Utah State University DigitalCommons@USU

All Graduate Theses and Dissertations Graduate Studies

12-2012

Mechanisms and Signal Pathways Involved in Bovine Activation

Ammon Hanson Bayles Utah State University

Follow this and additional works at: https://digitalcommons.usu.edu/etd

Part of the Animal Sciences Commons

Recommended Citation Bayles, Ammon Hanson, "Mechanisms and Pathways Involved in Bovine " (2012). All Graduate Theses and Dissertations. 1380. https://digitalcommons.usu.edu/etd/1380

This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. MECHANISMS AND SIGNAL TRANSDUCTION PATHWAYS INVOLVED IN

BOVINE OOCYTE ACTIVATION

by

Ammon Hanson Bayles

A dissertation submitted in partial fulfillment of the requirements for the degree

of

DOCTOR OF PHILOSOPHY

in

Animal, Dairy, and Veterinary Sciences Approved:

______Kenneth L. White Thomas D. Bunch Major Professor Committee Member

______Lee F. Rickords Chris J. Davies Committee Member Committee Member

______Gregory J Podgorski Kenneth L. White Committee Member ADVS Department Head

______Mark R. McLellan Vice President for Research and Dean of the School of Graduate Studies

UTAH STATE UNIVERSITY Logan, Utah

2012 ii

Copyright © 2012 All Rights Reserved iii

ABSTRACT

Mechanisms and Signal Transduction Pathways Involved in Bovine Oocyte Activation

by

Ammon Hanson Bayles, Doctor of Philosophy

Utah State University, 2012

Major Professor: Dr. Kenneth L. White Department: Animal, Dairy, and Veterinary Sciences

In addition to contributing genes at fertilization, the sperm induces the oocyte to leave its arrested state and resume in the process of activation. A hallmark

2+ of oocyte activation is a release of intracellular (Ca i) from the . The mediators of oocyte activation have been studied in many animal models, while little is known in the bovine model. Both Src Family (SFK) and

Phospholipase C (PLC) have been reported to be involved in oocyte activation in other animal models. In this dissertation are described experiments that define the role

2+ of SFK and PLC enzymes in the pathway leading to Ca i and calcium induced calcium release in bovine oocyte activation. Western blotting was used to discover that SFKs Src,

Hck, and Lck are present in matured bovine , and Src, Blk, and Yes are present in acrosome reacted bovine spermatozoa. The PLC δ1 and δ3 are present in both matured bovine oocytes and spermatozoa. PLC δ4, γ2, and η2 are present in matured bovine oocytes. Microinjecting a known general SFK inhibitor, PP2, significantly decreases iv

2+ both Ca i and rates. Microinjecting a 13 peptide that mimics the

2+ phosphorylated carboxyl terminal region of pp60c-src decreases both Ca i and cleavage rates. Microinjecting a downstream substrate of pp60c-src sequestered any signal

2+ produced by Src and decreased Ca i and cleavage rates. Microinjecting primary

2+ raised against PLC isotypes blocked both Ca i and cleavage rates, giving insight to the mechanism of calcium induced calcium release in the bovine model. The

2+ PLC isotypes δ3, δ4, and γ2 decreased Ca i oscillations and cleavage rates, indicating

2+ they are involved in both IP3R and RyR activation. PLC δ4 and η2 did not impact Ca i but did significantly decrease cleavage rates. The data presented in this dissertation increase the understanding of the pathway leading to bovine oocyte activation and further confirm that the detailed pathway differs among animal models.

(125 pages) v

PUBLIC ABSTRACT

Mechanisms and Signal Transduction Pathways Involved in Bovine Oocyte Activation

by

Ammon Hanson Bayles, Doctor of Philosophy

Utah State University, 2012

Major Professor: Dr. Kenneth L. White Department: Animal, Dairy, and Veterinary Sciences

Being able to create a genetically identical, living replicate of a prized animal sounds impossible. Through advanced scientific methods and the wonders of mother nature it can be accomplished by a process called cloning. In 2003, the USU Animal,

Dairy, and Veterinary Science department and Dr. Kenneth L. White gained world wide recognition when they, along with a team for Idaho, cloned the first equine species.

Cloning certain animals could be very advantageous to the agricultural industry.

Prized animals that have died could be cloned, producing the best meat and milk supply.

Animals that are sterile or endangered can be cloned, restoring the genetics and population. Although there are benefits to cloning there are also drawbacks. One of the biggest hurdles is the success rate. Cloning works correctly less than 8% of the time.

Methods that are not biological must be used to produce a clone, and these harsh methods could cause a decrease in success. If there was a better, more biological way to produce a clone the success rate would improve. vi

Through funding provided by the National Institute of Food and Agriculture, U.S.

Department of Agriculture, under Agreement No. 08-34526-19199 and 09-34526-19808, ways to improve the methods of producing a clone have been studied. This dissertation outlines a couple pathways to possibly improving cloning. The that takes place is complex and many additional studies will need to be conducted before some significant improvements are made.

If the success of cloning improves, there may come a time when these methods are used to improve human health by producing human tissue and organs that are genetically perfect. The possibility of curing life threatening illness through therapeutic cloning is exciting. As the success rate of cloning animals improves, so does the chance that cloning will affect and improve human lives. vii

ACKNOWLEDGMENTS

This dissertation was supported by the National Institute of Food and Agriculture,

U.S. Department of Agriculture, under Agreement No. 08-34526-19199 and

09-34526-19808.

I would like to thank my advisor Dr. Kenneth L. White for all his help and patience throughout my graduate career. I thank my committee members Dr. Gregory J.

Podgorski, Dr. Thomas D. Bunch, Dr. Chris J. Davies, and Dr. Lee F. Rickords for all their help. I thank my lab mates Ben Sessions, Justin Hall, Aaron Davis, and Kira

Morgado for their assistance with lab work. Also, a special thank you to my wife,

Natalie, and our children Daxton, Makenna, Tage, Paige, and Korvin for their love and support.

Ammon Bayles viii

CONTENTS

Page ABSTRACT ...... iii

PUBLIC ABSTRACT...... v

ACKNOWLEDGMENTS ...... vii

LIST OF TABLES...... x

LIST OF FIGURES ...... xi

CHAPTER

1. REVIEW OF LITERATURE ...... 1

Oocyte activation: An Overview ...... 1 Mechanisms of Oocyte Activation ...... 2 Src Family Kinase ...... 4 C ...... 5 Summary ...... 7 Impact of the Project ...... 8

2. SRC FAMILY KINASE IN THE ACTIVATION OF BOVINE OOCYTES ...... 23

Abstract ...... 23 Introduction ...... 24 Materials and Methods ...... 28 Results ...... 34 Discussion ...... 36

3. INVOLVEMENT OF ISOTYPES IN THE ACTIVATION OF BOVINE OOCYTES ...... 62

Abstract ...... 62 Introduction ...... 63 Materials and Methods ...... 66 Results ...... 73 Discussion ...... 75 ix

4. SUMMARY ...... 103

CURRICULUM VITAE...... 108 x

LIST OF TABLES

Table Page

2-1 Primary Src family kinase antibodies used for western blotting ...... 56

2-2 Effect of peptides, substrates, and inhibitors on oocyte activation as determined by release of intracellular Ca2+transients ...... 57

2-3 Effect of peptides, substrates, and inhibitors on oocyte activation as determined by the rate of cleavage ...... 58

3-1 Primary PLC antibodies used for western blotting ...... 93

3-2 Effect of microinjected PLC antibodies of oocyte activation as determined by release of intracellular Ca2+ transients ...... 94

3-3 Effect of microinjected PLC antibodies on oocyte activation as determined by the rate of cleavage ...... 95

3-4 The effect of microinjected antagonist, agonist, and antibodies on initial IP3R Ca2+ transients and RyR CICR of IVF produced as determined by the rate of Ca2+ transients ...... 96

3-5 The effect of microinjected antagonist, agonist, and antibodies on initial IP3R Ca2+ transients and RyR CICR of IVF produced embryos as determined by the rate of cleavage ...... 97 xi

LIST OF FIGURES

Figure Page

2-1 Western blot images of Src family kinase in bovine gametes ...... 59

2-2 Effect of peptides, substrates, and inhibitors on oocyte activation as determined by release of intracellular Ca2+transients ...... 60

2-3 Effect of peptides, substrates, and inhibitors on oocyte activation as determined by the rate of cleavage ...... 61

3-1 Western blot images of PLC proteins in bovine gametes ...... 98

3-2 Effect of microinjected PLC antibodies of oocyte activation as determined by release of intracellular Ca2+ transients ...... 99

3-3 Effect of microinjected PLC antibodies on oocyte activation as determined by the rate of cleavage ...... 100

3-4 The effect of microinjected antagonist, agonist, and antibodies on initial IP3R Ca2+ transients and RyR CICR of IVF produced embryos as determined by the rate of Ca2+ transients ...... 101

3-5 The effect of microinjected antagonist, agonist, and antibodies on initial IP3R Ca2+ transients and RyR CICR of IVF produced embryos as determined by the rate of cleavage ...... 102 CHAPTER 1

REVIEW OF LITERATURE

Bovine oocytes are ovulated at an immature state of metabolic arrest at metaphase

II (MII). In order for the oocyte to exit this it must undergo oocyte activation. The biochemical pathway leading to oocyte activation has been studied extensively in animal models such as , zebrafish, ascidian, starfish, , and murine models. To date, there is very little data available explaining the pathways seen in bovine oocyte activation.

Oocyte Activation: An Overview

Oocyte activation is a term given to a set of critical events that takes place when sperm and unite. It is initiated by the fertilizing sperm and is essential for proper . The defining feature of oocyte activation is intercellular calcium

2+ (Ca i) release from the endoplasmic reticulum (ER) (reviewed by Whitaker [1]). Other features of oocyte activation include exocytosis, resumption and completion of II, second extrusion, pronuclear formation, and DNA synthesis [2]. Some of the events associated with of oocyte activation begin within seconds of sperm-egg binding, while others last for hours after binding [3]. The rise and

2+ fall of Ca i lasts up to several hours in mammalian species [4-6]. One early event in

2+ mammalian oocyte activation is the exocytosis of cortical granuals. Ca i release plays an important role in this process. Cortical granuals are located along the cortical region of the oocyte [7] and contain enzymes that can modify the zona 2 pellucida (ZP) [8, 9]. Modifications to the ZP are an important mechanism that provide a block to polyspermy [1, 10-14].

2+ The increase of Ca i also plays an important role in the resumption of meiosis II.

Bovine oocytes are arrested at MII at , due to the presence of maturation promoting factor (MPF) [15]. MPF remains active when cytosolic factor B1 is

2+ present. The increase in Ca i shortly after sperm-oocyte binding induces cyclin degradation, which leads to a down regulation of MPF [16]. A decrease in MPF prompts the oocyte to reenter the cell , thus leaving MII arrest. The extrusion of the second polar body is a distinct morphological change that occurs during the resumption of meiosis. Upon extrusion of the second polar body, the DNA from the oocyte becomes

2+ haploid [17]. Several hours after sperm-oocyte binding, Ca i transients cease, the pronuclei form, and DNA synthesis is initiated [18].

Mechanisms of Oocyte Activation

The signal transduction pathway leading to oocyte activation has been described by two competing theories, a mediated pathway and soluble sperm-borne oocyte-activating factor [19]. The receptor mediated hypotheses postulates that as sperm undergo the , ligands on the inner acrosomal membrane are exposed.

Following penetration and migration across the peri-vitelline space, these inner acrosomal sperm ligands are available to bind to receptors located on the vitelline membrane. There have been several proposed sperm ligands, including, disintegrins

[20-22], fertilin [23, 24], Izumo [25, 26], cyritestin [27], and GPI-anchored proteins [28, 3

29]. All have been shown to play a role in fertilization. In addition, there are likely oocyte receptors on the oocyte that include, [21, 22, 30-33] and tetraspannins

[34] which function in synchrony with integrins. There are two other likely candidate receptors on the oocyte. G- coupled receptors and/or receptor kinase.

These two are potential candidates due to their downstream effects. Integrins, G-protein coupled receptors and receptor are all known to influence downstream Src

Family (SFK) and/or Phospholipase C (PLC) family enzymes. These cell surface receptors activate Phopholipase C (PLC), which cleaves phosphotidylinsoitol 4,5- bisphosphate (PIP2), producing membrane-bound diacyglycerol (DAG) and cytosolic soluble 1,4,5- trisphosphate (IP3). The binding of IP3 to the IP3 receptor (IP3R)

2+ on the endoplasmic reticulum leads to the release of Ca i within the of the oocyte

[35, 36].

The second theory states that a sperm-borne oocyte activating factor (SOAF) is responsible for oocyte activation [37]. This theory follows the logic that as the sperm’s inner acrosomal membrane fuses with the oocyte vitelline membrane, a soluble protein is released into the oocyte’s and is free to act as a SOAF. There have been multiple reports that indicate PLC ζ is the SOAF [38-43] and that in murine oocytes PLC

β1 may work along side[44]. Once in the oocyte cytoplasm, SOAF could act on cytosolic

2+ proteins such as PIP2, PI3 kinase, PLC, and/or SFK to initiate Ca i release through IP3R

2+ activation. It is accepted that Ca i comes from stores within the ER of the oocyte

2+ [45-52]. receptors (RyR) also play a role with IP3R in Ca i and calcium-

2+ induced calcium release (CICR). It is accepted that Ca i can come from cellular stores 4 through two separate channels, IP3R and RyR [53, 54]. Both IP3R and RyR are involved

2+ in Ca i in many different cell types and have been shown to be involved in sea urchin oocyte activation [55]. IP3R and RyR are both present in murine [56-58], porcine [59], human [60-62], and bovine [54, 63] oocytes. IP3R appears to be involved in the initial

2+ Ca i release that is seen at the early stages of activation, while the RyR is involved in later stages as a CICR mechanism [64]. Both IP3R and RyR are able to produce repetitive

2+ Ca i transients [65-67], and specifically, RyR operating primarily in CICR in bovine

2+ oocyte activation [54, 63]. Regardless of its source, Ca i is a hallmark of oocyte activation for all animal models studied to date.

Src Family Kinases

This literature review will focus on the history, structure, and activation of Src

Family Kinases (SFK), and their involvement in oocyte activation in different animal models. SFKs are present in almost every animal cell type. They are activated by growth factors, cell-, , and antigen receptors [68, 69]. Src was first discovered in 1977 by Burgge and Erikson [70]. Shortly thereafter, it was discovered that

Src had protein tyrosine kinase activity [71, 72]. There are nine SFK members: Src, Hck,

Lck, Fyn, Lyn, Blk, Fgr, Yrk, and Yes. All SFKs have very similar structural domains, consisting of SH3, SH2, and tyrosine kinase domains [73]. They also contain an SH4 membrane-targeting region, located near the N-terminus. This SH4 domain is always myristoylated and often palmitoylated [74].

SFKs participate in oocyte activation in several animal models. Sea urchin, ascidian, and starfish share a similar activation pathway involving SFK. Following 5 sperm-oocyte binding and/or fusion, SFK is activated, which then activates downstream

2+ PLC γ and leads to a release of Ca i through IP3R [35, 75, 76]. To date, it is unclear which SFK member(s) are involved in these models. Zebrafish use a similar signal transduction pathway in which Fyn and Src are both activated prior to PLC γ activation

[77]. A Src-related protein has been isolated from Xenopus oocytes [78], and it appears that SFK activation of PLC γ is required for fertilization [45]. The murine model appears quite different from the previously mentioned animal models, as it seems that SFK is not

2+ involved at fertilization. There are reports that SFK is not required for Ca i release and oocyte activation [79, 80]. Supposedly it has been shown that female mice lacking the

SFK Fyn, exhibit a reduction in fertility. This decrease in fertility appears to result from

2+ cytoskeletal defects and is not due to a reduction in Ca i transients [81]. SRC is present in pre-acrosome reacted bovine sperm and is involved in the acrosome reaction during sperm-zona pellucida binding [82]. It is unknown if other SFKs are involved in the acrosome reaction or if they are involved in bovine oocyte activation.

Phospholipase C

Phospholipase C (PLC) is an effector molecule in signal transduction. PLC is responsible for the hydrolysis of 4,5-bisphosphate (PIP2) to produce two second messengers, membrane bound diacylglycerol (DAG) and soluble inositol

1,4,5-trisphosphate (IP3). PLC was first recognized in 1953 from work done on pigeon

2+ pancreas [83] and PLC’s role as Ca i mobilizer was first described in 1983 [84]. Since then, multiple PLC types of different molecular weight, isoelectric points, and calcium dependency have been identified. To date, there are six known PLC isotypes, β, γ, δ, ε, ζ, 6 and η, that have been identified in various mammalian tissues. Within each isotype, there are potential isozymes, these are β1, β2, β3, β4, γ1, γ2, δ1, δ3, δ4, ε, ζ, η1, and η2 [85].

PLC isozymes contain two very consered regions called the catalytic X and Y domains.

These domains are located between an EF-hand domain and a C2 domain [86]. PLC isozymes also contain a PH domain. It has been demonstrated the the PLC δ1 PH domain binds PIP2 and promotes the availability of PLC δ1 to the plasma membrane [87].

PLC γ1 has been implicated in sea urchin, ascidian, zebrafish and starfish oocyte

2+ activation as being responsible for IP3 production and subsequent Ca i release [35,

75-77, 88]. No other PLC isotypes have been implicated in oocyte activation for these model systems. Following fertilization in Xenopus, there is an increase in IP3

2+ concentration that is involved in Ca i release [89, 90]. This spike in IP3 is likely due to the involvement of one or more PLC isotypes. Conflicting results show that neither

PLCγ1 nor PLCβ are required for IP3 production [90], while other studies support that

PLCγ is required [91] and is made active by SFK [92]. To date, it remains unclear if other

2+ PLC isotypes are involved in the production of IP3 and Ca i release in Xenopus.

Many studies have been done on the signal transduction pathway active in murine oocyte activation. Integrins mediate sperm-oocyte binding [93-101]. Following binding, it appears that mice utilize PLC ζ as a SOAF [38, 39, 42, 43]. PLC ζ is proposed to cleave

2+ PIP2, producing the IP3 that causes Ca i release from the ER via IP3R. Another PLC

2+ isotype, PLC β1, has been proposed to function in synchrony with PLCζ to produce Ca i transients and oocyte activation through IP3 production [102]. Recently, the involvement of PLC ζ in oocyte activation has been called into question. Aarabi et al claim that the 7 expression profile and localization of PLC ζ within the sperm head do not support its role as the SOAF [103]. They state that following ZP penetration, PLC ζ is no longer present

2+ within the sperm head and thus could not be involved in Ca i.

In bovine fertilization, the microinjection of bovine PLC ζ cRNA has been shown to activate oocytes at a 0.1 µg/µl [104]. More recently, it has been shown that

2+ microinjecting 0.1 µg/µl of bovine PLC ζ cRNA did not induce Ca i transients [105]. The completion of additional studies should resolve these disparities. While PLC ζ may be

2+ involved in the production of murine Ca i transients, its role in bovine oocyte activation remains unclear.

Summary

Mechanisms of oocyte activation are complex and differ among animals models.

The biochemical pathways have been well defined in sea urchin, zebrafish, ascidian, starfish, and Xenopus. There is very little data available explaining the pathways in bovine oocyte activation, while considerable data attempts to explain oocyte activation in the murine model. Global conclusions about mammalian activation maybe made mistakenly from work done in mice. This dissertation aims to better define the signal transduction pathway seen at bovine oocyte activation.

Oocyte activation is initiated by the fertilizing sperm and is essential for proper

2+ embryonic development. A hallmark of oocyte activation is the release of Ca i from the

ER. The IP3R located on the ER are activated by IP3. IP3 and DAG are produced when

PIP2 is cleaved by PLC. The source of enzymatically active PLC is unknown in the bovine model. 8

Impact of the Project

This dissertation describes experiments that define the role of SFK and PLC

2+ enzymes in Ca i and CICR in bovine oocyte activation. I microinjected antagonists and substrates of SFK, antibodies against the isozymes of PLC, and agonist and antagonists of IP3R and RYR followed by in vitro fertilization to better describe the pathway leading

2+ to Ca i and development in bovine oocytes. Western blotting was used to identify endogenous SFKs and PLC isotypes within bovine gametes. These data increase the understanding of the pathway leading to bovine oocyte activation and confirms that the detailed pathway of activation differs among animal models.

References

1. Whitaker M. Calcium at Fertilization and in Early Development. Physiological

Review 2006; 86.

2. Xu Z, Kopf GS, Schultz RM. Involvement of inositol 1,4,5-trisphosphate-

mediated Ca2+ release in early and late events of mouse egg activation.

Development 1994; 120:1851-1859.

3. Yanagimachi R. Mammalian Fertilization In: Knobil E, Neill JD (eds.), The

Physiology of Reproduction. New York Raven Press Ltd; 1994: 189-317.

4. Miyazaki S, Shirakawa H, Nakada K, Honda Y. Essential role of the inositol

1,4,5-trisphosphate receptor/Ca2+ release channel in Ca2+ waves and Ca2+

oscillations at fertilization of mammalian . Dev Biol 1993; 158:62-78.

5. Cuthbertson KS, Cobbold PH. Phorbol ester and sperm activate mouse oocytes by

inducing sustained oscillations in cell Ca2+. Nature 1985; 316:541-542. 9

6. Miyazaki S, Igusa Y. Fertilization potential in golden hamster eggs consists of

recurring hyperpolarizations. Nature 1981; 290:702-704.

7. Abbott AL, Ducibella T. Calcium and the control of mammalian cortical granule

exocytosis. Frontiers in bioscience: a journal and virtual library 2001;

6:D792-806.

8. Endo Y, Schultz RM, Kopf GS. Effects of phorbol esters and a diacylglycerol on

mouse eggs: inhibition of fertilization and modification of the zona pellucida. Dev

Biol 1987; 119:199-209.

9. Bleil JD, Beall CF, Wassarman PM. Mammalian sperm-egg interaction:

fertilization of mouse eggs triggers modification of the major zona pellucida

, ZP2. Deve Biol 1981; 86:189-197.

10. Sun Q-Y. Cellular and molecular mechanisms leading to and

polyspermy block in mammalian eggs. Microscopy Res and Technique 2003;

61:342-348.

11. Tae JC, Kim EY, Jeon K, Lee KS, Lee CH, Kim YO, Park SP, Kim NH. A MAPK

pathway is involved in the control of cortical granule reaction and mitosis during

bovine fertilization. Molecular Repro and Dev 2008; 75:1300-1306.

12. Swann K, McCulloh DH, McDougall A, Chambers EL, Whitaker M. Sperm-

induced currents at fertilization in sea urchin eggs injected with EGTA and

neomycin. Dev Biol 1992; 151:552-563. 10

13. McAvey BA, Wortzman GB, Williams CJ, Evans JP. Involvement of calcium

signaling and the in the membrane block to polyspermy in

mouse eggs. Biol of Reprod 2002; 67:1342-1352.

14. Ducibella T. The cortical reaction and development of activation competence in

mammalian oocytes. Hum Reprod Update 1996; 2:29-42.

15. Verlhac MH, de Pennart H, Maro B, Cobb MH, Clarke HJ. MAP kinase becomes

stably activated at metaphase and is associated with -organizing

centers during meiotic maturation of mouse oocytes. Deve Biol 1993;

158:330-340.

16. Lorca T, Cruzalegui FH, Fesquet D, Cavadore JC, Mery J, Means A, Doree M.

Calmodulin-dependent II mediates inactivation of MPF and CSF

upon fertilization of Xenopus eggs. Nature 1993; 366:270-273.

17. Williams CJ. Signalling mechanisms of mammalian oocyte activation. Hum

Reprod Update 2002; 8:313-321.

18. Jones KT, Carroll J, Merriman JA, Whittingham DG, Kono T. Repetitive sperm-

induced Ca2+ transients in mouse oocytes are cell cycle dependent. Development

1995; 121:3259-3266.

19. Whitaker M, Swann K. Lighting the fuse at fertilization. Development 1993;

117:1-12.

20. White KL, Passipieri M, Bunch TD, Campbell KD, Pate B. Effects of -

glycine-aspartic acid (RGD) containing snake venom peptides on parthenogenetic 11

development and in vitro fertilization of bovine oocytes. Molecular Reprod and

Deve 2007; 74:88-96.

21. Sessions BR, Aston KI, Davis AP, Pate BJ, White KL. Effects of amino acid

substitutions in and around the arginine-glycine-aspartic acid (RGD) sequence on

fertilization and parthenogenetic development in mature bovine oocytes.

Molecular Reprod and Deve 2006; 73:651-657.

22. Campbell KD, Reed WA, White KL. Ability of integrins to mediate fertilization,

intracellular calcium release, and parthenogenetic development in bovine oocytes.

Biol Reprod 2000; 62:1702-1709.

23. Nishimura H, Cho C, Branciforte DR, Myles DG, Primakoff P. Analysis of loss of

adhesive function in sperm lacking cyritestin or fertilin beta. Dev Biol 2001;

233:204-213.

24. Frayne J, Hall L. Mammalian sperm-egg recognition: does fertilin beta have a

major role to play? BioEssays: news and reviews in molecular, cellular and

1999; 21:183-187.

25. Inoue N, Ikawa M, Isotani A, Okabe M. The immunoglobulin superfamily protein

Izumo is required for sperm to fuse with eggs. Nature 2005; 434:234-238.

26. Sleight SB, Miranda PV, Plaskett NW, Maier B, Lysiak J, Scrable H, Herr JC,

Visconti PE. Isolation and proteomic analysis of mouse sperm detergent-resistant

membrane fractions: evidence for dissociation of lipid rafts during capacitation.

Biol Reprod 2005; 73:721-729. 12

27. Yuan R, Primakoff P, Myles DG. A role for the disintegrin domain of cyritestin, a

sperm surface protein belonging to the ADAM family, in mouse sperm-egg

plasma membrane adhesion and fusion. J cell Biol 1997; 137:105-112.

28. Coonrod SA, Naaby-Hansen S, Shetty J, Shibahara H, Chen M, White JM, Herr

JC. Treatment of mouse oocytes with PI-PLC releases 70-kDa (pI 5) and 35- to

45-kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma

binding and fusion. Dev Biol 1999; 207:334-349.

29. Coonrod S, Naaby-Hansen S, Shetty J, Herr J. PI-PLC releases a 25-40 kDa

protein cluster from the hamster oolemma and affects the sperm penetration assay.

Molecular human reproduction 1999; 5:1027-1033.

30. Fusi FM, Vignali M, Gailit J, Bronson RA. Mammalian oocytes exhibit specific

recognition of the RGD (Arg-Gly-Asp) tripeptide and express oolemmal integrins.

Mol Reprod Dev 1993; 36:212-219.

31. Almeida EA, Huovila AP, Sutherland AE, Stephens LE, Calarco PG, Shaw LM,

Mercurio AM, Sonnenberg A, Primakoff P, Myles DG, White JM. Mouse egg

alpha 6 beta 1 functions as a sperm receptor. Cell 1995; 81:1095-1104.

32. Bronson RA, Fusi F. Evidence that an Arg-Gly-Asp adhesion sequence plays a

role in mammalian fertilization. Biol Reprod 1990; 43:1019-1025.

33. Bronson RA, Fusi F. Sperm-oolemmal interaction: role of the Arg-Gly-Asp

(RGD) adhesion peptide. Fertility and Sterility 1990; 54:527-529.

34. Chen MS, Tung KS, Coonrod SA, Takahashi Y, Bigler D, Chang A, Yamashita Y,

Kincade PW, Herr JC, White JM. Role of the integrin-associated protein CD9 in 13

binding between sperm ADAM 2 and the egg integrin alpha6beta1: implications

for murine fertilization. Proc Natl Acad Sci USA 1999; 96:11830-11835.

35. Jaffe LA, Giusti AF, Carroll DJ, Foltz KR. Ca2+ signalling during fertilization of

echinoderm eggs. Semin Cell Dev Biol 2001; 12:45-51.

36. Malcuit C, Knott JG, He C, Wainwright T, Parys JB, Robl JM, Fissore RA.

Fertilization and inositol 1,4,5-trisphosphate (IP3)-induced calcium release in

type-1 inositol 1,4,5-trisphosphate receptor down-regulated bovine eggs. Biol

Reprod 2005; 73:2-13.

37. Kuretake S, Kimura Y, Hoshi K, Yanagimachi R. Fertilization and development of

mouse oocytes injected with isolated sperm heads. Biol Reprod 1996; 55:789-795.

38. Kouchi Z. Recombinant Phospholipase C Has High Ca2+ Sensitivity and Induces

Ca2+ Oscillations in Mouse Eggs. J Bio Chem 2003; 279:10408-10412.

39. Swann K, Larman MG, Saunders CM, Lai FA. The cytosolic sperm factor that

triggers Ca2+ oscillations and egg activation in is a novel

phospholipase C: PLCzeta. Reproduction 2004; 127:431-439.

40. Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann

K, Lai FA. PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and

development. Development 2002; 129:3533-3544.

41. Kouchi Z, Fukami K, Shikano T, Oda S, Nakamura Y, Takenawa T, Miyazaki S.

Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+

oscillations in mouse eggs. J Bio Chem 2004; 279:10408-10412. 14

42. Kurokawa M. Mammalian Fertilization: From Sperm Factor to Phospholipase Cζ.

Biol of the Cell 2004; 96:37-45.

43. Fujimoto S, Yoshida N, Fukui T, Amanai M, Isobe T, Itagaki C, Izumi T, Perry

ACF. Mammalian phospholipase Cζ induces oocyte activation from the sperm

perinuclear matrix. Dev Biol 2004; 274:370-383.

44. Igarashi H, Knott JG, Schultz RM, Williams CJ. Alterations of PLCβ1 in mouse

eggs change calcium oscillatory behavior following fertilization. Dev Biol 2007;

312:321-330.

45. Sato K-i, Tokmakov AA, Iwasaki T, Fukami Y. Tyrosine kinase-dependent

activation of phospholipase Cγ is required for calcium transient in Xenopus egg

fertilization. Dev Biol 2000; 224:453-469.

46. Crossley I, Swann K, Chambers EL, Whitaker M. Activation of sea urchin eggs

by inositol phosphates is independent of external calcium. Biochem J 1988;

252:257-262.

47. Terasaki M, Jaffe LA. Organization of the sea urchin egg endoplasmic reticulum

and its reorganization at fertilization. J Cell Biol 1991; 114:929-940.

48. Kline D. Attributes and dynamics of the endoplasmic reticulum in mammalian

eggs. Curr Top Dev Biol 2000; 50:125-154.

49. Galione A, Lee HC, Busa WB. Ca(2+)-induced Ca2+ release in sea urchin egg

homogenates: modulation by cyclic ADP-ribose. Science 1991; 253:1143-1146.

50. Stricker SA. Comparative Biology of during Fertilization and

Egg Activation in Animals. Dev Biol 1999; 211:157-176. 15

51. Rickords LF, White KL. Electroporation of inositol 1,4,5-triphosphate induces

repetitive calcium oscillations in murine oocytes. J Exp Zool 1993; 265:178-184.

52. Mitalipov SM, White KL, Farrar VR, Morrey J, Reed WA. Development of

Nuclear Transfer and Parthenogenetic Rabbit Embryos Activated with Inositol

1,4,5-Trisphosphate. Biol Rep 1999; 60:821-827.

53. Balakier H, Dziak E, Sojecki A, Librach C, Michalak M, Opas M. Calcium-

binding proteins and calcium-release channels in human maturing oocytes,

pronuclear and early preimplantation embryos. Hum Reprod 2002;

17:2938-2947.

54. Yue C, White KL, Reed WA, Bunch TD. The existence of inositol 1,4,5-

trisphosphate and ryanodine receptors in mature bovine oocytes. Development

1995; 121:2645-2654.

55. McPherson SM, McPherson PS, Mathews L, Campbell KP, Longo FJ. Cortical

localization of a calcium release channel in sea urchin eggs. J cell Biol 1992;

116:1111-1121.

56. Ayabe T, Kopf GS, Schultz RM. Regulation of mouse egg activation: presence of

ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose

on uninseminated and inseminated eggs. Development 1995; 121:2233-2244.

57. Mehlmann LM, Mikoshiba K, Kline D. Redistribution and increase in cortical

inositol 1,4,5-trisphosphate receptors after meiotic maturation of the mouse

oocyte. Dev Biol 1996; 180:489-498. 16

58. Fissore RA, Longo FJ, Anderson E, Parys JB, Ducibella T. Differential

distribution of receptor isoforms in mouse oocytes. Biol of

Repro 1999; 60:49-57.

59. Machaty Z, Funahashi H, Day BN, Prather RS. Developmental changes in the

intracellular Ca2+ release mechanisms in porcine oocytes. Biol of Repro 1997;

56:921-930.

60. Tesarik J, Sousa M, Mendoza C. Sperm-induced calcium oscillations of human

oocytes show distinct features in oocyte center and periphery. Molecular Repro

and Dev 1995; 41:257-263.

61. Sousa M, Barros A, Tesarik J. The role of ryanodine-sensitive Ca2+ stores in the

Ca2+ oscillation machine of human oocytes. Molecular human reproduction

1996; 2:265-272.

62. Goud PT, Goud AP, Van Oostveldt P, Dhont M. Presence and dynamic

redistribution of type I inositol 1,4,5-trisphosphate receptors in human oocytes

and embryos during in-vitro maturation, fertilization and early cleavage divisions.

Molecular human reproduction 1999; 5:441-451.

63. Yue C, White KL, Reed WA, King E. Localization and regulation of ryanodine

receptor in bovine oocytes. Biol Reprod 1998; 58:608-614.

64. Fill M, Copello JA. calcium release channels. Physiol Rev

2002; 82:893-922. 17

65. Berridge MJ, Galione A. Cytosolic calcium oscillators. FASEB J: official

publication of the Federation of American Societies for Experimental Biology

1988; 2:3074-3082.

66. Missiaen L, Taylor CW, Berridge MJ. Spontaneous calcium release from inositol

trisphosphate-sensitive calcium stores. Nature 1991; 352:241-244.

67. Taylor CW, Marshall IC. Calcium and inositol 1,4,5-trisphosphate receptors: a

complex relationship. Trends in biochemical sciences 1992; 17:403-407.

68. Brown MT, Cooper JA. Regulation, substrates and functions of src. Biochimica et

Biophysica Acta 1996; 1287:121-149.

69. Thomas SM, Brugge JS. Cellular functions regulated by Src family kinases.

Annual Rev Cell and Dev 1997; 13:513-609.

70. Brugge JS, Erikson RL. Identification of a transformation-specific antigen

induced by an avian sarcoma virus. Nature 1977; 269:346-348.

71. Collett MS, Erikson RL. Protein kinase activity associated with the avian sarcoma

virus src gene product. Proc Natl Acad Sci USA 1978; 75:2021-2024.

72. Levinson AD, Oppermann H, Levintow L, Varmus HE, Bishop JM. Evidence that

the transforming gene of avian sarcoma virus encodes a protein kinase associated

with a phosphoprotein. Cell 1978; 15:561-572.

73. Boggon TJ, Eck MJ. Structure and regulation of Src family kinases.

2004; 23:7918-7927. 18

74. Resh MD. Fatty acylation of proteins: new insights into membrane targeting of

myristoylated and palmitoylated proteins. Biochimica et Biophysica Acta 1999;

1451:1-16.

75. Runft L. Egg Activation at Fertilization: Where It All Begins. Dev Biol 2002;

245:237-254.

76. Giusti AF, Carroll DJ, Abassi YA, Foltz KR. Evidence that a starfish egg Src

family tyrosine kinase associates with PLC-gamma1 SH2 domains at fertilization.

Dev Biol 1999; 208:189-199.

77. Giusti AF, Carroll DJ, Abassi YA, Terasaki M, Foltz KR, Jaffe LA. Requirement

of a Src family kinase for initiating calcium release at fertilization in starfish eggs.

J Biol Chem 1999; 274:29318-29322.

78. Kinsey WH, Wu W, Macgregor E. Activation of Src-family PTK activity at

fertilization: role of the SH2 domain. Dev Biol 2003; 264:255-262.

79. Sato K, Aoto M, Mori K, Akasofu S, Tokamakov AA, Sahara S, Fukami Y.

Purification and Characterization of a Src-related p57 Protein-tyrosine Kinase

from Xenopus Oocytes. J Biol Chem 1996; 271:13250-13257.

80. Kurokawa M. Evidence that activation of Src family kinase is not required for

fertilization-associated [Ca2+]i oscillations in mouse eggs. Reproduction 2004;

127:441-454.

81. Mehlmann LM. SH2 domain-mediated activation of an SRC family kinase is not

required to initiate Ca2+ release at fertilization in mouse eggs. Reproduction

2005; 129:557-564. 19

82. Lou J, McGinnis LK, Kinsey WH. Role of Fyn kinase in oocyte developmental

potential. Reprod, Fertility and Dev 2010; 22:966-976.

83. Etkovitz N, Tirosh Y, Chazan R, Jaldety Y, Daniel L, Rubinstein S, Breitbart H.

Bovine sperm acrosome reaction induced by G-protein-coupled receptor agonists

is mediated by epidermal receptor . Dev Biol 2009;

334:447-457.

84. Hokin MR, Hokin LE. secretion and the incorporation of P32 into

phospholipides of pancreas slices. J Biol Chem 1953; 203:967-977.

85. Streb H, Irvine RF, Berridge MJ, Schulz I. Release of Ca2+ from a

nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-

trisphosphate. Nature 1983; 306:67-69.

86. Suh PG, Park JI, Manzoli L, Cocco L, Peak JC, Katan M, Fukami K, Kataoka T,

Yun S, Ryu SH. Multiple roles of phosphoinositide-specific phospholipase C

isozymes. BMB Reports 2008; 41:415-434.

87. Essen LO, Perisic O, Cheung R, Katan M, Williams RL. Crystal structure of a

mammalian phosphoinositide-specific phospholipase C delta. Nature 1996;

380:595-602.

88. Paterson HF, Savopoulos JW, Perisic O, Cheung R, Ellis MV, Williams RL, Katan

M. Phospholipase C delta 1 requires a pleckstrin homology domain for interaction

with the plasma membrane. Biochem J 1995; 312 ( Pt 3):661-666. 20

89. Snow P, Yim DL, Leibow JD, Saini S, Nuccitelli R. Fertilization stimulates an

increase in inositol trisphosphate and inositol lipid levels in Xenopus eggs. Dev

Biol 1996; 180:108-118.

90. Runft LL, Watras J, Jaffe LA. Calcium release at fertilization of Xenopus eggs

requires type I IP3 receptors, but not SH2 domain-mediated activation of PLCg or

Gq-mediated activation of PLCb. Dev Biol 1999; 214:399-411.

91. Sato K. Tyrosine Kinase-Dependent Activation of Phospholipase Cγ Is Required

for Calcium Transient in Xenopus Egg Fertilization. Developmental Biology

2000; 224:453-469.

92. Tokmakov AA, Sato KI, Iwasaki T, Fukami Y. Src kinase induces calcium release

in Xenopus egg extracts via PLCgamma and IP3-dependent mechanism. Cell

Calcium 2002; 32:11-20.

93. Boissonnas CC, Montjean D, Lesaffre C, Auer J, Vaiman D, Wolf J-P, Ziyyat A.

Role of sperm αvβ3 integrin in mouse fertilization. Dev Dynamics 2010;

239:773-783.

94. Evans JP, Kopf GS, Schultz RM. Characterization of the binding of recombinant

mouse sperm fertilin beta subunit to mouse eggs: evidence for adhesive activity

via an egg beta1 integrin-mediated interaction. Dev Biol 1997; 187:79-93.

95. Evans JP, Schultz RM, Kopf GS. Characterization of the binding of recombinant

mouse sperm fertilin alpha subunit to mouse eggs: evidence for function as a cell

adhesion molecule in sperm-egg binding. Dev Biol 1997; 187:94-106. 21

96. Evans JP, Schultz RM, Kopf GS. Identification and localization of integrin

subunits in oocytes and eggs of the mouse. Molec Reprod and Dev 1995;

40:211-220.

97. Evans JP, Schultz RM, Kopf GS. Mouse sperm-egg plasma membrane

interactions: analysis of roles of egg integrins and the mouse sperm homologue of

PH-30 (fertilin) beta. J Cell Sci 1995; 108 ( Pt 10):3267-3278.

98. Vjugina U, Zhu X, Oh E, Bracero NJ, Evans JP. Reduction of mouse egg surface

integrin alpha9 subunit (ITGA9) reduces the egg's ability to support sperm-egg

binding and fusion. Biol of Reprod 2009; 80:833-841.

99. Barraud-Lange V, Naud-Barriant N, Saffar L, Gattegno L, Ducot B, Drillet A-S,

Bomsel M, Wolf J-P, Ziyyat A. Alpha6beta1 integrin expressed by sperm is

determinant in mouse fertilization. BMC Dev Biol 2007; 7:102.

100. Tatone C, Carbone M. Possible involvement of integrin-mediated signalling in

oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate

and cortical granule exocytosis in mouse oocytes. Reprod Biol

and Endocrinol 2006; 4:48.

101. Chen H, Sampson NS. Mediation of sperm-egg fusion: evidence that mouse egg

alpha6beta1 integrin is the receptor for sperm fertilinbeta. Chem Biol 1999;

6:1-10.

102. Igarashi H, Knott J, Schultz R, Williams C. Alterations of PLCβ1 in mouse eggs

change calcium oscillatory behavior following fertilization. Deve Biol 2007;

312:321-330. 22

103. Aarabi M, Yu Y, Xu W, Tse MY, Pang SC, Yi YJ, Sutovsky P, Oko R. The

testicular and epididymal expression profile of PLCzeta in mouse and human does

not support its role as a sperm-borne oocyte activating factor. PloS one 2012;

7:e33496.

104. Ross PJ, Beyhan Z, Iager AE, Yoon S-Y, Malcuit C, Schellander K, Fissore RA,

Cibelli JB. Parthenogenetic activation of bovine oocytes using bovine and murine

phospholipase C zeta. BMC Deve Biol 2008; 8:16.

105. Cooney MA, Malcuit C, Cheon B, Holland MK, Fissore RA, D'Cruz NT. Species-

specific differences in the activity and nuclear localization of murine and bovine

phospholipase C zeta 1. Biol of Reprod 2010; 83:92-101. 23

CHAPTER 2

SRC FAMILY KINASE IN

THE ACTIVATION OF BOVINE OOCYTES

Abstract

In addition to contributing genes at fertilization, the sperm cell induces the oocyte to leave its MII arrested state and resume metabolism in the process of activation. A hallmark of oocyte activation is a release of intracellular calcium stores that results in repetitive transients. The mediators of oocyte activation have been studied extensively in sea urchin, zebrafish, ascidian, starfish, Xenopus, and murine models, while much less is known in the bovine model. Src Family Kinases (SFKs) has been reported to be involved in oocyte activation in many animal models. In this study we investigated the role of

SFKs in bovine oocyte activation. Western blotting was used to identify endogenous

SFKs within bovine gametes. Specific SFKs, notably SRC, were then targeted by the microinjection of antagonists and substrates to determine the role of individual SFKs on

2+ intracellular Ca release following fertilization and cleavage rates 48 hours post- fertilization. SRC was detected in both bovine oocytes and spermatozoa. Inhibition of

SRC significantly decreased oocyte activation, as assessed by decreased intracellular

Ca2+ transients and cleavage rates. These data indicate involvement of SFKs, especially

SRC, in the bovine oocyte activation pathway, which supports our hypothesis that the pathways leading to oocyte activation differ among animal models. 24

Introduction

Bovine oocytes are ovulated in a state of metabolic arrest at metaphase II (MII).

Activation allows the oocyte to exit this developmental checkpoint. Oocyte activation is initiated by the fertilizing sperm and is essential for initiating embryonic development.

2+ Activation includes repetitive intracellular calcium (Ca i) transients, cortical granule exocytosis, resumption and completion of meiosis II, pronuclear formation, second polar

2+ body extrusion, DNA synthesis, and subsequent embryonic development [1]. Ca i transients are involved in cortical granule exocytosis, which initiates a block to

2+ polyspermy [2-7]. Although the additional biological impacts of Ca i transients are not fully understood, these transients are believed to be the second messenger responsible for initiating down-regulatation of metaphase promoting factor (MPF) activity [8]. This directs the oocyte to re-enter the cell cycle and exit metaphase arrest.

Two hypotheses have been proposed to explain the signal transduction pathway

2+ that results in Ca i [9]. One is that spermatozoa bind to a receptor on the vitelline membrane of the oocyte. There are three potential receptor candidates: G-protein coupled receptors, receptor tyrosine kinases, and/or integrins. These receptors are thought to activate phospholipase C (PLC) either directly or by an indirect pathway that involves SFK activation. PLC cleaves phosphtidylinositol 4,5-bisphosphate (PIP2), yielding plasma membrane-bound diacylglycerol (DAG) and soluble cytosolic 1,4,5- inositol trisphosphate (IP3). IP3 then binds to the endoplasmic reticulum (ER) membrane-spanning IP3 receptor (IP3R). IP3R activation results in the releases of calcium from the ER into the cytosol of the oocyte. The second hypothesis proposes that 25 when spermatozoa fuse with the vitelline membrane, a sperm-borne oocyte-activating factor (SOAF) [10] is released from the sperm into the oocyte. SOAF interacts with cytosolic targets, such as PI3 kinase, SFK, and/or PLC isoforms to generate downstream

2+ Ca i transients via the production of IP3. It is generally accepted for animal models that

2+ calcium released during Ca i transients comes from the ER [11-20]. In many fish and echinoderm oocytes ryanodine receptors (RyR) are also a substantial trigger for Ca2+ release, acting in parallel with IP3R to facilitate calcium efflux and calcium-induced calcium release [21-25]. RyR pathways operate within bovine [26] and murine oocytes

2+ [27]. Whether through IP3R or RyR pathways, Ca i is the hallmark of oocyte activation for all animal models studied to date.

Oocyte activation pathways have been studied extensively in sea urchin, zebrafish, ascidian, starfish, Xenopus, and murine models, but there is comparatively little information available in the bovine model. Sea urchin, ascidian, and starfish share a similar oocyte activation pathway following spermatozoa and oocyte plasma membrane binding and/or fusion. This involves the activation of a SFK, which in turn activates

PLCγ. This produces IP3 which leads to Ca2+ release through the ER bound IP3R

[28-31]. It is not yet clear which member of the Src kinase family is activated in these species and whether more SFK than one plays a role in activation. Zebrafish share a similar pathway as the spermatozoa activates the SFK Fyn and/or Src prior to PLCγ activation [32]. Oocyte activation in Xenopus can be facilitated through contact with cathepsin B [33], peptides containing the integrin-binding RGD sequence [34], or the disintegrin region of testies-derived cDNA that codes for a metalloprotease/disintegrin 26 termed xMDC16 [35]. It appears that the Xenopus spermatozoa post-acrosomal sheath

2+ WW domain binding protein is capable of eliciting Ca i [36]. Following fertilization in

Xenopus, there is a rise in IP3 levels that is necessary for calcium efflux [37, 38]. Some studies indicate that in Xenopus neither PLCγ1 or PLCβ are necessary for IP3 production

2+ and the resulting Ca i transients [38], while other studies support the idea that PLCγ is required at fertilization [39]. PLCγ activity is triggered by SFK activation [40].

In the murine model, several membrane proteins are involved in the sperm-oocyte interaction. CD9 is a member of the tetraspanin superfamily of proteins. Knockout CD9

-/- female mice ovulate oocytes that exhibit a severe reduction in fertility, suggesting a key role for CD9 in fertilization [41-43]. Another protein with a role in fertilization is

Izumo, an immunoglobulin superfamily member. Izumo -/- male mice produce morphologically normal sperm, but are infertile because they cannot fuse with the oocyte vitelline membrane [44]. Substantial data supports the hypothesis that integrins mediate sperm-oocyte binding in mice [45-51]. Following sperm binding and fusion, it is believed that mouse oocyte activation is facilitated by PLCζ, a soluble sperm PLC isoform

[52-55]. An endogenous mouse oocyte PLCβ1 has been proposed to work in synchrony

2+ with sperm PLCζ to drive Ca i and subsequent oocyte activation via IP3R [56]. In many of the animal models, members of SFK are used to relay the activation signal downstream to PLC. In the murine model, SRC activation appears not to be required for

2+ downstream Ca i transients and oocyte activation [57, 58]. In female mice lacking the

SFK Fyn, there was a reduction of fertility, but this was attributed to cytoskeletal defects

2+ rather than a reduction in Ca i [59]. 27

Intracytoplasmic sperm injection (ICSI) may be used as tool to provide insight into the signal transduction pathway leading to oocyte activation among various animal models. ICSI consists of directly injecting an intact spermatozoa head into the ooplasm of an MII oocyte. This technique has been successfully used in hamsters for the past 34 years [60]. Since 1992, ICSI has been successful in men with major oligospermia and asthenozoospermia [61]. ICSI has been performed in rabbits [62], hamsters [60], sheep

[63, 64], cats [65, 66], mice [67, 68], goats [69-72], horses [73-76], pigs [77-79], cattle

[80-82], humans [83-85], and baboons [86], with varying degrees of success. Although

ICSI often must be accompanied by an activation step, usually in the form of a chemical activator such as ethanol, ionomycin, cycloheximide, or 6-dimethylaminopurine, this is not always essential. ICSI alone can induce oocyte activation in humans [83, 87], mice

[88], rabbits [62], and hamsters [60]. In these species, oocyte activation is solely facilitated either by the sperm head and its contents, such as a SOAF, or by the ISCI injection procedure that mechanically triggers activation. ICSI alone, however, has not stimulated proper oocyte activation in the bovine [89]. The success of ICSI supports the hypothesis that oocyte activation occurs via a SOAF. This suggests that signal transduction pathways needed for oocyte activation can function in the absence of spermatozoa-vitelline membrane binding [89, 90]. The SOAF model appears sound for species in which ICSI alone results in oocyte activation. But in species, such as cattle [80,

91-97], that require an additional exogenous activator, receptor-mediated events appear to be required in addition to SOAF. One report indicates that less than 10% of bovine 28

2+ oocytes subjected to ICSI alone showed Ca i and that normal signal transduction pathways seen during natural fertilization were not replicated following ICSI [97].

Previous work and findings reported here indicate that bovine oocyte activation is quite different from the mouse and may be more similar to activation in Xenopus and the zebrafish. To better understand the signal transduction pathways of bovine oocyte activation and the role of SFK, the present study was conducted to: 1) learn if endogenous SFKs are present within bovine oocytes and spermatozoa; 2) determine the

2+ effect of SFKs antagonists and substrates on Ca i following fertilization, and 3) assess how SFKs antagonists and substrates affect cleavage rates 48 hours post fertilization.

Materials and Methods

Reagents

All reagents were purchased from MP (ICN) Biomedical (Costa Mesa, CA) unless otherwise specified.

Oocyte Collection and

All procedures were performed according to published methods [27, 98-101].

Briefly, bovine ovaries were collected from a local abattoir (JBS Swift & Company,

Hyrum, UT) and transported to the laboratory for processing. Oocytes from 3-8 mm follicles were aspirated into 50-mL centrifuge tubes using an 18-gauge needle connected to a vacuum pump. Oocytes with intact multiple layers of cumulus cells and evenly shaded cytoplasm were selected and washed with PB1+ (Ca2+ and Mg2+ containing phosphate buffered saline, 0.32 mM sodium pyruvate, 5.55 mM glucose, 3 mg/mL BSA). 29

Oocytes were then transferred into 500 µL of maturation medium (MM), M199 containing 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), 0.5 µg/mL

FSH (Sioux Biochemical, Sioux City, IA), 5 µg/mL LH (Sioux Biochemical), 100 units/ mL penicillin (Hyclone), and 100 µg/mL streptomycin (Hyclone) in 4-well culture dishes

(Nunc, Milwaukee, WI) and cultured at 39ºC in a humidified atmosphere of 5% CO2 and air for 20-22 h.

Cumulus Cell Removal

Cumulus cells were completely removed from mature oocytes by vortexing vigorously for 4 min 20 seconds in PB1+ containing 10 mg/mL hyaluronidase. Denuded oocytes were evaluated by microscopy and only oocytes with good morphology were used. Selected oocytes were washed through 4 drops of PB1+ then randomly assigned to a treatment group and placed back in MM at 39ºC in a humidified atmosphere of 5% CO2 to recover prior to microinjection.

Microinjection

Thin-bore borosilicate glass capillaries were pulled into microinjection needles using a Narishige (East Meadow, NY) PB-7 pipette puller. Holding pipettes were similarly pulled and crafted using a Narishige MF-9 micro forge. Manipulation dishes were made using 100 µL PB1- (Ca2+ and Mg2+ free) drops under mineral oil. Cumulus- free mature oocytes were microinjected using a Nikon Diaphot inverted microscope and

Narishige IM 300 pneumatic micro-injector connected to compressed N2 gas with appropriate treatment. A typical bovine oocyte has a volume of ~800 pL. The 30 microinjection volume was calculated by assessing the formation of a bubble under oil immersion and then measuring it by an ocular micrometer. A volume of ~8 pL (1/100 of the oocyte volume) was microinjected into each oocyte. Microinjection was assumed to result in a 100-fold dilution of any injected material. All data is represented as this 100- fold diluted concentration. Groups of up to 20 oocytes were microinjected in batch to minimize the time oocytes were out of incubator. Microinjection volume was validated every 10 injections to ensure consistent injection volume. Sham controls were injected with vehicle medium, either ddH2O or DMSO. There were no observable differences between the vehicle medium groups (data not shown). Oocytes randomly assigned to IVF and No sperm groups were removed from incubation conditions and placed in a manipulation dish out of the incubator for equal time as other treatment groups.

In Vitro Fertilization of Oocytes and Culture of Embryos

After maturation, oocytes were fertilized and cultured in vitro (IVF) according to our standard laboratory procedures [100]. Frozen-thawed semen (Hoffman AI Breeders,

Logan, UT) obtained from the same bull on multiple collection days, was separated by centrifugation for 30 min at 400-x gravity on a 45% over 90% Percoll (Sigma, St. Louis,

MO) gradient. The resulting pellet was washed with Sperm TL with 6 mg/ml BSA and centrifuged again at 65x gravity for 10 min. The final pellet was diluted in Fertilization

Medium (FM) containing 1 µg/mL heparin and 4% (v/v) PHE (2mM penacillamine, 1 mM hypotaurine, 250 µM epinephrine). Oocytes were washed through 4 drops of PB1+, and incubated in 4-well culture dishes containing 500 µL of sperm/FM for 6 h at 39ºC in a humidified atmosphere of 5% CO2 and air. Fertilized oocytes were then removed from 31 sperm/FM and vortexed vigorously in PB1+ for 1 min to remove zona pellucida bound spermatozoa cells. Denuded oocytes were cultured in 500 µL CR2 [101] containing 3%

FBS over a monolayer of bovine cumulus cells at 39ºC in a humidified atmosphere of 5%

CO2 and air. Activation and development were assessed by identifying percent cleavage at day 2 of culture.

Visualization of Intracellular Calcium Transients

After microinjection, oocytes were assigned to treatment groups. Where calcium visualization was necessary, oocytes were loaded with Ca2+ indicator by incubation in 2

µM Fura-2 AM ester (Molecular Probes, Eugene, OR) and 0.02% Pluronic F-127

(Molecular Probes, Eugene, OR) in PB1- for 45 min in darkness at 39°C. Fura-2 loaded oocytes were washed extensively in 9 drops of PB1-, then 10 oocytes were placed in a 10

µL micro drop of sperm/FM under mineral oil and incubated at 39ºC in a humidified atmosphere of 5% CO2 and air for 6 h. The control group with no sperm was placed in a

2+ 10 µL micro-drop of FM. Following incubation with sperm, Ca i in the oocytes was measured for 30 minutes on an a Nikon Diaphot inverted microscope, coupled with a

Hamamatsu ORCA-ER digital camera and analyzed using Metafluor Imaging Series 7.6 software (Universal Imaging Corp, Downingtown, PA). The oocytes were placed back in

2+ 2+ culture conditions for 1.5 hours, then Ca i visualization was repeated. Ca i transients were evaluated by ratio changes in florescence intensity at 340 nm and 380 nm. Oocytes assigned to the ionomycin control treatment were visualized for only the first interval. At

2+ that time all oocytes exhibiting Ca i transients were removed and 1 µL of 1 mg/ml

2+ ionomycin was added to the drop containing remaining oocytes and Ca i transients were 32 then immediately measured for 30 minutes. Oocytes assigned to the IP3 treatment group

2+ were similarly measured for the first interval, then all oocytes exhibiting Ca i transients

2+ were removed. The remaining ooyctes were microinjected with 0.0025 µM IP3 and Ca i transients were immediately measured for 30 minutes.

Western Blot- Sperm sample preparation

Sperm were capacitated with heparin and the acrosomal reaction with lysophosphatidylcholine was performed according to published protocols [102]. Briefly, frozen-thawed semen (Hoffman AI Breeders, Logan, UT) was washed in Sperm TL with

6 mg/m BSA for 10 min at 400-x gravity. The resulting pellet was resuspended in 5ml

Sperm TL and 1 µg/ml heparin and vortexed vigorously for 20 seconds. The suspension was separated into 500 µL aliquots and incubated with 5 mg/ml Lysophophotidly for 15 minutes at 39°C. Aliquots were then centrifuged at 10,000 RPM for 10 minutes and the resulting pellet resuspended in 100 µL lysis buffer with protease inhibitor, vortexed and placed on a shaker at 4°C overnight. The aliquots were then centrifuged at

10,000 RPM for 10 minutes at 4°C, further aliquoted into 25 µL volumes and then frozen at -80°C until Western blot analysis.

Oocytes sample preparation

Cumulus cells were removed as previously described, then 50 bovine oocytes were added to 6.5 µL double-strength sample buffer and 5 µL ddH2O in a 0.5 ml

Eppendorf tube and frozen at -80ºC until use. 33

To confirm that primary antibodies would cross-react with predicted bovine proteins, protein was extracted from bovine brain. Briefly, 100 mg of ground brain tissue was placed in 10 ml 1X SDS buffer (1.25 M rTis HCl, 2% SDS, 10% glycerol, 0.001%

Bromophenol Blue, 5% 2-mercaptoethanol), incubated in an Eppendorf Thermomixer® for 10 min at 70°C with maximum shaking, centrifuged at 16,000 x g at room temperature for 10 minutes. The supernatant was placed into a fresh tube and frozen at

-20°C until use. Primary antibodies used are listed in Table 2-1.

Western blot analysis of bovine SFKs was performed according to the procedures used by Sun and Fan [103]. Samples were boiled for 4 minutes, placed on ice for 5 minutes, and centrifuged at 12,000g for 3 minutes. Total proteins were separated by

SDS-page with a 4% stacking gel and a 10% separating gel for 45 minutes at 110 V and

115 mA (Pierce Precise Protein Gels), while the nitrocellulose membrane and filters were incubated in the transfer buffer at 4ºC for 45 minutes. After electrophoresis, the gel was washed with transfer buffer for 15 minutes and then the total protein was transferred electrophoretically onto the nitrocellulose membrane for 60 minutes at 20 V and 350 mA.

Upon transfer, the membrane was washed twice with Tris-buffered saline (TBS; 20mM

Tris-HCL, 137mM NaCL, pH 7.4) for 5 minutes each time and then the membrane was blocked overnight at 4ºC in Tris-buffered saline with Tween (TBST; 20mM Tris-HCL,

137mM NaCL, 0.1% Tween-20, pH 7.4) with 5% skim milk. To detect bovine SFK- the membrane was incubated for 1 hour with a specific primary diluted 1:100 in

TBST, washed 3 times in TBST for 10 minutes each, incubated for 1 hour at 37ºC with appropriate HRP-labeled secondary antibody diluted 1:1000 in TBST, and finally 34 processed as described below. The nitrocellulose membrane was washed twice with

TBST, 5 minutes each time. For protein detection one part Opti-4CN diluent concentrate was mixed with nine parts ddH2O (BioRad Opti-4CN Substrate Kit). A 0.2 ml volume of

Opti-4CN substrate was added to 10 mls of diluent, mixed thoroughly, and poured onto the membrane. The membrane was incubated with gentle agitation in the substrate for 30 minutes and washed in ddH2O for 15 minutes.

Results

SFKs in Bovine Oocytes and Spermatozoa

Western blotting determined that SRC, BLK, YES, and FYN are present in both

IVM matured bovine oocytes and acrosome-reacted bovine spermatozoa. LYN and HCK are present in acrosome-reacted bovine spermatozoa only. Neither LCK, or FGR were detectable in either bovine gamete (Figure 2-1). These findings were confirmed by 2-4 replications and through a secondary antibody control experiment, where no primary antibody was used (data not shown).

Impacts of Injectants on Intracellular Calcium Transients

2+ IP3 control groups were processed as follows; Ca i was measured for 30 minutes,

2+ any oocytes that exhibited Ca i were removed. Remaining oocytes were microinjected

2+ with 0.0025 µM IP3, and Ca i transients were immediately measured for 30 minutes.

2+ The IP3 control validated that the signal transduction pathway leading to Ca i was functional and in operation. It further confirmed that inhibitors were functioning upstream of IP3 involvement. IVF control groups were removed from the incubator and 35 placed on the microscope stage while treatment groups were injected to mimic conditions. No sperm negative control groups were placed in fertilization medium with no sperm. Microinjection of SRC Peptide non-phosphorylated (SRCPnp), followed by

2+ IVF, had no impact (P > 0.05) on Ca i at any treatment concentration when compared to

IP3 and IVF positive controls.

Microinjection of PP2, a known inhibitor of Src, Lck, Fyn, and FAK resulted in a

2+ significant (P < 0.05) reduction in Ca i at all treatment concentrations relative to positive controls. There was no difference (P > 0.05) between the 3 µM PP2 treatment and the no sperm control (Figure 2-2 and Table 2-2). Microinjection of the phosphorylated version

2+ of SRC Peptide (SRCPP) prior to IVF had a dose-dependent impact on Ca i. The concentrations of 2 µM and 0.02µM SRCPP showed no difference (P > 0.05) from the no sperm control (Figure 2-2 and Table 2-2). Microinjection of Src Substrate II non-

2+ phosphorylated (SSIInp) resulted in a significant (P < 0.05) reduction in Ca i response at all concentrations as compared to positive controls. No difference (P > 0.05) was observed among any treatment concentration when compared to the negative control

(Figure 2-2 and Table 2-2). Microinjection of the phosphorylated version of SSII (SSIIP)

2+ also resulted in a significant (P < 0.05) reduction in Ca i response at all treatment concentrations. The concentrations of 2µM and 0.2µM SSIIP showed no difference from the no sperm control (Figure 2-2 and Table 2-2).

Impact of Injectants on Cleavage Rates

Using cleavage rate as a marker for activation provided helpful insight into the impacts of injectants. Oocytes within treatment groups were injected with various 36 compounds followed by IVF for 6 hrs, and then cleavage rates were then assessed on day

2 of culture. The positive controls, IVF and sham, controls were not injected (IVF) or injected with vehicle medium (sham) followed by 6 hr fertilization and 48 hr culture prior to cleavage assessment.

Microinjection of PP2 had a significant (P < 0.05) decrease on cleavage.

Treatment 3 µM PP2 showed no significant difference (P > 0.05) from the negative no sperm controls (Figure 3 and Table 2). Microinjecting SRCnPp prior to IVF showed no decrease in cleavage rates compared to positive controls. Microinjecting the phosphorylated version, SRCPP, showed a significant (P < 0.05) decrease in cleavage rate with the 0.02µM and 2µM SRCPP groups (Figure 2-3 and Table 2-2).

Discussion

2+ A rise in Ca i is a hallmark of oocyte activation, although the specific pathway leading to oocyte activation differs between species. To better understand the signal transduction pathway for bovine oocyte activation and to examine the potential involvement of SFKs, the present study was carried out to: 1) identify the specific endogenous SFKs present within bovine oocytes; 2) identify the specific endogenous

SFKs found within bovine spermatozoa, 3) determine the impact of different SFK

2+ antagonists and substrates on Ca i following fertilization; and 4) determine the impact of

SFK antagonists and substrates on cleavage 48 hours post fertilization. 37

Endogenous SFKs in Bovine Oocyte and Spermatozoa

A previous study reported that SRC is present in pre-acrosome reacted bovine spermatozoa and plays a role in the acrosome reaction [104]. It was unknown if other

SFKs are present in bovine spermatozoa, and if they were involved in oocyte activation.

SFK’s are associated with the cytosolic side of plasma membranes through its myristoylated N-terminus [105]. In this study, capacitated and acrosome reacted spermatozoa were lysed for Western blot analysis of SRKs. This preparation of spermatozoa reflects the physiological state of a sperm as it contacts the vitelline membrane. Because these spermatozoa were acrosome-reacted, any SFKs associated with the outer acrosome membrane were eliminated. Bovine brain extracts were used in conjunction with positive controls to confirm that all the SFK antibodies cross-reacted with bovine proteins.

The major SFKs in both bovine oocytes and spermatozoa were identified. BLK,

YES, SRC, and FYN were all present within both bovine oocytes and spermatozoa, as shown in Figure 2-1 (A). BLK showed strong signals in all Western blot lanes and YES showed moderate signal strength across all samples. Both BLK and YES showed a size shift relative to the bovine brain extract lane. This size difference might be due to differential or other post-translational modifications. LYN and HCK were observed in bovine spermatozoa, but absent in bovine oocytes (Figure 2-1, B) but at low signal strength. No SFK’s were detected in bovine oocytes alone. 38

2+ The SFK Inhibitor, PP2, Significantly Decreased Ca i and Cleavage Rates

PP2, a known inhibitor of multiple SFKs, was used to evaluate potential roles of

2+ for SFKs in oocyte activation. Microinjecting 3µM PP2 abolished Ca i transients and cleavage. This indicates that one or more SFKs are involved in bovine oocyte activation.

It is interesting to note that PP2 inhibits the activation of FAK, which is known to associate with integrin receptor molecules. Integrin-mediated cell adhesion typically induces FAK to become highly phosphorylated and transduce a signal based on its ability to interact with a number of signaling effectors from the SFK [106]. Integrins have been detected in bovine oocytes [107] and have been shown to play a role in bovine oocyte activation [98, 108].

SRC is Involved in Bovine Oocyte Activation

We selected specific peptides that target SRC protein and used these to evaluate

2+ SRC’s involvement in Ca i and subsequent cleavage following IVF. SRCPnp is a thirteen amino acid peptide that mimics the non-phosphorylated C-terminal phosphoregulatory region of SRC. Microinjection of this peptide had no effect on SRC protein’s ability to

2+ induce Ca i and cleavage. However, a phosphorylated from of this same peptide

(SRCPP) that is phosphorylated within the regulatory region at Tyr527, blocked signal

2+ transduction. Experiments evaluating Ca i indicate that SRCPP has a major impact in blocking signal transduction leading to bovine oocyte activation. The SRCPP 2µM treatment blocked calcium-signaling resulting in only 8/56 (14.3%)d oocytes exhibiting a

2+ Ca I. There was no difference between this treatment and the no sperm control 3/25

(12.0%)d. The percent cleavage observed in the SRCPnp treatments were consistent with 39

2+ the Ca i results, which indicated no difference between IVF and sham injection controls.

The percent cleavage for SRCPP treated oocytes indicated that 31/72 (43.1%)b cleaved at

0.02µM and 40/118 (33.9%)b at 2µM levels. Both treatments were different from controls

884/1200 (73.7%)a IVF, 372/492 (75.6%)a sham injection, and 90/639 (14.1%)c*,d no

2+ sperm controls. It is unclear why a difference was observed between Ca i and cleavage

2+ data following the 0.02µM SRCPP injection. Ca i is measured after a 6 hour fertilization and cleavage rates are evaluated after a 6 hour fertilization and a 48 hour culture period.

2+ This slight increase observed in cleavage over Ca i response may result from the increased time required for assessing these two different activation parameters. The increased incubation time required to evaluate cleavage rate may provide a window for endogenous oocyte to remove the inhibitory phosphate from Tyr527, thus facilitating signal transduction and cleavage. There was no delay observed in activation

2+ when evaluating Ca i.

2+ Based upon the combination of Ca i responses and cleavage rate data using

SCRPnp and SRCPP, I hypothesize that as the inhibitory phosphate is removed from the endogenous SRC protein in the phosphoregulatory region at Tyr527, the SH2 domain becomes unbound from the C-terminal phosphoregulatory region, making it available for the microinjected SRCPP to bind to the SH2 domain, and thus inhibit downstream signal transduction. The data indicates that SRC protein is involved in the signal transduction

2+ events leading to Ca i and subsequent cleavage in bovine oocytes. 40

IVF Activates SRC Protein and Injection of SRC Substrate Sequesters the Signal

As SRC is activated, it induces activation of downstream Src Substrate II (SSII) via phosphorylation. Microinjection of Src Substrate II non-phosphorylated (SSIInp) had

2+ a significant impact on Ca i response at all treatment concentrations, as none were statistically different from the no sperm control. Cleavage rates also decreased at treatments 0.2µM, 31/90 (34.4)c and 2µM, 29/87 (33.3)c , indicating that SSIInp was able to sequester any signal produced by upstream SRC activation.

Micro-injecting SSIIP, a phosphorylated form of the SSII peptide, had significant

2+ effects on Ca i responses at all concentrations. Oocytes injected at concentrations 0.2µM and 2µM SSIIP behaved indistinguishably from the no sperm control. SSIIP induced a concentration-dependent decrease in cleavage rates. Microinjection of SSIIP at 2µM provided the greatest decrease in activation, as assessed by cleavage rate. Based upon the

2+ combined results of SSIInp and SSIIP Ca i responses and cleavage data, we conclude that endogenous bovine SRC protein is activated following IVF and injected SSIInp and

SSIIP was available to sequester the signal transduction leading to oocyte activation. The data supports the involvement of SRC protein in the signal transduction pathway leading to bovine oocyte activation. Taken together, all of this data support the conclusion that the

SRC protein is involved in bovine oocyte activation.

SFKs play a role in oocyte activation in sea urchin, ascidian, starfish, Xenopus, and zebrafish [28-32, 39]. In the murine model, SFKs have been reported as unnecessary

2+ for Ca i at oocyte activation in mice [57, 58]. These findings have caused many researchers to conclude SFKs are therefore not necessary for fertilization of other 41 mammals. Generalizing from one mammalian model without conclusive evidence from other models can be misleading. For example, a comparative evaluation of the success of

ISCI support the idea that pathways leading to oocyte activation may differ among species. ISCI alone is sufficient to properly activate hamster [60], rabbit [62], human [83,

87], and murine oocytes [88], but bovine oocytes require an additional activation step

[89]. In this report we provide evidence that, unlike the mouse, SRC plays a vital role in bovine oocyte activation. These findings reinforce the conclusion that not all mammalian species are similar in their biochemical mechanisms of fertilization and urge caution against generalizing from research on fertilization in any single model system.

References

1. Xu Z, Kopf GS, Schultz RM. Involvement of inositol 1,4,5-trisphosphate-

mediated Ca2+ release in early and late events of mouse egg activation.

Development 1994; 120:1851-1859.

2. Sun Q-Y. Cellular and molecular mechanisms leading to cortical reaction and

polyspermy block in mammalian eggs. Microscopy Research and Tech 2003;

61:342-348.

3. Whitaker M. Calcium at Fertilization and in Early Development. Physiological

Review 2006; 86.

4. Tae JC, Kim EY, Jeon K, Lee KS, Lee CH, Kim YO, Park SP, Kim N-H. A MAPK

pathway is involved in the control of cortical granule reaction and mitosis during

bovine fertilization. Molec reprod and dev 2008; 75:1300-1306. 42

5. Swann K, McCulloh DH, McDougall A, Chambers EL, Whitaker M. Sperm-

induced currents at fertilization in sea urchin eggs injected with EGTA and

neomycin. Dev Biol 1992; 151:552-563.

6. McAvey BA, Wortzman GB, Williams CJ, Evans JP. Involvement of calcium

signaling and the actin cytoskeleton in the membrane block to polyspermy in

mouse eggs. Biol Reprod 2002; 67:1342-1352.

7. Ducibella T. The cortical reaction and development of activation competence in

mammalian oocytes. Hum Reprod Update 1996; 2:29-42.

8. Lorca T, Cruzalegui FH, Fesquet D, Cavadore JC, Mery J, Means A, Doree M.

Calmodulin-dependent protein kinase II mediates inactivation of MPF and CSF

upon fertilization of Xenopus eggs. Nature 1993; 366:270-273.

9. Whitaker M, Swann K. Lighting the fuse at fertilization. Development 1993;

117:1-12.

10. Kuretake S, Kimura Y, Hoshi K, Yanagimachi R. Fertilization and development of

mouse oocytes injected with isolated sperm heads. Biol Reprod 1996; 55:789-795.

11. Sato K, Tokmakov AA, Iwasaki T, Fukami Y. Tyrosine kinase-dependent

activation of phospholipase Cgamma is required for calcium transient in Xenopus

egg fertilization. Dev Biol 2000; 224:453-469.

12. Crossley I, Swann K, Chambers EL, Whitaker M. Activation of sea urchin eggs

by inositol phosphates is independent of external calcium. Biochem J 1988;

252:257-262. 43

13. Terasaki M, Jaffe LA. Organization of the sea urchin egg endoplasmic reticulum

and its reorganization at fertilization. J Cell Biol 1991; 114:929-940.

14. Kline D. Attributes and dynamics of the endoplasmic reticulum in mammalian

eggs. Curr Top Dev Biol 2000; 50:125-154.

15. Galione A, Lee HC, Busa WB. Ca(2+)-induced Ca2+ release in sea urchin egg

homogenates: modulation by cyclic ADP-ribose. Science 1991; 253:1143-1146.

16. Stricker SA. Comparative biology of calcium signaling during fertilization and

egg activation in animals. Dev Biol 1999; 211:157-176.

17. Marangos P, Carroll J. Fertilization and InsP3-induced Ca2+ release stimulate a

persistent increase in the rate of degradation of cyclin B1 specifically in mature

mouse oocytes. Dev Biol 2004; 272:26-38.

18. Rickords LF, White KL. Electroporation of inositol 1,4,5-triphosphate induces

repetitive calcium oscillations in murine oocytes. J Exp Zool 1993; 265:178-184.

19. Mitalipov SM, White KL, Farrar VR, Morrey J, Reed WA. Development of

Nuclear Transfer and Parthenogenetic Rabbit Embryos Activated with Inositol

1,4,5-Trisphosphate. Biol of Reprod 1999; 60:821-827.

20. Yue C, White KL, Reed WA, Bunch TD. The existence of inositol 1,4,5-

trisphosphate and ryanodine receptors in mature bovine oocytes. Development

1995; 121:2645-2654.

21. Fluck R, Abraham V, Miller A, Galione A. Microinjection of cyclic ADP-ribose

triggers a regenerative wave of Ca2+ release and exocytosis of cortical alveoli in

medaka eggs. 1999; 7:285-292. 44

22. Galione A, McDougall A, Busa WB, Willmott N, Gillot I, Whitaker M.

Redundant mechanisms of calcium-induced calcium release underlying calcium

waves during fertilization of sea urchin eggs. Science 1993; 261:348-352.

23. Miyazaki S, Shirakawa H, Nakada K, Honda Y. Essential role of the inositol

1,4,5-trisphosphate receptor/Ca2+ release channel in Ca2+ waves and Ca2+

oscillations at fertilization of mammalian eggs. Dev Biol 1993; 158:62-78.

24. Polzonetti V, Cardinali M, Mosconi G, Natalini P, Meiri I, Carnevali O. Cyclic

ADPR and calcium signaling in sea bream (Sparus aurata) egg fertilization. Mol

Reprod Dev 2002; 61:213-217.

25. Santella L, De Riso L, Gragnaniello G, Kyozuka K. Cortical granule translocation

during maturation of starfish oocytes requires cytoskeletal rearrangement

triggered by InsP3-mediated Ca2+ release. Exp Cell Res 1999; 248:567-574.

26. Yue C, White KL, Reed WA, King E. Localization and regulation of ryanodine

receptor in bovine oocytes. Biol Reprod 1998; 58:608-614.

27. Ayabe T, Kopf GS, Schultz RM. Regulation of mouse egg activation: presence of

ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose

on uninseminated and inseminated eggs. Development 1995; 121:2233-2244.

28. Jaffe LA, Giusti AF, Carroll DJ, Foltz KR. Ca2+ signalling during fertilization of

echinoderm eggs. Semin Cell Dev Biol 2001; 12:45-51.

29. Runft LL, Jaffe LA, Mehlmann LM. Egg activation at fertilization: where it all

begins. Dev Biol 2002; 245:237-254. 45

30. Giusti AF, Carroll DJ, Abassi YA, Foltz KR. Evidence that a starfish egg Src

family tyrosine kinase associates with PLC-gamma1 SH2 domains at fertilization.

Dev Biol 1999; 208:189-199.

31. Giusti AF, Carroll DJ, Abassi YA, Terasaki M, Foltz KR, Jaffe LA. Requirement

of a Src family kinase for initiating calcium release at fertilization in starfish eggs.

J Biol Chem 1999; 274:29318-29322.

32. Kinsey WH, Wu W, Macgregor E. Activation of Src-family PTK activity at

fertilization: role of the SH2 domain. Dev Biol 2003; 264:255-262.

33. Mizote A, Okamoto S, Iwao Y. Activation of Xenopus eggs by proteases: possible

involvement of a sperm protease in fertilization. Dev Biol 1999; 208:79-92.

34. Iwao Y, Fujimura T. Activation of Xenopus eggs by RGD-containing peptides

accompanied by intracellular Ca2+ release. Dev Biol 1996; 177:558-567.

35. Shilling FM, Kratzschmar J, Cai H, Weskamp G, Gayko U, Leibow J, Myles DG,

Nuccitelli R, Blobel CP. Identification of metalloprotease/disintegrins in Xenopus

laevis testis with a potential role in fertilization. Dev Biol 1997; 186:155-164.

36. Aarabi M, Qin Z, Xu W, Mewburn J, Oko R. Sperm-borne protein, PAWP,

initiates zygotic development in Xenopus laevis by eliciting intracellular calcium

release. Molec Reprod and Dev 2009; 77:249-256.

37. Snow P, Yim DL, Leibow JD, Saini S, Nuccitelli R. Fertilization stimulates an

increase in inositol trisphosphate and inositol lipid levels in Xenopus eggs. Dev

Biol 1996; 180:108-118. 46

38. Runft LL, Watras J, Jaffe LA. Calcium release at fertilization of Xenopus eggs

requires type I IP3 receptors, but not SH2 domain-mediated activation of PLCg or

Gq-mediated activation of PLCb. Dev Biol 1999; 214:399-411.

39. Sato K. Tyrosine Kinase-Dependent Activation of Phospholipase Cγ Is Required

for Calcium Transient in Xenopus Egg Fertilization. Dev Biol 2000; 224:453-469.

40. Tokmakov AA, Sato KI, Iwasaki T, Fukami Y. Src kinase induces calcium release

in Xenopus egg extracts via PLCgamma and IP3-dependent mechanism. Cell

Calcium 2002; 32:11-20.

41. Kaji K, Oda S, Shikano T, Ohnuki T, Uematsu Y, Sakagami J, Tada N, Miyazaki

S, Kudo A. The gamete fusion process is defective in eggs of Cd9-deficient mice.

Nat Genet 2000; 24:279-282.

42. Le Naour F, Rubinstein E, Jasmin C, Prenant M, Boucheix C. Severely reduced

female fertility in CD9-deficient mice. Science 2000; 287:319-321.

43. Miyado K, Yamada G, Yamada S, Hasuwa H, Nakamura Y, Ryu F, Suzuki K,

Kosai K, Inoue K, Ogura A, Okabe M, Mekada E. Requirement of CD9 on the

egg plasma membrane for fertilization. Science 2000; 287:321-324.

44. Inoue N, Ikawa M, Isotani A, Okabe M. The immunoglobulin superfamily protein

Izumo is required for sperm to fuse with eggs. Nature 2005; 434:234-238.

45. Boissonnas CC, Montjean D, Lesaffre C, Auer J, Vaiman D, Wolf J-P, Ziyyat A.

Role of sperm αvβ3 integrin in mouse fertilization. Dev Dynamics 2010;

239:773-783. 47

46. Evans JP, Schultz RM, Kopf GS. Mouse sperm-egg plasma membrane

interactions: analysis of roles of egg integrins and the mouse sperm homologue of

PH-30 (fertilin) beta. J Cell Sci 1995; 108 ( Pt 10):3267-3278.

47. Evans JP, Kopf GS, Schultz RM. Characterization of the binding of recombinant

mouse sperm fertilin beta subunit to mouse eggs: evidence for adhesive activity

via an egg beta1 integrin-mediated interaction. Dev Biol 1997; 187:79-93.

48. Vjugina U, Zhu X, Oh E, Bracero NJ, Evans JP. Reduction of mouse egg surface

integrin alpha 9 subunit (ITGA9) reduces the egg's ability to support sperm-egg

binding and fusion. Biol of Reprod 2009; 80:833-841.

49. Barraud-Lange V, Naud-Barriant N, Saffar L, Gattegno L, Ducot B, Drillet A-S,

Bomsel M, Wolf J-P, Ziyyat A. Alpha6beta1 integrin expressed by sperm is

determinant in mouse fertilization. BMC Dev Biol 2007; 7:102.

50. Tatone C, Carbone M. Possible involvement of integrin-mediated signalling in

oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate

protein kinase C and cortical granule exocytosis in mouse oocytes. Reprod Biol

and Endocrinol 2006; 4:48.

51. Chen H, Sampson NS. Mediation of sperm-egg fusion: evidence that mouse egg

alpha6beta1 integrin is the receptor for sperm fertilinbeta. Chem Biol 1999;

6:1-10.

52. Swann K, Larman MG, Saunders CM, Lai FA. The cytosolic sperm factor that

triggers Ca2+ oscillations and egg activation in mammals is a novel

phospholipase C: PLCzeta. Reproduction 2004; 127:431-439. 48

53. Kouchi Z. Recombinant phospholipase C has high Ca2+ sensitivity and induces

Ca2+ oscillations in mouse eggs. J of Biol Chem 2003; 279:10408-10412.

54. Kurokawa M. Mammalian Fertilization: From Sperm Factor to Phospholipase Cζ.

Biol of the Cell 2004; 96:37-45.

55. Fujimoto S, Yoshida N, Fukui T, Amanai M, Isobe T, Itagaki C, Izumi T, Perry

ACF. Mammalian phospholipase Cζ induces oocyte activation from the sperm

perinuclear matrix. Dev Biol 2004; 274:370-383.

56. Igarashi H, Knott J, Schultz R, Williams C. Alterations of PLCβ1 in mouse eggs

change calcium oscillatory behavior following fertilization. Dev Biol 2007;

312:321-330.

57. Kurokawa M. Evidence that activation of Src family kinase is not required for

fertilization-associated [Ca2+]i oscillations in mouse eggs. Reproduction 2004;

127:441-454.

58. Mehlmann LM. SH2 domain-mediated activation of an SRC family kinase is not

required to initiate Ca2+ release at fertilization in mouse eggs. Reproduction

2005; 129:557-564.

59. Lou J, McGinnis LK, Kinsey WH. Role of Fyn kinase in oocyte developmental

potential. Reprod, Fertility and Dev 2010; 22:966-976.

60. Uehara T, Yanagimachi R. Microsurgical injection of spermatozoa into hamster

eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol

Reprod 1976; 15:467-470. 49

61. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after

intracytoplasmic injection of single into an oocyte. Lancet 1992;

340:17-18.

62. Keefer CL. Fertilization by sperm injection in the rabbit. Gamete Res 1989;

22:59-69.

63. Gou KM, An XR, Tian JH, Chen YF. [Sheep transgenic embryos produced by

intracytoplasmic sperm injection]. Shi Yan Sheng Wu Xue Bao 2002; 35:103-108.

64. Gomez MC, Catt JW, Evans G, Maxwell WM. Cleavage, development and

competence of sheep embryos fertilized by intracytoplasmic sperm injection and

in vitro fertilization. Theriogenology 1998; 49:1143-1154.

65. Gomez MC, Pope CE, Harris R, Davis A, Mikota S, Dresser BL. Births of kittens

produced by intracytoplasmic sperm injection of domestic cat oocytes matured in

vitro. Reprod Fertil Dev 2000; 12:423-433.

66. Pope CE, Johnson CA, McRae MA, Keller GL, Dresser BL. Development of

embryos produced by intracytoplasmic sperm injection of cat oocytes. Anim

Reprod Sci 1998; 53:221-236.

67. Lyu QF, Deng L, Xue SG, Cao SF, Liu XY, Jin W, Wu LQ, Kuang YP. New

technique for mouse oocyte injection via a modified holding pipette. Reprod

Biomed Online 2010.

68. Stein P, Schultz RM. ICSI in the mouse. Methods Enzymol 2010; 476:251-262. 50

69. Keskintepe L, Morton PC, Smith SE, Tucker MJ, Simplicio AA, Brackett BG.

Caprine formation following intracytoplasmic sperm injection and

defined culture. Zygote 1997; 5:261-265.

70. Wang B, Baldassarre H, Pierson J, Cote F, Rao KM, Karatzas CN. The in vitro

and in vivo development of goat embryos produced by intracytoplasmic sperm

injection using tail-cut spermatozoa. Zygote 2003; 11:219-227.

71. Jimenez-Macedo AR, Izquierdo D, Anguita B, Paramio MT. Comparison between

intracytoplasmic sperm injection and in vitro employing oocytes

derived from prepubertal goats. Theriogenology 2005; 64:1249-1262.

72. Jimenez-Macedo AR, Anguita B, Izquierdo D, Mogas T, Paramio MT. Embryo

development of prepubertal goat oocytes fertilised by intracytoplasmic sperm

injection (ICSI) according to oocyte diameter. Theriogenology 2006;

66:1065-1072.

73. Dell'Aquila ME, Cho YS, Minoia P, Traina V, Fusco S, Lacalandra GM, Maritato

F. Intracytoplasmic sperm injection (ICSI) versus conventional IVF on abattoir-

derived and in vitro-matured equine oocytes. Theriogenology 1997;

47:1139-1156.

74. Li X, Morris LH, Allen WR. Influence of co-culture during maturation on the

developmental potential of equine oocytes fertilized by intracytoplasmic sperm

injection (ICSI). Reproduction 2001; 121:925-932. 51

75. Choi YH, Love CC, Varner DD, Hinrichs K. Equine blastocyst development after

intracytoplasmic injection of sperm subjected to two freeze-thaw cycles.

Theriogenology 2006; 65:808-819.

76. Cochran R, Meintjes M, Reggio B, Hylan D, Carter J, Pinto C, Paccamonti D,

Godke RA. Live foals produced from sperm-injected oocytes derived from

pregnant mares. J of Equine Vet Sci 1998; 18:736-740.

77. Lai L, Sun Q, Wu G, Murphy CN, Kuhholzer B, Park KW, Bonk AJ, Day BN,

Prather RS. Development of porcine embryos and offspring after intracytoplasmic

sperm injection with liposome transfected or non-transfected sperm into in vitro

matured oocytes. Zygote 2001; 9:339-346.

78. Nakai M, Kashiwazaski N, Takizawa A, Hayashi Y, Nakatsukasa E, Fuchimoto D,

Noguchi J, Kaneko H, Shino M, Kikuchi K. Viable piglets generated from porcine

oocytes matured in vitro and fertilized by intracytoplasmic sperm head injection.

Biol Reprod 2003; 68:1003-1008.

79. Yong HY, Hao Y, Lai L, Li R, Murphy CN, Rieke A, Wax D, Samuel M, Prather

RS. Production of a transgenic piglet by a sperm injection technique in which no

chemical or physical treatments were used for oocytes or sperm. Mol Reprod Dev

2006; 73:595-599.

80. Galli C, Vassiliev I, Lagutina I, Galli A, Lazzari G. Bovine embryo development

following ICSI: effect of activation, sperm capacitation and pre-treatment with

dithiothreitol. Theriogenology 2003; 60:1467-1480. 52

81. Fujinami N, Hosoi Y, Kato H, Matsumoto K, Saeki K, Iritani A. Activation with

ethanol improves embryo development of ICSI-derived oocytes by regulation of

kinetics of MPF activity. J Reprod Dev 2004; 50:171-178.

82. Suttner R, Zakhartchenko V, Stojkovic P, Mtiller S, Alberio R, Medjugorac I,

Brem G, Wolf E, Stojkovic M. Intracytoplasmic sperm injection in bovine: effects

of oocyte activation, sperm pretreatment and injection technique. Theriogenology

2000; 54.

83. Tesarik J, Sousa M, Testart J. Human oocyte activation after intracytoplasmic

sperm injection. Hum Reprod 1994; 9:511-518.

84. Shang XM, Li ZP. [The establishment of in vitro fertilization-embryo transfer

(IVF-ET) and the development of its derivative techniques]. Zhonghua Yi Shi Za

Zhi 2009; 39:93-99.

85. Kashir J, Heindryckx B, Jones C, De Sutter P, Parrington J, Coward K. Oocyte

activation, phospholipase C zeta and human infertility. Hum Reprod Update 2010.

86. Simerly CR, Castro CA, Jacoby E, Grund K, Turpin J, McFarland D, Champagne

J, Jimenez JB, Jr., Frost P, Bauer C, Hewitson L, Schatten G. Assisted

reproductive technologies (ART) with baboons generate live offspring: a

nonhuman primate model for ART and reproductive sciences. Reprod Sci 2010;

17:917-930.

87. Catt JW, Rhodes SL. Comparative intracytoplasmic sperm injection (ICSI) in

human and domestic species. Reprod Fertil Dev 1995; 7:161-166; discussion 167. 53

88. Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol

Reprod 1995; 52:709-720.

89. Garcia-Rosello E, Garcia-Mengual E, Coy P, Alfonso J, Silvestre MA.

Intracytoplasmic sperm injection in livestock species: an update. Reprod Domest

Anim 2009; 44:143-151.

90. Williams CJ. Signalling mechanisms of mammalian oocyte activation. Hum

Reprod Update 2002; 8:313-321.

91. Bevacqua RJ, Pereyra-Bonnet F, Fernandez-Martin R, Salamone DF. High rates of

bovine blastocyst development after ICSI-mediated gene transfer assisted by

chemical activation. Theriogenology 2010; 74:922-931.

92. Abdalla H, Shimoda M, Hirabayashi M, Hochi S. A combined treatment of

ionomycin with ethanol improves blastocyst development of bovine oocytes

harvested from stored ovaries and microinjected with spermatozoa.

Theriogenology 2009; 72:453-460.

93. Galli C, Lazzari G. The manipulation of gametes and embryos in farm animals.

Reproduction in domestic animals = Zuchthygiene 2008; 43 Suppl 2:1-7.

94. Goto K, Kinoshita A, Takuma Y, Ogawa K. Fertilisation of bovine oocytes by the

injection of immobilised, killed spermatozoa. The Veterinary record 1990;

127:517-520.

95. Rho GJ, Wu B, Kawarsky S, Leibo SP, Betteridge KJ. Activation regimens to

prepare bovine oocytes for intracytoplasmic sperm injection. Molec Reprod and

Dev 1998; 50:485-492. 54

96. Chung JT, Keefer CL, Downey BR. Activation of bovine oocytes following

intracytoplasmic sperm injection (ICSI). Theriogenology 2000; 53:1273-1284.

97. Malcuit C, Maserati M, Takahashi Y, Page R, Fissore RA. Intracytoplasmic sperm

injection in the bovine induces abnormal [Ca2+]i responses and oocyte activation.

Reprod Fertil Dev 2006; 18:39-51.

98. Campbell KD, Reed WA, White KL. Ability of integrins to mediate fertilization,

intracellular calcium release, and parthenogenetic development in bovine oocytes.

Biol Reprod 2000; 62:1702-1709.

99. Yue C, White KL, Reed WA, Bunch TD. The existence of inositol 1,4,5-

trisphosphate and ryanodine receptors in mature bovine oocytes. Development

1995; 121:2645-2654.

100. Goto K, Iwai N, Takuma Y, Nakanishi Y. Co-culture of in vitro fertilized bovine

embryos with different cell monolayers. J of Animal Sci 1992; 70:1449-1453.

101. Rosenkrans CFJ, First NL. Effect of free amino acids and vitamins on cleavage

and developmental rate of bovine zygotes in vitro. J of Animal Sci 1994;

72:434-437.

102. Parrish JJ, Susko-Parrish JL, Handrow RR, Ax RL, First NL. Effect of sulfated

glycoconjugates on capacitation and the acrosome reaction of bovine and hamster

spermatozoa. Gamete Res 1989; 24:403-413.

103. Sun QY, Fan HY. Activity of MAPK/p90rsk during fertilization in mice, rats, and

pigs. Methods Mol Biol 2004; 253:293-304. 55

104. Etkovitz N, Tirosh Y, Chazan R, Jaldety Y, Daniel L, Rubinstein S, Breitbart H.

Bovine sperm acrosome reaction induced by -coupled receptor agonists

is mediated by receptor transactivation. Dev Biol 2009;

334:447-457.

105. Sato K, Aoto M, Mori K, Akasofu S, Tokamakov AA, Sahara S, Fukami Y.

Purification and Characterization of a Src-related p57 Protein-tyrosine Kinase

from Xenopus Oocytes. J of Biol Chem 1996; 271:13250-13257.

106. Chong Y, Ia K, Mulhern T, Cheng H. Endogenous and synthetic inhibitors of the

Src-family protein tyrosine kinases. Biochimica et Biophysica Acta (BBA) -

Proteins & Proteomics 2005; 1754:210-220.

107. Pate BJ, White KL, Chen D, Aston KI, Sessions BR, Bunch TD, Weimer BC. A

novel approach to identify bovine sperm membrane proteins that interact with

receptors on the vitelline membrane of bovine oocytes. Molec Reprod and Dev

2008; 75:641-649.

108. Sessions BR, Aston KI, Davis AP, Pate BJ, White KL. Effects of amino acid

substitutions in and around the arginine-glycine-aspartic acid (RGD) sequence on

fertilization and parthenogenetic development in mature bovine oocytes. Molec

Reprod and Dev 2006; 73:651-657. 56

Table 2-1. Primary antibodies used for western blotting. Amino acid numbers from Santa Cruz antibodies are not available, except for LYN (H-6): sc.-7274.

SFK Antibody (source) Epitope SRC mAB327 (EMD OP07) SH3 domain BLK K-23: sc-329 (Santa Cruz) N-terminus YES 3: sc-14 (Santa Cruz) N-terminus FGR C-1: sc-17 (Santa Cruz) C-terminus FYN FYN3: sc-16 (Santa Cruz) N-terminus HCK M-28: sc-1428 (Santa Cruz) N-terminus LCK 2102: sc-13 (Santa Cruz) C-terminus LYN H-6: sc-7274 (Santa Cruz) Amino acids 40-70, near N-terminus 57

Table 2-2. Effect of peptides, substrates, and inhibitors on oocyte activation as 2+ a-d determined Ca i. Unlike superscripts are significantly different (P<0.05). Comparisons are made within treatments and against controls.

Number of oocytes showing Treatment Number of matured oocytes 2+ Ca i Inomyocin 31 31 (100)a IP3 46 38 (82.6)a,b IVF Control 346 241 (69.7)b No Sperm 25 3 (12.0)d SrcPnp 0.02 µM 23 15 (65.2)b SrcPnp 0.2 µM 26 17 (65.4)b SrcPnp 2 µM 31 22 (71.0)b SrcPP 0.002 µM 25 13 (52.0)b,c SrcPP 0.02 µM 27 9 (33.3)c,d SrcPP 2 µM 56 8 (14.3)d PP2 0.004 µM 47 24 (51.1)c PP2 0.25 µM 30 11 (36.7)c PP2 3 µM 51 11 (21.6)c,d SSIInp 0.02 µM 30 10 (33.3)c,d SSIInp 0.2 µM 40 12 (30.0)c,d SSIInp 2 µM 63 18 (28.6)c,d SSIIP 0.02 µM 28 14 (50.0)c SSIIP 0.2 µM 25 9 (36.0)c,d SSIIP 2 µM 43 8 (18.6)d 58

Table 2-3. Effect of peptides, substrates, and inhibitors on development as determined by cleavage after IVF. a-d Unlike superscripts are significantly different (P < 0.05). Comparisons are made within treatments and against controls. c* makes comparison between these groups only.

Treatment Number of matured oocytes Number of oocytes cleaved IVF Control 1200 884 (73.7)a Sham Injection 492 372 (75.6)a No Sperm 639 90 (14.1)c*,d SrcPnp 0.02 µM 23 15 (65.2)a SrcPnp 0.2 µM 26 17 (65.4)a SrcPnp 2 µM 31 22 (71.0)a SrcPP 0.002 µM 47 35 (74.5)a SrcPP 0.02 µM 72 31 (43.1)b SrcPP 2 µM 118 40 (33.9)b PP2 0.004 µM 89 37 (41.6)b PP2 0.25 µM 185 61 (33.0)b PP2 3 µM 190 37 (19.5)c* SSIInp 0.02 µM 122 61 (50.0)b SSIInp 0.2 µM 90 31 (34.4)c SSIInp 2 µM 87 29 (33.3)c SSIIP 0.02 µM 172 122 (70.9)a SSIIP 0.2 µM 200 115 (57.5)b SSIIP 2 µM 285 96 (33.7)c 59

Figure 2-1. Western blots of SFK proteins in bovine gametes and bovine negative and positive control cell lysates. The bovine control was bovine brain lysate and the postive controls were Jurkat, RAW 364.7, Namalwa, 293T, or BJAB cells. (A) SFKs detected in both bovine oocytes and bovine spermatozoa. (B) SFKs detected in bovine spermatozoa only. (C) SFKs not detected in bovine oocytes or spermatozoa. 293Tµg) (5 ladder sperm brain (A) egg Jurkatµg) (5 Jurkatµg) (5 Namalwaµg) (5 ladder sperm brain ladder sperm brain ladder egg sperm brain egg egg

100 - 70 -

55

35

BL YES SRC FY BJAB (5 µg) (5 BJAB RAWµg) (5 (B) ladder egg sperm brain ladder egg sperm brain

100 - 70 -

55

35

LY HC Jurkatµg) (5 RAWµg) (5 (C) ladder egg sperm brain ladder egg sperm brain

100 - 70 -

55

35

LC FG 60

% Calcium Transients a 100.0

a,b

80.0 b b b b

60.0 b,c c c

c c,d 40.0 c,d c,d c,d c,d

c,d d 20.0 d d

0

IP3 IVF 2µM 2µM 3µM 2µM 2µM 0.2µM 0.2µM 0.25µM 0.02µM 0.2µM 0.02µM 0.02µM 0.02µM NoSperm 0.002µM 0.004µM Ionomyocin

Controls SRCPnp SRCPP PP2 SSIInp SSIIP 61

Cleavage % 80.0 a a a a a a a

64.0 b

b

48.0 b b

c c b b c 32.0

c* c*, d 16.0

0

IVF 3µM Sham 2µM 2µM 2µM 2µM 0.02µ 0.2µM 0.25µ 0.2µM 0.2µM 0.002µ 0.02µM 0.004µ 0.02µM 0.02µM No Sperm Controls SRCPnp SRCPP PP2 SSIInp SSIIP 62

CHAPTER 3

INVOLVEMENT OF PHOSPHOLIPASE C ISOTYPES IN THE ACTIVATION

OF BOVINE OOCYTES

Abstract

At fertilization, a sperm cell induces the oocyte to leave its state of metabolic arrest and resume metabolism in the process of activation. A defining feature of

2+ activation is the release of intercellular calcium (Ca i) from the endoplasmic reticulum

2+ (ER) that results in repetitive Ca i transients. There is little known about the mediators of bovine oocyte activation, while sea urchin, zebrafish, ascidian, starfish, Xenopus, and murine models are better defined. It is well accepted that the activation of Phospholipase

C (PLC) results in the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2), producing membrane-bound diacylglycerol (DAG) and cytosolic soluble 1,4,5-inositol

2+ trisphosphate (IP3). The binding of IP3 to its ER receptor leads to the release of Ca i within the oocyte. In this study we investigated the six PLC isotypes and their potential

2+ role in bovine oocyte activation. To determine the role of PLC isotypes in Ca i release and cleavage, PLC antibodies were microinjected into mature bovine oocytes followed by in vitro fertilization (IVF). Western blotting was used to confirm the presence of specific

PLC proteins within bovine oocytes and spermatozoa. The inhibition of several PLC

2+ isotypes was shown to significantly decrease Ca i and cleavage rates. These data identify which PLC isotypes and isozymes are involved in bovine oocyte activation and further confirm that the detailed pathway leading to oocyte activation differs among animal models. 63

Introduction

Bovine oocytes are ovulated immature at metaphase II (MII) and must undergo activation to exit this state of metabolic arrest. The fertilizing sperm initiates activation, which is critical for embryonic development. Oocyte activation includes cortical granule

2+ exocytosis, repetitive intercellular calcium (Ca i) transients, pronuclear formation, second polar body extrusion, DNA synthesis, resumption and completion of meiosis II,

2+ and embryonic development [1, 2]. Ca i plays an important role in cortical granule

2+ exocytosis, which provides a block to polyspermy [3-5]. The role that Ca i transients play in activation is not fully understood, but is believed to act as a second messenger that initiates down-regulation of maturation promoting factor (MPF) activity [6]. A decrease in MPF induces the oocyte to re-enter the cell cycle and exit MII arrest.

2+ The signal transduction pathway leading to Ca i has been explained by two competing hypotheses [7]. The receptor mediated hypothesis proposes a sperm binds to a receptor on the vitelline membrane of the oocyte. There are three likely receptor candidates, G-protein coupled receptors, integrins, and/or . These receptors are known to activate Phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2), producing membrane-bound diacylglycerol

(DAG) and cytosolic soluble 1,4,5-inositol trisphosphate (IP3). The binding of IP3 to its

2+ endoplasmic reticulum IP3 receptor (IP3R) leads to the release of Ca i within the cytosol of the oocyte [8, 9].

The sperm-borne oocyte-activating factor (SOAF) [10] proposes that when spermatozoa fuse with the oocyte vitelline membrane, SOAF is deposited into the oocyte, 64

2+ which then interacts with cytosolic proteins to produce downstream Ca i through the

2+ pathway described above. It is well accepted that Ca i stores come from the ER [11-18].

2+ Ryanodine receptors (RyR) play a parallel role with IP3R in Ca i and calcium-induced calcium release (CICR) in many fish and echinoderm oocytes. RyR have also been shown to operate in CICR in bovine [19, 20] and within murine oocytes [21]. Whether through

2+ IP3R alone or IP3/RyR combined pathways, Ca i is a hallmark of oocyte activation for all animal models studied to date.

Oocyte activation pathways in models such as sea urchin, zebrafish, ascidian, starfish, Xenopus, and murine models have been extensively studied, but little information is available for the bovine model. Similar activation pathways are found for sea urchin, ascidian, and starfish activation. This involves the activation of Src Family

Kinase (SFK), which then activates PLCγ. Enzymatically active PLCγ can then cleave

2+ PIP2 to produce IP3, which leads to Ca i release from the ER [2, 8, 22, 23]. The activation pathway in zebrafish is more clearly defined as the specific SFK Fyn and/or

Src are activated prior to PLCγ activation [24]. Xenopus oocyte activation is facilitated through multiple pathways. It has been shown that contact with cathepsin B [25], peptides containing the integrin-binding RGD sequence [26], or a disintergrin region of testes derived cDNA that codes for a metalloprotease/disintegrin termed xMDC16 [27] can all activate Xenopus oocytes. Further, the Xenopus spermatozoa post-acrosomal

2+ sheath WW domain binding protein produces Ca i [28]. After fertilization there is an

2+ increase in IP3 concentration that is essential for Ca i release in Xenopus [29, 30].

Conflicting results indicate that neither PLCγ1 nor PLCβ are necessary for IP3 65

2+ production and the resulting Ca i transients [30], while other studies support that PLCγ is required [11] and is made active by SFK [31].

Considerable data supports the idea that in murine, integrins mediate sperm- oocyte binding [32-40] followed by oocyte activation by SOAF PLCζ isotype [41-44].

Endogenous oocyte PLCβ1 has been proposed to function in synchrony with PLCζ to

2+ produce Ca i transients and oocyte activation via IP3R [45].

Additional insight into the signal transduction pathway can be provided by intracytoplasmic sperm injection (ICSI), which involves the direct injection of spermatozoa into the cytoplasm of a MII oocyte. This procedure was first demonstrated in hamsters [46] and is routinely used in human reproductive medicine, with the first cases dating back to 1992 [47]. ICSI has been performed in multiple species, hamsters

[46], rabbits [48], cats [49, 50], mice [51, 52], goats [53-55], pigs [56-58], baboons [59], horses [60-62], cattle [63-65], and humans [66, 67] with varying degrees of success.

Just as in somatic cell nuclear transfer, ICSI must often be accompanied by an activation step, typically using a chemical activator such as ethanol, cycloheximide, 6- dimethylaminopurine, or ionomyocin. However, humans [66, 68], mice [69], hamsters

[46], and rabbits [48] do not require activation as ICSI alone induces oocyte activation.

For these species, oocyte activation is accomplished by the spermatozoa and its contents, possibly a SOAF, or mechanically by the ICSI procedure. The SOAF hypothesis is supported by the success of ICSI without the use of exogenous agents to induce activation in these species, since there is no need for spermatozoa-vitelline membrane binding and all membrane receptors are bypassed by injection. This implies that that 66 proper signal transduction can occur without receptor binding [70]. The SOAF model of oocyte activation is reasonable for species in which ICSI alone initiates oocyte activation.

This is not true for bovine, since ICSI alone does not induce proper oocyte activation and subsequent development without an additional exogenous activator to induce activation

[63, 71-77]. It appears that in cattle a receptor interaction between spermatozoa-vitelline membrane is required for oocyte activation. One report indicates that < 10% of bovine

2+ oocytes subjected to ICSI alone showed Ca i transients and that typical signal transduction pathways seen during natural fertilization were not replicated [77].

Previous work and data reported here indicate that bovine oocyte activation is different from other mammalian models and may more closely resemble pathways found in Xenopus and the zebrafish. To better understand the signal transduction pathway in bovine oocyte activation and the role of PLC isotypes this study was conducted to: 1) determine which PLC isotypes are present in bovine gametes, 2) determine the impact

2+ microinjecting antibodies against PLC isotypes have on Ca i transients following IVF, 3) determine the impact microinjecting antibodies against PLC isotypes have on cleavage rates 48 hr post IVF.

Materials and Methods

Reagents

Reagents were purchased from MP Biomedical (Costa Mesa, CA) unless otherwise specified. 67

Oocyte Collection and In Vitro Maturation

All procedures were performed according to published methods [20, 21, 78-80].

Briefly, bovine ovaries were collected from a local abattoir (JBS Swift & Company,

Hyrum, UT) and immediately processed. Oocytes were aspirated from follicles 3-8mm in size into 50-mL tubes using a 18-gauge needle and a vacuum pump. Oocytes were washed with PB1+ (Ca2+ and Mg2+ containing phosphate buffered saline, 0.32 mM sodium pyruvate, 5.55 mM glucose, 3 mg/mL BSA), transferred into 500 µL of maturation medium (MM), M199 containing 10% fetal bovine serum (FBS; Hyclone

Laboratories, Logan, UT), 0.5 µg/mL FSH (Sioux Biochemical, Sioux City, IA), 5 µg/ mL LH (Sioux Biochemical), 100 units/mL penicillin (Hyclone), and 100 µg/mL streptomycin (Hyclone) in 4-well culture dishes (Nunc, Milwaukee, WI) and cultured at

39ºC in a humidified atmosphere of 5% CO2 and air for 20-22 h.

Cumulus Cell Removal

Cumulus cells were removed from in vitro matured (IVM) oocytes by vortexing for 5 mins in PB1+ containing 10 mg/mL hyaluronidase. Oocytes were then washed through 4 drops of PB1+, randomly assigned to treatment groups and placed back in fresh

MM to recover prior to microinjection.

Microinjection

Microinjection needles were pulled using thin-bore borosilicate glass capillaries and a Narishige (East Meadow, NY) PB-7 pipette puller. Holding pipettes were crafted using a Narishige MF-9 micro forge. Micro-manipulation dishes were made with 100 µL 68

PB1- (Ca2+ and Mg2+ free) drops under mineral oil. IVM cumulus-free oocytes were microinjected with appropriate treatment using compressed N2 gas, a Narishige IM 300 pneumatic, and a Nikon Diaphot inverted microscope. The microinjection volume was determined by measuring a bubble formed under oil immersion by an ocular micrometer.

Each oocyte was microinjected with ~8 pL (1/100 of a typical oocyte volume) of appropriate treatment, resulting in a 100-fold dilution of any injected material. All data is represented at this 100-fold diluted working concentration. Groups of 20 oocytes were injected at a time to minimize time out of the incubator. After 10 oocytes, the microinjection volume was checked to ensure consistent injection volume. All sham controls were injected with ddH2O. All oocytes in IVF and no sperm control groups were removed from the incubator and placed in a manipulation dish for an equal amount of time as other treatment groups.

In Vitro Fertilization of Oocytes and Culture of Embryos

Between 21-24 h maturation, oocytes were fertilized according to our standard laboratory procedures. Frozen-thawed semen (Hoffman AI Breeders, Logan, UT) was separated on a 45% over 90% Percoll (Sigma, St. Louis, MO) at 400-x gravity for 30 min. The pellet was then washed with Sperm TL containing 6 mg/ml BSA and centrifuged at 65x gravity for 10 min. The final pellet was resuspended in Fertilization

Medium (FM) containing 1 µg/mL heparin and 4% (v/v) PHE (2mM penacillamine, 1 mM hypotaurine, 250 µM epinephrine). Oocytes were fertilized in 4-well culture dishes containing 500 µL of sperm/FM at 39ºC in a humidified atmosphere of 5% CO2 and air for 6 h at which time were removed and vortexed vigorously in PB1+ for 1 min to 69 remove bound spermatozoa cells. Oocytes were then cultured in 500 µL CR2 [80] at

39ºC in a humidified atmosphere of 5% CO2 and air. On day 2 of culture proper activation and development were assessed by identifying percent cleavage.

Visualization of Intracellular Calcium Transients

Oocytes assigned to calcium visualization were incubated in 2 µM Fura-2 AM ester (Molecular Probes, Eugene, OR) and 0.02% Pluronic F-127 (Molecular Probes,

Eugene, OR) in PB1- for 45 min in darkness at 39°C. Groups of 10 oocytes were then washed extensively in 9 drops of PB1- and placed in a 10 µL micro drop of sperm/FM under mineral oil and incubated at 39ºC in a humidified atmosphere of 5% CO2 and air for 6 h. The no sperm control group were placed in a 10 µL micro-drop containing FM

2+ only. Following fertilization, Ca i was measured for a first 30 min interval on an a Nikon

Diaphot inverted microscope, coupled with a Hamamatsu ORCA-ER digital camera and analyzed using Metafluor Imaging Series 7.6 software (Universal Imaging Corp,

Downingtown, PA). After visualization, oocytes were placed back in incubation

2+ conditions for 1.5 h, then Ca i visualization was repeated for an second 30 min interval.

2+ Ca i transients were determined by ratio changes in florescence intensity at 340 nm and

380 nm wavelengths. Oocytes in the ionomycin control treatment were only visualized

2+ during the first interval, at which time oocytes showing Ca i transients were removed

2+ from the microdrop and 1 µL of 1 mg/ml ionomycin was added and Ca i transients were then immediately measured for 30 min. Oocytes used in the IP3 treatment group were

2+ similarly evaluated for the first interval only. All oocytes exhibiting Ca i transients were 70

2+ removed. The remaining ooyctes were then microinjected with 0.0025 µM IP3 and Ca i transients were immediately measured for 30 min.

CICR, IP3R, and RyR Experiments Using PLC η2 and PLC δ4

2+ To better understand the role of PLC η2 and PLC δ4 in both Ca i transients and cleavage rates the following was conducted. Following IVM and cumulus cell removal, oocytes were allowed to recover for 1-2 h. At 21-22 h post maturation cumulus free oocytes were micro-injected with ~8pL of the given PLC isotype. For those randomly

2+ assigned to Ca i visualization they were incubated in 2 µM Fura-2 AM ester (Molecular

Probes, Eugene, OR) and 0.02% Pluronic F-127 (Molecular Probes, Eugene, OR) in PB1- for 45 min in darkness at 39°C. Groups of 5 oocytes were then washed extensively in 9 drops of PB1- and placed in a 500 µL of sperm/FM in a 4-well dish and incubated at

39ºC in a humidified atmosphere of 5% CO2 and air for 6 h. Oocytes were then removed from sperm/FM, vortexed for 1 min, washed through 3 drops of PB1- and placed in a 10

µL drop of PB1- under mineral oil. Oocytes were then micro-injected with either 200 µM

2+ procaine or 1 mg/ml heparin and immediately Ca i was visualized as described above.

For oocytes randomly assigned to cleavage groups they were micro-injected with the given PLC isotype 21-22 h post maturation, fertilized for 6 h, vortexed for 1 min, micro- injected with either 200 µM procaine or 1 mg/ml heparin, placed in 500 µL CR2 and incubated at 39ºC in a humidified atmosphere of 5% CO2 and air for 48 h prior to cleavage assessment. 71

Western Blot

Sperm and oocyte sample preparation

Sperm were capacitated with heparin and acrosome reacted with lysophosphatidylcholine according to published protocols [81]. Quickly, frozen-thawed semen (Hoffman AI Breeders, Logan, UT) was washed suspended in Sperm TL with 6 mg/m BSA and centrifuged at 400-x gravity for 10 min. The pellet was resuspended in

5ml Sperm TL and 1 µg/ml heparin and vortexed for 20 seconds. The suspension was divided into 500 µL volumes with 5 mg/ml Lysophosphatidly Choline, and incubated for

15 min at 39°C. The suspension was then centrifuged at 10,000 RPM for 10 min and the resulting pellet resuspended in 100 µL lysis buffer with protease inhibitor, vortexed and placed on a shaker at 4°C overnight. The aliquots were then centrifuged at 10,000 RPM for 10 min at 4°C, further aliquoted into 30 µL volumes and frozen at -80°C.

Cumulus cells were removed as previously described, then 50 bovine oocytes were washed through 4 drops of PBS and added to 5 µL double-strength sample buffer and frozen at -80ºC until use.

Bovine tissue sample preparation

To confirm that primary antibodies would cross-react with bovine, protein was extracted from bovine brain, lung, liver, lymph node, and testis using the same procedure.

Briefly, 1g of frozen bovine tissue was diced into small thin pieces using a razor blade and disrupted and homogenized in 5 ml 1X SDS buffer (1.25 M rTis HCl, 2% SDS, 10% glycerol, 0.001% Bromophenol Blue, 5% 2-mercaptoethanol) and protease inhibitor, then incubated on ice for 30 min. The mixture was then transferred to micro-centrifuge tubes 72 and centrifuged at 10,000g for 10 min at 4ºC. The supernatant was removed and placed in a clean centrifuge tube and again centrifuged at 10,000g for 10 min at 4ºC. Centrifugation was repeated until a clear lysate was obtained. Primary antibodies used are listed in Table

3-1.

Western blot analysis of bovine PLC’s was performed according to the procedures used by Sun and Fan [82]. Samples were boiled for 5 min, placed on ice for 5 min, and centrifuged at 10,000g for 1 min. Total proteins were separated by SDS-page with a 8% -

16% Pierce Precise Protein Gel for 45 min at 150 V and 120 mA at 4ºC for 45 min. After electrophoresis, the gel was washed with transfer buffer (39 mM glycine, 48 mM rTis,

0.037% SDS, and 20% methanol) for 15 min and then the total protein was transferred electrophoretically onto the nitrocellulose membrane for 60 min at 20 V and 350 mA.

Upon transfer, the membrane was washed twice with Tris-buffered saline (TBS; 20mM

Tris-HCL, 137mM NaCL, pH 7.4) for 5 min each time and then the membrane was blocked overnight at 4ºC in TBS with 5% skim milk. To detect bovine PLC’s- the membrane was incubated for 1 h with a specific primary antibody diluted 1:200 in Tris- buffered saline with Tween (TBST; 20mM Tris-HCL, 137mM NaCL, 0.1% Tween-20, pH 7.4) and 5% skim milk, washed 3 times in TBST for 10 min each, incubated for 1 h at

37ºC with appropriate HRP-labeled secondary antibody diluted between 1:1000 - 1:5000 in TBST with 5% skim milk. The nitrocellulose membrane was then washed twice with

TBST and once in TBS for 5 min. For protein detection, 1.1 ml Opti-4CN diluent concentrate was mixed with 11 ml ddH20 and 0.242 ml volume of Opti-4CN substrate

(BioRad Opti-4CN Substrate Kit), mixed thoroughly, and poured onto the membrane. 73

The membrane was incubated with gentle agitation in the substrate for 30 - 60 min and washed in ddH2O for 15 min.

Results

PLC Isotypes in Bovine Oocytes and Spermatozoa

Western blotting indicated that PLC δ1 and PLC δ3 are present in both bovine oocytes and acrosome-reacted bovine spermatozoa. PLC δ4, PLC γ2, and PLC η2 are present in bovine oocytes, but not detected in bovine spermatozoa. PLC ζ is present in bovine

2+ sperm but did not have any impact on Ca i or the cleavage rates (Figure 3-1). PLC β1,

PLC β2, PLC β3, PLC β4, PLC ε, and PLC γ1 were not detected in either bovine oocytes or bovine spermatozoa (data not shown).

Impact of PLC Isotypes on Intracellular Calcium Transients

Micro-injecting PLC β1, PLC β2, PLC β3, PLC β4, PLC ε, PLC η2, PLC δ4, and

2+ PLC γ1 had no effect on Ca i when compared to IVF and Sham controls (P>0.05). PLC

2+ δ3, PLC δ1, and PLC γ2 all had significant effects on Ca i when compared to IVF and

Sham injected controls (P>0.05) but was not different from the no sperm control group

2+ (P<0.05). Oocytes in treatments of PLC δ3, PLC δ1, and PLC γ2 that showed no Ca i were further treated with additional controls of Ionomycin and/or IP3 to confirm calcium release mechanisms were functional in these oocytes (Figure 3-2 and Table 3-2).

Impact of PLC Isotypes on Cleavage Rates

Micro-injecting PLC β1, PLC β2, PLC β3, PLC β4, PLC ε, and PLC γ1 had no effect on cleavage rates when compared to IVF and Sham controls (P>0.05). PLC δ3, 74

PLC δ1, PLC η2, PLC δ4, and PLC γ2 all had significant effects on cleavage rates when compared to IVF and Sham injected controls (P<0.05). PLC δ3 was not different from no sperm control (P<0.05) (Figure 3-3 and Table 3-3).

2+ PLC η2 and PLC δ4 had no effect on Ca i but did have a significant impact on cleavage rates. It was hypothesized that they were involved in CICR and RyR functionality. To test this, PLC η2 and PLC δ4 isotypes were investigated in conjunction with known IP3R and RyR antagonists in a set of experiments.

Impact of PLC η2 and PLC δ4 on CICR as Determined by Intracellular Calcium Transients

2+ Micro-injecting PLC η2 and PLC δ4 had no impact on Ca i following 6 hours of

IVF (P>0.05). Micro-injecting the know IP3R antagonist heparin (HP), 6 h post

2+ fertilization, had a significant impact on Ca i when compared to IVF and sham controls

2+ (P<0.05). Similarly the known RyR antagonist procaine (PC) greatly effected Ca i when compared to IVF and sham controls (P<0.05). The combinations of PC/PLC δ4, PC/PLC

2+ η2, HP/PLC η2, and HP/PLC δ4 significantly decreased Ca i from IVF and showed no significant difference from the no sperm control group (Figure 3-4 and Table 3-4).

Impact of PLC η2 and PLC δ4 on CICR as Determined by Cleavage Rates

All treatments were significantly different from IVF and Sham control groups.

Treatments IP3 + PLC η2/No Sperm, IP3 + PLC δ4/No Sperm, HP/PLC η2, and HP/PLC

δ4 showed no difference from the No Sperm control group. The remaining groups of HP, 75

PC, PLC η2, PLC δ4, PC/PLC η2, and PC/PLC δ4 were statistically different from IVF,

Sham and the No Sperm control groups (Figure 3-5 and Table 3-5).

Discussion

2+ A hallmark of oocyte activation is the rise of Ca i from internal stores. All models

2+ studied to date display Ca i at oocyte activation, but the specific pathway leading to this even differs between species. To better understand the pathway leading to bovine oocyte activation the present study was conducted to: 1) determine which PLC isotypes are present in bovine gametes, 2) determine the impact microinjecting antibodies against

2+ PLC isotypes will have on Ca i transients following IVF, 3) determine the impact microinjecting antibodies against PLC isotypes will have on cleavage rates 48 hourst post

IVF.

PLC Isotypes in Bovine Oocytes and Spermatozoa

Several PLC Isotypes have been reported to be present in spermatozoa. One study found δ4, β1, β3, and γ2 all present in mouse sperm and that δ4 was involved in the acrosome reaction and zona pellucida penetration [83]. Others have also reported that

PLC γ [84], β1 [85], and ζ [86] are also present in mouse sperm and are either involved in the acrosome reaction and/or fertilization. PLC ζ has drawn much attention as the proposed mammalian SOAF [41, 86-89]. While others state that the epididymal and testicular expression profiles in mouse and human do not support its role as SOAF [90].

Mouse oocytes have been shown to have mRNA coding for β1, β3, and γ [91]. PLC γ1

[91] and β1 [92] have also been detected by western blotting. 76

Here we report the first evidence of endogenous PLC isotypes in bovine spermatozoa and oocytes. Sperm were acrosome reacted, then lysed to represent the state at sperm-vittelline membrane binding and to eliminate any PLC isotypes that would be removed during the acrosome reaction. We determined that PLC δ1 and δ3 are present in both bovine oocytes and spermatozoa. PLC δ4, γ2, and η2 were detected in bovine oocytes and ζ is present in sperm (Figure 3-1). The other PLC isotypes were not detected in either bovine oocytes or sperm (data not shown). Although PLC ζ is present in sperm, it did not impact bovine oocyte activation.

PLC Isotypes Involed in Bovine Oocyte Activation

2+ From the combination of Ca i and cleavage rates we conclude that the PLC isotypes involved in bovine oocyte activation are δ1, δ3, δ4, γ2, and η2. PLC δ1, δ3, and

2+ γ2 significantly impact both Ca i and cleavage rates, leading to the conclusion that they are potentially involved in both IP3R and RyR activation. The isotypes δ4 and η2 did not

2+ 2+ impact Ca i visualization and only impacted cleavage rates. Ca i was visualized 6 hours post fertilization, while cleavage rates were calculated 48 hours post fertilization. We hypothesized that δ4 and η2 are involved in CICR and the impact of the microinjected

2+ antibodies are not seen until after Ca i had been measured. To answer this hypothesis we used the known antagonist of IP3R, Heparin (HP) and the known antagonist of RyR,

Procaine (PC), to track the effects of these PLC isotypes on CICR. It is accepted that

2+ Ca i can come from two cellular stores and by two separate channels [20, 93]. Both IP3R

2+ and RyR are involved in Ca i in many different cell types and have been shown to be involved in sea urchin oocyte activation [94]. IP3R and RyR are both present in murine 77

[21, 95, 96], porcine [97], humans [98-100], and bovine [19, 20] oocytes. IP3R is

2+ involved in the initial Ca i release seen at activation, while the RyR functions more with

2+ a CIDR mechanism [101]. Both IP3R and RyR appear to produce repetitive Ca i transients [102-104]. A control group of IP3 + δ4/No Sperm and IP3 + η2/No Sperm were included to validate functionality of IP3R and confirm that the PLC isotypes were not

2+ effecting Ca i release from IP3R. Ionomycin and IP3 controls were used to verify that the calcium-releasing machinery of the oocyte was functional. All treatments used

2+ significantly impacted Ca i transients (Figure 3-4 and Table 3-4). Cleavage rates were similary effected by the treatments, with HP + δ4 and HP + η2 showing no difference

2+ from the No Sperm control group.(Figure 3-5 and Table 3-5). The combination of Ca i and cleavage rates leads to the conclusion that both δ4 and η2 are involved in the

2+ propagation of Ca i transients and most likely function in a pathway involved with RyR activation. When IP3R was block with HP and either δ4 or η2 was present, cleavage rates

2+ dropped drastically, indicating that both mechanisms for Ca i release were inhibited.

It is well accepted in the literature that PLC ζ is involved in murine oocyte activation [41-44]. Here we report that unlike the murine model, PLC ζ is not required for bovine oocyte activation, further validating our hypothesis that the detailed pathway leading to mammalian oocyte activation differs among models.

The process of ICSI alone is sufficient to activate human [66, 68], mice [69], hamster [46], and rabbits [48]. There is no need for membrane binding or an additional exogenous activation procedure with these species. This is not true for bovine. ICSI alone does not activate bovine oocytes [63, 71-77]. It appears that bovine oocytes are not 78 activated by a SOAF, require sperm-egg binding for oocyte activation, and involve PLC

2+ δ1, δ3, δ4, γ2, and η2 in pathways leading to IP3R and RyR Ca i transients. These PLC

2+ isotypes may function in a redundant manner or in tandem. A combination of Ca i and cleavage data leads to the hypothesis that PLC δ1, δ3, and γ2 may function in the early

2+ 2+ IP3R Ca i release. If this is sufficiently blocked, then Ca i visualization at 6 h post fertilization is significantly decreased (Figure 3-2 and Table 3-2). It is reasonable to

2+ conclude that if the initial Ca i is blocked that the later events of CICR would also be reduced. If CICR is hindered, MPF levels would remain elevated and evaluating cleavage rates at 48 h post fertilization would be decreased, as was seen (Figure 3-3 and Table

3-3).

Previously reported results, along with the data reported here, reinforce the conclusion that mammalian species differ in the specific signal transduction pathways used at fertilization that lead to activation and induction of subsequent development.

References

1. Whitaker M. Calcium at Fertilization and in Early Development. Physiological

Rev 2006; 86.

2. Runft L. Egg Activation at Fertilization: Where It All Begins. Dev Biol 2002;

245:237-254.

3. Sun Q-Y. Cellular and molecular mechanisms leading to cortical reaction and

polyspermy block in mammalian eggs. Microscopy Research and Tech 2003;

61:342-348. 79

4. Tae JC, Kim EY, Jeon K, Lee KS, Lee CH, Kim YO, Park SP, Kim N-H. A MAPK

pathway is involved in the control of cortical granule reaction and mitosis during

bovine fertilization. Molec Reprod and Dev 2008; 75:1300-1306.

5. Gardner AJ, Williams CJ, Evans JP. Establishment of the mammalian membrane

block to polyspermy: evidence for calcium-dependent and -independent

regulation. Reproduction 2007; 133:383-393.

6. Lorca T, Cruzalegui FH, Fesquet D, Cavadore JC, Mery J, Means A, Doree M.

Calmodulin-dependent protein kinase II mediates inactivation of MPF and CSF

upon fertilization of Xenopus eggs. Nature 1993; 366:270-273.

7. Whitaker M, Swann K. Lighting the fuse at fertilization. Development 1993;

117:1-12.

8. Jaffe LA, Giusti AF, Carroll DJ, Foltz KR. Ca2+ signalling during fertilization of

echinoderm eggs. Semin Cell Dev Biol 2001; 12:45-51.

9. Malcuit C. Fertilization and inositol 1,4,5-trisphosphate (IP3)-induced calcium

release in type-1 inositol 1,4,5-trisphosphate receptor down-regulated bovine

eggs. Biol of Reprod 2005; 73:2-13.

10. Kuretake S, Kimura Y, Hoshi K, Yanagimachi R. Fertilization and development of

mouse oocytes injected with isolated sperm heads. Biol Reprod 1996; 55:789-795.

11. Sato K. Tyrosine kinase-dependent activation of phospholipase Cγ is required for

calcium transient in Xenopus egg fertilization. Dev Biol 2000; 224:453-469. 80

12. Crossley I, Swann K, Chambers EL, Whitaker M. Activation of sea urchin eggs

by inositol phosphates is independent of external calcium. Biochem J 1988;

252:257-262.

13. Terasaki M, Jaffe LA. Organization of the sea urchin egg endoplasmic reticulum

and its reorganization at fertilization. J Cell Biol 1991; 114:929-940.

14. Kline D. Attributes and dynamics of the endoplasmic reticulum in mammalian

eggs. Curr Top Dev Biol 2000; 50:125-154.

15. Galione A, Lee HC, Busa WB. Ca(2+)-induced Ca2+ release in sea urchin egg

homogenates: modulation by cyclic ADP-ribose. Science 1991; 253:1143-1146.

16. Stricker SA. Comparative biology of calcium signaling during fertilization and

egg activation in animals. Dev Biol 1999; 211:157-176.

17. Rickords LF, White KL. Electroporation of inositol 1,4,5-triphosphate induces

repetitive calcium oscillations in murine oocytes. J Exp Zool 1993; 265:178-184.

18. Mitalipov SM, White KL, Farrar VR, Morrey J, Reed WA. Development of

nuclear transfer and parthenogenetic rabbit embryos activated with inositol 1,4,5-

trisphosphate. Biol of Reprod 1999; 60:821-827.

19. Yue C, White KL, Reed WA, King E. Localization and regulation of ryanodine

receptor in bovine oocytes. Biol Reprod 1998; 58:608-614.

20. Yue C, White KL, Reed WA, Bunch TD. The existence of inositol 1,4,5-

trisphosphate and ryanodine receptors in mature bovine oocytes. Development

1995; 121:2645-2654. 81

21. Ayabe T, Kopf GS, Schultz RM. Regulation of mouse egg activation: presence of

ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose

on uninseminated and inseminated eggs. Development 1995; 121:2233-2244.

22. Giusti AF, Carroll DJ, Abassi YA, Foltz KR. Evidence that a starfish egg Src

family tyrosine kinase associates with PLC-gamma1 SH2 domains at fertilization.

Dev Biol 1999; 208:189-199.

23. Giusti AF, Carroll DJ, Abassi YA, Terasaki M, Foltz KR, Jaffe LA. Requirement

of a Src family kinase for initiating calcium release at fertilization in starfish eggs.

J Biol Chem 1999; 274:29318-29322.

24. Kinsey WH, Wu W, Macgregor E. Activation of Src-family PTK activity at

fertilization: role of the SH2 domain. Dev Biol 2003; 264:255-262.

25. Mizote A, Okamoto S, Iwao Y. Activation of Xenopus eggs by proteases: possible

involvement of a sperm protease in fertilization. Dev Biol 1999; 208:79-92.

26. Iwao Y, Fujimura T. Activation of Xenopus eggs by RGD-containing peptides

accompanied by intracellular Ca2+ release. Dev Biol 1996; 177:558-567.

27. Shilling FM, Kratzschmar J, Cai H, Weskamp G, Gayko U, Leibow J, Myles DG,

Nuccitelli R, Blobel CP. Identification of metalloprotease/disintegrins in Xenopus

laevis testis with a potential role in fertilization. Dev Biol 1997; 186:155-164.

28. Aarabi M, Qin Z, Xu W, Mewburn J, Oko R. Sperm-borne protein, PAWP,

initiates zygotic development in Xenopus laevis by eliciting intracellular calcium

release. Molec Reprod and Dev 2009; 77:249-256. 82

29. Snow P, Yim DL, Leibow JD, Saini S, Nuccitelli R. Fertilization stimulates an

increase in inositol trisphosphate and inositol lipid levels in Xenopus eggs. Dev

Biol 1996; 180:108-118.

30. Runft LL, Watras J, Jaffe LA. Calcium release at fertilization of Xenopus eggs

requires type I IP3 receptors, but not SH2 domain-mediated activation of PLCg or

Gq-mediated activation of PLCb. Dev Biol 1999; 214:399-411.

31. Tokmakov AA, Sato KI, Iwasaki T, Fukami Y. Src kinase induces calcium release

in Xenopus egg extracts via PLCgamma and IP3-dependent mechanism. Cell

Calcium 2002; 32:11-20.

32. Boissonnas CC, Montjean D, Lesaffre C, Auer J, Vaiman D, Wolf J-P, Ziyyat A.

Role of sperm αvβ3 integrin in mouse fertilization. Dev Dynamics 2010;

239:773-783.

33. Evans JP, Kopf GS, Schultz RM. Characterization of the binding of recombinant

mouse sperm fertilin beta subunit to mouse eggs: evidence for adhesive activity

via an egg beta1 integrin-mediated interaction. Dev Biol 1997; 187:79-93.

34. Evans JP, Schultz RM, Kopf GS. Characterization of the binding of recombinant

mouse sperm fertilin alpha subunit to mouse eggs: evidence for function as a cell

adhesion molecule in sperm-egg binding. Dev Biol 1997; 187:94-106.

35. Evans JP, Schultz RM, Kopf GS. Identification and localization of integrin

subunits in oocytes and eggs of the mouse. Molec Reprod and Dev 1995;

40:211-220. 83

36. Evans JP, Schultz RM, Kopf GS. Mouse sperm-egg plasma membrane

interactions: analysis of roles of egg integrins and the mouse sperm homologue of

PH-30 (fertilin) beta. J of cell sci 1995; 108 ( Pt 10):3267-3278.

37. Vjugina U, Zhu X, Oh E, Bracero NJ, Evans JP. Reduction of mouse egg surface

integrin alpha 9 subunit (ITGA9) reduces the egg's ability to support sperm-egg

binding and fusion. Biol of Reprod 2009; 80:833-841.

38. Barraud-Lange V, Naud-Barriant N, Saffar L, Gattegno L, Ducot B, Drillet A-S,

Bomsel M, Wolf J-P, Ziyyat A. Alpha6beta1 integrin expressed by sperm is

determinant in mouse fertilization. BMC Developmental Biology 2007; 7:102.

39. Tatone C, Carbone M. Possible involvement of integrin-mediated signalling in

oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate

protein kinase C and cortical granule exocytosis in mouse oocytes. Reprod Biol

and Endocrin 2006; 4:48.

40. Chen H, Sampson NS. Mediation of sperm-egg fusion: evidence that mouse egg

alpha6beta1 integrin is the receptor for sperm fertilinbeta. Chem Biol 1999;

6:1-10.

41. Swann K, Larman MG, Saunders CM, Lai FA. The cytosolic sperm factor that

triggers Ca2+ oscillations and egg activation in mammals is a novel

phospholipase C: PLCzeta. Reproduction 2004; 127:431-439.

42. Kouchi Z. Recombinant phospholipase C has high Ca2+ sensitivity and induces

Ca2+ oscillations in mouse eggs. J of Biol Chem 2003; 279:10408-10412. 84

43. Kurokawa M. Mammalian fertilization: from sperm factor to phospholipase Cζ.

Biol of the Cell 2004; 96:37-45.

44. Fujimoto S, Yoshida N, Fukui T, Amanai M, Isobe T, Itagaki C, Izumi T, Perry

ACF. Mammalian phospholipase Cζ induces oocyte activation from the sperm

perinuclear matrix. Dev Biol 2004; 274:370-383.

45. Igarashi H, Knott J, Schultz R, Williams C. Alterations of PLCβ1 in mouse eggs

change calcium oscillatory behavior following fertilization. Dev Biol 2007;

312:321-330.

46. Uehara T, Yanagimachi R. Microsurgical injection of spermatozoa into hamster

eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol

Reprod 1976; 15:467-470.

47. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after

intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;

340:17-18.

48. Keefer CL. Fertilization by sperm injection in the rabbit. Gamete Res 1989;

22:59-69.

49. Pope CE, Johnson CA, McRae MA, Keller GL, Dresser BL. Development of

embryos produced by intracytoplasmic sperm injection of cat oocytes. Anim

Reprod Sci 1998; 53:221-236.

50. Gomez MC, Pope CE, Harris R, Davis A, Mikota S, Dresser BL. Births of kittens

produced by intracytoplasmic sperm injection of domestic cat oocytes matured in

vitro. Reprod Fertil Dev 2000; 12:423-433. 85

51. Lyu QF, Deng L, Xue SG, Cao SF, Liu XY, Jin W, Wu LQ, Kuang YP. New

technique for mouse oocyte injection via a modified holding pipette. Reprod

Biomed Online 2010.

52. Stein P, Schultz RM. ICSI in the mouse. Methods in enzymology 2010;

476:251-262.

53. Keskintepe L, Morton PC, Smith SE, Tucker MJ, Simplicio AA, Brackett BG.

Caprine blastocyst formation following intracytoplasmic sperm injection and

defined culture. Zygote 1997; 5:261-265.

54. Wang B, Baldassarre H, Pierson J, Cote F, Rao KM, Karatzas CN. The in vitro

and in vivo development of goat embryos produced by intracytoplasmic sperm

injection using tail-cut spermatozoa. Zygote 2003; 11:219-227.

55. Jimenez-Macedo AR, Anguita B, Izquierdo D, Mogas T, Paramio MT. Embryo

development of prepubertal goat oocytes fertilised by intracytoplasmic sperm

injection (ICSI) according to oocyte diameter. Theriogenology 2006;

66:1065-1072.

56. Lai L, Sun Q, Wu G, Murphy CN, Kuhholzer B, Park KW, Bonk AJ, Day BN,

Prather RS. Development of porcine embryos and offspring after intracytoplasmic

sperm injection with liposome transfected or non-transfected sperm into in vitro

matured oocytes. Zygote 2001; 9:339-346.

57. Nakai M, Kashiwazaski N, Takizawa A, Hayashi Y, Nakatsukasa E, Fuchimoto D,

Noguchi J, Kaneko H, Shino M, Kikuchi K. Viable piglets generated from porcine 86

oocytes matured in vitro and fertilized by intracytoplasmic sperm head injection.

Biol Reprod 2003; 68:1003-1008.

58. Yong HY, Hao Y, Lai L, Li R, Murphy CN, Rieke A, Wax D, Samuel M, Prather

RS. Production of a transgenic piglet by a sperm injection technique in which no

chemical or physical treatments were used for oocytes or sperm. Molec Reprod

and Dev 2006; 73:595-599.

59. Simerly CR, Castro CA, Jacoby E, Grund K, Turpin J, McFarland D, Champagne

J, Jimenez JB, Jr., Frost P, Bauer C, Hewitson L, Schatten G. Assisted

reproductive technologies (ART) with baboons generate live offspring: a

nonhuman primate model for ART and reproductive sciences. Reprod Sci 2010;

17:917-930.

60. Dell'Aquila ME, Cho YS, Minoia P, Traina V, Fusco S, Lacalandra GM, Maritato

F. Intracytoplasmic sperm injection (ICSI) versus conventional IVF on abattoir-

derived and in vitro-matured equine oocytes. Theriogenology 1997;

47:1139-1156.

61. Cochran R, Meintjes M, Reggio B, Hylan D, Carter J, Pinto C, Paccamonti D,

Godke RA. Live foals produced from sperm-injected oocytes derived from

pregnant mares. J of equine vet sci 1998; 18:736-740.

62. Choi YH, Love CC, Varner DD, Hinrichs K. Equine blastocyst development after

intracytoplasmic injection of sperm subjected to two freeze-thaw cycles.

Theriogenology 2006; 65:808-819. 87

63. Galli C, Vassiliev I, Lagutina I, Galli A, Lazzari G. Bovine embryo development

following ICSI: effect of activation, sperm capacitation and pre-treatment with

dithiothreitol. Theriogenology 2003; 60:1467-1480.

64. Suttner R, Zakhartchenko V, Stojkovic P, Mtiller S, Alberio R, Medjugorac I,

Brem G, Wolf E, Stojkovic M. Intracytoplasmic sperm injection in bovine: effects

of oocyte activation, sperm pretreatment and injection technique. Theriogenology

2000; 54.

65. Fujinami N, Hosoi Y, Kato H, Matsumoto K, Saeki K, Iritani A. Activation with

ethanol improves embryo development of ICSI-derived oocytes by regulation of

kinetics of MPF activity. J Reprod Dev 2004; 50:171-178.

66. Tesarik J, Sousa M, Testart J. Human oocyte activation after intracytoplasmic

sperm injection. Hum Reprod 1994; 9:511-518.

67. Shang XM, Li ZP. [The establishment of in vitro fertilization-embryo transfer

(IVF-ET) and the development of its derivative techniques]. Zhonghua Yi Shi Za

Zhi 2009; 39:93-99.

68. Catt JW, Ryan JP, Pike IL, O'Neill C, Saunders DM. Clinical intracytoplasmic

sperm injection (ICSI) results from Royal North Shore Hospital. Reprod Fertil

Dev 1995; 7:255-261; discussion 261-252.

69. Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol

Reprod 1995; 52:709-720.

70. Williams CJ. Signalling mechanisms of mammalian oocyte activation. Hum

Reprod Update 2002; 8:313-321. 88

71. García-Roselló E, García-Mengual E, Coy P, Alfonso J, Silvestre MA.

Intracytoplasmic sperm injection in livestock species: an update. Reprod in

Domestic Animals 2009; 44:143-151.

72. Bevacqua RJ, Pereyra-Bonnet F, Fernandez-Martin R, Salamone DF. High rates of

bovine blastocyst development after ICSI-mediated gene transfer assisted by

chemical activation. Theriogenology 2010; 74:922-931.

73. Abdalla H, Shimoda M, Hirabayashi M, Hochi S. A combined treatment of

ionomycin with ethanol improves blastocyst development of bovine oocytes

harvested from stored ovaries and microinjected with spermatozoa.

Theriogenology 2009; 72:453-460.

74. Galli C, Lazzari G. The manipulation of gametes and embryos in farm animals.

Reproduction in domestic animals = Zuchthygiene 2008; 43 Suppl 2:1-7.

75. Rho GJ, Wu B, Kawarsky S, Leibo SP, Betteridge KJ. Activation regimens to

prepare bovine oocytes for intracytoplasmic sperm injection. Molec Reprod and

Dev 1998; 50:485-492.

76. Chung JT, Keefer CL, Downey BR. Activation of bovine oocytes following

intracytoplasmic sperm injection (ICSI). Theriogenology 2000; 53:1273-1284.

77. Malcuit C, Maserati M, Takahashi Y, Page R, Fissore RA. Intracytoplasmic sperm

injection in the bovine induces abnormal [Ca2+]i responses and oocyte activation.

Reprod Fertil Dev 2006; 18:39-51. 89

78. Campbell KD, Reed WA, White KL. Ability of integrins to mediate fertilization,

intracellular calcium release, and parthenogenetic development in bovine oocytes.

Biol Reprod 2000; 62:1702-1709.

79. Goto K, Iwai N, Takuma Y, Nakanishi Y. Co-culture of in vitro fertilized bovine

embryos with different cell monolayers. J of Animal Sci 1992; 70:1449-1453.

80. Rosenkrans CFJ, First NL. Effect of free amino acids and vitamins on cleavage

and developmental rate of bovine zygotes in vitro. J of Animal Sci 1994;

72:434-437.

81. Parrish JJ, Susko-Parrish JL, Handrow RR, Ax RL, First NL. Effect of sulfated

glycoconjugates on capacitation and the acrosome reaction of bovine and hamster

spermatozoa. Gamete Res 1989; 24:403-413.

82. Sun QY, Fan HY. Activity of MAPK/p90rsk during fertilization in mice, rats, and

pigs. Methods Mol Biol 2004; 253:293-304.

83. Fukami K, Nakao K, Inoue T, Kataoka Y, Kurokawa M, Fissore RA, Nakamura

K, Katsuki M, Mikoshiba K, Yoshida N, Takenawa T. Requirement of

phospholipase Cdelta4 for the zona pellucida-induced acrosome reaction. Science

2001; 292:920-923.

84. Spungin B, Breitbart H. Calcium mobilization and influx during sperm

exocytosis. Journal of cell science 1996; 109 ( Pt 7):1947-1955.

85. Choi D, Lee E, Hwang S, Jun K, Kim D, Yoon BK, Shin HS, Lee JH. The

biological significance of phospholipase C beta 1 gene mutation in mouse sperm 90

in the acrosome reaction, fertilization, and embryo development. J of assist

Reprod and Genetics 2001; 18:305-310.

86. Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann

K, Lai FA. PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and

embryo development. Development 2002; 129:3533-3544.

87. Larman MG, Saunders CM, Carroll J, Lai FA, Swann K. Cell cycle-dependent

Ca2+ oscillations in mouse embryos are regulated by nuclear targeting of

PLCzeta. J Cell Sci 2004; 117:2513-2521.

88. Kouchi Z, Fukami K, Shikano T, Oda S, Nakamura Y, Takenawa T, Miyazaki S.

Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+

oscillations in mouse eggs. J of Biol Chem 2004; 279:10408-10412.

89. Young C, Grasa P, Coward K, Davis LC, Parrington J. Phospholipase C zeta

undergoes dynamic changes in its pattern of localization in sperm during

capacitation and the acrosome reaction. Fertility and Sterility 2009;

91:2230-2242.

90. Aarabi M, Yu Y, Xu W, Tse MY, Pang SC, Yi YJ, Sutovsky P, Oko R. The

testicular and epididymal expression profile of PLCzeta in mouse and human does

not support its role as a sperm-borne oocyte activating factor. PloS one 2012;

7:e33496.

91. Dupont G, McGuinness OM, Johnson MH, Berridge MJ, Borgese F.

Phospholipase C in mouse oocytes: characterization of beta and gamma isoforms 91

and their possible involvement in sperm-induced Ca2+ spiking. Biochem J 1996;

316 ( Pt 2):583-591.

92. Avazeri N, Courtot AM, Pesty A, Duquenne C, Lefevre B. Cytoplasmic and

nuclear phospholipase C-beta 1 relocation: role in resumption of meiosis in the

mouse oocyte. Molec Biol Cell2000; 11:4369-4380.

93. Balakier H, Dziak E, Sojecki A, Librach C, Michalak M, Opas M. Calcium-

binding proteins and calcium-release channels in human maturing oocytes,

pronuclear zygotes and early preimplantation embryos. Hum Reprod 2002;

17:2938-2947.

94. McPherson SM, McPherson PS, Mathews L, Campbell KP, Longo FJ. Cortical

localization of a calcium release channel in sea urchin eggs. Journal of Cell Biol

1992; 116:1111-1121.

95. Mehlmann LM, Mikoshiba K, Kline D. Redistribution and increase in cortical

inositol 1,4,5-trisphosphate receptors after meiotic maturation of the mouse

oocyte. Dev Biol 1996; 180:489-498.

96. Fissore RA, Longo FJ, Anderson E, Parys JB, Ducibella T. Differential

distribution of inositol trisphosphate receptor isoforms in mouse oocytes. Biol of

Reprod 1999; 60:49-57.

97. Machaty Z, Funahashi H, Day BN, Prather RS. Developmental changes in the

intracellular Ca2+ release mechanisms in porcine oocytes. Biol of Reprod 1997;

56:921-930. 92

98. Tesarik J, Sousa M, Mendoza C. Sperm-induced calcium oscillations of human

oocytes show distinct features in oocyte center and periphery. Molec Reprod and

Dev 1995; 41:257-263.

99. Sousa M, Barros A, Tesarik J. The role of ryanodine-sensitive Ca2+ stores in the

Ca2+ oscillation machine of human oocytes. Molec Hum Reprod 1996;

2:265-272.

100. Goud PT, Goud AP, Van Oostveldt P, Dhont M. Presence and dynamic

redistribution of type I inositol 1,4,5-trisphosphate receptors in human oocytes

and embryos during in-vitro maturation, fertilization and early cleavage divisions.

Molec Hum Reprod 1999; 5:441-451.

101. Fill M, Copello JA. Ryanodine receptor calcium release channels. Physiological

reviews 2002; 82:893-922.

102. Berridge MJ, Galione A. Cytosolic calcium oscillators. FASEB journal: official

publication of the Federation of American Societies for Experimental Biology

1988; 2:3074-3082.

103. Missiaen L, Taylor CW, Berridge MJ. Spontaneous calcium release from inositol

trisphosphate-sensitive calcium stores. Nature 1991; 352:241-244.

104. Taylor CW, Marshall IC. Calcium and inositol 1,4,5-trisphosphate receptors: a

complex relationship. Trends in biochem sci 1992; 17:403-407. 93

Table 3-1. Primary antibodies used for micro-injection and western blotting. All antibodies were purchased from Santa Cruz Biotechnology Inc.

PLC Antibody Target Epitope

β1 G-12 sc-205 C-terminus

β2 Q-15 sc-206 C-terminus

β3 L-17 sc-31760 N-terminus

β4 C-18 sc-404 C-terminus

ε T-20 sc-28403 C-terminus

δ1 H-140 sc-30062 Amino acids 1-140 mapping at the N- terminus δ3 L-15 sc-30826 Internal Region

δ4 Q-15 sc-30830 C-terminus

δ4 H-250 sc-30063 Amino acids 1-250 mapping at the N- terminus γ1 E-12 sc-7290 Amino acids 1243-1262 near the C-terminus

γ2 B-10 sc-5283 Amino acids 826-985

η D-14 sc-104620 Internal Region

ζ C-12 sc-131752 C-terminus 94

2+ a-d Table 3-2. Effect of micro-injected antibodies on activation as determined by Ca i. Unlike superscripts are significantly different (P < 0.05).

Number of Mature Number of Oocytes Showing Treatment 2+ Oocytes Ca i (%) Ionomycin 41 41 (100)a IP3 30 27 (91.2)a,b ε 30 22 (73.5)b,c γ1 30 21 (70.6)b,c δ4 40 27 (67.3)c η2 30 20 (66.8)c Sham 62 41 (66.1)c β3 32 21 (65.7)c IVF 239 155 (65.2)c β1 31 20 (64.5)c β2 30 19 (63.3)c ζ 52 32 (63.0)c β4 31 18 (58.2)c δ3 50 16 (32.2)d δ1 31 10 (32.1)d γ2 56 11 (20.1)d No Sperm 59 10 (16.9)d 95

Table 3-3. Effect of micro-injected antibodies on development as determined by rate of cleavage. a-c Unlike superscripts are significantly different (P < 0.05)

Number of Cleaved Oocytes Treatment Number of Mature Oocytes (%) ζ 153 104 (66.8)a IVF 973 659 (66.8)a Sham 189 125 (66.7)a β3 96 63 (65.6)a β2 68 43 (62.7)a ε 79 49 (62.3)a β1 99 62 (61.3)a γ1 72 44 (60.7)a β4 62 36 (57.9)a δ1 133 43 (32.6)b δ4 92 29 (31.7)b γ2 125 39 (29.6)b η2 83 22 (26.0)b δ3 79 19 (24.3)b,c No Sperm 131 17 (12.8)c 96

Table 3-4. The effect of micro-injected antagonist, agonist, and antibodies on initial IP3R 2+ Ca i and RyR CICR of IVF produced embryos as determined by the rate of calcium transients. a-d Unlike superscripts are significantly different (P < 0.05).

Number of Mature Number of Oocytes Showing Treatment 2+ Oocytes Ca i (%) Ionomycin 41 41 (100)a IP3 30 27 (91.2)a,b IP3 + δ4 NO 13 11 (85.0)a,b,c Sperm IP3 + η2 NO 11 9 (83.3)b,c Sperm δ4 40 27 (67.3)c η2 30 20 (66.8)c Sham 62 41 (66.1)c IVF 239 155 (65.2)c Heparin 19 7 (36.5)d Procaine + δ4 14 5 (35.0)d Procaine 15 5 (32.8)d Procaine + η2 13 4 (30.0)d Heparin + η2 15 4 (26.1)d Heparin + δ4 14 3 (21.7)d No Sperm 59 10 (16.9)d 97

Table 3-5. The effect of micro-injected antagonist, agonist, and antibodies on initial IP3R 2+ a-f Ca i and RyR CICR of IVF produced embryos as determined by the rate of cleavage. Unlike superscripts are significantly different (P < 0.05).

Number of Cleaved Oocytes Treatment Number of Mature Oocytes (%)

IVF 973 659 (66.8)a Sham 189 125 (66.7)a

Heparin 51 21 (41.1)b

Procaine 54 21 (38.8)b,c

δ4 92 29 (31.7)b,c,

Procaine + δ4 59 16 (28.1)b,c,d,e

Procaine + η2 46 12 (26.1)b,c,d,e

η2 83 22 (26.0)b,c,d,e

IP3+ η2 NO sperm 27 6 (21.8)b,c,d,e,f IP3 + δ4 NO 24 4 (16.7)c,d,e,f sperm Heparin + η2 45 7 (15.5)d,e,f

Heparin + δ4 80 12 (15.4)e,f No Sperm 131 17 (12.8)f

Table 3-5. The effect of micro-injected antagonist, agonist, and antibodies on 2+ initial IP3R Ca i and RyR CICR of IVF produced embryos as determined by the rate of cleavage. a-f Unlike superscripts are significantly different (P < 0.05). 98 ladder sperm Bovine Testis BovineBrain Rat Testisµg) (30 ladder egg sperm BovineBrain RIPA BovineBrain SDS µg) (30 F9 ladder egg sperm Bovine Testis Rat Testisµg) (30 egg

100 - δ1, δ3, 70 -

55 -

35 -

δ1 δ3 δ4 ladder egg sperm BovineLymph γ PLC (h): 2 293T Lysate (20 ladder egg sperm Bovine Testis Rat Testisµg) (30 ladder egg sperm Bovine Testis Rat Testisµg) (30

170- γ2, 130-

100 - ζ 70 -

55 -

35 -

γ2 η2 ζ

Figure 3-1. Western blots of PLC Isotypes in bovine oocytes, sperm, bovine control, and positive control lysates. 99

Calcium Transients (%) a 100 a,b

80 b,c b,c c c c c c c c c c 60

40 d d

d 20 d

0 IM IP3 IVF Sha NS ε ζ η2 β1 β2 β3 β4 δ1 δ3 δ4 γ2 γ1

CONTROLS ANTIBODIES

Figure 3-2. The effect of micro-injected antibodies on oocyte activation of IVF produced embryos as determined by the rate of calcium transients. Error bars represent standard deviation within treatment group. a-d Unlike superscripts are significantly different (P < 0.05) IM (Ionomycin control) NS (No Sperm control) 100

Cleavage Rate (%) 75 a a a a a a a a a 60

45

b b b 30 b b,c

c 15

0 IV Sham NS ε ζ η2 β1 β2 β3 β4 δ1 δ3 δ4 γ1 γ2 CONTROLS ANTIBODIES

Figure 3-3. The effect of micro-injected antibodies on development of IVF produced embryos as determined by the rate of cleavage. Error bars represent standard deviation within treatment group. a-c Unlike superscripts are significantly different (P < 0.05). NS (No Sperm control) 101

CICR Calcium Transients (%) a 100 a,b

a,b,c b,c

80 c c c c

60

d d 40 d d d d d 20

0 IM IP3 IVF Sham NS IP3/η2 IP3/δ4 η2 δ4 PC PC/η2 PC/δ4 HP HP/η2 HP/δ4 /NS /NS

CONTROLS ANTIBODIE PROCAINE / RyR HEPARIN / IP3R

Figure 3-4. The effect of micro-injected antagonist, agonist, and antibodies on initial 2+ IP3R Ca i and RyR CICR of IVF produced embryos as determined by the rate of calcium transients. Error bars represent standard deviation within treatment group. a-d Unlike superscripts are significantly different (P < 0.05) IM (Ionomycin) PC 102

CICR Cleavage Rate (%) 75 a a

60

45 b b,c

b,c,d b,c,d,e 30 b,c,d,e b,c,d,e b,c,d,e,f c,d,e, d,e,f e,f 15 f

0 IVF Sham NS IP3/η2 IP3/δ4 η2 δ4 PC PC/η2 PC/δ4 HP HP/η2 HP/δ4 NS NS CONTROLS ANTIBODIE PROCAINE / RyR HEPARIN / IP3R

Figure 3-5. The effect of micro-injected antagonist, agonist, and antibodies on initial 2+ IP3R Ca i and RyR CICR of IVF produced embryos as determined by the rate of cleavage. Error bars represent standard deviation within treatment. a-f Unlike superscripts are significantly different (P < 0.05) PC (Procaine), HP (Heparin), NS (No Sperm control) 103

CHAPTER 4

SUMMARY

In addition to contributing genes at fertilization, the sperm cell induces the oocyte to leave its state of metabolic arrest through a process called activation. A hallmark of

2+ oocyte activation is a release of intracellular calcium (Ca i) from the endoplasmic reticulum stores that results in repetitive transients. The mediators of oocyte activation have been studied extensively in sea urchin, zebrafish, ascidian, starfish, Xenopus, and murine models. To date, little is known about the biochemical pathway in bovine oocyte activation. Global conclusions regarding mammalian are often attributed to work in the murine model. Caution must be used in making generalizations from one animal model to another. This research dissertation delineates and defines putative roles of Src Family

2+ Kinase (SFKs) and Phopholipase C (PLC) enzymes in the pathway leading to Ca i and calcium-induced calcium-release (CICR) in bovine oocyte activation and establishes specific differences among mammalian models. Based upon the results in these studies bovine and murine models follow different oocyte activation pathways.

A brief overview of activation pathways among all animal models indicates that both SFK and/or PLC enzymes are involved. SFK has been proposed to active PLC which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2), producing membrane bound diacylglycerol, and cytosolic soluble 1,4,5-inositol trisphosphate (IP3). Cytosolic IP3 can then bind to IP3 receptors located on the membrane of the endoplasmic reticulum and initiate the release of stored calcium. The release of Ca2+ leads to a second source of calcium release, through CICR and ryanodine receptors (RYR). 104

The first facet of the research reported in this dissertation was understanding the role SFKs play in bovine oocyte activation. The SFKs present in both bovine oocytes and spermatozoa are BLK, YES, SRC and FYN. LYN and HCK were detected in bovine spermatozoa. Micro-injection of PP2, a known inhibitor of multiple members of SFK, was used to determine that one or more SFKs are involved in activation. An injection of 3

2+ µM PP2 abolished Ca i release and cleavage rates. This points to the involvement of one or more members of SFKs. Also, PP2 inhibits the activation of kinase

(FAK), which associates with integrin receptor molecules found on the vitelline membrane. Integrin binding induces FAK activation and downstream signal transduction, specifically SFKs. Integrins are present in bovine oocytes and have been shown to play a role in their activation. Microinjection of SRC peptide non-phosphorylated (SRCnPp), a thirteen amino acid peptide mimics the C-terminal phosphoregulatory region of SRC, had

2+ no effect on Ca i release and cleavage rates. However, microinjection of a phophorylated version of this peptide (SRCPP) blocked signal transduction significantly. Based on these results, it was hypothesized that endogenous phosphatase removes the inhibitory phosphate from endogenous SRC at the phosphoregulatory residue, Tyr527, causing the

SH2 domain to release from the C-terminal region. In this state, SH2 is free to bind to the microinjected SRCPP, and thus inhibits downstream signal relay. The potential role of

SFK was further validated by microinjection of a substrate of SRC, Src Substrate II

(SSII). As SRC is activated, it further relays a signal to downstream substrates. The microinjected SSII acted to sequester any signal produced by endogenous SRC. These 105 data implicate SRC and potentially other members of SFKs in the process of bovine oocyte activation.

The second facet of this research focused on the involvement of PLC isotypes.

PLC δ1 and δ3 are present in both bovine oocytes and spermatozoa. PLC δ4, γ2, and η2 are also present in bovine oocytes and ζ is present only in sperm. The PLC isotypes β1,

β2, β3, β4, γ1, and ε were not detected in either bovine oocytes or sperm. Although PLC ζ is present in sperm, it did not appear to be involved in bovine oocyte activation, as

2+ determined by both Ca i release and cleavage rates.

The PLC isotypes involved in bovine oocyte activation are δ1, δ3, δ4, γ2, and η2,

2+ as determined by both Ca i release and cleavage. The PLCs δ1, δ3, and γ2 significantly

2+ decreased both Ca i release and cleavage rate, while PLC δ4 and η2 only impacted cleavage. These results lead to a further investigation as to why δ4 and η2 only effected

2+ cleavage and their potential involvement with the specific Ca i RyR. The possible role of

2+ PLC δ4 and η2 seems to be in CICR and RyR induced release of Ca i. PLC δ1, δ3, and

2+ γ2 are involved in initial Ca i release through IP3R. Their involvement with RyR was not within the scope of this study. Further experiments would need to be designed to answer this question.

From a combination of the work carried out in this study and past work by other colleagues point to why a generalized conclusion regarding mammalian fertilization should not be made from one single animal species. Bovine oocyte activation differs from that reported in the murine and other animals. 106

It appears that the biochemical signal transduction pathway in bovine oocyte activation is receptor mediated. It is likely that a disintegrin on the inner acrosomal membrane ligand of a spermatozoa binds to a integrin on the oocyte vitelline membrane, causing the clustering of FAK. This would recruit SRC, and potentially other SFKs, to the . As SRC is activated, its affects work downstream to activate the PLCs δ1,

δ3, and/or γ2, most likely in a redundant fashion to ensure successful signal transduction.

These PLCs induce the cleavage of PIP2, producing DAG and IP3. IP3 then binds to

2+ 2+ IP3R located on the ER resulting in the release of Ca i. The presence of cytosolic Ca i activates RyR. Endogenous oocyte PLC δ4 and η2 interact with RyR to produce CICR

2+ and propagate Ca i over several hours, thus facilitating proper oocyte activation.

Together these data provide significant information that establishes the involvement of SFK and PLC isotypes in bovine oocyte activation. Studies involving the selective SFK inhibitor PP2 target Src, Lck, and Fyn. Microinjecting PP2 had significant

2+ impacts on Ca i and cleavage rates, implicating one of PP2‘s targeted SFKs in bovine oocyte activation. To further study the involvement of Src in bovine oocyte activation, peptides mimicking the C-terminal region of Src were injected in both a phosphorylated and non-phosphorylated form. Microinjecting Src-peptide phosphorylated significantly

2+ decrease Ca i and cleavage rates, solidifying the involvement of Src in the pathway leading to bovine oocyte activation. By microinjecting primary antibodies raised against the PLC family of enzymes it was determined that δ1, δ3, and γ2 play a role in the release

2+ 2+ of Ca i and by blocking their function a significant decrease in Ca i was observed.

Evaluating the impact of PLC antibodies on cleavage rate showed the involvement of δ1, 107

δ3, δ4, η2 and γ2. These data raised the question, why δ4 and η2 were involved in

2+ cleavage rates but not the preceding Ca i release. Additional experiments postulate that

δ4 and η2 are likely involved in CICR and RyR activation. These data increase the understanding of the pathway leading to bovine oocyte activation and further confirm that the detailed pathway differs from one animal models to another. 108

CURRICULUM VITAE

Ammon Hanson Bayles 4815 Old Main Hill Logan, UT 84322 (Cell) 435-881-5576 (Lab) 435-797-1395 [email protected]

EDUCATION Ph.D Utah State University, Logan, UT 2005-2012 Major: Animal Science Speciality: Reproductive Biology Dissertation: Mechanisms and Signal Transduction Pathways Involved in Bovine Oocyte Activation Advisor: Dr. Kenneth L. White

B.S. Utah State University, Logan, UT 2001-2005 Major: Animal Science Minor: Chemistry

A.A. Ricks College, Rexburg, ID 1998-2000 Major: Pre-Medicine

CAREER INTEREST Assisted reproductive technologies (ART) in human infertility

Receive training and clinical experience in all aspect of ART

Human

Receive an American Board of Bioanalysis certificate for High-complexity Clinical Laboratory Director

TECHNICAL EXPERIENCE PhD Candidate, Animal, Dairy, and Veterinary Science 2005-2012 Department, Utah State University, Logan, UT Determined the presence of Src family kinases and phospholipase C isotypes in both bovine gametes using western blotting. Outlined the involvement of Src family kinase, specifically SRC, in bovine oocyte activation by microinjecting peptides, substrates, and inhibitors into oocytes prior to in vitro fertilization. 109

TECHNICAL EXPERIENCE (continued) Analyzed the involvement of phospolipase C isotypes in bovine oocyte activation by microinjecting function blocking antibodies into oocytes prior to in vitro fertilization. Evaluated proper oocyte activation by monitoring intracellular calcium oscillation following in vitro fertilization. Assessed proper fertilization and early embryonic development through in vitro culture of bovine embryos. Investigated three stages of a novel lysis protocol to extract inner acrosomal membrane bound proteins by 2D-gel electrophoresis comparison. Analyzed unique spots in the fraction containing integral membrane proteins by mass spectrometry and the Mascot database. Used ion exchange chromatography to isolate sperm membrane proteins that are potentially involved in binding, fusion, and/or activation of bovine oocytes. Initiated a collaboration between Diamond G Rodeo Company and the ADVS department to clone superior bucking bulls.

Professional Practice, Hoffman, A.I. Breeders, Logan, UT 2004-2012 Responsible for the collection, evaluation, and cryopreservation of bovine and equine semen. Additional duties included estrous synchronization, artificial insemination, breeding soundness exams, and animal handling.

Undergraduate Work Study, Animal, Dairy, and Veterinary 2003-2005 Science Department, Utah State University, Logan, UT Reproductive Biology Lab: Dr. Kenneth L. White Collected bovine ovaries from local abattoir. Aspirated bovine oocytes from ovaries for somatic cell nuclear transfer and in vitro fertilization experiments. Prepared media for in vitro maturation and fertilization. Performed in vitro fertilization for control groups.

PUBLICATIONS Src Family Kinases in the Activation of Bovine Oocytes. Ammon H. Bayles, Thomas D. Bunch, Aaron P. Davis, Christopher J. Davies, Justin S. Hall, Benjamin R. Sessions, Kenneth L. White. Final revisions prior to submission.

Involvement of Phospholipase C in the Activation of Bovine Oocytes Ammon H. Bayles, Thomas D. Bunch, Aaron P. Davis, Christopher J. Davies, Justin S. Hall, Benjamin R. Sessions, Kenneth L. White. Final revisions prior to submission. 110

RESEARCH / TEACHING ASSISTANTSHIP Departmental Assistantship, Animal, Dairy and Veterinary 2006-2012 Science Department, Utah State University, Logan, UT Awarded a annual stipend of $15,000. Duties included 10/h week research and 10/h week teaching assistant.

TEACHING EXPERIENCE Lab Instructor, Animal, Dairy and Veterinary 2006-2011 Science Department, Utah State University, Logan, UT Course: Physiology of Reproduction and Lactation (2 sections)

Guest Lecturer, Animal, Dairy and Veterinary Fall 2010 Science Department, Utah State University, Logan, UT Course: Dairy Production Practices Topic: Maximizing Fertility in Dairy Cows Course: Swine Management Topic: Reproductive Management in Swine

Teachers Assistant, Animal, Dairy and Veterinary 2009-2010 Science Department, Utah State University, Logan, UT Course: Undergraduate Seminar Course: Swine Management

Course Instructor, Animal, Dairy and Veterinary 2009-2010 Science Department, Utah State University, Logan, UT Course: Physiology of Reproduction and Lacation Developed curriculum in all areas including lectures, exams research project, and two lab sections.

Guest Lab Instructor, Animal, Dairy and Veterinary Fall 2009 Science Department, Utah State University, Logan, UT Course: Animals Cell Culture Methods Topic: Nuclear Transfer Techniques

Guest Lecturer, Center for Integrated Biosystems Teacher 2006-2008 Symposium, Utah State University, Logan, UT Lecture: Animal Cloning and Nuclear Transfer

Teachers Assistant, Animal, Dairy and Veterinary 2006-2007 Science Department, Utah State University, Logan, UT Course: Physiology of Reproduction and Lactation 111

TEACHING EXPERIENCE (continued) Teachers Assistant, Animal, Dairy and Veterinary 2010-2011 Science Department, Utah State University, Logan, UT Course: Live Animal and Carcass Evaluation

AWARDS College of Agriculture Graduate Teaching Instructor of the Year 2010 College of Agriculture, Utah State University, Logan, UT Awarded to the top graduate teaching instructor among all academic department with in the college.

Graduate Student Senate Travel Award, Graduate Student 2006-2009 Senate, Utah State University, Logan, UT A financial award to graduate student who demonstrate outstanding research findings. Funds used for travel to national scientific meetings in San Antonio, TX, San Francisco, CA and San Diego, CA.

Graduate Student Senate Enhancement Award Fall 2006 Graduate Student Senate, Utah State University, Logan, UT One of seventeen graduate students awarded a $4,000 scholarship. Awarded to graduate students who demonstrate outstanding achievements and contributions to both USU and their respective departments.

PRESENTATIONS Poster Presentation, Annual meeting of the American 2009 Society for Cell Biology, San Diego, CA Identifying Endogenous PLC Isoforms Involved in the Signal Transduction Pathway at Bovine Oocyte Activation.

Oral and Poster Presentations, Intermountain Graduate 2009 Symposium, Logan, UT Identifying Endogenous PLC Isoforms Involved in the Signal Transduction Pathway at Bovine Oocyte Activation.

Poster Presentation, Annual meeting of the American 2008 Society for Cell Biology, San Francisco, CA Identifying Molecules Involved in the Signal Transduction Pathway at Bovine Oocyte Activation Using Activators and Inhibitors of Src Family Kinases. 112

PRESENTATIONS (continued) Oral and Poster Presentations, Intermountain Graduate 2008 Identifying Molecules Involved in the Signal Transduction Pathway at Bovine Oocyte Activation Using Activators and Inhibitors of Src Family Kinases.

Poster Presentation, Society for the Study of Reproduction, 2007 San Antonio, TX Purification of Bovine Sperm Membrane-Proteins Involved In Sperm-Oocyte Interactions: Adhesion, Fusion, and Activation.

Oral Presentation, Center for Integrated Biosystems Research 2007 Student Presentation, Logan, UT Sperm Ligands Involved In Sperm-Oocyte Interaction and Oocyte Pathways Involved In Activation

Poster Presentation, Intermountain Graduate Symposium 2007 Logan, UT Purification of Bovine Sperm Membrane-Proteins Involved In Sperm-Oocyte Interactions: Adhesion, Fusion, and Activation

Poster Presentation, Intermountain Graduate Symposium 2007 Logan, UT Involvement of Tyrosine Kinases, Specifically SRC Family Kinase, Focal Adhesion Kinase, and Agonist-Induced PLC In the Activation And Development of Bovine Oocytes.

ACADEMIC SERVICE College of Agriculture Senator, Graduate Student Senate, Utah 2008-2009 State University, Logan, UT Organized events to promote graduate student involvement, served on several committees, and relayed senate information to College of Agriculture graduate students.

Stipend Enhancement Award Commitee, Graduate Student Spring 2009 Senate, Utah State University, Logan, UT Determined eligibility, contributions, achievements, and need of applicants interested in receiving a $4,000 stipend enhancement award.

ADVS Departmental Representative, Graduate Student 2007-2008 Senate, Utah State University, Logan, UT Relayed information from college senator to ADVS students. 113

SPECIAL TRAINING Laboratory Safety, Utah State University, Logan, UT 2006 & 2009 Environmental Health & Safety Certificate of Training

Protein Purification: Isolation & Characterization, 2006 Center for Integrated Biosystems, Utah State University, Logan, UT

Artificial Insemination, Synchronization of Estrus 2005 and Animal Set-up for Embryo Transfer, Hoffman A.I. Breeders, Logan, UT 114

REFERENCES

Dr. Ken White 4815 Old Main Hill Department of Animal, Dairy, and Veterinary Science Logan, UT 84322 Office: 435-797-2162 [email protected]

Dr. Tom Bunch 4815 Old Main Hill Department of Animal, Dairy, and Veterinary Science Logan, UT 84322 Office: 435-797-2148 [email protected]

Brett Bowman M.S. 4815 Old Main Hill Department of Animal, Dairy, and Veterinary Science Logan, UT 84322 Office: 435-797-2141 [email protected]