role of osteopontin in bovine sperm capacitation and

The Pennsylvania State University

The Graduate School

Intercollege Graduate Program in Physiology

ROLE OF OSTEOPONTIN IN BOVINE SPERM CAPACITATION AND

FERTILIZATION

A Thesis in

Physiology

by

David William Erikson

© 2006 David William Erikson

Submitted in Partial Fulfillment

of the Requirements for the Degree of

Doctor of Philosophy

December 2006

ii

The thesis of David William Erikson was reviewed and approved* by the following: Gary J. Killian Distinguished Professor of Reproductive Physiology Thesis Adviser Chair of Committee

Guy F. Barbato Associate Professor of Physiological Genetics

Daniel R. Hagen Professor of Animal Science

Ronald S. Kensinger Professor of Animal Nutrition and Physiology

Ramesh Ramachandran Associate Professor of Neuroendocrinology and Transgenics

Leonard S. Jefferson, Jr. Evan Pugh Professor Chair of Cellular and Molecular Physiology Chair of Intercollege Graduate Program in Physiology

*Signatures are on file in the Graduate School.

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ABSTRACT

Osteopontin (OPN) is an acidic, secreted phosphoprotein found in various tissues

including bone, milk, kidney and the male and female reproductive tracts. Bovine OPN

has a calcium binding site, a thrombin cleavage site and binds to various integrins

through which it promotes cell adhesion and intracellular signaling. OPN has also been

positively correlated to fertility in the seminal plasma of Holstein bulls and has been

identified in bovine oviductal fluid. The purpose of this research was to investigate the

presence of OPN on Holstein bull sperm, the role of OPN in bovine fertilization and the

effects of OPN on sperm capacitation, intracellular calcium content, mitochondrial

activity and viability to gain a better understanding of its role in fertility. Semen was

collected by artificial vagina and solubilized sperm membranes were subjected to SDS-

PAGE and Western blot analysis. A polyclonal rabbit antibody to purified bovine OPN

(anti-OPN) detected OPN on sperm membranes, in testis homogenates and in cauda

epididymal fluid. Results indicated that ejaculated sperm had approximately 50% more

OPN than epididymal sperm and the protein was localized to the post-acrosomal region

of the sperm head as well as the midpiece. Fertilization rates were reduced and incidence

of polyspermy increased when sperm were incubated in anti-OPN prior to exposure to

oocytes. OPN was localized to the same postacrosomal region on sperm in which the

acrosome reaction had been induced. Integrin subunits known to associate with OPN

were identified on bovine sperm and oocytes using SDS-PAGE and Western blot

analysis. Other studies have shown that antibodies to these integrins decreased

fertilization rates in the same manner as anti-OPN. These results suggest that OPN

interacts with integrins during bovine fertilization, and may be involved in a block to

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polyspermy. OPN was also able to induce sperm capacitation and positively influenced

sperm viability, but had no effect on sperm intracellular calcium content or mitochondrial

activity. Taken together these results suggest exciting new roles for OPN in male

reproductive physiology.

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TABLE OF CONTENTS LIST OF FIGURES ................................................................................................... viii ACKNOWLEDGEMENTS...........................................................................................x CHAPTER ONE: INTRODUCTION ..........................................................................1 CHAPTER TWO: REVIEW OF THE LITERATURE ..............................................3

The Spermatozoon .....................................................................................................3 The Sperm Head ......................................................................................................5 The Sperm Tail.........................................................................................................6 The Surface of the Spermatozoon .............................................................................7

Capacitation ...............................................................................................................8 The Acrosome Reaction...........................................................................................10 Oviductal Fluid ........................................................................................................11

The Effect of Oviductal Fluid on Sperm..................................................................12 Oviductal Fluid Proteins Associate with Sperm......................................................13

Fertilization ..............................................................................................................13 Sperm Interaction with the Zona Pellucida.............................................................16 Sperm Interaction with the Oocyte Plasma Membrane ...........................................17

Integrins ...................................................................................................................18 Integrins in Reproduction ......................................................................................19

The Oocyte at Fertilization......................................................................................21 Osteopontin ..............................................................................................................22

Osteopontin in Reproduction..................................................................................23 Research Objectives.................................................................................................25 Animals Used in These Studies................................................................................25 Collection of Oviductal Fluid ..................................................................................25 Antibody to Bovine Osteopontin .............................................................................26

CHAPTER THREE: DETECTION OF OSTEOPONTIN ON HOLSTEIN BULL SPERMATOZOA, IN CAUDA EPIDIDYMAL FLUID AND TESTIS HOMOGENATES, AND ITS POTENTIAL ROLE IN BOVINE FERTILIZATION......................................................................................................................................27

Introduction .............................................................................................................27 Materials and Methods............................................................................................28

Isolation of Sperm Membranes from Ejaculated Sperm ..........................................28 Isolation of Cauda Epididymal Sperm Membranes and Preparation of Cauda Epididymal Fluid ...................................................................................................29 Collection and Preparation of Testis Tissue Samples .............................................29 1D SDS-PAGE and Western Blot Analysis .............................................................30 Immunocytochemistry of Ejaculated and Cauda Epididymal Spermatozoa .............31 Adsorption of Osteopontin Antibody with Osteopontin ...........................................31 Oocyte Collection and Maturation .........................................................................32 Sperm Preparation.................................................................................................33 Sperm � Oocyte Binding.........................................................................................33

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In vitro Fertilization of Oocytes with Sperm Incubated in Osteopontin Antibody ....33 Statistical Analysis.................................................................................................34

Results ......................................................................................................................34 Osteopontin on Sperm and in Cauda Epididymal Fluid and Testis Homogenates ...34 Immunocytochemistry of Ejaculated and Epididymal Sperm...................................35 Analysis of Sperm Treated with OPN-Adsorbed Anti-OPN .....................................36 In vitro Fertilization of Oocytes with Sperm Incubated in Osteopontin Antibody ....36 Binding of Sperm Incubated with Osteopontin Antibody to Oocytes .......................37

Discussion .................................................................................................................47 CHAPTER FOUR: EVIDENCE FOR THE ROLES OF OSTEOPONTIN AND αV AND α5 INTEGRIN SUBUNITS IN BOVINE FERTILIZATION ...........................52


Preparation of Capacitated and Acrosome-Reacted Sperm ....................................54 Detection of Osteopontin on Sperm ........................................................................54 Detection of Acrosome-Reacted Sperm...................................................................55 Detection of Osteopontin on Acrosome-Reacted Sperm ..........................................55 Detection of Osteopontin in Bovine Oocytes...........................................................56 Detection of Integrins in Solubilized Sperm Membranes and Bovine Oocytes.........56

Results ......................................................................................................................57 Localization of Osteopontin on Acrosome-Reacted Sperm......................................57 Detection of Osteopontin on Western Blots of Bovine Oocytes and of Integrin Subunits αv and α5 on Western Blots of Sperm Membranes and Bovine Oocytes .....58

Discussion .................................................................................................................61 CHAPTER FIVE: INFLUENCE OF OSTEOPONTIN, CASEIN AND OVIDUCTAL FLUID ON BOVINE SPERM CAPACITATION, INTRACELLULAR CALCIUM CONTENT, MITOCHONDRIAL ACTIVITY AND VIABILITY ........................................................................................................65


Capacitation and Acrosome Reaction of Sperm......................................................66 Detection of the Acrosome Reaction and Sperm Viability .......................................67 Detection of Sperm Intracellular Calcium and Cell Viability..................................68 Detection of Mitochondrial Activity in Sperm.........................................................68 Binding of Biotinylated Osteopontin and Casein to Sperm .....................................68 Statistical Analysis.................................................................................................69

Results ......................................................................................................................69 Effect of Treatments on Capacitation and Acrosome Reaction................................69 Effect of Treatments on Mitochondrial Activity ......................................................70 Effect of Treatments on Intracellular Calcium........................................................70 Effect of Treatments on Sperm Viability .................................................................70 Osteopontin and Casein Binding to Sperm .............................................................71

Discussion .................................................................................................................80

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CHAPTER SIX: CONCLUSIONS AND FUTURE STUDIES .................................84 REFERENCES ............................................................................................................88

viii

LIST OF FIGURES Figure 2.1 Structure of mammalian sperm Figure 2.2 Sequence of mammalian fertilization Figure 3.1 Representative Western blot of osteopontin in ejaculated and cauda

epididymal Holstein bull sperm Figure 3.2 Representative Western blot of osteopontin in cauda epididymal fluid and

homogenates of testicular parenchyma from Holstein bulls Figure 3.3 Densitometric analysis of osteopontin in ejaculated and cauda epididymal

Holstein bull sperm membranes Figure 3.4 Immunofluorescent localization of osteopontin on ejaculated Holstein bull

sperm Figure 3.5 Immunofluorescent localization of osteopontin on cauda epididymal

Holstein bull sperm Figure 3.6 Side-by-side comparison of osteopontin localization on Holstein bull

sperm Figure 3.7 Negative controls omitting osteopontin antibody Figure 3.8 Determination of specificity of osteopontin antibody to osteopontin on

sperm with osteopontin-adsorbed polyclonal rabbit antibody to purified bovine milk osteopontin

Figure 3.9 Mean percentage ± SEM of oocytes fertilized, including normal and

polyspermic fertilization Figure 3.10 Mean percentage ± SEM of oocytes fertilized normally, excluding

polyspermic fertilizations Figure 3.11 Mean percentage ± SEM of polyspermic fertilizations, expressed as a

percentage of total fertilizations Figure 3.12 Mean number ± SEM of sperm bound per zona pellucida Figure 4.1 Immunofluorescent localization of osteopontin on acrosome-reacted and

intact sperm Figure 4.2 Representative Western blot of integrin subunits in bovine sperm

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Figure 4.3 Representative Western blot of integrin subunits in bovine oocytes Figure 4.4 Representative Western blot of osteopontin in bovine oocytes Figure 4.5 Interactions between extracellular matrix components and integrins at

fertilization Figure 5.1 Effect of osteopontin treatment on sperm capacitation, represented as the

mean percentage ± SEM of acrosome-reacted live sperm in each treatment

Figure 5.2 Effect of oviductal fluid treatment on sperm capacitation, represented as

the mean percentage ± SEM of acrosome-reacted live sperm in each treatment

Figure 5.3 Effect of osteopontin treatment on mitochondrial activity in sperm,

represented as mean fluorescence ± SEM Figure 5.4 Effect of oviductal fluid treatment on mitochondrial activity in sperm,

represented as mean fluorescence ± SEM Figure 5.5 Effect of osteopontin treatment on intracellular calcium content in sperm,

represented as mean fluorescence ± SEM Figure 5.6 Effect of oviductal fluid treatment on intracellular calcium content in

sperm, represented as mean fluorescence ± SEM Figure 5.7 Effect of osteopontin treatment on sperm viability, represented as the

mean percentage ± SEM of viable sperm Figure 5.8 Effect of oviductal fluid treatment on sperm viability, represented as the

mean percentage ± SEM of viable sperm Figure 5.9 Association of biotinylated osteopontin and casein with sperm,

represented as mean fluorescence ± SEM Figure 6.1 Interaction of osteopontin and integrins during fertilization.

x

ACKNOWLEDGEMENTS

I would like to thank my adviser, Dr. Gary Killian, and my committee members,

Drs. Guy Barbato, Dan Hagen, Ron Kensinger and Ramesh Ramachandran for their

advice and assistance in the preparation of this thesis.

I thank Dr. Amy Way, Dave Chapman, Jackie Pollock and the rest of the staff,

both past and present, at the Almquist Research Center for their technical assistance.

I thank the numerous undergraduate students I had the pleasure to supervise over

the last six years for their friendship and invaluable help in the completion of these

experiments.

In addition, I thank the graduate students in the Department of Dairy & Animal

Science and Intercollege Graduate Degree Program in Physiology for their friendship and

assistance. I would also like to acknowledge the many visiting scientists employed at the

Almquist Research Center for their contributions to this work.

I would like to thank Travis Edwards, Bill Weaver and Les Carlson for sample

collection and animal care, and Elaine Kunze, Susan Magargee and Nicole Bem at the

Cell Analysis and Imaging Center for their friendship and assistance with flow cytometry

analysis.

Most importantly I would like to thank my family for their support while I

completed my degree.

Thank you all.

CHAPTER ONE

INTRODUCTION

Artificial insemination has contributed greatly to the success of the dairy industry

for the past 50 years. This technique has allowed for continued improvement in milk

production and genetic merit in cattle herds worldwide. In fact, an entire industry

focused on genetic selection of animals as well as semen evaluation and cryopreservation

has been built around selecting sires of high genetic value with superior semen quality.

As a result of careful monitoring and data collection, the fertility of individual

sires can be determined using non-return rates of inseminated females. Studies have

shown that factors in bull seminal plasma, the fluid component of semen, can both

positively and negatively affect fertility [1, 2]. In an analysis of seminal plasma from

Holstein bulls of varying fertility, four proteins were found to be correlated to fertility

[1]. Two of these proteins, later identified as lipocalin-type prostaglandin D synthase [3]

and osteopontin [4] were correlated to high fertility.

Products of the male reproductive tract are not the only factors capable of

affecting male fertility. Sperm are in contact with male fluids for a very brief time before

they are disseminated into the female reproductive tract during natural mating, where

they may reside for nearly a day before fertilizing an ovum [5]. Studies using oviductal

fluid indicate that the oviduct can have positive effects on sperm motility, capacitation

and fertilizing ability [6-8]. Oviduct-specific glycoprotein, which is actively produced

and secreted by the oviduct at estrus has demonstrated such positive effects on sperm

physiology [9].

2

Despite the success of semen cryopreservation and artifical insemination, an

interest in improving the fertility of frozen-thawed sperm still remains. Fertility has

emerged as a growing challenge in high-producing dairy herds around the world [10].

Successful selection for production traits has negatively impacted reproductive

performance in dairy cattle [10]. It is therefore important to identify potential fertility-

related factors in both the male and female and investigate their effects on sperm before

they can be used for practical means. By understanding how these proteins may impact

fertility and interact in vivo, new therapeutic methods to improve reproductive

performance can be developed [11].

The objectives of this study were to identify the secreted phosphoprotein

osteopontin on Holstein bull sperm and determine the effects of the protein on sperm

physiology. Identified in oviductal fluid [12] and as a factor correlated to high fertility in

Holstein bull seminal plasma [4], it is important to elucidate the potential positive effects

of this protein on sperm.

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CHAPTER TWO

REVIEW OF THE LITERATURE

The Spermatozoon

Spermatozoa are the haploid germ cells of the male, produced in the seminiferous

tubules of the mammalian testis during a process called spermatogenesis. In the bull,

spermatogenesis takes approximately 61 days [13] and consists of three stages:

proliferation of spermatogonia, meiosis, which produces spermatids from primary and

secondary spermatocytes and differentiation, which marks the production of a fully

differentiated sperm cell [14]. It requires 4.5 cycles, of 13.5 days each for spermatogonia

to develop into fully developed spermatozoa [13]. Each cycle is the interval between two

consecutive cellular associations appearing at the same location in the seminiferous

epithelium.

Mammalian sperm share a similar structure (Figure 2.1): a 2-5 µM diameter head,

which contains the nucleus and the acrosome; a tail which is 10-100 µM in length,

comprised of a �9+2� complex of microtubules characteristic of eukaryotic flagella and

cilia; and a midpiece containing the mitochondria. The head is joined to the midpiece by

the connecting piece and the midpiece is joined to the principal piece of the tail by the

annulus. The cytoplasmic volume of sperm is extremely small and primarily located in

the sperm head. Sperm lack the cellular organelles to synthesize proteins or nucleic acids

and serve only to deliver genetic material to the oocyte [15].

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Figure 2.1. Structure of mammalian spermatozoa. Sperm cells are polarized cells with a head, flagellum and midpiece (A, schematic surface drawing). The sperm head can be subdivided in four regions: apical, pre-equatorial, equatorial and post-equatorial regions. The acrosome (large secretory vesicle, 3) is situated apical to the nucleus (B). After binding of the sperm cell to the oocyte with its apical plasma membrane, the plasma membrane fuses with the underlying acrosomal membrane at multiple sites (C). The acrosomal content (hydrolytic enzymes) will be secreted, which enables the sperm cell to digest the egg extracellular matrix (ZP). After the acrosome reaction has been completed, the inner acrosomal membrane forms a continuum with the remaining plasma membrane (D). This hairpin structure is involved in the primary binding of the sperm cell to the oolemma. Note that the representations (B), (C) and (D) are cross-sections through a flattened cell. 1: plasma membrane; 2: outer acrosomal membrane; 3: acrosomal content; 4: inner acrosomal membrane; 5: nuclear envelope; 6: nucleus containing highly condensed DNA; 7: posterior ring; 8: midpiece; 9: mitochondrion; 10: annular ring; 11: flagellum; 12: mixed vesicle (i.e. plasma membrane fused with outer acrosomal membrane); 13: acrosomal secretion; 14: hairpin structure. Source: FM Flesch, BM Gadella, Biochim Biophys Acta 1469 (2000) 197-235.

5

The Sperm Head

The head of a mammalian sperm consists of the nucleus, acrosome and

postacrosomal region; the postacrosomal region includes the equatorial segment. The

nucleus of the sperm cell contains highly condensed chromatin, which is extremely stable

due to extensive disulfide bonds [16-18]. The high levels of condensation leave the

sperm nucleus at metabolic rest and highly resistant to digestion [18]. This condensation

of the nucleus as well as the tapered-shape of the sperm head may aid in penetration of

the zona pellucida (ZP) in mammalian species [18]. Uniformity between individual

sperm produced by a single animal is significant, as hundreds of millions of sperm are

produced on a daily basis with little to no developmental differences or variation in head

shape [18].

The acrosome is a cap-like structure closely associated with the anterior portion of

the sperm head [19, 20]. Three layers are present in the bovine acrosome: an inner

membrane, an outer membrane and a middle layer. The inner and outer membranes are

continuous with one another at the posterior edge of the acrosome, with the acrosomal

contents contained completely within this single membrane [21]. The outer membrane

lies close to the cell plasma membrane, while the inner membrane is closely associated

with the nuclear envelope [18]. Analyses of the acrosome suggest it is rich in

carbohydrates, including galactose, mannose, fucose, galactosamine, glucosamine and

sialic acid [22, 23]. Acrosomal contents, released upon completion of the acrosome

reaction, include acid phosphatase, β-glucuronidase, n-acetylglucosaminidase and

acrosin, a trypsin-like protease [24-29]. Because these contents lead to digestion and

6

penetration of the ZP by sperm, the acrosome has been characterized as a specialized

lysosome [18].

The postacrosomal region of the sperm head lies behind the posterior portion of

the acrosome, contains the equatorial segment which forms after the acrosome reaction,

and is the region on sperm where binding and fusion to the oocyte occurs. Not much is

known of the chemical makeup of the equatorial region, as its location makes it difficult

to isolate for analysis [18]. Formation of the equatorial segment requires loss of the

anterior portion of the acrosome during the acrosome reaction; the outer acrosomal

membrane fuses with the cell plasma membrane, leaving the inner acrosomal membrane

present in the anterior portion of the sperm head. This results in the formation of the

equatorial segment in the intact posterior region of the acrosome [21].

The Sperm Tail

The mammalian sperm tail is composed of three parts: the middle piece or

midpiece, principal piece and endpiece. The midpiece contains mitochondria which

generate the necessary energy for sperm motility [18]. The mitochondria are arranged

end to end in a helix [30].

The major functional unit of the sperm tail is the axial filament complex or

axoneme. The axoneme consists of two central microtubules surrounded by a row of

nine evenly spaced doublet microtubules [18, 30]. Called a �9+2� pattern, this

arrangement of microtubules occurs in flagella throughout plant and animal species [31,

32]. On the midpiece and anterior portion of the principal piece the axoneme is

surrounded by nine outer dense fibers, creating a �9+9+2� pattern [18, 30]. The function

of the axoneme is to produce movement of the flagella or sperm tail, thus creating

7

forward movement of the sperm. This is accomplished by a sliding mechanism similar to

that found in skeletal muscle and powered by the protein dynein. Dynein is responsible

for converting chemical energy into mechanical movement through its ATPase activity

[18, 33, 34].

The Surface of the Spermatozoon

The surface of ejaculated spermatozoa may have as many as 300 different

proteins [35]. Some of these proteins are glycosylated and form the sperm glycocalyx, a

dense-coating of carbohydrate-rich molecules located outside the membrane bilayer [36,

37]. The sperm glycocalyx is composed of lipid-bound carbohydrates, anchored in the

lipid-bilayer of the cell as well as carbohydrates loosely bound to the sperm via

superficial polar groups or hydrophobic interactions [37]. Most attention given to the

sperm membrane has focused on the specific make-up of the glycocalyx, as many

interactions between sperm and other cells, such as species-specific recognition of

oocytes, is carbohydrate-dependent.

Three classes of glycoproteins can be found on the sperm surface. The first class

is comprised of proteins produced in the testis with transmembrane domains integrated

into the sperm membrane. A second class of glycoproteins consists of proteins that

become associated with the sperm either in the testis or epididymis through polar protein-

protein or lectin-carbohydrate interactions. These interactions may be of high affinity

and specificity, but may also include low affinity interactions with proteins released from

the sperm surface during capacitation [38] such as the spermadhesins [39]. The final

class of sperm glycoproteins are those proteins that undergo a secondary integration into

the sperm membrane [37]. These interactions include those facilitated by a

8

glycophosphatidylinositol (GPI) anchor, but may also include membrane-membrane

interactions between the sperm and epididymal or accessory sex gland proteins [37, 40,

41].

During transport through the epididymis, sperm membrane alterations occur and

sperm acquire the ability to be motile and to fertilize [42-44]. At ejaculation, sperm

undergo further alterations after they are mixed with seminal plasma, a mixture of

testicular, epididymal and accessory sex gland secretions [45]. Several proteins from

seminal plasma, including BSP-A1, BSP-A2, BSP-A3 and BSP-30-kDa (collectively

referred to as the BSP proteins) are products of the vesicular glands in bulls [46] and bind

to ejaculated sperm [45, 47]. The BSP proteins, as well as other proteins of seminal

plasma, may serve as �decapacitation factors� [48, 49] to prevent sperm from

capacitating or undergoing the acrosome reaction [50-53]. These proteins must be

removed or altered before the sperm is able to fertilize an oocyte [52-56].

Capacitation

Following spermiation, or the release of the sperm into the lumen of the

seminiferous tubule, sperm are carried through the rete testis in the testicular effluent to

the epididymis where they undergo the final stages of maturation. Sperm found in the

distal or cauda epididymides are capable of fertilization [57], but only after a period of

incubation in the female reproductive tract. The changes that occur to sperm during this

period in the female tract are collectively referred to as capacitation. First described

independently by Austin [58] and Chang [49], capacitation includes a number of

molecular, biochemical and physiological changes to sperm which enables them to

undergo the acrosome reaction and fertilization when presented with the appropriate

9

stimulus. Capacitated sperm from many rodent species may also become very actively

motile, a condition referred to as hyperactivation.

While the in vivo location of capacitation has not been determined, it can occur in

the uterus [58-61] and is accelerated by sperm exposure to the uterus and then oviduct

[55, 60, 62, 63]. More recent studies utilizing regional and staged oviductal fluid and

conditioned medium from cultured oviductal epithelium explants have shown that the

isthmus of the oviduct is the likely site of sperm capacitation [6, 64-67].

During capacitation, sperm undergo substantial alterations. These changes

include the loss or modification of surface antigens [68-70], changes in the distribution of

intramembranous particles [71-74], alterations in net surface charge [22, 75-78],

alterations in the carbohydrate moieties of surface glycoproteins [79-84] and alteration of

membrane sterols [85-88], changes in intracellular ions, the adenylate cyclase cAMP

system and plasma membrane [89].

One of the most important advances in our understanding of capacitation is the

discovery that a heparin-like glycosaminoglycan in oviduct fluid is a potential

capacitating agent [90] and that sperm can be artificially capacitated using heparin [91].

While other methods of artificial capacitation exist [92], capacitation by heparin is the

preferred method [93]. The mechanism by which heparin capacitates sperm has been

characterized [91, 94-97] and includes regulation of protein tyrosine phosphorylation

[94], increase in intracellular pH [95] and an increase in intracellular calcium [93].

Changes in intracellular calcium are regulated by a Ca2+-ATPase extrusion system [98], a

Na2+/Ca2+ antiporter [99], voltage-activated calcium channels [100] and intracellular

calcium stores [101], including influx of calcium into an acrosomal reservoir [93, 102].

10

The increase in intracellular pH may be due to movement of bicarbonate ions into sperm.

Bicarbonate has been identified as essential to sperm capacitation, and while little is

known about its transport across the sperm membrane, it regulates sperm cAMP

metabolism by stimulating adenylyl cyclase [102-107]. Changes in the adenylyl cyclase

system have been demonstrated in sperm capacitation [89].

Capacitation also involves cholesterol efflux from the sperm membrane, with

albumin present in both the oviduct and in vitro capacitation media acting as a �sink� for

cholesterol by removing it from the sperm membrane [53, 108-114]. Partial induction of

cholesterol efflux can be stimulated by BSP proteins in vitro [115]. The removal of

cholesterol leads to changes in the cholesterol/phospholipids ratio of the sperm

membrane, likely resulting in redistribution of other membrane components.

Characteristic of this redistribution is increased methylation of membrane phospholipids,

concentration of phosphatidylcholine to the inner portion of the membrane and

production of fusogenic lysophospholipids [89, 116, 117].

The Acrosome Reaction

Sperm that have been capacitated are able to undergo the acrosome reaction. The

acrosome reaction involves fusion of the plasma membrane overlying the acrosome with

the outer acrosomal membrane, formation of vesicles and time-dependent release of

hydrolytic enzymes from the acrosome and finally the disappearance of acrosomal

contents and hybrid vesicles held together by the acrosomal matrix. Fusion of the plasma

and outer acrosomal membranes coupled with the release of hydrolytic enzymes allows

the sperm to penetrate the zona pellucida and fertilize an oocyte [118, 119].

11

The acrosome reaction is a contact-mediated event, as ZP3, a ZP protein,

stimulates the reaction in capacitated sperm. It is believed that oligosaccharide chains

polyvalently bound to ZP3 actually account for this stimulation, crosslinking and forming

multimers with sperm receptors, possibly β1-4 galactosyltransferase-1, to initiate

exocytosis of acrosomal contents [120, 121].

A major event in the acrosome reaction is a sustained influx of calcium that is

directly responsible for exocytosis. Studies have shown that exocytosis of acrosomal

contents shares many similarities with formation of exocytic vesicles in secretory cells

[122]. The binding of sperm to ZP3 results in the opening of low voltage-activated

calcium channels, causing a transient calcium influx and activation of the G-proteins Gi1

and Gi2. Phospholipase C (PLC) is then activated, elevating intracellular pH and

producing the sustained calcium flux [120, 121]. Activation of adenylyl cyclase and an

increase in cAMP also occurs, leading to activation of protein kinases and Ca2+ and

phospholipid-dependent kinases [123]. The rise in calcium stimulates phospholipase A2

(PLA2), an enzyme which cleaves fatty acids from phospholipids, forming

lysophospholipids [124]. This promotes fusion and vesiculation of the sperm

membranes, forming hybrid vesicles of the plasma and outer acrosomal membranes.

Ultimately, this allows the release of acrosomal contents at the site of sperm-zona

binding, resulting in penetration of the sperm through the ZP [124].

Oviductal Fluid

After ejaculation, sperm are disseminated into the fluids of the female

reproductive tract. Sperm are present in the oviduct in as little as fifteen minutes, and

may reside in the oviductal isthmus for up to 20 h before fertilization [5] in an oviductal

12

sperm reservoir [125-127]. The oviduct provides the environment for sperm capacitation,

the acrosome reaction, fertilization and early embryo development. During these

processes, both gametes are bathed in oviductal fluid (ODF), a combination of serum

transudate and components produced and actively secreted by the oviductal epithelium

[128]. Collection of ODF to study its effects on sperm physiology can be accomplished

by a variety of methods, including placement of indwelling catheters into the oviduct

[129]. Modification of an existing procedure allows for collection of daily isthmic and

ampullary fluid samples by ligating the oviduct between these two regions [130]. By

measuring serum progesterone levels, the stage of the estrous cycle (luteal stage:

progesterone > 1.5 ng ml-1; non-luteal: progesterone ≤ 1.5 ng ml-1) of daily ODF samples

can also be determined [131]. Collection of regional (ampullary or isthmic) and staged

(luteal or non-luteal) ODF is possible through these methods. ODF contains

approximately 20-30% of the total protein found in blood serum. Maximal production of

ODF occurs during the non-luteal phase and decreases during the luteal phase [132]. In

addition, protein concentration is lower in the non-luteal phase of the estrous cycle, but

total protein is higher during the non-luteal phase [132].

The Effect of Oviductal Fluid on Sperm

Functional studies utilizing regional and staged ODF revealed sperm are more

likely to undergo the acrosome reaction after exposure to isthmic non-luteal ODF

(INLODF), while sperm incubated in ampullary non-luteal ODF (ANLODF) are more

likely to fertilize oocytes suggesting the isthmus is the site of capacitation and the

ampulla is the site of fertilization [6-8, 125]. In addition, conditioned medium (CM)

from cultured estrual isthmic oviduct explants showed increased levels of capacitated

13

sperm [67] and the secretory products of the oviduct, especially at estrus, increased sperm

viability and maintained motility [133]. The capacitating effect of estrual isthmic CM

may be associated with proteins, glycosaminoglycans or proteoglycans [67].

Regional and staged ODF also differentially affects the ability of sperm to bind to

the ZP. Sperm exposed to isthmic ODF bind in higher numbers to oocytes than sperm

treated with ampullary ODF. In addition, sperm treated with ANLODF have greater

fertilization rates than isthmic ODF, suggesting sperm exposed to ampullary fluid in vivo

may be best prepared for fertilization [6]. Non-luteal ODF also capacitates more sperm

than luteal ODF, suggesting a stage-related effect of the oviduct on sperm [134].

Oviductal Fluid Proteins Associate with Sperm

In addition to affecting sperm physiology, components of oviductal secretions

associate with sperm. Several ODF proteins are known to associate with sperm [133,

135-138], including oviduct-specific glycoprotein (OSG), a protein of oviductal origin

most prevalent during the peri-ovulatory period [136]. The effects of OSG on sperm are

well-documented and include maintenance of motility and viability, increased

capacitation, increased fertilization rates, and improved development of embryos derived

from OSG-treated sperm [133, 139-141]. Despite its apparent positive effects on sperm-

related events in the oviduct, OSG-knockout mice are fertile, questioning the necessity of

OSG for normal reproductive function [142].

Fertilization

Fertilization is defined as the successful union of a sperm and an oocyte to create

a one-celled embryo called a zygote. The process of fertilization (Figure 2.2) can be

separated into seven distinct events that occur in a necessary order: 1) binding of the

14

sperm to the ZP; 2) the acrosome reaction; 3) penetration of the ZP; 4) sperm binding to

the oocyte plasma membrane: 5) fusion of sperm and oocyte membranes; 6)

incorporation of the sperm head into the ooplasm and formation of the male pronucleus;

7) syngamy, the fusion of the male and female pronuclei. After successful completion of

these steps, an oocyte has been fertilized and becomes a zygote, and additional sperm can

no longer interact with the ZP [143].

Sperm associate with oocytes in three distinct events. First the sperm contacts the

cumulus cells and hyaluronic extracellular matrix of the egg; second the sperm contacts

the structure surrounding the oocyte, the ZP; finally, the sperm binds to and fuses with

the oocyte plasma membrane.

15

Figure 2.2. Sequence of mammalian fertilization. (A) Freshly ejaculated sperm cells are activated in the female genital tract during a process called capacitation. (B) Capacitated sperm cells are hypermotile and are able to bind to the egg extracellular matrix (ZP). (C) Binding of sperm cells to the ZP triggers the acrosome reaction and acrosomal enzymes are secreted. (D) Hydrolytic enzymes secreted from the acrosome degrade the ZP and subsequent sperm cells penetrate the ZP, enter the perivitelline space and bind to the oolemma with the apical tip. (E) Subsequent to apical tip binding, oolemmal binding changes to the hairpin structure of the acrosome reacted sperm cell. (F) After hairpin structure binding to the oolemma, the sperm cell fuses with the oocyte and the sperm cell is subsequently incorporated in the oocyte. 1: perivitelline space; 2: ZP; 3: oolemma (egg plasma membrane). Source: FM Flesch, BM Gadella, Biochim Biophys Acta 1469 (2000) 197-235.

16

Sperm Interaction with the Zona Pellucida

The ZP is a filamented structure composed of three noncovalently bound proteins

synthesized and secreted by the oocyte called ZP1, ZP2 and ZP3. Research in the mouse

has revealed that carbohydrate moieties of ZP3 participate in sperm-oocyte binding while

ZP1 and ZP2 provide structural support to maintain the integrity of the ZP [144, 145].

During the acrosome reaction, however, the affinity of sperm for the ZP changes from

ZP3 to ZP2, possibly as a means to keep the sperm anchored during rearrangement of the

sperm membrane and ZP [146]. High levels of conservation across species (50-98%

nucleic acid similarity) suggest similar functions for the proteins, but species-specific

functional differences are present [147-150]. ZP3 is the most highly conserved ZP

protein: the protein in the mouse, pig and rabbit are 70% identical to human ZP3, while

ZP3 in various primate species is over 90% identical to that of the human [148, 150].

Evidence points to the initial attachment of sperm to the ZP to be a carbohydrate-

mediated event, possibly involved in species-specificity of the sperm-ZP interaction

[143]. While numerous sperm proteins are thought to bind to ZP3, only β1,4 �

galactosyltransferase-1 fulfills all of the criteria required of a ZP3 receptor [144].

However, the following tight binding of the sperm and ZP is the result of carbohydrate-

carbohydrate, carbohydrate-protein and protein-protein interactions, leading to the

acrosome reaction [151-153]. Most of the work done to elucidate this sequence of events

has been performed in the mouse, with additional investigation in pig and rabbit sperm-

ZP interactions. In the mouse, O-linked oligosaccharides covalently linked to ZP3 have

been identified as the ZP-ligand responsible for sperm-ZP binding [154, 155]. There is,

however, disagreement over which specific carbohydrate moiety is involved [147], and

17

no fewer than four (α-galactose, β-N-acetylglucosamine, fucose and mannose) have been

identified as potential participants in this event [144]. It is more likely that multiple

ligands on the ZP interact with multiple sperm proteins for optimal regulation of the

binding events preceding fertilization [147].

Acrosome-reacted sperm bind to the ZP and must penetrate the membrane in

order to fuse with the plasma membrane of the oocyte. This penetration is accomplished

through a combination of sperm motility and enzymatic hydrolysis of the ZP by acrosin,

an acrosomal serine protease. Following zona penetration the sperm enters the

pervitelline space where it will bind to and eventually fuse with the oocyte plasma

membrane [143].

Sperm Interaction with the Oocyte Plasma Membrane

The molecules involved in sperm-egg plasma membrane binding and fusion have

been studied intensely in recent years [120, 122, 143, 144, 156-161] and a number of

sperm proteins have been identified as potential players in species-specific binding of

sperm to oocytes [150, 162, 163]. The most highly investigated of these are oocyte

integrins (α6β1 and others) and sperm disintegrin-like proteins, namely several ADAM (a

disintegrin and metalloprotease domain) proteins [156] including fertilin α, fertilin β and

cyritestin.

The sperm disintegrins partially share a domain structure with the snake venom

metalloproteases, including a metalloprotease domain, a disintegrin domain and/or a

cysteine-rich domain [164]. The discovery of fertilin, or PH30, on guinea pig sperm

resulted in the classification of the ADAM protein family [165]. The identification of the

disintegrin domains in ADAM proteins led to the hypothesis that a protein on sperm

18

containing such a domain could potentially mediate sperm-egg binding by interacting

with an oocyte integrin [156].

Fertilin is a heterodimer of α and β subunits and its discovery led to the idea that

an oocyte integrin is needed for membrane fusion during fertilization [166]. Fertilin β,

which contains a disintegrin-like domain, has been implicated in sperm-egg binding [167-

170], namely to the oocyte plasma membrane integrin α6β1 [170]. Fertilin α contains a

disintegrin domain [171, 172], but also contains a short sequence similar to viral fusion

peptides [173], and is thought to function in fusion of the sperm and oocyte membranes

[163, 174, 175]. Cyritestin, another ADAM protein found on sperm may also have a role

in sperm-egg binding [121].

Integrins

Integrins are important cell membrane receptors for cell adhesion to extracellular

matrix (ECM) proteins and play major roles in cell-cell adhesion [176]. Heterodimers of

α and β subunits, integrins are specific to metazoan species [177] and there are currently

18 known α subunits and eight known β subunits which can specifically associate to form

24 integrin heterodimers [176]. These integrins can be divided into four groups based on

their ligand binding characteristics: RGD receptors, laminin receptors, collagen receptors

and leukocyte-specific receptors [176]. Based on studies with knockout mice, each of the

24 identified integrins appears to have a nonredundant function, and phenotypes range

from a complete block in preimplantation development (β1), to developmental defects (α4,

α5, αv, β8), defects in leukocyte function (αL, αM, αE, β2, β7), bone remodeling (β3),

inflammation (β6), hemostasis (αIIb, β3, α2) and angiogenesis (α1, β3) [176, 178-181]. The

α and β subunits combine with each other in a noncovalent complex, and contain a small

19

C-terminal cytoplasmic domain, a transmembrane region and a larger N-terminal

extracellular domain [182]. Many integrins are expressed on cell surfaces in an inactive

state, and must be activated through other signaling pathways before they engage in

ligand binding or signaling [176]. Most of the known integrins recognize short peptide

sequences, many of which contain an acidic amino acid such as the RGD (Arg-Gly-Asp)

motif [176].

Integrins in Reproduction

Found on nearly all mammalian cells and in the ECM of most tissues, integrins

serve important functions in reproductive events. All mammalian oocytes express

integrins on their surface [170, 183-185], and there is evidence that the α6β1 integrin

serves as a sperm receptor during fertilization [170]. Several integrin subunits have been

localized to the human sperm equatorial segment [186, 187] with regulation of α5β1 and

αvβ3 integrins varying depending on the capacitation status of the sperm. Integrins

containing recognition sites for an RGD-containing ligand have been investigated and

this sequence does not appear to be important for mouse or human fertilization [170], but

the use of RGD-peptides does inhibit bovine fertilization [188]. Thus, there appear to be

some species-related differences in how integrins and their ligands cooperate during

fertilization.

Integrins are also important players in the ruminant implantation cascade. The

best characterized of these integrins is αvβ3, which recognizes the RGD sequence. This

sequence has been demonstrated to modulate trophoblast attachment and outgrowth [189,

190] and blocking either the integrin or amino acid binding sequence on the ligand

reduces implantation in the mouse [191]. Osteopontin (OPN), an RGD containing

20

glycoprotein, is produced and secreted by the endometrium at the time of implantation,

localized to the apical surfaces of luminal and glandular epithelium as well as the

trophoblast in sheep [192]. Integrins that could assemble to form αvβ3, αvβ1, αvβ5, α4β1,

and α5β1 heterodimers were detected on trophoblast and the apical surface of endometrial

luminal epithelium, suggesting that integrins are involved in the attachment of the

conceptus to the uterine wall via RGD containing proteins such as OPN [192, 193].

ECM proteins, particularly integrins, are also involved in embryonic development

by affecting cell growth, migration and differentiation [194-196]. In mice, the integrins

α5β1, α6β1 and αvβ3 are expressed by the embryo throughout early development and other

β1-associating α subunits are developmentally regulated [185]. Regulation of integrins is

most evident at the late blastocyst stage prior to implantation, and the number of integrin

receptors and ligands expressed may be important in basement membrane formation on

the inner surface of the trophectoderm [185, 194-201]. Integrins also contribute to cell

migration during gastrulation in early embryos, aiding in the formation of the

mesodermal and endodermal tissue layers inside the embryo [202].

A number of integrins have been observed on oocyte plasma membranes in

various mammalian species, including α3β1, α5β1, α6β1, α2β1, α9β1, αvβ1, αvβ3, and αvβ5

[184, 203]. Oocytes from all mammalian species analyzed exhibit integrins on the

membrane surface, suggesting that integrin-mediated sperm-egg binding is a general

mechanism for gamete interaction [170, 188].

In addition to interacting with sperm ADAM proteins, another class of proteins

containing an RGD peptide sequence has been shown to play a role in fertilization. Many

integrins have demonstrated the ability to recognize this ligand-binding sequence [204]

21

and coincubation of RGD-containing peptides with gametes in IVF systems resulted in a

significant decrease in oocyte-adherent sperm and fertilization [205]. The presence of an

RGD-binding molecule on oocytes was also demonstrated with the use of RGD-

containing immunobeads which bound to the surface of oocytes [205]. RGD peptides

have also been shown to inhibit fertilization in bovine oocytes [188]. The same study

demonstrated the ability of the RGD peptides to initiate parthenogenetic development and

generate intracellular calcium transients into bovine oocytes [188], the latter a necessity

for resumption of meiosis and embryogenesis [206-208].

The Oocyte at Fertilization

At fertilization, sperm binding to, and/or penetration of the oocyte causes

increases in intracellular calcium. The resultant calcium oscillations involve intracellular

calcium stores. While the significance of changes in oocyte intracellular calcium is not

fully understood, calcium does play a role in cortical granule release that leads to a

polyspermy block and progression of the cell cycle in oocytes [206-208]. These events

are collectively referred to as egg activation, which occurs after fertilization and initiates

embryonic development [209].

At ovulation, oocytes are arrested at metaphase of meiosis II in the cell cycle

[120]. An unknown factor from sperm is able to initiate egg activation, inducing

intracellular calcium oscillations and resumption of the cell cycle after fusion of the

gamete membranes [210]. The egg-activating component in sperm seems to be

associated with the nucleus [211] and is present on the sperm following spermatogenesis

[210].

22

Once the oocyte has been activated, the sperm nucleus enters the cytoplasm of the

egg, becoming the male pronucleus, and undergoes changes including decondensation

which will allow male and female chromosomes to properly align. At this point, the male

and female pronuclei fuse and fertilization is complete. The resulting zygote will then

enter the initial stages of embryonic development in the oviduct [145].

Osteopontin

Osteopontin (OPN) is an acidic, secreted, glycosylated phosphoprotein member of

the small integrin binding ligand N-linked glycoprotein (SIBLING) family of

extracellular proteins. Originally identified in the nonmineralized matrix of bovine bone

[212], OPN has since been identified in numerous tissues and fluids [213-215]. A protein

rich in aspartic acid, glutamic acid and serine residues, OPN contains a polyaspartic acid

motif through which it can bind to hydroxyapatite and calcium ions [216, 217] and an

RGD (Arg-Gly-Asp) amino acid sequence which can mediate cell attachment to

integrins. A monomer of approximately 300 amino acids in length, OPN is subject to

extensive posttranslational modifications including phosphorylation, glycosylation and

cleavage, yielding isoforms ranging in size from 25-75 kDa [218]. Other characteristics

of the protein include a hydrophobic leader sequence typically found in secreted proteins,

a thrombin cleavage site and two glutamines that serve in transglutaminase-supported

multimer formation [219]. Ovine and bovine OPN share a 22 amino acid deletion that

contains a potential calcium binding site and substitution of KS at the RS thrombin

cleavage site found in OPN of other species [219].

Genes encoding OPN show moderate levels of sequence conservation among

species [220], the highest level of which is found near the N and C terminals and in 50

23

amino acids bracketing the RGD integrin binding sequence [219]; a polyaspartic acid

sequence, thrombin cleavage sites, and phosphorylation sites also show higher levels of

conservation [221]. In the bovine, two related genes have been identified, with a cDNA

for bovine kidney OPN differing significantly from a bovine bone cell OPN cDNA [213].

Also known as eta-1, secreted-phosphoprotein-1 (SPP1), 2ar and bone sialoprotein, OPN

is involved in cell adhesion, biomineralization, regulation of normal bone resorption,

tumor invasion and metastatic spreading, cell survival, leukocyte recruitment and

function and regulation of calcium [213, 218, 219, 221].

OPN can bind to integrins via its RGD amino acid sequence as well as other

known and cryptic motifs. Integrins are transmembrane receptors found on an increasing

number of cell types. Consisting of heterodimers of noncovalently bound α and β

subunits, integrins participate in cell-cell and cell-ECM interactions and are common in

adhesion cascades [218].

OPN interacts with αvβ1, αvβ5, and α8β1 integrins through its RGD motif with

similar affinity to the �classic� RGD/OPN receptor, integrin αvβ3 [222, 223]. OPN can

bind to integrins through non-RGD mediated means as well, shown by its association

with the α4β1 integrin in leukocyte adhesion through an LDV (Leu-Asp-Val) peptide

sequence [224]. OPN also binds to variants of the cell surface molecule CD44

(hyaluronic acid receptor) [214], acting as a proadhesion molecule to recruit leukocytes

and convert integrins to an active configuration [225-227].

Osteopontin in Reproduction

The functions of OPN in reproduction are becoming clear, especially in the

female. In the sheep, OPN is the most highly upregulated protein in the pregnant

24

ruminant uterus and a major component of uterine histotroph, endometrial gland

secretions containing enzymes, growth factors, cytokines, lymphokines, hormones,

transport proteins and other substances [192, 218]. OPN in the histotroph is likely

involved in the implantation cascade, secreted by the endometrial glands and binding to

luminal epithelium and conceptus trophectoderm to facilitate attachment of the

conceptus. Similar observations have been made in the cow [228], rabbit [229], pig [230]

and human [231]. In the OPN knockout mouse, there were no significant differences in

the number of embryos carried by pregnant females, but knockout animals were

impregnated at roughly half the rate of wild-type mice and embryos were significantly

smaller at all stages of gestation in the knockout animal [232].

In the bovine oviduct, OPN isoforms were identified at 55, 48 and 25 kDa in both

isthmic and ampullary ODF in luteal and non-luteal stages of the estrous cycle [12].

While gene expression remained constant, the 25 kDa isoform was prevalent in ODF

except in isthmic non-luteal (INL) ODF, suggesting a changed role for the protein in

INLODF [12].

The role of OPN in the male reproductive tract has been less explored. OPN has

been linked to fertility in the seminal plasma of Holstein bulls, as seminal plasma of

higher fertility bulls contains roughly 2.5 times more of a 55 kDa form of OPN than

seminal plasma of lower fertility bulls [1, 4]. Protein localization studies identified the

major sources of OPN in seminal plasma as the ampulla and vesicular glands [233], while

gene expression was primarily localized to the epithelium of the ampullae and germ cells

in seminiferous tubules containing elongated spermatids [234]. Studies in the rat

revealed OPN in the epididymis and testis, potentially as an adhesive protein binding

25

early germ cells to the basement membrane of the seminiferous tubule and adjacent

Sertoli cells [235, 236] and may participate in regulation and communication of these cell

types during spermatogenesis. OPN was also identified on ejaculated and epididymal rat

sperm and small amounts of protein were found to be associated with the plasma

membranes of developing germ cells in mice [236, 237], but the protein was not

identified on bull sperm [233].

Research Objectives

The goals of this research were to investigate the association of OPN with

Holstein bull sperm and determine the effects of OPN on sperm capacitation,

mitochondrial activity, intracellular calcium and viability. Further, the potential role of

OPN and integrins in fertilization was investigated as was the ability of OPN to bind to

sperm. With these data, we hoped to obtain functional insight into the correlation of

OPN to high fertility in Holstein bulls.

Animals Used in These Studies

Mature Holstein bulls obtained from Genex, Inc. (Ithaca, NY) were used in the

experiments described in this work. All bulls were of sound body with good quality

semen as determined by evaluation of normal semen parameters including volume, color,

concentration, motility and morphology. These bulls were utilized in all studies in which

semen was collected via artificial vagina.

Collection of Oviductal Fluid

Oviductal fluid was collected daily from indwelling ampullary and isthmic

cannulae in six cows as described previously [130]. The stage of the estrous cycle was

determined by progesterone radioimmunoassay using serum samples taken daily at the

26

time of oviductal fluid collection. Samples with serum progesterone greater than 1.5 ng

ml-1 were considered to be luteal and samples with serum progesterone equal to or less

than 1.5 ng ml-1 were considered to be non-luteal. Daily fluid samples stored in liquid

nitrogen were thawed and combined from at least three cows into single pools for each

oviductal region collected during the luteal or non-luteal phase. Pools were filtered (0.45

µm) and stored in liquid nitrogen until use.

Antibody to Bovine Osteopontin

A polyclonal antiserum was produced in rabbits to purified bovine milk OPN.

The IgG fraction of serum harvested from immunized rabbits was affinity purified

resulting in an affinity-purified polyclonal rabbit antibody to bovine milk OPN. This

antibody was used in all experiments requiring an antibody to OPN.

27

CHAPTER THREE

DETECTION OF OSTEOPONTIN ON HOLSTEIN BULL SPERMATOZOA, IN

CAUDA EPIDIDYMAL FLUID AND TESTIS HOMOGENATES, AND ITS

POTENTIAL ROLE IN BOVINE FERTILIZATION

Introduction

Osteopontin (OPN) is a secreted phosphoprotein first identified in the mineralized

matrix of bovine bone [212]. It has since been detected in many tissues and fluids

including urine [238], milk [239, 240], kidney [241], uterine endometrium of rabbits

[229], sheep [218, 228], cows [228] and pigs [230], the bovine oviduct [12] and on the

luminal surfaces of epithelial cells of human gastrointestinal and reproductive tracts, gall

bladder, pancreas, lung, breast, urinary tract, salivary glands and sweat glands [215].

OPN may be glycosylated, phosphorylated and sulfated, and its expression and post-

translational modifications are tissue-specific and regulated by many hormones and

growth factors [213]. Bovine OPN contains a thrombin cleavage site, a calcium binding

site and binds to various integrins via an RGD amino acid sequence which allows OPN to

participate in cell adhesion and intracellular communication [213]. Integrin binding may

also be RGD-independent via the SVVYGLR motif, and OPN binds to different isoforms

of the hyaluronic acid receptor CD44 [214].

OPN has been detected in the seminal plasma of Holstein bulls, where it was

positively correlated to fertility [233]. OPN has been detected in bull accessory sex gland

fluid, seminal vesicle fluid and ampullary fluid [233], the epithelium of the male

reproductive tract in humans [215], and rat testis, epididymis [235, 236] and sperm [236].

Although previous studies failed to detect OPN on bovine sperm by

28

immunocytochemistry [233], its presence in the male reproductive tract and seminal

plasma and correlation with male fertility suggests that OPN has some association and

function in ejaculated bovine spermatozoa. In support of this notion, OPN has been

described as a sperm surface molecule in rats [236], as associated with sperm during

development in the testis [235], and while sperm are transported and stored in the

epididymis [235, 236], and is present in accessory sex gland fluid [233] at ejaculation. In

addition, proteins from accessory sex gland fluid are known to bind to sperm during

ejaculation [45].

The goals of this study were to characterize OPN on the plasma membrane of

bovine spermatozoa and to assess the functional role of OPN as a sperm ligand during

sperm-egg binding and fertilization using in vitro fertilization (IVF).

Materials and Methods

Isolation of Sperm Membranes from Ejaculated Sperm

Semen was collected via artificial vagina from eight Holstein bulls housed at the

Almquist Research Center. Following evaluation of sperm concentration and motility,

each sample was centrifuged (600 x g) for 10 min at room temperature (RT). Seminal

plasma was removed by aspiration and the sperm pellet was washed twice by

centrifugation in warm (37ºC), sterile phosphate buffered saline (PBS). Sperm

membrane solubilization was performed as previously described [242, 243]. Sperm were

incubated in sperm membrane solubilization buffer (0.4% sodium deoxycholate, 0.26 M

sucrose, 10 mM Tris, pH 8.5) with 200 µM phenyl methyl sulfonyl flouride (PMSF) at a

concentration of 2.5 x 108 sperm ml-1 for 1 h at 4°C. Samples were then centrifuged

(10,000 x g) for 30 min at 4°C and the supernatant containing sperm membrane proteins

29

was dialyzed overnight at 4°C against 50 mM ammonium bicarbonate and vacuum-

concentrated. Protein concentration was determined [244] and sperm membrane proteins

were stored at -80°C until use in SDS-PAGE and Western blot analysis.

Isolation of Cauda Epididymal Sperm Membranes and Preparation of Cauda Epididymal

Fluid

Testes and epididymides from five Holstein bulls were collected at slaughter and

transported on ice to the laboratory. Sperm and fluid from the cauda region of each

epididymis were recovered by back-flushing the epididymis via the vas deferens with

PBS [245]. Flushes from each epididymis were centrifuged (600 x g) for 10 min at RT.

Cauda epididymal fluid (CEF) was aspirated from the sperm pellet and centrifuged

(10,000 x g) for 60 min at 4ºC to remove remaining sperm. The supernatant containing

the CEF was aspirated, the protein concentration was determined [244] and samples were

stored at -80ºC until use. Cauda epididymal sperm were washed twice in cold, sterile

PBS and membranes were solubilized and stored as described above.

Collection and Preparation of Testis Tissue Samples

Whole testes and epididymides from five Holstein bulls were obtained at

slaughter and transported on ice to the laboratory. Sections of testicular parenchyma

were excised from the testes of all five bulls, snap frozen in liquid nitrogen and stored at -

80ºC. Testis samples were later homogenized in protein extraction buffer (10 mM Tris,

pH 8.0, 0.2 M sucrose, 0.2 mM EDTA, pH 8.0, 50 mM NaCl, 1% Triton X-100, 200µM

PMSF) for 30 sec, using 10 ml of buffer per 1 g of tissue. The homogenates were then

centrifuged (10,000 x g) for 30 min at 4ºC and the supernatant containing testis proteins

was dialyzed overnight against 50 mM ammonium bicarbonate at 4ºC. Following

30

determination of protein concentration [244], the samples were frozen at -80ºC until use

in SDS-PAGE and Western blot analysis.

1D SDS-PAGE and Western Blot Analysis

Ejaculated sperm membrane (100 µg), cauda sperm membrane (100 µg) and

epididymal fluid proteins (100 µg), and testicular parenchyma homogenates (100 µg)

were separated by 1D SDS-PAGE (10-17.5% gradient gels) under denaturing conditions

as previously described [233] and transferred to nitrocellulose (Schleicher and Schuell

Bioscience, Keene, NH) at 208 mA for 1 h using a Multiphor II NovaBlot (Amersham

Pharmacia Biotech, Uppsala, Sweden). Blots were incubated overnight at 4°C in PBS

containing 0.5% Tween 20 (v/v), 5% heat-inactivated normal goat serum (HINGS)(v/v)

and 3% bovine serum albumin (BSA)(w/v)(blocking buffer 1) with gentle rocking to

reduce non-specific antibody binding. Blots were incubated with an affinity-purified

polyclonal rabbit antibody to bovine milk OPN (anti-OPN)(1:2000 w/v in blocking buffer

1)[12] or normal rabbit serum (1:2000 w/v in blocking buffer 1) for 2 h at room

temperature and then washed in PBST (3 x 20 min). After washing, blots were incubated

in anti-rabbit IgG peroxidase conjugate (Sigma, St. Louis, MO)(1:7500 w/v in blocking

buffer 1) for 1 h. Following washes in PBST (3 x 20 min), blots were visualized using

enhanced chemiluminescence (ECL)(Amersham Biosciences, Buckinghamshire,

England) and developed onto radiography film (Kodak, Rochester, NY). The developed

film was subsequently scanned using an imaging densitometer (BioRad, Hercules, CA).

31

Immunocytochemistry of Ejaculated and Cauda Epididymal Spermatozoa

Semen from four Holstein bulls housed at the Almquist Research Center was

collected and evaluated as described above. A volume of semen containing 5 x 107

sperm was washed twice by centrifugation (1,000 x g, 5 min) with warm (37°C), sterile

PBS. Sperm were fixed in warm (37°C) 2% paraformaldehyde (PFA) for 10 min at 4°C,

washed twice in PBS (10,000 x g, 5 min) and incubated in 1 ml of PBS containing 5%

BSA (w/v) and 0.1% Tween 20 (v/v)(blocking buffer 2) for 2 h with gentle rocking.

After blocking, sperm were incubated in 1 ml anti-OPN (1:100 w/v in blocking buffer 2),

1 ml normal rabbit serum (1:100 w/v in blocking buffer 2) or blocking buffer 2 alone

overnight at 4ºC with gentle rocking. Following two washes in PBS, sperm were

incubated in 1 ml PBS containing 1% BSA (w/v) and 0.1% Tween 20 (v/v)(blocking

buffer 3) with FITC-labeled anti-rabbit IgG (Sigma, St. Louis, MO; 1:300 w/v) for 1 h

with gentle rocking. Sperm were then washed twice with PBS, smeared onto slides,

mounted with Antifade (Molecular Probes, Eugene, OR) and analyzed using fluorescent

microscopy. Alternatively, cauda epididymal sperm from five bulls were obtained as

described above and subjected to the same immunocytochemical staining procedure as

ejaculated sperm.

Adsorption of Osteopontin Antibody with Osteopontin

OPN was purified as described previously [246] from bovine skim milk. A 1:100

(w/v) dilution of anti-OPN was incubated in 0 µg ml-1, 10 µg ml-1, 25 µg ml-1, 50 µg ml-1

or 100 µg ml-1 purified OPN in blocking buffer 2 overnight at 4°C with gentle rocking,

then centrifuged at 4°C for 1 h (10,000 x g) to remove any immune complexes that may

have formed. The supernatants containing OPN-adsorbed anti-OPN were recovered and

32

stored at 4°C for no more than 4 h. Semen was collected from three Holstein bulls,

pooled and washed twice in PBS (700 x g). From the pooled sample, 5 x 107 sperm were

fixed in 2% PFA, washed twice in PBS (10,000 x g) and incubated in blocking buffer 2

for 2 h at RT before an overnight incubation at 4°C in one of the prepared OPN-adsorbed

anti-OPN solutions with gentle rocking. Cells were then washed in PBS (10,000 x g) and

incubated in FITC-labeled anti-rabbit IgG (1:1500 w/v in blocking buffer 3) for 1 h at

RT, washed twice in PBS and analyzed on a Beckman-Coulter EPICS XL-MCL flow

cytometer using System II software. Specificity of anti-OPN for OPN on the sperm

membrane was determined by the ability of purified OPN to inhibit antibody binding to

sperm.

Oocyte Collection and Maturation

Bovine ovaries were harvested at an abattoir and placed in Dulbecco�s PBS

(Invitrogen, Carlsbad, CA) (35°C) prior to an approximately 2 h transport to the

laboratory. At the laboratory, ovaries were rinsed with Dulbecco�s PBS (39°C) and

oocytes were aspirated from visible ovarian follicles and washed in low bicarbonate

HEPES medium [247]. Those with at least one intact cumulus cell layer were matured in

vitro in medium M199 containing 10% fetal bovine serum (v/v), LH (6 µg ml-1), FSH (8

µg ml-1), and penicillin (100 units ml-1)/streptomycin (100 µg ml-1) for 22-24 h at 39ºC in

5% C02/air [248]. After maturation oocytes were prepared for sperm binding and

fertilization experiments.

33

Sperm Preparation

Semen from three fertile Holstein bulls was collected via artificial vagina, pooled

and the sperm washed twice (700 x g) in Modified Tyrode�s medium (MTM)[91]. Sperm

(5 x 107 ml-1) were incubated in MTM containing: a) no anti-OPN; b) 2.5 µg ml-1 anti-

OPN; c) 5 µg ml-1 anti-OPN or d) 10 µg ml-1 anti-OPN for 2 h at 39ºC in 5% C02/air

(v/v).

Sperm � Oocyte Binding

In vitro-matured oocytes were vortexed for 2 min to remove cumulus cells,

washed twice in low bicarbonate Hepes medium and placed in Nunclon 4-well culture

dishes (Fisher Scientific, Pittsburgh, PA) containing 0.5 ml fertilization medium. Twenty

to twenty-five oocytes were inseminated with 125,000 spermatozoa from the a) no anti-

OPN; b) 2.5 µg ml-1 anti-OPN; c) 5 µg ml-1 anti-OPN or d) 10 µg ml-1 anti-OPN sperm

treatments. Heparin (2 µg) was added to each well at the time of insemination [248].

Oocytes and spermatozoa were co-incubated for 20 h at 39°C in 5% CO2/air (v/v). After

co-incubation, oocytes were washed once in low bicarbonate HEPES and stained with

Hoechst fluorescent dye 33342 (Sigma #B-2261)[8]. The number of sperm bound to each

zona pellucida was evaluated by fluorescence microscopy.

In vitro Fertilization of Oocytes with Sperm Incubated in Osteopontin Antibody

In vitro-matured oocytes were washed twice in low bicarbonate Hepes medium,

placed in fertilization medium with 2 µg heparin and inseminated as described above.

After 20 h of co-incubation oocytes were vortexed to remove cumulus cells and

accessory spermatozoa and washed in low bicarbonate Hepes medium. Oocytes were

34

fixed in acid alcohol for 24 h and stained with aceto-orcein [249]. The presence of two

pronuclei in the cytoplasm of the oocyte indicated normal fertilization.

Statistical Analysis

Densitometry data comparing OPN on ejaculated and epididymal sperm was

analyzed using Student�s T-test. The significance level for this test was P < 0.05.

For flow cytometry data, three replicates of each experiment were performed and

data were pooled. Data were analyzed using the Kolmogorov-Smirnov Goodness of Fit

Test to compare means between different treatments. The significance level for this test

was P < 0.001.

For fertilization and sperm binding, each experiment was repeated three times and

data from each experiment were pooled. Analysis of variance using a general linear

model was performed using mean number of spermatozoa bound per zona pellucida for

each treatment in the sperm-oocyte binding experiments, and a weighted mean based on

the number of oocytes per treatment in the fertilization experiments. Least square means

comparisons were used to assess sperm binding and weighted least square means were

used to analyze fertilization data [8]. The significance level for these tests was P < 0.05.

Results

Osteopontin on Sperm and in Cauda Epididymal Fluid and Testis Homogenates

Solubilized sperm membrane proteins subjected to 1D SDS-PAGE and Western

blot analysis showed that anti-OPN recognized a 35 kDa protein in ejaculated and cauda

epididymal sperm (Figure 3.1). A 55 kDa protein and 25 kDa protein in CEF and in

testicular parenchyma (TP) were also recognized by the antibody to OPN (Figure 3.2).

While a majority of immunoreactivity occurred at 55 kDa and 25 kDa in CEF and TP,

35

immunoreactive bands also appeared at 60, 40 and 22 kDa in TP and 45 kDa in CEF. A

similar pattern of OPN detection at multiple molecular weights was previously reported

in rat testis homogenates [236], perhaps due to enzymatic breakdown of the protein

during homogenization. Densitometry analysis of OPN in ejaculated and epididymal

sperm from eight bulls revealed that, while not significantly different (P = 0.06),

ejaculated sperm contained approximately 50% more OPN than epididymal sperm

(Figure 3.3).

Immunocytochemistry of Ejaculated and Epididymal Sperm

Immunocytochemical analysis of ejaculated sperm showed that the same antibody

that reacted to a 35 kDa protein on Western blots of sperm membrane proteins recognized

a protein on intact sperm membranes from four Holstein bulls (Figures 3.4 and 3.5).

Immunofluorescence occurred in a well-defined band in the postacrosomal region on the

sperm head, as well as on the midpiece. Not all sperm in a given sample showed

consistent staining for OPN and the possibility of different populations of OPN-positive

sperm exists in a given ejaculate: 93% ± 0.82% of ejaculated sperm exhibited staining on

the head and midpiece, 3% ± 1.63% exhibited staining on the head alone, and 4% ±

2.16% exhibited staining on the midpiece alone. In addition, fluorescence appeared to be

more intense on ejaculated than epididymal sperm (Figure 3.6). Immunofluorescence

was detected using a 1:100 dilution of OPN antibody; sperm incubated in normal rabbit

serum as a control exhibited no fluorescence at the same dilution (1:100), indicating that

the binding of OPN antibody to sperm was specific. Negative controls using no OPN

antibody and no normal rabbit serum exhibited no fluorescence, indicating that no non-

specific binding of the FITC-labeled secondary antibody occurred (Figure 3.7).

36

Analysis of Sperm Treated with OPN-Adsorbed Anti-OPN

Pooled sperm from three bulls were analyzed by flow cytometry after treatment

with OPN-adsorbed anti-OPN. Adsorption of the antibody with as little as 5 µg OPN ml-

1 was enough to reduce fluorescence by an average of 63.5% (0.759 fluorescent units for

0 µg ml-1 OPN, 0.277 fluorescent units for 5 µg ml-1 OPN) over three replicates. Sperm

treated with anti-OPN adsorbed with 10 µg OPN ml-1 (64.0%), 25 µg OPN ml-1 (63.6%),

50 µg OPN ml-1 (67.5%) and 100 µg OPN ml-1 (66.5%) also showed reduced

fluorescence compared to the control (Figure 3.8A). The percentage of sperm exhibiting

fluorescence as compared to an unstained control was also decreased in cells treated with

OPN-adsorbed anti-OPN (Figure 3.8B). Adsorption of anti-OPN with 5 µg ml-1 OPN

reduced the percentage of fluorescent sperm by 84.2% compared to sperm treated with

anti-OPN adsorbed with 0 µg ml-1 OPN. Antibody adsorbed with 10 µg ml-1 OPN

(85.2%), 25 µg ml-1 OPN (85.1%), 50 µg ml-1 OPN (90.0%) and 100 µg ml-1 OPN

(89.8%) also exhibited reduced numbers of fluorescent sperm compared to the unstained

control.

In vitro Fertilization of Oocytes with Sperm Incubated in Osteopontin Antibody

Due to high levels of polyspermic fertilization in some treatment groups, analysis

of fertilization was presented as percent total fertilization, percent normal fertilization and

percent polyspermic fertilization. Fewer oocytes were fertilized by sperm treated with

anti-OPN than sperm treated in control medium, with sperm incubated in 5 µg ml-1 of

antibody fertilizing the smallest percentage of oocytes (P < 0.05, Figure 3.9). This was

also true when normal fertilization rates were evaluated, excluding polyspermic

fertilization (P < 0.05, Figure 3.10). The highest percent of polyspermic fertilizations

37

occurred when sperm were incubated in 10 µg ml-1 anti-OPN (P < 0.05), with

polyspermy occurring in nearly 22% of all fertilizations by sperm treated with this

antibody dilution (Figure 3.11).

Binding of Sperm Incubated with Osteopontin Antibody to Oocytes

Fewer sperm bound to oocytes treated with a 2.5 µg ml-1 of anti-OPN than sperm

incubated in control medium (Figure 3.12, P < 0.05). However, sperm treated with 5 µg

ml-1 or 10 µg ml-1 of antibody actually bound more sperm per oocyte than sperm

incubated with 2.5 µg ml-1 anti-OPN or control medium.

38

Figure 3.1. Representative Western blot of osteopontin (OPN) in ejaculated and cauda epididymal solubilized sperm plasma membranes from two Holstein bulls. Ejaculated and epididymal sperm were washed, solubilized, and 100 µg of each sample were separated by 1D SDS-PAGE under denaturing conditions and transferred to nitrocellulose. Resultant blots were probed with an affinity purified polyclonal rabbit antibody to bovine milk OPN (1:2000 w/v) and visualized using enhanced chemiluminescence (ECL) and developed onto radiography film. ES: ejaculated sperm; CS: cauda epididymal sperm; Mr x 10-3: molecular weight markers.

39

Figure 3.2. Representative Western blot of osteopontin in cauda epididymal fluid and homogenates of testicular parenchyma from Holstein bulls. Cauda epididymal fluid and sections of testicular parenchyma were obtained from bulls at slaughter. Sections of testicular parenchyma were homogenized in protein solubilization buffer. Testis homogenates and cauda epididymal fluid were separated by 1D SDS-PAGE (100 µg of each sample) under denaturing conditions and transferred to nitrocellulose. Resultant blots were probed with an affinity purified polyclonal rabbit antibody to bovine milk OPN (1:2000 w/v) and visualized using enhanced chemiluminescence (ECL) and developed onto radiography film. CEF: cauda epididymal fluid; TP: testicular parenchyma; Mr x 10-3: molecular weight markers.

40

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Ejaculated Sperm Cauda Epididymal Sperm

Den

sito

met

ric U

nits

Figure 3.3. Densitometric analysis of osteopontin in ejaculated Holstein bull sperm membranes and cauda epididymal Holstein bull sperm membranes. Solubilized ejaculated and cauda epididymal sperm plasma membrane proteins from five bulls were separated by 1D SDS-PAGE (100 µg each sample) and transferred to nitrocellulose. Resultant blots were probed with an affinity purified polyclonal rabbit antibody to bovine milk OPN (1:2000 w/v), visualized using enhanced chemiluminescence (ECL) and developed onto radiography film. Film was then analyzed on an imaging densitometer. The absorbance for each is shown (mean ± SEM). There was no significant difference between the two values (P = 0.06).

41

Figure 3.4. Immunofluorescent localization of osteopontin (OPN) on ejaculated Holstein bull sperm. Ejaculated sperm were washed to remove seminal plasma, fixed and incubated with an affinity purified polyclonal rabbit antibody to bovine milk OPN (1:100 w/v). Sperm were then stained with FITC-labeled anti-rabbit IgG (1:300 w/v) to visualize OPN on the sperm. A: FITC-labeled sperm; B: brightfield image of same sperm.

42

Figure 3.5. Immunofluorescent localization of osteopontin (OPN) on cauda epididymal Holstein bull sperm. Cauda epididymal sperm were collected from dissected epididymides from bulls at slaughter and washed to remove epidydmal fluid. Cells were fixed and incubated with an affinity purified polyclonal rabbit antibody to bovine milk OPN (1:100 w/v). Sperm were then stained with FITC-labeled anti-rabbit IgG (1:300 w/v) to visualize OPN on the sperm. A: FITC-labeled sperm; B: brightfield image of same sperm.

Figure 3.6. Side-by-side comparison of osteopontin localization on Holstein bull sperm. Ejaculated and cauda epididymal sperm were washed, fixed and incubated with an affinity purified polyclonal rabbit antibody to bovine milk OPN (1:100 w/v). Sperm were then stained with FITC-labeled anti-rabbit IgG (1:300 w/v) to visualize OPN on the sperm. A: FITC-labeled ejaculated sperm; B: FITC-labeled cauda epididymal sperm.

43

Figure 3.7. Negative controls omitting osteopontin antibody. Ejaculated Holstein bull sperm were washed and fixed before incubation in normal rabbit serum (1:100 w/v) and FITC-labled anti-rabbit IgG (1:300 w/v), FITC-labeled anti-rabbit IgG(1:300 w/v) or control buffer. A: Sperm incubated with normal rabbit serum and FITC-labeled anti-rabbit IgG; B: brightfield image of sperm in A; C: Sperm incubated with FITC-labeled anti-rabbit IgG; D: brightfield image of sperm in C; D: Sperm incubated in control buffer; E: brightfield image of sperm in D. Figure 3.8 (next page). Determination of specificity of osteopontin antibody to osteopontin (OPN) on sperm with OPN-adsorbed affinity purified polyclonal rabbit antibody to purified bovine milk osteopontin (anti-OPN). Ejaculated sperm were washed, fixed and treated with: (1) anti-OPN (1:100 w/v); (2) anti-OPN (1:100 w/v) adsorbed with 0 µg ml-1 OPN; (3) anti-OPN (1:100 w/v) adsorbed with 5 µg ml-1 OPN; (4) anti-OPN (1:100 w/v) adsorbed with 10 µg ml-1 OPN; (5) anti-OPN (1:100 w/v) adsorbed with 25 µg ml-1 OPN; (6) anti-OPN (1:100 w/v) adsorbed with 50 µg ml-1 OPN; (7) anti-OPN (1:100 w/v) adsorbed with 100 µg ml-1 OPN; (8) FITC-labeled anti-rabbit IgG (1:1500 w/v); (9) control buffer. Cells were then analyzed by flow cytometry. A: Mean fluorescence ± SEM on sperm from three pooled samples; B: Mean percentage ± SEM of sperm exhibiting fluorescence compared to unstained cells from three pooled samples.

44

A:

B:

45

Figure 3.9. Mean percentage ± SEM of oocytes fertilized, including normal and polyspermic fertilizations. Oocytes were inseminated with spermatozoa incubated in MTM, 2.5 µg ml-1 anti-OPN, 5 µg ml-1 anti-OPN or 10 µg ml-1 anti-OPN prior to insemination. Bars with the same letter are not significantly different (P < 0.05).

Figure 3.10. Mean percentage ± SEM of oocytes fertilized normally, excluding polyspermic fertilizations. Oocytes were inseminated with spermatozoa incubated in MTM, 2.5 µg ml-1 anti-OPN, 5 µg ml-1 anti-OPN or 10 µg ml-1 anti-OPN prior to insemination. Bars with the same letter are not significantly different (P < 0.05).

0

20

40

60

80

100

Antibody Concentration

Perc

enta

ge T

otal

Fe

rtili

zatio

n 0 ug/ml

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5 ug/ml

10 ug/ml

a b

c b

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Perc

enta

ge N

orm

al

Fert

iliza

tion 0 ug/ml

2.5 ug/ml

5 ug/ml

10 ug/ml

a

bc c

46

Figure 3.11. Mean percentage ± SEM of polyspermic fertilizations, expressed as a percentage of total fertilizations. Oocytes were inseminated with spermatozoa incubated in MTM, 2.5 µg ml-1 anti-OPN, 5 µg ml-1 anti-OPN or 10 µg ml-1 anti-OPN prior to insemination. Bars with the same letter are not significantly different (P < 0.05).

Figure 3.12. Mean number ± SEM of sperm bound per zona pellucida (ZP). Oocytes were inseminated with spermatozoa incubated in MTM, 2.5 µg ml-1 anti-OPN, 5 µg ml-1 anti-OPN or 10 µg ml-1 anti-OPN prior to insemination. Bars with the same letter are not significantly different (P < 0.05).

0

20

40

60

80

100


Sper

m B

ound

per

Zon

a Pe

lluci

da

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a b

c c

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Perc

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47

Discussion

This is the first study to describe OPN on the bovine sperm membrane and relate

it to sperm function. Western blot analyses showed that OPN was present in ejaculated

and cauda epididymal solubilized sperm plasma membranes, CEF and TP homogenates.

Immunocytochemical analyses of ejaculated and cauda epididymal sperm resulted in

fluorescence of a distinct band on the post-acrosomal region of the sperm head.

Although earlier studies were unable to detect OPN on bovine sperm membranes [233],

that work used an antibody specific to the 55 kDa isoform of OPN found in Holstein bull

seminal plasma. Since the completion of that study the affinity-purified rabbit antibody

to bovine milk OPN used in the current study which reacts with multiple isoforms of

OPN was produced [12].

In addition to previous work on OPN protein localization [233] and gene

expression in the bull reproductive tract [234], OPN has been identified in the

reproductive tract of the male rat [235, 236] and human male [215]. Siiteri, et al [236]

identified OPN in rat epididymal fluid in a broad band at 27-32 kDa, and on the surface

of epididymal sperm and in sperm detergent extracts and testis homogenates. While not

significantly different in the present study, the amount of OPN exhibited on epididymal

bovine sperm was visibly less than that of ejaculated sperm. This suggests that some

OPN from accessory sex gland fluid (AGF) becomes associated with sperm during

ejaculation. It is also probable that sperm acquire OPN during testicular development or

during transport through the epididymis, since a unique molecular weight form of OPN

was detected on sperm membranes not present in accessory sex gland fluid, epididymal

fluid or seminal plasma. It is possible that the different isoforms of OPN detected in

48

AGF, CEF and sperm membranes have different functions in the bull reproductive tract,

although determining these roles was beyond the scope of this investigation.

OPN was previously identified in epididymal tissue and germ cells in the

spermatogonial stage of spermatogenesis in rats. The authors suggested that OPN

functioned as an adhesion molecule, binding these germ cells to the basement membrane

of the seminiferous tubule and to adjacent Sertoli cells [235]. Primordial germ cells

utilize integrins and extracellular matrix proteins to maintain contact with the Sertoli cells

and other primordial germ cells [250]; Siiteri, et al [236] suggested that Sertoli cells in

the rat testis secrete OPN based on its localization to the basal and adluminal region of

the seminiferous tubule. In the bull, OPN was expressed in the seminiferous tubule, but

only in tubules that contain elongated spermatids, suggesting a stage-related expression

pattern [234]. Data showing the presence of OPN protein in homogenates of bovine

testicular parenchyma along with OPN gene expression in the bovine seminiferous tubule

[234] suggest that OPN may be expressed by Sertoli cells in the later stages of

spermatogenesis in the Holstein bull.

The presence of OPN in the epididymis and on epididymal sperm may be

important in regulating calcium content of the sperm and epididymal lumen [235]. OPN

contains a calcium binding site and causes calcium release in osteoclasts via an integrin-

stimulated IP3 pathway [213]. When secreted into the proximal tubule of the mouse

nephron, OPN suppressed the accumulation of calcium oxalate crystals, most likely by

interfering with the crystallization process [251]. Calcium crystal deposits have also

been shown in human rete testis, efferent ducts and epididymis [252, 253]. Luedtke, et al

[235] suggested that OPN in epididymal fluid may prevent calcium crystallization that

49

can be detrimental to sperm motility and fertility. Our observations that OPN is present

in epididymal fluid and increased fertility of dairy bulls is correlated to greater amounts

of OPN in seminal plasma [4] support this claim.

Identification of OPN on ejaculated spermatozoa may signal its importance in

fertilization, as indicated by a decrease in fertilization by treating sperm with anti-OPN.

Abundant evidence points to the involvement of an RGD-mediated mechanism in binding

and fusion of sperm to oocytes in several mammalian species [120, 143, 156, 157].

Further, studies showed that RGD peptides can activate, induce calcium transients into

and induce parthenogenetic development in bovine oocytes [188]. It is possible that the

OPN localized to the postacrosomal region on sperm membranes may participate in

bovine fertilization by interacting with egg integrins. Integrin receptors are found on the

oolema of sea urchin, mouse, hamster, human [158] and bovine unfertilized oocytes [188,

254]. Integrins may act as co-receptors during fertilization by transducing a signal to

initiate and propagate calcium release via IP3 [158]. Integrins such as αvβ3 and α5β1

which recognize the RGD peptide that is characteristic of OPN binding are among those

integrins present on mammalian oocytes [156, 183, 255]. OPN is known to associate

with these integrin subunits and the RGD peptide can competitively inhibit fertilization

and induce intracellular calcium transients in oocytes and initiate parthenogenetic

development of oocytes [188]. It is likely that sperm-derived OPN plays a role in bovine

fertilization by promoting sperm-egg binding and oocyte activation.

OPN is also a known ligand for the CD44 family of plasma membrane receptors.

CD44 and its splice variants are members of the hyaluronic acid receptor family, are

ubiquitously expressed and can bind extracellular matrix proteins, such as OPN in

50

addition to its primary ligand hyaluronic acid [254]. While Smith, et al [256] claim that

CD44-OPN interactions may not be common in vivo and Katagiri, et al [257] have shown

that OPN binds only to CD44 variants in an RGD-independent manner, multiple studies

argue that OPN, CD44 and RGD-binding integrins (e.g. αvß3, ß1 subunit) may cooperate

in adhesion, signaling pathways and stimulation of motility in various cell types [258-

261]. CD44 is expressed on the acrosomal region of human sperm cells [262], on porcine

oocytes [263] and cumulus cells [264] and bovine oocytes and early embryos [265].

Sperm-associated OPN may interact with CD44 on bovine oocytes during fertilization to

facilitate adhesion and signaling.

The in vitro fertilization data suggest that OPN may be involved in a block to

polyspermy. A recent study indicates that OPN may be involved in preventing

polyspermy in porcine oocytes in vitro [266]. Mammalian eggs establish blocks to

polyspermy at both the level of the ZP and the plasma membrane. While the zona block

to polyspermy has been well-characterized [89] little is known of the molecular events

surrounding the plasma membrane block to polyspermy [267]. Sperm treated with anti-

OPN bound to oocytes in higher numbers than sperm incubated in control medium, and

the incidence of polyspermic fertilization also increased with antibody-treated sperm.

The localization of OPN on sperm in the postacrosomal region makes it unlikely that it

participates in zona interactions but a likely candidate for interaction with the plasma

membrane of oocytes. While it is possible that antibody-coated sperm bound in higher

numbers to oocytes than control sperm through IgG-ZP interactions, a decrease in

fertilization rates coupled with an increase in polyspermic fertilizations suggest that OPN

51

on sperm may participate in the induction of polyspermy blocks in bovine oocytes at the

level of the plasma membrane.

The results of this study show that OPN is present on bovine sperm membranes

and confirms previous results showing that multiple isoforms of OPN exist in the

Holstein bull reproductive tract. While the exact nature of its association with sperm is

not known, it is likely that sperm acquire OPN in the testis and that sperm-associated

OPN is involved in the fertilization process and a block to polyspermy. Other isoforms

present in the bull reproductive tract may have different functions such as calcium

regulation in the epididymis, although those roles were not investigated in this study.

52

CHAPTER FOUR

EVIDENCE FOR THE ROLES OF OSTEOPONTIN AND αV AND α5 INTEGRIN

SUBUNITS IN BOVINE FERTILIZATION

Introduction

The extracellular matrix phosophoprotein osteopontin (OPN) was previously

identified on ejaculated and cauda epididymal Holstein bull sperm, localized to the

postacrosomal region of the sperm head. OPN has also been identified on the dorsal

portion of the rat sperm head [236] and contains an RGD amino acid sequence [268]

through which it binds to integrins and other cell membrane receptors, inducing cell-cell

adhesion and intracellular signaling pathways [213]. The RGD sequence has also been

implicated in mammalian fertilization [188, 205, 269] and the induction of calcium into

oocytes [188], a necessary step in oocyte activation. Several proteins containing the

RGD sequence, including fibronectin [270] and vitronectin [271], have been identified on

sperm and have been implicated in fertilization. Decreased fertilization rates occurred

when sperm were treated with an affinity-purified polyclonal rabbit antibody to bovine

milk OPN (anti-OPN) prior to fertilization.

The process of capacitation prepares sperm for the acrosome reaction and is

believed to take place in the isthmus of the oviduct near the sperm reservoir, as

determined by research using oviductal epithelium explants [65, 66] and isthmic

oviductal fluid [6]. Sperm can be artificially capacitated using the glycosaminoglycan

heparin [91]; further treatment with lysophosphatidylcholine (LPC) will induce the

acrosome reaction in capacitated sperm [91, 134].

53

Acrosome-reacted sperm bind to oocytes via the plasma membrane on the

postacrosomal region of the sperm head. Because sperm can bind to the plasma

membrane of oocytes only after the acrosome reaction has occurred, it is important to

investigate binding proteins in this region of acrosome-reacted sperm. Recent studies

have focused on a sperm-integrin ligand and oocyte integrin as the molecular basis for

sperm-egg plasma membrane binding [156-161]. Members of the ADAM (a disintegrin

and metalloprotease domain) family of proteins, namely fertilin β [272], have been

identified on sperm [166, 171] and considered candidates for a sperm-integrin ligand in

sperm-egg fusion [144].

Interestingly, homologous regions of these ADAM proteins do not contain an

RGD sequence, although they do have a similar ECD sequence that has been implicated

in sperm-egg interactions [273]. Production of fertilin β knockout mice resulted in sperm

with dramatically reduced (~90%) binding to oocytes [121, 274]. However, these

knockout mice also had significant loss of fertilin α and cyritestin, both sperm integrin

ligands and ADAM proteins. Mice that were null for cyritestin showed significant

decreases in production of fertilin β (60% of wild-type protein levels) and fertilin α [121].

It was also reported that the human fertilin α gene is non-functional [275]. The

importance of one ADAM protein functioning both in sperm-egg binding and fertilization

is debatable, making the need to identify other potential sperm ligands in the process of

fertilization a necessity [158].

Because OPN is an RGD-containing protein on sperm, the objective of this study

was to localize OPN on acrosome-reacted sperm and investigate its potential role in

fertilization. In this study, OPN was localized on Holstein bull sperm that had been

54

artificially capacitated with heparin and in which the acrosome reaction was induced

using LPC. OPN was localized to the postacrosomal region of the sperm head on

acrosome-reacted sperm. This study also determined whether bovine oocytes and sperm

contained OPN and integrins known to associate with OPN to provide evidence for the

mechanism of sperm-oolemma binding.


Preparation of Capacitated and Acrosome-Reacted Sperm

Semen was collected from three fertile Holstein bulls housed at the Almquist

Research Center and pooled. Sperm were washed twice (700 x g) in Modified Tyrode�s

Medium (MTM)[91]. Sperm (5 x 107 ml-1) were incubated (39°C, 5% CO2 in air) for 4 h

in MTM or MTM with 10 µg heparin ml-1 (MTM-H) in a total volume of 1 ml. After 4 h,

sperm incubated in MTM and MTM-H were challenged with LPC to induce the

acrosome reaction (MTM-H-LPC). To each tube, 125 µl of 50 mg/ml BSA in MTM and

163 µl of 60 µg ml-1 LPC in MTM were added and incubated for 10 min in a 39°C water

bath. Sperm incubated in only MTM served as a control.

Following treatment with LPC, sperm were washed twice in MTM, fixed in warm

(37°C) 2 % paraformaldehyde (PFA) for 10 min at 4°C and finally washed twice in PBS.

At this point there were four groups of treated sperm: (1) MTM; (2) MTM-H; (3) MTM-

H-LPC; (4) MTM-LPC. Each of the four groups was subjected to the staining protocols

described below.

Detection of Osteopontin on Sperm

Fixed sperm were incubated in PBS containing 5% BSA (w/v) and 0.1% Tween

20 (v/v)(blocking buffer 1) for 2 h at room temperature with gentle rocking. After an

55

overnight incubation in an affinity-purified rabbit polyclonal antibody to bovine milk

OPN (anti-OPN)(1:100 w/v in blocking buffer 1), sperm were washed twice in PBS

(10,000 x g) and incubated in FITC-labeled anti-rabbit IgG (Sigma, St. Louis, MO)(1:300

w/v) in PBS containing 1% bovine serum albumin (BSA)(w/v) and 0.1% Tween 20

(v/v)(blocking buffer 2) for 1 h at room temperature with gentle rocking. Sperm were

then washed twice in PBS and viewed using fluorescence microscopy.

Detection of Acrosome-Reacted Sperm

The lectin Pisum sativum agglutinin (PSA) can be used to identify sperm that

have undergone the acrosome reaction in vitro [276]. PSA binds to the outer acrosomal

membrane of sperm, indicating sperm that have not undergone the acrosome reaction.

Fixed sperm were incubated for 30 min in 1 µg/ml fluorescein-labeled PSA (FITC-

PSA)(Sigma, St. Louis, MO) in PBS at room temperature with gentle rocking. Sperm

were then washed twice in PBS (10,000 x g) and viewed using fluorescence microscopy.

Binding of FITC-PSA to the outer-acrosomal membrane indicated that the sperm was

intact and had not undergone the acrosome reaction. Presence of the acrosomal ridge on

the apical portion of the sperm head in a brightfield image of sperm was used to confirm

that the acrosomal membrane was intact and the sperm had not undergone the acrosome

reaction.

Detection of Osteopontin on Acrosome-Reacted Sperm

Fixed sperm were first subjected to the FITC-PSA staining protocol and then to

the anti-OPN protocol, both of which are outlined above. Sperm without FITC-PSA

bound to the acrosome were considered to have undergone the acrosome-reaction.

Absence of the acrosomal ridge on the apical portion of the sperm head in a brightfield

56

image of sperm was used to confirm that the outer acrosomal membrane was no longer

present and the sperm had undergone the acrosome reaction. Fluorescence indicating

OPN localization was viewed on the postacrosomal region of these sperm using

fluorescence microscopy.

Detection of Osteopontin in Bovine Oocytes

Bovine ovaries were obtained from a local abattoir. Oocytes were retrieved by

follicular aspiration and washed twice in low bicarbonate HEPES medium [247]. Nude

oocytes were pooled into groups of 100 and solubilized in 100 µl of SDS sample-

treatment buffer containing β-mercaptoethanol (BME)[233] for 5 min in a 95°C water

bath. Samples were then separated by 1D SDS-PAGE [4] under denaturing conditions

and transferred to nitrocellulose. Blots were blocked overnight at 4°C in PBS containing

3% BSA (w/v), 5% heat-inactivated normal goat serum (HINGS)(v/v) and 0.1% Tween

20 (v/v)(blocking buffer 3) and then incubated for 2 h at room temperature in anti-OPN

(1:2000 w/v in blocking buffer 3). Blots were washed three times (20 min each) in PBS

containing 0.1% Tween 20 (v/v)(PBST) and incubated in anti-rabbit IgG peroxidase

conjugate (Sigma, St. Louis, MO)(1:5000 in blocking buffer 3). Following washes in

PBST (3 x 20 min), blots were visualized with enhanced chemiluminescence

(ECL)(Amersham, Buckinghamshire, UK) and developed onto radiography film.

Detection of Integrins in Solubilized Sperm Membranes and Bovine Oocytes

Semen was collected from four fertile Holstein bulls, evaluated for concentration

and motility and centrifuged (700 x g). Sperm pellets were washed twice in PBS (700 x

g) and sperm were incubated (2.5 x 108 ml-1) in sperm solubilization buffer (0.4%

deoxycholic acid, 0.26 M sucrose, 10 mM Tris, pH 8.5, 200 µM PMSF) for 60 min at

57

4°C [242] to solubilize sperm plasma membranes. Solubilized membranes were

recovered following centrifugation (10,000 x g) and dialyzed overnight against 4 L of 50

mM ammonium bicarbonate (4°C). Protein concentration was determined [244] and the

samples were stored at -80°C until use.

Bovine oocytes were collected and solubilized as described above, separated by

1D SDS-PAGE under denaturing conditions and transferred to nitrocellulose. Sperm

membrane proteins (200 µg) were separated by 1D SDS-PAGE [4] under denaturing

conditions and transferred to nitrocellulose. Blots of bovine oocytes and sperm

membranes were incubated overnight in PBS containing 5% HINGS (v/v) and 0.1%

Tween 20 (v/v)(blocking buffer 4) and then in affinity-purified polyclonal antibodies to

either human integrin αv or α5 (Santa Cruz Biotechnology, Santa Cruz, CA)(0.5 µg ml-1 in

blocking buffer 4) for 1 h at room temperature. After washing in PBST (3 x 20 min),

blots were incubated in anti-rabbit IgG peroxidase conjugate (1:5000 in blocking buffer

4) for 45 min and then washed in PBST (3 x 20 min). Blots were visualized using ECL

and developed onto radiography film.

Results

Localization of Osteopontin on Acrosome-Reacted Sperm

Sperm that were capacitated with heparin and induced to acrosome-react with

LPC exhibited localization of OPN on the postacrosomal region of the sperm head and

the sperm midpiece (Figure 4.1). This localization was the same on intact and acrosome-

reacted sperm. In control sperm that were treated with both PSA and anti-OPN, the OPN

antibody did not bind to the sperm if PSA was already bound to the acrosome. This was

most likely due to spatial interference, as it was the case if sperm were exposed to PSA

58

before or after anti-OPN exposure. PSA has been known to bind non-specifically to

other regions of the sperm [277] and may have prevented binding of anti-OPN to the

postacrosomal region.

Detection of Osteopontin on Western Blots of Bovine Oocytes and of Integrin Subunits αv

and α5 on Western Blots of Sperm Membranes and Bovine Oocytes

An antibody to human integrin αv showed immunoreactivity for fragments of 65

kDa and 38 kDa on Western blots of bovine oocytes and sperm membranes, respectively

(Figure 4.2). An antibody to human integrin α5 showed immunoreactivity for fragments

of 64 kDa and 37 kDa on Western blots of bovine oocytes and sperm membranes,

respectively (Figure 4.3). While these molecular weights are smaller than those

previously reported for integrins [182], the antibodies used in this study were specific for

the smaller, cytoplasmic C-terminal region of human integrins αv and α5. It is possible

that the denaturing conditions under which the gels were run denatured the subunits into

smaller protein fragments which were detected by the antibodies.

Osteopontin was detected at 30 kDa in three pools of 100 bovine oocytes (Figure

4.4). The oocytes used in this portion of the study were immature and cumulus-free,

indicating that OPN is either a constitutive protein in the oocyte or is bound to the ZP

during development in the ovarian follicle.

59

Figure 4.1. Immunofluorescent localization of osteopontin (OPN) on acrosome-reacted and intact sperm. Ejaculated sperm were washed and capacitated with heparin (10 µg ml-1) The acrosome reaction was induced with lysophosphatidylcholine (LPC) in capacitated sperm. Cells were then fixed and incubated in fluorescein-labeled Pisum sativum agglutinin (FITC-PSA) to detect intact acrosomes and an affinity-purified polyclonal rabbit antibody to bovine milk OPN (anti-OPN). Sperm were then incubated in FITC-labeled anti-rabbit IgG and viewed using fluorescence microscopy. A: brightfield image of acrosome-reacted sperm; B: localization of OPN on sperm in A using FITC; C: brightfield image of intact sperm; D: FITC-PSA staining of sperm in C.

A B

C D

60

Mr x 10-3

Figure 4.2. Representative Western blot of integrin subunits in bovine sperm. Ejaculated Holstein bull sperm were washed to remove seminal plasma and plasma membranes were solubilized. Solubilized sperm plasma membranes (200 µg each sample) were separated by 1D SDS-PAGE under denaturing conditions and transferred to nitrocellulose. Resultant blots were probed with affinity purified polyclonal rabbit antibodies to either human integrin α5 or αv. Blots were visualized using enhanced chemiluminescence (ECL) and developed onto radiography film. A: integrin α5; B: integrin αv. Mr x 10-3: molecular weight markers.

Figure 4.3. Representative Western blot of integrin subunits in bovine oocytes. Bovine oocytes were aspirated from follicles on bovine ovaries obtained at slaughter. Cumulus-free oocytes were pooled into groups of 100 and solubilized with sample treatment buffer containing β-mercaptoethanol and separated by 1D SDS-PAGE under denaturing conditions. Proteins were transferred to nitrocellulose and resultant blots were probed with affinity purified polyclonal rabbit antibodies to either human integrin α5 or αv. Blots were visualized using enhanced chemiluminescence (ECL) and developed onto radiography film. A: integrin αv; B: integrin α5. Molecular weight is indicated to the right.

A B

65 kDa A

64 kDa B

45 29

61

Figure 4.4. Representative Western blot of osteopontin (OPN) in bovine oocytes. Bovine oocytes were aspirated from follicles on bovine ovaries obtained at slaughter. Cumulus-free oocytes were pooled into groups of 100 and solubilized with sample treatment buffer containing β-mercaptoethanol and separated by 1D SDS-PAGE under denaturing conditions. Proteins were transferred to nitrocellulose and resultant blots were probed with an affinity purified polyclonal rabbit antibody to bovine milk OPN. Blots were visualized using enhanced chemiluminescence (ECL) and developed onto radiography film. Mr x 10-3: molecular weight markers.

Discussion

This study demonstrates that OPN, an extracellular matrix protein, is localized to

the postacrosomal region on acrosome-reacted bovine sperm. In addition, integrins

known to serve as receptors for OPN are present in Western blots of oocytes and sperm

plasma membrane proteins. Osteopontin is also present on Western blots of solubilized

bovine oocytes and using an antibody to OPN in a previous study, fertilization of bovine

oocytes was inhibited when sperm were treated with the antibody prior to fertilization.

OPN was previously identified on ejaculated and epididymal sperm, also

localized to the postacrosomal region. Siiteri, et al [236] identified OPN on rat

epididymal and ejaculated sperm, localized primarily to the dorsal region of the sperm

66 45 29

Mr x 10-3

62

head. OPN contains an RGD amino acid sequence, a sequence previously shown to

mediate fertilization, release of intracellular calcium, and parthenogenetic development in

bovine oocytes [188]. Fusi, et al [255] indicated a high binding affinity for the RGD

peptide in human and hamster oocytes and more recent work showed that alteration of the

amino acid sequence in or around the RGD sequence can negatively impact fertilization

in bovine oocytes, further underlining the importance of this sequence in fertilization

[278]. A current model of mammalian fertilization states that a sperm-integrin ligand

binds to an integrin on the oocyte plasma membrane [202]. Integrins have been

identified on the plasma membranes of oocytes from different species [184, 279, 280].

Of these integrins, OPN is known to associate with αvβ3, αvβ1, α4β1 and αvβ5 [255, 281,

282]. At the time of fertilization, α5β1, α6β1 and αv integrins, possibly αvβ3 and αvβ5 are

highly expressed, suggesting their importance in sperm-egg interactions [184]. While

most of these observations have not been reported in bovine species, the high-level of

specificity of the different integrins suggests these interactions and integrin expression

would be similar in the cow [176]. Gonçalves and Killian [283] demonstrated that

antibodies to integrins αv and α5 can inhibit fertilization of bovine oocytes. Oocytes

treated with antibodies to these integrin subunits were fertilized at 45-59% of the rate of

untreated oocytes, while sperm treated with these antibodies were only able to fertilize

48-60% of oocytes fertilized by untreated sperm. Sperm-egg binding also decreased

when oocytes and/or sperm were treated with antibodies to integrins αv and α5 to roughly

45-59% of control levels.

An antibody to OPN also recognized a 30 kDa protein on Western blots of

solubilized bovine oocytes. OPN mRNA has previously been detected in the bovine

63

ovarian follicle and corpus luteum [284], but this is the first report of OPN protein

detection in oocytes of any species. Previous work in our lab indicated that OPN is

associated with the ZP after exposure to oviductal fluid [285] and OPN mRNA has been

detected in the bovine follicle [284]. This suggests that OPN may associate with oocytes

through unknown mechanisms while in the follicle and oviduct. The role that OPN on

oocytes plays in gamete interactions and fertilization is unclear, but exposing oocytes to

anti-OPN prior to fertilization inhibits fertilization, indicating a role for OPN in this

process [286].

OPN can promote cell attachment by binding to integrins αvβ3, αvβ1, αvβ5 and α8β1

through its RGD sequence [176, 213, 218, 287, 288]. Intracellular calcium levels in

osteoclasts can be affected by the binding of OPN to the αvβ3 integrin; one study showed

an extrusion of calcium from osteoclasts in a calmodulin-dependent reaction involving an

activated plasma membrane Ca2+-ATPase [289], while a second study observed a

transient increase in intracellular calcium levels in mouse-derived osteoclast-like cells

exposed to OPN [290]. It is apparent that OPN affects intracellular calcium stores of the

cells to which it binds, and it is possible for the protein to cause a transient increase in

intracellular calcium in such cells. Campbell, et al [188] reported the ability of RGD

peptides to induce intracellular calcium transients in approximately 93% of observed

oocytes. Given its ability to induce calcium transients under certain conditions and the

inclusion of an RGD amino acid sequence, OPN is a good candidate for a protein that

will induce the effects of the RGD sequence on oocytes.

Based on the data gathered in this study, we propose a mechanism through which

OPN and integrins on oocytes interact with OPN and integrins on sperm during

64

fertilization utilizing the RGD amino acid sequence. OPN was localized to the

postacrosomal region of the sperm head after the acrosome reaction, the region of the

sperm which participates in binding to the oocyte plasma membrane; OPN is present on

Western blots of solubilized oocytes and the integrin subunits αv and α5, both of which

are known RGD receptors, are present in Western blots of sperm plasma membrane

proteins. Antibodies to OPN and αv and α5 integrin subunits all decreased rates of in

vitro fertilization of bovine oocytes in previous studies, and work in other laboratories

has shown the importance of the RGD sequence in bovine fertilization [188, 278]. This

leads us to believe that OPN on sperm and oocytes interacts with integrins on sperm and

oocytes to promote sperm-egg binding and fusion during fertilization of bovine oocytes

(Figure 4.5).

Figure 4.5. Interactions between extracellular matrix components and integrins at fertilization. The extracellular matrix proteins, fibronectin and vitronectin are localized in the region where sperm and egg interact. Spermatozoa and oocytes express αvβ3 and αvβ5 integrins at plasma membranes. It is possible that integrins and extracellular matrix components, including osteopontin, mediate sperm binding to eggs. Source: T Darribère M Skalski H Cousin A Gaultier C Montmory D Alfandair Biol Cell 92 (2000) 5-25.

65

CHAPTER FIVE

INFLUENCE OF OSTEOPONTIN, CASEIN AND OVIDUCTAL FLUID ON

BOVINE SPERM CAPACITATION, INTRACELLULAR CALCIUM CONTENT,

MITOCHONDRIAL ACTIVITY AND VIABILITY

Introduction

Before they are capable of fertilization, sperm must undergo a period of

preparation in the female reproductive tract known as capacitation [49, 58]. While the

specific changes undergone by the sperm during this process are not completely

understood, they include a number of cellular and molecular changes including removal

and alteration of sperm plasma membrane surface components and changes in

intracellular ion concentrations [89]. The exact site of sperm capacitation in vivo is still

undetermined, although it can occur in the uterus [58-61] and occurs more rapidly when

sperm are exposed to first the uterine and then oviductal environments [55, 60, 62, 63].

Studies with oviductal fluid and conditioned medium from oviductal epithelium explants

suggest that capacitation occurs in the isthmus of the oviduct [6, 65, 66].

Capacitation has been induced in vitro using heparin-like glycosaminoglycans

[91, 291] and oviduct-specific glycoprotein (OSG) [67, 134, 139], both of which can be

found in the bovine oviduct. In addition, sperm can be artificially capacitated using

heparin [91]. Oviductal fluid (ODF) can capacitate sperm [90, 134], with more sperm

capacitated in non-luteal ODF than in luteal ODF [134]. Binding of oviductal fluid

proteins to sperm has also been demonstrated [136-138, 243].

Osteopontin (OPN) is a secreted extracellular matrix phosphoprotein positively

correlated to Holstein bull fertility in seminal plasma [1, 4] and identified in ODF [12].

66

While only one OPN mRNA transcript was found in the oviduct and amounts did not

vary during the estrous cycle, three different protein isoforms were detected [12]. These

isoforms (55, 48 and 25 kDa) were present in ampullary and isthmic ODF during the

luteal and non-luteal stages. The 25 kDa isoform was the most prevalent isoform except

in the isthmus during the non-luteal phase of the estrous cycle, the time during which

sperm enter the oviduct and prepare for interaction with the oocyte. This difference in

isoform prevalence suggests a role for OPN in the events surrounding capacitation of

sperm in the oviduct.

The purpose of this study was to evaluate the effect of partially purified OPN on

sperm capacitation and sperm quality as indicated by mitochondrial activity, viability and

intracellular calcium levels. Because OPN was purified from bovine milk and some

residual casein remained in the OPN preparations, the effect of casein on sperm was also

evaluated as a control. The ability of OPN and casein to associate with sperm was

investigated as well, as these could be important to reproductive events and

cryopreservation of sperm with milk extender.


Capacitation and Acrosome Reaction of Sperm

Semen from three Holstein bulls housed at the Almquist Research Center was

collected by artificial vagina, pooled and washed twice in Modified Tyrode�s medium

(MTM)(700 x g)[91]. Sperm (5 x 107 ml-1) were incubated (39°C, 5% CO2/air (v/v), 4 h)

in the following treatments in MTM: 1) no treatment; 2) 1 µg ml-1 OPN; 3) 5 µg ml-1

OPN; 4) 10 µg ml-1 OPN; 5) 20 µg ml-1 OPN; 6) 1 µg ml-1 casein (Sigma, St. Louis,

MO); 7) 5 µg ml-1 casein; 8) 10 µg ml-1 casein; 9) 20 µg ml-1 casein; 10) 10 µg ml-1

67

heparin (Sigma, St. Louis, MO); 11) 60% ampullary non-luteal ODF (ANLODF); 12)

60% isthmic non-luteal ODF (INLODF); 13) 60% bovine skim milk. ODF was pooled

from three cows over three non-consecutive estrous cycles; ODF and skim milk were

adjusted to 10 mg total protein ml-1 using 50 mM sodium bicarbonate, pH 8.5 [138].

Following incubation, samples were divided in half and treated either with the fusogenic

lipid lysophosphatidyl choline (LPC)(Sigma, St. Louis, MO) to induce the acrosome

reaction or an MTM negative control [91, 134]. LPC was prepared as previously

described [134]. A 644 µl incubation volume was used for each treatment tube. A 500

µl volume of sperm suspension was incubated with 60 µg LPC ml-1 (81.5 µl LPC stock

solution) or the same volume of MTM. After LPC was added, 62.5 µl of bovine serum

albumin (BSA)(Sigma, St. Louis, MO)(50 mg ml-1 stock solution in MTM) was added to

each tube and the tubes were incubated for 10 min in a 39°C water bath. Sperm were

then washed twice and stained for viability and the acrosome reaction.

Detection of the Acrosome Reaction and Sperm Viability

Ethidium monoazide (EMA)(Molecular Probes, Carlsbad, CA) was used to detect

cell viability. Sperm (5 x 107 ml-1) were incubated in 10 µg ml-1 EMA in PBS for 10 min

on a white metal pan 20 cm below a fluorescent light to induce photochemical covalent

binding of EMA to nucleic acids in cells with compromised membranes [292]. After two

washes in PBS, cells were fixed in 2% paraformaldehyde (PFA) and washed twice more

in PBS. Sperm were then incubated in 1 µg ml-1 fluorescein-labeled Pisum sativum

agglutinin (FITC-PSA)(Sigma, St. Louis, MO) in PBS for 30 min to detect intact sperm

[293], washed in PBS and analyzed on a Beckman-Coulter EPICS XL-MCL flow

cytometer using System II software.

68

Detection of Sperm Intracellular Calcium and Cell Viability

Semen was collected and pooled as described above. After two washes in MTM,

5 x 107 sperm ml-1 were incubated (39°C, 5% CO2/air (v/v), 4 h) in the treatments listed

above. Sperm were washed twice in MTM and incubated (39°C, 5% CO2/air (v/v), 30

min) in 5 µM fluo-3 AM (Molecular Probes, Carlsbad, CA) in MTM. The cell permeant

fluo-3 AM is not fluorescent unless bound to Ca2+. The fluorescent emission of Ca2+-

bound fluo-3 is detected using the same methods as FITC. Following two washes with

MTM, sperm were suspended in PBS and stained with propidium iodide (PI) (Molecular

Probes, Carlsbad, CA) (10 µg ml-1) to measure sample viability. PI is a cell permeant

used routinely to detect dead or dying cells; it only enters compromised membranes and

binds to nucleic acids. The samples were then analyzed by flow cytometry. Only viable

cells that did not stain with PI were selected and analyzed for calcium content.

Detection of Mitochondrial Activity in Sperm

Semen was collected, pooled and washed in MTM as described above. Sperm (5

x 107 ml-1) were incubated (39°C, 5% CO2/air (v/v), 4 h) in the treatments listed above.

Sperm were washed twice in MTM and incubated (39°C, 5% CO2/air (v/v), 15 min) in 5

µM MitoTracker Red CMXRos (Molecular Probes, Carlsbad, CA) in MTM. Following

two washes with MTM, sperm were suspended in PBS and stained with PI (10 µg ml-1)

and analyzed by flow cytometry. Only viable cells that did not stain with PI were

selected and analyzed for mitochondrial activity.

Binding of Biotinylated Osteopontin and Casein to Sperm

OPN was partially purified from commercially available skim milk as previously

described [246]. OPN and casein were biotinylated using a method previously used to

69

label oviductal fluid proteins [138]. Sperm (5 x 107 ml-1) were incubated (39°C, 5%

CO2/air (v/v), 4 h) in the following treatments in MTM using biotinylated OPN and

casein: 1) no treatment; 2) 1 µg ml-1 OPN; 3) 5 µg ml-1 OPN; 4) 10 µg ml-1 OPN; 5) 25

µg ml-1 OPN; 6) 50 µg ml-1 OPN; 7) 100 µg ml-1 OPN; 8) 1 µg ml-1 casein; 9) 5 µg ml-1

casein; 10) 10 µg ml-1 casein; 11) 25 µg ml-1 casein; 12) 50 µg ml-1 casein or 13) 100 µg

ml-1 casein . After two washes in MTM, sperm were fixed in 2% PFA, washed twice in

PBS and incubated for 30 min in 100 µg ml-1 FITC-labeled streptavidin (Sigma, St.

Louis, MO). Following a wash in PBS, protein binding to sperm was analyzed by flow

cytometry.

Statistical Analysis

In each experiment, 104 sperm from each sample were analyzed by flow

cytometry. Three replicates of each experiment were performed and data were pooled.

Data were analyzed using the Kolmogorov-Smirnov Goodness of Fit Test to compare

means between different treatments. The significance level for all tests was P < 0.001.

Results

Effect of Treatments on Capacitation and Acrosome Reaction

Sperm capacitation was measured by the ability of LPC to induce the acrosome

reaction; sperm that had undergone the acrosome reaction and were still viable based on

lack of EMA staining were considered functionally capacitated, as dead sperm often

suffer membrane damage leading to removal of the acrosome. Therefore, the percent of

acrosome-reacted live (ARL) sperm were measured and compared in each treatment

(Figure 5.1). In all treatments, the percent of ARL sperm was greater than sperm

incubated in MTM (negative control). OPN capacitated significantly more sperm than

70

heparin (positive control) and casein at all concentrations without a dose-dependent

effect. This suggests that small amounts of OPN can have positive effects on sperm

capacitation. The largest percentage of ARL sperm occurred in INLODF, with skim milk

containing the second largest population of functionally capacitated sperm (Figure 5.2).

The effect of INLODF was expected, as sperm become capacitated in the isthmus of the

oviduct in vivo [6, 65, 66].

Effect of Treatments on Mitochondrial Activity

Addition of OPN to sperm had a significant effect on MitoTracker Red CMXRos

uptake at 1 µg ml-1 OPN versus the MTM control (Figure 5.3). OPN was not different

than casein or MTM at all other concentrations. Sperm treated with heparin and

INLODF were also not different than the control (Figure 5.4), but MitoTracker Red

CMXRos uptake was significantly increased when sperm were treated with ANLODF

and skim milk (P < 0.001).

Effect of Treatments on Intracellular Calcium

OPN had no effect on intracellular content in sperm versus the MTM control

(Figure 5.5). OPN caused a significant increase in intracellular calcium versus casein in

only the 10 µg ml-1 and 20 µg ml-1 treatments. Incubation of sperm in heparin, ANLODF

and INLODF all increased intracellular calcium levels, with INLODF showing the

greatest effect (Figure 5.6). Skim milk decreased the levels of calcium in sperm after

incubation.

Effect of Treatments on Sperm Viability

In all treatment groups, OPN significantly increased the percentage of viable

sperm versus the MTM control as measured by exclusion of PI (Figure 5.7). This was

71

different than the casein control in only the 1 µg ml-1 and 5 µg ml-1 treatment groups.

There was no dose-dependent effect of OPN on sperm viability, suggesting that very

small amounts of OPN may have positive effects on sperm survival. Sperm samples

treated with heparin, ANLODF, INLODF and skim milk all contained fewer viable sperm

than the MTM control (Figure 5.8). This may have been due to an artifact of capacitation

as the sperm membranes may have become permeable to PI and may not be indicative of

the true number of viable cells.

Osteopontin and Casein Binding to Sperm

More casein associated with sperm as the concentration of casein in which sperm

were incubated increased (Figure 5.9). All concentrations of biotinylated casein 10 µg

ml-1 and higher showed significantly more fluorescence on sperm than the negative

control. In addition, all concentrations of casein 10 µg ml-1 and higher associated with

sperm compared to OPN at the same incubation concentration. At no concentration did

OPN bind to sperm in greater amounts than the negative control.

72

Figure 5.1. Effect of osteopontin treatment on sperm capacitation, represented as the mean percentage ± SEM of acrosome-reacted live (ARL) sperm in each treatment. Sperm were incubated (39°C, 5% CO2/air, 4 h) in 1, 5, 10 or 20 µg ml-1 osteopontin (OPN) or casein, 10 µg ml-1 heparin in Modified Tyrode�s medium (MTM) or MTM alone and the acrosome reaction was induced in capacitated sperm using lysophosphatidylcholine (LPC). Sperm were then stained with ethidium monoazide (EMA) and fluorescein-labeled Pisum sativum agglutinin (FITC-PSA) to evaluate viability and acrosome status of sperm. Cells (104 per sample) were analyzed by flow cytometry. Three replicates were performed and data were pooled. The percentage of functionally capacitated sperm (acrosome-reacted live) from each treatment is represented. OPN = osteopontin; MTM = Modified Tyrode�s medium. Bars marked with asterisks are not significantly different (P < 0.001); all other bars are significantly different.

73

Figure 5.2. Effect of oviductal fluid treatment on sperm capacitation, represented as the mean percentage ± SEM of acrosome-reacted live (ARL) sperm in each treatment. Sperm were incubated (39°C, 5% CO2/air, 4 h) in 60% ANLODF, 60% INLODF, 60% skim milk or 10 µg ml-1 heparin in Modified Tyrode�s medium (MTM) or MTM alone. The acrosome reaction was induced in capacitated sperm using lysophosphatidylcholine (LPC). Sperm were then stained with ethidium monoazide (EMA) and fluorescein-labeled Pisum sativum agglutinin (FITC-PSA) to evaluate viability and acrosome status of sperm. Cells (104 per sample) were analyzed by flow cytometry. Three replicates were performed and data were pooled. The percentage of functionally capacitated sperm (acrosome-reacted live) from each treatment is represented. MTM = Modified Tyrode�s medium; ANLODF = ampullary non-luteal oviductal fluid; INLODF = isthmic non-luteal oviductal fluid. ANLODF, INLODF and skim milk were adjusted to 10 mg total protein ml-1 with 50 mM sodium bicarbonate, pH 8.5 prior to dilution in MTM. All bars are significantly different (P < 0.001).

74

Figure 5.3. Effect of osteopontin treatment on mitochondrial activity in sperm, represented as mean fluorescence ± SEM. Sperm were incubated (39°C, 5% CO2/air, 4 h) in 1, 5, 10 or 20 µg ml-1 osteopontin (OPN) or casein, 10 µg ml-1 heparin in Modified Tyrode�s medium (MTM) or MTM alone and stained with MitoTracker Red CMXRos to evaluate mitochondrial status and propidium iodide (PI) to ensure that only live cells were evaluated. Cells (104 per sample) were analyzed by flow cytometry. Three replicates were performed and data were pooled. OPN = osteopontin; MTM = Modified Tyrode�s medium. Bars marked with asterisks are significantly different (P < 0.001).

75

Figure 5.4. Effect of oviductal fluid treatment on mitochondrial activity in sperm, represented as mean fluorescence ± SEM. Sperm were incubated (39°C, 5% CO2/air, 4 h) in 60% ANLODF, 60% INLODF, 60% skim milk or 10 µg ml-1 heparin in Modified Tyrode�s medium (MTM) or MTM alone. Cells were stained with MitoTracker Red CMXRos to evaluate mitochondrial status and propidium iodide (PI) to ensure that only live cells were evaluated and analyzed (104 per sample) by flow cytometry. Three replicates were performed and data were pooled. MTM = Modified Tyrode�s medium; ANLODF = ampullary non-luteal oviductal fluid; INLODF = isthmic non-luteal oviductal fluid. ANLODF, INLODF and skim milk were adjusted to 10 mg total protein ml-1 with 50 mM sodium bicarbonate, pH 8.5 prior to dilution in MTM. Bars marked with asterisks are not significantly different from each other (P < 0.001); unmarked bars are not significantly different from each other.

76

Figure 5.5. Effect of osteopontin treatment on intracellular calcium content in sperm, represented as mean fluorescence ± SEM. Sperm were incubated (39°C, 5% CO2/air, 4 h) in 1, 5, 10 or 20 µg ml-1 osteopontin (OPN) or casein, 10 µg ml-1 heparin in Modified Tyrode�s medium (MTM) or MTM alone and stained with fluo-3 AM to evaluate calcium content. Sperm were also stained with propidium iodide (PI) to ensure that only live cells were evaluated. Cells (104 per sample) were analyzed by flow cytometry. Three replicates were performed and data were pooled. OPN = osteopontin; MTM = Modified Tyrode�s medium. Bars marked with the same letter are not significantly different (P < 0.001).

77

Figure 5.6. Effect of oviductal fluid treatment on intracellular calcium content in sperm, represented as mean fluorescence ± SEM. Sperm were incubated (39°C, 5% CO2/air, 4 h) in 60% ANLODF, 60% INLODF, 60% skim milk or 10 µg ml-1 heparin in Modified Tyrode�s medium (MTM) or MTM alone and stained with fluo-3 AM to evaluate calcium content. Sperm were also stained with propidium iodide (PI) to ensure that only live cells were evaluated. Cells (104 per sample) were analyzed by flow cytometry. Three replicates were performed and data were pooled. MTM = Modified Tyrode�s medium; ANLODF = ampullary non-luteal oviductal fluid; INLODF = isthmic non-luteal oviductal fluid. ANLODF, INLODF and skim milk were adjusted to 10 mg total protein ml-1 with 50 mM sodium bicarbonate, pH 8.5 prior to dilution in MTM. All bars are significantly different (P < 0.001).

78

Figure 5.7. Effect of osteopontin treatment on sperm viability, represented as mean percentage ± SEM of viable sperm. Sperm were incubated (39°C, 5% CO2/air, 4 h) in 0, 1, 5, 10 or 20 µg ml-1 osteopontin (OPN) or casein or 10 µg ml-1 heparin in Modified Tyrode�s medium (MTM) and stained with propidium iodide (PI) to evaluate sperm viability. Cells (104) were analyzed by flow cytometry. Three replicates were performed and data were pooled. OPN = osteopontin; MTM = Modified Tyrode�s medium. Bars marked with the same letter are not significantly different (P < 0.001).

79

Figure 5.8. Effect of oviductal fluid treatment on sperm viability, represented as mean percentage ± SEM of viable sperm. Sperm were incubated (39°C, 5% CO2/air, 4 h) in 60% ANLODF, 60% INLODF, 60% skim milk or 10 µg ml-1 heparin in Modified Tyrode�s medium (MTM) or MTM alone and stained with propidium iodide (PI) to evaluate sperm viability. Cells (104) were analyzed by flow cytometry. Three replicates were performed and data were pooled. MTM = Modified Tyrode�s medium; ANLODF = ampullary non-luteal oviductal fluid; INLODF = isthmic non-luteal oviductal fluid. ANLODF, INLODF and skim milk were adjusted to 10 mg total protein ml-1 with 50 mM sodium bicarbonate, pH 8.5 prior to dilution in MTM. Bars marked with asterisks are not significantly different (P < 0.001); all other bars are significantly different.

80

Figure 5.9. Association of biotinylated OPN and casein with sperm, represented as mean fluorescence ± SEM. Sperm (5 x 107 ml-1) were incubated (39°C, 5% CO2/air, 4 h) in 1, 5, 10, 20, 50 or 100 µg ml-1 biotinylated osteopontin (OPN) or biotinylated casein in Modified Tyrode�s medium (MTM) or MTM alone and stained with fluorescein-labeled streptavidin (FITC-SA). Cells (104) were analyzed using flow cytometry. Three replicates were performed and data were pooled. OPN = osteopontin; MTM = Modified Tyrode�s medium; FITC-SA = fluorescein-labeled streptavidin. Bars marked with different letters are significantly different (P < 0.001).

Discussion

These studies were conducted to examine the physiological effects of OPN on

sperm. Sperm were incubated in concentrations of OPN that we believed to be similar to

those of seminal plasma [4] and ODF. OPN was purified from commercially available

skim milk [246] with moderate success. While 50-75% pure OPN was obtained,

contaminating casein was never completely eliminated. For this reason, casein was

included as a control whenever partially purified OPN was used.

81

Sperm were incubated in the various treatments for 4 h, mimicking the

appropriate time for heparin to capacitate sperm [91]. Heparin capacitation was used as a

control in all experiments to compare the effect of OPN on capacitation and other

physiological parameters against a defined capacitating agent.

At all incubation concentrations, OPN produced more acrosome-reacted live

(ARL) sperm than heparin and casein controls as detected by our dual staining method.

However, there was not a dose-dependent effect of OPN on capacitation, suggesting that

the protein may not directly capacitate sperm, but may rather be part of a larger

mechanism. It is of interest to note that the percent of ARL sperm produced by OPN is

greater than that of heparin at the same time point, indicating a slightly more powerful or

different mechanism of affecting capacitation.

Previous studies have shown the ability of ODF to capacitate sperm. Estrual ODF

capacitated sperm at higher rates than heparin [90] and similar experiments using

regional-staged ODF showed that INLODF capacitated more sperm than ANLODF [6-8].

While the conditions of these experiments yielded slightly different numbers of

capacitated sperm with these treatments, the overall effect was the same with INLODF

capacitating more sperm than ANLODF and heparin.

Increased intracellular calcium is a characteristic of capacitated sperm [294], and

can be used as an indicator of the induction of sperm capacitation [93, 295, 296]. There

are two distinct waves of calcium uptake during capacitation [294], the first of which is

associated with an acrosomal calcium reservoir [93]. In these experiments, OPN showed

no positive effect on intracellular calcium levels in sperm, suggesting its effect on

capacitation is by different means.

82

The effect of OPN on sperm mitochondrial activity was also examined.

Mitochondria have a negative interior membrane potential, in which fluorescent cations

are able to accumulate [297]. Functional mitochondria enable the uptake of MitoTracker

Red CMXRos based on membrane potential [298]. These experiments showed that

increasing the concentration of OPN in the sperm environment had no direct effect on

MitoTracker Red CMXRos uptake by sperm, indicating that the presence of OPN has no

effect on mitochondrial membrane potential. Coupled with data showing increased

intracellular calcium in OPN-treated sperm, it is possible that an acrosomal store of

calcium is being facilitated by OPN, which may explain its effect on sperm capacitation.

This induction of calcium into the sperm could possibly trigger other membrane

alterations in the capacitation mechanism [294]. Sperm treated with heparin and

INLODF, which had the highest intracellular calcium levels, had the least active

mitochondria based on MitoTracker Red CMXRos uptake. This would suggest the sperm

have entered a quiescent state with reduced mitochondrial respiration, much as they

might in an oviductal sperm reservoir in vivo [299].

The possibility that OPN and casein could bind to sperm was also investigated in

this study. Previous studies have shown that proteins in ODF can associate with sperm

[133, 135-138], including oviduct-specific glycoprotein (OSG)[136]. According to these

data, OPN did not associate with sperm, but casein did in a dose-dependent manner. This

is the first report showing association of casein, the major protein in milk which is a

commonly used semen extender, with bovine sperm. The data presented here appear to

contradict previous data which suggested that OPN from accessory sex gland fluid (AGF)

appears to associate with sperm during ejaculation. It is possible that OPN alone is not

83

able to associate with sperm, but requires an additional protein or enzyme interaction to

bind. Another possibility is that ejaculated sperm used in these studies are already

saturated with OPN acquired from AGF during ejaculation, occupying available OPN

binding sites on the sperm membrane.

The final parameter studied was sperm viability. OPN has been shown to

stimulate cell survival by inhibiting apoptosis in various cell types [214, 287, 300].

While there was again no dose-dependent effect of OPN on cell survival, there was an

overall increase in sperm viability in samples incubated with OPN. This could indicate

an important function for OPN in oviductal fluid as sperm can reside in the oviduct for up

to 20 h before fertilization [5].

An interesting result from this study occurred with the inclusion of bovine skim

milk as a treatment in the experiments. While there was a high number of ARL sperm

after treatment in milk, intracellular calcium was decreased and mitochondrial activity

was increased. Cell viability was similar to that of sperm incubated in ANLODF, which

has been shown to enhance the ability of sperm to fertilize [6-8]. Milk has long been

used as an extending agent for bovine semen cryopreservation, and these data suggest

milk may help prepare sperm for fertilization through undetermined mechanisms.

In summary, the effect of OPN on sperm physiology was evaluated in the present

study. OPN promotes capacitation of sperm through undetermined means and enhances

sperm viability, possibly by blocking apoptotic pathways. OPN had no effect on sperm

mitochondrial membrane potential or intracellular calcium content and partially purified

OPN did not bind to sperm. These data provide further insight into the various roles

OPN seems to play in reproductive events.

84

CHAPTER SIX

CONCLUSIONS AND FUTURE STUDIES

The studies conducted here have provided a snapshot view into the presence of

OPN in sperm and the effect of OPN on sperm function. While it was demonstrated that

the capacitation of sperm is enhanced by OPN and that the association of OPN with

sperm is likely important in fertilization, the mechanisms through which the protein

operates are yet to be discovered.

Several components of ODF are able to capacitate sperm and induce the acrosome

reaction. Having redundancy in the capacitation mechanism will ensure a population of

sperm is always present in the oviduct to arrive at the site of fertilization at the proper

time. The effect of OPN on capacitation was positive, but the mechanism through which

the protein acts is still undetermined. OPN has been known to induce calcium flux into

certain cell types, but this was not observed in these studies. Although the role of

calcium in sperm capacitation is not completely understood it has been reported that

heparin regulates calcium through modulation of voltage-gated channels by possibly

binding to plasma membrane receptors [95, 301, 302]. OPN may affect calcium in a

similar way under the proper conditions by activating a mechanism which opens these

channels upon contact with sperm. Changes in intracellular calcium are known to

precede increases in cAMP, pH and tyrosine phosphorylation, which are necessary for

sperm capacitation [291, 303-305]. While OPN had no effect on sperm intracellular

calcium in the experiments described here, further investigation into the role of OPN,

intracellular calcium and capacitation is warranted.

85

The ligand-receptor mechanism involving OPN and integrins during fertilization

needs to be fully characterized. While a few potential sperm ligands containing an RGD

amino acid binding sequence have been identified, none has been definitively proven to

participate in such a mechanism. The specific integrin receptors involved in OPN-

mediated fertilization mechanism also need to be identified. Figure 4.5 shows a potential

mechanism for an RGD-containing protein, in this case fibronectin or vitronectin,

associating with integrins during fertilization. It is reasonable to assume that OPN works

in much the same way, although more data needs to be collected to define this

mechanism. Figure 6.1 depicts the way in which OPN and integrins may interact during

bovine fertilization.

A major question raised by these studies is the nature of the association of OPN

with sperm. These data showed that while OPN was present on cauda epididymal sperm,

a greater amount was visible on ejaculated sperm, suggesting that additional OPN is

bound from seminal plasma during ejaculation. This binding, however, was not

duplicated using washed ejaculated sperm and purified OPN. It would be interesting to

determine if a �cofactor� in seminal plasma enabled OPN to bind to sperm and, if so, to

what specific receptors on the sperm membrane the protein was binding. Answers to

these questions may allow for the use of OPN as a fertility enhancer during semen

cryopreservation.

The effect of OPN on the oocyte was not pursued in this work and should be

investigated to obtain a complete picture of the role of OPN in gamete interactions. It

appears that OPN, with its RGD binding sequence, could stimulate activation of the egg

as previous studies with RGD peptides have indicated [188]. The effect of OPN on

86

oocyte physiology should be demonstrated much as its effect on sperm was demonstrated

in this work.

These studies describe a role for OPN in sperm function and fertilization, and

suggest additional roles for this fascinating protein in the field of gamete physiology. By

further defining the mechanisms through which OPN affects sperm and ova, additional

understanding of the role this protein plays in reproductive physiology and its effects on

Holstein bull fertility will be obtained.

87

Figure 6.1. Interaction of osteopontin and integrins during fertilization. After penetration of the zona pellucida (ZP), sperm bind to and eventually fuse with the oocyte plasma membrane (PM). Osteopontin (OPN) on the postacrosomal region of the sperm head may adhere to integrins on the PM to facilitate binding of the sperm and egg. This diagram shows a proposed model for how sperm-associated OPN may interact with αvβ3 and α5β1 integrins on the oocyte PM. OPN: osteopontin; ZP: zona pellucida; PM: oocyte plasma membrane.

ZP PM

Integrins αvβ3 α5β1 OPN

OPN

OPN

αvβ3

α5β1

88

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Vita David W. Erikson

Education The Pennsylvania State University University Park, PA Ph.D. in Physiology December 2006 Delaware Valley College Doylestown, PA B.S. in Small Animal Science and Conservation May 2000 Employment and Research Experience The Pennsylvania State University University Park, PA Graduate Student and Research Assistant 2000 to 2006 Ph.D. Dissertation: Role of Osteopontin in Bovine Sperm Capacitation and Fertilization Thesis Adviser: Dr. Gary Killian The Pennsylvania State University University Park, PA Graduate Teaching Assistant 2001 to 2004 Taught upper-level reproductive physiology laboratories emphasizing reproductive gross anatomy and histology of domestic livestock species and concepts related to gamete biology and embryo development. Publications Bergqvist AS, Killian GJ, Erikson DW, Hoshino Y, Bage R, Sato E, Rodriguez-Martinez H. Detection of fas ligand in the bovine oviduct. Animal Reprod Sci 86: 71-88 (2005). Erikson DW, Golash CD, Killian GJ. Increased binding of the oviductal fluid glycoprotein osteopontin following treatment with the RCA-1 lectin. Biol Reprod 66 (Suppl 1): 174 (2002). Erikson DW, Chapman DA, Ealy AD, Killian GJ. Immunodetection of osteopontin on Holstein bull sperm and of αv and α5 integrins on bovine oocytes. Biol Reprod 68 (Suppl 1): 575 (2003). Erikson DW, Killian GJ. Evidence for binding of osteopontin from oviductal fluid to integrins on sperm plasma membranes. Proceedings of the 15th International Congress on Animal Reproduction, Porto Seguro, Brazil: 1 (2004). Goncalves RF, Erikson DW, Ealy AD, Killian GJ. Influence of arginine-glycine-aspartic acid (RGD) in bovine sperm-egg binding and fertilization in vitro. Reprod Fertil Devel 6: 256 (2004). Erikson DW, Killian GJ. Localization of osteopontin on ejaculated and epididymal Holstein bull sperm and flow cytometric analysis of osteopontin on ejaculated Holstein bull sperm. Biol Reprod 73 (Suppl 1): 528 (2005). Fellowships and Awards NIH Training Fellowship (Physiological Adaptations to Stress): 2002-2005 John and Norma Almquist Graduate Scholarship in Dairy and Animal Science 2002- 2003; 2004-2005; 2005-2006 Professional Affiliations Society for the Study of Reproduction American Association for the Advancement of Sciences

role of osteopontin in bovine sperm capacitation and

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