corona radiata cells form numerous intracellular

processes penetrating through the zona pellucida and maintaining their communication

with the oocyte via gap junctions (Anderson and Albertini, 1976; Hyttel, 1987; de Loos

et a l.t 1991; Allworth and Albertini, 1993). This intercellular communication allows

metabolite transfer (Gilula et al., 1987) and maturation (see review by Buccione et al.,

1990). The breakdown of the cumulus-oocyte gap junction is correlated with oocyte

meiotic resumption (Larsen et al., 1986). Ultrastructural studies demonstrated that

complete disconnection between the cumulus cells and the oocyte occurs prior to oocyte

maturation in vivo (Larsen et al., 1987) or in vitro (Hyttel, 1987; Suzuki et al., 1994).

The exact role of the gap junctions on meiotic resumption and oocyte maturation,

however, is not clearly understood (Wert and Larsen, 1989).

Numerous reports on oocyte in vitro maturation and fertilization (IVM/IVF) have

suggested that oocyte appears to acquire developmental competence only when the

cumulus cells are in direct contact with the oocyte (Leibfried-Rutledge et al., 1989).

Significantly more cumulus-enclosed oocytes were fertilized and then developed to the

73

74

blastocyst stage in vitro when compared with cumulus-free oocytes (Shioya et al., 1988;

Mochizuki et al., 1991). A high proportion of abnormal fertilization was found when

cumulus-free oocytes were used for in vitro fertilization (Vanderhyden and Armstrong,

1989). However, all these studies involved grouping oocytes into various categories

based on morphological characteristics. Some of those cumulus-free oocytes may have

come from atretic follicles and were possibly degenerating at the time of collection (de

Loos et al., 1991). Thus, these studies on the function of cumulus cells were likely

confounded with different initial quality of the oocytes. The purpose of this study was

to investigate the effect of cumulus cells and the cumulus-oocyte transzonal connections

on in vitro maturation, fertilization and subsequent embryo development in cattle. Only

those oocytes with compact layers of cumulus cells and homogeneous cytoplasm were

used, and the cumulus cells were removed at different developmental stages based on the

experimental design for the study. These oocytes have routinely resulted in 90 to 95%

maturation, 80 to 85% fertilization and 30 to 40% blastocyst development with standard

procedures in our laboratories (Zhang et al., 1992b; Yang et al., 1993).


Experimental design

Experiment 5.1. Cumulus cell removal and co-culture on bovine IVM/IVF/IVC

This experiment was conducted in a 4 x 2 factorial arrangement. The effects of

cumulus cell removal (at four different times) and culture condition (with or without co

culture) were investigated. Cumulus cells were removed before maturation (IVM),

before insemination (IVF), 7 h post-IVF and 48 h post-IVF. The fourth treatment served

75

as a control; as cumulus cells were normally removed at this stage (48 h) in our routine

IVF protocols (Zhang et al., 1992b; Yang et al., 1993). Denuded oocytes or embryos

were then either cultured in medium alone or co-cultured with detached cumulus cells to

test if the direct connection between cumulus cells and the oocyte is necessary at specific

developmental stages (Table 6).

Experiment 5.2. Complete and partial cumulus cell removal on bovine IVM/IVF/IVC

This experiment was further extended to include cumulus cell were either removal

at 7, 20, or 48 h post-IVF. Cumulus cells were either removed completely as in

Experiment 1 or incompletely, with only a few cells (8 to 12 cells) remaining attached

to the zona pellucida. These oocytes were either cultured in medium alone or co-cultured

with cumulus cells as described in Experiment 1. The experimental outline is shown in

Table 7.

Experimental oocytes

Ovaries were collected immediately after slaughter from cows or heifers at a local

abattoir. Excised ovaries were placed in physiological saline (.9% NaCl) or Dulbecco’s

phosphate-buffered saline (DPBS) and maintained at 23 to 28°C during transport to the

embryo laboratory. In the laboratory, ovaries were rinsed three times in DPBS

containing antibiotics (100IU penicillin, 100 /tg streptomycin/ml) and dried with a sterile

paper towel. Oocytes were aspirated from small antral follicles (2 to 8 mm in diameter)

using an 18-gauge needle attached to a 6 ml sterile, plastic syringe. Oocytes with intact

cumulus and homogeneous cytoplasm were selected and randomly assigned to various

treatments in these experiments.

76


Cumulus cell removal Co-culture No. of oocytes

Before IVM Yes 200No 200

Before IVF Yes 200No 200

7 h after IVF Yes 200No 200



Treatment group Cumulus cell removal Co-culture No. of oocytes

Complete cell removal7 h after IVF Yes 100

No 10020 h after IVF Yes 100

No 10048 h after IVF Yes 100

No 100Partial cell removal




78

Cumulus cell removal

Cumulus cells were removed from oocytes or early embryos by placing them in

2.9% sodium citrate solution for 2 min followed by manual shaking or vortexing. The

detached cumulus cells were recovered to prepare monolayers. Only those oocytes or

early embryos without any attached cumulus cells were used in these experiments.

Denuded oocytes or embryos were then processed for IVM/IVF and cultured in medium

alone or in co-culture with the detached cumulus cells (1 x 10s cells/ml) as indicated in

Experiments 5.1 and 5.2. The co-culture groups of oocytes or embryos were

continuously cultured on the cumulus cell monolayers except during in vitro fertilization.

In vitro maturation and fertilization

Standard successful procedures used routinely in our laboratories (Zhang et al.,

1992; Yang et al., 1993) were employed for experiments. Oocytes were placed in 500

jul medium in four-well tissue culture plates (Nunclon, 25 oocytes/well, Experiment 1)

or 100 p\ droplets (15 oocytes/droplet, covered with Dow Coming Medical fluid,

Experiment 2). Tissue Culture Medium-199 (TCM-199) supplemented with 5% fetal

bovine serum (FBS) was used throughout the study for maturation and embryo culture.

The oocytes were matured for 22 h and the culture environment was 5% C 02 in

humidified air at 39°C.

At 1 to 2 h prior to insemination, two straws of frozen semen from a fertile dairy

bull were thawed in a 25° C water bath for 1 min. The sperm cells were washed twice

in B-0 medium (Brackett and Oliphant, 1975) supplemented with 10 mM caffeine by

centrifugation (350 x g, 6 min). The sperm cell concentration was adjusted to 3 x 10s

79

motile sperm/ml for the experiments. Sperm cells were then exposed to .1 of Ca++

ionophore A23187 for 1 min to aid in capacitation. Approximately 50 fxl of the treated

sperm cells were quickly added to each 50 /xl fertilization droplet containing oocytes.

The fertilization medium was B-0 medium containing 20 mg/ml bovine serum albumin

(BSA). The final sperm concentration was 1.5 x 106 motile sperm/ml. Oocytes and

sperm cells were co-incubated at 39°C with 5 % COj in humidified air for 6 h.

Embryo culture

After 6 h of co-incubation, the inseminated oocytes were transferred to TCM-199

with 5% FBS in four-well tissue culture plates or microdroplets and cultured for an

additional 42 h under the same condition to assess cleavage. Cleaved embryos were

continuously cultured in the same conditions for 10 to 14 days. The culture medium was

changed every 2 days and embryo development was recorded. All uncleaved ova were

fixed and stained with 2% orcein to examine the arrested stages.


The criteria for evaluation included percentages of oocyte maturation, fertilization,

and embryo development to cleavage, morula, blastocyst and hatched blastocyst stages.

The results were analyzed with SAS categorical data modeling (CATMOD) procedure

(SAS Institute, Gary, NC) and the logit model was used to fit the observed data. A P

value of < .05 was regarded as statistically significant and the best-fitted model was used

for comparisons. The differences among individual treatments were assayed by

developing 95% confidence intervals.

80

Results

Experiment 1

In this experiment, the effect of cumulus cells and their cellular connection with

the oocyte on IVM/IVF was tested by removing the cumulus cells from the oocytes at

various stages and then culturing the oocytes or embryos with or without the detached

cumulus cells.

The results of oocyte in vitro maturation, fertilization and subsequent embryo

development rates are shown in Table 8. Overall, cumulus cell removal significantly

affected oocyte fertilization and embryo development (P< .0001). There was no problem

in removing the cumulus cells from the oocytes either before or after maturation with our

procedure. Removing cumulus cells with enzyme such as hyaluronidase treatment was

not successful for immature oocytes, thus, we abandoned this approach in the

experiment. When cumulus cells were removed before maturation culture, only 4 to

26% of the oocytes developed to the metaphase II (M-II) stage, and even poorer

fertilization and development resulted when compared with other treatment groups

(P< .05). No interaction (P> .05) between cumulus cell removal and developmental

stages was detected indicating that in this experiment, no matter at what stage that the

cumulus cells were removed, it affected negatively on fertilization and subsequent

development when compared with the control group (P C .05).

The culture condition also significantly affected embryo development (P < .0001).

Higher development rates to morula and blastocyst stages were obtained when denuded

oocytes or embryos were co-cultured with detached cumulus cells versus without cells

81

Table 8. Effects of cumulus cell removal at various times and co-culture on oocyte maturation, fertilization and embryo development*

Cumulus cells removed

Coculture

No. of oocytes

% of oocvtes develooed to*MATR FERT CLEAV MORL BLST HBLST

Before IVM Yes 219 26 9 5 1 1 0No 216 4 0 - - - -

Before IVF Yes 192 93 58 47 19 6 3No 196 92 60 40 12 0 -

7 h after IVF Yes 177 93 71 54 27 12 6No 188 93 71 53 21 2 0

48 h after IVF Yes 202 96 92 78 48 19 12No 162 93 91 74 42 9 4

♦Major results of analysis: effect of cumulus cell removal (P< .0001); co-culture effect (Pc.0001); interaction of cumulus cell removal and co-culture (P<.01).

'Abbreviations: MATR=matured, FERT=fertilized, CLEAV=cleaved, MORL=morula, BLST=blastocyst, HBLST=hatched blastocyst.

82

(P < .05, Table 8). The interaction between culture conditions and developmental stages

was significant (P C .01). The beneficial effect of cumulus cell co-culture was more

prominent at later embryonic stages than at early stages. The interaction between

cumulus cell removal and culture condition was also significant (P C .01). The

interaction was partially introduced by the experimental procedure. The earlier the

cumulus cells were removed from the oocytes, the stronger the co-culture effect.

To more clearly show the effect of cumulus removal on each stage of

development, the statistical analyses of the main effects are presented in Table 9 and 10.

The percentages of development showed in these tables were based on their previous

developmental stages. This helped eliminate the interference of early treatment effect on

later developmental stages. Table 9 shows the result of removing cumulus cells at

different stages in the presence of co-culture with cumulus cells. Removing cumulus

cells from oocytes before maturation significantly (PC.05) reduced the rates of oocyte

maturation, fertilization and embryo developmental potential in each progressive stage,

except for the development from morula to blastocyst stages (P< .05). When cumulus

cells were removed after maturation, but before IVF or 7 h after IVF, fewer oocytes

were fertilized (P< .05), but the subsequent development rates of the fertilized ova were

not affected (P < .05), More oocytes were fertilized when cumulus cells were removed

at 7 h after IVF compared with those with cumulus cells removed before IVF (P< .05).

The effects of cumulus cell removal in the absence of co-culture are shown in Table 10.

The overall developmental rates were lower compared with the corresponding treatments

of the co-culture groups (P< .05). Negative impact on development by cumulus cell

83

Table 9. Effect of cumulus cell removal at various times on the development of IVF-derived bovine embryos in the presence of co-culture*

Cumulus cells No. of % of oocvtes develooed tobremoved oocytes MATR FERT CLEAV MORL BLST HBLST

Before 1VM 219 26° 35c 5(T 20° 100* 0*

Before IVF 192 93d 62d Side 40d 31c 55d

7 h after IVF 177 93d 76e 76d 50“ 46° 50“

48 h after IVF 202 96d 95f 85° 61d 4 (f 64d

'Data presented were derived from Table 8. Denuded ova were co-cultured with detached cumulus cells.

bThe percentage of development is based on the previous stages.

e.d.e,fyaiues with different superscripts within columns are different (P < .05).

84

Table 10. Effect of cumulus cell removal at various times on the development of IVF-derived bovine embryos in the absence of co-culture?

Cumulus cells No. of % of oocvtes develotwd tobremoved oocytes MATR FERT CLEAV MORL BLST HBLST

Before IVM 216 4® 0® 0® 0® 0® 0®

Before IVF 196 92“ 66d 66d 29d 0® 0®

7 h after IVF 188 93d I T 74® 40d gde 0®

48 h after IVF 162 93d 97f 00 57® 22® 40"

•Data presented were derived from Table 8. Denuded ova were cultured in medium alone.

•The percentage of development is based on the previous stages.

c,<u.fvaiues with different superscripts within columns are significantly different (P < .05).

85

removal without co-culture was detected at practically all subsequent stages of

development (see Tables 9 and 10).

Experiment 2

This experiment was an extension of Experiment 5.1 by testing the effect of

cumulus cell removal (complete vs. partial) at 7, 20 and 48 h after IVF (Table 11).

When cumulus cells were partially removed but with only a few cumulus cell remaining

attached to the zona pellucida, no differences (P> .05) were noted among the treatment

groups at all stages examined, except at the hatched blastocyst stage where co-culture

enhanced the rate of embryo development (P<.05) and cell count (not shown). When

cumulus cells were completely removed (Experiment 5.2), significantly more embryos

developed to morula stage or beyond for the 20- and 48-h treatment groups compared

with the 7-h group. No difference (P> .05) was detected between the 20-h vs. 48-h

treatment groups in all stages examined, particularly when cumulus cell co-culture was

used during incubation. Cumulus cell co-culture did not affect fertilization or cleavage

development of embryos (P> .05), but did significantly improve embryo development to

morula stage or beyond for all three different cumulus cell removal time treatment groups

examined (P < .05). No interaction between stage of cumulus cell removal and co-culture

was found in this study (P> .05).

Because no difference was noted among replications within experiment and the

same procedures were used for both studies, except for the extent of cumulus cell

removal, data were pooled for a retrospective comparison between the extent of cumulus

cell removal on subsequent embryo development. The pooled results and their

86

Table 11. Effect of cumulus cells on fertilization and embryo development*

Cumulus Co No. of % of oocvtes developed toremoval culture oocytes FERT CLEAV MORL BLST HBLST

Complete removal

7 h IVF Yes 90 81 70 12 2 0No 86 81 64 14 0 0

20 h IVF Yes 93 88 73 30 15 4No 92 89 80 13 2 0

48 h IVF Yes 93 88 76 30 19 5No 83 84 77 25 7 0

Partial removal

7 h IVF Yes 91 89 80 34 21 3No 90 83 68 27 18 0

20 h IVF Yes 90 86 77 34 18 6No 92 88 75 38 24 0

48 h IVF Yes 97 89 79 38 24 5No 93 89 76 39 27 0

'Major result of analysis: for complete removal of cumulus cells, significant differences (P< .05) were noted at morula stage or beyond by cumulus cell removal and co-culture, and for incomplete removal of cumulus cells, no difference was detected among treatment groups at all stages evaluated, except for hatched blastocyst stage where coculture improved development (P C .05). Interactions between stage of cumulus cell removal and co-culture are not significantly different (P>.05).

87

comparisons are presented in Table 12. The extent of cumulus cell removal did not

affect the rate of fertilization or cleavage development (P> .05). However, partial

cumulus cell removal allowed more cleaved embryos to develop to the morula or

blastocyst stage (P c .05 ), particularly when removal was performed at 7 or 20 h after

IVF when compared with complete cumulus cell removal. Partial cumulus cell removal

resulted in similar rates of progressive embryo development regardless of the stages of

cumulus removal (P > .05).

Discussion

Previous reports have suggested the importance of cumulus cells during bovine

oocyte in vitro maturation and fertilization after studying different populations of oocytes

with different layers of cumulus cell. Significantly lower rates of maturation and

fertilization were obtained from the group of cumulus-free oocytes compared with

cumulus-enclosed oocytes (Fukui and Sakuma,1980; Shioya et al.,1988; Leibfried-

Rutledge, et al., 1989). However, the cumulus-free oocytes used in those studies may

have originated from atretic follicles and, therefore, could have already started to

degenerate prior to in vitro culture. In the present study, only cumulus intact,

morphologically normal-appearing oocytes were selected and used in experiments.

Cumulus cells were removed at different developmental stages and then replaced in the

culture medium to evaluate the effect of cumulus cells on oocyte maturation, fertilization

and early embryonic development in vitro. The results demonstrated that the intact state

of surrounding cumulus cells of oocytes are beneficial at least up to 7 h after IVF, but

not at 20 h after IVF.

88

Table 12. Main effect of partial and complete cumulus cell removal

Cumulus cell removal No. of oocytes

_ Proeressive % of ova develoDed toTime Extent FERT CLEAV MORL BLST

7 h IVF Complete 176 143 (81)' 118 (83)* 23 (19)* 2(9)*Partial 181 156 (86)* 134 (86)* 56 (42)b 36 (64)b

20 h IVF Complete 185 164 (89)* 142 (87)* 40 (28)° 16 (40)'Partial 182 158 (87)* 138 (87)* 66 (48)b 38 (58)b

48 h IVF Complete 176 152 (86)* 135 (89)* 49 (36)* 24 (49)*Partial 186 169 (91)* 148 (88)* 73 (49)b 48 (66)b

■.b.eyaiues with different superscripts within columns are significantly different (P < .05).

89

Cumulus cell removal before maturation appears to be detrimental to bovine

oocytes. Only 4% of the denuded oocytes developed to M-II stage after 22 h of culture

in medium alone, and most of the oocytes degenerated during this culture period. When

denuded oocytes were co-cultured with detached cumulus cells, the maturation rate was

significantly improved to 26%. However, when intact cumulus-oocyte complexes were

matured in the same condition, on the average, 93% of oocytes developed to M -n stage,

similar to the results of previous studies (Zhang et al., 1992b; Yang et al., 1993).

Because the evaluation of maturation was conducted until after fertilization, sufficient

time may have elapsed to allow oocytes to degenerate and therefore appear not to have

reached metaphase II. Whereas it is possible to underestimate the "true" maturation

rates, the fact that all groups were evaluated at the same time makes the comparison valid

and meaningful. This result thus indicates that the intact stage of the cumulus-oocyte

complex is essential and important during oocyte maturation in cattle.

Ultrastructural analysis of bovine cumulus-oocyte complexes also demonstrate that

a complete breakdown of the cytoplasmic processes occurs prior to oocyte maturation at

13 to 20 h of in vitro maturation (Hyttel, 1987; Laurincik et al., 1992; Suzuki et al.,

1994). In contrast, a more recent study (Allworth and Albertini, 1993) with laser

scanning confocal and fluorescence microscopy demonstrated that complete breakdown

of cytoplasmic processes did not occur even at 24 h of maturation. However, the

transition between actin-rich and microtube-rich processes was evident during different

stages of maturation. It has been hypothesized that follicular cells produce meiosis-

arresting factors and that the breakdown of these junctional complexes causes meiotic

90

resumption. The findings of this study demonstrated that complete cumulus cell removal

blocked maturation and co-culture of oocytes with detached cumulus cells partially

restored maturation suggesting other possible factors may be involved in oocyte

maturation.

Research in cattle and other species also showed a beneficial effect of cumulus

cells on in vitro maturation and fertilization (Kennedy and Donahue, 1969; Cross and

Brinster, 1970; Vanderhyden and Armstrong, 1989; Chian and Niwa, 1994). Although

some denuded oocytes can complete the meiotic resumption, fertilization and

development to normal cleaved embryos is significantly reduced as shown in the present

study and by others in previous reports (Staigmiller and Moor, 1984; Sirard et al., 1988).

It has also been noted that a higher proportion of fertilized oocytes that matured without

cumulus cells showed evidence of abnormal fertilization (Vanderhyden and Armstrong,

1989; Chian and Niwa, 1994).

Our results showed a significant decrease in fertilization rate when cumulus cells

were removed prior to insemination. This negative effect was also noted when cumulus

cells were removed 7 h after insemination in Experiment S .l, but was not evident in

Experiment 5.2. Interestingly, even though cumulus cells removed at this time decreased

the fertilization rate compared with those in the control groups, subsequent development

of the fertilized ova seemed not to be affected. Similar rates of embryo development to

blastocyst and hatched blastocyst stages were obtained when compared with the control

groups, particularly in the co-culture groups (P> .05, Table 9). This indicates that the

overall low development rates in these treatment groups (Table 8) were primarily due to

91

the reduced rates of fertilization. However, the beneficial effect of cumulus cells on

fertilization and its mechanisms are still unclear. Possible explanations include: 1)

cumulus cells are able to facilitate sperm cell penetration and fertilization (Brackett et a l.,

1989; Vanderhyden and Armstrong, 1989; Fukui, 1990; Mochizuki, 1991; Younis and

Brackett, 1991); 2) cumulus cells are able to induce sperm capacitation and acrosome

reaction in cattle (Fukui, 1990; Boatman and Robbins, 1991a; Takahashi and First,

1993); and 3) cumulus cells facilitate sperm penetration and fertilization by preventing

zona hardening (Downs et al., 1986; Mattioli et al., 1988).

Despite all the supporting evidence on the beneficial effect of cumulus cells, two

recent studies require some attention. Hawk et al.(1992) reported that partially detaching

cumulus cells from bovine oocytes either before or after maturation significantly

increased fertilization rate and the proportions of oocytes developing to expanded

blastocysts. Dominko and First (1991) suggested that maturation of cumulus-removed

oocytes may result in normal fertilization and embryo development when cultured on a

cumulus cell monolayer. One possible explanation for these findings is incomplete

removal of cumulus cells from oocytes in these experiments. These remaining cumulus-

oocyte connections may have been sufficient to provide the communication between the

oocyte and cumulus cells, as suggested by the present study (Experiment 5.2). To what

extent the cumulus cells should be removed remains a question for further investigation.

Presence of cumulus cells in the culture system significantly improved embryo

development to morula, blastocyst and hatched blastocyst stages in this study. This

supports previous reports on the beneficial effects on mammalian embryos of co-culture

92

or conditioned medium (Eyestone and First, 1989; Kane et al., 1992; see review by

Thibodeaux and Godke, 1992). However, Bavister et al. (1992) have reported similar

rates of morula/blastocyst development in TCM-199 alone and conditioned medium. Our

present study suggests that a few remaining cumulus cells on the zona may mask the

difference and support embryo development beyond the in vitro culture block. In our

laboratories, cumulus cells are routinely used as co-culture cells for culturing bovine IVF

embryos (Zhang et al., 1992b; Yang et al., 1993). Live calves have been bom after

transfer of frozen-thawed IVF-derived cumulus cell co-cultured embryos (Zhang et al.,

1993). These experiments and those of other investigators (Goto et al., 1988; Fukuda

et al., 1990) have shown that the cumulus cell co-culture system at present, remains

better in supporting embryonic development over the control medium alone.

In conclusion, this study demonstrated that cumulus cells are important and

necessary for oocyte maturation and acquisition of full embryonic development

competence. The intact stage of the surrounding cumulus cells was demonstrated to be

beneficial to the oocyte and subsequent embryo development at least up to 7 h, but not

at 20 h after insemination of oocytes. The efficiency of producing IVF-derived bovine

embryos was comparable to the controls when cumulus cells were completely removed

» 20 h post-insemination. The results also showed that co-culture of denuded oocytes

improved the rates of maturation and fertilization through some unknown mechanism(s).

However, this improvement did not overcome the detrimental effect caused by

breakdown of the direct connection. The results with embryo co-culture confirmed

previous findings of the beneficial effect of somatic cells on overcoming the 8- to 16-cell

in vitro "developmental block" and enhanced subsequent embryo development.

CHAPTER 6

EVALUATING BOVINE OVIDUCT CELLS USED IN COMBINATION WITH BOVINE CUMULUS CELLS TO CO-CULTURE IVF-DERIVED

BOVINE EMBRYOS IN VITRO

Introduction

Although the biochemical processes during co-culture and the interaction between

embryo and "helper" cells are not yet fully understood, various types of somatic cells

have been shown to benefit early bovine embryos development in vitro (Kuzan and

Wright, 1982; Camous et al., 1984; Lu et al., 1987b; Goto et al., 1988; Scodras et al.,

1991). Pregnancies and normal calves have been produced after transfer of in vitro

matured/m vitro fertilized (IVM/IVF) oocytes subsequently in vitro cultured (TVC) with

bovine oviduct cells (Lu et al., 1988; Xu et al., 1990; Monson et al., 1992) and bovine

cumulus cells (Goto et al., 1988; Zhang et al., 1992a). However, the proportion of

IVM/IVF embryos that develop to blastocyst stages in different in vitro culture systems

remains variable, suggesting that the optimum culture system for IVF-derived bovine

embryos has yet to be discovered.

Conventional culture systems for in vitro development of early stage bovine

embryos are somewhat complicated, involving a complex culture medium (e.g. TCM-199

or Ham’s F-10), serum, and somatic cells. Recently, several research groups reported

using simple chemically defined, serum-free medium to support the in vitro development

of early stage in vivo fertilized (Ellington et al., 1990a) or IVF-derived (Pinyopummintr

and Bavister, 1991) bovine embryos. It would be beneficial to eliminate undefined

94

95

biochemical components in standard complex culture media so that the embryotrophic

components produced by co-culture cells could be analyzed and the requirements for

early embryo development could be identified.

The objective of this study was to evaluate the use of bovine oviduct cells, bovine

cumulus cells and a combination of both of these cells to co-culture IVF-derived bovine

embryos. In addition, two different culture media, modified CZB medium, a chemically

defined serum-free medium containing 20 amino acids and TCM-199, a complex

medium, supplemented with 5% fetal bovine serum (FBS) were also compared for

supporting IVF-derived embryos in both co-culture and medium alone culture.


IVF-derived embryos

Cumulus-intact oocytes were harvested from 3 to 8 mm follicles from abattoir

ovaries of dairy and beef females. The oocytes with compacted cumulus cell were

selected and washed two times in Dulbecco’s phosphate-buffered saline (DPBS)

containing . 1 % Polyvinyl alcohol (PVA) and two times in Tissue Culture Medium 199

(TCM-199) plus 5% fetal bovine serum (FBS). Oocytes were then incubated in .5 ml

of TCM-199 with 5% FBS at 39°C for 24 h. At 1 h prior to insemination, two straws

of frozen semen of a fertile dairy bull were thawed in a 25°C water bath for 1 min. The

sperm cells were washed with Brackett-Oliphant (B-O) medium containing 10 mM of

caffeine and adjusted to a concentration of 3 x 10s motile sperm cells/ml. Sperm cells

were then exposed to .1 j*M of Ca++ ionophore A23187 for 1 min to aid in capacitation.

Approximately 50 /il of a sperm cell suspension was then quickly added to each 50 fil

96

fertilization droplet containing 20 to 25 oocytes. The fertilization medium was B-O

medium containing 20 mg/ml bovine serum albumin (BSA). Oocytes and sperm cells

were co-incubated at 39°C in 5% C02 in humidified air. After 6 h of incubation, the

oocytes (with their cumulus cells) were moved to TCM-199 with 5 % FBS and incubated

for * 4 0 h until they reached the 2- to 8-cell stage. Cumulus cells were removed from

these early IVM/IVF embryos by culturing them in 2.9% sodium citrate solution for 2

min followed by manual shaking. Only early stage embryos without any attached

cumulus cells were used in this experiment.

Experimental design

This experiment was conducted according to a 2 x 4 factorial arrangement. The

effects of two culture media: 1) CZB medium; 2) TCM-199 plus 5% FBS and four

culture systems: 1) medium alone as a control; 2) bovine oviduct epithelia cell co-culture

(BOEC); 3) bovine cumulus cell co-culture (BCC); and 4) BOEC + BCC co-culture

were evaluated in this experiment. Two- to 8-cell stage IVF-derived bovine embryos

were randomly assigned into each of eight treatment groups. The experimental outline

is shown in Table 13.

Monolayer preparation and co-culture

Bovine oviducts (obtained from midcycle mature beef females at a local abattoir)

were transported at 25 °C in PBS with antibiotics (1000 IU penicillin and 1000 #ig

streptomycin/ml PBS). Oviducts were washed twice in PBS, dried with a sterile paper

towel, trimmed free from surrounding connective tissue, briefly dipped in 70% ethanol,

rinsed in PBS and again dried. The infundibulum was excised from each oviduct.

97

Table 13. Experimental design for Experiment 6

Culture medium Culture system No. of 2- to 8-cell embryosCZB Medium alone 100

CZB BOEC 100

CZB BCC 100

CZB BOEC+ BCC 100

TCM-199 Medium alone 100

TCM-199 BOEC 100

TCM-199 BCC 100

TCM-199 BOEC+ BCC 100

98

Epithelial cells were manually expressed into a sterile test tube by gently squeezing the

oviduct between the jaws of a pair of sterile forceps while pulling from the isthmus

toward the infundibulum. The harvested cells were washed three times with TCM-199

plus 5 % FBS by centrifugation (360 x g , 4 min). The cell pellets were resuspended in

the same medium, and the cells were seeded in four-well tissue culture plate (1 x 106

cells/ml) to develop monolayers for BOEC co-culture . The culture plates that used for

developing early stage IVF embryos (cumulus cells monolayer formed at this time) were

used in BCC cell and BOEC+ BCC cell co-culture treatments (Figure 3). The oviduct

cell clumps was used in BOEC+ BCC cell co-cultures.

Early stage embryos free of cumulus cells (n = 800) were equally and randomly

allotted to one of the eight treatment groups. Embryos were incubated in .5 ml of CZB

medium (Appendix C) or TCM-199 (25 Mm HEPES, Earle's salts, GIBCO) plus 5%

FBS either alone or with medium plus cells. The culture media were replaced with fresh

medium at 48-h intervals and embryos were evaluated using an inverted microscope

immediately after changing the medium. There were four replications in this experiment.


Chi-square (x2) analysis was used to test the difference in the proportion of

oocytes developed to morula, blastocyst, expanded blastocyst and hatched blastocyst

stages. A P-value of < .05 was regarded as statistically significant in this experiment.

Results

In this experiment, the effect of two culture media (CZB medium and TCM-199)

and two type of "helper" cells (BOEC and BCC) on supporting IVF-derived 2- to 8- cell

99

Figure 3. Somatic cell monolayers used for co-culture of IVF-derived bovine embryos. Bovine cumulus cell (BCC) monolayer (a). Bovine oviduct epithelial cell (BOEC) monolayer (b). Magnification: X600.

100stage bovine embryos was tested. Within each culture medium, embryos were either

cultured in medium alone or co-cultured with BOEC, BCC and BOEC + BCC. The

results of embryo development rates at morula, blastocyst, expanded blastocyst and

hatched blastocyst stages are shown in Table 14.

When CZB medium was used as a culture medium, more 2- to 8-cell stage

embryos developed to blastocysts and expanded blastocysts after co-culturing with BOEC,

BCC and BOEC + BCC treatment groups (PC.05) than culturing in CZB medium

alone. When TCM-199 with 5% FBS was used, more embryos developed to blastocysts

and expanded blastocysts (P< .05) in the BCC cell and the BOEC+BCC cell co-culture

treatment groups when compared with embryos in the BOEC cell co-culture or in

medium alone culture. This pattern continued through to the embryo hatching stage.

To compare overall medium effect, data from both co-cultures and medium alone

culture were pooled according the culture medium that was used. Result showed that

more 2- to 8-cell stage embryos developed to morula stage in CZB (PC.05) than in

TCM-199 with serum (74% vs. 66%). However, fewer embryos hatched in vitro in

CZB medium without serum (PC.05) than TCM-199 with serum (6% vs. 14%). No

significant differences were found between CZB and TCM-199 on supporting IVF-

derived embryo development at blastocyst and expanded blastocyst stages (P> .05).

Discussion

The in vitro development of IVF-derived bovine embryos in co-culture with

bovine oviduct epithelial cell, bovine cumulus cell and the combination of these two types

of cells using both simple serum-free medium (CZB medium) and complex serum-

Table 14. Effect of cell types and culture media on in vitro development ofIVF-derived bovine embryos*

Treatmentgroup 2-

No. of No. of to 8-cells MORL {%)

No. of No. of No. of BLST (%) ExBLST (%) HBLST (%)

CZB (control) 100 68* 14* 9* 2*

CZB+BOEC 100 68* 26b 20b 4*,b

CZB+ BCC 100 81b 35b 29b g..b

CZB+BOEC+BCC 100 78.,b 31b 26b l l b

TCM-199 (control) 100 68*,b 18* 15*,b 6*

TCM-199+BOEC 100 55* 16* 9* 7*

TCM-199+BCC 100 69b 28b TP 22b

TCM-199+BOEC+ BCC 100 71b 26b 26b 22b

Total CZB combined 400 (74)* (27)* (21)* (6)’

Total TCM combined 400 (66)b (22)* (19)* (14)b*MORL=morulae; BLST=blastocysts; Ex BLST—expanded blastocysts;HBLST=hatched blastocysts.

^X 2 analysis was used to compare treatment difference within each column for each culture medium (PC.05).

102containing medium (TCM-199 plus 5% FBS) were studied in this experiment. The

results clearly showed the evidence of the beneficial effect of co-culture on embryo

development. Significantly more embryos developed to morula, blastocyst and hatched

blastocyst stages from all three co-culture treatment groups compared with medium alone

culture when CZB medium was used. However, when TCM-199 plus 5% FBS was used

for embryo culture, more embryos developed to morulae, blastocysts and hatched

blastocysts in cumulus cells co-culture and oviduct cell plus cumulus cell co-culture

compared with in oviduct cell co-culture and in medium alone culture. These data

suggest that bovine cumulus cells with or without bovine oviduct cells would be an

acceptable choice for co-culturing IVF-derived bovine embryos.

Studies on bovine in vitro fertilization have also shown the importance of

cumulus/granulosa cells on oocyte in vitro maturation and fertilization (Leibfried-

Rutledge et al, 1989; Younis and Brackett, 1991). Greater maturation and fertilization

rates were observed with the use of cumulus-enclosed bovine oocytes than cumulus-

denuded oocytes (Shioya et al., 1988; Mochizuki et al., 1991; Cox et al., 1993).

Removal of cumulus cells before oocyte maturation and fertilization resulted in lower

maturation and fertilization rates (see Chapter 5), whereas addition of granulosa cells

to culture medium improved the fertilization rates (Mochizuki et al., 1991).

The in vitro development of IVF-derived bovine embryos were also enhanced by

using cumulus cell co-culture. Goto et al. (1988) reported the establishment of

pregnancies after nonsurgical transfer of IVF-derived blastocyst stage bovine embryos

that were co-cultured with cumulus cells. Younis and Brackett (1991) reported that 20%

of IVF-derived embryos developed to morula and blastocyst stages when co-cultured with

103

cumulus cells compared with only 2.8% when cultured in medium alone. The results

from the present study further confirmed those previous findings, and showed the

supporting effect of cumulus cell co-culture on the in vitro development of IVF-derived

2- to 8-cell stage embryos to morula and blastocyst stages in both simple serum-free

medium (CZB) and complex serum-containing medium (TCM-199).

Oviduct epithelial cells are a wildly used "helper" cell for supporting early stage

embryos to past the in vitro developmental block (see reviews, Rexroad, 1989;

Thibodeaux and Godke, 1992). Positive results have been reported with oviduct cell

monolayers in sheep (Rexroad and Powell, 1986; Gandolfi and Moor, 1987), cattle

(Eyestone and First, 1989; Ellington et al., 1990b) and pig (White et al., 1989).

However, in the present study, no significant improvement on blastocyst development

was detected using oviduct cell co-culture compared with medium alone culture when

TCM-199 plus 5% FBS was used as culture medium. It has been proposed that helper

cells benefit early embryos developing in vitro by producing an embryotrophic factor(s)

or removing toxic substances from the culture medium (Kane et al., 1992). In this

experiment, oviduct cells used with complex serum-containing medium (TCM-199 plus

5 % FBS) seems not as effective as those used with simple serum-free medium (CZB) on

promoting embryo development in vitro. The reason for this difference is not known.

Controversial results with the use of bovine oviduct cell co-culture have also been

reported previously. Aoyagi et al. (1990) reported that bovine oviduct cells were more

effective for stimulating blastocyst development of IVF-derived early stage bovine

embryos in vitro than bovine cumulus cells. Whereas, Berg and Brem (1990) reported

that significantly more bovine IVF embryos developed to morula and blastocyst stages

with cumulus cell co-culture (32%) compared with oviduct cell co-culture (17%).

Eyestone and First (1989) reported that both oviduct cell co-culture and oviduct cell

conditioned medium culture enhanced the development of IVF-derived embryos to the

morula and blastocyst stages (22% and 22%, respectively). However, Ellington et al.

(1990b) reported that better embryo quality (as evidenced by the percentage of poor-

quality nuclei number of cells per embryo and embryo quality score) and greater hatching

rates resulted with fresh oviduct cell co-culture compared with frozen-thawed oviduct

cells and conditioned medium when culturing 1- to 2-cell stage bovine embryos in vitro.

Bavister et al. (1992) also reported that the bovine oviduct cell conditioned medium did

not increase morula and blastocyst formation compared with medium alone. A possible

explanation for these controversial results may be that the stage of the bovine estrous

cycle has an effect on the performance of oviduct cells during co-culture. In the present

study, oviducts were harvested from the animals at midcycle (presence of luteal tissue).

Xu et al. (1992a) reported that co-culture with bovine oviduct cells harvested from

preovulatory (presence of a corpus haemorrhagicum or a mature follicle in the absence

of corpus luteum) animal significantly improved the in vitro development of IVF-derived

bovine embryos compared with medium alone. Further study is needed to investigate the

cell source, culture condition, as well as the mechanism of the beneficial role of oviduct

cell on embryo development.

When CZB medium (without serum) was compared with TCM-199 (with serum),

significant more embryos developed to morula stage in CZB than in TCM-199.

However, significant fewer embryos hatched in vitro in CZB than TCM-199. This

indicates that fetal bovine serum inhibited embryo cleavage at early development stages

105

but stimulated in vitro development at later stages, especially near the time of hatching.

This developmental pattern on in vitro development of IVF-derived bovine embryos with

bovine calf serum has also been reported by Pinyopummintr and Bavister (1991). They

indicated that the inhibition effect of serum was at or before the first cleavage division.

In our study, however, only 2- to 8 -cell stage embryos were used, and significantly fewer

embryos developed to compacted morula stage when serum was present, which indicates

the inhibition effect of the serum on early stage bovine embryo development was not only

at first cell division but also several subsequent cell divisions and blastomere compaction.

At the embryo hatching stage, significant improvement was observed when serum was

present in the culture system, which is in agreement with the results reported by

Pinyopummintr and Bavister (1991). Although the mechanism of this biphasic effect of

serum on bovine embryo development remains unclear, this finding suggests that a

culture system that starts with serum-free medium and then systematically increases the

serum concentration of the medium to fit the embryo developmental stages may be more

suitable for in vitro producing bovine embryos.

In this study, there was a marked reduction in the number of morulae that

developed into blastocysts during in vitro culture in all treatment groups. This result

suggests there may be another in vitro development block at morula to blastocyst

transition in addition to the 8 - to 16-cell in vitro block in bovine prehatched embryos.

If this is the case, morula stage development in vitro may not be a good indicator for

embryo culture effectiveness. Further studies are needed to verify the existence of a

secondary "block'' and to further develop efficient in vitro culture systems for bovine

embryos.

CHAPTER 7

EFFECT OF GROWTH FACTORS AND GROWTH MODULATORS ON BOVINE OOCYTE IVM/IVF/IVC

Introduction

The regulatory effect of peptide growth factors on somatic cell proliferation and

differentiation during in vitro culture has been well established (see review by

Gospodarowicz and Moran, 1976). However, how growth factors affect the development

of in vitro cultured early stage mammalian embryos remains to be determined. Although

in some species, such as mouse (Whitten, 1956; Cholewa and Whitten, 1970), rabbit

(Kane and Foote, 1970; Kane, 1972) and human (Caro and Trounson, 1986), early stage

( 1 - to 2 -cell) embryos can develop to blastocysts and hatch in defined culture medium

without the addition of growth factors, it has become evident that early embryos may

require endogenous as well as exogenous growth factors for normal development.

First, in vitro culture system have been shown not to be as efficient as in vivo on

the development of early stage mammalian embryos. In vitro developmental block has

been reported in several mammalian species, including hamster at 2- to 4-cell stage

(Whittingham and Bavister, 1974), rat (Bavister, 1988) and pig (Davis and Day, 1978)

at 4- to 8 -cell stage, and sheep (Wintenberger et al., 1953), goat (Betteridge, 1977) and

cow (Thibault, 1966) at 8 - to 16-cell stage. Bowman and McLaren (1970) studied the

growth rate of in vitro and in vivo developed mouse embryos and found that the cleavage

rates of in vitro developed embryos were slower than that of in vivo developed embryos.

Iwasaki et al. (1990) reported that at the same developmental stages, the cell number of

in vitro cultured IVF-derived bovine embryos was lower than that of in vivo cultured

106

107

IVF-derived embryos and in vivo produced embryos. However, no difference was found

between in vivo cultured IVF-derived embryos and in vivo produced embryos. These

findings suggest that some paracrine factor(s) from the oviduct was involved in the early

stage embryo development, and this factor(s) was missing in the in vitro culture systems.

Several growth factors, such as epidermal growth factor (EGF), transforming

growth factor (TGF)-or, TGF-/31, colony-stimulating factor (CSF)-l, platelet-derived

growth factor (PDGF), insulin-like growth factor (IGF)-I, IGF-II, fibroblast growth

factor (FGF) and interferon- 7 have been detected in human uterine endometrium (see

review by Giudice, 1994). Transcripts encoding for IGF-I, IGF-II, TGF-a, TGF-01,

TGF-/32, bFGF, PDGF-A and the receptors for IGF-I, IGF-II, insulin and PDGF-a have

also been detected in ovine and bovine oviductal epithelial cell cultures (Watson et

al.,1994), which may explain the supportive effect of oviduct cell co-culture on early

embryo development.

Secondly, improved in vitro development of mouse embryos has been reported

by decreasing the volume of culture medium or group culturing the embryos (Paria and

Dey, 1990). This finding suggests that autocrine factor(s) may also participated in early

embryo development. Recent studies have shown that the early stage mammalian

embryos were able to produce a number of growth factors. Rappolee et al. (1988)

reported the expression of PDGF, TGF-a, and TGF-jSl in mouse embryos. Zhang et al.

(1994) detected the gene expression of IGF-I, IGF-II, IGF-I receptor, IGF-II receptor

and insulin receptor in rat prehatched embryos. The expression of similar growth factor

ligand and receptor genes have also been reported in cow embryos (Watson et al., 1992),

sheep embryos (Watson et al.,1994) and human embryos (Schultz and Heyner).

108

The results of the experiments in which growth factors were applied exogenously

to cultured embryos suggest that some growth factors may facilitate early embryo

development in vitro. Paria and Dey (1990) reported that the addition of EGF or TGF-a

or TGF-/J1 to the culture medium markedly improved the development of single cultured

mouse embryos. Harvey and Kaye (1992) reported that IGF-I increased the number of

cells in the inner cell mass (ICM) of in vitro cultured mouse blastocysts when added to

the medium for culturing 2-cell stage mouse embryos. The in vitro development of rat

(Zhang and Armstrong, 1990) embryos and IVF-derived bovine embryos (Harper and

Brackett, 1993) were also enhanced by adding growth factors to culture media.

The objective of this study was to evaluate growth factors and growth modulators

on in vitro maturation (IVM), in vitro fertilization (IVF) and subsequent embryo

development during in vitro culture (IVC) of bovine oocytes by adding purified products

to culture medium. These include: insulin, growth hormone (GH), EGF, FGF, IGF-1,

PDGF, prostaglandin (PGF3a) and arachidonic acid (AA).


Ovary collection and oocvte aspiration

Ovaries were collected from mixed breed cows or heifers at a local abattoir

immediately after slaughter. Excised ovaries were placed in physiological saline (.9%

NaCl) containing antibiotics (100IU penicillin, 100 fig streptomycin/ml) and maintained

at 25 to 28°C during transportation to the embryo laboratory. In the laboratory, ovaries

were rinsed three times in saline and dried with a sterile paper towel. Oocytes were

aspirated from small antral follicles ( 2 to 8 mm in diameter) using an 18-gauge needle

attached to a 6 ml sterile, plastic syringe. Follicular fluid was placed in a 50-ml conical

109

centrifuge tube. After a sedimentation interval (3-5 minutes), supernatant was discarded

and 10 ml of PBS with . 1 % PVA was added to the centrifuge tube. The oocyte-PBS

suspension was poured into a 1 0 0 mm sterile plastic petri dish for oocyte searching and

evaluation. Only those oocytes with intact cumulus and homogeneous cytoplasm were

used in this study. Ovary washing and oocyte collecting were performed at room

temperature (25 to 28 °C).

Experimental design

Experiment 7.1. Effect of insulin on bovine IVM/IVF/IVC

This experiment was designed to evaluate the effect of insulin on bovine oocytes

in vitro maturation (IVM), in vitro fertilization (IVF) and subsequent embryo

development during in vitro culture (IVC). Insulin was added into culture medium

during oocyte maturation incubation. The culture medium used in this study was Tissue

Culture Medium 199 (TCM-199) supplemented with 5% fetal bovine serum (FBS), which

is the culture medium used in the standard bovine IVF protocol (see Chapter 3). The

insulin concentration was .1 ^g/ml. Selected oocytes (n=800) were randomly assigned

to two treatment groups. Oocytes were either cultured in the medium containing insulin

or in medium alone (control) for in vitro maturation. Cumulus cell expansion scores

were recorded after maturation incubation. A score of 1 to 4 was assigned to each

oocyte: 1 ) cumulus cells had almost no expansion; 2 ) cumulus cells had moderate

expansion, corona radiata cells had no expansion; 3) cumulus cells were fully expanded,

corona radiata cells had no expansion; and 4) both cumulus cells and corona radiata cells

were fully expanded.

110Matured oocytes were inseminated with Ca++ ionophore A23187 treated sperm

cells according to the standard IVF protocol (Chapter 3). The cleaved embryos were

then co-cultured on cumulus cell monolayers for 8 days for in vitro development. The

embryo development culture medium was TCM-199 plus 5% FBS. The proportion of

oocytes matured, fertilized, cleaved and subsequently developed to morula, blastocyst,

and hatched blastocyst stages were recorded. By day- 8 of co-culture, embryos that

developed to morulae, blastocysts and hatched blastocysts were fixed and stained with

Hoechst stain 33342 for cell count. The experimental design of this experiment is

outlined in Table 15.

Experiment 7.2. Effect of GH, EGF, FGF, IGF-1 and PG F^ on embryo development

This experiment was designed in a 6 x 2 factorial arrangement. The effect of the

five growth factors (GH, EGF, FGF, IGF-I and PG FjJ, fetal bovine serum (FBS) and

interactions were investigated. Oocytes were randomly assigned to each of 12 treatment

groups for maturation, fertilization and embryo development culture. Embryo

development was supported with cumulus cell co-culture. The growth factors were added

to culture medium throughout the in vitro culture interval. The basal medium used in

this experiment was TCM-199 supplemented with .1% polyvinyl alcohol (PVA). The

concentration of growth factors are: 50 ng/ml GH, 10 ng/ml EGF, 50 ng/ml FGF, 100

ng/ml IGF-I and 50 ng/ml PGF2a. Each growth factor was added into either the basal

culture medium or the basal culture medium plus 5% FBS. The experimental outline is

shown in Table 16.

Ill


Treatment No. of Oocytes

Insulin* 400

Control 400

♦Insulin concentration was .1 Atg/ml of medium.

112


Growth Factor*

No. of oocytes

with FBS without FBS

GH 2 0 0 2 0 0

EGF 2 0 0 2 0 0

FGF 2 0 0 2 0 0

IGF-1 2 0 0 2 0 0

PGF2a 2 0 0 2 0 0

Control 2 0 0 2 0 0

* GH= growth hormone; EGF= epidermal growth factor; FGF= fibroblast growth factor; IGF-1= insulin-like growth factor-I; PG F^= prostaglandin Fja*

113

Experiment 7.3. Effect of AA and PDGF on IVF embryo development

Experiment 7.3a. This experiment was designed to evaluate the effects of

arachidonic acid (AA) and platelet-derived growth factor (PDGF) on bovine oocyte in

vitro maturation, fertilization and subsequent development. Oocytes were cultured in the

basal culture medium (TCM-199 plus . 1 % PVA) containing: 1) 50 ng/ml AA; 2) 5 ng/ml

PDGF; 3) 5% FBS; and 4) basal medium alone for in vitro maturation, fertilization and

early development. Cleaved embryos were then randomly divided into two subgroups.

They were cultured continuously in the same medium alone or co-cultured on a cumulus

cell monolayer to test if the presence of somatic cells is necessary for embryos to utilize

AA and PDGF. The cumulus cell monolayers were developed from the cumulus cells

that attached to the bottom of culture plates during oocyte maturation and fertilization.

The experimental design is shown in Table 17.

Experiment 7.3b. Based on the result of Experiment 7.3a, adding AA into culture

medium had significant effect on blastocyst formation, expansion and hatching compared

with other treatment groups in both co-culture and medium alone culture. This effect

was equivalent to FBS when embryos were cultured in medium alone, and was greater

than the presence of FBS when embryos were co-cultured with cumulus cells. However,

adding PDGF had no effect at all. In Experiment 7.3b, the effect of AA on embryo

development was further investigated. A A was added into the culture medium 1) for

oocyte maturation; 2) for embryo development; 3) throughout in vitro culture period; and

4) no A A addition as control. The cleaved embryos were subsequently co-cultured on

cumulus cell monolayer as in Experiment 7.3a. The uncleaved oocytes were fixed and

stained to examine the arrested stages. The experimental outline is shown in Table 18.

114

Table 17. Experimental design for Experiment 7.3a

Treatment groups* No. of oocytes Co-cultureAA 2 0 0 +

PDGF 2 0 0 +

FBS 2 0 0 +

Control 2 0 0 +-

* AA= arachidonic acid; PDGF= platelet-derived growth factor; FBS= fetal bovine serum.

115

Table 18. Experimental design for Experiment 7.3b

Treatment group AA* No. of oocytes

Maturation + 1 0 0

Development + 1 0 0

Throughout + 1 0 0

Control 1 0 0

*AA=arachidonic acid (50 ng/ml added to the medium).

116

Experiment 7.3c. Based on the result of Experiment 7.3b, adding AA into culture

medium during early embryo development had significant stimulating effect at expanded

blastocyst and hatched blastocyst stages. In Experiment 7.3c, the dose response of AA

on the development of IVF-derived embryos was tested. Two- to 8 -cell stage IVF-

derived embryos were cultured using basal culture medium supplemented with 1 ) 0 ng/ml

AA (control); 2) 10 ng/ml AA; 3) 50 ng/ml AA; 4) 100 ng/ml AA; and 5) 500 ng/ml

AA. Cumulus cell co-culture system was used in this experiment. The experimental

design outline is shown in Table 19.

Source of growth factors

The peptide growth factors, including EGF, FGF, IGF-1 were obtained from

Collaborative Research Incorporated. Insulin, GH, PDGF, PG F^ , and AA were

purchased from Sigma. Tissue Culture Medium 199 (TCM-199) (25 mM HEPES,

Earle’s salts), Dulbecco’s phosphate buffered saline (PBS) and fetal bovine serum (FBS)

were purchased from Gibco. Bovine serum albumin (BSA, fraction V), Polyvinyl

alcohol (PVA) and other chemicals were obtained from sigma. All reagents used in this

study were cell culture tested.

Oocyte in vitro maturation and fertilization

The in vitro maturation (IVM), in vitro fertilization (IVF) procedure used in this

study was the same as described previously (Chapter 3). Briefly, selected oocytes were

washed two times in PBS containing .1% PVA, two times in culture medium, and

placed into four-well tissue culture plate containing 500 n 1 of culture medium in each

well (20-25 oocyte/well). The basal culture medium used in this study was TCM 199

117

Table 19. Experimental design for Experiment 7.3c

Treatment group No. of 2 to 8 -cell embryos

0 ng/ml AA* 1 0 0

10 ng/ml A A 1 0 0

50 ng/ml AA 1 0 0

100 ng/ml AA 1 0 0

500 ng/ml AA 1 0 0

*AA=arachidonic acid.

118

plus .1% PVA or TCM 199 plus 5% FBS (Experiment 7.1). Oocytes were incubated

22 to 24 h for maturation at 39°C in 5% C0 2 in humidified air.

At 1 h prior to insemination, two straws of frozen semen from a fertile Holstein

dairy bull were thawed in a 25 °C water bath for 1 min. The sperm cells were washed

twice in B-O medium (Brackett and Oliphant, 1975) supplemented with 10 mM caffeine

by centrifugation (350 x g, 6 min), and adjusted to a concentration of 3 x 10s motile

sperm cells/ml. Sperm cells were then exposed to Ca++ ionophore A23187 (final

ionophore concentration of . 1 ^M) for 1 min to aid in capacitation. Approximately 50

Atl of treated sperm cell suspension were then added to each 50 jxl fertilization droplet

containing 20 to 25 oocytes. The fertilization medium was B-O medium containing 20

mg/ml BSA. Oocytes and sperm cells were co-incubated at 39°C in 5% COj in

humidified air for 6 h.

Embrvo culture and evaluation

After 6 h of incubation, oocytes with attached cumulus cells were washed with

culture medium and transferred either in 500 pi of culture medium in four-well tissue

culture plate (25 oocytes/well) or into 100 pi of culture medium in 8 x 12-well tissue

culture plate (10 oocytes/well). Oocytes were then cultured for an additional 43 h to

evaluate the rate of cleavage in vitro. At the time of cleavage evaluation, cumulus cells

surrounding the embryos were removed manually with a small pore glass pipette. The

cells that already attached to the bottom of the culture plate were maintained as co-culture

feeder cells. The cleaved embryos were subsequently cultured on cumulus cell

monolayer or in medium alone at 39°C in 5% C 0 2 in air until reached the hatched

blastocyst stage. The culture medium was replaced with fresh medium at a 48-h

119

intervals, and the embryo development was recorded immediately after medium change.

Uncleaved oocytes were fixed at 48 h post-insemination in ethanol:acetic acid (3:1) for

24 h, and stained with 2% aceto-orcein to examine the arrested stages.


For those experiments with factorial arrangement, the results were analyzed with

SAS categorical data modeling (CATMOD) procedure (SAS Institute, Gary, NC), and

the logit model was used to fit the observed data. For single factor designed

experiments, Chi-squire analysis was used to test the difference in the proportion of

oocytes matured, fertilized and developed to certain developmental stages among

treatment groups. The cumulus cell expansion score and cell count were analyzed using

Student-t test. A P-value of < .05 was regarded as statistically significant.

Results

Experiment 7.1

In this experiment, the effect of adding insulin to culture medium during oocyte

maturation incubation on bovine oocyte IVM/IVF/IVC was evaluated. The results of

cumulus cell expansion score, the proportion of oocyte matured, fertilized and developed

to various stages, as well as, the cell count (Figure 4) for morula, blastocyst and hatched

blastocyst stage embryos are shown in Table 20. Insulin addition had a significant effect

on cumulus cell expansion. The mean cumulus cell expansion score in insulin treated

group was 3.61, which was significant higher than score of 3.20 from control group

(P C .05). The fertilization rate was also significantly greater for the insulin treated

group compare with the control group (P C .05). There were no detectable difference

between two treatment groups on the proportion of oocytes that cleaved, and

120

Figure 4. A blastocyst (a) and a hatched blastocyst (b) developed from insulin treatment group after Hoechst 33342 staining of the nuclei. The appearance of meiotic figures and the lack of fragmented nuclei were suggestive of healthy viable embryo. Magnification: X400.

121

Table 20. Bovine oocytes matured in vitro with and without insulin*

Treatment No. of CCE FERT CLEAV MORL BLST HBLSTgroup oocytes score % % %(cell no.) %(cell no.) % (cell no.)

Control 411 3.20“ 6 6 a 54 41 (34)a 27 (75) 19 (509)

Insulin 401 3.61b 77b 55 42 (49)b 23 (93) 17 (8 8 8 )'Abbreviations: CCE=cumulus cell expansion; FERT=fertilized; CLEAV=cleaved; MORL=morula; BLST=blastocyst; HBLST=hatched blastocyst.

, b Values in the same column with different superscripts are significantly different (P C .05).

122subsequently developed to morula, blastocyst and hatched blastocyst stages. However,

the number of cells per embryo was significantly different at morula stage. Embryos

developed from insulin treatment group had greater cell number per embryo than control

group (P< .05). A similar trend was noted at blastocyst and hatched blastocyst stages,

but there were no statistically different, due to a large standard deviation.

Experiment 7.2

The results of Experiment 7.2 are presented in Table 21. All growth factors

tested in this experiment including growth hormone, epidermal growth factor, fibroblast

growth factor, insulin-like growth factor-I, and prostaglandin F*. had no effect on

IVM/IVF/IVC of bovine oocytes in both serum-free medium and serum-containing

medium compared with control (P>.05). When compare serum-containing and serum-

free treatment effect, overall development rates were the same except serum-containing

groups had higher cumulus cell expansion score (3.74 vs. 3.20) and fertilization rate

(90.8% vs. 86,4%) (P < .05). Among treatment groups, 83 to 95% oocytes fertilized,

6 8 to 77% cleaved, 38 to 55% developed to morulae, 9.4 to 20.4% developed to

expanded blastocyst and 4.7 to 12.4% developed to hatched blastocyst stage.

Experiment 7.3a

In this experiment, the effects of AA, PDGF and FBS on bovine oocyte

IVM/IVF/IVC were tested. Cleaved embryos were either co-cultured on cumulus cell

monolayer or cultured in medium alone. A summary of the results are given in Table

22. Overall, growth factor addition had a significant positive treatment effect (P < .001)

on embryo development, which is due to the effect of AA according to the analysis of

weighted-least-square estimates. Culture condition also had significant effect (P < .0005),

123

Table 21. Effect of growth factors on IVF-derived bovine embryos'

Growth No. of CCES FERT CLEAV MORL ExBLAST HBLASTfactor FBS oocytes % % % % % %GH + 200 3.82 95.1 73.7 38.4 10.6 6.1

- 200 3.40 94.7 76.0 50.0 13.2 6.6EGF + 200 3.71 87.8 76.9 52.3 13.3 9.7

- 200 3.31 83.0 71.6 40.0 9.4 4.7FGF + 200 3.69 90.2 69.2 39.5 14.3 7.2

- 200 3.09 87.6 71.4 44.4 13.2 8.5IGF-I + 200 3.66 86.8 69.0 42.3 14.2 6.3

- 200 3.01 84.8 68.7 40.3 14.9 6.5PGFza + 200 3.79 93.2 72.6 49.7 13.7 4.1

- 200 3.20 81.5 68.0 55.0 10.1 5.6Control + 200 3.81 92.0 77.7 47.5 19.3 12.4

- 200 3.22 .86.5 76.7 52.2 20.4 10.8

Total + 1,200 3.74* 90.8* 73.7 44.7 14.2 7.6- 1,200 3.20b 86.4b 72.0 46.6 13.4 7.0

'Abbreviations: GH=growth hormone (50 ng/ml); EGF=epidermal growth factor (10 ng/ml); FGF=fibroblast growth factor (50 ng/ml); IGF-1= insulin-like growth factor-1 (100 ng/ml); PGF2a=prostaglandin F20( (50 ng/ml); FBS=fetal bovine serum (5%); CCES=cumulus cell expansion score; FERT=fertilized; CLEAV=cleaved;MORL=morula; ExBLST= expanded blastocyst; HBLST=hatched blastocyst.

*,b Values in the same column with different superscripts are significantly different (P C .05).

124

Table 22. Arachidonic acid and PDGF on bovine oocyte in vitro maturation, fertilization and development*

Trt Co- No. ofMATR FERTCLEAV No. of MORLBLAST ExBLASTHBLASTgroup cultureoocytes % % % 2 - 8 cell % % % %AA + 224 93 83 81b 75 72 28 25 23b

PDGF + 205 90 80 75* 62 73 18 13 I I

FBS + 229 92 83 71* 52 65 21 21 S'

Control + 209 92 82 75* 58 71 22 19 10-

AA - - - - - 75 76b 23b 20** 9

PDGF - - - - - 62 45* 8* 7* 5

FBS - - - - - 52 65b 29b 23b 4

Control - - - - - 58 48* 9" 5* 3

‘Abbreviations: Trt= treatment; AA=arachidonic acid (50 ng/ml); PDGF=platelet- derived growth factor (5 ng/ml); FBS= fetal bovine serum (5%). MATR= matured; FERT=fertilized; CLEAV=cleaved; MORL=morula; BLST=blastocyst;ExBLST= expanded blastocyst; HBLST=hatched blastocyst.

■>b Values in the same column with different superscripts are significantly different (P < .05).

125

more embryos developing to the later developmental stages when co-cultured with

cumulus cells. There was a interaction between growth factor effect and embryo

developmental stages, which indicates that the growth factor had stronger stimulating

effect at later developmental stages.

There were no differences on oocyte maturation rates and fertilization rates among

four treatment groups. However, more oocytes cleaved from AA group compared with

FBS group (P < .05), and no detectable difference either between AA and control or FBS

and control groups. Pairwise comparison within each developmental stage showed that

significant more embryos developed to hatched blastocyst stage from AA group compared

with other treatment groups when embryos were co-cultured (P< .05), and significantly

more embryos developed to morula, blastocyst and expanded blastocyst stages from AA

and FBS groups compared with other two treatment groups when embryos were cultured

in medium alone (P< .05).

Experiment 7.3b

This experiment evaluated the effect of adding AA at different in vitro culture

intervals on bovine oocyte IVM/IVF/IVC (Table 23). AA was added into the basal

culture medium during 1) maturation incubation, 2) embryo development incubation, 3)

throughout in vitro culture period, and 4) no A A addition as a control. The rates of

oocyte maturation, fertilization and cleavage were not affected by adding AA at all

different culture periods compared with the control (P> .05). However, there was a

trend that more embryos developed to expanded blastocysts and hatched blastocysts when

AA was present in the culture medium compared with the control, although significant

126

Table 23. Adding A A at different time intervals on bovine oocytesIVF and development

Treatment No. of group oocytes

MATR FERT CLEAV% % %

MORUCLEAV

BLAST/CLEAV

ExBLAST/ HBLAST/ CLEAV CLEAV

Maturation 108 8 6 34 30 63" 53 53* 2 2 *

Developmentl08 91 35 27 69* 59 59b 31b

Throughout 108 94 37 34 92b 46 46* 24*

Control 108 93 38 29 74* 42 32“ 1 0 *

'Abbreviations: AA=arachidonic acid (50 fig/ml); MART=matured; FERT=fertilized; CLEAV=cleaved; MORL=morula; BLST=blastocyst; ExBLST= expanded blastocyst; HBLST=hatched blastocyst.

*,bValues in the same column with different superscripts are significantly different (P < .05).

127

difference was detected only between adding A A during embryo development culture and

the control (P< .05).

When A A was added throughout the whole in vitro culture period, more cleaved

embryos developed to morula stage than that of other AA treatment groups and control

group (P< .05). No significant difference were found among three AA addition groups

at blastocyst, expanded blastocyst and hatched blastocyst stages (P> .05).

Experiment 7.3c

This experiment was to investigate the dose response of adding AA on the

development of IVF-derived bovine embryos. Four concentration levels of AA (10, 50,

100 and 500 ng/ml) were evaluated in this experiment. A A was added to culture medium

during embryo development culture. Two- to 8 -cell stage IVF-derived embryos were

randomly assigned to each treatment groups. Overall, adding 10 ng/ml, 50 ng/ml, 100

ng/ml, but 500 ng/ml of A A to the culture medium had a beneficial effect on in vitro

embryo development (Table 24). The difference among treatment groups started to show

up at blastocyst stage. More embryos developed to the blastocysts from 50 ng/ml group

compared with the control (P< .05), and no difference was detected at this stage among

10 ng/ml, 50 ng/ml, and 100 ng/ml groups (P> .05). At expanded blastocyst and

hatched blastocyst stages, the embryo development were significantly improved in 1 0

ng/ml, 50 ng/ml and 100 ng/ml groups compared with the control group (P C .05).

The AA concentration of 500 ng/ml had no effect on embryo development at all

stages evaluated in this study compared with the control (P> .05). The treatment group

that had the highest developmental rates at various stages was 50 ng/ml group,

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Table 24. Dose response of AA on the development of IVF-derived bovine embryos*

Treatment No. of group 2 to 8 cell

No. of MORL (%)

No. of BLAST (%)

No. of ExBLAST (%)

No. of HBLAST (%)

0 ng/ml 96 61 (64) 30 (31)* 25 (26)* 2 (2 )-

1 0 ng/ml 96 64 (67) 40 (42)be 27 (28)b 9 (9)*

50 ng/ml 96 70 (73) 43 (45)' 35 (36)b 15 (16)'

1 0 0 ng/ml 96 67 (70) 38 (40)* 29 (30)b 1 0 ( 1 0 )*

500 ng/ml 96 70 (73) 26 (27)' 16 (17)" 4 (4)*

’Abbreviations: AA=arachidonic acid; MORL=morula; BLST=bIastocyst; ExBLST=expanded blastocyst; HBLST=hatched blastocyst.

•■b‘cValues in the same column with different superscripts are significantly different (P< .05).

129

with the development rates at blastocyst, expanded blastocyst and hatched blastocyst

stages of 45, 36 and 16%, respectively.

Discussion

The role of peptide growth factor on preimplantation embryo development has

attracted a great attention to the developmental biologists in resent years. The biological

effect of growth factors have been demonstrated in intact animals, organ cultures and cell

cultures (Gospodrowicz and Moran, 1976). However, how growth factor affect

fertilization and early embryo development is unclear. The development of in vitro

embryo culture systems and the success on in vitro fertilization in many mammalian

species make it possible to investigate the effect of growth factor on early development

in vitro. One approach to study growth factor on early development is by adding growth

factors to culture media and measuring embryo developmental rate in culture. This

approach may give a clue for further biochemical and molecular studies on growth

factors and their receptors gene expression and protein synthesis by early embryos. It

may also give some information that helps to reveal the growth factor reaction passway.

In the present study, this approach was used to evaluate six common peptide growth

factors (insulin, GH, EGF, FGF, IGF-1, PDGF) and two fatty acids (PGF^, A A) on

bovine oocyte in vitro maturation, fertilization and subsequent embryo development.

The addition of insulin

The data from present study demonstrated that insulin had a significant effect on

cumulus cell expansion during oocyte in vitro maturation. This finding suggests that

insulin was able to stimulate the functional hyaluronic acid synthesis and secretion within

the cumulus mass (Chen et al., 1993). It has been reported that in several species, the

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optimal growth and expression of function of granulosa cells require the presence of

insulin (Channing et al., 1976; Orly et al., 1980; May and Schonbery, 1981; Savion et

al., 1981). In addition, the quality of cumulus cell expansion has been shown closely

related to the success of in vitro fertilization in cattle (Ball et al., 1983) and mouse (Chen

et al., 1993). The results of present study are in agreement with those of previous

findings that showed a significant improvement of in vitro fertilization in the insulin-

treated oocyte compared with the control (P < .05). This evidence further demonstrates

that insulin is able to modulate the metabolism and function of cumulus cells, which may

affect the sperm penetration of the oocyte and subsequently fertilization.

Another interesting finding from this experiment was that although fertilization

rate was higher in insulin treated group, the proportion of the oocytes that cleaved and

developed to morula and blastocyst stages were not different. Furthermore, the number

of cells per embryo was significantly greater in the insulin-treated group at morula stage

(P< .05), and the same trend was found at blastocyst and hatched blastocyst stages. A

possible explanation for these results is that at early maturation stage, insulin mainly

associated with cumulus cell activity instead of oocyte itself. This causes cumulus cell

expansion and facilitates sperm penetration, but does not have an effect on early

embryonic cell division. After maternal-embryonic genomic transition, which has been

proved at 8 to 16-cell stage in bovine (King et al., 1989), the embryos start to synthesize

proteins and peptides that required for cell perliferation and differeciation. Insulin may

be taken up by embryo and involved in the embryonic metabolism at this time. In

contrast, insulin is a well known anabolic hormone, which promotes the synthesis of

glycogen, protein, and lipid while inhibits the degradation of these substances (Czech,

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1977). It have been reported that the protein, DNA and RNA synthesis in

preimplantation mouse embryos were enhanced by insulin (Harvey and Kaye, 1988;

Heyner et al., 1989). Therefore, in present study, the insulin supplementation of the

culture medium may have had stimulatory effect on mitotic division of the embryonic

cells after maternal-embryonic genomic transition and resulted in greater number of cells

at morula and blastocyst stages.

One thing that should be mentioned in this study, is that insulin was added to the

culture medium only during the time of oocyte maturation incubation, while the primary

effects of insulin was on cell division at the morula and blastocyst stages. This may be

an indication that insulin can be stored and then used by embryos. Correspondingly,

Heyner et al. (1989) have reported that insulin receptors are expressed in early morula

stage mouse embryos and increased in numbers through the blastocyst stages. Their

study demonstrated that insulin was present in mouse oviductal fluid, and the insulin that

was internalized by the early embryo was of maternal origin. They also reported that

insulin could be absorbed nonspecifically in the zona pellucida and bound specifically to

its receptors on cytoplasmic membrane of the blastomeres. The ability of zona pellucida

to accumulate proteins was also reported by Schlafke and Enders (1973), which may

explain the later on effect of insulin on embryonic cell division observed from the present

study.

Ceil number and the mitotic index of in v/Vro-cultured embryos are now used by

developmental biologists as an indicator of embryo quality. It has been reported that in

v/vo-produced bovine embryos had greater cell number compared with in wVro-produced

embryos at the morula blastocyst and expanded blastocyst stages (Iwasaki et al., 1990),

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and in v/vo-cultured IVF-derived embryos had similar cell number as in vivo produced

embryos (Harlow and Quinn, 1982; Iwasaki and Nakahara, 1990). This finding indicates

that existing in vitro culture system is not as efficient as in vivo, although oocytes can

be matured, fertilized and cultured to morula, blastocyst, even hatched blastocyst stages

in vitro.

The viability of in v/'rro-produced embryos may not be as good as in vivo-

produced embryos when pregnancy after transfer to recipient animals is used as the

indicator of embryo quality (Bowman and McLaren, 1970; Gandolfi and Moor, 1978).

The present study showed a significant improvement in the number of cells per embryo

by adding insulin into the maturation medium, which confirmed previous findings that

insulin is involved in mammalian early stage embryo development by Heyner et al.

(1988,1989). This further indicates that insulin may play a role in early bovine

embryonic development.

The addition of GH. EGF. FGF. IGF-1. PGF7-

Experiment 7.2 was designed to investigate the effect of primary growth factors

on IVM/IVF/IVC of bovine oocytes. The only difference detected in the results was that

the overall cumulus cell expansion scores and fertilization rates were greater when serum

was presented in the culture medium (P < .05). This would suggest a positive effect of

serum on cumulus cell expansion and sperm penetration. However, except cumulus cell

expansion and fertilization rates, the proportion of oocyte matured, cleaved and

developed to morula, blastocysts and hatched blastocysts were not different when

compared with control medium either with or without serum supplementation. This

findings suggest that serum may not be necessary for IVM/IVF/IVC of bovine oocytes.

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Exogenous serum has long been used as a cell culture additive to stimulate cellular

proliferation, cell attachment and biological production (Jayme et al., 1988). Beneficial

effect of serum supplementation has been demonstrated in oocyte in vitro maturation,

fertilization and subsequent embryo development (Choi et al., 1987; Vanderhyden and

Armstrong, 1989; Leibfried-Rutledge et al., 1987). Serum has been used as a medium

supplement in most bovine IVM/IVF/IVC systems with concentration in the media

ranging from 5%(Goto et al., 1989; Zhang et al., 1993) to 20%(Younis and Brackett,

1991; Xu et al., 1992). However, serum is an undefined fluid that exhibits lot-to-lot

variability in biochemical composition and culture performance (Art to Science in Tissue

Culture Vol. 5:3-4, Hyclone Laboratories, 1986).

Efforts have been made to improve the culture system by adding growth factors

(Paria and Dey, 1990; Das et al., 1991; Harvey and Kaye, 1992), gonadotrophins

(Younis and Brackett, 1992) or both (Happer and Brackett, 1993) to the serum-free

culture medium to stimulate oocyte and embryo in vitro development. The results of the

present study indicate that when cumulus cells were used as feeder cells for co-culture,

oocyte maturation and embryo development were not affected by the presence of serum.

Similar results have been reported by Saeki et al. (1991), who reported successful oocyte

maturation, fertilization and embryo development to blastocyst stage in cattle using

serum-free medium and cumulus cell co-culture. This finding allows us to eliminate the

time consuming work on serum lot screening and possible contamination of the culture

system from serum. Removing the serum provides for a more defined culture system to

study the mechanism of cumulus cell co-culture on early mammalian development.

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Recent studies have shown the evidences of the involvement of growth factors in

early mammalian embryonic development. The expression of growth factor genes

(Rappolee et al., 1988), the presence of growth factor receptors (Rappolee et a!., 1990;

Adamson, 1990; Wiley et al., 1992) and the growth promoting effect of exogenous

growth factors on mammalian oocyte and preimplantation embryo development (Yoshida

and Kanagawa, 1984; Paria and Dey, 1990; Das et al., 1991; Harvey and Kaye, 1992;

Harper and Brackett, 1993) have been reported. However, some research groups also

reported the lack of effect of exogenous growth factors on bovine oocyte IVM/IVF/IVC

(Colver et al., 1991; Flood et al., 1993).

The lack of effect of growth factors on oocytes maturation, fertilization and

embryo development from the present study may be explained as follows. First, in

present study, oocytes and embryos were cultured in groups (20 to 25/group in .5 ml

culture medium) instead of individually. Therefore, the effect of exogenous growth

factors could be covered by the effect of embryonic origin growth factors that participate

in embryo development in an autocrine manner. Paria and Dey (1990) reported a

cooperative interaction among embryos in culture, and indicated that this interaction is

mediated by specific growth factors released by embryos. They found that 2-cell mouse

embryos cultured singly had low development to blastocyst stage and lower cell number

per blastocyst compared with those cultured together in groups. Their study also showed

that the low development of single-cultured embryos was markedly improved by addition

of EGF or TGF-/31 to the culture medium. The beneficial effect of group embryo culture

has also been reported by Wiley et al. (1986).

135

Secondly, since the cumulus cell co-culture system was used in the present

experiment, the effect of exogenous growth factors could be masked by the interaction

between embryos and the helper cells. One hypotheses on the beneficial effect of co

culture is that helper cells may produce substance(s) that promote embryonic cell

proliferation and differentiation (Kane et al., 1992). There is a wide variety of somatic

cell types that have been shown to enhance embryo development in vitro (Camous et al.,

1984; Voelkel et al., 1985; Rexroad and Powell, 1986, Goto et al., 1989, Kim et al.,

1991).

In summary, this experiment showed no effect of GH, EGF, FGF, IGF-1, and

PGF2„ on in vitro development of IVF-derived bovine embryos with or without serum

supplementation under our current IVM/IVF/IVC system. This study also demonstrated

that bovine oocytes can be matured, fertilized and cultured to hatched blastocyst in

serum-free medium when co-cultured with cumulus cells originated from oocyte cumulus

complex.

The addison of AA and PDGF

The effect of arachidonic acid and platelet derived growth factor were evaluated

in Experiment 7.3a. Based on the finding of Experiment 7.2, the co-culture system may

mask the effect of exogenous growth factors. In the present experiment, cleaved

embryos from each treatment group were randomly divided into two groups and were

either co-cultured with cumulus cells or cultured in medium alone. The major finding

from this experiment was that the addition of AA to culture medium had significant effect

on blastocyst hatching when embryos were co-cultured compared with other treatment

groups. The proportion of embryos developed to morula, blastocyst and expanded

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blastocyst were enhanced with AA addition when embryos were cultured in medium

alone. This finding suggests that AA was able to promote bovine embryo development

by affecting blastomere compaction, embryonic cell differentiation, blastocyst expansion

and hatching. Furthermore, these results further verified that the effect of exogenous

growth factors could be masked by co-culture. The results showed the equivalency of

AA to cumulus cell co-culture and serum supplementation on morula, blastocyst

formation and blastocyst expansion.

A A is a C20 essential fatty acid and a precursor of prostaglandins (PG). The

uptake of AA by prehatched embryos (mainly blastocyst stage embryos) from the culture

medium has been reported in several mammalian species including cattle (Menezo et al.,

1982; Hwang et al., 1988), sheep (Marcus, 1981; Sayer and Lewis, 1993) and primates

(Neulen et al., 1991). Furthermore, it has been shown AA can be incorporated into the

prehatched embryos and converted inside the embryo into a number of compounds,

mainly prostaglandin and thromboxanes (Hwang et al., 1988; Neulen et al., 1991; Sayre

and Lewis, 1993). It is reported that the conversion of AA to PG by embryos may have

the role in embryo hatching from the zona pellucida (Biggers et al., 1978; Chida et al.,

1986; Sayre and Lewis, 1993). The results from the present study are further

demonstrated a hatching promoting effect of AA on the development of in vitro cultured,

IVF-derived bovine embryos both in co-culture and medium alone culture systems.

Beside hatching, the results of present study also provided an evidence that AA

is able to stimulate embryo development at the stage of first cell division, morula and

blastocyst formation. For many years, research had been focused on how AA convert

to PG and then affect mammalian development. However, resent studies showed that AA

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is directly involved in the signal transduction system that regulates the cell metabolism.

It has been proposed that AA is a second messenger generated from membrane

phospholipid, which actives the enzymes and regulates the ion channels (O’Neill et al.,

1990; Hansel et al., 1991). Chien et al. (1986) reported that in progesterone induced

amphibian oocyte maturation, AA incorporation was increased after an increased Ca2+

efflux. AA was found to be able to trigger starfish oocyte maturation, which is normally

induced by nature hormone 1-methyladenine (Meijer et al., 1986).

Using radiolabeling technique, Waterman and Wall (1988) found that rabbit

zygotes were able to concentrate A A to a 170 fold of the initial concentration in culture

medium during 6 h of incubation. These findings indicate that AA could be involved in

maturation, oocyte activation during fertilization and early embryo development. In these

events, AA may have a direct effect on development as a second message instead of

converting into PG in the cells. The evidence of the direct action of A A is that A A was

shown to be able to active enzymes and ion channels (O’Neill et al., 1990).

A study on luteal cell function by Lukaszewska and Hansel (1980) demonstrated

that A A stimulate luteal cell secretory function by inducing oxytocin and progesterone

release. They also showed that this stimulatory effect do not result from PG formation

(Lafrance and Hansel, 1992). This is further evidence of second message role of AA in

modulating cell function.

In Experiment 7.3b, AA was added into culture medium during maturation,

embryo development, or throughout the whole in vitro culture period. The result of this

experiment showed significant improvement of blastocyst expansion in all AA treated

groups compared with the control (P < .05). Also, there was no difference among AA

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treatment groups, which indicated that A A can be taken up by oocyte, zygote or early

stage embryos. The effect of AA appeared at blastocyst stages as the embryos were co-

cultured with cumulus cells in this experiment.

In Experiment 7.3c, the dose response of AA on IVF-derived bovine embryo

development was evaluated. The concentration of 10 ng/ml, 50 ng/ml and 100 ng/ml of

AA had a significant promoting effect on blastocyst formation, blastocyst expansion and

embryo hatching (P < .05). The concentration of 500 ng/ml AA tested in this experiment

did not improve embryo development when compared with the control group. The

developmental rates at blastocyst and expanded blastocyst stages were found to be even

lower than that of control group. This finding suggests a high level of AA had a

negative effect on embryo development in vitro. Excess A A in the culture system may

not only be unable to stimulate but also inhibit the development by blocking or altering

other metabolism passway. The best embryo development rates obtained from this

experiment was the group with the addition of 50 ng/ml of AA to the culture medium.

In this study, no effect was found when PDGF was added to culture medium

when compared with the control. PDGF is one of the growth factors that its transcripts

encoding ligand and receptor were found in oocyte and embryos, therefore, is probably

synthesized by prehatched embryos (Rappolee et al., 1988; Watson et al., 1992, 1994).

It is proposed that PDGF may have effect on embryonic cell proliferation and

differentiation. The possible explanation for the results of the present study could be that

embryos do not need exogenous PDGF at early developmental stages. Secondly, PDGF

of embryo origin is enough to support the early development. As mentioned earlier, the

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cooperative interaction among embryos may exist in this study because embryos were

cultured in groups.

This study showed a strong evidence of arachidonic acid stimulating embryo

development in vitro. The positive effect was mainly on the development of morula,

blastocyst and hatching stages. This effect was significant even when embryos were

cultured together in group and co-cultured with somatic cells. Furthermore, these results

indicate that AA can be taken up by oocytes, zygotes or early cleavage stage embryos

from culture media. In this study, the optimal AA concentration was SO ng/ml of

medium; whereas, the 500 ng/ml concentration of AA appeared to be toxic to cultured

embryos.

In conclusion, adding growth factor(s) into culture media is a useful approach to

study the effect of growth factor on preimplantation embryo development. Previous

studies provided the evidence of synthesizing and releasing growth factors by early

embryos, and the presence of growth factor receptors on early embryos. The present

study further demonstrated that insulin and arachidonic acid are involved in bovine

oocyte maturation, fertilization and early embryo development. This study also showed

the overlapping effect of growth factor with co-culture or the presence of serum in

culture medium. Some growth factors tested in this study had no effect on early

development, which may reflect the presence of cooperative interaction among those

group cultured embryos in our culture system. Further study is needed to evaluate the

growth factors on embryo in vitro development in a well controlled culture system. This

is needed to reveal the regulating passway of growth factors on mammalian early

development.

CHAPTER 8

BIRTH OF LIVE CALVES AFTER TRANSFER OF FROZEN-THAWED BOVINE EMBRYOS FERTILIZED IN VITRO

Introduction

The first successful in vitro fertilization procedure producing a live calf was

reported in the USA in the early 1980s (Brackett et al., 1982a). Since then, the methods

for in vitro maturation and fertilization have markedly advanced. Bovine morulae and

blastocysts can now be obtained routinely from in vitro matured/m vitro fertilized and

in vitro cultured oocytes collected from abattoir ovaries. Although pregnancies and live

calves have been obtained from the transfer of embryos matured, fertilized and cultured

in vitro in several countries (Goto et al., 1988; Lu et al., 1988; Xu et al., 1990; Jiang

et al., 1991; Monson et al., 1992), the question remains whether in vitro fertilized

embryos are as viable in utero as in v/vo-derived embryos. Furthermore, it is not known

whether bovine embryos fertilized in vitro can survive freezing and then produce viable

offspring after being transferred into a recipient cow. Iwasaki et al. (1990) have reported

that in vitro fertilized bovine embryos often have a smaller inner cell mass and fewer

cells at the blastocyst stage than embryos of a similar age produced in vivo. In this case,

culturing embryos fertilized in vitro could increase the chance of success after in vitro

fertilization by transferring embryos that are more developed and from a more selected

embryo population. If culturing an embryo after it has been thawed offers an advantage,

then it should be considered for developing frozen-thawed in vitro fertilized embryos

before they are transferred to recipient animals.

140

141

The objective of this study was to evaluate the viability of in vitro fertilized

bovine embryos after freezing and thawing and their subsequent pregnancy rates in

recipient cattle.


Experimental oocytes

Ovaries from beef and dairy cattle were collected at a local abattoir and

transported to the laboratory in Dulbecco’s-phosphate buffered saline (PBS) at 23 to

25°C. In the laboratory, ovaries were washed three times in PBS containing antibiotics

(100 IU penicillin and 100 /zg streptomycin/ml). The oocytes were aspirated from 3 to

8 mm follicles using an 18-gauge needle attached to a 6 ml sterile, plastic syringe. The

oocytes were evaluated for the number of layers of cumulus cells and washed twice in

PBS containing . 1 % polyvinyl alcohol (PVA). The oocytes with intact cumulus cells and

homogenous cytoplasm were used in this study.

In vitro maturation and fertilization procedures

Good quality cumulus-intact oocytes were placed in four-well tissue culture plates

(Nunclon) (25 oocytes/well) and incubated at 39°C for 24 h in .5 ml of Tissue Culture

Medium 199 (TCM-199) supplemented with 5% fetal bovine serum (FBS) in 5% CO*

in an atmosphere of air for maturation. Matured oocytes were then exposed to the

standard bovine in vitro fertilization procedure used in this laboratory (Chapter 3).

Briefly, two straws of frozen semen from a fertile Holstein dairy bull were

thawed in a 25°C water bath for 1 min. The sperm cells were washed in Brackett-

Oliphant (B-O) medium supplemented with 10 mM caffeine and adjusted to a

concentration of 3 x 106 motile sperm cells/ml and then exposed to Ca++ ionophore

142

A23187 (ionophore concentration of . 1 pM) for 1 min to aid in capacitation. Sperm and

oocytes were then incubated in a 100 pi drop of B-O medium with 10 mg/ml bovine

serum albumin (BSA) at 39°C in 5% C 02 in an atmosphere of air. After 6 h of

incubation, the oocytes (with their cumulus cells) were moved to TCM-199 with 5% FBS

and cultured for an additional 43 h at 39°C in 5% C 02 in air.

Cumulus cells surrounding the embryos were removed manually with a glass

pipette. These cells and those already attaching to the culture dish were then maintained

as a co-culture system. The fertilized embryos were then cultured with the attached

cumulus cells in TCM-199 with 5% FBS at 39°C in 5% CO2 in air. The culture medium

was replaced with fresh medium at 48-h intervals. The embryos were evaluated

immediately after the medium change using an inverted microscope to establish the

development of morula, blastocyst and expanded blastocyst stages in vitro.

Embryo freezing and thawing

When 300 contemporary in v/rro-fertilized embryos reached either the 16-cell,

morula or early blastocyst stage they were placed in .25 ml plastic straws (2

embryos/straw) and frozen in 10% glycerol using a standard embryo freezing procedure

(Leibo, 1986). The embryos were plunged into liquid nitrogen (-196°C) when they had

reached -33 °C. After approximately 1 year of storage in liquid nitrogen, 49 straws

(containing 98 embryos at various morphological stages) were selected and thawed in air

at room temperature (22°C). The cryoprotectant was removed in three 5-min steps with

mixtures of sucrose and glycerol (.3 M sucrose with 10% glycerol, 5% glycerol and no

glycerol) and then into PBS medium. The embryos were then evaluated in terms of their

143

morphological development and placed into in vitro culture treatment groups before being

transferred to recipient females.

Post thaw embryo culture and transfer

Frozen-thawed embryos were cultured either for 4 h in TCM-199 or cultured for

2 days on cumulus cells with TCM-199, as previously described by Zhang et al. (1992b).

Embryos from the group cultured for up to 4 h, which appeared viable, were then

transferred nonsurgically to recipient females (1 or 2 embryos/female) ipsilateral to the

corpus luteum on days 6 or 7 of their estrous cycle (estrus=day 0). Similarly, morulae

and blastocysts from the group cultured for 2 days were then transferred to recipient

animals (1 or 2 embryos/female) on days 7 or 8 of their estrous cycle. Either one

expanded blastocyst or one expanded blastocyst plus one morula were transferred to each

recipient females in 2-day culture group.

Results

Of 2,297 bovine oocytes exposed to in vitro maturation during a 30-day

experiment period, 2,108 (92%) matured, 1,917 (83%) were fertilized, 1,674 (73%)

cleaved, 1,048 (47%) developed to the morula stage and 317 (14%) developed to the

expanded blastocyst stage. An additional 300 embryos produced during this 30-day

period were cultured in TCM-199 to 16-cell, morula or early blastocyst stages on bovine

cumulus cells and then frozen using a standard laboratory freezing procedure (Figure 5).

These embryos were stored in liquid nitrogen at -196°C for later use.

The results of thawing, culturing and transferring the frozen IVF-derived embryos

are summarized in Table 25. Of the 98 frozen embryos that were randomly selected and

thawed, 58 were incubated for up to 4 h, of which 36 (62%) were determined to be

144

Figure 5. IVF-derived embryos produced from this study. A group of 300 embryos were frozen at 8- to 12-cell stage (a), 16-cell stage (b), morula stage (c) and early blastocyst stage (d). Ninety eight of these frozen embryos at 16-cell, morula and blastocyst stages were thawed, cultured and transferred to the recipient females. Magnifications: a, X100; b, c and d, X600.

145

Table 25, Results of culturing and transferring frozen-thawed in vitrofertilized bovine embryos

Cultureinterval

No. of embryos No. of embryos thawed transferred (%)

No. of recipients

No. of pregnancy* (%)

Offspring born (sex)

<£4 h 58 36 (62) 20 4(20) 2 (1F,1M)

^ 2 days 40 12b (30) 10 5(50) 3 (3M)

Total 98 48 (49) 30 9 (30) 5 (IF, 4M)

‘Pregnancy diagnosed 75 days after transfer.

'Transferred at least one expanded blastocyst/recipient.

146

viable. Of the 40 embryos cultured for 2 days, 12 (30%) appeared viable after the

culture interval, and 10 (25%) of these developed to the expanded blastocyst stage.

The 36 viable embryos cultured for up to 4 h were transferred to 20 recipients,

and the 12 viable embryos that co-cultured for 2 days were transferred to 10 recipient

females. Nine (30%) of the 30 recipients receiving frozen-thawed embryos were

diagnosed pregnant. Only 4 of the 20 (20%) recipients received embryos cultured for

up to 4 hours were diagnosed pregnant; whereas, 5 of the 10 (50%) recipients with

embryos cultured for 2 days were determined to be pregnant.

Five live healthy calves were bom after a 278- to 295-day gestation interval, with

body weights ranging from 37.3 to 54.5 kg (Figure 6). The 4 embryos that implanted

and then failed to survive, were lost after 75 days of pregnancy. One calf died of

unknown causes shortly after birth, but the remaining calves gave every indication of

being normal, and nursed without difficulty. These calves continued to grow and

develop normally to adulthood.

The survival rates of the 40 frozen-thawed embryos that cultured for 2 days are

shown in Table 26. Of the embryos that were co-cultured, 67% of the early blastocyst

stage embryos and 46% of the morulae developed to expanded blastocysts; whereas, only

8.3% of the 16-cell stage embryos developed to expanded blastocyst after the co-culture

interval.

Discussion

This high maturation and fertilization rates achieved demonstrated that the in vitro

fertilization procedure used for bovine oocytes was efficient for the production of pre

blocked embryos. The cumulus cell co-culture system was able to provide a suitable

147

Figure 6. First frozen-thawed IVF-derived embryo resulting in live birth (a), and other calves and recipient cows from the same study (b). Although the breed types of the calves contributed to the range in birth weights, all calves were vigorous and developed normally during the post partum nursing period.

148

Table 26. Development of frozen-thawed in vitro fertilized bovine embryosafter co-culture with cumulus cells

Stage of thawed embryos No. of embryos cultured No. of expanded blastocysts (%)

16-cell 24 2 (8.3)

Morulae 13 6 (46)

Early blastocysts 3 2(67)

Total = 40 10 (25)

149

environment for the early embryos to overcome the 8- to 16-cell in vitro developmental

block and develop to the 16-cell and the early morula stages. However, Zhang et al.

(1992c) have recently suggested that there is another decline in embryo development

between the morula stage and the blastocysts stage during the in vitro culture of in vitro

fertilized bovine embryos, and further study may therefore be needed to develop a better

in vitro culture system in order to overcome this secondary in vitro block.

The results of culturing frozen-thawed in vitro fertilized bovine embryos (Table

26) clearly indicated that more later stage embryos (morulae and early blastocysts)

survived freezing and culturing (>46% ) than similarly frozen-thawed 16-cell stage

embryos (< 10%). This finding suggests that when culturing frozen-thawed, in vitro-

produced embryos, late morula or early blastocyst stages are likely to result in a greater

pregnancy rate after transfer.

The pregnancies and the birth of healthy calves after the transfer of frozen-thawed

in vitro fertilized embryos verifies that embryos that are matured, fertilized and cultured

in vitro are viable after freezing. One advantage may be the highly selected embryos

provided by using embryo culture after thawing. In the present study, a higher

pregnancy rate (50%) resulted when frozen-thawed embryos were co-cultured for 2 days

and developed to the blastocyst stage before being transferred than when embryos were

cultured for up to 4 hours before being transferred. This pregnancy rate is similar to that

achieved by frozen-thawed bovine embryos (48.4%) derived in vivo at commercial

transplant units (Munar et al., 1990). Long term culture increased the selection effect

for embryos of transferable quality when compared with short term culture (30% vs.

62%), however, the overall efficiency (number of pregnancies per number of embryos

ISO

thawed) was also considered higher for long term cultured embryos. One possible

explanation may be that transferring embryos at the blastocyst stage may produce a

stronger embryonic signal to trigger the maternal recognition of pregnancy than

transferring embryos at the 16-cell and morula stages.

The normal calves bom during this study indicate that embryos that have been

matured, fertilized and cultured in vitro can survive freezing, and culturing and finally

develop to term after being transferred to recipient cattle. This success with in vitro

fertilization has encouraged the authors to make use of the oocyte resource from

slaughterhouse animals to produce a laboratory supply of bovine embryos for research

purposes. Producing calves from frozen-thawed in vitro fertilized embryos also affords

opportunities to enhance the reproductive efficiency of farm animals and of rare,

endangered species.

CHAPTER 9

CONCLUSION

The in vitro fertilization technology for cattle has developed rapidly during the

past decade. Laboratories have been successful with their IVF protocols with proof of

viability resulting with embryos producing live young after embryo transfer. However,

the success rates of many IVF procedures are variable and the overall efficiency of

current in vitro systems is still low. To evaluate factors affecting the successes of in

vitro production of bovine embryos, a series of experiments were conducted in the

present study.

A simple and efficient IVM/IVF/IVC procedure was developed, which produced

a large number of prehatched bovine embryos for research and for embryo transfer.

Oocytes that harvested from abattoir ovaries were matured in hormone-free medium,

inseminated with Ca++ ionophore A23187 treated sperm and co-cultured with endogenous

cumulus cells. Embryos derived from this procedure progressed rapidly through the 8-

to 16-cell in vitro developmental block and resulted normal calves after being frozen,

thawed and transferred into recipient animals. With this procedure, maturation rate of

90 to 95%, fertilization rate of 85 to 90%, cleavage rate of 65 to 75%, and morula and

blastocyst development of 45 to 50% were consistently resulted after processing over

6,000 bovine oocytes in this study.

An experiment that compared three ovary holding temperature (0°C, 18°C and

25 °C) verified the detrimental effects of cooling ovaries after collection on the

development competence of oocytes. When ovaries were held at 0°C 3 for hours during

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152

transportation to the embryo laboratory, the rates of oocyte maturation and fertilization

were significantly reduced, and none of the oocytes cleaved from that group. When

ovaries were held at 18°C for 3 hours, only 8% of oocytes developed to morula stage

and none hatched, which was significantly lower than that of 25°C (50% and 20%). It

appears that holding temperature of 25 to 30°C is suitable for bovine ovary transportation

than lower temperatures based on the results from the present study. Therefore, in our

IVF protocol, ovary transportation and subsequent handling (oocyte aspiration, evaluation

and washing) are now preformed at room temperature (25-28°C).

The optimal incubation temperature and C 02 level were found very important for

the in vitro maturation and fertilization of bovine oocytes. An experiment of comparing

different combinations of two temperatures (37°C vs. 39°C) and two C 02 levels (2.5%

vs. 5%) during maturation and fertilization showed that only 8% of 600 oocytes cleaved

when matured and fertilized both at 37°C under 2.5% COj. The cleavage rate was

significantly increased when oocytes were matured at 37°C, but fertilized at 39°C in

2.5% C 02 (27.6%). A similar cleavage rate was obtained when oocytes were both

matured and fertilized at 39°C in 2.5% C02 (24.7%). The highest cleavage rate was

resulted when oocytes were matured and fertilized at 39 °C with the CC^ level raised to

5% (78.7%). These results indicate that the incubation temperature of 39°C is critical

for bovine oocyte fertilization, but not for maturation. Furthermore, low CO* levels may

have a negative effect on both maturation and fertilization of bovine oocytes.

In an experiment on cumulus cell function during IVM/IVF/IVC, cumulus cells

were removed before and after maturation and after fertilization for 7, 20 and 48 hours.

The cumulus-free oocytes or embryos were either cultured in medium alone or co

153

cultured on cumulus cell monolayers that prepared on the day of maturation culture.

Cumulus cell removal before maturation significantly reduced the rates of oocyte

maturation (4 to 26% vs. 93 to 96%) fertilization (0 to 9% vs. 91 to 92%), and in vitro

development at all stages evaluated. Cumulus cell removal immediately prior fertilization

or 7 hours after fertilization reduced the rates of fertilization (58 to 71 % vs. 91 to 92%),

cleavage (40 to 54% vs. 74 to 78%), and morula plus blastocyst development (15 to

24% vs. 45%). Cumulus cells removal at 20 hours after IVF resulted in similar rates

of development to that of control (cumulus cells removal at 48 hours after IVF) at all

stages evaluated in this study.

The results from this study demonstrated that cumulus cells are important and

necessary for oocyte maturation and acquisition of full embryonic development

competence. The intact state of surrounding cumulus cells are beneficial to the oocyte

and subsequent embryo development at least up to 7 hours but not at 20 hours after

insemination of oocytes. The results also showed that the co-culture of denuded oocytes

improved the rates of maturation and fertilization. However, this improvement did not

overcome the detrimental effect caused by breakdown of the intercellular connections

between cumulus cells and oocytes before and after maturation. This study verified the

relationship of cumulus cells and oocytes at early developmental stages, and provide

important information for in vitro fertilization and gamete micromanipulation research.

Bovine oviduct epithelial cells (BOEC) and bovine cumulus cells (BCC) are most

commonly used helper cells for co-culture of IVF-derived bovine embryos. In the

experiment of evaluating these two co-culture systems, results showed that cumulus cell

co-culture consistently supported more 2- to 8-cell stage embryos developing to morula

154

and blastocyst stages. This was also the case in either simple defined serum-free medium

(CZB) or in complex serum-containing medium (TCM-199). When BOEC and BCC was

used in combination for embryo co-culture, the development rates were similar to that

of BCC co-culture at all stages tested. This result further established that BCC co-culture

is stable in supporting early bovine embryo development in vitro. However, the effect

of BOEC on early development may depend to some degree on the stage of estrous cycle

of the cow from which the cells are harvested.

When two culture media were compared in this study (CZB vs. TCM-199), the

results showed that more early stage embryos developed to morulae in serum-free CZB

than in serum-containing TCM-199, but the embryo development in CZB medium was

slowed down thereafter and significantly fewer embryos hatched in CZB than in TCM-

199. It appears that serum inhibited embryo cleavage at early development stages but

stimulated the blastocyst formation, expansion and hatching. Findings from this study

suggest that a culture system that starts with serum-free medium and then systematically

increases the serum concentration to fit the embryo developmental stage may be more

suitable for in vitro production IVF-derived bovine embryos.

Although there are evidences that growth factors involved in pre-hatched embryo

development, how this factors regulate this early developmental process remains to be

determined. In the present study, eight growth factors/mitogens (insulin, GH, EGF,

FGF, IGF-1, PDGF, PGF^ and AA) were investigated by adding them to the culture

medium and monitoring the embryo development in vitro. Among these growth factors,

arachidonic acid was found to have a marked stimulating effect on IVF-derived bovine

embryos. Arachidonic acid promoted early stage embryos developed to morula,

155

blastocyst and hatching stages, and this effect was significantly greater than the other

treatments both in co-culture or medium alone culture. The optimal arachidonic acid

concentration was found to be 50 ng/ml of culture medium, and high concentration (500

ng/ml) actually inhibited blastocyst formation. This was the first time that direct

evidence of arachidonic acid stimulating bovine embryo development has been reported.

Further study is necessary to reveal the mechanism of this response.

Beside arachidonic acid, insulin was found to be able to stimulate cumulus cell

expansion during maturation and increase the rate of in vitro fertilization for bovine

oocytes. Insulin also stimulated the embryonic cell proliferation by increasing the

number of cells/embryo at the morula, blastocyst and hatched blastocyst stages when

compared with the control group. This finding suggests that exogenous insulin

participated in the metabolism of incubated oocytes and further indicated that insulin is

involved in early embryo development.

Other growth factors evaluated in this study had no detectable effect on in vitro

embryo development when compared with the control treatment. This may be explained

by the presence of a cooperative interaction among cultured bovine embryos. It has been

found that embryos can produce growth factors as well as express receptors for those

autocrine/paracrine growth factors. Since the embryos were group cultured in these

experiments, the effect of exogenous growth factors could have been masked by embryo

origin growth factors that participate in embryo development. Therefore, a more

controlled culture system is necessary (e.g. continuous flow system) to study the function

of growth factor during early embryo development. The other possible explanations are

the exogenous growth factors may be not needed for prehatched bovine embryo

156

development, or the concentrations of exogenous growth factors used in this study were

too low in those experiments to affect the embryo development.

Research and commercial use of prehatched embryos ultimately requires embryo

cryopreservation. Although bovine embryos can be produced in the laboratory, it has

been found that in vitro produced embryos are much more sensitive to freezing than in

vivo produced embryos. To test the viability of embryos produced from the present

study, 300 IVF-derived embryos were frozen at 16-cell, morula or early blastocyst

stages. Some of this frozen embryos were thawed 10 to 12 month later and either

transferred after 4 hours or co-cultured for 2 days to allow embryos develop to the

expanded blastocyst stage and then transferred into recipient animals.

The survival and development of frozen-thawed IVF-derived embryos were much

greater when embryos were frozen at relatively later developmental stages than at an

early stage. The expanded blastocyst development rate was 8.3% for 16-cell stage

embryos, 46% for morulae and 67% for early blastocysts. Higher pregnancy rate (50%)

resulted when frozen-thawed embryos were co-cultured and developed to expanded

blastocysts before transfer compared with those of cultured for only a few hours and

transferred (20%). Extended in vitro culture increased the selection effect for embryos

of transferable quality when compared with a shorter term culture (30% vs. 62%).

Furthermore, the overall efficiency (number of pregnancies/number of thawed embryos)

was also greater for embryos cultured » 2 days. Nine pregnancies were established from

embryo transfer of 30 recipient cows and 5 calves were bom after a normal gestation

period. These results demonstrate that in vitro-produced bovine embryos are viable in

terms of producing live young. These results also showed that the survival of the frozen-

157

thawed IVF-derived embryos that developed to term after embryo transfer are likely to

be obtained from blastocyst-stage embryos. These calves were the first frozen-thawed,

IVF-derived offsprings in the USA.

The methodology of in vitro production of bovine preimplantation embryos has

been established and now being used for both research and commercial purposes.

However, the underlying mechanism of gamete interaction and early mammalian

development is far from fully understood. Further studies on the requirement and

regulation of these early development are needed to better understand the early

development, as well as finding of an optimal in vitro system for laboratory embryo

production. The ability of producing viable prehatched embryos will, in turn, provide

a continuous laboratory supply. Thus, stimulating the basic studies especially molecular

biology research on early embryos that is now difficult without large number of bovine

embryos. The progresses in resent years are very encouraging. The successes on in

vitro fertilization and embryo production will also afford opportunities to enhance the

reproductive efficiency of farm animals and of rare, endangered species.

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APPENDIX A

BRACKETT-OLIPHANT MEDIUM

Item mM 100 ml

B-O Stock Solution (10X)NaCl 112.00 6.550 gKC1 4.02 0.300 gNaH2P 04*H20 0.83 0.115 gMgCl2-6H20 0.52 0.106 gCaCl2-2H20 2.25 0.331 gD-Glucose 13.90 2.518 gPenicillin-G — 100 000 IUStreptomycin S04 — 100 0 00 /tg0.5 % Phenol red _ _ 1.6 mlDistilled Milli-Q water raise to 100 ml total

B-O Medium (100 ml)NaHC03 37.00 0.3104 gNa pyruvate 1.25 0.0138 gB-O Stock Solution (10X) — 10 mlDistilled Milli-Q water raise to 100 ml total

To prepare B-O Stock Solution:

1. In 60 ml of distilled Milli-Q water, dissolve NaCl, KC1, NaH2P 0 4*H20 , D- glucose, penicillin-G, potassium, and streptomycin sulfate.

2. In 10 ml of Milli-Q water, dissolve CaCl2-2H20 and MgCl2*6H20 . Then slowly add solution from step 2 to the solution prepared in step 1.

3. Add 1.6 ml phenol red (optional).

4. Add Milli-Q water to make a final volume of 100 ml and then filter through a0.2-fim filter, and aliquot into sterile containers.

5. Store at 4°C for up to 3 mo.

To prepare B-O Medium:

1. In 80 ml of Milli-Q water dissolve NaHCO, and sodium pyruvate.

2. Add 10 ml of B-O Stock Solution and then q.s. with Milli-Q water to make a final volume of 100 ml.

186

187

3. Adjust pH to between 7.6 and 7.8 and osmolarity to 270 and 290 mOsm.

4. Filter through a 0.2-/xm filter and aliquot into sterile vessels.

5. Store at 4°C for up to 1 mo.

APPENDIX B

PREPARATION OF Ca++ IONOPHORE A23187 FOR SPERM CAPACITATION

Ca++ ionophore A23187 solution (1 mM)

1. In .1.9 ml of dimethylsulfoxide dissolve 1 mg Ca++ ionophore A23187.

2. Aliquot 30 /*1 of the ionophore solution into sterile containers (microvials).

3. Store the aliquots in the dark at -20CC for up to 6 mo.

188

APPENDIX C

COMPOSITION OF CZB MEDIUM

Component mg/ml mM

NaCl 4767 81.62KC1 360 4.83k h 2p o 4 161 1.18MgS04 • 7HzO 291 1.18NaHCOj 2110 25.12CaCl2 • 2H20 250 1.70Na pyruvate 29.7 0.27Na lactate 3609 31.30EDTA (disosium salt) 41.8 0.11Glutamine 146 1.00BSA (mg/ml) 5.0Na penicillin-G (IU/ml) 100Streptomycin (mg/ml) 0.7

(Chatot et al., 1989)

189

VITA

Li Zhang, the daughter of Jingshan Zhang and Qingmei Liang, was bom in

Beijing, China on January 17, 1956. She received her elementary and secondary

education at Beijing Tielu No. 5 Elementary School and Beijing Tielu No. 3 Middle

School in Beijing, China. After passing the national examination to enter the college in

August, 1978, she enrolled in Beijing Normal University where she received the degree

of Bachelor of Science with a major in biology in August, 1982. For the following year,

she was employed as an instructor in Beijing Fengtai Agricultural Technology School.

In August, 1983 she entered the Graduate School at Chinese Academy of

Agricultural Sciences. She was awarded the degree of Master of Science in August,

1986 with a major in Animal Reproductive Physiology, and at the same time she

accepted a research associate position in the Institute of Animal Science, Chinese

Academy of Agricultural Sciences. In April, 1989 she become a visiting scientist in the

Department of Animal Science, Cornell University. The same year in September, she

moved to Louisiana State University and worked in Animal Science Department as a

research associate.

In August of 1990, she started her doctor training program in Reproductive

Physiology under the guidance of Dr. Robert A. Godke in the Department of Animal

Science at Louisiana State University. She was awarded the degree of Doctor of

Philosophy in December of 1995.

190

DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: Li Zhang

Major Field; Animal Science

Title of Dissertation: In Vitro Maturation, In Vitro Fertilizationand Development of Bovine Oocytes

Approved:

Major Professor and Chairman

Graduate School

EXAMINING COMMITTEE:

1 f t ' t s

Date of Examination:

October 19, 1995

in vitro maturation, in vitro fertilization and

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