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EQUINE EMBRYOS PRODUCED IN VITRO: HOW MUCH DO THEY MISS A MARE?

Katrien Smits

Thesis submitted in fulfillment of the requirements for the degree of Doctor in Veterinary

Sciences (PhD), Faculty of Veterinary Medicine, Ghent University, 2010

Promotor: Prof. Dr. A. Van Soom

Co-Promotor: Prof. Dr. L. Peelman

Faculty of Veterinary Medicine

Department of Reproduction, Obstetrics and Herd Health

Cover photo and design: Maarten Hoogewijs

ISBN: 978-90-5864-235-6

Live as if you were to die tomorrow.

Learn as if you were to live forever.

Mahatma Gandhi

TABLE OF CONTENTS LIST OF ABBREVIATIONS ............................................................................................................................... 1

CHAPTER 1 GENERAL INTRODUCTION ..................................................................................................... 5

CHAPTER 2 AIMS OF THE THESIS ........................................................................................................... 33

CHAPTER 3 THE IN VITRO PRODUCTION OF EQUINE EMBRYOS ............................................................ 37

3.1 A DIFFERENT APPROACH TO THE IN VITRO PRODUCTION OF HORSE EMBRYOS: A PILOT STUDY

OF LASER-ASSISTED VERSUS PIEZO DRILL ICSI ............................................................................ 39

3.2 BIRTH OF THE FIRST ICSI-FOAL IN THE BENELUX ........................................................................ 49

CHAPTER 4 DETERMINATION OF A SET OF RELIABLE REFERENCE GENES FOR RT-QPCR IN IN VIVO

DERIVED AND IN VITRO PRODUCED EQUINE BLASTOCYSTS ............................................... 59

CHAPTER 5 IN VIVO DERIVED HORSE BLASTOCYSTS SHOW TRANSCRIPTIONAL UPREGULATION OF

DEVELOPMENTALLY IMPORTANT GENES COMPARED TO IN VITRO PRODUCED HORSE

BLASTOCYSTS ...................................................................................................................... 77

CHAPTER 6 INFLUENCE OF THE UTERINE ENVIRONMENT ON THE DEVELOPMENT OF IN VITRO

PRODUCED EQUINE EMBRYOS.......................................................................................... 107

CHAPTER 7 GENERAL DISCUSSION ....................................................................................................... 135

SUMMARY ................................................................................................................................................. 157

SAMENVATTING ........................................................................................................................................ 163

DANKWOORD/ACKNOWLEDGEMENTS ..................................................................................................... 169

CURRICULUM VITAE .................................................................................................................................. 173

BIBLIOGRAPHY .......................................................................................................................................... 175

ADDENDUM 1 PROTOCOL IN VITRO PRODUCTION EQUINE EMBRYOS ............................................ 179

ADDENDUM 2 SUPPRESSION SUBTRACTIVE HYBRIDIZATION .......................................................... 185

ADDENDUM 3 REVERSE TRANSCRIPTION QUANTITATIVE POLYMERASE CHAIN REACTION . .......... 187

1

LIST OF ABBREVIATIONS ACTB

AI

ART

BEX2

cDNA

COC

Cqvalue

CZB medium

DMEM-F12

DPBS

ET

FABP3

FAF-BSA

FCS

GAPDH

H2A/I

hCG

HEPES

HPRT

HSP90AA1

ICSI

IVC

IVF

IVM

IVP

KSOM

MII

MCM7

MEM

MOBKL3

beta actin

artificial insemination

artificial reproductive technologies

brain expressed X-linked 2

complementary deoxyribonucleic acid

cumulus oocyte complex

quantification cycle value

Chatot-Ziomek-Bavister medium

Dulbecco's modified Eagle medium: nutrient mixture F-12

Dulbecco's phosphate buffered saline

embryo transfer

fatty acid binding protein 3

fatty acid-free bovine serum albumin

fetal calf serum

glyceraldehyde-3-phosphate dehydrogenase

histone H2A type 1-C

human chorionic gonadotropin

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

hypoxanthine phosphoribosyltransferase 1

heat shock protein 90kDa alpha, class A member 1

intracytoplasmic sperm injection

in vitro culture

in vitro fertilization

in vitro maturation

in vitro production

simplex optimization medium with elevated K+ concentration

metaphase of the second meiotic division

minichromosome maintenance complex component 7

Eagle's minimal essential medium

mps one binder kinase activator-like 3

2

ODC

OPU

PBS

PCR

pFSH

pLH

POU5F1

RNA

RPL32

RT

RT-qPCR

SDHA

SOF

SSH

SR

TALP

TCM199

TUBA4A

UBC

ornithine decarboxylase

ovum pick-up

phosphate buffered saline

polymerase chain reaction

porcine follicle-stimulating hormone

porcine luteinizing hormone

POU domain, class 5, transcription factor 1

ribonucleic acid

ribosomal protein L32

reverse transcription

reverse transcription quantitative real time polymerase chain reaction

succinate dehydrogenase complex, subunit A

synthetic oviduct fluid

suppression subtractive hybridization

serum replacement

Tyrode’s albumin-lactate-pyruvate

tissue culture medium 199

tubulin, alpha 4a

ubiquitin C

CHAPTER 1

GENERAL INTRODUCTION

Chapter 1

7

1.1 ARTIFICIAL REPRODUCTIVE TECHNOLOGIES IN THE HORSE

Up to the end of the nineteenth century, the vast majority of horses were bred by natural

cover. The introduction of artificial reproductive technologies (ART) started with the first

reported equine pregnancy produced by artificial insemination (AI), which is the placement of

stallion sperm into a mare’s uterus (Heape, 1898). Nowadays, the majority of mares are

impregnated by AI using fresh, cooled or frozen-thawed sperm, thereby providing the

opportunity for genetically valuable stallions to produce more offspring. On the female side,

genetic selection can be accelerated by recovering an embryo from the uterus of a valuable

donor mare and subsequent embryo transfer (ET) to a recipient mare, which carries the foal to

term (for review see Stout, 2006). The first successful ET in horses was achieved in 1974 (Oguri

and Tsutsumi, 1974) and ET is now a routine procedure in practice (Scherzer et al., 2008). In

both of these techniques, early embryonic development occurs in the female reproductive

tract, i.e. in vivo. This is in contrast to in vitro production (IVP) of embryos, in which fertilization

and early development occur under laboratory conditions outside the mare. In vitro production

of equine embryos only evolved recently and is the subject of this thesis.

1.2 EARLY EQUINE EMBRYONIC DEVELOPMENT IN VIVO

As for all mammalian oocytes, the equine oocyte is arrested in the prophase of the first meiotic

division during foetal development, and only a few hundred selected oocytes ever reach the

second metaphase (MII) stage prior to ovulation (King et al., 1987; Pierson, 1992). Fertilization

of the mature oocyte occurs at the ampulla-isthmus junction of the oviduct, where the

developing embryo remains during its subsequent cleavage divisions (Betteridge et al., 1982;

McKinnon et al., 1992; Weber et al., 1996). After 5 days at the ampulla-isthmus junction,

transport through the oviductal isthmus occurs rapidly and the late morula or early blastocyst

enters the uterus through the utero-tubal junction 144-156 h after ovulation (Oguri and

Tsutsumi, 1972; Weber et al., 1996; Battut et al., 1997) (Figure 1). Unfertilized eggs on the

other hand are retained in the oviduct (Van Niekerk and Gerneke, 1966). This selective

oviductal transport is linked to the stage specific production of prostaglandin E2 by the equine

Chapter 1

8

conceptus, an example of extremely early embryo-maternal communication or interaction

(Weber et al., 1991).

Figure 1 Equine embryonic development in vivo. Fertilization (1,2) and early cleavage (3,4,5)

occur at the ampulla-isthmic junction. After rapid transport through the istmus, the morula (6)

or early blastocyst (7) reaches the uterus. A glycoprotein capsule is formed between the

trophectoderm and the zona pellucida, which is subsequently shed from the rapidly expanding

blastocyst (8).

Chapter 1

9

Another exceptional feature of the equine is the formation of an acellular glycoprotein capsule.

This structure was first described over a century ago (Bonnet, 1889; Krölling, 1937), but most of

the research has been performed in the past 40 years (Marrable and Flood, 1975; Betteridge et

al., 1982; Flood et al., 1982). Upon arrival of the embryo in the uterus, the capsule is formed

between the trophectoderm and the zona pellucida of the equine blastocyst and it surrounds

the conceptus until around day 21 of gestation (Betteridge et al., 1982; Enders and Liu, 1991)

(Figure 2). Embryonic tertiary coats are described in several species (Betteridge, 1989; Denker,

2000). The glycoprotein capsule as present in equids shows the most similarities with the

neozona in rabbits (Betteridge, 1989; Denker, 2000).

Figure 2 Equine embryonic capsule in vivo. This figure displays the capsule of two in vivo

derived blastocysts in a different stadium of the embryonic development. A: Slightly collapsed

hatched horse blastocyst surrounded by a loose, overlarge capsule; B: Tight capsule between the

trophectoderm and the zona pellucida of an expanding blastocyst.

Up to day 16, the spherical equine embryo is very mobile and migrates through the uterus. This

migration is necessary to obtain maternal recognition of the pregnancy. While migrating

through the uterus, the equine conceptus signals its presence and prevents cyclical luteolysis

(Allen, 2000). The strong, elastic capsule has been suggested to protect the preimplantation

embryo during this migratory phase (Betteridge et al., 1982). The capsule also appears to be

Chapter 1

10

involved in subsequent fixation (day 16-17) and orientation of the embryo (Oriol, 1994).

Throughout embryonic development, the capsule is thought to function as a ‘mailbox’,

incorporating endometrial components and transporting these to the developing embryo

(Herrler and Beier, 2000). In this way, it can be involved in the early embryo-maternal

communication. Even though the precise role is not entirely clear, the capsule has been shown

to be essential for the continuance of pregnancy (McKinnon et al., 1989; Stout et al., 2005). The

equine trophoblast is the primary source of the capsular material (Albihn et al., 2003).

However, absence of normal capsule formation in vitro implies an essential role of the maternal

environment (Hinrichs et al., 1990a; Tremoleda et al., 2003). It remains to be determined which

aspects of the uterine surroundings and which mechanisms are involved in the formation of this

intriguing component of the equine embryo.

Endometrial secretions (‘histotrophe’) are particularly important in the horse, because of the

exceptionally long pre-implantation period, during which the developing embryo totally

depends on these secretions for its nutrition (Stewart et al., 2000). In this regard, uterocalin, a

component of the uterine secretions, might be involved in capsule formation. Uterocalin, a 19

kDa protein, was first isolated using SDS-PAGE of equine embryonic capsules (Stewart et al.,

1995). Structural analysis classifies uterocalin as a member of the lipocalin family and suggests

that the primary function of uterocalin is to act as a carrier of biologically important lipids and

as a source of essential amino acids for the developing conceptus (Suire et al., 2001; Kennedy,

2004). Substantial concentrations of uterocalin have been associated with the capsule, and

passage through the capsule to the developing conceptus has been evidenced by the presence

of uterocalin on the trophoblast cells (Crossett et al., 1996; Ellenberger et al., 2008).

Furthermore, uterocalin is positively charged. This facilitates its binding to the negatively

charged sialic acid residues of the capsule (Oriol et al., 1993; Crossett et al., 1998). Uterocalin is

secreted by the endometrial glands in a progesterone dependant way during both dioestrus

and early pregnancy (Stewart et al., 1995). Interestingly, the high concentrations of uterocalin

in the uterus, which are prevailing during early pregnancy (day 6-day 23), coincide exactly with

the period during which the equine embryo is surrounded by the capsule (Crossett et al., 1998).

In summary, structural, functional and temporal associations have been made between the

Chapter 1

11

maternal uterocalin and the embryonic capsule in the horse, but whether uterocalin effectively

supports the capsule formation remains to be determined.

The examples mentioned above show the important interaction between the developing

embryo and the maternal genital tract. In vivo derived horse embryos have been exposed to the

maternal genital tract and are supposed to have developed normally. Such equine embryos

represent therefore interesting study material and can be used as a gold standard, to which in

vitro produced embryos that have been cultured in the absence of the maternal genital tract

can be compared. Unfortunately, in vivo derived horse embryos are relatively difficult to obtain.

From the zygote to the early morula stage, they remain for a relatively long stay in the oviduct

and can only be harvested surgically. After its arrival in the uterus, a horse blastocyst can be

recovered atraumatically by uterine flushing. But even then, generally only one embryo can be

obtained per mare, since the mare is mono-ovulatory and responds only moderately to

superovulatory treatments (for review see McCue, 1996; Stout, 2006). In the experiments

described in this thesis, equine in vivo embryos were flushed at day 7, at which time they have

reached the blastocyst stage. These in vivo derived blastocysts represented the gold standard

for comparison with the in vitro produced blastocysts. When equine embryos are produced and

cultured in vitro, the absence of these maternal interactions have morphological and

developmental consequences, which will be discussed in the next chapters of this thesis.

1.3 EARLY EQUINE EMBRYONIC DEVELOPMENT IN VITRO

The in vitro production (IVP) of equine embryos consists of several important steps.

First of all, oocytes need to be collected. In the living mare, the mature oocyte can be obtained

from a preovulatory follicle by aspiration using a long needle placed through the flank of the

sedated standing mare (Vogelsang et al., 1983; Palmer et al., 1986; Hinrichs et al., 1990b).

Alternatively, several follicles can be aspirated through ultrasound guided transvaginal ovum

pick up (OPU) (Brück et al., 1992; Cook et al., 1993; Bezard et al., 1995; Meintjes et al., 1995;

Goudet et al., 1997; Galli et al., 2001). Post mortem, immature oocytes can be collected from

ovaries through either aspiration (Desjardins et al., 1985; Shabpareh et al., 1993) or scraping of

Chapter 1

12

the follicles (Del Campo et al., 1995; Dell’Aquila et al., 2001). Next, all immature oocytes must

undergo maturation in vitro (IVM). Finally, the mature oocytes must be fertilized. Conventional

in vitro fertilization (IVF), which implies the co-incubation of mature COCs with capacitated

sperm, is largely unsuccessful in horses. Therefore, intracytoplasmic sperm injection (ICSI), a

micromanipulation technique during which a single sperm cell is injected into the cytoplasm of

a mature oocyte, is the method of choice. These fertilized oocytes are then cultured in vitro

(IVC) up to the blastocyst stage, which can be transferred to the uterus of a recipient mare (ET).

Specific problems during these various steps of the IVP of equine embryos have hindered rapid

progress towards large scale equine IVP and will be covered in the subjoined paragraphs. The

general protocol that was followed throughout the thesis is described in Addendum 1.

1.3.1 Oocyte collection

Oocytes available for research are scarce, since the access to abattoir ovaries is limited. In some

countries, such as the USA, all horse abattoirs have been closed, and in these countries all

oocytes must be collected using the time consuming technique of in vivo collection through

OPU (McPartlin et al., 2007). Furthermore, the very tight connection between the equine

oocyte and the follicle wall requires scraping or vigorous flushing of the follicle to recover the

oocyte (Hawley et al., 1995).

Figure 3 Ex vivo collection of horse oocytes by means of aspiration. The follicular fluid is

aspirated (-100 mm Hg) (A), the follicles are scraped with the aspirating needle and

simultaneously flushed (B) with heparin (25IU/ml). After aspiration of the superficial follicles,

the ovary is cut to reach the follicles inside (C).

Chapter 1

13

Oocytes can be recovered ex vivo either by incising the follicle and scraping the wall with a

bone curette or by aspiration of the follicular contents (Figure 3). An experiment conducted in

our laboratory comparing scraping with aspiration, indicated that the scraping technique is

associated with more cumulus cells surrounding the recovered oocytes, when compared to

aspiration (Table 1). Our observations are in agreement with Dell’Aquila et al. (2001), who

reported 53% of the aspirated oocytes to have only a partial cumulus, while this was only 15%

for the scraped oocytes. The presence of several layers of cumulus cells favors the classification

of the oocytes into expanded (Figure 4A) versus compact (Figure 4B) cumulus oocyte complexes

(COCs) (Hinrichs, 2010b). It is useful to be able to make an accurate distinction between the

expanded and the compact COCs, because the optimal maturation time is different for both

populations (Hinrichs et al., 1993). In other species, expanded COCs are discarded, as they

mostly originate from atretic follicles and are associated with a low developmental capacity. In

contrast, the equine expanded COCs have been shown to result in similar blastocyst rates,

when compared to compact COCs (Galli et al., 2007; Hinrichs, 2010a). When oocytes are

collected by aspiration, a significant proportion of oocytes is only surrounded by a few layers of

corona cells, which complicates an accurate classification. Despite the difficult classification of

oocytes collected by aspiration, the aspiration technique is the preferred technique for oocyte

recovery in our laboratory. This preference is largely based on the higher oocyte recovery rate

(73%) for aspiration compared to scraping (51%) in our laboratory, and by the fact that

aspiration is up to 4 times less time consuming (Using the aspiration methodology developed in

our laboratory, a trained technician can collect the same number of oocytes in one hour as two

technicians using scraping technique in two hours). It should however be pointed out that in

order to obtain as much oocytes as possible, the follicle wall is scraped repeatedly with the

point of the aspirating needle, and thus our aspiration technique is in fact a combination of the

scraping and the aspiration method. As a whole, oocyte recovery in the horse is far more time

consuming than it is in other species and for example over 10-fold more time consuming than

oocyte recovery in cattle (Galli et al., 2007). Galli et al. (2007) give the example of 4 technicians

needing 3-4 hours to collect 100 equine oocytes, while 2 technicians can collect the same

number of bovine oocytes in only 30-40 minutes.

Chapter 1

14

Table 1 Oocyte collection technique. Oocytes were classified based on the expansion and

extensiveness of the surrounding cumulus cells. Oocytes were categorised as expanded COCs,

compact COCs and oocytes with only a few layers from the corona radiata. Recovery rates for

each category of oocytes were compared for two different collection techniques: scraping of the

follicle wall with a bone curette and follicular aspiration.

Total Expanded Compact Corona

Scraping 200 50 (25%) 105 (52.5%) 45 (22.5%)

Aspiration 430 68 (16%) 169 (39%) 193 (45%)

Figure 4 Immature equine cumulus oocyte complexes (magnification 100x). A: expanded COC;

B: compact COC

1.3.2 In vitro maturation

In equine, compared to bovine, a larger proportion of degenerate oocytes can be identified

after IVM of abattoir-derived oocytes (Galli et al., 2007). This post mortem degeneration affects

around 30% of the collected oocytes and substantially decreases the maturation rate.

Maturation of horse oocytes is judged after IVM and removal of the cumulus cells by assessing

the extrusion of the polar body (Figure 5), which indicates progression to metaphase II (MII).

Chapter 1

15

Figure 5 Polar body (magnification 300x). The mature MII oocyte is characterized by an

extruded polar body (arrow).

However, nuclear maturation which is characterized by the extrusion of a polar body, does not

necessarily reflect the cytoplasmic maturation of the oocyte. During cytoplasmic maturation,

proteins, RNA, substrates and nutrients are accumulated, rendering the oocyte competent for

further development (Watson, 2007). This process is crucial for further embryonic development

and it is greatly influenced by the oocyte environment in vivo or in vitro. Therefore, the

maturation conditions can have a major influence on the blastocyst rate. Replacement of Tissue

Culture Medium199 (TCM199) by Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12

(DMEM-F12) for equine IVM has been shown to result in higher cleavage and blastocyst rates

without affecting the proportion of MII oocytes (Galli et al., 2007). For this reason, DMEM-F12-

based maturation medium is used in our laboratory.

1.3.3 Fertilization

Conventional in vitro fertilization (IVF), which implies the co-incubation of mature COCs with

capacitated sperm, is largely unsuccessful in horses, primarily because it is difficult to

adequately stimulate horse sperm to penetrate the zona pellucida in vitro (Alm et al., 2001;

Roasa et al., 2007). Only two foals produced by means of conventional IVF have been born to

date (Palmer et al., 1991). Even though acceptable in vitro fertilization rates were recently

Chapter 1

16

reported following hyperactivation of stallion sperm with procaine (McPartlin et al., 2009), this

procedure has not yet been repeated in other laboratories.

To circumvent the problem of sperm activation in vitro, intracytoplasmic sperm injection (ICSI)

is now used for the IVP of equine embryos. This technique, using micromanipulation to inject a

single sperm cell into the cytoplasm of a mature oocyte (Figure 6), was initially developed in

Belgium for treatment of male factor infertility in humans (Palermo et al., 1992). The first ICSI

foal was produced by Squires et al. (1996), who injected in vitro matured oocytes and

transferred these oocytes to the oviduct of recipient mares.

Figure 6 Intracytoplasmic sperm injection in the horse (magnification 300x). The mature

oocyte is immobilized with the polar body at the 6 o’clock position (arrow) by aspiration with a

holding pipette at 9 o’clock. An immobilized sperm cell is injected using a fine injection pipette

at 3 o’clock.

After this initial success, several laboratories tried to implement the ICSI technique, with

variable results. In 2002, the piezo drill, a device that creates minute vibrations of the injection

pipette, was introduced for equine ICSI and, at the same time, increased cleavage rates and

more consistent results were reported (Choi et al., 2002, Galli et al., 2002). This has led to

general use of the piezo drill, even though a causal relationship with the improved results has

never been proven. Various aspects can explain the positive effect of piezo assisted ICSI. In

mice, the piezo drill was applied for ICSI and resulted in the first ICSI offspring (Kimura and

Chapter 1

17

Yanagimachi, 1995). Advantages compared to conventional ICSI include a higher probability of

oolemma breakage and reduced damage to the oocyte. Moreover, the piezo might cause

increased permeabilization of the sperm membrane, which could facilitate oocyte activation

(Choi et al., 2002). The best results are obtained when a small amount of mercury is present in

the tip of the injection pipette (Ediz and Olgac, 2004). Mercury reduces the lateral oscillations

of the pipette, which reduces oocyte damage during the piercing (Ediz and Olgac, 2005). On the

other hand, mercury is a cumulative neurotoxin. Furthermore, piezo assisted

micromanipulation in mice has been associated with DNA damage (Yu et al., 2007). The possible

influence of the injection method on subsequent embryonic developmental competence,

including the advantages and disadvantages, needs to be investigated further. In this respect, it

might be interesting to consider other sperm injection technologies, like laser-assisted ICSI.

During laser-assisted ICSI, a hole is made in the zona pellucida prior to sperm injection. This

causes less oocyte disturbance than conventional ICSI. In human, the laser has been shown to

be advantageous for patients with fragile oolemmas or patients with high rates of oocyte

degeneration after conventional ICSI (Abdelmassih et al., 2002; Rienzi et al., 2004).

1.3.4 In vitro culture

As fertilized oocytes reside in the oviduct under physiological circumstances, initially sperm

injected horse oocytes were transferred to a recipient mare’s oviduct (Squires et al., 1996;

Cochran et al., 1998; McKinnon et al., 2000), with all the disadvantages associated with the

required surgery. Therefore, efforts were made to develop in vitro conditions to culture the

embryos up to the blastocyst stage, allowing transcervical transfer into the uterus. Firstly, co-

culture of embryos with different types of cells was performed. In vivo derived embryos were

cultured with oviduct cells (Battut et al., 1991) or uterine cells (Ball et al., 1993) and IVP

embryos with Vero cells (Guignot et al., 1998), cumulus cells (Li et al., 2001) or granulosa cells

(Rosati et al., 2002). Then, several defined media, like G1.2 (Choi et al., 2002) and CZB (Choi et

al., 2004) were evaluated, but blastocyst rates remained variable and below 20%. Eventually,

important progress was obtained by the finding that using DMEM-F12 as an equine embryo

culture medium yielded blastocyst development rates of up to 34% (Choi et al., 2006b).

Chapter 1

18

Originally developed for cell culture, this medium contains a high glucose concentration when

compared to regular embryo culture media. Contrary to other species, equine embryos

apparently benefit from high glucose concentrations during early development (Herrera et al.,

2008). Despite these advances, the percentage of injected oocytes reaching the blastocyst stage

in clinical practice is only 10%, as recently published by one of the most experienced groups in

the field (Barbacini et al., 2010) and temporary culture in sheep oviducts still appears to yield

better results than total IVP (Lazzari et al., 2010), illustrating further the necessity for optimizing

IVC conditions for equine embryos.

1.3.5 Embryo transfer and foals

Several ICSI foals have been born to date (Squires et al., 1996; Cochran et al., 1998; McKinnon

et al., 2000; Li et al., 2001; Hinrichs et al., 2007; Galli et al., 2007). Pregnancy rates after transfer

of IVP embryos are comparable to those obtained after transfer of in vivo derived embryos

(Galli et al., 2007; Hinrichs, 2010b; Farin et al., 2010). In cattle, initial pregnancy rates are also

similar for in vivo and in vitro produced embryos, but subsequent survival is compromised by

considerable embryonic and fetal losses and the occurrence of the large offspring syndrome

(Willadsen et al., 1991; Walker et al., 1996; Niemann and Wrenzycki, 2000). The large offspring

syndrome, as observed in cattle after IVP and cloning, has not been reported in the horse. As

the application of ICSI in horses has only evolved recently and the number of foals born to date

is limited, data on possible long term influences are scarce. Initially, abnormal pregnancies

without an embryo proper were reported (Hinrichs et al., 2007). However, normal development

has been reported recently and this improved development has been contributed to more

consistent IVC conditions (Hinrichs, 2010b). An evaluation of 14 cloned foals revealed the need

for intensive support during the first week, but if the foals survived this critical period, further

development appeared to be normal (Johnson et al., 2010). As the number of IVP foals is still

limited, further research is necessary to evaluate long term consequences. Moreover, the

ability of IVP embryos to develop to normal foals remains the method of choice to evaluate the

quality of in vitro embryo production in horses: it is the ultimate proof that the equine embryos

which are produced after ICSI are not parthenogenetic or chromosomally abnormal blastocysts.

Chapter 1

19

Despite an initially slow progress, IVP of equine embryos has evolved rapidly in the last decade.

Nowadays the combination of OPU, IVM, ICSI, IVC and ET is clinically available (Colleoni et al.,

2007). Since only a few normal sperm cells are required, ICSI can be applied for subfertile

stallions and even for semen which has been frozen-thawed, diluted and frozen again or for

sexed semen (Lazzari et al., 2002; Choi et al., 2006a; Squires et al., 2008). ICSI has also been

shown to be helpful in case of problems on the female side such as advanced age of the

breeding mare, degenerative endometrosis and cervical laceration (Colleoni et al., 2007).

Moreover, ICSI can be beneficial for creating offspring from valuable animals post mortem

(Hinrichs, 2005).

1.4 WHAT TO LOOK FOR WHEN EVALUATING EQUINE EMBRYOS: IN VITRO VERSUS IN VIVO

Even though the capability of establishing pregnancies is comparable for IVP and in vivo derived

equine embryos (Colleoni et al., 2007), differences between both types of embryos remain

present. Compared to their in vivo counterparts, equine IVP embryos display several

morphologic and developmental aberrations.

The kinetics of development differ, being slower in vitro than in vivo (Tremoleda et al., 2003;

Pomar et al., 2005; Rambags et al., 2005). Most equine in vivo embryos recovered 7 days after

ovulation have reached the blastocyst stage and are larger and contain more cells than their in

vitro counterparts 7 days after ICSI, which are predominantly still at the morula stage (Pomar et

al., 2005). In order to compare embryos at the same stage, Tremoleda et al. (2003) and

Rambags et al. (2005) used day 7 in vivo embryos and day 9-10 IVP embryos. Figure 7

represents an equine IVP blastocyst 9 days after ICSI and an in vivo derived blastocyst flushed 7

days after ovulation.

Chapter 1

20

Figure 7 Equine blastocysts (magnification 200x). An in vitro produced blastocyst cultured for 9

days after ICSI (A) and an in vivo derived blastocyst recovered 7 days after ovulation (B).

Further morphological analysis using specific stains has revealed higher incidences of apoptotic

(Tremoleda et al., 2003; Pomar et al., 2005) and chromosomally abnormal cells (Rambags et al.,

2005) in IVP (compared to in vivo) equine embryos. Moreover IVP embryos exhibit a disturbed

microfilament distribution. A very intriguing abnormality is the failure of normal capsule

formation; even though capsular glycoproteins are formed in vitro, they fail to coalesce into the

distinct continuous capsule seen around in vivo embryos (Tremoleda et al., 2003). Two

hypotheses have been formulated to explain this phenomenon (Oriol et al., 1993; Tremoleda et

al., 2003). A first explanation is the simple dispersal of the glycoproteins in the culture medium,

impeding the critical concentration for assembling to be reached. Another possibility is the

failure of hydration and cross-linking of the capsular glycoproteins in the absence of a uterine

component. Recently, a positive effect of temporary transfer to the mare’s uterus for 2-3 days

on capsule formation of equine IVP blastocysts has been shown (Choi et al., 2009). Further

experiments are needed to reveal what is the mechanism behind this observation.

The importance of the early embryo-maternal interaction is illustrated above. When equine

embryos are produced in vitro, and therefore in the absence of the maternal tract, they differ

markedly from their in vivo counterparts. In order to bridge this difference, the IVP process

Chapter 1

21

needs to be optimized. This requires fundamental insight into the aberrations in vitro and

sensitive evaluation of the impact of environmental changes. A valuable approach to achieve

this, is the assessment of the impact of the embryonic environment on gene expression levels.

It is commonly accepted that the suboptimal in vitro culture environment of embryos exerts

negative short-term effects, such as aberrations in genes involved in development and

metabolism up to the blastocyst stage. Even long-term effect with implications for the resulting

offspring have been described (Khosla et al., 2001; Fleming et al., 2004). These genetic

aberrations have been related to the use of suboptimal culture media (Fleming et al., 2004).

Differential gene expression in IVP versus in vivo derived embryos has been described in several

species, including cattle (Mohan et al., 2004; Corcoran et al., 2006; Goossens et al., 2007), pigs

(Magnani and Cabot, 2008) and mice (Fernández-González et al., 2009). In these species, the

expression level of specific genes has also been used to evaluate the effect of different embryo

culture media. The expression level of developmentally important genes can also be used to

assess the effect of specific components, like serum or cytokines in the embryo culture medium

(McElroy et al. 2008; Chin et al. 2009; Purpera et al. 2009). Some recent studies examined the

expression of specific genes in horse embryos, including the pluripotency marker POU5F1 (Choi

et al., 2009) and the embryonic receptors for estrogen and progesterone (Rambags et al.,

2008). However, large studies, evaluating gene expression in horse embryos produced in vivo

and in vitro, have not yet been reported. The genetic techniques used in this thesis, namely SSH

and RT-qPCR, are illustrated in Addendum 2 and 3.

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102. Willadson S.M., Janzen R.E., McAlister R.J., Shea B.F., Hamilton G., McDermand D. 1991.

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CHAPTER 2

AIMS OF THE THESIS

Chapter 2

35

The general aim of this research is to test the hypothesis that development is altered in in vitro

produced blastocysts as compared to in vivo derived equine blastocysts, with gene expression

and capsule formation as a parameter, and that this altered development can be influenced by

the mare’s uterine environment, more specifically by uterocalin.

In order to test this general hypothesis, in a first part of the thesis we needed to optimize the

procedure for in vitro embryo production in the horse. This was achieved by evaluating

different methods for ICSI (3.1) and by confirming the developmental competence of in vitro

produced equine blastocysts after embryo transfer (3.2) (CHAPTER 3).

Next, we continued by investigating the aforementioned general hypothesis, which was

subdivided in three specific aims, namely :

1. To develop a reliable method for evaluating gene expression in equine blastocysts

(CHAPTER 4).

2. To identify genes which are differentially expressed between in vivo derived and in vitro

produced equine blastocysts (CHAPTER 5).

3. To examine the influence of the maternal environment on embryonic development,and

more specifically, to determine the influence of uterocalin on equine embryonic capsule

development and gene expression (CHAPTER 6).

CHAPTER 3

THE IN VITRO PRODUCTION OF EQUINE EMBRYOS

3.1 A DIFFERENT APPROACH TO THE IN VITRO PRODUCTION OF HORSE EMBRYOS: A PILOT STUDY OF LASER-ASSISTED VERSUS PIEZO DRILL ICSI

Adapted from Smits K., Govaere J., Hoogewijs M., Piepers S., Van Soom A. Reproduction

in Domestic Animals submitted.

3.2 BIRTH OF THE FIRST ICSI-FOAL IN THE BENELUX

Adapted from Smits K., Govaere J., Hoogewijs M., De Schauwer C., Van Poucke M.,

Peelman L., Van Soom A. 2010. Vlaams Diergeneeskundig Tijdschrift 70 134-138.

Chapter 3

39

3.1 A DIFFERENT APPROACH TO THE IN VITRO PRODUCTION OF HORSE EMBRYOS: A PILOT STUDY OF LASER-ASSISTED VERSUS PIEZO DRILL ICSI

3.1.1 ABSTRACT

Intracytoplasmic sperm injection (ICSI) is the method of choice for the in vitro production of

equine embryos. Conventional ICSI has been associated with mechanical damage to the oocyte

due to deformation of the zona and exposure of the oolemma to negative pressure during

injection. The introduction of the less traumatic and more efficient piezo drill-assisted ICSI

yielded higher cleavage rates and more consistent results. This device is also associated with

disadvantages such as the use of mercury and its association with DNA damage in the mouse.

The in vitro production of equine embryos would profit from further optimization in order to be

efficiently applicable on large scale like in cattle. This has led us to explore an alternative

method avoiding oocyte trauma, namely laser-assisted ICSI, which involves creating a hole in

the zona pellucida prior to ICSI. In this pilot study both the piezo drill and the laser were

compared for ICSI in the horse. A higher cleavage rate was achieved in the laser group, but no

significant influences on subsequent blastocyst development were observed.

3.1.2 INTRODUCTION

Conventional in vitro fertilization (IVF) is of limited success in the horse, mainly because stallion

spermatozoa have difficulties in penetrating the zona pellucida in vitro (Roasa et al., 2007,

McPartlin et al., 2009). Therefore ICSI has been introduced as an alternative to circumvent this

problem and it is used routinely for the in vitro production of equine embryos. Conventional

ICSI is characterized by the crushing of a sperm tail on the bottom of the petri dish, thus causing

immobilization of the spermatozoon, followed by mechanical breakage of the zona pellucida

and the oolemma with a beveled injection pipette and subsequent injection of the immobilized

sperm into the cytoplasm of a mature oocyte. The first successful pregnancy after ICSI in horses

was announced in 1996 (Squires et al., 1996) and was followed by a period in which different

laboratories tried to implement the technique, but unfortunately with variable results. In 2002,

Chapter 3

40

the piezo drill was introduced for ICSI in horses and at the same time, increased cleavage rates

were reported during in vitro production of equine embryos (Choi et al., 2002; Galli et al.,

2002). Whether the introduction of the piezo drill had a causal relationship with the increased

cleavage rates was not clear, since no comparative studies have been published on this topic.

Acceptable results with the piezo led to a generalized use of the device for ICSI in horses.

Possible explanations for the positive effect of piezo-assisted over conventional ICSI include the

higher probability of oolemma breakage, the reduction of damage to the oocyte since

mechanical suction of the oolemma is replaced by a single pulse and the increased

permeabilization of the sperm membrane facilitating oocyte activation (Yanagida et al., 1998;

Choi et al., 2002). The piezo produces pulses on the injection pipette resulting in micro-

oscillations of the blunt pipette of ≥0.1 µm at ≤40 µm s -1, which allows efficient and precise

penetration of the zona pellucida and the oolemma (Ediz and Olgac, 2004; Yoshida et al., 2007).

However, research to reveal the exact reason has not been performed yet. Another aspect

which needs further investigation is the favorable effect of mercury during piezo-assisted ICSI

(Ediz and Olgac, 2005). It leads to significant improvement of the success rate, but its toxicity

implies an important drawback (Ediz and Olgac, 2004). Another possible disadvantage that

should be considered, is that piezo-assisted micromanipulation has been associated with DNA

damage in mice (Yu et al., 2007).

An alternative advanced approach could be offered by laser-assisted ICSI. Drilling a hole in the

zona pellucida by means of a diode laser prior to ICSI can avoid the mechanical compression

and oocyte distortion as it occurs in the initial phase of zona penetration during conventional

ICSI (Rienzi et al., 2001). This less traumatic approach has been described in human to be

beneficial in case of patients which suffered of high rates of oocyte degeneration or fragile

oolemmas after conventional ICSI (Abdelmassih et al., 2002; Rienzi et al., 2004), although a

subsequent study reported no advantage (Richter et al., 2006).

In horses the in vitro production of embryos happens on a small scale and in most laboratories

piezo-assisted ICSI is preferred. However, a direct comparison of different ICSI methods has not

been performed yet. The aim of this technical study was to introduce a new method for horse

Chapter 3

41

ICSI and to evaluate some of the advantages and disadvantages of piezo- versus laser-assisted

ICSI in the horse.

3.1.3 MATERIALS AND METHODS

3.1.3.1 General procedure for the in vitro production of equine embryos

Horse embryos were produced in vitro as described in Addendum 1. Briefly, oocytes were

aspirated from ovaries that were recovered at the slaughterhouse. The oocytes were matured

in vitro during 28h in DMEM-F12 based medium, containing 10% serum replacement, 10 µg/ml

pFSH and 2 µg/ml pLH (Stimufol, ULg FMV PhR, Sart-Tilman, Belgium) in 5% CO2 in air (Galli et

al., 2007). In this experiment, only compact cumulus-oocyte-complexes (n=253) were used.

After denudation, oocytes with visible polar bodies were selected and fertilized by means of

piezo- (Prime Tech, Ibaraki, Japan) or laser- (XYClone, Hamilton Thorne, Beverly, USA) assisted

ICSI. Six replicates were performed, in half of them piezo-assisted ICSI preceded laser-assisted

ICSI and in the other half the sequence was reversed. Injected oocytes were cultured in vitro up

to the blastocyst stage in DMEM-F12 with 10% fetal calf serum at 38.5°C in 5% CO2, 5% O2 and

90% N2. On day 2.5, cleavage was evaluated, the embryos that did not cleave were removed

and half of the medium was changed. Part of the cleaved embryos resulting from the piezo-

assisted ICSI was used for another experiment (CHAPTER 6) (Table 1). On day 6, half of the

medium was refreshed again and on day 9 blastocyst formation was evaluated.

3.1.3.2 Piezo-assisted ICSI

Piezo-assisted ICSI was performed with a blunt injection pipette (piezo-6-25, Humagen,

Charlottesville, VA, USA). A small amount of mercury was put in the tip of the pipette. A

progressive motile sperm was immobilized by piezo pulses and subsequently the zona and the

oolemma were penetrated with a piezo intensity setting of 5 and 4 respectively and a speed of

4 and 3 respectively, after which the sperm was injected into the ooplasm.

Chapter 3

42

3.1.3.3 Laser-assisted ICSI

A beveled injection pipette (MIC-50-35, Humagen, Charlottesville, VA, USA) was used and one

progressively motile sperm cell was immobilized by crushing its tail. A hole was drilled in the

zona pellucida using the laser. In all replicates maximal power was used. In a preliminary

experiment, 250 µs pulses were given, but this resulted in cytoplasm leakage. Therefore, this

was reduced to 200 µs in the first replicate. Because some cytoplasm leakage remained, this

was further reduced to 150 µs in the other replicates. Several adjacent small holes were made

until the zona was fully penetrated. The oolemma was penetrated mechanically in conformity

with conventional ICSI without the ‘squeezing’ of the oocyte.

3.1.3.4 Statistical analysis

The cleavage and blastocyst rates in both groups were compared by means of a Pearson Chi-

square test (SPSS 16.0, SPSS Inc., Headquarters, Chicago, Illinois, US). The mean injection times

were compared using a t-test. A p-value <0.05 was considered significant.

3.1.4 RESULTS

Of the 96 oocytes which were injected using laser, 74 cleaved (cleavage rate: 77%). The

cleavage rate of the piezo injected oocytes was a little lower (65%), a difference which was

significant (p=0.0421). In contrast, in the piezo drill-assisted group, 11.3 % of the cleaved

oocytes developed to blastocysts, and 6.8 % for the laser-assisted ICSI, but this was not

significant (p>0.05). The mean time for ICSI, including the handling of the oocytes before and

after the procedure, was longer for the laser-assisted ICSI (4.0 minutes/oocyte) than for the

piezo-assisted ICSI (2.9 minutes/oocyte). This difference was not significant. The details on

development are listed in Table 1 and summarized in Figure 1.

Chapter 3

43

Figure 1 Developmental competence of equine embryos after piezo- versus laser-assisted ICSI.

For both groups the mean cleavage rate (cleaved per injected) and the blastocyst rate

(blastocyst per cleaved) are displayed as well as the corresponding standard errors. *significant

difference (p=0.0421).

Table 1 Development of equine embryos following piezo- versus laser-assisted ICSI. For the six

conducted replicates, cleavage and blastocyst development was recorded for the oocytes

fertilized by piezo (P) and laser (L). Half of the cleaved embryos resulting from piezo-assisted ICSI

was destined to another experiment. The number of cleaved embryos used in this experiment

are listed under ‘used P’.

ICSI P ICSI L

Cleaved P

(% of injected)

Cleaved L

(% of injected) Used P

Blastocyst P

(% of cleaved)

Blastocyst L

(% of cleaved)

1 53 18 31 (58%) 11 (61%) 13 1 (7%) 1 (9%)

2 20 23 16 (80%) 19 (83%) 7 1 (14%) 0 (0%)

3 20 22 10 (50%) 16 (72%) 5 1 (20%) 2 (12.5%)

4 35 16 26 (74%) 12 (75%) 15 0 (0%) 1 (8.3%)

5 23 12 14 (61%) 12 (100%) 8 2 (25%) 1 (8.3%)

6 6 5 5 (83%) 4 (80%) 5 1 (20%) 0 (0%)

Total 157 96 102 (65%) 74 (77%) 53 6 (11.3%) 5 (6.8%)

0%10%20%30%40%50%60%70%80%90%

100%

Cleavage rate Blastocyst rate

Piezo

Laser

*

Chapter 3

44

Figure 2 Day 2.5 embryo resulting from laser-assisted ICSI. After laser-assisted ICSI the hole in

the zona pellucida (arrow) was still apparent at the cleavage stage. This resulted in blastomere

leakage in some embryos.

The hole in the zona after laser-assisted ICSI was persistent in later stages of development. This

resulted in what we describe as blastomere leakage at day 2.5 (Figure 2) and prominent

hatching at the blastocyst stage (Figure 3) when compared to the piezo group. However, this

became less frequent when the pulse duration was diminished.

Figure 3 Blastocyst resulting from laser-assisted ICSI (magnification 150x). A substantial part

of the blastocyst has hatched through the hole in the zona. This hatching was more prominent

when compared to the blastocysts resulting from piezo-assisted ICSI.

Chapter 3

45

3.1.5 DISCUSSION

This study was performed to compare the piezo drill and the laser during ICSI in the horse. The

developmental competence of the embryos produced in both ways is comparable. A smaller

cleavage rate is observed in the group where the piezo was used. The obtained cleavage rate of

65% is nevertheless comparable to results of 62-79%, reported in literature (Galli et al., 2007). It

must be remarked that the 65% cleavage rate was lower than average results (75%) with the

piezo in concurrent experiments (unpublished data). This may be related to the fact that the

injection pipette had to be changed for both procedures, which resulted in small variations in

the angle of the injection pipette. For piezo-assisted injection this angle is critical, since the

holding pipette, the oolemma and the injection pipette need to be in the same plane. In one

replicate the angle appeared suboptimal during the piezo-assisted injection, resulting in a few

more piezo pulses required to penetrate the oocyte and subsequently a reduced cleavage rate

(50%). The precise alignment of both pipettes (holding and injection) in the 3 dimensional

aspect of this technique appears to be less critical when laser-assisted ICSI is performed. This

could be possibly explained by the fact that the laser acts in a vertical plane, acting on the full

height of the zona at a particular point. Subsequently, the injection in the horizontal plane

becomes less critical.

Generally, the laser is very easy to handle and allows precise working. However, the sperm

needs to be captured and the oolemma is penetrated in the same way like in conventional ICSI.

Combined with the fact that several adjacent holes need to be made to penetrate the zona

pellucida, the laser-assisted ICSI remains rather time consuming when compared to piezo-

assisted ICSI where the penetration of the zona is followed fluently by the penetration of the

oolemma. Concerning the oolemma breakage, this study represents a comparison between

piezo-assisted and conventional ICSI, since the laser does not interact at this level. The suction

of the oolemma and the ooplasm into the injection pipette during conventional ICSI is

considered to be more traumatic than the oolemma breakage through piezo drill. Whether this

or the other piezo associated advantages as previously described contributed to the tendency

of a higher blastocyst rate is not clear. Anyhow, no significant differences were obtained.

Chapter 3

46

Another peculiarity is the apparent hole in the zona after laser-assisted ICSI. Although the

blastomere leakage and the early hatching were reduced by minimizing the laser settings, it

remained present in some embryos. This might be associated with an increased chance of

monozygotic twinning and it was suggested that the zona pellucida can be thinned instead of

fully penetrated by the laser prior to ICSI when applying it to human embryos (Moser et al.,

2004). Also, we did not attempt reducing the power of the laser, which might be worth

exploring as an option with the laser method.

While interpreting these results, it must be taken into account that the lab had no prior

experience with the laser, while piezo-assisted ICSI had been performed routinely for a year.

The ease of laser use permitted these preliminary results, indicating the laser as a valid

alternative for equine ICSI. This opens new avenues for research into the molecular effects and

events that result from these methods.

3.1.6 CONCLUSIONS

The in vitro production of equine embryos is based upon ICSI. To minimize oocyte damage

associated with conventional ICSI, piezo- or laser-assisted ICSI can be used. In this study both

techniques were compared and present specific advantages and disadvantages, but no major

differences in embryonic development were observed. Further exploration of these methods is

required to define their value for equine ICSI.

REFERENCES

1. Abdelmassih S., Cardoso J., Abdelmassih V., Dias J.A., Abdelmassih R., Nagy Z.P. 2002.

Laser-assisted ICSI: a novel approach to obtain higher oocyte survival and embryo

quality rates. Hum Reprod 17 2694-2699.





Chapter 3

47

3. Ediz K., Olgac N. 2004. Microdynamics of the piezo-driven pipettes in ICSI. IEEE Trans

Biomed Eng 51 1262-1268.

4. Ediz K., Olgac N. 2005 Effect of mercury column on the microdynamics of the piezo-

driven pipettes. J Biomech Eng 127 531-535.

5. Galli C., Crotti G., Turini R., Duchi G., Mari G., Zavaglia G., Duchamp G., Daels P., Lazzari

G. 2002. Frozen-thawed embryos produced by ovum pickup of immature oocytes and

ICSI are capable to establish pregnancies in the horse. Theriogenology 58 705-708

(abstract).








8. Moser M., Ebner T., Sommergruber M., Gaisswinkler U., Jesacher K., Puchner M.,

Wiesinger R., Tews G. 2004. Laser-assisted zona pellucida thinning prior to routine ICSI.

Hum Reprod 19 573-578.

9. Oguri N., Tsutsumi Y. 1972. Non-surgical recovery of equine eggs, and an attempt at

non-surgical transfer in horses. J Reprod Fertil 31 187-195.

10. Richter K.S., Davis A., Carter J., Greenhouse S.J., Mottla G.L., Tucker M.J. 2006. No

advantage of laser-assisted over conventional intracytoplasmic sperm injection: a

randomized controlled trial [NCT00114725]. J Exp Clin Assist Reprod 3 1-7.

11. Rienzi L., Greco E., Ubaldi F., Iacobelli M., Martinez F., Tesarik J. 2001. Laser-assisted

intracytoplasmic sperm injection. Fertil Steril 76 1045-1047.

12. Rienzi L., Ubaldi F., Martinez F., Minasi M.G., Lacobelli M., Ferrero S., Tesarik J., Greco E.

2004. Clinical application of laser-assisted ICSI: a pilot study. Eur J Obstet Gynecol

Reprod Biol 115 S77-S79.

Chapter 3

48

13. Roasa L.M., Choi Y.H., Love C.C., Romo S., Varner D.D., Hinrichs K. 2007. Ejaculate and

type of freezing extender affect rates of fertilization of horse oocytes in vitro.


14. Smits K., Govaere J., Hoogewijs M., De Schauwer C., Vanhaesebrouck E., Van Poucke M.,

Peelman L.J., Van den Berg M., Vullers T., Van Soom A. 2010. Birth of the first ICSI foal in

the Benelux. Vlaams Diergeneeskundig Tijdschrift 79 134-138.

15. Squires E.L., Wilson J.M., Kato H., Blaszczyk A. 1996. A pregnancy after intracytoplasmic

sperm injection into equine oocytes matured in vitro. Theriogenology 45 306 (abstract).

16. Yanagida K., Katayose H., Yazawa H., Kimura Y., Konnai K., Sato A. 1998. The usefulness

of a piezo-micromanipulator in intracytoplasmic sperm injection in humans. Hum

Reprod 14 448-453.

17. Yoshida N., Perry A.C.F. 2007. Piezo-actuated mouse intracytoplasmic sperm injection

(ICSI). Nature Protocols 2 296-304.

18. Yu Y., Ding C., Wang E., Chen X., Li X., Zhao C., Fan Y., Wang L., Beaujean N., Zhou Q,

Jouneau A., Ji W. 2007. Piezo-assisted nuclear transfer affects cloning efficiency and may

cause apoptosis. Reproduction 133 947-954.

Chapter 3

49

3.2 BIRTH OF THE FIRST ICSI-FOAL IN THE BENELUX

3.2.1 ABSTRACT

This manuscript describes the creation of a foal using conventional intracytoplasmic sperm

injection (ICSI) and embryo transfer. Oocytes were aspirated from ovaries from slaughtered

mares. After in vitro maturation, the oocytes were fertilized by ICSI and cultured in vitro for 9

days. Two embryos reached the blastocyst stage and they were transferred to the uterus of a

synchronized mare. Six days later a single embryonic vesicle was diagnosed by ultrasound. After

a normal pregnancy a healthy foal was born the 27th of October 2009. Parentage testing via

microsatellite genotyping confirmed that the foal originated from the transferred embryo.

3.2.2 INTRODUCTION

Conventional in vitro fertilization (IVF), which implies culture of matured oocytes with

capacitated sperm, is not efficient in horses. Only two IVF foals, produced from in vivo matured

oocytes, have been born up till now (Palmer et al., 1991). It is not yet completely clear why the

in vitro fertilization of horse oocytes is so difficult. One theory states that it may be due to a

defective capacitation of stallion sperm, which impairs the normal hyperactivation process of

the sperm (McPartlin et al., 2009). Hyperactivation is a typical motility pattern which is

exhibited by capacitated sperm and it is generally characterized by an increased lateral head

displacement and beat asymmetry. Hyperactivation is believed to be necessary for the sperm

cell to penetrate the equine zona pellucida (McPartlin et al., 2009). To circumvent this problem

of defective hyperactivation and fertilization in vitro, ICSI has been used for the in vitro

production (IVP) of equine embryos. This technique involves injecting a single sperm cell into

the cytoplasm of a mature oocyte using a fine glass needle. It was initially developed for

treatment of male factor infertility in humans and the first ICSI baby was born in Belgium in

1992 (Palermo et al., 1992).

The first ICSI foals resulted from in vitro matured equine oocytes, which were surgically

transferred to the oviduct after ICSI (Squires et al., 1996; Cochran et al., 1998; McKinnon et al.,

Chapter 3

50

2000). Subsequently also embryos which were cultured in vitro up to the blastocyst stage and

transferred to the uterus resulted into healthy foals (Li et al., 2001; Hinrichs, 2005; Galli et al.,

2007). Although ICSI is now commercially available for infertility treatment of both mares and

stallions, only a few laboratories perform this practice. In this case report, the birth of the first

ICSI foal in the Benelux is described.

3.2.3 MATERIALS AND METHODS

Equine ovaries were collected in the abbatoir and all follicles larger than 5 mm were aspirated

with a vacuum pump (-100 mm Hg), scraped with the aspirating needle and flushed with

heparin in phosphate buffered saline (PBS) (25IU/ml). The oocytes were matured during 26

hours in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM-F12) based medium

in an atmosphere containing 5% CO2 (Galli et al., 2007). After removal of the surrounding

cumulus cells by means of gentle pipetting, the oocytes with an extruded polar body were

fertilized by conventional ICSI. Frozen sperm from a stallion of proven fertility was thawed,

centrifuged at 750 x g during 40 minutes over a 90%/45% Percoll® gradient, washed with

calcium free Tyrode’s Albumin-Lactate-Pyruvate (TALP) solution and centrifuged again at 400x

g for 10 minutes. The sperm pellet was resuspended in Synthetic Oviductal Fluid (SOF) medium

and stored at 38.5 °C in 5% CO2. During ICSI the oocytes were kept in 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES) buffered SOF medium and the sperm in 9%

polyvinylpyrrolidone in PBS. All manipulations were performed on a heated plate (38.5 °C) of an

inverted microscope. A progressively motile sperm cell was immobilized by crushing the tail on

the bottom of the scale and injected into the cytoplasm of a mature oocyte. The injected

oocytes were cultured in groups of 10-20 embryos in 20µl droplets of DMEM-F12 with 10% fetal

calf serum at 38.5°C in 5% CO2, 5% O2 and 90% N2. On day 2.5 after fertilization, the embryos

which were not cleaved were removed and half of the medium was changed. On day 6 again

half of the medium was changed and on day 9 the embryonic development was evaluated and

blastocysts were selected. Two blastocysts were washed in Emcare Holding Medium® and put

in a 2 ml tube filled with Emcare Holding Medium®. During the 3 hour transport to the embryo

transfer centre, this tube was kept in 50 ml preheated saline and surrounded by 15 l infusion

Chapter 3

51

bags at 38.5 °C in an isothermal box. Upon arrival, the embryos were washed again in Emcare

Washing Medium® and they were both transferred to the uterus of a recipient mare at day 6

post ovulation. Due to recipient availability, both embryos were transferred to the same mare.

3.2.4 RESULTS

Of the 52 collected oocytes, 27 (52%) extruded the first polar body at 26 h of maturation and

were subsequently injected with a spermatozoon (Figure 1).

Figure 1 Intracytoplasmic sperm injection (ICSI).

This resulted in 23 cleaved embryos (Figure 2), of which 2 (8.7%) reached the blastocyst stage at

day 9 (Figure 3).

Figure 2 Cleaved equine embryo (day 2.5). Figure 3 Equine IVP blastocyst (day 9).

Chapter 3

52

On December 18th 2008 the embryos were transferred to the uterus of a mare that had

ovulated 6 days before. Six days after transcervical transfer one single embryonic vesicle of 0.7

cm was diagnosed by rectal ultrasound. The 12th of January 2009 one embryonic heart beat was

detected and regular ultrasound examinations revealed a normal development of a singleton

conceptus. The recipient mare was transported at 10 months of pregnancy to the Veterinary

Faculty at Merelbeke, where she gave birth the 27th of October 2009. A chestnut mare foal of

44 kg was born without complications and was called SMICSI (Figure 4,5).

Figure 4 Birth of SMICSI.

Figure 5 SMICSI 3 days old.

Chapter 3

53

A DNA profile, based on 12 microsatellite markers, was determined for the foal, the sperm

donor and the recepient mare. DNA profile comparison assigned the sperm donor as the father

and excluded the receptor mare as the biological mother of the foal. The filly is now over one

month old and is developing normally.

3.2.5 DISCUSSION

The IVP of equine embryos has lagged behind that of other large domestic animals. This can be

explained by the scarce availability of equine oocytes, the inefficiency of conventional IVF and

the suboptimal culture conditions and low blastocyst rates. However, after the introduction of

ICSI, the IVP of equine embryos developed into a reasonably efficient and repeatable technique

(Hinrichs, 2005). Especially the use of the piezo drill, a device which causes minute vibrations of

the injection pipette during ICSI, seemed to be less traumatic for the oocyte and resulted in a

rapid evolution in recent years (Choi et al., 2002; Galli et al., 2007).

Nowadays the combination of ovum pick up, in vitro maturation, ICSI, in vitro culture and

embryo transfer is not only used for research purposes, but also clinically. Since only a few

normal sperm cells are required, it can be applied for subfertile stallions, for semen which has

been frozen-thawed, diluted and frozen again and for sexed semen (Lazzari et al., 2002; Choi et

al., 2006; Squires et al., 2008). It has also been successful in case of problems on the female

side like advanced age, degenerative endometriosis and cervical laceration (Colleoni et al.,

2007). Moreover, ICSI can be beneficial in creating offspring of valuable animals post mortem.

These prosperous results are only achieved in a few laboratories in the world. In general the

technique is labour intensive, expensive and not yet optimally efficient. Problems concerning

superovulation in horses and a very tight connection between the equine oocyte and the follicle

wall imply only limited oocyte collection. Moreover the IVP process is still suboptimal in horses

when compared to other species. The application of IVP of equine embryos on a larger scale,

like it happens in cattle, has not been achieved yet in the horse (Blanco et al., 2009).

In this study a cleavage rate of 85% was obtained, which is comparable to the results in other

recent publications and which is rather good when it is considered that the ICSI procedure was

Chapter 3

54

performed in the conventional way without the use of the piezo drill. However, only 2 out of 23

injected oocytes (8.7%) reached the blastocyst stage, a fairly low number when compared to

other reports of 15.2% (Colleoni et al., 2007) and 23% (Hinrichs et al., 2007). Possible

explanations might include differences in oocyte source and culture conditions as well as in the

ICSI technique and experience. Even though spectacular blastocyst rates up to 38% have been

obtained during in vitro culture of equine embryos in DMEM/F12 (Hinrichs et al., 2005), there

are still quantitative and qualitative differences when (temporary) in vivo culture is performed.

Recent studies illustrated a higher percentage of equine ICSI embryos developing to the

compact morula or blastocyst stage in sheep oviducts (56%) when compared to culture in vitro

(20%) (Lazzari et al., 2010). Another intriguing finding was that aberrant expression of a

pluripotency gene in IVP blastocysts when compared to in vivo derived embryos could be

normalized by 2-3 days culture in an equine uterus (Choi et al., 2009). Studying gene expression

can reveal fundamental differences between equine in vivo and in vitro embryos (Smits et al.,

2009). When compared to in vivo derived equine blastocysts, IVP horse embryos have been

shown to be retarded in the kinetics of development, with more apoptosis and higher levels of

chromosomal abnormalities (Tremoleda et al., 2003; Pomar et al., 2005; Rambags et al., 2005).

Despite these differences, pregnancy rates after intra-uterine transfer of IVP equine blastocysts

are comparable to those after transfer of their in vivo counterparts and the possibility of

freezing IVP embryos at rather early stages provides the opportunity of successful

cryopreservation (Galli et al., 2007).

3.2.6 CONCLUSION

Although the IVP of equine embryos has evolved only recently and is not yet optimally efficient

when compared to other species, it provides important possibilities for research and clinical

application. This first ICSI foal of the Benelux is an important step in the improvement of the IVP

processes, which opens possibilities for more research in the domain of equine embryonic

development and valuable applications of the technique.

Chapter 3

55

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8. Hinrichs K. 2005. Update on equine ICSI and cloning. Theriogenology 64 535-541.

9. Hinrichs K., Choi Y.H., Love L.B., Varner D.D., Love C.C., Walckenaer B.E. 2005. Chromatin

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11. Lazzari G., Crotti G., Turini P., Duchi R., Mari G., Zavaglia G., Barbacini S., Galli C. 2002.

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oviduct versus in vitro culture for different domestic species. Theriogenology 73 748-

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13. Li X., Morris L.H., Allen W.R. 2001. Influence of co-culture during maturation on the

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20. Smits K., Goossens K, Van Soom A., Govaere J., Hoogewijs M., Vanhaesebrouck E., Galli

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21. Squires E.L., Suh T.K., Graham J.K., Carnevale E.M. 2008. Use of sexed, refrozen

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CHAPTER 4

DETERMINATION OF A SET OF RELIABLE REFERENCE GENES

FOR RT-QPCR IN IN VIVO DERIVED AND IN VITRO

PRODUCED EQUINE BLASTOCYSTS

Adapted from BMC Research Notes Smits K., Goossens K., Van Soom A., Govaere J., Hoogewijs

M., Vanhaesebrouck E., Galli C., Colleoni S., Vandesompele J., Peelman L. 2009. 2 246

Chapter 4

61

4.1 ABSTRACT

Background

Application of reverse transcription quantitative real-time polymerase chain reaction is very

well suited to reveal differences in gene expression between in vivo and in vitro produced

embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques.

However, for a correct interpretation of the real-time PCR results, all data must be normalized,

which is most reliably achieved by calculating the geometric mean of the most stable reference

genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh

and frozen-thawed in vitro embryos.

Findings

The expression stability of 8 candidate reference genes (ACTB, GAPDH, H2A/I, HPRT1, RPL32,

SDHA, TUBA4A, UBC) was determined in 3 populations of equine blastocysts (fresh in vivo, fresh

and frozen-thawed in vitro embryos). Application of geNorm indicated UBC, GAPDH, ACTB and

HPRT1 as the most stable genes in the in vivo embryos and UBC, RPL32, GAPDH and ACTB in

both in vitro populations. When in vivo and in vitro embryos were combined, UBC, ACTB, RPL32

and GAPDH were found to be the most stable. SDHA and H2A/I appeared to be highly

regulated.

Conclusions

Based on these results, the geometric mean of UBC, ACTB, RPL32 and GAPDH is to be

recommended for accurate normalization of quantitative real-time PCR data in equine in vivo

and in vitro produced blastocysts.

4.2 BACKGROUND

Conventional IVF has been rather unsuccessful in horses. To overcome this barrier ICSI has been

introduced and has resulted in transferable blastocysts that were used for research and

Chapter 4

62

commercial purposes (Galli et al., 2007; Hinrichs et al., 2007; Stokes et al., 2009). The ability of

in vitro produced blastocysts to establish normal pregnancies is comparable to that of the in

vivo derived ones and, contrary to other species, in vitro produced equine blastocysts tolerate

freezing better than in vivo produced ones and they are able to establish pregnancies after

thawing (Galli et al., 2007). Despite these successes, there is a great variability in oocyte quality

and culture conditions resulting in a low percentage of blastocyst formation in vitro with great

variations amongst laboratories. Compared to their in vivo counterparts, in vitro blastocysts are

retarded in the kinetics of development, are smaller with fewer cells, show more apoptosis and

higher levels of chromosomal abnormalities (Tremoleda et al., 2003a; Rambags et al., 2005;

Pomar et al., 2005). Moreover intra-uterine transfer of in vitro produced embryos can give rise

to abnormal pregnancies with the development of a trophoblastic vesicle without an embryo

proper (Hinrichs et al., 2007). Understanding the fundamental difference between equine in

vivo versus in vitro embryos may prove beneficial in the development of equine assisted

reproductive techniques.

RT-qPCR is a highly specific and sensitive tool to compare mRNA expression levels of specific

genes (Bustin et al., 2005). Not only the RNA quality, the RT, the reagents and the protocol are

critical factors, the analytical method can also influence the results dramatically (Bustin et al.,

2004; Bustin et al., 2005). All data must be normalized for technical differences between

samples. The method of choice consists in normalization to internal reference genes, which

should be constitutively expressed without influence of the experimental treatment. Since

there is no universal reference gene with a constant expression in all tissues, optimal reference

genes need to be selected for each system (Kubista et al., 2006). The use of a single reference

gene or of multiple unstable reference genes may lead to erroneous normalization (Dheda et

al., 2005). Therefore, the geometric mean of carefully selected genes is recommended for

reliable normalization (Vandesompele et al., 2002).

Reference genes have been validated in preimplantation embryos of cattle (Goossens et al.,

2005), mice (Mamo et al., 2007), pigs (Kuijk et al., 2007) and rabbits (Mamo et al., 2008).

However, gene stability in mammalian embryos turned out to be species specific, which implies

Chapter 4

63

the need of reference gene selection for the study of equine embryos. Gene expression studies

in equine species are relatively scarce. Real-time PCR has been conducted on equine

conceptuses to evaluate expression of progesterone and oestrogen receptors and on equine

blastocysts to identify POU5F1 expression, but in these studies only one reference gene, β-

actin, was used (Rambags et al., 2008; Choi et al., 2009). While sets of reference genes have

been identified for equine skin (Bogaert et al., 2006) and lymphocytes (Capelli et al., 2008), the

most reliable reference genes in equine embryos have not been studied before.

The aim of this study was to identify a set of stable reference genes for equine in vivo derived

embryos and fresh and frozen-thawed in vitro embryos.

4.3 METHODS

The in vivo blastocyst collection procedures used were approved by the ethics committee of the

faculty of veterinary medicine (reference number EC 2007/009). Cycling mares were followed

up by transrectal ultrasound. When the follicle size exceeded 35 mm in the presence of an

edematous uterus, 3000 IU hCG (Chorulon, Intervet, Belgium) was injected intravenously. The

next day the mare was inseminated with fresh semen. The time of ovulation was determined by

daily ultrasonography of the genital tract.. If the mare did not ovulate within 48 hours after AI,

insemination was repeated. The mares’ uterus was flushed 7 days after ovulation and

recovered embryos were washed in DPBS (14190, Gibco, Invitrogen, Belgium) and conserved

individually at -80 °C in lysis buffer (10 % RNasin Plus RNase inhibitor (Promega, The

Netherlands), 5 % dithiothreitol (Promega, The Netherlands), 0.8 % Igepal CA-630 (Sigma,

Belgium) in RNase free water) until further analysis.

For the production of fresh in vitro blastocysts, ovaries were recovered at the slaughterhouse

and follicles ≥ 5 mm were aspirated with a vacuum pump at a negative pressure of 100 mm Hg.

The follicle wall was scraped with the aspirating 16 gauge needle and flushed with 0.5 % (v/v)

heparin (H 1027, Sigma, Belgium) in DPBS (14190, Gibco, Invitrogen, Belgium). Oocytes were

matured for 28 h in DMEM-F12 based medium (Galli et al., 2007) at 38.5 °C in 5% CO2 in air. The

MII oocytes were fertilized by conventional ICSI as described by Tremoleda et al. (2003b).

Chapter 4

64

Frozen-thawed sperm was used from the same fertile stallion that was used for production of in

vivo embryos of this experiment. In vitro culture was performed for 8.5 to 9.5 days in 20 µl

drops of DMEM-F12 (D 2906, Sigma, Belgium) with 10% FCS (F 9665, Sigma, Belgium) or 5% FCS

and 5% SR (10828-028, Gibco, Invitrogen, Belgium) at 38.5 °C in 5% CO2, 5% O2 and 90% N2.

Blastocysts were stored individually at -80 °C in lysis buffer until further analysis.

The frozen-thawed in vitro embryos were produced in Italy (Galli et al., 2007). Briefly, oocytes

were recovered from abbatoir ovaries by scraping and washing all the follicles between 5 and

30 mm. Oocytes were washed in HEPES buffered TCM199 and transferred for 24 h into DMEM-

F12 based maturation medium as previously described (Galli et al., 2007). After removal of all

cumulus cells, the oocytes with a polar body were fertilized by ICSI with frozen-thawed sperm

from stallions of proven fertility. Injected oocytes were cultured up to day 8 in 20 µl drops of

modified SOF (from day 6 half SOF: half DMEM-F12) at 38.5 °C in 5% CO2, 5% O2 and 90% N2.

Embryos that reached the blastocyst stage were loaded individually into 0.25 ml straws, frozen

with a slow cooling protocol (0.5 °C/min up to -32°C) in glycerol and stored in liquid N2. The

embryos were shipped to Belgium in dry ice and upon arrival they were immediately

transferred into lysis buffer with a minimum of medium and conserved at - 80 °C until further

analysis.

In all groups only blastocysts which were not hatched and had good morphological

characteristics were retained.

For each of the 3 groups, 8 embryos were analysed separately. Total RNA was extracted from

single embryos with the PicoPure RNA-isolation Kit (Arcturus, Mountain View, CA, USA), treated

with RQ1 DNAse (Promega, The Netherlands) and purified over a spin column (Microcon YM-

100, Millipore, Belgium). After minus RT control with primers for GAPDH to check for

contaminating genomic DNA, RNA was concentrated by precipitation with 3M sodium acetate

and ethanol. RNA amplification and conversion into cDNA was performed by means of the WT-

Ovation RNA Amplification System (NuGEN, USA) according to the manufacturers’ instructions

and the cDNA was purified again over a spin column. Amplified cDNA samples were diluted 10-

20 times, depending on the yield, in 10 mM Tris HCL pH 8.0 and stored at - 80 °C.

Chapter 4

65

Eight reference genes were selected based on previous studies (Goossens et al., 2005; Bogaert

et al., 2006; Capelli et al., 2008). The selected genes (ACTB, GAPDH, H2A/I, HPRT, RPL32, SDHA,

TUBA4A and UBC) belong to different functional classes, which reduces the chance of co-

regulation.

Primers for ACTB, HPRT1, RPL32, TUBA4A and UBC were provided by Bogaert and colleagues

(2006). The other primers were designed by means of Primer3 software

(http://frodo.wi.mit.edu/primer3/), based on horse sequences found in the NCBI GenBank

(http://www.ncbi.nlm.nih.gov/). Primers were selected over intron-exon boundaries, tested

using a BLAST analysis against the NCBI database and verified using MFold

(http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/dna-form1.cgi). The optimal primer

annealing temperatures were determined on cDNA of equine mixed tissues. A melt curve

analysis followed by agarose gel electrophoresis was performed to test for primer-dimer

formation and specificity of the amplicons. All primers are listed in Table 1.

All reactions were executed in duplicate for 8 embryos in each of the three groups and a blank

was included in each run.

The diluted cDNA (2.5 µl), 0.33 µM of both forward and reverse primers and 4µl of RNAse free

water were added to 7.5 µl of KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems) to a final

volume of 15 µl. The reactions were performed on the iCycler iQ Real-Time PCR Detection

System (Bio-Rad, Belgium).

The PCR program started with an initial denaturation at 95°C for 3 minutes to activate the DNA

polymerase. Then 45 cycles were performed with a denaturation step at 95°C for 20 seconds

followed by an annealing/extension step at the primer specific annealing temperature for 40

seconds, during which fluorescence was measured. A dilution series with pooled cDNA from all

24 embryos was included for each gene to acquire PCR efficiencies based on a relative standard

curve. Calculation of the Cq values, PCR efficiencies, correlation coefficients and analysis of the

melting curves was performed by means of iCycler iQ Optical System Software Version 3.0a. The

standard errors of the PCR efficiencies were obtained by the qBasePlus software

(http://www.qbaseplus.com/).

Chapter 4

66

Table 1 Primers. For each reference gene, the NCBI GenBank accession number, the sequence of

both forward and reverse primer, the size of the amplicon and the optimal primer annealing

temperature are listed. The RTPrimerDB ID (http://rtprimerdb.org/) is also reported, except for

the primers for which there is not yet an official reference sequence available in the database

(*).

Gene GenBank accession number

Sequence Amplicon size (bp)

Ta (°C)

RTprimerDB ID

Cq range

ACTB AF035774 CCAGCACGATGAAGATCAAG

GTGGACAATGAGGCCAGAAT

88 60 7848 16.3-23.8

GAPDH XM_001496020.1 CAGAACATCATCCCTGCTTC

ATGCCTGCTTCACCACCTTC

187 59 7849 14.2-24.5

H2A/I XM_001497311.2 ATATTCAGGCCGTGCTGCT

TTTGGGTTTCAAAGCGTTTC

105 60 * 23.5-37.3

HPRT1 AY372182 GGCAAAACAATGCAAACCTT

CAAGGGCATATCCTACGACAA

163 57 7850 17.6-25.8

RPL32 XM_001492042.2 AGCCATCTACTCGGCGTCA

TCCAATGCCTCTGGGTTTC

149 60 * 16.8-25.9

SDHA XM_001490889 TCCATCGCATAAGAGCAAAG

GGTGGAACTGAACGAACTCC

159 59 7851 20.7-33.3

TUBA4A XM_001491910.2 GCCCTACAACTCCATCCTGA

ATGGCTTCATTGTCCACCA

78 60 * 16.6-27.9

UBC AF506969 GCAAGACCATCACCCTGGA CTAACAGCCACCCCTGAGAC

206 60 7874 15.5-23.7

The expression stabilities were evaluated using the geNorm software for Microsoft Excel

(Vandesompele et al., 2002). This program ranks the genes based on the internal control gene

stability parameter M. Stepwise exclusion of the gene with the highest M value and

recalculation results in a ranking of the reference genes. Lower M values represent higher

Chapter 4

67

expression stabilities. Furthermore the minimum number of genes required for the calculation

of a reliable normalization factor is determined.

4.4 RESULTS AND DISCUSSION

For the recovery of the in vivo embryos (n=8), 13 uterine flushes were performed in a

population of 8 mares (recovery rate: 61.5 %). To collect the fresh in vitro embryos (n=8), 123

ovaries were recovered in 5 experiments, which gave rise to a total number of 365 oocytes.

Maturation was completed and ICSI was performed in 209 oocytes (57 %) and 74 % of these

injected oocytes cleaved. Of the cleaved oocytes 5.8 % reached the blastocyst stage. The 8 in

vitro blastocysts which were used for genetic analysis were produced in 2 of those 5 batches

where the blastocyst percentage was 7.3 %.

Frozen embryos were produced in experiments for quality control purposes. They were frozen

on day 8 at the early blastocyst or blastocyst stage.

Prior to the start of the experiment, PCR was tested on one of the frozen-thawed embryos to

evaluate possible effects of remnants of the cryoprotectant. Both undiluted and 25x diluted

cDNA resulted in clear electrophoretic bands of expected size for UBC and ACTB. Only a minimal

amount of cryoprotectant accompanying the embryo was transferred to and diluted in the lysis

buffer. Importantly Cq-values of the fresh and frozen-thawed embryos remained in the same

range.

Because only a small quantity of RNA can be extracted from one embryo, an RNA pre-

amplification step was included in the protocol. Ribo-SPIA, the isothermal messenger RNA

amplification method used, has been shown to be accurate and reproducible (Dafforn et al.,

2004). High sensitivity permits quantification of low abundance transcripts and there is a good

correlation in differential gene expression between amplified and nonamplified cDNA (Dafforn

et al., 2004).

Chapter 4

68

Dilution series of all candidate reference genes gave PCR-efficiencies between 97.1 % and 100

% and linear correlation coefficients that varied from 0.973 to 1.000. In table 2 the efficiencies

and their standard errors are listed.

One in vivo and one fresh in vitro embryo consistently resulted in Cq-values around 40. Probably

the amplification went wrong and both samples were deleted from further analysis.

The selection of appropriate reference genes can be achieved by evaluating RT-qPCR data with

statistical algorithms. The uniformity in gene ranking between geNorm (Vandesompele et al.,

2002), NormFinder (Andersen et al., 2004) and BestKeeper (Pfaffl et al., 2004) has been shown

to be high (Cappelli et al., 2008; Willems et al., 2006).

Table 2 PCR efficiency and the respectively standard error. Results of the reference gene

stability, as determined by geNorm, are shown in Figure 1, 2, 3 and 4. When all three groups of

embryos were included, the most stable genes were UBC, ACTB, RPL32 and GAPDH. The M

values of these genes range between 0.6 and 0.9, which indicates relatively good stability.

Gene Efficiency (%) Standard error ACTB 98.1 0.01 GAPDH 100 0.021 H2A/I 100 0.067 HPRT1 100 0.037 RPL32 100 0.071 SDHA 99.3 0.033 TUBA4A 100 0.017 UBC 97.1 0.017

Chapter 4

69

Figure 1 Average expression stability values of equine in vivo and in vitro embryos. The average

stability values of the control genes were calculated with geNorm. When in vivo embryos and fresh and

frozen-thawed in vitro embryos were combined, UBC, ACTB, RPL32 and GAPDH were found to be the

most stable.

Figure 2 Average expression stability values of equine in vivo embryos. The average stability values of

the control genes were calculated with geNorm. In the population of the equine in vivo embryos UBC,

GAPDH, ACTB and HPRT1 were found to be the most stable.

0,6

0,8

1

1,2

1,4

1,6

1,8

2

2,2

2,4

SDHA H2A/I TUBA4A HPRT1 GAPDH RPL32 ACTBUBC

Aver

age

expr

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<::::: Least stable genes Most stable genes ::::>

0,3

0,8

1,3

1,8

2,3

2,8

H2A/I SDHA TUBA4A RPL32 HPRT1 ACTB UBCGAPDH

Aver

age

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Chapter 4

70

Figure 3 Average expression stability values of fresh equine in vitro embryos. The average stability

values of the control genes were calculated with geNorm. In the population of the fresh equine in vivo

embryos UBC, RPL32, GAPDH and ACTB were found to be the most stable.

Figure 4 Average expression stability values of frozen-thawed equine in vitro embryos. The average

stability values of the control genes were calculated with geNorm. In the population of the fresh equine

in vivo embryos UBC, RPL32, GAPDH and ACTB were found to be the most stable.

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

SDHA HPRT1 H2A/I TUBA4A ACTB GAPDH UBCRPL32

Aver

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0,5

0,7

0,9

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1,3

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SDHA TUBA4A H2A/I HPRT1 ACTB GAPDH UBCRPL32

Aver

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Chapter 4

71

The order of the 4 most stable genes, UBC, RPL32, GAPDH and ACTB, is identical in both in vitro

groups. This is an interesting finding as there was a difference in embryo culture conditions and

as the cryopreservation could have affected the results. Three of those 4 genes, UBC, GAPDH

and ACTB, represent the most stable genes in the in vivo embryos. In murine embryos the order

of stability of reference genes has also been found to be similar in both in vitro and in vivo

embryos (Mamo et al., 2007). In the latter study however, the stability measure values M were

described to be higher in the in vitro samples compared to the in vivo samples, which was not

the case in the equine embryos. SDHA and H2A/I generally turned out to be highly regulated.

These results differ from those in embryos from other species and from those in other equine

tissues. SDHA, which was highly regulated in equine embryos, appeared to be very stable in

bovine embryos (Goossens et al., 2005) and equine lymphocytes (Cappelli et al., 2008). This is

also the case for H2A/I, which was preferred as a reference gene in embryos of mice (Mamo et

al., 2007) and rabbits (Mamo et al., 2008). ACTB on the other hand appears fairly stable in

equine embryos, although it is highly regulated in bovine embryos (Goossens et al., 2005). This

again demonstrates the necessity to validate the genes according to the species and the tissue

type.

The use of the geometric mean of several internal control genes as a normalization factor (NF)

is more accurate than the use of a single reference gene. Inclusion of unstable genes on the

other hand negatively influences the NF and the number of reference genes used to calculate

this NF is a trade-off between practical considerations and accuracy. The optimal number of

genes was determined with geNorm by means of the pairwise variations (Vn/n+1) between the

sequential normalization factors (NFn and NFn+1) after successive inclusion of less stable

reference genes (Figure 5). The value of the pairwise variations reduces until 0.207 for V3/4. This

suggests that the inclusion of a fourth reference gene contributes to the stability. Therefore it is

recommended to use the 4 most stable genes, UBC, ACTB, RPL32 and GAPDH. The high values

of V6/7 and V7/8 represent the instability of H2A/I and SDHA, which is also clear in the steep rise

in Figure 1.

Chapter 4

72

Figure 5 Determination of the optimal number of control genes for normalization. The optimal

number of control genes for normalization was calculated by geNorm. The value of the pairwise

variations reduces until 0.207 for V3/4, which indicates that the inclusion a fourth reference gene

contributes to the stability. Therefore the average of the 4 most stable genes is recommended to

determine a reliable NF.

In conclusion, the geometric mean of ACTB, UBC, RPL32 and GAPDH is recommended for

normalization of RT-qPCR data in experiments with equine in vivo and in vitro blastocysts.

REFERENCES

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2. Bogaert L., Van Poucke M., De Baere C., Peelman L., Gasthuys F., Martens A. 2006.

Selection of a set of reliable reference genes for quantitative real-time PCR in normal

equine skin and in equine sarcoids. BMC Biotechnol 6 24.

3. Bustin S.A., Nolan T. 2004. Pitfalls of quantitative real-time reverse-transcription

polymerase chain reaction. J Biomol Tech 15 155-166.

4. Bustin S.A., Benes V., Nolan T., Pfaffl M.W. 2005. Quantitative real-time RT-PCR: a

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21. Tremoleda J.L., Stout T.A. Lagutina I., Lazzari G., Bevers M.M., Colenbrander B., Galli C.

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J Dev Biol 50 627-635.







CHAPTER 5

IN VIVO DERIVED HORSE BLASTOCYSTS SHOW

TRANSCRIPTIONAL UPREGULATION OF DEVELOPMENTALLY

IMPORTANT GENES COMPARED TO IN VITRO PRODUCED

HORSE BLASTOCYSTS

Adapted from Smits K., Goossens K., Van Soom A., Govaere J., Hoogewijs M., Peelman L. 2010.

Reproduction Fertility and Development in press.

Chapter 5

79

5.1 ABSTRACT

In vitro produced (IVP) equine blastocysts can give rise to successful pregnancies, but their

morphology and developmental rate differ from that of in vivo derived equine blastocysts. The

aim of this study was to evaluate this difference at the genetic level. Therefore suppression

subtractive hybridization (SSH) was used to construct a cDNA library enriched for transcripts

preferentially expressed in in vivo derived equine blastocysts compared to in vitro produced

ones. Of the 62 different genes identified in this way, 6 genes involved in embryonic

development (BEX2, FABP3, HSP90AA1, MOBKL3, MCM7 and ODC) were selected for the

confirmation of this differential expression by means of reverse transcription quantitative real-

time PCR (RT-qPCR). For 5 of these genes the higher expression in vivo was proven by RT-qPCR

to be significant (FABP3 and HSP90AA1) or highly significant (ODC, MOBKL3 and BEX2),

confirming the results of the SSH. For MCM7 the difference was not significant. In conclusion 5

genes which are transcriptionally upregulated in in vivo derived equine blastocysts as compared

to IVP blastocysts have been identified. Because of their possible importance in embryonic

development, the expression of these genes can be used as a marker to evaluate in vitro

embryo production systems in the horse.

5.2 INTRODUCTION

Compared to other species the in vitro production (IVP) of equine embryos has been hampered

by the scarce availability of oocytes and by the inefficiency of conventional in vitro fertilization

(IVF). The introduction of intracytoplasmic sperm injection (ICSI) induced a rapid progress over

the last decade and transferable equine in vitro blastocysts have been successfully produced for

research as well as for commercial purposes (Galli et al., 2007; Hinrichs et al., 2007; Smits et al.,

2010). The IVP of equine embryos is a very valuable technique to study early stages of embryo

development, which used to be quite inaccessible because of their location in the oviduct.

Moreover in vitro embryos can be used to optimize the different steps of the IVP process itself.

Further research is required to obtain large scale clinical applications like in cattle (Blanco et al.,

2009), but promising results illustrating the great value of IVP in the horse have been published

Chapter 5

80

recently (Colleoni et al., 2007). The combination of OPU and IVP represents in some cases the

only opportunity to obtain foals from valuable subfertile mares and stallions. It can be used for

mares with reproductive disorders or for aged mares and since the requirements for sperm

which is selected for ICSI are minimal, it is also applicable for stallions with poor sperm (Colleoni

et al., 2007). In this way IVP provides the production of offspring from genetically valuable

horses, which is of considerable benefit to the horse breeding industry.

Even though the capability of establishing pregnancies is comparable for IVP and in vivo derived

equine embryos (Colleoni et al., 2007), differences between both types of embryos remain

present. Compared to their in vivo counterparts equine IVP embryos have fewer cells with more

apoptosis and chromosomal abnormalities (Tremoleda et al., 2003; Pomar et al., 2005;

Rambags et al., 2005). In in vivo derived equine blastocysts a distinct glycoprotein capsule is

formed between the trophectoderm and the zona pellucida. In vitro produced blastocysts have

a deficient capsule formation and show only scattered patches of glycoproteins (Tremoleda et

al., 2003). Suboptimal culture conditions are a possible cause of this aberrant differentiation

since even temporary exposure of IVP embryos to an in vivo environment can have a major

influence on embryonic development. Lazzari et al. (2010) reported that 56 % of cleaved equine

in vitro embryos were developing to compact morulae and blastocysts after temporary culture

in sheep oviducts, which differed significantly from the 20 % which was achieved after total in

vitro culture. Another example was the finding that impaired differentiation of the equine

trophectoderm in vitro, which was determined by aberrant expression of the pluripotency

marker POU5F1 when compared to equine in vivo blastocysts, could be normalized by culturing

the IVP blastocysts in vivo for 2-3 days in an equine uterus (Choi et al., 2009).

In vivo derived horse embryos are relatively difficult to obtain, because the mare does not

respond adequately to superovulatory treatments and because the horse embryo arrives only

in the uterus at day 6.5, which limits the time period during which equine embryos can be

obtained by non-surgical flushing (Scherzer et al., 2008). As such, horse embryos are mostly

flushed during a natural cycle at day 7, at which time they have developed to the blastocyst or

expanded blastocyst stage. Such horse blastocysts represent the gold standard for early equine

Chapter 5

81

embryonic development. Comparing IVP and in vivo derived horse blastocysts at the genetic

level can reveal fundamental differences responsible for the observed morphological and

developmental changes. In other mammalian species the expression level of specific genes has

been used to evaluate the effect of different embryo culture media as well as the effect of

adding specific components like serum or cytokines to these media on embryonic

differentiation (McElroy et al., 2008; Chin et al., 2009; Purpera et al., 2009). In the horse, the

expression of genes involved in cumulus expansion has been used to assess different oocyte

maturation conditions (Dell’Aquila et al., 2004). Evaluation of horse embryos by means of RT-

qPCR may provide the basis for improving the culture conditions for horse embryos.

Little is known about gene expression in equine embryos. At the start of this project, no

commercial horse microarray was available and annotation of the horse genome was poor.

Microarray is very useful if one wants to investigate a known panel of genes but it is not able to

detect novel or unknown genes. Therefore we decided to use suppression subtractive

hybridization (SSH), a powerful method for the detection of differentially expressed genes

when prior knowledge is limited (Diatchenko et al., 1996). This technique provides a selective

amplification of target cDNA fragments which are specifically expressed in one of the two cDNA

populations and in this way can be applicable for the comparison of the gene expression in in

vivo versus in vitro embryos. Results of this SSH however need to be confirmed by reverse

transcription quantitative real-time polymerase chain reaction (RT-qPCR), a highly sensitive and

specific tool, which can evaluate and quantify the difference in expression of the genes of

interest selected from the SSH.

The aim of this study was to evaluate gene expression in equine blastocysts at the

transcriptional level. SSH was used to identify genes which were expressed at a higher level in in

vivo derived equine blastocysts when compared to IVP equine blastocysts. Six of these genes

were selected for the confirmation and quantification of this difference in expression by RT-

qPCR. The choice of these genes was based upon their involvement in embryonic development

in other species.

Chapter 5

82

5.3 MATERIALS AND METHODS

5.3.1 Collection of embryos

The procedures performed on the mares were approved by the Ethics Committee of the Faculty

of Veterinary Medicine (EC 2007/009). The in vivo embryos were collected by uterine flushing

of inseminated mares 7 days after detection of ovulation as described previously (Smits et al.,

2009). The in vitro embryos were produced following the protocol as previously described by

Smits et al. (2010). Briefly slaughterhouse oocytes were matured in vitro during 26h in DMEM-

F12 based medium in an atmosphere containing 5% CO2 (Galli et al., 2007). MII oocytes were

fertilized by conventional ICSI and cultured in vitro during 9-9.5 days in DMEM-F12 with 10%

fetal calf serum at 38.5°C in 5% CO2, 5% O2 and 90% N2.

In order to compare similar developmental stages, rather than embryos of equal chronological

age, the in vitro blastocysts were collected later than the in vivo derived blastocysts. This choice

was based upon previous research on equine in vivo and in vitro produced embryos. Tremoleda

et al. (2003) and Rambags et al. (2005) described day 7 IVP embryos to be smaller, to contain

fewer cells and to be retarded in the kinetics of development when compared to their in vivo

derived counterparts of the same age (day 7). In a subsequent study by Pomar et al. (2005) in

vivo embryos were collected 7 days after ovulation and in vitro embryos 9 days after ICSI and in

a recent publication by Choi et al. (2009) similarities in cell count and embryonic diameter were

found between IVP blastocysts on day 10 and the smallest in vivo derived blastocysts (day 7). In

both populations in our study the blastocyst stage was characterized by a blastocoele cavity

surrounded by a layer of trophoblast cells. Early expansion was displayed by a thinned zona

pellucida. However, considering rapid expansion in vivo, blastocysts in the in vivo group were

collected rather early and variation was minimized through regular ultrasound to detect

ovulation (1-2 times daily). For the SSH 10 in vivo embryos and 12 in vitro embryos were

collected. The number of in vitro embryos exceeded the number of in vivo embryos to provide

sufficient cDNA in the driver group, i. e. the in vitro embryos, in order to achieve hybridization

of common strands with the in vivo embryos. The RT-qPCR was performed with 8 in vivo and 8

in vitro embryos.

Chapter 5

83

5.3.2 SSH

To identify the genes that were preferentially expressed in the in vivo embryos, 10 in vivo

embryos were pooled and composed the tester population. The driver population consisted of

12 pooled in vitro embryos. In both groups total RNA was extracted with the Pico Pure RNA

Isolation Kit (Arcturus, Mountain View, CA, USA), treated with RQ1 DNAse (Promega, The

Netherlands) and purified over a spin column (Microcon YM-100, Millipore, Belgium). After

concentration by precipitation with ammonium acetate and 95 % ethanol, conversion to and

amplification of cDNA was performed with the SMART PCR cDNA Synthesis Kit (Takara Bio Inc.,

France).

The SSH was accomplished with the PCR-Select cDNA Subtraction Kit (Takara Bio Inc., France)

following the manufacturer’s instructions. Briefly, Rsa I digestion of the cDNA and adaptor

ligation to the tester population was followed by 2 hybridizations and 2 PCR amplifications. This

resulted in enrichment of the differentially expressed high- and low-abundance tester

sequences. These were cloned into a T/A cloning vector (Invitrogen, Belgium) and transformed

into competent DH5α E. coli cells (Invitrogen, Belgium). The DNA-inserts were sequenced with

the BigDye Terminator v.3.1 cycle sequencing kit (Applied Biosystems, Belgium) on the Applied

Biosystems 3710xI DNA Analyzer and identified by BLAST analysis against the NCBI gene

database (Altschul et al., 1990) (http://www.ncbi.nlm.nih.gov/). Functional and pathway

analysis of the identified genes was performed by means of Ingenuity software

(http://www.ingenuity.com/products/pathways_analysis.html).

5.3.3 RT-qPCR

In both the in vivo and the in vitro group 8 embryos were analysed individually. The RNA

extraction, purification, concentration, amplification and conversion into cDNA were performed

as described previously (Smits et al., 2009).

The primers (Integrated DNA Technologies, Belgium) were designed by means of Primer3

software (Rozen and Skaletsky 2000), based on horse RNA and DNA sequences found in the

NCBI GenBank. To distinguish genomic DNA amplification, to provide specificity and to avoid

Chapter 5

84

secondary structures in the primer region, primers were respectively selected over intron-exon

boundaries, tested using a BLAST analysis against the NCBI database and characterized with

MFold (Zuker, 2003). Primers were tested and optimal annealing temperature was determined

on cDNA of equine mixed tissues. The amplicons were run on a 2% agarose gel and confirmed

by nucleotide sequencing. All primers are listed in Table 1.

All RT-qPCR reactions were performed in duplicate with the KAPA SYBR FAST qPCR Master Mix

(Kapa Biosystems, USA) as previously described in Smits et al. (2009).

A blank, a melting curve and a 5- or 10-fold serial dilution series of pooled amplified embryonic

cDNA were included for each gene to check for contamination and specificity and to acquire

PCR efficiencies (Table 1) based on a relative standard curve. All Cq values were converted into

raw data using these PCR efficiencies and normalized by dividing them by their respective

normalization factor. This normalization factor was determined per embryo by calculating the

geometric mean of the validated reference genes ACTB, UBC, RPL32 and GAPDH (Smits et al.,

2009). For each gene the difference in expression level between the in vivo derived embryos

and the in vitro produced ones was analyzed by means of a Mann Whitney test and p-values

smaller than 0.05 were considered statistically significant.

Table 1 Primers RT-qPCR (Equus caballus). For each gene the GenBank accession number, the

primer sequences, the size of the amplicon, the primer annealing temperature and the qPCR

efficiency with its respective standard error are listed.

Gene Accession number

Sequence 5’→3’ Amplicon size (bp)

Ta (°C)

Efficiency (%)

Standard error

BEX2 XM_001503074.1 AAGCTGGTGAATGCTGTGTG AACTGCCCGCAAACTATGAC

209 63 99.5 0.064

FABP3 NM_001163885.1 CTGCTCTCTTGGCTCTTCTTTG CGATGATTGTGGTAGGCTTG

173 60 94.5 0.036

HSP90AA1 NM_001163955.1 GGATCTGGTCATCCTGCTCTAC ACGTGTCGTCATCTCCTTCA

197 63 96.1 0.112

MCM7 XM_001505047.1 GAGGATGATGAGGCTGGTG TCAGGCAGATGTTGATGTTG

211 63 93.1 0.040

MOBKL3 XM_001917847.1 GGCCGAAACTGTAACCAAAG CCAACCTTCACGCTAACTGG

138 60 94.8 0.012

ODC XM_001502323.2 CATGGGCGCTTATACTGTTG GAAGTCGTGGTTCTGGATTTG

121 63 97.2 0.018

Chapter 5

85

5.4 RESULTS

5.4.1 Collection of equine blastocysts

For the recovery of the 10 in vivo blastocysts for the SSH 20 uterine flushes were performed

(recovery rate: 50 %), the 8 blastocysts for RT-qPCR were recovered in 13 flushes (recovery

rate: 61.5 %). These were the same blastocysts as those, used in CHAPTER 4.

To produce the 12 in vitro blastocysts needed for the SSH 466 mature oocytes were injected

and 44 % cleaved. Of the cleaved oocytes 5.8 % reached the blastocyst stage. To produce the 8

in vitro blastocysts necessary for RT-qPCR 123 ovaries were recovered in the course of 5

replicates, which gave rise to a total of 365 oocytes. ICSI was performed in 209 mature oocytes

(57 %) and 74 % of these injected oocytes cleaved. Of the cleaved oocytes 5.8 % reached the

blastocyst stage. The 8 in vitro blastocysts used for genetic analysis were derived from 2 out of

5 replicates, with a mean blastocyst percentage of 7.3 %. Parallel experiments revealed mean

cell counts of around 500 and the developmental compentence of the in vitro blastocysts was

shown by embryo transfer of 2 in vitro blastocysts resulting in a foal (Smits et al., 2010).

5.4.2 SSH

A total of 84 clones was sequenced, but some genes were represented by more than one clone.

This lead to the identification of 62 different genes preferentially expressed in the in vivo

blastocysts when compared to the in vitro produced blastocysts. Table 2 represents the

accession numbers of the SSH sequences and the respective genes which were identified

through BLAST.

Analysis of these genes by means of Ingenuity Pathway Analysis revealed the most important

functions in which these genes were involved (Figure 1). A majority of genes identified play a

role in protein synthesis, including many ribosomal genes, and in energy production, including

many mitochondrial genes. Furthermore several of the genes with higher expression in in vivo

embryos could be mutually linked in a functional network. The networks are added in Figure 2.

Chapter 5

86

Table 2 Genes upregulated in in vivo derived blastocyst when compared to in vitro produced

blastocysts as determined by SSH (Equus caballus). This table describes the 62 genes that were

expressed at a higher level in the in vivo equine blastocysts than in the in vitro equine

blastocysts as determined by SSH. For each gene the NCBI accession number (AN) of the

sequence resulting from the SSH is listed. This sequence was identified through BLAST analysis

and the details on the homologous gene which was found in the NCBI database are also

included in the table. Finally the query coverage and the % of homology between both

sequences are described. * Predicted sequence, Ecab: Equus caballus, sim: similar to

AN SSH AN reference Gene GeneID

Query coverage (%)

Max identity (%)

GW820038 XM_001492988.2 *: Ecab sim tyrosine 3/tryptophan 5 -monooxygenase activation protein, zeta polypeptide (LOC100056060), mRNA YWHAZ 18 100

GW820037 XM_001505047.1 *: Ecab sim Minichromosome maintenance complex component 7 (LOC100068673), mRNA MCM7 39 100

GW820039 XM_001505158.2 *: Ecab sim HNRPC protein, transcript variant HNRPC 85 97

GW820040 XM_001494978.2 *: Ecab sim ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 (LOC100059401), mRNA ATP5J2 37 99

GW820041 NM_001163880.1 Ecab ribosomal protein L35a (RPL35A), mRNA RPL35A 36 99

GW820042 XR_044465.1 *: Ecab misc_RNA (LOC100054512), miscRNA / 40 89

GW820043 XM_001503074.1 *: Ecab sim brain expressed X-linked 2 (LOC100059298), mRNA BEX2 31 89

GW820044 NM_001163950.1 Ecab ribosomal protein S4 (RPS4), mRNA RPS4 72 99

GW820045 XM_001494060.2 *: Ecab sim mCG7602, transcript variant 1 (LOC100051535), mRNA mCG7602 77 98

GW820046 XM_001490466.2 *: Ecab sim Saccharopine dehydrogenase SCCPDH 56 100

GW820047 NM_001163890.1 Ecab ribosomal protein S26 (RPS26), mRNA RPS26 79 99

GW820048 XM_001917666.1 *: Ecab mitochondrial ATP synthase, H+ transporting F1 complex beta subunit (ATP5B), mRNA ATP5B 40 100

GW820029 XM_001918118.1 *: Ecab sim tat-associated protein (LOC100072802 / 56 99

GW820030 XM_001488883.2 *: Ecab actin, gamma 1 (ACTG1), mRNA ACTG1 43 99

GW820031 XM_001504949.1 *: Ecab sim ribosomal protein S13 (LOC100056386), mRNA RPS13 57 99

GW820032 XM_001500201.2 *: Ecab sim 60S ribosomal protein L12 (LOC100070538), mRNA RPL35A 31 98

GW820033 XM_001495020.2 *: Ecab sim ferritin heavy chain (LOC100062546 FTH1 73 98

GW820034 XM_001504725.2 *: Ecab sim transmembrane protein 93 ( TMEM93 57 99

GW820035 NM_001081781.1

Ecab eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), mRNA >dbj|AB292108.1| Ecab EEF1A1 mRNA for eukaryotic translation elongation factor 1 alpha 1, complete cds EEF1A1 28 99

GW820036 XM_001494582.2 *: Ecab actin related protein 2/3 complex, subunit 1A, 41kDa (ARPC1A), mRNA ARPC1A 37 98

GW820049 XM_001493016.1 *: Ecab sim myosin regulatory light chain MYL12B 74 98

GW820050 XM_001492042.2 *: Ecab sim ribosomal protein L32 (LOC100056766 RPL32 79 100

GW820051 XM_001918320.1 *: Ecab sim proteasome (prosome, macropain PSMD11 47 98

GW820052 XM_001499248.2 *: Ecab sim ribosomal protein L23a (LOC100055584), mRNA RPL23A 47 98

GW820053 XM_001498955.2 *: Ecab sim ribosomal protein L37 (LOC100066211), mRNA RPL37 68 100

GW820054 XM_001503344.2 *: Ecab serine carboxypeptidase 1 (SCPEP1), mRNA SCPEP1 35 82

Chapter 5

87

GW820055 XM_001493543.1 *: Ecab sim heat shock 105kDa/110kDa HSPH1 30 99

GW820056 XM_001916408.1 *: Ecab sim rCG50488 (LOC100067336), mRNA YBX1 88 99

GW820057 XM_001496510.2 *: Ecab sim rab11 (LOC100066116), mRNA RAB11A 58 99

GW820058 XM_001917617.1 *: Ecab sim HLA-B associated transcript 1 (LOC100146384), mRNA BAT1 66 100

GW820059 XM_001499910.2 *: Ecab Tax1 (human T-cell leukemia virus type I) binding protein 1 (TAX1BP1), mRNA TAX1BP1 42 99

GW820060 XM_001494880.2 *: Ecab G-beta-like protein (LOC100057771), mRNA GNB2L1 76 99

GW820061 XM_001916244.1 *: Ecab sim ATP synthase subunit alpha, mitochondrial (LOC100053178), mRNA ATP5A1 68 99

GW820062 XM_001496761.2 *: Ecab sim ribosomal protein S8 (LOC100065845 RPS8 61 99

GW820063 XM_001499070.2 *: Ecab sim caspase 12 (LOC100069290), mRNA CASP12 21 91

GW820064 XM_001502980.2 *: Ecab DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A), mRNA DNMT3A 63 100

GW820065 XM_001500732.2 *: Ecab sim aldose reductase (LOC100065145 AKR1B1 87 99

GW820066 XM_001493571.2

*: Ecab sim 60S ribosomal protein L6 (TAX-responsive enhancer element-binding protein 107) (TAXREB107) (Neoplasm-related protein C140) (LOC100051509), mRNA RPL6 92 99

GW820067 XM_001493775.2 *: Ecab sim endoplasmic reticulum protein 29 (LOC100057258), mRNA ERP29 78 99

GW820068 XM_001502323.2 *: Ecab sim Ornithine decarboxylase (ODC) (LOC100072405), mRNA ODC1 83 99

GW820069 XM_001504903.2 *: Ecab sim ribosomal protein L27a-like (LOC100071004), mRNA RPL27A 62 100

GW820070 NM_001163885.1 Ecab fatty acid binding protein 3, muscle and heart (mammary-derived growth inhibitor) (FABP3), mRNA FABP3 50 99

GW820071 XM_001502665.2

*: Ecab sim 60 kDa heat shock protein, mitochondrial precursor (Heat shock protein 60) (HSP-60) (Hsp60) (60 kDa chaperonin) (Chaperonin 60) (CPN60) (Mitochondrial matrix protein P1) (P60 lymphocyte protein) (HuCHA60) (LOC100055147), mRNA HSPD1 52 98

GW820072 XM_001500153.2 *: Ecab septin 7 (SEPT7), mRNA SEPT7 43 98

GW820073 XM_001490339.1 *: Ecab sim zinc ribbon domain containing 1 (LOC100056703), mRNA ZNRD1 6 97

GW820074 XM_001503115.2 *: Ecab sim ribosomal protein S24 (LOC100073005 RPS24 77 100

GW820075 XM_001491121.2 *: Ecab sim Isoleucyl-tRNA synthetase, cytoplasmic (Isoleucine--tRNA ligase) (IleRS) (IRS) (LOC100054721), mRNA IARS 74 99

GW820076 XM_001503108.2 *: Ecab sim 14-3-3 protein beta/alpha (Protein kinase C inhibitor protein 1) (KCIP-1) (Protein 1054) (LOC100056084), mRNA YWHAB 88 99

GW820077 XM_001492385.2 *: Ecab sim COX6B protein (LOC100051249), mRNA COX6B 42 99

GW820078 XM_001499935.1

*: Ecab sim ATP synthase lipid-binding protein, mitochondrial precursor (ATP synthase proteolipid P3) (ATPase protein 9) (ATPase subunit c) (LOC100053347), mRNA ATP5G3 89 99

GW820079 XM_001501901.2 *: Ecab sim Fructose-bisphosphate aldolase A (Muscle-type aldolase) (Lung cancer antigen NY-LU-1) (LOC100066121), mRNA ALDOA 84 100

GW820080 NM_001163955.1 Ecab heat shock protein 90kDa alpha (cytosolic), class A member 1 (HSP90AA1), mRNA HSP90AA1 38 99

GW820081 XM_001915319.1 *: Ecab laminin, beta 1 (LAMB1), mRNA LAMB1 29 100

GW820082 XM_001491034.2 *: Ecab sim proteasome (prosome, macropain) subunit, alpha type 7 (LOC100057828), mRNA PSMA7 40 98

GW820083 XM_001494265.1 *: Ecab sim Malate dehydrogenase, cytoplasmic (Cytosolic malate dehydrogenase) (LOC100051612), mRNA MDH1 61 99

GW820084 XM_001493077.2 *: Ecab sim Protein tweety homolog 2 (hTTY2) (LOC100060986), mRNA TTYH2 68 100

GW820085 XM_001496363.2 *: Ecab sim ribosomal protein S6 (LOC100052368), mRNA RPS6 59 99

GW820086 XM_001498163.2 *: Ecab sim ribosomal protein L9 (LOC100055158 RPL9 83 99

GW820087 XM_001917847.1 *: Ecab sim Mps One Binder kinase activator MOBKL3 51 100

GW820088 XM_001498694.1 *: Ecab sim FGF-binding protein (LOC100068876), mRNA FGFBP1 64 89

GW820089 XM_001504562.1 *: Ecab heterogeneous nuclear ribonucleoprotein HNRNPA1 84 98

GW820090 XM_001501228.1 *: Ecab hypothetical LOC100066131 (LOC100066131 65 99

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Figure 1 Functions genes SSH.The genes upregulated in in vivo equine blastocysts resulting from

SSH analysis were divided into functional classes by means of Ingenuity Pathway Analysis

software. The 13 most significant functions (p<0.003) are listed in this figure. The legend of the

X axis is –log (p-value). This p-value is calculated through Ingenuity Pathway analysis by means

of a right tailed Fisher’s Exact test and determines the probability that the association between

the genes in the dataset and the function is explained by chance alone.

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A

B

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C

D

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Figure 2 Network classification according to Ingenuity Pathway Analysis.The genes resulting

from the SSH were mutually linked in several networks as determined by Ingenuity Pathway

Analysis (http://www.ingenuity.com/products/pathways_analysis.html), namely A: energy

production, nucleic acid metabolism, small molecule biochemistry B: organ development, gene

expression, cancer C: cardiovascular system development and function, hair and skin

development and function, organ development and D: organismal development, cell death,

nervous system development and function. The genes resulting from the SSH are colored grey,

the other genes involved in the networks are white. The genes selected for RT-qPCR are

indicated with a red circle. Solid lines imply direct relationships between proteins; dotted lines

imply indirect interactions.

5.4.3 RT-qPCR

Of the 62 genes identified, 6 which were previously shown to be involved in embryo

development in other species were selected for further evaluation by RT-qPCR. For 5 (brain

expressed X-linked 2 (BEX2), fatty acid binding protein 3 (FABP3), heat shock protein 90kDa

alpha, class A member 1 (HSP90AA1), mps one binder kinase activator-like 3 (MOBKL3), and

ornithine decarboxylase (ODC)) of the 6 genes that were selected from the SSH, the higher

expression in the in vivo derived embryos could be confirmed with RT-qPCR. Both FABP3 and

HSP90AA1 were significantly more expressed in the in vivo derived embryos compared to the in

vitro produced embryos (p<0.05). For ODC, MOBKL3 and BEX2 this difference was highly

significant (p<0.005). For the sixth gene (minichromosome maintenance complex component 7

(MCM7) the mean expression was higher in the in vivo population than in the in vitro group, but

this was not statistically significant. The results are summarized in Figure 3.

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Figure 3 Differential gene expression in vivo versus in vitro as determined by RT-qPCR.This

figure presents the mean expression of 6 genes as determined by RT-qPCR in in vivo derived and

in vitro produced equine blastocysts. The standard errors are indicated by bars. For BEX2,

FABP3, HSP90AA1, MOBKL3 and ODC the mean expression was significantly higher in the in vivo

derived embryos. *significant (p<0.05) **higly significant (p<0.005)

5.5 DISCUSSION

Until recently the equine zygote and cleaving embryo were relatively inaccessible as a research

specimen without using surgery or slaughter (Betteridge, 2007), but since the establishment of

ICSI in the horse, it is possible to produce horse embryos outside the genital tract. As such, IVP

of equine embryos represents a valuable tool for research as well as for clinical purposes.

However, IVP embryos show marked differences when compared to embryos derived in vivo.

Research in other species revealed the influence of different steps in the IVP process on the

embryonic expression of developmentally important genes. Not only the fertilization through

IVF (Giritharan et al., 2007) or ICSI (Fernández-González et al., 2008), but also the IVC conditions

(Niemann and Wrenzycki, 2000; Rizos et al., 2002, Goossens et al., 2007) affect gene expression

levels. Even simple modifications of the embryo culture media, like the presence or absence of

**

**

**

**

0

0,5

1

1,5

2

2,5

BEX2 FABP3 HSP90AA1 MCM7 MOBKL3 ODC

Mean in vivo

Mean in vitro

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serum, can profoundly influence the gene expression, which is reflected in the developmental

competence and the quality of the IVP embryos and even in long-term consequences (McElroy

et al., 2008; Fernández-González et al., 2009). Therefore the expression of specific genes has

been monitored to evaluate how different fertilization techniques and culture media can

influence embryonic differentiation.

In horses very little is known about gene expression in early embryos, with just a few recent

papers focusing on this topic (Rambags et al., 2008; Choi et al., 2009; Smits et al., 2009).

Comparative analysis of embryonic gene expression in the presence or absence of the maternal

genital tract is however a very valuable technique to get more insight into embryo-maternal

interaction in the horse and as such it can provide the basis for the optimisation of culture

conditions for horse embryos. Since the expression of specific genes can be stage specific, care

was taken to use similar developmental stages in the in vivo and the in vitro group. For the

reasons stated earlier, suppression subtractive hybridization (SSH) was used for the detection

of differentially expressed genes. In this study the genes expressed at a higher level in in vivo

derived equine blastocysts (tester) when compared to IVP equine blastocysts (driver) were

selectively amplified. This comparison, which has been shown to be valuable in other species,

has not yet been performed in the horse.

Ingenuity Pathway Analysis revealed the networks and functional categories in which these 62

differentially expressed genes are involved (Figure 1, Figure 2). Remarkable and on the other

hand logical is their implication in embryonic development, which shows clearly from the

different networks. Furthermore both the networks and the function categories illustrate the

involvement of these genes in energy production, nucleic acid metabolism and small molecule

biochemistry. The most important functional role appeared to be protein synthesis. This is in

agreement with the upregulation of protein synthesis genes in bovine in vivo blastocysts when

compared to in vitro blastocysts as determined by SSH (Mohan et al., 2004). In another

comparison of bovine in vivo versus in vitro blastocysts using microarrays the authors suggested

that phenotypic differences between both types of embryos might be associated with

inefficient transcription and translation in IVP embryos when compared to in vivo derived

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embryos (Corcoran et al., 2006). The differentially expressed genes involved in protein

synthesis contained several ribosomal proteins. Knockdown studies in zebrafish and allelic

inactivation in mice illustrated the importance of ribosomal proteins in embryonic development

(Panić et al., 2006; Chakraborty et al., 2009).

The occurrence of false positive results in SSH analysis, as in microarray analysis, requires

confirmation of the differential expression by a very sensitive and specific method, RT-qPCR. Six

genes were selected from the SSH results based upon evidence of their involvement in

embryonic development in other species. For 5 of these genes the higher expression in in vivo

derived equine blastocysts when compared to IVP equine blastocysts was proven by RT-qPCR to

be significant (FABP3 and HSP90AA1) or highly significant (ODC, MOBKL3 and BEX2), confirming

the results of the SSH. Subsequently the biological relevance of the RT-qPCR results will be

discussed for ODC, HSP90AA1, BEX2, MOBKL3, FABP3 and MCM7.

Ornithine decarboxylase (ODC) is an enzyme which decarboxylates L-ornithine to putrescine,

the first step in the biosynthetic pathway of polyamines, which are regulators of cell growth

and differentiation. Polyamines stimulate DNA repair and they prevent DNA damage by

stabilizing the chromatin structure and by acting as antioxidant as they scavenge reactive

oxygen species (ROS) (Pendeville et al., 2001). Several studies illustrate the importance of

polyamines, proline, a major substrate for the polyamine synthesis, and ODC, the key

regulatory enzyme in this synthesis, throughout embryonic and fetal development in different

species (Wu et al., 2008; Gao et al., 2009; Lopez-Garcia et al., 2009). The essential role of ODC

in embryonic development has been demonstrated by the lethality of Odc-deficient murine

embryos, caused by a deficient expansion of the inner cell mass (ICM) of these embryos, which

was found to be mediated through increased apoptosis in the ICM (Pendeville et al., 2001). The

lower expression of ODC in equine in vitro blastocysts could be a possible factor influencing the

indistinct separation between the ICM and the trophectoderm as observed in in vitro produced

horse blastocysts. A possible way to improve embryonic expression of ODC was investigated in

a study of pig parthenotes: by means of addition of exogenous polyamines to the embryo

culture medium, the mRNA expression of ODC could be enhanced and this improved porcine

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embryonic development to the blastocyst stage, with increased cell counts and decreased

apoptosis (Cui and Kim 2005). Although the exact molecular mechanisms are not clear, the

authors state that the induced transcription of ODC through the exogenous polyamines might

regulate cell cycle and/or apoptosis related gene expression in the embryos, resulting in

enhanced embryo viability. This anti-apoptotic effect of ODC was also found during oocyte

maturation and appeared to be mediated through the suppression of ROS since an increase of

ROS was demonstrated in ODC-deficient oocytes (Zhou et al., 2009).

Another group of proteins exhibiting an anti-apoptotic role during embryonic development are

the heat shock proteins (HSP) (Esfandiari et al., 2007). Under physiological circumstances these

molecular chaperones exhibit several of the functions indicated in Figure 1, like protein folding.

Increased synthesis is induced in response to stressful conditions, including rapid cell growth

and differentiation, toxicity and inflammation. Their important role in embryonic development

starts at the early onset of zygotic genome activity (Bensaude et al., 1983; Christians et al.,

1995). The supplementation of mouse in vitro culture media with mononoclonal antibodies to

HSP60, HSP70 or HSP90 has been shown to result in impaired embryonic development with

reduced blastocyst formation and higher rates of apoptosis (Neuer et al., 1998; Esfandiari et al.,

2007). This anti-apoptotic effect of HSPs is valuable for in vitro embryo production systems

since suboptimal culture conditions can induce a higher degree of apoptosis when compared to

conditions prevailing in the maternal genital tract. Therefore Esfandiari et al., (2007) suggest

that overexpression of HSP might be beneficial for embryonic development. A similar

conclusion was drawn in cattle in vivo embryos in which a positive correlation was found

between HSP70 expression and embryo quality (Pretheeban et al., 2009), which is in agreement

with our results. In the SSH several HSPs, namely HSP60, HSP90AA1 and HSPH1, were expressed

at a higher level in the in vivo derived equine blastocysts when compared to the IVP blastocysts.

For HSP90AA1 this difference was further evaluated by RT-qPCR and confirmed as significant.

However, it must be noted that some authors interpret a higher HSP expression as a negative

sign illustrating increased stress for the embryo (Chin et al., 2009).

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One more group of genes which is intriguing with regard to embryonic development is the BEX

family. It consists of 4 to 7 genes, according to the species, with BEX1 and BEX2 highly diverged

from the others (Zhang, 2008). BEX1, BEX2 and BEX3 have been discovered in mice in an

experiment where parthenogenetic and normal blastocysts were compared to identify new

imprinted genes. The expression of BEX1 appeared to be upregulated in parthenogenetic

blastocysts (Brown and Kay, 1999). However, Williams et al., (2002) found indications that this

elevated expression did not result from genomic imprinting of the BEX1 gene since no

difference in expression was observed between androgenotes (two paternal genomes),

gynogenotes (two maternal genomes) and control embryos. On the other hand the BEX1

expression seemed to be dependent on the developmental stage and the cell type with an

increased expression at the expanding blastocysts stage, which was moreover trophectoderm

specific. Therefore a more likely explanation for the higher expression in the parthenogenotes

as observed by Brown and Kay (1999) could be the combination of the effects of the

trophectoderm specific expression of BEX1 and the differences in timing of trophectoderm

differentiation between the different classes of embryos. In our study the expression of BEX2

was found to be higher in equine in vivo blastocysts than in IVP blastocysts. Possibly this might

be explainable in a similar way since the differentiation of the equine trophectoderm, as

indicated by loss of expression of the pluripotency marker POU5F1, has been shown to be

impaired in IVP equine blastocysts (Choi et al., 2009). Recent studies however do support X-

linked imprinting, as they describe a predominant expression in female embryos of BEX1 in

mice (Kobayashi et al., 2010) and BEX1 and BEX2 in cattle (Bermejo-Alvarez et al., 2010).

Bermejo-Alvarez et al. (2010) found a higher expression of BEX1 and BEX2 in female bovine

blastocysts produced in vitro when compared to their male and parthenogenetic counterparts,

suggesting preferential expression of the paternal allele. Even though little is known about X-

chromosome inactivation during early embryonic development, several studies illustrate the

importance of genes involved in imprinting for the long term phenotype as well as a substantial

influence of the in vivo or in vitro environmental conditions on the expression of these genes

(Young et al., 2001; Fernàndez-Gonzàlez et al., 2009). Further research is needed to elucidate

the role and the mode of action of the BEX genes in horse embryos.

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Another gene expressed at a significantly higher level in the in vivo derived equine blastocysts is

FABP3. The role of fatty acids in embryonic development is controversial. Compared to mice

and human, oocytes and embryos of domestic animals contain high amounts of lipids. A

hypothesis to explain this is that the lipids function as endogenous energy reserve during the

time that the embryos remain unattached in the uterus, which is relatively long when

compared to the early attachment in human and mice, which occurs immediately after hatching

(Sturmey et al., 2009). Previous research indicates the ability of oocytes and early embryos to

use fatty acids as energy substrates (Sturmey et al., 2009). Several studies illustrate the

essential role of FABP during embryonic development (Gentili et al., 2004; Arai et al., 2005).

However, abundance of fatty acids in the embryonic environment negatively influences the

development. Supplementation of hyperlipidaemic sera to bovine embryo culture medium

resulted in a reduction of blastocyst development (Leroy et al., 2010).

Mps One Binder kinase activator-like 3 (MOBKL3, MOB1) is also known as preimplantation

protein 3 because of its expression during oocyte maturation and preimplantation embryo

development following embryonic genome activation. Research in mice revealed a relatively

high expression through the one cell stage, a decline at the two cell stage and a subsequent rise

at either the eight cell or blastocyst stage, indicating a role in preimplantation embryogenesis

(Temeles et al., 1994). In pigs the polymorphism of this gene was associated with litter size

traits and suggested to be useful for marker assisted selection (Niu et al., 2006). In this study

this preimplantation protein was expressed at a higher level in the in vivo derived equine

blastocysts when compared to the IVP blastocysts. Information about this gene in horses is

absent, but an important role in the coordination of mitotic exit and cytokinesis has been found

in yeast and mammalian homologues appear to play similar roles (Hergovich et al., 2008;

Wilmeth et al., 2010).

Another gene involved in the guidance of the accurate execution of cell division is MCM7. For

normal embryonic development it is important that complete duplication of the genome occurs

exactly once per cell cycle (Blow and Laskey, 1988). This crucial event is ensured through DNA

licensing by the assembly of a prereplication complex to the replication origins (Donaldson and

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Blow, 1999). As a part of this complex the minichromosome maintenance (MCM) 2-7 helicase

plays a central role in DNA replication initiation and elongation (Labib et al., 2000). The

expression of MCM genes can serve as a sensitive marker for proliferation zones during

embryogenesis (Ryu and Driever, 2006). A member of this MCM family, MCM7, which is

involved in the oogenesis and the first embryonic cell cycle in mice (Sweich et al., 2007), was

indicated by the SSH to be preferentially expressed in in vivo equine embryos when compared

to their in vitro counterparts. However, evaluation of this differential expression by RT-qPCR

revealed it to be not significant.

In conclusion 62 genes which were expressed at a higher level in in vivo derived equine

blastocysts when compared to in vitro produced equine blastocysts were identified by means of

SSH. Ingenuity Pathway analysis revealed the functional categories and networks in which these

genes are involved and indicated an important role in protein synthesis. For 5 of these SSH

sequences, namely ODC, HSP90AA1, BEX2, MOBKL3 and FABP3, the higher expression in vivo

was confirmed by RT-qPCR. The evaluation of embryos at the genetic level has appeared to be

valuable in other species. This study contains the first step of a similar approach in the horse,

which may enable progress in the field of assisted reproductive techniques. Further research

will be focused on environmental influences on the embryonic gene expression, finally aiming

to improve knowledge and valuable clinical applications for the subfertile horse.

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CHAPTER 6

INFLUENCE OF THE UTERINE ENVIRONMENT ON THE

DEVELOPMENT OF IN VITRO PRODUCED EQUINE EMBRYOS

Smits K., Govaere J., Peelman L.J., Goossens K., de Graaf D.C., Vercauteren D., Vandaele L.,

Hoogewijs M., Stout T., Van Soom A. Adapted from Reproduction submitted.

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6.1 ABSTRACT

After an unusually long stay in the oviduct, the equine embryo passes through the utero-tubal

papilla on day 6 after ovulation. Soon after its arrival in the uterus, the embryo becomes

enveloped by a glycoprotein tertiary coat (the ‘capsule’); during the first 5 weeks of intrauterine

development the conceptus is entirely dependent on endometrial secretions for its nutrition.

The necessity for early interaction between the embryo and the oviductal and/or uterine

environment in the horse is reflected by several striking differences between equine embryos

that develop in vivo and those produced in vitro. Better understanding of the salient

interactions may help to improve the efficiency of in vitro equine embryo production. In an

initial experiment, cleavage-stage in vitro produced (IVP) equine embryos were transferred into

the uterus of recently ovulated recipient mares to determine whether premature placement in

this in vivo environment would improve subsequent development. In a second experiment, an

important element of the uterine environment was mimicked by adding uterocalin, a major

component of the endometrial secretions during early pregnancy, to the culture medium. Intra-

uterine transfer of cleavage-stage equine IVP embryos yielded neither ultrasonographically

detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not a suitable

environment for per-compact morula stage horse embryos. By contrast, exposure to uterocalin

during IVP improved capsule formation, although it did not measurably affect development or

expression of a panel of genes known to differ between in vivo and in vitro embryos.

Nevertheless, the positive effect of uterocalin on capsule formation in IVP horse blastocysts

illustrates that adding a specific endometrial protein to embryo culture medium can help

embryos develop more physiologically; further studies are required to evaluate whether

uterocalin serves purely as a carrier protein or more directly promotes improved capsule

development.

6.2 INTRODUCTION

Early embryonic development in the horse is characterized by a number of peculiarities

(Betteridge, 2007). Firstly, the equine embryo doesn’t enter the uterus via the prominent utero-

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tubal papilla until as late as 144-156 h after ovulation (Battut et al., 1997). Moreover,

unfertilized eggs are not capable of stimulating passage through the ampullary-isthmic junction

and are instead retained in the oviduct. This selective oviductal transport has been shown to be

a stage specific function of the production of prostaglandin E2 by day 4-5 equine conceptuses

(Weber et al., 1991), and is a clear example of very early embryo-maternal interaction in the

horse.

Another enigmatic feature exemplifying early embryo-maternal interaction in the horse is the

formation of an acellular glycoprotein tertiary embryo coat (‘capsule’: Flood et al., 1982) very

soon after the arrival of the horse embryo in the uterus; the capsule completely envelopes the

equine conceptus until around day 21 of gestation (Betteridge, 1982). Although the precise

functions of the capsule are not clear, it has been proposed to function as a ‘mailbox’,

incorporating endometrial components such as signalling molecules and nutrients and

transporting them to the embryo (Herrler & Beier, 2000), and to physically protect the mobile

embryo and maintain its spherical shape while migrates around the uterus to signal its presence

to its dam and prevent luteolysis (Ginther, 1985; Allen and Stewart, 2001; Stout and Allen,

2001). In addition, structural changes in the capsule are thought to be instrumental to the

process of fixation and orientation of the conceptus within the mares’ uterus (Oriol, 1994).

Since a functional chorioallantoic placenta is not formed until as late as days 40-45 of gestation,

equids have the longest pre-implantation period of all mammals studied to date (Allen and

Stewart, 2001); moreover, during this prolonged pre-implantation period the embryo is entirely

dependent on endometrial secretions (‘histotrophe’) for its nutrition. An undoubtedly

important component of this histotrophe is the 19 kDa progesterone-dependent protein,

uterocalin, that is secreted by the endometrial glands during both dioestrus and early

pregnancy (Crossett et al., 1996). The marked drop in uterocalin production that coincides with

the disappearance of the capsule at around day 21, and the fact that uterocalin is one of the

most abundant proteins in the capsule (Quinn et al., 2007), suggests that there may be a

functional correlation between uterocalin production and the presence of the capsule (Crossett

et al., 1996). Moreover, detection of uterocalin in the trophoblast and yolk-sac fluid of equine

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conceptuses implies passage through the capsule and absorption by the conceptus proper. On

the basis of its structure, uterocalin has been classified as a member of the lipocalin family,

which contains several transport proteins that bind small hydrophobic molecules (Crosset et al.,

1996). Moreover, in depth structural analysis of uterocalin, suggests a putative function as a

carrier of essential lipids and amino acids for the developing conceptus (Kennedy, 2004).

All of the above illustrate the importance of embryo-maternal interaction during early

embryonic development in the horse. Furthermore, when equine embryos are produced in

vitro, and therefore in the absence of the maternal tract, they differ markedly from their in vivo

counterparts in terms of the kinetics of development, incidence of apoptotic cells, inner cell

mass morphology and gene expression patterns (McKinnon, 1989; Hinrichs, 1990; Tremoleda et

al., 2003; Pomar et al., 2005; Smits et al., 2010a). One of the more striking irregularities of IVP

horse embryos is the failure of normal capsule formation (Tremoleda et al., 2003); even though

capsular mucin-like glycoproteins are produced in vitro, they fail to coalesce into the distinct

continuous capsule observed around in vivo equine embryos from the early blastocyst stage.

The reason(s) for the failure of capsular glycoprotein coalescence in vitro are not known but

may involve simple dispersion of the glycoproteins into the culture medium, preventing

attainment of the critical concentration required for capsule assembly, or failure of hydration

and cross-linking of the capsular glycoproteins in the absence of a specific uterine

component(s) (McKinnon, 1989; Hinrichs, 1990; Tremoleda et al., 2003). In either case, the

presence of the mare’s uterus appears to be essential to the process of capsule formation; to

confirm that the uterine environment and/or specific uterine components are central to

capsule formation, we exposed IVP embryos to either the complete uterine environment or to

the endometrial protein, uterocalin, which is known to contribute substantially to the

substance of the capsule of day 10-18 blastocysts. A recent study demonstrated that temporary

transfer of IVP horse blastocysts to the mare’s uterus for 2-3 days had a positive effect on

capsule formation, as assessed by light microscopy (Choi et al., 2009). In this study, we wanted

to further determine whether ‘premature’ transfer of day 2-3 embryos to the uterus could not

only enhance capsule formation but also improve equine blastocyst development rates and

quality, as compared to culture in vitro. In this latter respect, while it is common practice to

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culture embryos to the blastocyst stage prior to intra-uterine transfer in most domestic species,

in human medicine premature intra-uterine transfer of day 2 and 3 embryos is a routine

procedure that yields good results (Younis et al., 2009) and circumvents the potential

downsides of prolonged in vitro culture, or the difficulty of transferring early embryos to the

oviduct. If transfer of cleavage stage IVP embryos to the uterus of the mare was successful, it

would considerably simplify the IVP process even if it didn’t have additional beneficial effects

on embryonic development and capsule formation. To more specifically investigate the putative

role of uterocalin in capsule formation and early development of equine embryos, recombinant

uterocalin was added to culture medium for 5-10 days, and the effect on subsequent

development was examined in terms of capsule formation and expression of a panel of genes

known to be differentially expressed by in vivo versus IVP horse embryos.

6.3 MATERIALS AND METHODS

6.3.1 Experiment 1: Intra-uterine transfer of cleavage stage equine in vitro embryos

All animal procedures were approved by the ethics committee of the Faculty of Veterinary

Medicine at Ghent University. In vitro embryos were produced as previously described by Smits

et al. (2010b). Briefly, slaughterhouse oocytes were matured in vitro for 24h in a DMEM-F12

based medium in an atmosphere containing 5% CO2 (Galli et al., 2007). MII oocytes were

fertilized by conventional ICSI and cultured in vitro in DMEM-F12 with 10% fetal calf serum at

38.5°C in 5% CO2, 5% O2 and 90% N2. On day 2-3 after ICSI, cleaved embryos were transferred

by means of a transcervical pipet (IMV Technologies, France) to the uterus of a recipient mare

that had ovulated 2-3 days previously. A total of 99 cleaved embryos were transferred to the

uterus of 12 synchronized mares (average of 8.25 embryos per mare). On the day of transfer,

the recipient mare was injected intravenously with 1.1 mg/kg of the non-steroidal anti-

inflammatory agent, flunixine meglumine (Emdofluxin, Emdoka, Belgium) and daily per os

treatment with 0.044mg/kg of the synthetic progestagen, altrenogest (Regumate, Intervet,

The Netherlands) was initiated and continued until evaluation for embryo development. Half of

the mares were examined by per rectum uterine ultrasound 14 days after ICSI, and half were

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subjected to embryo recovery by transcervical uterine lavage on day 7 after ICSI. Briefly,

flushing was performed with 6 liters of Lactated Ringer’s solution using a Bivona-catheter

(Minitüb, Germany) and the recovered fluid was passed through an EZ filter (Bioniche, Ireland).

Any embryos recovered were stained with Hoechst 33342 (Molecular Probes, The Netherlands)

to assess cell viability and number.

6.3.2 Experiment 2: IVP in the presence of recombinant uterocalin

Production of recombinant uterocalin

A recombinant uterocalin clone and an anti-uterocalin-antibody were kindly provided by

Professor MW Kennedy (University of Glasgow, UK). The recombinant uterocalin was purified

mainly as described by Suire et al. (2001) using the ProfinityTM IMAC Ni-Charged Resin (Bio-

Rad), to produce a working concentration of 7.88 mg/ml.

Estimation of the physiological concentration of uterocalin

Since no absolute concentrations of uterocalin in the uterine environment are reported in the

literature, the physiological concentration of uterocalin was estimated using a uterine secretion

sample recovered from a day 7 pregnant mare. Sampling of uterine secretions was performed

by means of aspiration through a pipette for deep intra-uterine insemination as described by

Velazquez et al. (2010), while subsequent uterine lavage resulted in the recovery of an embryo,

thereby confirming that the mare was pregnant. A dot blot technique was used to compare a

dilution series of recombinant uterocalin with the recovered uterine secretion and indicated

that the concentration of uterocalin in the uterine secretions was approximately 4 mg/ml.

In vitro production of equine blastocysts

In vitro embryos were produced as described for experiment 1, except that only oocytes with a

compact cumulus complex were used, the maturation time was 28h and ICSI was performed

using a Piezo Drill (Prime Tech Ltd., Ibaraki, Japan). The embryos were cultured in groups of 10-

20 in 20 µl droplets of DMEM-F12 with 10% fetal calf serum at 38.5°C in 5% CO2, 5% O2 and 90%

N2. On day 2.5, half of the medium was refreshed and the embryos that had not cleaved were

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removed. On day 6, half of the medium was refreshed again and, in half of the culture droplets,

2.54 µl of the medium was replaced by the recombinant uterocalin solution, resulting in a final

concentration of 1 mg/ml recombinant uterocalin. On day 9-9.5, the embryos that had reached

the blastocyst stage were recovered for further analysis.

Immunofluorescent staining of the capsule

Immunofluorescent staining of the equine capsule was performed as described by Tremoleda et

al. (2003) using the monoclonal anti-capsule antibody OC-1 (Oriol et al., 1993), which was kindly

provided by Professor KJ Betteridge (University of Guelph, Canada). Day 9.5 blastocysts were

fixed in 4% paraformaldehyde (P6118, Sigma-Aldrich, Bornem, Belgium) and stored at 4°C until

analysis. Twelve blastocysts that had been cultured with uterocalin and 14 blastocysts from the

control group were stained simultaneously. After permeabilisation by exposure to 0.5% (v/v)

Triton X-100 for 30 minutes at room temperature, the blastocysts were washed 3 times in PBS

containing 1 mg/ml PVP. Non-specific staining was blocked by incubation in 10% (v/v) goat

serum (16210-064, Invitrogen, Merelbeke, Belgium) for 30 minutes at 37°C. The blastocysts

were then washed again and incubated with the primary antibody (mouse monoclonal anti-

capsule OC-1: 1/200 dilution) for 1.5h at 37°C. In both groups, a negative control blastocyst was

incubated in 10% goat serum without primary antibody. After a washing step, incubation with

the secondary antibody (goat-anti-mouse FITC: Molecular Probes, Leiden, The Netherlands),

1/100 dilution) was performed for 1h at 37°C, followed by another washing step. Nuclei were

then stained by incubation with 2% propidium iodide (Molecular Probes, Leiden, The

Netherlands) for 30 minutes at room temperature, after which the embryos were fixed in

Dabco (Acros, Ghent, Belgium) on siliconized glass and enclosed under a cover slip supported by

small vaseline bridges to prevent crushing of the embryos. All embryos were evaluated in one

session using a Nikon C1 confocal laser scanning module attached to a motorized Nikon

TE2000-E inverted microscope (Nikon Benelux, Brussels, Belgium) and identical settings.

Subsequent fluorescence measurements were performed using Nikon EC-V1 FreeViewer

software. Since some blastocysts were a little squeezed by the cover slip, intact capsules of 6

uterocalin blastocysts and 8 control blastocysts in similar condition were evaluated. For each

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embryo, the total fluorescence of 3 areas in different, randomly selected, spots of the capsule

were measured and the mean of these 3 measurements was recorded (Figure 1).

Figure 1 Measurement of the capsule fluorescence (Equus caballus). For each blastocyst

capsular fluorescence was measured in 3 areas of at least 10 µm2 as represented in the figure.

Random places on different sides of the blastocyst showing an intact capsule were assessed. For

each embryo the mean of these 3 measurements was calculated.

RT-qPCR

For both the uterocalin and the control group, 11 blastocysts were selected on day 9. After

washing in DPBS, individual blastocysts were transferred to cryotubes with 2 µl lysis buffer,

frozen in liquid nitrogen for 3 minutes and stored at -80°C. RNA-extraction was performed using

the RNeasy Micro Kit (Qiagen, Venlo, The Netherlands) and, after RT minus control, the RNA

was converted into cDNA using the iScriptTMcDNA synthesis Kit (Bio-Rad, Nazareth Eke,

Belgium). The expression of the 5 development ‘marker’ genes (BEX2, FABP3, HSP90AA1,

MOBKL3 and ODC) was quantified by RT-qPCR as described by Smits et al. (2010a).

Normalization of data was performed using UBC, ACTB, RPL32 and GAPDH as reference genes

(Smits et al., 2009).

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Statistical analysis

The blastocyst development rates for the uterocalin and control groups were compared using a

Pearson Chi-square test (SPSS 16.0, SPSS Inc., Headquarters, Chicago, Illinois, US). Cell number

and capsular fluorescence were compared between the groups using t-tests, while gene

expression between the uterocalin and control groups was compared with a Mann-Whitney

test using GraphPadInStat3. A p-value <0.05 was considered statistically significant.

6.4 RESULTS

6.4.1 Experiment 1: Intra-uterine transfer of cleavage stage IVP embryos

A total of 99 cleaved (2-8 cell stage) embryos were transferred to the uterus of 12 synchronized

(day 2-3 after ovulation) mares (average of 8.25 embryos / mare). Six of these mares were

subsequently examined for pregnancy by transrectal ultrasound on day 14 after ovulation, but

no conceptus vesicles were detected. The uterus of the remaining six mares was flushed on day

7 after ovulation. Disappointingly, embryos were recovered from only 3 of the 6 mares and no

mare yielded more than a single embryo (overall recovery rate 6%). Moreover, none of the 3

recovered embryos had developed to the blastocyst stage; instead all 3 were clearly degenerate

(Figure 2).

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Figure 2 Intra-uterine transfer of cleavage stage embryos (Equus caballus). After intra-uterine

transfer of day 2-3 equine cleavage stage embryos and subsequent flushing on day 7 only

degenerated embryos were recovered. The embryos were stained with Hoechst 33342.

6.4.2 Experiment 2: Addition of recombinant uterocalin to embryo culture medium

No significant differences in overall development were observed between embryos that had or

had not been exposed to uterocalin during IVP (Table 1). In total, 60% of recovered oocytes

reached the MII stage and 76% of sperm injected oocytes had cleaved 48h after ICSI. In the

control group, 25 of 198 cleaved embryos developed to the blastocyst stage (12.6%); in the

group cultured with uterocalin, 22 of 165 cleaved embryos (13.3%) reached the blastocyst stage

(Figure 3 A, B). Mean cell counts and embryo diameters (± S.E.M.) were respectively 579 (± 40)

and 247 (±15) µm for the blastocysts cultured with uterocalin (n=11) and 551 (± 47) and 270

(±12) µm for the control group (n=11) (p>0.05).

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Table 1: Influence of uterocalin on development and capsule formation in equine in vitro

produced blastocysts (Equus caballus). Addition of uterocalin during IVC did not affect

blastocyst rate, cell count and embryonic diameter. Immunofluorescence of the embryonic

capsule was significantly increased in the presence of uterocalin. Mean values and their

respective standard errors are displayed.

Control Uterocalin p-value

Blastocyst percentage (%) 12.6 (±2.5) 13.3 (±3.5) 0.842

Mean cell count D9.5 551 (±47) 579 (±40) 0.660

Mean diameter (µm) 270 (±12) 247 (±15) 0.251

Fluorescence capsule 2123 (±117) 2745 (±208) 0.0312

By contrast, total fluorescence after immunofluorescent labeling of the embryos with the

capsule specific antibody OC-1 (Oriol et al., 1993) was significantly higher in blastocysts that had

been cultured in the presence of uterocalin (2745 ±208; n=6), than in those cultured in control

medium (2123 ±117; n=8) (p=0.0312) (Table 1). Penetration of capsular glycoproteins into the

transzonal channels was observed in both groups, and was more obvious in smaller blastocysts

(Figure 3 C, D). In the larger blastocysts, the capsular material appeared to form a more or less

continuous layer (Figure 3 E, F), although this did not extend over the part of the embryo that

had herniated through the hole in the zona created during ICSI; instead the glycoprotein over

the protruding trophectoderm was visible in patches in both groups (Figure 4). Interestingly, in

the uterocalin group, an apparently continuous area of capsule associated with the

trophectoderm was observed in an area where the zona had become loosened locally around

one blastocyst and in a zona-free area of another blastocyst (Figure 5); similar findings were not

observed in control embryos.

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Figure 3 Day 9.5 blastocysts produced in vitro and cultured in the absence (A,C,E) or presence

(B,D,F) of uterocalin (Equus caballus). A,B: Equine blastocysts before fixation. Small blastocysts

display a thin capsular line (OC1-staining) and penetration of capsular material in the transzonal

channels in both the embryos cultured in the absence (C) or presence (D) of uterocalin. Larger

blastocysts present a more developed and continuous capsule in both the control (E) and the

uterocalin (F) group, but still show penetration of the glycoproteins in the thinned zona. White

scale bar = 10 µm.

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Figure 4 Hatching blastocyst produced in vitro (Equus caballus).This immunofluorescent

staining of the capsular OC-1 illustrates the capsular glycoproteins in the hatched part of an in

vitro produced blastocyst of the control group (A) and the uterocalin group (B). No confluent

capsule is apparent.

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Figure 5 Confluent capsule adjacent to trophectoderm (Equus caballus). This day 9.5 horse

blastocyst was cultured in the presence of uterocalin and capsule formation was assessed by

immunofluorescent OC1-staining. Interruption in the zona pellucida reveals a confluent capsule

which is attached to the trophectodermal surface. White scale bar = 10 µm.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a highly

specific and sensitive tool for comparing expression of mRNA for specific genes between

experimental groups. The 5 genes analysed, BEX2, FABP3, HSP90AA1, MOBKL3 and ODC were

chosen as markers for developmental quality, because a previous study indicated

downregulation of these genes in in vitro produced compared to in vivo derived equine

blastocysts (Smits et al., 2010a). It was hypothesized that adding the endometrial protein,

uterocalin, might induce an expression pattern in the in vitro embryos more closely resembling

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that of in vivo embryos. In fact, no differences in expression levels were found between the

blastocysts which were cultured with uterocalin (n=11) and the blastocysts from the control

group (n=11) (Figure 6). In this experiment, qPCR efficiencies of ≥ 99% and correlation efficients

≥ 0.992 were obtained, indicating that the results were reliable.

Figure 6 Expression of BEX2, FABP3, HSP90AA1, MOBKL3 and ODC in blastocysts cultured with

uterocalin and control blastocysts as determined by RT-qPCR (Equus caballus). The expression

of these 5 genes was previously found to be downregulated in in vitro produced equine

blastocysts when compared to in vivo derived equine blastocysts. However, this figure shows

that the addition of uterocalin, an important protein in the uterine secretions during early

pregnancy, to the in vitro culture medium from day 6 up to day 9 did not influence the

expression levels of these genes. The error bars represent the S.E.M.

6.5 DISCUSSION

The ability to efficiently produce equine embryos in vitro is of interest for both research

purposes and the clinical treatment of (equine) infertility. Presently, acceptable rates of embryo

production and levels of embryo quality can be obtained using intracytoplasmic sperm injection

of in vitro matured oocytes followed by culture in DMEM/F12 supplemented with serum;

however, there is still room for further optimisation of the in vitro culture process to improve

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

BEX2 HSP90AA1 ODC FABP3 MOBKL3

RT-qPCR

control

uterocalin

Chapter 6

123

the overall efficiency of the procedure (Galli et al., 2007; Hinrichs et al., 2007; Blanco et al.,

2009). Understanding the influence of the equine oviductal and uterine environments, and

specific components thereof, on early embryonic development could lead to targeted

adaptations of the in vitro environment to mimic aspects of the maternal environment

identified as beneficial.

In this study, intra-uterine transfer of cleavage stage IVP equine embryos did not yield any

pregnancies and, since no healthy blastocysts were recovered on day 7 (i.e. 4-5 days after

transfer), it appears that the transferred cleavage stage embryos do not develop in the uterine

environment. There are several possible explanations for this failure of development. Firstly, it

is possible that closure of the cervix 2-3 days after ovulation was suboptimal when compared to

the day 4-9 period during which commercial embryo transfers are usually performed. This could

result in loss of embryos through the cervix soon after transfer. On the other hand, pregnancy

after transfer of day 10 embryos to recipient mares on days 1 or 3 after ovulation has been

described by Wilsher et al. (2010) and, although none of these embryo’s subsequently

developed into normal pregnancies (none developed an embryo proper), it does illustrate that

the uterus should be capable of mechanically retaining embryos introduced soon after

ovulation. A second possible explanation is simple absence of intrinsic developmental potential

of the transferred in vitro embryos. However, using identical preliminary steps, standard in vitro

production yielded 5-10 % blastocysts in our hands, and transfer of one of these blastocysts has

resulted in pregnancy and the birth of a live foal (Smits et al., 2010b); in short, at least some (5-

10) of the cleaved embryos should have been capable of further development.

The remaining possible interpretation is that the mare’s uterus does not provide an adequate

or appropriate environment for these early cleavage stage embryos. A similar failure to

establish pregnancy following premature intrauterine transfer was reported for 292 day 2

mouse embryos (Goto et al., 1993), and for previous small studies that described the intra-

uterine transfer of early in vivo derived horse embryos. For example, Weber et al. (1993)

achieved no pregnancies following intra-uterine transfer of 7 day 2 horse embryos and while

Allen and Rowson (1975) did describe a single pregnancy after transfer of 5 day 3 equine

Chapter 6

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embryos, the age of the embryo was estimated from daily examination for ovulation by

transrectal palpation; the embryo that resulted in pregnancy could thus easily have been closer

to 4 days. Indeed, day 4 equine embryos have been reported to be sufficiently mature to

survive in the mare’s uterus (Peyrot et al., 1987).

In human medicine, cleavage stage embryos are routinely transferred to the uterus and, while

the procedure is considered to entail both specific advantages and disadvantages when

compared to blastocyst transfer, overall favourable pregnancy rates of 30-40% are common

(Bromer & Seli, 2008; Papanikolaou et al., 2008). The reason for the marked differences in the

success of intrauterine transfer of day 2-3 embryos in women and some primates compared

with other domestic species might be due to marked anatomical differences. In the mare,

there is a distinct, tightly closed uterotubal papilla, which presumably helps to maintain the

marked differences in fluid composition observed in different parts of the oviduct and uterus in

both horses and other domestic and laboratory species. This is in marked contrast to the lack of

an anatomically distinct utero-tubal junction in women, in which the oviductal and uterine

fluids appear to mix freely (Hunter, 1998).

During the initial intrauterine period, the equine embryo migrates continually, surrounded by a

protective glycoprotein capsule and bathed in nourishing endometrial secretions, which contain

several progesterone dependent proteins (Ellenberger et al., 2008). The addition of one of the

major progesterone dominated proteins, uterocalin, to embryo culture medium had a positive

effect on capsule formation around IVP blastocysts. Blastocysts cultured in the presence of

uterocalin showed more intense fluorescence following labelling with a capsule specific

antibody (OC-1) than control embryos. Furthermore, where the zona pellucida was locally

absent around blastocysts cultured in the presence of uterocalin, a distinct and confluent

capsule was found associated with the trophectoderm (Figure 5); such zona-independent areas

of continuous capsule were not seen on control embryos, and have not been described

previously for IVP equine embryos. Indeed, when Tremoleda et al. (2003) separated the zona

pellucida from a day 10 in vitro produced embryo, they found the capsular material to be stuck

to the zona instead of forming a separate layer between zona and trophectoderm. This

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apparent effect on capsular glycoprotein coalescence may reflect an additional function of

uterocalin and, together with previous studies illustrating that uterocalin makes a considerable

contribution to the total mass of the capsule, it suggests that uterocalin may be a maternally-

derived structural component of the capsule rather than just a transiently associated transport

molecule. Previous studies, have demonstrated that OC-1 reactive capsular glycoproteins are

secreted by trophectoderm cells (Albihn et al., 2003), while the failure of normal capsule

formation in vitro suggests the need for an additional maternal component (Tremoleda et al.,

2003). In this respect, the 19kDa endometrial protein uterocalin was first isolated as one of the

dominant proteins detected by SDS-PAGE of equine embryonic capsules (Stewart et al., 1995).

Subsequent studies confirmed the presence of uterocalin in the capsule in a temporal pattern

that suggested a functional correlation between the two (Crossett et al., 1996; Herrler & Beier,

2000; Quinn et al., 2007). Subsequently, and as a result of its molecular structure, uterocalin

has been proposed to function primarily as a carrier of biologically important lipids and a source

of essential amino acids for the developing conceptus (Suire et al., 2001, Kennedy, 2004),

where the positive charge of uterocalin (Crossett et al., 1996) is thought to facilitate its binding

to the negatively charged sialic acid residues of the capsule (Oriol et al., 1993). Since uterocalin

has also been found in trophoblast cells (Crossett et al., 1996; Ellenberger et al., 2008), some of

the molecule clearly passes through the capsule and presumably fulfils a role as a carrier

protein. In addition, it seems likely that uterocalin contributes to the structure of the capsule,

and plays a role in the initial aggregation and cross-linking of trophectoderm-produced OC-1

reactive glycoproteins. In summary, uterocalin appears to be instrumental in initial capsular

glycoprotein coalescence, contributes significantly to the substance and structure of the

capsule and plays an important role in transporting essential nutrients and or signalling

molecules to the developing conceptus during the initial intra-uterine period.

The dynamics of embryonic covering formation and loss and, in particular, the addition of tubal

and uterine secreted materials during development has been described in several species (for

Review see Denker, 2000). In this respect, the equine embryonic capsule has been proposed to

be most analogous to the neozona of the rabbit (Betteridge, 1989; Herrler & Beier, 2000). For

example, while trophoectodermal secretions are important for neozona formation, maternal

Chapter 6

126

components also appear to be critical (Fisher et al., 1991; Denker, 2000). Indeed, in vitro culture

of rabbit embryos is associated with the deposition of granular material on the inside of the

mucoprotein layer, but failure of normal neozona formation, aberrant herniation of embryonic

cells through the zona, and incomplete or failed dissolution of the zona pellucida (Fisher et al.,

1991). These features are reminiscent of the aberrations of capsule formation and hatching

observed in IVP equine embryos (Tremoleda et al., 2003; Stout et al., 2005). However, in both

species, these deviations are not necessarily irreversible and most can be corrected by

exposure to uterine components. In this respect, addition of uterine flushings to rabbit embryo

culture medium, or intra-uterine transfer of in vitro cultured embryos, has been shown to allow

reactivation and completion of zona dissolution, although uterine flushings alone did not induce

neozona formation in vitro (Fisher et al., 1991). In the horse, intra-uterine transfer of in vitro

produced blastocysts results in successful pregnancies (Galli et al., 2007; Hinrichs et al., 2007;

Smits et al., 2010b) with an apparently normal capsule (Choi et al., 2009), illustrating that the

degree of initial aberration in capsule formation during IVP is not so severe as to preclude

normal establishment of pregnancy, at least as long as exposure to the uterine environment is

sufficiently early to remedy the aberrations; by contrast, total removal of the blastocyst capsule

from day 6.5 embryos has been reported to be incompatible with embryonic survival after

transfer (Stout et al., 2005); clearly the disruption to capsule formation suffered during IVP is

not equivalent to removal.

In the current study, some aspects of aberrant equine embryonic capsule formation in vitro

appeared to be ameliorated by the presence of uterocalin. However, it is not known how the

uterocalin had this effect, and neither was uterocalin alone sufficient to completely normalize

capsule formation; e.g. the ‘hatched’ areas of trophectoderm still exhibited dispersed patches

of OC-1 reactive glycoproteins that did not coalesce into a confluent layer (Figure 4). In

addition, capsular material still penetrated into the transzonal channels as previously reported

for IVP embryos (Tremoleda et al., 2003) but not seen in in vivo embryos; the transzonal

penetration of capsular glycoproteins in this study was particularly evident for smaller embryos

(Figure 3). While it is possible that there was insufficient uterocalin provided in the current

study to completely normalize capsule production, it is more likely that this partial correction

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reflects the continued absence of other components of the complex intra-uterine environment

that are required for capsule formation.

Surprisingly, no clear influence of uterocalin on the development of equine IVP blastocysts was

observed, as illustrated by equal blatocyst rates, embryo diameters and cell counts between

control and uterocalin groups (Table 1). Moreover, exposure to uterocalin did not alter

expression of 5 genes (BEX2, FABP3, HSP90AA1, MOBKL3 and ODC: Figure 6), previously found

to be down-regulated in IVP embryos as compared to in vivo derived embryos (Smits et al.,

2010a). A similar failure of endometrial proteins to influence early equine embryonic

development was reported by Bøgh et al. (2002) who exposed day 8 in vivo derived embryos to

a p19 (i.e. uterocalin) homologue during a 3h incubation, and found no obvious influence on

subsequent embryonic growth and metabolism. The absence of effects on embryonic growth,

metabolism (Bøgh et al., 2002), gene expression or quality (this study) might be an artefact due

to measurement of factors not influenced by uterocalin. However, it is also possible that a

crucial ligand(s) necessary for uterocalin to influence gene expression or metabolism was

absent in the in vitro culture medium; this would seem logical if uterocalin is primarily a carrier

protein, as suggested by its ability to bind several essential small lipids (Suire et al., 2001),

rather than a stimulator of embryonic growth or development per se.

6.6 CONCLUSION

Several unusual features of early embryonic development in the horse illustrate the importance

of embryo-maternal interaction. To optimize equine IVP it may well be necessary to further

clarify, and develop methods to mimic, particular aspects of this interaction. In the current

study, an essential role of the oviductal environment was illustrated by the failure of premature

intrauterine transfer to yield any viable embryos. In addition, the need for the uterine

environment of slightly older embryos was illustrated by the positive effect on capsule

development of exposing IVP embryos to the maternal endometrial protein, uterocalin.

However, since capsule formation in the presence of uterocalin was not completely normal,

while embryo development and gene expression still differed markedly from in vivo embryos, it

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is clear that many aspects of the maternal environment necessary to support optimal early

embryonic development still need to be identified.

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CHAPTER 7

GENERAL DISCUSSION

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7.1 PRODUCTION OF EQUINE BLASTOCYSTS IN VIVO AND IN VITRO

This research focused on the equine blastocyst, a clinically relevant developmental phase

because it is the physiological stage associated with presence in the uterus. Compared to

oviductal stages, blastocysts can be obtained easily by flushing of the uterine cavity and can

also be transferred by means of a non-invasive transcervical technique into the uterus. Equine

embryonic development in vitro is retarded compared to in vivo development (Pomar et al.,

2005). In this study, the in vivo derived blastocysts were recovered 7 days after ovulation

whereas the in vitro produced blastocysts were harvested 9-9.5 days after ICSI in order to

obtain comparable developmental stages rather than embryos of similar chronological age.

However, considerable individual variation in the kinetics of development in vitro was

observed, as illustrated in Figure 1.

Figure 1 Equine IVP blastocysts 9.5 days after ICSI (magnification 60x). Even though the 4

blastocysts have exactly the same age, there are differences in grade of expansion and hatching.

The lower two blastocysts are more expanded than the upper two. The right blastocyst starts

hatching.

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Historically, research on equine blastocyts has been hampered by the difficulty of obtaining

sufficient samples. Recovery of large numbers of in vivo embryos is hindered by the fact that

the horse is a seasonal breeder with little or no ovarian activity during the winter months and,

moreover, the mare is mono-ovulatory and does not react favourably to superovulatory

treatments. In our studies, embryo recovery rates ranged around 50-60% (CHAPTER 4,5), which

is in agreement with published figures (Squires et al., 2003).

Neither is IVP of equine blastocysts widespread nor efficient. Maturation (50%) and cleavage

rates (70-80%) in our study were comparable to results published elsewhere (Galli et al., 2007).

However, our blastocyst production rate was low and variable and improved from less than 2%

in initial experiments up to more than 20% in later experiments. Even though media were

constant throughout the period, there are some possible explanations for this improvement.

First of all, the original maturation time of 24h (Galli et al., 2007) was increased to 28h, which

has been reported to improve cleavage rates for compact COCs (Choi et al., 2004). In this

regard, it must be noted that the expanded COCs, for which a shorter maturation time might be

preferential, matured to a high degree (up to 70%), which is in agreement with Galli et al.

(2007) and Hinrichs and Schmidt (2000). However, the development of oocytes from expanded

COCs up to the blastocyst stage was generally very poor when compared to that of compact

COCs. This is in contrast to the results of Galli et al. (2007), who found a similar developmental

competence for oocytes from both compact and expanded COCs. It should however be noted

that these authors collected the oocytes by scraping the follicle wall, while in our laboratory

aspiration of the follicles was performed. The latter is associated with the collection of a

significant proportion of oocytes surrounded by only a few layers of cumulus cells, and they

were classified as compact COCs, leaving the expanded COCs as a minority of the collected

COCs (CHAPTER 1, Table 1). This difference in classification might have influenced the results

observed.

A second factor that might have had a positive influence on the blastocyst production rate was

the introduction of the piezo drill in December 2008. The introduction of the piezo drill for ICSI

in the horse has been associated with better cleavage rates and more consistent results (Choi et

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139

al., 2002). Possible explanations for the positive effect of piezo-assisted over conventional ICSI

include the higher probability of oolemma breakage, reduced damage to the oocyte because

mechanical suction of the oolemma is replaced by a single pulse and better permeabilization of

the sperm membrane, which facilitates oocyte activation (Yanagida et al., 1998, Choi et al.,

2002). Considering the possible influence of the injection technique on subsequent embryonic

development, the laser was introduced as an alternative for the piezo drill. The pilot study

presented here, determined the laser as a valuable option for ICSI in the horse.

A final consideration is the acquisition of experience over the years, coinciding with faster

manipulation of the oocytes and reduced damage during subsequent IVP procedures.

The low blastocyst rate could be considered suboptimal. It could be criticized that IVP

blastocysts, cultured in suboptimal conditions, will have more aberrations in gene expression.

However, the focus of the research was on the identification of genes that were more highly

expressed in the golden standard, which were in vivo derived equine blastocysts. Moreover,

several stains (Figure 2) and the successful transfer of two IVP blastocysts leading to the birth of

a foal (CHAPTER 3.2) proved the developmental competence and vitality of the produced

blastocysts.

Figure 2 Equine IVP blastocysts (day 9) (magnification 400x). Fluorescent staining with Hoechst

33342 (A) and propidium iodide (B) illustrate the vital nuclei of horse blastocysts produced in

vitro.

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Despite the relatively low blastocyst rate, the foal rate is comparable to the results of an established

laboratory in this field (Barbacini et al., 2009). The Italian group obtained 103 blastocysts out of 1553

collected oocytes. Considering the 3 ongoing pregnancies delivered a foal, 17 foals were produced out of

84 transferred blastocysts (20%). In summary, an average of 0.013 foals per collected oocyte can be

calculated. In our study, one foal resulted from 52 collected oocytes (0.019). This is a good result, even

though it only concerns a single experiment.

7.2 EMBRYO-MATERNAL INTERACTION

The early post-fertilization stages of embryonic development in the horse are supported by

very specific micro-environments, offered by the oviduct and the uterus. Several aspects of this

embryo-maternal interaction have been highlighted during the thesis and are summarized in

Table 1.

Table 1 Embryo-maternal interaction in the horse. Several unusual aspects, both maternal and

embryonic, contribute to specific interactions between the oviduct and the early embryo on the

one hand and between the uterus and the later embryo on the other hand.

Maternal environment Embryo

Oviduct – Cleavage/morula Separated from the uterus by a

distinct uterotubal papilla

Specific transport of embryos, while

oocytes are retained

Long stay (~6 days)

Stage specific production of

prostaglandin E2 inducing relaxation of

the oviduct

Uterus - Blastocyst Nourishing histotrophe

Progesterone dependent proteins,

including uterocalin

Long pre-implantation period

Capsule

Mobile, signaling its presence to

prevent luteolysis

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Absence of the physiological maternal environment results in major or minor effects on the

embryos. The oviductal environment supporting the early cleavage stage equine embryo is

distinct to uterine effects, as illustrated by the failure of development after premature intra-

uterine transfer of cleavage stage embryos (CHAPTER 6). On the other hand, the uterine

environment is essential to attainment of specific features of a normal blastocyst. Blastocysts

produced in vitro differ from their in vivo counterparts with regard to the kinetics of

development, morphology and gene expression (Tremoleda et al., 2003; Pomar et al., 2005;

CHAPTER 5). The addition of a specific component of the in vivo environment, for example

uterocalin, to the in vitro culture medium appeared to partially restore one aspect, namely

capsule formation, although other morphological and developmental features and expression

of specific genes remained unaffected (CHAPTER 6). The absence of effects on embryonic

growth, gene expression or quality might be an artefact due to measurement of factors not

influenced by uterocalin. However, it is also possible that a crucial ligand(s) necessary for

uterocalin to influence gene expression or metabolism was absent in the in vitro culture

medium. More research is needed to better define the pieces of this complex embryo-maternal

interaction.

7.3 GENE EXPRESSION

7.3.1 Gene expression in mammalian embryos

Several reciprocal interactions exist between embryonic development, embryonic gene

expression and embryo culture conditions (Figure 3). The embryonic environment affects

embryonic development, which is in turn reflected by the genes expressed by the embryo. On

the other hand, environmental conditions can induce changes in gene expression, which can in

their turn interfere with embryonic developmental competence. Altered embryo quality can

then induce secondary changes in gene expression.

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Figure 3 Reciprocal interactions between embryonic development, gene expression and

environmental circumstances.

Marked morphological and developmental differences between in vivo derived and IVP

embryos have been shown in several species, and they have been associated to culture

environment induced changes in mRNA abundance (Corcoran et al., 2006). This differential

gene expression is evident at several levels. Firstly, IVP embryos exhibit a different gene

expression pattern when compared to their in vivo derived counterparts, as shown in various

mammals, including cattle (Mohan et al., 2004; Corcoran et al., 2006; Goossens et al., 2007),

pigs (Magnani and Cabot, 2008) and mice (Fernández-González et al., 2009). Furthermore the

gene expression level is influenced by IVF per se (Giritharan et al., 2007), by the type of embryo

culture medium (Rinaudo and Schultz., 2004) and by embryo density (Hoelker et al., 2009).

Therefore the expression of specific developmentally important genes has been used to

evaluate the effects of different culture media and of the addition of particular components to

the embryo culture medium (McElroy et al., 2008; Chin et al., 2009). For example, the presence

of FCS during IVC has been associated with the upregulation of the pro-apoptotic Bax-gene in

bovine (Rizos et al., 2003) and porcine (Cui et al., 2004) embryos. Moreover FCS in murine IVC

medium modifies the expression of genes related to epigenetic mechanisms (Fernández-

González et al., 2009). Since early epigenetic modifications affect the later phenotype, aberrant

expression of the related genes might provide the link between suboptimal embryonic culture

conditions and long-term phenotype perturbation (Fernández-González et al., 2009). Epigenetic

changes due to suboptimal culture conditions have also been associated with the ‘large

offspring syndrome’ in sheep (Young et al., 2001). This illustrates the importance of gene

expression analysis as a tool to understand the link between the early embryonic environment

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and the long-term phenotype on the one hand, and to evaluate IVC conditions in order to

optimize the IVP of embryos on the other.

7.3.2 Gene expression in equine embryos

As gene expression reflects embryo quality and the effects of embryo culture conditions, we

aimed to identify genes that are expressed at a higher level in golden standard in vivo derived

equine blastocysts than in IVP equine blastocysts, with the ultimate purpose of inducing similar

expression patterns in vitro by optimization of embryo culture conditions. The most suitable

technique at that moment to attain this purpose was SSH. The SSH method as developed and

described in detail by Diatchenko et al. (1996) uses cDNA of a tester (in vivo blastocysts) and a

driver (in vitro blastocysts) and combines several hybridizations and amplifications resulting in

suppression of non-target DNA amplification and the selective amplification of the tester

specific sequences (Addendum 2). Suppression subtractive hybridization has proven extremely

useful for studying early embryonic development in other mammals including cattle (Mohan et

al., 2004; Goossens et al., 2007), mice (Wen and Guang-xiu, 2007) and monkeys (Sun et al.,

2004) because of a number of particular advantages (Bui et al., 2005; Pfeffer et al., 2007).

1. No prior knowledge about the genes of interest is required. At the start of this project in

2007, little was known about gene expression in equine embryos and the equine

genome was poorly annotated. Therefore SSH was preferred over micro-array, which

requires specific probes.

2. The genetic material can be pre-amplified without biasing the outcome of the SSH. This

is very valuable considering the difficulty of obtaining blastocysts in the horse and the

very low amount of RNA in each embryo.

3. It enriches lowly abundant tester-specific transcripts, while common sequences are

blocked, leading to the identification of tester-specific genes of interest in very small

amounts of initial cDNA.

Even though initial identification of the amplified target sequences via BLAST analysis against

the NCBI database (www.ncbi.nlm.nih.gov) was hindered by the poor annotation of the horse

Chapter 7

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genome and frequent changes through updates, final identification of 62 genes with a higher

expression in in vivo derived equine blastocysts was achieved. Functional analysis revealed

particular involvement of these genes in protein synthesis and energy metabolism. This is in

agreement with reports in other species about impaired mitochondrial function (Mtango et al.,

2008) and transcription/translation (Corcoran et al., 2006) in vitro, when compared to the

situation in vivo.

As for other large scale screening techniques such as microarray, SSH is at risk of false positive

results. Therefore, differentially expressed genes require confirmation by means of a very

sensitive and specific method, as offered by RT-qPCR (Addendum 3). RT-qPCR permits accurate

quantification of the difference in expression between in vivo derived and IVP equine

blastocysts, but in order to ensure reliability of the results, the RT-qPCR assays must be

carefully conducted. Most importantly, accurate normalization is obligatory for correct

interpretation (Vandesompele et al., 2002; Bustin and Nolan, 2004; Bustin et al., 2005). Since

no RT-qPCR normalisation studies had been reported for horse embryos, a preliminary trial was

conducted to optimize the RT-qPCR standard operating procedure for equine embryos (Smits et

al., 2008) and reference genes for comparing in vivo and in vitro produced equine blastocysts

were evaluated (CHAPTER 4). Similarities in reference gene stability between the 3 different

types of equine blastocysts and differences to reference genes reported for other species

supported respectively the reliability and the need for the experiment. Meanwhile, RT-qPCR

has been used in equine embryos to examine expression of progesterone and oestrogen

receptors (Rambags et al., 2008), POU5F1 (Choi et al., 2009) and HSPA1A (Mortensen et al.,

2010). However, in these studies, only one reference gene was used, namely ACTB (Rambags et

al., 2008; Choi et al., 2009) or 18S rRNA (Mortensen et al., 2010). Even though ACTB appeared

stable in our study, the use of a single reference gene can lead to erroneous normalization

(Dheda et al., 2005).

Of the six genes selected from the SSH, five (ODC, HSP90AA1, BEX2, MOBKL3 and FABP3) were

confirmed to be expressed more highly in the in vivo derived horse blastocysts by means of RT-

qPCR (83%). This is a good result, as it is lower than the reported incidence of false positive

Chapter 7

145

sequences in SSH, being of the order of 30% (Mohan et al., 2004) and 25% (Goossens et al.,

2007).

Even though statistical significance was shown, biological relevance remains hard to interpret.

Although several transcriptomic analyses have been performed in different species, data on the

function of many identified genes are much scarcer. To obtain this functional information,

several methods can be applied, including:

1. Knocking out specific genes (Guan et al., 2010). Through knocking out a specific gene

and subsequent observation of the phenotype, the function of this gene can be

deduced. For practical and ethical reasons this method is mainly restricted to mice.

2. RNA interference (Schellander et al., 2007; Perrimon et al., 2010). Posttranscriptional

gene silencing includes the introduction of siRNA, dsRNA or shRNA, leading to

degradation of the target mRNA. This technique has proven valuable in functional

genomics not only in murine, but also in bovine and porcine oocytes and embryos in

vivo and in vitro.

3. Systems biology (Romero et al., 2006). The central dogma describes the transcription of

genes to mRNA and further translation to the protein, which actually exerts the

biological function. Accordingly, combining genomics, transcriptomics, proteomics and

metabolomics is essential to understand the role of specific genes in the associated

biological system. This approach was followed for example to clarify the preterm

parturition syndrome in human (Romero et al., 2006). Genomics was used to determine

a genetic predisposition for spontaneous preterm labour. Then, mRNA changes in

reproductive tissues, associated with preterm birth, were determined by

transcriptomics. By means of proteomics, differential protein profiles in the amniotic

fluid from patients with premature labour were established. Finally, metabolic profiling

of amniotic fluid was used to evaluate the risk for preterm delivary. As a whole, the

combination of all these techniques, called high-dimensional biology, can provide insight

into a complex biological phenomenon.

Chapter 7

146

The involvement in embryonic development of the six genes that were selected out of the 62

genes shown to be differentially expressed by SSH, has been documented in several other

species as discussed extensively in CHAPTER 5. However, a lack of data on gene expression in

horse embryos hinders comparative analysis in this field. A recent study describing the

expression of heat shock protein 70 (HSP1A1) in equine in vivo and in vitro blastocysts

(Mortensen et al., 2010) is interesting considering that this was one of the genes shown to be

differentially expressed by SSH. Mortensen et al. (2010) found an increase of HSP1A1 mRNA

relative to 18S rRNA in IVP equine blastocysts when compared to in vivo derived grade 1 equine

blastocysts, but this was primarily due to lower concentrations of 18S rRNA in the IVP

blastocysts. This difference in analysis compared to the methods used in this thesis makes it

difficult to compare and interpret the results.

The purpose of examining gene expression includes fundamental understanding of early

development on the one hand and implementation of the results in order to improve IVP

efficiency on the other. Regarding the latter, it needs to be considered that the expression of a

gene can either reflect a direct influence of the embryo culture medium or it can be secondary

through induction by a downstream effect (embryonic environment -> deregulation of certain

genes -> altered embryo quality -> deregulation of other genes) (Figure 3). The expression of a

particular gene does not necessarily mean that the addition of its substrate to the culture

medium will benefit embryonic development. Okawara et al. (2009) detected the expression of

glycerol kinase in bovine oocytes and embryos, indicating a physiological role of glycerol;

however, the addition of glycerol to the maturation medium appeared to negatively influence

subsequent maturation rates. On the other hand, changes in the culture medium are reflected

by gene expression patterns. For example, during IVP in mice KSOM resulted in higher

blastocyst rates when compared to Whitten’s medium and when, in a later microarray

experiment, gene expression was compared to that of in vivo derived embryos, culture in KSOM

induced far less aberrant gene expression than the suboptimal Whitten’s medium (Gardner and

Lane, 2005).

Chapter 7

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To improve understanding of both the meaning of the genes identified in CHAPTER 5 and the

influence of uterocalin on IVP embryos, the expression of ODC, HSP90AA1, BEX2, MOBKL3 and

FABP3 was evaluated in equine IVP blastocysts cultured in the presence or absence of

uterocalin. It was hypothesized that the addition of a maternal component to the embryo

culture medium could result in a more ‘in vivo-like’ gene expression pattern. However,

uterocalin did not appear to affect the examined genes.

7.4 GENERAL CONCLUSIONS

In a first part of the thesis, the procedure for in vitro embryo production in the horse was

optimized.

1. IVP of equine blastocysts was successfully introduced to our laboratory. After an initial

period of conventional ICSI, piezo drill assisted ICSI was introduced and subsequently

compared with laser assisted ICSI. Both devices have specific advantages and

disadvantages, but no major effect on subsequent embryonic development was

observed (CHAPTER 3.1).

2. The viability of the IVP equine blastocysts was illustrated by successful embryo transfer,

resulting in the birth of the first ICSI-foal in the Benelux (CHAPTER 3.2).

Based on the results presented in this thesis, the following conclusions can be drawn:

1. A method for reliable determination of gene expression in equine blastocysts was

developed by the optimization of a standard operating procedure for RT-qPCR and

through the determination of the most stable reference genes for normalization. The

geometric mean of UBC, ACTB, RPL32 and GAPDH is recommended as a normalization

factor for RT-qPCR in equine in vivo and in vitro derived blastocysts (CHAPTER 4).

2. Upregulation of genes in in vivo derived equine blastocysts, when compared to IVP

equine blastocysts, was determined by SSH. A total of 62 upregulated genes was

identified, with major involvement in protein synthesis and energy metabolism. The

differential expression was confirmed by RT-qPCR for five out of six developmentally

important genes, namely ODC, HSP90AA1, BEX2, MOBKL3 and FABP3 (CHAPTER 5).

Chapter 7

148

3. The premature intra-uterine transfer of cleavage stage equine IVP embryos (day 2-3) did

not result in successful embryo development (CHAPTER 6).

4. The addition of recombinant uterocalin to the embryo culture medium on day 6

improved capsule formation in equine IVP blastocysts, but did not influence embryonic

development nor the expression of the examined genes (CHAPTER 6).

7.5 FUTURE PERSPECTIVES

The IVP of equine embryos has evolved greatly in recent years, creating the possibility to obtain

foals through the combination of OPU, IVM, ICSI, IVC and ET for research (Jacobson et al., 2010)

as well as for clinical (Colleoni et al., 2007) purposes. However, each of these steps needs

optimization through further research in order to allow large scale application as it happens

already in cattle (Blanco et al., 2009). Recent publications stress the limitations of

superovulation and OPU techniques (Blanco et al., 2009; Jacobson et al., 2010), IVM (Galli et al.,

2007), ICSI (Choi et al., 2002), IVC (Choi et al., 2004) and ET (Panzani et al., 2009). Taken into

account a current blastocyst rate of 10% in one of the most experienced commercial labs

(Barbacini et al., 2009), there is considerable room to improve both the quantity and the quality

of transferable blastocysts by optimizing all these crucial steps.

A clear influence of the maternal environment on embryonic development was observed. An

important role of the oviductal environment was also shown (CHAPTER 6), and further

information on the mechanism involved or on the composition of equine oviductal fluid and the

influence of oviduct epithelial cell secretions on early embryonic stages would be very

interesting. Considering the blastocyst and its uterine environment, a link was made between

the maternal protein uterocalin and the embryonic capsule. However, elucidating underlying

mechanisms of this phenomenon, influence of the addition of ligands for uterocalin and the

role of other components of the uterine histotrophe all require further research.

One way to evaluate embryos is to look at gene expression. Since genetic research in equine

embryos is still in its infancy, plenty of opportunities exist to expand this field in order to

achieve a level of information, which is comparable to that in other species. Growing

Chapter 7

149

knowledge of the equine genome and advances in genetic technologies are opening new

avenues for rapid generation of large amounts of data. Recent work on endometrial gene

expression was performed with a home-made microarray (Klein et al., 2010). Microarray

analysis has generated substantial information on murine (Fernández-González et al., 2009) and

bovine (Vigneault et al., 2009) embryonic gene expression. Embryonic biopsy, combined with

microarray and DNA fingerprinting can contribute to the identification of markers to predict

embryonic developmental competence (Jones et al., 2008). Currently, microarray is in its turn

being replaced by even more advanced techniques like next generation sequencing (deep

sequencing), providing massive amounts of information in a short time (Hurd and Nelson,

2009). Combining knowledge from genomics and transcriptomics with proteomics and

metabolomics is crucial in order to achieve functional insights (Romero et al., 2006; Werner et

al., 2010) in the complex puzzle called ‘early embryonic development’.

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http://www.ncbi.nlm.nih.gov/pubmed?term=%22Carnevale%20EM%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22McCue%20PM%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Bruemmer%20JE%22%5BAuthor%5D�







http://www.ncbi.nlm.nih.gov/pubmed?term=%22Vigneault%20C%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Gravel%20C%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Vall%C3%A9e%20M%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22McGraw%20S%22%5BAuthor%5D�

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Chapter 7

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54. Young L.E., Fernandes K., McEvoy T.G., Butterwith S.C., Gutierrez C.G., Carolan C.,

Broadbent P.J., Robinson J.J., Wilmut I., Sinclair K.D. 2001. Epigenetic change in IGF2R is

associated with fetal overgrowth after sheep embryo culture. Nat Genet 27 153-154.

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http://www.ncbi.nlm.nih.gov/pubmed?term=%22Fernandes%20K%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22McEvoy%20TG%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Butterwith%20SC%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Gutierrez%20CG%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Carolan%20C%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Broadbent%20PJ%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Robinson%20JJ%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Wilmut%20I%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Sinclair%20KD%22%5BAuthor%5D�

Summary

157

SUMMARY

In vitro produced equine embryos are valuable, not only to investigate aspects of early

embryonic development, but also from a clinical point of view. The low success of conventional

IVF contributed to the initial slow development and implementation of IVP in the horse.

However, once ICSI was introduced almost 15 years ago progress became much more rapid.

Despite this advance, the other steps of the IVP process (oocyte collection - IVM – ICSI – IVC)

still pose specific problems, and need optimization to enhance the efficiency of the overall

procedure.

The blastocyst stage embryo is particularly interesting, being on the one hand the endpoint of

the IVP process and on the other hand the first embryonic stage, which is unique to the uterus,

allowing the possibility to atraumatically collect in vivo blastocysts from donor mares as well as

to transfer in vivo or in vitro produced blastocysts to recipient mares. Comparing IVP blastocysts

with their counterparts derived in vivo reveals differences in the kinetics of development and in

morphology, reflecting the influence of the unique equine maternal environment. CHAPTER 1

represents a general introduction.

The purpose of this thesis (CHAPTER 2) is to contribute to the optimization of the IVP of equine

embryos by considering some technical aspects of the process (CHAPTER 3) and through

fundamental understanding of differences between in vitro produced and in vivo derived

equine blastocysts, with the emphasis on gene expression (CHAPTER 4,5) and on the influence

of the maternal environment on early equine embryonic development (CHAPTER 6).

The method of choice for fertilizing equine oocytes is ICSI. After an initial period of conventional

ICSI, the piezo drill was introduced and was associated with higher cleavage rates and more

consistent results, even though no direct comparison with conventional ICSI had been

performed. Since the piezo drill also has some disadvantages, like the toxicity of mercury, other

advanced injection technologies should also be considered. Therefore in CHAPTER 3.1 piezo-

assisted ICSI was compared with laser-assisted ICSI. No major influence on subsequent

embryonic development was observed; a slightly higher cleavage rate was obtained after laser-

Summary

158

assisted ICSI, but blastocyst rates were not significantly affected. Further technical advantages

of the laser included the absence of mercury and the ease of use, since it was less sensitive to

exact 3-dimensional positioning than the piezo drill. On the other hand, laser-assisted ICSI

requires the making of several adjacent holes in the zona pellucida, after which conventional

ICSI needs to be applied anyway to penetrate the oolemma. This occurs less fluently than in

piezo-assisted ICSI, in which both the zona and the oolemma are penetrated in one

manipulation. However, the difference in duration of the procedure was not significant.

The IVP of equine embryos was introduced to the laboratory. The feasibility of the IVP protocol

used in this thesis and the viability and developmental competence of the resulting IVP

blastocysts was confirmed through the birth of the first ICSI foal in the Benelux, as described in

CHAPTER 3.2.

The major part of this thesis was focusing on the differences between equine blastocyts

produced in vitro and those derived in vivo. The evaluation of gene expression was selected as

an approach to gain fundamental insight into these differences. The aim was to identify genes

which were upregulated in golden standard in vivo derived blastocysts, when compared to in

vitro produced embryos. Considering the limited knowledge on embryonic gene expression in

the horse and the scarcity of embryonic RNA, SSH was at the time the method of choice for

initial genetic analysis (CHAPTER 5). Using this technique, 62 genes were identified that were

expressed at a higher level in in vivo derived equine blastocysts than in their in vitro produced

counterparts. Functional analysis revealed substantial involvement of these genes in protein

synthesis and energy metabolism. The possibility to generate false positive results by SSH

required confirmation of these results using a very sensitive and specific technique, as offered

by RT-qPCR. Six genes, resulting from the SSH and described in the literature as being involved

in embryonic development, namely BEX2, FABP3, HSP90AA1, MOBKL3, MCM7 and ODC, were

selected for RT-qPCR.

In order to obtain reliable results by RT-qPCR, careful assay and data analysis is crucial. An

important aspect is correct normalization of the data. The method of choice to achieve this

consists of the calculation of a normalization factor, from the geometric mean of the most

Summary

159

stably expressed reference genes in the tissue of interest. To this end, the expression of eight

reference genes was evaluated in in vivo derived and in fresh and frozen-thawed in vitro

produced equine blastocysts. This analysis indicated the geometric mean of ACTB, UBC, RPL32

and GAPDH as a reliable normalization factor for RT-qPCR data in experiments with equine in

vivo and in vitro blastocysts (CHAPTER 4).

This normalisation factor was subsequently used for the analysis of the RT-qPCR data of the 6

developmentally important genes, generated from SSH. For 5 of these genes, namely BEX2,

FABP3, HSP90AA1, MOBKL3, and ODC, a higher level of expression in in vivo derived horse

blastocysts, when compared to in vitro produced blastocysts, was confirmed by means of RT-

qPCR (CHAPTER 5).

In CHAPTER 6 the influence of the maternal environment during embryonic development was

examined. A first experiment examined the necessity of the oviduct for normal early embryonic

development through the premature intra-uterine transfer of day 2-3 equine embryos

produced in vitro. Flushing of the recipient mares on day 7 yielded no viable blastocysts and

pregnancy diagnosis on day 14 revealed no pregnancies, suggesting that the uterus cannot

replace the oviductal environment as a support for the development of early cleavage stage

horse embryos.

In a second experiment the influence of the uterus was assessed, and more specifically the

influence of an important protein component of endometrial secretions, uterocalin.

Recombinant uterocalin was added to IVC medium on day 6 and subsequent in vitro produced

blastocysts were evaluated on day 9-9.5. No differences in blastocyst production, blastocyst

diameter or cell number were found between the embryos cultured in the presence or absence

of uterocalin. Uterocalin did not affect the expression of the genes shown to be upregulated in

vivo embryos (CHAPTER 5) either. However, the presence of uterocalin in the IVC medium

appeared to stimulate embryonic capsule formation.

CHAPTER 7 represents a general discussion and the conclusions of this thesis:

Summary

160

In a first part of the thesis, the procedure for in vitro embryo production in the horse was

optimized.

1. IVP of equine blastocysts was successfully introduced to our laboratory. After an

initial period of conventional ICSI, piezo drill assisted ICSI was introduced and

subsequently compared with laser assisted ICSI. Both devices have specific

advantages and disadvantages, but no major effect on subsequent embryonic

development was observed (CHAPTER 3.1).

2. The viability of the IVP equine blastocysts was proven by successful embryo transfer,

resulting in the birth of the first ICSI-foal in the Benelux (CHAPTER 3.2).

Based on the results presented in the second part of this thesis, the following conclusions can

be drawn:

1. A method for reliable determination of gene expression in equine blastocysts was

developed by the optimization of a standard operating procedure for RT-qPCR and

through the determination of the most stable reference genes for normalization.

The geometric mean of UBC, ACTB, RPL32 and GAPDH is recommended as a

normalization factor for RT-qPCR in equine in vivo and in vitro derived blastocysts

(CHAPTER 4).

2. Upregulation of genes in in vivo derived equine blastocysts, when compared to IVP

equine blastocysts, was determined by SSH. A total of 62 upregulated genes was

identified, with major involvement in protein synthesis and energy metabolism. The

differential expression was confirmed by RT-qPCR for five out of six developmentally

important genes, namely ODC, HSP90AA1, BEX2, MOBKL3 and FABP3 (CHAPTER 5).

3. The premature intra-uterine transfer of cleavage stage equine IVP embryos (day 2-3)

did not result in successful embryo development (CHAPTER 6).

4. The addition of recombinant uterocalin to the embryo culture medium on day 6

improved capsule formation in equine IVP blastocysts, but did not influence

embryonic development nor the expression of the examined genes (CHAPTER 6).

Samenvatting

163

SAMENVATTING

In vitro geproduceerde paardenembryo’s zijn waardevol, niet alleen voor onderzoek met als

doel fundamenteel inzicht te verwerven in de vroege embryonale ontwikkeling, maar ook

vanuit een klinisch standpunt. Het beperkte succes van conventionele IVF heeft bijgedragen tot

een initieel trage opgang van de IVP bij het paard. ICSI werd echter met succes geïntroduceerd

en tijdens het laatste decennium werd een snelle vooruitgang geboekt. Ondanks deze

progressie stellen de verschillende stappen van het IVP proces (eicelverzameling – IVM – ICSI -

IVC) nog steeds meerdere specifieke problemen, waarbij optimalisatie vereist is om de

efficiëntie van de techniek te verbeteren.

Het blastocyststadium is in het bijzonder interessant omdat het enerzijds het eindpunt van het

IVP proces betekent en omdat het anderzijds het stadium is dat zich onder fysiologische

omstandigheden in de baarmoeder bevindt, waardoor het mogelijk is om op atraumatische

wijze zowel in vivo blastocysten te verzamelen van donormerries, als om in vivo of in vitro

geproduceerde blastocysten over te planten in receptormerries. Wanneer IVP blastocysten met

hun in vivo verzamelde tegenhangers worden vergeleken, zijn er verschillen in ontwikkeling en

morfologie, die de invloed van de maternale omgeving weerspiegelen. Een algemene inleiding

wordt gegeven in HOOFDSTUK 1.

Het doel van deze thesis (HOOFDSTUK 2) is om bij te dragen aan de optimalisatie van de IVP van

paardenembryo’s door bepaalde technische aspecten van het proces te onderzoeken

(HOOFDSTUK 3) en door de fundamentele verschillen tussen in vivo en in vitro geproduceerde

paardenblastocysten trachten te begrijpen, met de nadruk op genexpressie (HOOFDSTUK 4,5)

en op de invloed van de maternale omgeving op de vroege embryonale ontwikkeling bij het

paard (HOOFDSTUK 6).

De voorkeurstechniek om paardeneicellen te bevruchten is ICSI. Na een initiële periode van

conventionele ICSI werd de piëzo drill geïntroduceerd voor ICSI bij het paard, wat werd

geassocieerd met hogere delingspercentages en meer herhaalbare resultaten. Een directe

vergelijking met conventionele ICSI werd echter nooit gemaakt. Aangezien de piëzo drill ook

Samenvatting

164

problemen stelt, zoals de toxiciteit van kwik, moeten andere vooruitstrevende technologieën

ook overwogen worden. Daarom werd in HOOFDSTUK 3.1 piëzo-geassisteerde ICSI vergeleken

met laser-geassisteerde ICSI. Er werden geen grote invloeden op de daaropvolgende

embryonale ontwikkeling vastgesteld. Een iets hoger delingspercentage werd bekomen na

laser-geassisteerde ICSI, maar de blastocystpercentages werden niet significant beïnvloed.

Verdere technische voordelen van de laser behelsen de afwezigheid van kwik en het

gebruiksgemak, waarbij de laser ook minder gevoelig is voor de 3-dimensionale positionering

van de micromanipulatoren dan de piëzo. Aan de andere kant moeten er bij laser-geassisteerde

ICSI verschillende gaten in de zona pellucida gemaakt worden, waarna alsnog conventionele

ICSI toegepast dient te worden om de eicelmembraan te penetreren. Dit verloopt minder vlot

dan bij piëzo-geassisteerde ICSI, waarbij zowel de zona als de eicelmembraan in één vloeiende

manipulatie kunnen worden gepenetreerd. Er was echter geen significant verschil in tijd tussen

beide technieken.

De IVP van paardenembryo’s werd in het laboratorium geïntroduceerd. De kwaliteit van het

protocol voor IVP dat in deze thesis gebruikt werd, wordt bevestigd door de vitaliteit en het

ontwikkelingspotentieel van de resulterende IVP blastocysten en door de geboorte van het

eerste ICSI-veulen van de Benelux, die beschreven wordt in HOOFDSTUK 3.2.

In deze thesis lag de nadruk echter op de verschillen tussen in vitro versus in vivo

geproduceerde paardenblastocysten. De evaluatie van hun genexpressie werd gekozen als

benadering om fundamenteel inzicht te verwerven in deze verschillen. Het doel was om genen

te identificeren die opgereguleerd worden bij in vivo blastocysten, die als gouden standaard

gebruikt worden, in vergelijking met in vitro blastocysten. Rekening houdend met de beperkte

kennis over embryonale genexpressie bij het paard en de schaarsheid aan embryonaal RNA,

was SSH de voorkeurstechniek voor een initiële genetische analyse (HOOFDSTUK 5). Op deze

manier werden 62 genen geïdentificeerd, die meer tot expressie kwamen bij in vivo

geproduceerde paardenblastocysten dan bij hun in vitro geproduceerde tegenhangers.

Functionele analyse onthulde een belangrijke betrokkenheid van deze genen in de

eiwitsynthese en het energiemetabolisme. De mogelijkheid van het genereren van vals

Samenvatting

165

positieve resultaten met SSH vereist echter een extra bevestiging door middel van een zeer

gevoelige en specifieke techniek, namelijk RT-qPCR. Uit de SSH werden zes genen geselecteerd

op basis van hun in de literatuur beschreven betrokkenheid bij de embryonale ontwikkeling,

namelijk BEX2, FABP3, HSP90AA1, MOBKL3, MCM7 en ODC.

Om betrouwbare resultaten te verkrijgen met RT-qPCR is een zorgvuldige methode en data-

analyse cruciaal. Hierbij is een correcte normalisatie van de data van belang. De

voorkeursmethode om dit te bereiken bestaat uit de berekening van een normalisatiefactor,

zijnde het geometrisch gemiddelde van de stabielste referentiegenen in het weefsel van

interesse. Daarom werd de expressie van 8 referentiegenen geëvalueerd zowel in in vivo

geproduceerde als in verse en ontdooide in vitro geproduceerde paardenblastocysten. Deze

analyse bepaalde het geometrisch gemiddelde van ACTB, UBC, RPL32 en GAPDH als een

betrouwbare normalisatiefactor voor RT-qPCR data in experimenten met zowel in vivo als in

vitro paardenblastocysten (HOOFDSTUK 4).

De op deze manier bepaalde normalisatiefactor werd vervolgens gebruikt voor de analyse van

de RT-qPCR data van de zes geselecteerde genen. Voor vijf van deze genen, namelijk BEX2,

FABP3, HSP90AA1, MOBKL3, and ODC, werd de hogere expressie in in vivo geproduceerde

paardenblastocysten in vergelijking met in vitro geproduceerde paardenblastocysten bevestigd

door middel van RT-qPCR (HOOFDSTUK 5).

In HOOFDSTUK 6 werd de invloed van de maternale omgeving tijdens de embryonale

ontwikkeling onderzocht. Een eerste experiment stelde de cruciale rol van de eileider in vraag

door gedeelde embryo’s van 2 à 3 dagen oud vroegtijdig over te planten naar de baarmoeder.

Er werden geen blastocysten gevonden na baarmoederspoeling van de receptormerries op dag

7 en er werden ook geen drachten vastgesteld na echografie op dag 14 wat suggereert dat de

baarmoeder de eileideromgeving niet kan vervangen om de ontwikkeling van vroege gedeelde

embryonale stadia bij het paard te ondersteunen.

In een tweede experiment werd de invloed van de baarmoeder nagegaan en meer specifiek de

invloed van een belangrijk eiwit in de baarmoedersecreties, uterocaline. Recombinant

uterocaline werd toegevoegd aan het IVC medium op dag 6 en de in vitro geproduceerde

Samenvatting

166

blastocysten werden 9 tot 9,5 dagen na ICSI geëvalueerd. Geen verschillen in

blastocystpercentage, diameter of celaantal werden gevonden tussen de embryo’s die werden

gekweekt in de aan- of afwezigheid van uterocaline. Uterocaline had ook geen invloed op de

expressie van de genen die opgereguleerd zijn in vivo (HOOFDSTUK 5). De aanwezigheid van

uterocaline in het cultuurmedium bleek echter wel de embryonale kapselvorming te

stimuleren.

HOOFDSTUK 7 bestaat uit een algemene discussie, gevolgd door de conclusies van deze thesis:

In een eerste deel van de thesis werd de procedure voor de in vitro productie van

paardenembryo’s geoptimaliseerd.

1. De IVP van paardenembryo’s werd in ons laboratorium ingevoerd. Na een initiële

periode van conventionele ICSI werd piëzo-geassisteerde ICSI geïntroduceerd en

vergeleken met laser-geassisteerde ICSI. Beide apparaten hadden specifieke voor- en

nadelen, maar geen groot effect op de daaropvolgende embryonale ontwikkeling werd

vastgesteld.

2. De leefbaarheid van de IVP paardenblastocysten werd bewezen door succesvolle

embryotransplantatie die resulteerde in de geboorte van het eerste ICSI-veulen van de

Benelux.

De conclusies op basis van het onderzoek in het tweede deel van deze thesis zijn:

3. Een methode voor betrouwbare bepaling van genexpressie bij paardenblastocysten

werd op punt gesteld door middel van de optimalisatie van het protocol voor RT-qPCR

en door de bepaling van de stabielste referentiegenen voor datanormalisatie. Het

geometrisch gemiddelde van ACTB, UBC, RPL32 en GAPDH wordt aanbevolen als

normalisatiefactor voor RT-qPCR van in vivo en in vitro geproduceerde

paardenblastocysten.

4. Opregulatie van genen in in vivo geproduceerde paardenblastocysten, in vergelijking

met in vitro geproduceerde paardenblastocysten, werd bepaald met SSH. In totaal

werden 62 genen geïdentificeerd, die een belangrijke betrokkenheid bij eiwitsynthese

Samenvatting

167

en energiemetabolisme vertonen. De differentiële expressie werd bevestigd door RT-

qPCR voor vijf van de zes belangrijke ontwikkelingsgenen, namelijk BEX2, FABP3,

HSP90AA1, MOBKL3 en ODC.

5. De voortijdige transplantatie naar de baarmoeder van IVP gedeelde paardenembryo’s

ondersteunde de verdere embryonale ontwikkeling niet.

6. De toevoeging van recombinant uterocaline aan het embryocultuurmedium op dag 6

stimuleerde de kapselvorming bij IVP paardenblastocysten, maar had geen invloed op

de embryonale ontwikkeling of op de expressie van de onderzochte genen.

169

DANKWOORD/ACKNOWLEDGEMENTS

Een woord van dank begint bij het begin. Mijn ouders nemen dan ook de eerste plaats in. Papa, mama, jullie hebben me steeds alle kansen gegeven om mijn eigen weg te zoeken, Me laten kiezen tussen mensen en dieren, tussen kleine en grote, tussen praktijk en onderzoeken. Enkel bij die laatste keuze heb ik enige beïnvloeding ervaren, toen ik zei: ‘Die voorgestelde onderwerpen boeien me niet zo, doctoreren is wellicht niet echt iets voor mij. Ik wil iets met paarden, iets klinisch en tof.’ ‘Wel,’ zei mijn papa, ‘neem dan zelf het initiatief en schrijf naar je prof.’ Professor de Kruif, toen kwam ik bij u aankloppen. U kwam met het meest fantastische onderwerp op de proppen. ‘IVF bij het paard, wat is je gedacht?’ ‘Prima voor mij!’ en zo werd ik aangenomen op de DI08. Ik begin mijn professionele bedanking nu ongegeneerd met een man, maar de meeste dank ben ik verschuldigd aan mijn promotor, Ann. Bedankt om zo in het paardenonderzoek te geloven. Na 2 jaar geploeter, kwam mijn kopje toch boven. Bedankt voor het enthousiasme, de ideeën en contacten, Bedankt voor de steun als de moed even zakte. Mijn co-promotor, professor Peelman, Luc, uw genetische inbreng werd zeer geapprecieerd. Bedankt voor uw hulp, ik heb op genetica veel geleerd. Karen, zonder jou was ik er nooit geraakt. Jij hebt mijn onzichtbare pellets en mijn real time-initiatie meegemaakt. Was het een labotechniek, een genetisch programma of een reviewer-commentaar, Vier jaar lang stond je altijd voor mij klaar. I would also like to express my gratitude towards the members of the examination committee. Prof. Stout, Prof. Deleuze, Prof. Martens, Dr. Palmer, Dr. Daels, thank you for improving my PhD. A special thanks goes to Prof. Galli, Silvia Colleoni and the rest of the wonderful Avantea crew. Prof. Betteridge, Prof. Kennedy, Aline et Thomas de Cryozootech, it was very nice to collaborate with you. Home sweet home, daarom een dikke merci aan mijn bureaugenoten, Catharina, Maarten en Jan, Bedankt voor de opleiding in de kliniek, jullie kunnen er wat van! Bedankt ook om mij zo veel tijd te geven voor mijn onderzoek en mijn doctoraat. Ik vind het jammer dat ik jullie niet meer heb kunnen teruggeven, nu ik jullie al verlaat. Jan, heel veel succes met de eicellen en met de dvd! Catharina, ik zal de vrouwengesprekken missen tussen ons twee. Maarten, nog een extra merci voor mijn kaft, en ik weet Dat jij dagelijks de kleur van mijn bh zal raden, ook al zit ik aan de andere kant van de planeet. Op onze rommelige bureau (bedankt om hier af en toe orde te scheppen, Nicole en co) voelde ik me prima thuis, Al werd die op tijd en stond ingeruild voor een etentje of een sangria in het Middenstandshuis.

170

Ook de andere collega’s van paard, bedankt voor de merrie-opvolging, Katrien, Emilie en Kim, en voor jullie deelname aan de embryoverzameling. De nieuwere generatie staat alweer klaar, Hilde, Sarah en Bart, zorg goed voor het paardenteam en voor elkaar. Verder werd de samenwerking met de grote embryotransfercentra in de buurt geapprecieerd Een woord van dank is dan ook voor Diergaerdhof, Keros en Hof ter Leeuwe gereserveerd. Initieel was ik de enige paardentrut in het IVF-labo, Naast Leen, Mirjan, Mohamed en Mohammad van rund, Muriel en Tom met de kleine en voor de varkens Josine en Jo. Eline, aan jou kon ik mijn mensenvragen stellen. Bedankt allemaal voor de hulp en de multi-species tips. En Muriel, Jo en Mirjan (en natuurlijk ook leider Philippe), merci voor de gezellige ski-trips! Isabel en Petra, jullie hebben me in alle staten gezien. Kwaad als mijn tijdsschema eraan ging omdat ik iets niet vond, ronddansend bij het vinden van een blastocyst of tien! Bedankt voor de lange en trouwe hulp van jullie zijde. Het IVF-labo heeft geluk met jullie beiden. Ook hartelijk dank aan de mensen van het IVF-labo van het UZ in Gent. Ik ben meer dan eens voor ICSI-pipetjes naar daar gerend. Het slachthuis in Rekkem mag ik ook niet vergeten. Ik heb quasi al hun paardeneierstokken gekregen, hoewel ik waarschijnlijk nooit een paard zal eten. De nachtelijke keizersnedes heb ik eigenlijk altijd graag gedaan, Al moest ik soms toch dringend een wacht ruilen om naar de zee te gaan. Gelukkig had ik super collega’s met wie dat omruilen vlot ging. Bedankt Davy, Miel, Sebastiaan, Alfonso, Vanessa, Josine, Hilde, Cyriel, Ellen en Liesbeth voor jullie respect voor mijn surfverslaving. Sebastiaan, je bent intussen een echte zakenman die kalkoenen promoot, Rijzig, sereen en lankmoedig worden kleine jongens groot. De vele andere collega’s van verloskunde, evenals de collega’s van weleer, Wil ik ook bedanken voor hun hulp en voor de aangename sfeer. Willy, Dirk, Wilfried, Marnik en Véronique, bedankt om de proefmerries en mijn Smicsi’tje zo goed te soigneren. Bedankt ook Els, Ria, Leïla, Sandra en tante Nadine; Steven en Miel, van jullie kan ik nog veel computergeheimen leren. Mijn 2de vakgroep bevond zich aan de overkant. UNO en ijsjes, het was daar plezant! Verder heb ik ook hulp gehad van Kelly en Jip voor de dot blot en van Prof. De Graaf en Marleen Brunain voor de uterocalineconcentratie, Evenals van Prof. Vandesompele voor mijn genetische statistiek en van Dries voor de microscopie-evaluatie.

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‘Of all the things which wisdom provides to make us entirely happy, much the greatest is the possession of friendship’ zei Epicurus zo goed. Het zijn mijn familieleden en vrienden aan wie ik een diepe dankbaarheid richten moet. Mama en papa, jullie heb ik in het begin al vermeld. Het is dan ook een ‘dank u’ die dubbel telt. Ines, mijn allerliefste zus, aan u kan ik al mijn avonturen vertellen, We zullen onze paardrijritjes tijdelijk moeten vervangen door Skypegewijs bellen. Wouter en Margo, jullie hebben steeds een vrolijke aanhang mee, Als ik terugkom van Aruba, is ons Lennertje al twee. Oma, bomama en bonpapa, zo’n fiere grootouders hebben, doet me veel plezier. Het rijmseltje is dan ook speciaal voor jullie vertier. Pascale, wij hebben samen het doctoraatsleven gedeeld, Samen gestudeerd, gereisd, gewoond, gefeest, en ons alleszins zelden verveeld. Katrien en Charlotte, jullie hebben me een prima studententijd gegeven. Nele, Sabine en Karen, jullie zijn ook vriendinnen voor het leven. Alle andere vrienden die mijn leven kleur geven opsommen, daar ga ik niet toe geraken. Voor mijn surfmaatjes zal ik nog even een uitzondering maken. Geen betere compensatie voor een overdosis wetenschap dan een lekkere zuid-west. Stein, Pieter, Seppe, Sven,… jullie begrijpen dit als geen ander, merci voor de zalige tijden op ‘t water, you’re the best! Bedankt allemaal, ik ga jullie missen!

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CURRICULUM VITAE Katrien Smits werd geboren op 11 april 1982 te Zoersel. Na het behalen van het diploma hoger

secundair onderwijs aan het Sint-Jan Berchmanscollege (Latijn-Wetenschappen) te Westmalle,

begon zij in 2000 met de studie Diergeneeskunde aan de Universiteit Gent. In 2006 behaalde ze

het diploma van dierenarts (optie Paard) met grote onderscheiding.

Op 1 oktober 2006 trad ze in dienst van de Vakgroep Voortplanting, Verloskunde en

Bedrijfsdiergeneeskunde als doctoraatsbursaal (BOF). Daar verrichtte ze onderzoek naar de

ontwikkeling en genexpressie van in vitro geproduceerde paardenembryo’s. Naast haar

onderzoek was Katrien Smits ook werkzaam in de kliniek verloskunde, waar ze participeerde in

de nacht- en weekenddiensten.

Katrien Smits is auteur of mede-auteur van verschillende publicaties in internationale en

nationale wetenschappelijke tijdschriften en nam actief deel aan diverse internationale en

nationale congressen.

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BIBLIOGRAPHY

PUBLICATIONS

Govaere J., Hoogewijs M., De Schauwer C., Van Zeveren A., Smits K., Cornillie P., de Kruif A.

2009. An abortion of monozygotic twins in a warmblood mare. Reprod Domest Anim 44 852-

854.

Govaere J., Maes S., Saey V., Hoogewijs M., De Schauwer C., Smits K., Roels K., Vercauteren G.,

de Kruif A. 2010. Uterine fibrosarcoma in a Warmblood mare. Reprod Domest Anim in press.

Hoogewijs M., De Vliegher S., De Schauwer C., Govaere J., Smits K., Hoflack G.G., de Kruif A.,

Van Soom A. 2010. Validation and usefulness of the Sperm Quality Analyzer V equine for equine

semen analysis. Theriogenology in press.

Moyaert H., Decostere A., Pasmans F., Baele M., Ceelen L., Smits K., Ducatelle R., Haesebrouck

F. 2007. Acute in vivo interactions of Helicobacter equorum with its equine host. Equine Vet J 39

370-372.

Smits K., Goossens K., Van Soom A., Govaere J., Hoogewijs M., Vanhaesebrouck E., Galli C.,

Colleoni S., Vandesompele J., Peelman L. 2009. Selection of reference genes for quantitative

real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. BMC Res Notes

Smits K., Govaere J., Hoogewijs M., De Schauwer C., Van Haesebrouck E., Van Poucke M., van

den Berg M., Vullers T., Van Soom A. 2010. Birth of the First ICSI Foal in the Benelux. Vlaams

Diergeneesk Tijdschr 79 134-138.

2 246.

Smits K., Goossens K., Van Soom A., Govaere J., Hoogewijs M., Vandesompele J., Peelman L.J.

2010. In vivo derived horse blastocysts show upregulation of developmentally important genes

compared to in vitro produced horse blastocysts. Reprod Fertil Dev in press.





176

Smits K., Govaere J., Peelman L.J., Goossens K., de Graaf D.C., Vercauteren D., Vandaele L.,

Hoogewijs M., Stout T., Van Soom A. 2010c. Influence of the uterine environment on the

development of equine in vitro embryos. Reproduction submitted.

Smits K., Govaere J., Hoogewijs M., Piepers S., Van Soom A. 2010. A different approach to the in

vitro production of horse embryos: a pilot study of laser-assisted versus piezo drill ICSI. Reprod

Domest Anim submitted.

Vanhaesebrouck E., Govaere J., Smits K., Durie I., Vercauteren G., Martens A., Schauvliege S.,

Ducatelle R., Hoogewijs M., De Schauwer C., De Kruif A. 2010. Ovarian teratoma in the mare: a

review and two cases. Vlaams Diergeneesk Tijdschr 79 32-41.

ORAL PRESENTATIONS AND POSTERS AT NATIONAL AND INTERNATIONAL CONFERENCES

Smits K., De Graaf D., Lemahieu I., Van Damme P., Piepers S., Peelman L., Van Soom A. 2008.

Influence of uterocalin on bovine in vitro blastocyst rate: a preliminary trial. FNRS meeting,

Mons, Belgium.

Smits K., Goossens K., Van Poucke M., Peelman L., Van Soom A. 2008. Optimization of standard

operating procedure for quantitative real-time PCR in equine embryos. Benelux qPCR

symposium, Ghent, Belgium.

Smits K., Goossens K., Van Soom A., Peelman L. 2009. Selection of reference genes for

quantitative real-time PCR in equine in vivo derived and in vitro produced blastocysts. Fertility

2009 Conference, Edinburgh, Scotland.

Smits K., Goossens K., Van Soom A., Peelman L. 2009. Identification of differentially expressed

genes in in vivo versus in vitro equine blastocysts by suppression subtractive hybridization

(SSH). ESDAR, Ghent, Belgium.

Smits K., Govaere J. Van Soom A. 2009. Intra-uterine transfer of equine cleaved embryos. COST

meeting, Alghero, Italy.

177

Smits K., Goossens K., Van Soom A., Peelman L. 2010. Differential gene expression in in vivo

derived versus in vitro produced equine blastocysts as determined by RT-qPCR. IETS, Cordoba,

Argentina.

179

ADDENDUM 1 PROTOCOL IN VITRO PRODUCTION

EQUINE EMBRYOS Based on Galli et al., 2007; Lagutina et al., 2005

MEDIA

- Flush medium

o DPBS, 50 µg/ml BSA, 25 IU/ml heparin

- Dissection medium

o HEPES buffered TCM199, 1 mg/ml bovine serum albumin (BSA), 1 mg/ml

polyvinyl alcohol (PVA), 10 µg/ml heparin, 50 µg/ml gentamicin

o pH: 7.45; 280 mosm

- Maturation medium

o DMEM-F12, 10% (v:v) serum replacement, 50 ng/ml epidermal growth factor, 10

µg/ml pFSH, 2 µg/ml pLH, 0.1 µg/ml cystine, 50 ng/ml cysteamine, 0.5 µl/ml

lactic acid, 0.1 µg/ml glutamine, 0.075 µg/ml ascorbic acid, 25 ng/ml PVA, 5

ng/ml myoinositol, 1 mM sodium pyruvate, 1 µl/ml insulin transferring sodium

selenite, 50 µg/ml gentamicin

o For DMEM-F12: pH:7.43; 286 mosm

- SOF-IVF

o Synthetic oviductal fluid, no glucose, 1µg/ml heparin

o pH: 7.45; 280 mosm

- Ca++free TALP

o pH: 7.4; 280 mosm

- H-SOF

o HEPES-buffered synthetic oviductal fluid

o pH: 7.38; 275 mosm

- Culture medium

o DMEM-F12, 10% fetal calf serum

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o For DMEM-F12: pH:7.43; 286 mosm

OOCYTE COLLECTION

- Preparations before going to the slaughterhouse:

o Put 3l of sterile NaCl-solution in the heating bath (38°C)

o Media

Prepare flush medium and keep at room temperature

Put dissection medium in the incubator without CO2

Prepare maturation medium and put 500 µl of it in each well of a 4-well-

plate. Put the 4-well plate in the incubator with 5% CO2. The remaining

medium is also placed in the incubator with unscrewed lid.

- Collection ovaries at slaughterhouse

- Transport: 1 hour, ambient temperature, isolated box

- Removal adherent tissues

- Ovaries rinsed twice in sterile NaCl-solution (38°C)

- Ovaries kept in clean sterile NaCl-soution (38°C) on heated surface (37°C) during further

procedure

- Aspiration of follicle fluid into sterile recipient with 16-G needle connected to vacuum

pump and flushing of the follicles with flush medium, using a 20ml-syringe with 18-G

needle

- Recipient with follicle fluid on heated surface (37°C) during entire procedure

- 10 ml of follicle fluid from the bottom of the recipient is pipetted into large Petri dish

and evaluated under stereomicroscope

- Oocytes collected in dissection medium; only oocytes with at least 3 layers of cumulus

cells

- Last 2 steps are repeated until no more oocytes are found

- Oocytes washed twice in dissection medium

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MATURATION

- Maturation medium

- Wash oocytes in first 3 wells of maturation medium

- 20-50 oocytes / 500 µl medium

- 24 h-28h

- 5% CO2

- 38.5 °C

ICSI

Preparation sperm

- Put Percoll 90% and Ca++free TALP at room temperature 1h before preparing sperm

- Thaw SOF-IVF at room temperature, vortex and leave in incubator at 38.5°C

- Preparation Percoll discontinuous density gradient (45% over 90%)

o 2 ml of Percoll 90% in tube A

o 1.5 ml of Percoll 90% mixed with 1.5 ml of Ca++free TALP in tube B

o 2 ml of 45% solution of tube B is gently pipetted on top of the 2 ml of 90%

Percoll in tube A

- Thawing sperm 1h before ICSI in 26°C H2O

- Sperm is gently put on top of Percoll gradient with 20-G needle on 1 ml syringe

- Centrifugation

o 750 x g = 2041 t/min

o 40 min

o 26°C

- Removal supernatans

- Resuspension sperm pellet in 5 ml of Ca++free TALP

- Centrifugation

o 400 x g = 1490 t/min

o 10 min

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o 26°C

- Removal supernatans

- Resuspension sperm pellet in 0.3 ml of SOF-IVF

- Just before ICSI: little bit of sperm solution is pipetted on left bottom side of drop of 9%

polyvinylpyrrolidone (PVP)

Preparation oocytes

- Decumulation

o Plate with 2 droplets of 45 µl hyaluronidase (1µg/ml) and 10 droplets of H-SOF

under mineral oil

o In hyaluronidase (1µg/ml)

3 min

Partial denudation with 170 µm pipette

o In H-SOF

Denudation by gently pipetting with 130µm pipette

MII and MI are separated

- Maturation medium

o MII oocytes and MI oocytes in separate droplets of 50 µl of maturation medium

under mineral oil at 38.5°C and 5% CO2 until ICSI

Time scheme preparation ICSI

- 4 h before ICSI: Thaw culture medium at RT, make a 4-well with 20µl

droplets under mineral oil

Make a 4-well with 50 µl droplets of maturation medium

under mineral oil for denuded oocytes

- 2.5 h before ICSI: take out Percoll, Ca++free TALP and SOF-IVF

- 1.5 h prepare plates

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o 2 decumulation plates: plate with 2 droplets of 45 µl of

hyaluronidase and 10 droplets of 45 µl of H-SOF under

mineral oil

o 2 ICSI plates: plate with 13 droplets of 5 µl of H-SOF under

mineral oil

- 1 h Prepare sperm

- 50 min 1st centrifugation

Denudation oocytes

- 10 min 2nd centrifugation

Preparation microscope and micromanipulation

ICSI

- Preparation plate

o 1 blue droplet of H-SOF is replaced by 9% PVP

o Orange droplets are numbered and +/- 10 oocytes are divided over numbered

droplets (MII oocytes first)

o Sperm is pipetted on left bottom site of PVP-droplet

o The 3 steps are repeated until all mature oocytes are injected

- ICSI

o Heated plate

o Olympus inverted microscope

o Narishige micromanipulation

o Injection of 10 oocytes takes about half an hour

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o Injected oocytes: culture medium at 38.5 °C and 5% CO2 until all oocytes are

injected

CULTURE

- Culture medium

- Prepare 4-well plate with 20 µl droplets under mineral oil

- Put in the CO2incubator for at least two hours to equilibrate

- 10-20 embryos/20µl-droplet

- 5% CO2, 5% O2, 90 % N2: Put the plates in a turtle, let the gas mix flow through the turtle

during 2 minutes and then close the turtle thoroughly

- 38.5 °C

- Day 2,5:

o Change ½ of medium by adding culture medium

o Only cleaved embryos are kept

- Day 6: Change ½ of medium

- Day 8-9: Evaluation embryos

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ADDENDUM 2 SUPPRESSION SUBTRACTIVE

HYBRIDIZATION (SSH)

This scheme represents the principle of suppression subtractive hybridization, based on

Diatchenko et al., (1996). The tester cDNA is subdivided into two portions, and each is ligated

with a different adaptor. The SSH involves two hybridizations. In the first one, an excess of

driver cDNA is added to each tester sample. The samples are denatured and allowed to anneal,

generating the type a, b, c and d molecules in each sample. In the second hybridization, the two

samples from the first hybridization are mixed together without prior denaturation, again with

an excess of driver cDNA. Only the remaining equalized and subtracted ss tester cDNAs can

reassociate and form new type e hybrids. After the ends are filled in, the type e molecules have

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two different primer annealing sites. Subsequently, all molecules are subjected to PCR to

amplify the desired differentially expressed sequences. Type a and d molecules lack primer

annealing sites and can not amplify during the PCR. Most type b molecules form a ‘pan-like’

structure. This suppression effect prevents amplification of the type b molecules. Type c

molecules, having only one primer annealing site, amplify linearly. Only the type e molecules,

representing the differentially expressed sequences, have two different adaptors and can

amplify exponentially. Finally, a second PCR is performed to further reduce background and

enrich the differentially expressed tester specific sequences.

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ADDENDUM 3 REVERSE TRANSCRIPTION QUANTITATIVE POLYMERASE CHAIN REACTION (RT-QPCR)

Figure 1 qPCR. This scheme represents the PCR reaction, using SYBR Green for quantification.

The 3 PCR steps, denaturation, primer annealing and elongation, are repeated in a cyclical

pattern. This results in exponential amplification of the target sequence. Binding of SYBR Green

to the double stranded DNA results in fluorescence, which is measured during each cycle of the

qPCR.

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When working with RNA, the RNA strand is first transcribed into complementary DNA (cDNA),

using the enzyme reverse transcriptase (RT). The cDNA can be amplified by means of the

polymerase chain reaction (PCR) (Peake et al., 1989; Mullis, 1990). The PCR cycle consists of 3

different steps. Firstly, DNA denaturation is caused by heating to 95°C. The second step involves

the annealing of primers, specifically developed for the region of interest. In third instance, the

complementary strands are synthesized, using a thermostable DNA polymerase enzyme.

Cyclical repeat of this process results in exponential amplification of the region between the

two target specific primers.

Reverse transcription quantitative PCR (RT-qPCR) is based upon this PCR reaction (Morrison et

al., 1998; Bustin, 2000; for review see Van Guilder et al., 2008). Not only can specific sequences

be detected and amplified, qPCR also allows quantification of the genetic material in the initial

samples. Quantification can be achieved through different methods. In this thesis, a DNA

binding fluorescent dye, namely SYBR Green, was used (Figure 1). Unbound dye exhibits little

fluorescence. Binding to double stranded DNA, makes the dye fluoresce. During qPCR, this

fluorescence is measured during the elongation step of each cycle. The sample fluorescence is

plotted against the qPCR cycle (Figure 2). For each sample, the quantification cycle (cq) value is

determined. This is the point where the sample fluorescence exceeds the background

fluorescence, as represented by the threshold line. A lower cq value means that the threshold

fluorescence is achieved in few cycles, so that the original sample contained a large number of

copies of the target sequence. Vice versa, a high cq value equals many cycles needed to reach

the threshold, representing little material in the initial sample. A difference of 3.3 cq reflects a

10-fold difference in the original template.

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Figure 2 qPCR amplification graph. Sample fluorescence is measured during each cycle and

plotted against the qPCR cycle. Five samples were evaluated. The orange line represents the

threshold line. Lower levels of fluorescence are considered as background. The quantification

cycle (cq) value represents the point where the sample fluorescence exceeds this threshold line,

for example around 17 for the green sample.

equine embryos produced in vitro how much · pdf filereported equine pregnancy produced by...

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