MOLECULAR MECHANISMS UNDERLYING THE

MOLECULAR MECHANISMS UNDERLYING THE MAMMALIAN MEMBRANE BLOCK TO POLYSPERMY

by Brent Colin Turner

A thesis submitted to the Bloomberg School of Public Health of The Johns Hopkins University in conformity with the requirements for the degree of Master of Science Baltimore, MD June 2012

© 2012 Brent C. Turner All Rights Reserved

ABSTRACT Upon fertilization, the egg undergoes a series of events known as the egg-toembryo transition, or egg activation. Successful completion of development requires the fertilization of the egg by only one sperm, and therefore, the egg has evolved with two preventative mechanisms that occur during egg activation known as the blocks to polyspermy: the zona pellucida (ZP) block and the membrane block to polyspermy. The establishment of the membrane block to polyspermy in mammalian eggs is partially dependent on calcium signaling and actin, and likely results from a culmination of multiple post-fertilization events. The goal of this thesis is to elucidate additional molecular mechanisms and cellular processes involved in the egg’s establishment of the membrane block. This research tested three distinct hypotheses: (1) ZP-free ovulated eggs would differ in the extent of polyspermy from ZP-free in vitro matured eggs, (2) the Src family kinase Fyn plays a role in membrane block, and (3) endocytosis plays a role in the membrane block. Direct, side-by-side comparisons of sperm incorporation during in vitro fertilization (IVF) of ZP-free ovulated and in vitro matured eggs reveal a slight trend of in vitro matured eggs toward greater extents of polyspermy over increased time post-insemination, possibly indicative of a less robust membrane block. Loss of Fyn kinase activity has been linked to the disruption of actin-associated events in the mouse oocyte and egg, providing rationale to study the effect of Fyn kinase inhibition using pharmacological inhibition (SKI-606) on the membrane block. Studies here reveal a modest increase in polyspermy in ZP eggs treated with SKI-606, setting the stage for additional studies to examine more closely if Fyn kinase functions in the membrane block. Lastly, endocytosis can result in membrane remodeling and thus could play role in

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internalizing receptors necessary for maintained egg receptivity to sperm. Experiments here used two different pharmacological inhibitors of endocytosis (dynasore and monodansylcadaverine [MDC]) and revealed statistically higher extents of polyspermy in MDC-treated eggs. These data lend support to the hypothesis that endocytosis is involved in the membrane block to polyspermy.

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ACKNOWLEDGMENTS I share each of my successes with those who have encouraged me along the way. In particular, I dedicate this thesis to my aunt, Donna Monseur. Reflecting on the journey that led me to public health, it seems inevitable that I reached this point. From a young age, Donna fostered the intellectual curiosity that continues to define an integral part of the way I experience the world around me. It was because of her selflessness that I was able to immerse myself in a variety of educational opportunities. These endeavors provided an escape from the marital discord that plagued my childhood and helped sustain the fortitude necessary to cope with the loss of my great aunt, Mae (Tiggy). The unfortunate diagnosis of a neuromuscular disease forced Donna to leave her master’s program, just a few credits short of completion. As she grew stronger, I believe that she sacrificed her return to graduate school in order to nurture the dreams of those around her. Her constant reaffirmation dared me to continue believing that I had what it took to overcome any adversity. The completion of this thesis marks the fulfillment of my degree requirements for a Master of Science. Donna, this is your master’s degree, too, and I am honored to share the accomplishment with you. Of course, Donna has not been the only inspiration throughout this master’s program. Janice Evans, your enthusiasm for science is contagious. As much as I love fertilization biology, I deferred medical school because I wanted to work with you. With your mentorship, I have grown to appreciate and better understand the scientific process and the critical thinking behind experimental design. The skills I’ve gained will be instrumental in my future as both a clinician and a researcher and for that, I thank you. Oh, and rest assured, “the Gordon” hasn’t seen the last of me!

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And then there’s my 6 A.M. “gammers” partner and science-sister, Lauren “McG” McGinnis. Where would I’ve been without an early morning Britney Spears dance partner and hospital breakfast buddy!? I truly can’t imagine my Sc.M. year without you by my side. Your work ethic and positive attitude kept me afloat even after the worst collections. I wish you all the best and hope you will continue to stay in touch. No matter what, I could always count on Hyo “Hyocyte” Lee for positive reassurance and ideas to maintain solidarity in the laboratory. Her commitment to other lab mates and teamwork is admirable and doesn't go unnoticed. Our trip to H Mart for seaweed crisps and ingredients for the elusive purple rice will live on in Evans lab history; I entrust her with the care of the new lab rice cooker. I feel very fortunate to have also had the opportunity to work alongside Mindy Christianson during my Sc.M. year. Attending to clinical, research, and familial responsibilities is a testament to her ability to maintain a healthy work-life balance. I admire Mindy as a role model and hope to one day emulate her qualities. I greatly appreciate all of her personal and professional advice as well as her encouragement to pursue a career in reproductive endocrinology and infertility. I would also like to express my gratitude to Adam Farra for providing unwavering emotional support during the past year and a half. With his influence, I continue to grow more and more into the man I hope to become. Lastly, this thesis project would not have been possible without the comments and guidance of my thesis readers: Terry Brown, Sabra Klein, and Anne Burke. Each of you left memorable impressions during my academic career at the JHSPH and I am honored that you participated in the successful completion of this thesis.

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TABLE OF CONTENTS ABSTRACT ....................................................................................................................... ii ACKNOWLEDGMENTS ............................................................................................... iv TABLE OF CONTENTS ................................................................................................ vi TABLE OF FIGURES.................................................................................................... vii LITERATURE REVIEW ................................................................................................ 1 MATERIALS & METHODS ........................................................................................ 27 RESULTS ........................................................................................................................ 34 DISCUSSION .................................................................................................................. 42 REFERENCES................................................................................................................ 80 CURRICULUM VITAE................................................................................................. 99

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TABLE OF FIGURES Figure 1: Scoring Eggs for Fused Sperm: Monospermy vs. Polyspermy ........................ 53 Figure 2: Schematic diagram of the steps of fertilization ................................................ 55 Figure 3: Diagrammatic illustration of normal membrane block establishment and perturbed membrane block establishment using the sperm incorporation over time assay ........................................................................................................................... 57 Figure 4: Diagrammatic illustration of normal membrane block establishment and perturbed membrane block establishment using the reinsemination assay ................ 59 Figure 5: Sperm Incorporation Over Time in Ovulated vs. In Vitro Matured Eggs ........ 61 Figure 6: Sperm incorporation over time in eggs treated with 25 µM of the Src-family kinase inhibitor, SKI-606 ........................................................................................... 63 Figure 7: Analysis of the extent of sperm-egg fusion (average number of sperm fused per egg) at 3 hours post-insemination in control and SKI-606 treated eggs .................... 65 Figure 8: Sperm incorporation over time in eggs treated with the dynamin inhibitor, dynasore ..................................................................................................................... 67 Figure 9: Analysis of the extent of sperm-egg fusion (average number of sperm fused per egg) at 3 hours post-insemination in control, 40 µM dynasore, and 60 µM dynasore treated eggs ................................................................................................. 69 Figure 10: Sperm incorporation over time in eggs treated with the endocytosis inhibitor, monodansylcadaverine (MDC) .................................................................................. 70 Figure 11: Analysis of the extent of sperm-egg fusion (average number of sperm fused per egg) at 3 hours post-insemination in control, 5 µM MDC, and 20 µM MDC treated eggs ................................................................................................................. 73

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Figure 12: Labeling sperm with Alexa Fluor succinimidyl esters fluorescent dyes ........ 75 Figure 13: Reinsemination of eggs treated with the endocytosis inhibitor, monodansylcadaverine (MDC) .................................................................................. 77

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LITERATURE REVIEW A. Introduction Understanding the molecular basis for fertilization presents multiple opportunities for public health researchers to advance reproductive health: to explore new ideas for contraceptive technology and to develop new tools for the prevention and treatment of infertility. The Guttmacher Institute reports that almost half (3.2 million) of all U.S. births are unintended (http://www.guttmacher.org/pubs/FB-Unintended-PregnancyUS.html, 2012). Development of contraceptives with specific molecular targets could have fewer side effects than hormonal contraception and play a role in decreasing the risk of unintended pregnancy (Natraj 2001). Another large proportion of Americans, approximately 2 million couples, suffer from infertility, an inability to conceive after 12 months of unprotected sex (Macaluso et al., 2010). As the burgeoning field of assisted reproductive technologies (ART) continues to make advances, there are even more unanswered questions regarding the consequences of disrupting the natural physiological process of fertilization (Florman and Ducibella, 2006). According to the Centers for Disease Control and Prevention’s (CDC’s) National ART Surveillance System (NASS), over 60,000 births in 2009 were due to the use of ART, approximately 1% of the entire U.S. birth cohort (http://www.cdc.gov/ reproductivehealth/drh/activities/art.htm, 2012). The European Society of Human Reproduction and Embryology (ESHRE) estimates that approximately 3.75 million babies worldwide have been born using ART (http://www.eshre.eu/ESHRE/ English/Guidelines-Legal/ART-fact-sheet/page.aspx/1061, 2012). One public health concern is recurrent pregnancy loss, or the repeat occurrence of

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spontaneous abortion. Of note for this thesis research topic, triploidy is diagnosed in ~10% of early spontaneous abortions (Jacobs et al., 1978; McFadden and Langlois, 2000). While the cause of triploidy is often debated, most triploidies are thought to be due to fertilization by more than one sperm (polyspermic fertilization; Jacobs et al., 1978; Hassold et al., 1980; Michelmann et al. 1986, McFadden and Langlois, 2000; Gardner and Evans, 2006; Fig. 1). Triploid embryos typically die early in embryogenesis, which may limit clinical detection of this condition, particularly for cases of early pregnancy loss (Hassold et al., 1980). Elucidating molecular mechanisms underlying cellular events associated with fertilization, including the prevention of polyspermic fertilization, should remain a priority in the fields of medicine and public health. Further research can help ensure a continuation of the availability of safe, reproductive health services to enhance chances of reproductive success. This Literature Review will discuss a series of events, collectively termed the egg-to-embryo transition, that occur as a result of sperm-egg fusion. The primary focus will be on one of two events that attempt to ensure fertilization by one and only one sperm, thereby preventing polyspermic fertilization. These “blocks to polyspermy” occur at two levels: the egg’s external coat, the zona pellucida (ZP), and the egg’s plasma membrane (Fig. 2). The latter is the focus of this thesis project. It is estimated that even with these blocks in place, 1-3% of conceptuses are reported to have polyspermy (Boué et al., 1975; Golbus 1981). While these risks of polyspermy are of concern to the general population at risk of infertility and pregnancy loss, ART uses artificial conditions that expose eggs to higher concentrations of sperm than the in normal physiological environment in the oviduct. Reports of polyspermy as a

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result of conventional IVF range from 2-9%, but certain protocol adjustments can result in percentages as high as 18% (Xiong et al., 2011). These findings further stress the need to enhance the understanding of the mechanisms of preventing polyspermic fertilization, particularly at the level of the egg’s plasma membrane, which still remains an enigmatic process in mammalian species. B. Egg-to-Embryo Transition Following fertilization, a series of events known as “egg activation” take place that constitute the egg-to-embryo transition. The events following sperm entry into the egg cytoplasm include cortical granule exocytosis, establishment of the blocks to polyspermy, and the onset of paternal chromatin decondensation. Other events include completion of maternal meiosis, progression into embryonic interphase (i.e., pronuclear formation), and the recruitment of maternal mRNAs for the translation of necessary proteins (Florman and Ducibella 2006). Intracellular calcium release and oscillations provide the basic mechanism for the cellular events necessary to convert the egg into an embryo (Ducibella et al., 2002; Jaffe 1985; Runft et al., 2002). B1. Post-Fertilization Role of Calcium Release and Oscillations Fertilization is known to induce a large, prolonged calcium signal followed by smaller transient increases (also known as oscillations) (Runft et al., 2002; Ducibella et al., 2002; Malcuit et al., 2006). These increases in cytosolic calcium concentrations are a downstream event from the production of inositol triphosphate (IP3) catalyzed by the sperm-specific phospholipase, phospholipase C zeta (PLCζ) (Swann et al., 2006; Knott et al., 2005). IP3 interacts with its receptor, IP3-R1, on the endoplasmic reticulum where calcium is stored, which then leads to release of calcium ions from the endoplasmic

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reticulum into the cytoplasm (Kurokawa et al., 2004; Malcuit et al., 2006). Significantly, while the first calcium transient can initiate the events of egg activation, the completion of events is dependent on the continuation of these transients, which occur in a distinct spatiotemporal fashion (Ducibella et al., 2002). By mimicking calcium oscillations using electric field (EF) pulses, it has been demonstrated that the quantity of pulses differentially initiates the various cellular events involved in egg activation (Ducibella et al., 2002). Unlike other post-fertilization egg activation events where calcium transients are both necessary and sufficient, the establishment of the membrane block is only partly dependent on calcium. While delayed, the membrane block does establish in eggs with attenuated or completely suppressed calcium transients (Gardner et al., 2007b). Additional stimuli associated with the membrane block establishment are thought to be dependent on some other fertilization event(s) (Gardner et al., 2007b). B2. Cell Cycle Progression and Resumption of Meiosis Fertilization of metaphase II-arrested eggs typically occurs in the oviduct and induces a completion of meiosis after sperm fusion (Ducibella and Florman 2006). Calcium signal transduction occurs through the calmodulin (CaM)-dependent protein kinase II (CaMKII) pathway, which has been shown to oscillate concurrently with sperminduced calcium transients in fertilized eggs (Markoulaki et al., 2004). Injection of eggs with constitutively active CaMKII (CA-CaMKII) has also been demonstrated to cause meiotic resumption but not trigger sperm-induced calcium transients implicating CaMKII activity downstream of sperm-induced calcium increase (Knott et al., 2006; Madgwick et al., 2005). Building on this finding, studies have shown that even in the presence of normal calcium oscillations, CaMKIIγ knock down or targeted deletion of Camk2g in

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eggs results in failed meiotic resumption after fertilization. (Chang et al., 2009; Backs et al., 2009). As previously mentioned, the majority of cases of triploidy occur due to fertilization by two sperm (dispermy); however, it is worth noting that polyploidy in embryos can also occur as a result of errors during completion of female meiosis (Jacobs et al., 1978; Hassold et al., 1980; Michelmann et al. 1986, McFadden and Langlois, 2000; Gardner and Evans, 2006). C. Polyspermy In order to prevent the fertilization by more than one sperm, the female reproductive tract has evolved barriers to prevent low quality sperm from entering the cervix. Of the millions of sperm ejaculated into the vagina, only a few thousand will travel through the anatomically complex uterotubal junctions to reach the oviducts where fertilization ultimately occurs (Suarez and Pacey, 2006). Still, as noted above, 1-3% of eggs fertilized in vivo will be fertilized by more than one sperm (Boué et al., 1975; Golbus 1981), underscoring the importance of the egg's own levels of protection to prevent fertilization by more than one sperm: the zona pellucia (ZP) block to polyspermy and the membrane block to polyspermy (Fig. 2). There is variability between mammalian species in which of these two egg-based blocks to polyspermy is more heavily utilized to prevent polyspermic fertilization. This is based on studies examining the numbers of sperm that gain access to the space in between the egg plasma membrane and the zona pellucida, also known as the perivitelline space (PVS; Fig. 2). These numbers of sperm in the PVS differ across mammalian species and are related to which block to polyspermy is utilized. In species with high levels of sperm in the PVS such as the rabbit or mole, the ZP block is less effective than

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the membrane block. Conversely, species such as the dog or sheep with a rare occurrence of sperm in the PVS, have a very effective ZP block; experiments have not been done with these species to ascertain if they also use a membrane block. Mice and humans fall into a category of intermediate levels of sperm in PVS, which make the mouse model appropriate for the study of the prevention of polyspermic fertilization. In these species, the use of both the ZP block and the membrane block are observed (Ducibella and Florman 2006; Lewis and Wright 1935; Odor and Blandau 1949; Austin 1961; Hunter 1994; Wolf et al., 1981; Hunter et al., 1998). C1. The Zona Pellucida (ZP) Block to Polyspermy The zona pellucida (ZP) block to polyspermy occurs over the course of several hours following fertilization to prevent additional sperm from binding to the zona pellucida (Inoue and Wolf 1975, Baibakov et al., 2007). The mouse ZP is comprised of three glycoproteins (ZP1, ZP2, ZP3), which interact to form an extracellular matrix around the egg (Bleil and Wassarman, 1980). By contrast, the human ZP is comprised of four glycoproteins (ZP1, ZP2, ZP3, ZP4) (Florman and Ducibella 2006). The establishment of the ZP block has been linked to the exocytosis of cortical granules, which are Golgi-derived, membrane-bound vesicles lying just underneath the plasma membrane (Barros and Yanagimachi 1971; Wolf 1974; Jaffe and Gould 1985; Hedrick and Hardy 1991; Gadella and Evans 2011; Fig. 2). As stated earlier, the initiation of the exocytosis of cortical granules is one of the earliest events of egg activation and is dependent on calcium oscillations (Abbott and Ducibella 2001). While the contents of cortical granules are presently being characterized, it has been concluded that a factor released induces the cleavage of ZP2 to its post-fertilization form ZP2f, which results in a

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conversion of the ZP, making it unreceptive to further sperm binding; this is also referred to as "hardening" of the ZP, since it makes the ZP more resistant to protease digestion in in vitro assays (Hoodbhoy and Talbot 1994, 2001; Gwatkin et al., 1973; Wolf and Hamada 1977; Visconti and Florman 2010). Recent work suggests that an oocyteenriched metalloendoprotease, ovastacin, is responsible for the proteolytic cleavage of ZP2 to ZP2f, though it should be noted that another group reported that this protein (also known as SAS1B) is a candidate-binding partner for a sperm acrosomal ligand (Burkart et al., 2012; Sachdev et al., 2012). The conversion ZP3 to a post-fertilization form has also been reported; it has been hypothesized that the conversion of ZP3 to ZP3f is due to a modification in an O-glycan, although there is no direct biochemical evidence to support this (Visconti and Florman 2010). While still being debated, current models of sperm-ZP binding suggest that the converted ZP3f form may interfere with initial spermZP binding and that ZP2f prevents sperm-ZP penetration (Visconti and Florman 2010). C2. Membrane Block to Polyspermy As mentioned in the introduction above (Section C), evidence of supernumerary sperm in the perivitelline space provides the evidence of a membrane block to polyspermy (Lewis and Wright 1935; Odor and Blandau 1949; Austin 1961; Hunter 1994; Wolf et al., 1981; Hunter et al., 1998; Gadella and Evans 2011). Notably, these sperm in the PVS appear to be unable to fuse with the plasma membrane of the egg. ZPfree eggs inseminated with sperm have demonstrated a plateau in the number of fertilizing sperm in a matter of hours (Wolf 1978; Binor et al, 1982; McAvey et al., 2002). This supports the notion that the egg’s membrane changes its receptivity to sperm over time. Additionally, ZP-free, already fertilized eggs, typically do not get fertilized if

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exposed to a second batch of sperm, which suggest that the egg membrane is no longer supportive of sperm-egg fusion (Wolf 1978; Zuccotti et al., 1991; Horvath et al., 1993; Maluchnik and Boruk 1994; Sengoku et al., 1995). Unlike in mammals, the membrane block in non-mammalian species (e.g., sea urchins and frogs) involves a depolarization of the egg plasma membrane, which has not been observed in various mammalian models (Jaffe and Gould 1985; Miyazaki and Igusa 1981; Igusa et al. 1983; Jaffe et al., 1983; McCulloh et al., 1983; Gardner and Evans 2006). While biochemical differences in the ZP before and after fertilization have been detected and characterized, the difference(s) between the zygote membrane and the egg membrane that could prevent additional sperm from binding or fusing are less obvious (Gadella and Evans 2011). Some work has investigated membrane lipids and protein diffusion post-fertilization; however, more studies need to be performed to functionally link these observations to the membrane block (Wolf et al., 1981; Wolf and Ziomek 1983). Mechanical differences have also been documented showing that the cortical tension is higher in zygotes than in unfertilized eggs; however, this, too, needs to be further investigated to demonstrate a direct link to the membrane block (Larson et al., 2010). Interestingly, observed tension measures in immature, prophase I oocytes, which are more prone to polyspermy, were even higher than that of metaphase II eggs (Larson et al., 2010, Kryzak, ScM Thesis, 2008). This may suggest a possible link between cortical tension and the membrane block; successful membrane block establishment would not merely be dependent on high or low tension, but rather certain ranges of tension would be more effective than others.

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Still, fundamental pieces of the understanding of the establishment of the membrane block have been uncovered. Studies in which ZP-free, fertilized eggs are exposed to sperm at various time points post insemination demonstrate that the ability of additional sperm to penetrate already-fertilized eggs declines with increasing time after the insemination with the first batch of sperm, indicating that the membrane block is established by 1-1.5 hours post-insemination (Wolf et al., 1978; Gardner et al., 2007b). This time frame is roughly similar to the time it takes to establish the ZP block (0.5-1 hr) (Gardner and Evans 2006). Relationships between the membrane block establishment and calcium signaling, parthenogenesis, sperm, and actin have also been investigated; these studies will be reviewed below. C2.a Calcium and the membrane block to polyspermy As noted above (Section C), calcium plays a role in initiating the series of events referred to as egg activation, among which is establishment of the membrane block. A common experimental approach used to study intracellular calcium signaling is use of the cell-permeable calcium chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethaneN,N,N’N’-tetra-acetic acid acetoxymethyl ester). This calcium chelator can be loaded into cells, and suppress downstream calcium signaling events by binding calcium ions in the cytoplasm, thus preventing the calcium ions from interacting with Ca2+ effector molecules. Eggs that are loaded with 5-10 µM BAPTA-AM and then fertilized show no sperm-induced calcium oscillations, and as a result, do not undergo cortical granule exocytosis, do not emit a second polar body, and do not resume meiosis (Kline and Kline 1992). These data suggest that an increase in calcium is necessary and sufficient for these various post-fertilization events; however, these studies did not investigate the membrane

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block to polyspermy. Based on these observations related to other egg activation events, the hypothesis that increases in cytosolic calcium would also be necessary for successful membrane block establishment was tested by examining levels of polyspermy observed in BAPTA-AM-treated eggs. In these studies, the extent of sperm-egg fusion (average number of sperm fused per egg) at various time points post insemination was examined (this experimental design, which is also used in this thesis research, is referred to as assessing sperm incorporation over time; Fig. 3; McAvey et al., 2002). This work revealed that BAPTA-AM-treated eggs yielded significantly higher extents of polyspermy and thus supported a role for calcium increase in another post-fertilization event, the membrane block to polyspermy (McAvey et al., 2002). Based on these results of complete suppression of calcium transients (McAvey et al., 2002) as well as earlier findings suggesting that calcium levels differentially initiated certain post-fertilization events (Ducibella et al., 2002), it was unclear whether partial suppression of calcium would have an effect on membrane block establishment. Since treatment of ZP-free eggs with 10 µM of BAPTA-AM results in no calcium oscillations, additional experiments were performed looking at the effects of varying concentrations of BAPTA-AM (0.5, 1, 2, and 5 µM; Gardner et al., 2007b). These lower concentrations of BAPTA-AM reduce the frequency and amplitude of calcium transients. Even in the presence of attenuated calcium transients, increasingly higher levels of average numbers of sperm fused per egg are observed for each of the increasing BAPTA-AM doses used (Gardner et al., 2007b). This dose responsiveness suggests that the membrane block is not simply a digital switch from an unreceptive versus a receptive state, and that the establishment is a dynamic process associated with the extent of calcium signaling.

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While these data support the role of calcium signaling in membrane block establishment, they do not provide information on whether the increased extents of polyspermy are, in fact, due to a defect in the membrane block and, if so, how the block is perturbed by reduced calcium signaling. In order to assess calcium’s role, BAPTAAM-treated fertilized eggs (zygotes) were created and then allowed to recover in the absence of sperm so that the membrane block was given adequate time to establish, then exposed to additional sperm. The expectation for these experiments is that fertilization by these additional sperm will be unsuccessful if the membrane block has established. Conversely, if BAPTA-AM perturbs the membrane block, then additional sperm would be expected to fertilize the zygote (reinsemination assay; Fig. 4). BAPTA-AM-treated eggs that were allowed to recover for 0.75 hr after the first insemination incorporated an average number of sperm similar to sperm penetration with unfertilized, naïve eggs; this result is consistent with a perturbed membrane block. However, BAPTA-AM-treated eggs that recover for 1.5 hr showed significantly reduced amount of sperm penetration, which is consistent with an established membrane block. These observations suggest that calcium’s role in the membrane block is related to the timing of its establishment. By delaying the establishment of the membrane block, sperm were allowed additional time to fuse with the egg resulting in the higher averages of sperm fused per egg (Gardner et al., 2007b). As previously mentioned (Section B.2), CaMKII activity is strongly correlated with the calcium oscillations seen in eggs post fertilization (Markoulaki et al., 2004). Additionally, experiments demonstrated that expression of constitutively active CaMKII (CA-CaMKII) in eggs does not cause calcium oscillations, but does induce cycle

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resumption (Madgwick et al., 2005; Knott et al., 2006). Studies that knocked down CaMKIIγ in eggs further demonstrated CaMKII’s role in cell cycle progression since these eggs were unable resume meiosis even in the presence of a fertilizing sperm (Chang et al., 2009). The evidence for a role of CaMKII as a downstream calcium effector in egg activation (Madgwick et al., 2005; Knott et al., 2006; Chang et al., 2009; Backs et al., 2009) provided the foundation for examining whether CaMKII played a role in the establishment of the membrane block to polyspermy. Two approaches were performed to test this hypothesis by assessing the extent of polyspermy. One approach involved a reinsemination assay (Fig. 4), taking early embryos that had been treated with the CaMKII inhibitor myristoylated autocamtide-2 related inhibitory peptide (myrAIP) and challenging them with additional sperm to determine if these sperm can fertilize. A second approach involved parthenogenetically activating eggs by injecting them with CA-CaMKII-encoding mRNA, and then challenging these activated egg with sperm (Gardner et al., 2007a). If inhibition of CaMKII activity with myrAIP perturbed the membrane block, myrAIP-treated eggs would be expected to have high extents of polyspermy. Conversely, if CaMKII is sufficient, then injection with CaMKII would cause eggs to establish the membrane block to polyspermy and thus prevent high extents of polyspermy. While CA-CaMKII was not sufficient to establish the membrane block, inhibition of CaMKII with myrAIP resulted in eggs with a partially effective membrane block (Gardner et al., 2007a). These findings suggest that while CaMKII activity participates in the successful establishment of the membrane block, it is not sufficient to induce the membrane block.

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Suppression of calcium oscillation in fertilized eggs provides evidence that calcium is necessary for normal membrane block establishment (McAvey et al., 2002). To address whether calcium is sufficient to establish the membrane block, studies with parthenogenetically activated eggs were used. Parthenogenetic activation refers to egg activation in the absence of fertilization by sperm through treatment of eggs with agents such as SrCl2 or calcium ionophore that cause parthenogenetic increases in cytosolic calcium; these treatments result in cortical granule exocytosis and cell cycle resumption (Gardner and Evans 2006). Considering that parthenogenetic activation can induce egg activation events that mimic those occurring with fertilization, it was hypothesized that the parthenogenetic increase in calcium could cause the establishment of the membrane block. Treating eggs with parthenogenetic agents; however, did not result in a mounting of the membrane block to polyspermy (Wolf et al., 1979; Horvath et al., 1993; Wortzman-Show et al., 2007; Gardner et al., 2007a). These data suggest that the membrane block to polyspermy is distinct from other events of egg activation in that calcium stimulation alone will not cause its establishment, rather that another component of fertilization is necessary. Since sperm induce calcium oscillations, the additional component could be the sperm-egg fusion event or some factor(s) deposited by the sperm during fertilization. C2.b Sperm and the membrane block to polyspermy Eggs that have been injected with sperm into their cytoplasm (intracytoplasmic sperm injection) are known to undergo cortical granule exocytosis and meiotic resumption (Madgwick et al., 2005; Knott et al., 2006; Gardner et al., 2007a; Bedford et al., 2004; Yangimachi 2005), but early studies suggested that eggs post-ICSI did not

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induce establishment of the membrane block (Maleszewski et al., 1996; Sengoku et al. 1999). Since calcium appears to be necessary, but not sufficient, for normal membrane block establishment, studies with ICSI were optimized to induce fertilization-like calcium transients (Wortzman-Show et al., 2007). A lysate of sperm proteins (also known as sperm factor) contains the sperm-specific PLCz (Section B1), and when injected into eggs, can recapitulate fertilization-induced calcium transients and cause parthenogenetic egg activation. Sperm factor was microinjected into eggs and extents of polyspermy assessed after exposure to sperm; if this injection caused the membrane block to establish, then sperm would be unsuccessful in fertilizing eggs. Instead, eggs microinjected with sperm factor were fertilized to extents comparable to unfertilized eggs (Wortzman-Show et al., 2007). This suggests that the contents of sperm, while able to induce calcium transients with similar spatio-temporal characteristics as seen in normal fertilization, are not sufficient to cause a mounting of the membrane block (Wu et al., 1998a, 1998b, Oda et al, 1999; Wortzman-Show et al., 2007). These results indicate that some other event associated with fertilization, not occurring during ICSI, may be necessary for membrane block establishment. Since eggs undergoing ICSI bypass spermegg binding and sperm-egg fusion, it is possible that one of these steps induces certain signaling cascades important for the membrane block (Wortzman-Show et al., 2007). Since sperm-egg binding, but not sperm-egg fusion, can occur in calcium-deficient medium, this provided an opportunity to study sperm-egg binding in conjunction with injection of sperm factor (Miyamoto and Ishibashi, 1975; Yanagimachi, 1978; Fraser, 1987; Fujimoto et al., 1994; Evans et al., 1995; Wortzman-Show et al., 2007). However, the additional stimulus of sperm-egg binding did not result in an establishment of the

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membrane block as evidenced by similar extents of fertilization as seen in unfertilized eggs. These data suggest that there may still be other differences that arise as a result of ICSI versus conventional IVF that could affect the membrane block, one being the remodeling of the cortical cytoskeleton (Wortzman-Show et al., 2007). Interestingly, another difference observed in eggs fertilized by ICSI when compared to conventional IVF is a delay in the formation of an actin-rich fertilization cone that lies over the sperm DNA (Wortzman-Show et al., 2007; see also below, Section C2.c). C2.c Egg actin and the membrane block to polyspermy Fertilization is known to induce a remodeling of the egg’s cortical cytoskeleton, which results in an actin-rich area over the sperm DNA, known as the fertilization cone (Lopata et al. 1980; Brunet and Maro, 2005). Evidence for a link between actin and the membrane block to polyspermy comes from increased extents of polyspermy observed in eggs treated with a particular actin-disrupting drug, cytochalasin D (McAvey et al., 2002). These experiments were based on data that microfilament disruptors had adversely affected other fertilization events such as spindle rotation, polar body emission, fertilization cone formation, sperm tail incorporation, and pronuclear migration (Longo 1978; Maro et al., 1984; Schatten et al., 1986, 1989; Le Guen et al., 1989; Sutkovsky et al., 1996; Terada et al., 2000; Sun et al., 2001; McAvey et al., 2002). After treatment with various actin-disrupting drugs (cytochalasin B, cytochalasin C, cytochalasin D, jasplakinolide, and latrunculin B), only cytochalasin D was shown to increase extents of polyspermy, which was attributed to mechanistic differences in how disruption occurs (McAvey et al., 2002). Unlike the other actin-disrupting drugs, cytochalasin D prevents actin polymerization by binding to the growing microfilament and preventing assembly

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(McAvey et al., 2002). Another observation was a dose-dependent effect of cytochalasin D where only lower doses of drug (2.5 and 5 µg/ml) caused an increase in polyspermy while a higher concentration (10 µg/ml) did not. Notably, treatment with cytochalasin D did not disrupt calcium oscillations previously reported to be necessary for membrane block establishment (McAvey et al., 2002). These data suggest a function for actin in the establishment of the membrane block to polyspermy. D. Genetic mouse models with abnormalities in the extent of polyspermy Studies of mouse knockout models do not always report subfertile phenotypes; however, some studies of particular genetic mouse models have provided insight into reproductive dysfunction, and noted that eggs were observed to have increased extents of polyspermy. This section will review reports of two knockout mice with subfertile female phenotypes, as well as two knockout models investigating if sperm would generate embryos with increased extents of polyspermy. This section will highlight work on the function of the SRC-family kinases, in particular Fyn kinase, for its potential role in the prevention of polyspermic fertilization. D1. Ubiquitin C-terminal hydrolase (UCH-L1) Ubiquitin C-terminal hydrolase L1 (UCH-L1) is an enzyme involved in the pathway of post-translation modification of ubiquitination (also called ubiquitylation). Ubiquitination is the conjugation of a small protein known as ubiquitin to substrate proteins. This in turn modifies the fates and/or functionality of substrate proteins, including targeting the substrate protein for degradation by the 26S proteasome, affecting the localization or binding partners of the substrate protein, or changing the activity of the substrate protein. Ubiquitination can also be a signal for internalization and endocytic

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sorting of membrane proteins (Acconia et al., 2009); this is of note because endocytosis in eggs was one mechanism examined in this thesis research as a cellular process involved in the membrane block to polyspermy (see below, Section E). Ubiquitination is mediated by a three-step process involving three enzymes, culminating in the conjugation of ubiquitin to its substrate. Ubiquination can be reversed through a process known as deubiquitination, mediated by deubiquitinating enzymes. UCH-L1 is one of these deubiquitinating enzymes (Sekiguchi et al., 2006). UCH-L1 became of interest to reproductive biology based on the findings that the protein was only expressed in neurons, testis, ovary, and placenta (Sekiguchi et al., 2006). Furthermore, various studies reported a role for the ubiquitin-proteasome system (UPS) in the degradation of various proteins during gametogenesis and fertilization (Baarends et al., 1999; Sutovsky et al., 2003; Sutovsky et al., 2004). More recent studies demonstrated UCH-L1 localization underlying the egg plasma membrane and reported increased polyspermy in Uchl1-deficient mice (Sekiguchi et al., 2006). Based on these findings, it was concluded that UCH-L1 could play a role in the membrane block to polyspermy. However, ZP-intact eggs were used in these experiments, and without using ZP-free eggs, increased extents of polyspermy may be due to disruption of the ZP block and/or the membrane block to polyspermy (Sekiguchi et al., 2006). Recent data have complemented the work with eggs from the Uchl1-deficient mice with studies using a competitive, cell permeable UCH-L1 inhibitor, LDN-57444 (also known as C30) to treat oocytes (Susor et al., 2010; Gerolstein, ScM thesis, 2011). Bovine, prophase I oocytes were treated with LDN-57444 during maturation to metaphase II and then washed free of inhibitor prior to insemination (Susor et al., 2010).

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These eggs showed higher extents of polyspermy as well as an impairment in cortical granule migration to the egg cortex (Susor et al., 2010). It was proposed that this impairment in cortical granule migration is linked to impaired cortical granule exocytosis and, as a result, a defect in the establishment of the ZP block to polyspermy (Susor et al., 2010). While chronic depletion of UCHL-1 resulted in increased polyspermy as shown in the Uchl1 deficient mice (Sekiguchi et al., 2006), this study of bovine oocytes using pharmacological inhibition during meiotic maturation demonstrated that acute depletion of UCHL-1 also resulted in higher extents of polyspermic fertilization (Susor et al., 2010). Based on these results, it is possible that the increase in polyspermy is due to a disruption of normal meiotic maturation from prophase I to metaphase II and not specific to a perturbation in one or both of the blocks to polyspermy. Building on these observations, ZP-free metaphase II eggs treated with either 5 or 10 µM LDN-57444 immediately prior to and during insemination showed higher extents of polyspermy, which supports the hypothesis that UCH-L1 is implicated in the membrane block to polyspermy (Gerolstein, ScM thesis, 2011). This study more precisely defined the time window when UCH-L1 activity may be important for successful establishment of the membrane block to polyspermy. D2. A Disintegrin and A Metalloprotease 24 (ADAM24) ADAMs (A Disintegrin and A Metalloproteases), a family of transmembrane proteins with a metalloprotease and a disintegrin domain, have been implicated in having both enzymatic activity and adhesive functions, specifically in spermatogenesis and fertilization (Zhu et al., 2009). ADAM24 (also known as testase 1) is a protein on the surface of mouse sperm, and Adam24-null mice have a subfertile phenotype (Zhu et al.,

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2009). Additionally, sperm from these Adam24-null mice were shown to generate polyspermic embryos in IVF assays using ZP-free eggs by 40 minutes post-insemination (Zhu et al., 2009). No further studies, such as examining sperm incorporation over time with wild-type and Adam24-null sperm, have examined the role of ADAM24 in the membrane block to polyspermy. Additionally, it is unclear if Adam24-null sperm are more efficient at fertilization resulting in higher extents of polyspermy unrelated to a defect in the membrane block (Zhu et al., 2009). Thus, it is difficult to conclude if ADAM24 on the sperm plays a role in triggering the egg's establishment of the membrane block. D3. Tyrosylprotein sulfotransferase 2 (Tpst2) Tyrosine O-sulfation is a type of post-translational modification involving two tyrosylprotein sulfotransferases (TPST-1 and TPST-2). Tpst2-null females have normal fertility, but Tpst2-null males are infertile (Borghei et al., 2006). IVF studies using ZPfree wild-type eggs and sperm from Tpst2-null males revealed that embryos generated by Tpst2-null sperm have increased extents of polyspermy as compared embryos generated sperm of a wild-type male (Marcello et al., 2011). To address whether this increased polyspermy was due to a defect in the establishment of the membrane block to polyspermy, a re-insemination assay where fertilized eggs (zygotes) were exposed to a second batch of sperm was performed (Fig. 4). If the Tspt2-null sperm caused a mounting of the membrane block, then sperm from the second batch should not be able to fertilize the zygote. The results of this assay indicated that sperm from Tpst-2-null males were, in fact, able to establish the membrane block since 84% of the zygotes created with Tpst2null did not show penetration by this second batch of sperm, indicating that fertilization

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by Tpst2-null sperm had induced membrane block establishment (Marcello et al., 2011). These findings suggest that the increased extents of polyspermy may be due to the fact that Tpst2-null sperm are more efficient at sperm-egg fusion and not linked to perturbation in the establishment of the membrane block. D4. Fyn Kinase Fyn kinase is a member of the Src-family protein tyrosine kinases (PTKs), which are cytosolic kinases containing Src homology 2 (SH2) and Src homology 3 (SH3) domains. These protein interaction domains, coupled with an N-terminal fatty acid acylation site, make this PTK family candidate molecules for various signaling cascades, including cell surface receptors and actin-based cytoskeleton changes (Luo et al., 2009). Fyn-/- mice were originally characterized to have deficiencies in memory and learning and have normal fertility (Grant et al., 1992; Stein et al., 1994), but more recently shown to have various defects related to female fertility, including impaired oocyte maturation, reduced developmental potential of eggs, and smaller litter size (McGinnis et al., 2009; Luo et al., 2009). Among the Src-family PTKS, Fyn kinase was shown to have the highest levels of expression in MII oocytes (Luo et al., 2009). In spite of this, little was known regarding Fyn kinase function in oocytes until relatively recently. Fyn knock outs as well as inhibition with Src-family kinase pharmacological inhibitor, SKI-606 (also known as Bosutinib) were performed to assess the effects of inhibition of the Src-family PTK activity in wild-type eggs prior to exposure to sperm (Luo et al., 2009). After pharmacological inhibition, there was a trend of increased extents of polyspermy in the SKI-606 treated eggs (23 ± 15.6% polyspermic embryos) compared to the control eggs (5

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± 3.3% polyspermic embryos; Luo et al. 2009). Since these experiments used ZP-intact eggs, it cannot be concluded whether or not the increased extent of polyspermy would be exacerbated in eggs once the zona pellucida was removed or if the trend in increased polyserpmy could be due to a defect in the membrane block (Luo et al., 2009) A series of experiments by the same group concluded that Fyn activity was required for normal morphology and distribution of microvilli on oocytes, establishment and maintenance of the cortical granule-free zone, and maintenance of the cortical actin filamentous network (Luo et al., 2009). Importantly, treatment with SKI-606 or in Fynnull eggs was noted to disrupt filamentous actin content seen in wild-type eggs (Luo et al., 2009). This trend towards increased polyspermy in SKI-606-treated eggs as well as the noted disruption of the actin cytoskeletal network role in the blocks to polyspermy (see Section C2.c) warranted further investigation into Fyn kinase’s role in regulating the membrane block to polyspermy (McAvey et al., 2002). E. Endocytosis in oocytes and eggs Endocytosis is a mechanism by which the plasma membrane of a cell can be changed and remodeled, through removal of surface proteins and trafficking of those to an endocytotic pathway. Once endocytosed, surface proteins may be recycled back to the membrane or degraded (Reiter & Lefkowitz, 2006; Moore et al., 2007). Internalization of a protein or complex of proteins through post-fertilization endocytosis could be a mechanism by which an egg converts its membrane from a receptive to an unreceptive state. This possibility was examined in this thesis research, and therefore, a review of what is known about endocytosis in oocytes and eggs will be presented here.

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Endocytosis occurs in the eggs of a variety of species, including echinoderms, amphibians, and mammals (Bernardi et al., 1987; Fisher et al., 1983; Kline and StewartSavage, 1994). Mouse oocytes have recently been shown to express dynamin 2 and the clathrin heavy chain, known components of the endocytic machinery (Lowther et al., 2011). Endocytosis occurs with fertilization, and may be, at least in part, a compensatory membrane retrieval process following cortical granule exocytosis (Fisher et al., 1987; Bement et al., 2000; Smith et al., 2000). Endocytosis also occurs in prophase I mouse oocytes, apparently with a role in regulating the amount of the surface-localized of the Gprotein-coupled receptor GPR3 (Lowther et al., 2011). GPR3 and a related protein GPR12 are important for maintaining levels of cyclic AMP and thus arrest of the oocyte in prophase of meiosis I (Mehlmann et al., 2004; Hinckley et al., 2005). Inhibition of endocytosis in prophase I oocytes through treatment of oocytes with pharmacological inhibitors of endocytosis, monodansylcadaverine (MDC) or dynasore, led to spontaneous exit from prophase I arrest (Lowther et al., 2011). These drugs block endocytosis through different mechanisms involved in this cellular process. Dynasore, a noncompetitive inhibitor, inhibits GTPase activity of dynamin, while MDC is thought to impair clathrin function by inhibiting transglutaminase (Schlegel et al., 1982; Macia et al., 2006). The inhibitory effects of these drugs on endocytosis in mouse oocytes were confirmed using a variety of assays for endocytosis (Lowther et al., 2011). The membrane dye FM 1-43 was used for a membrane uptake assay. FM 1-43 dye fluoresces after being incorporated into membranes, and comparison of plasma membrane to cytoplasmic fluorescence ratios of MDC-treated and untreated eggs revealed that MDC-treated eggs had less FM 1-43

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uptake (Lowther et al., 2011). Staining control, MDC-treated, and dynasore-treated oocytes with an antibody against the early endosome marker, early endosome antigen 1 (EEA1), showed that drug-treated oocytes had less EEA1 staining (Lowther, et al., 2011). It was thus concluded that endocytosis occurs at the plasma membrane in mouse oocytes and this process can be inhibited by either MDC or dynasore (Lowther et al., 2011). F. Background and Introduction for this thesis research This thesis research examined multiple aspects of egg biology relevant to the process of establishment of the membrane block to polyspermy. This research had three distinct parts. The first part of this thesis presents work testing the hypothesis that ovulated eggs would differ in the extent of polyspermy from eggs that reached metaphase II as a result of in vitro maturation. The rationale for examining this hypothesis is as follows. In studies using an assay that examined sperm incorporation into eggs over time, recent work in the Evans lab has demonstrated a less robust plateau in the average number of sperm fused per egg over time (Gerolstein, ScM thesis, 2011) compared to previous published work (McAvey et al., 2002; Gardner et al., 2007a, Gardner et al., 2007b, Wortzman-Show et al., 2007). Interestingly, the only noted difference in the protocols was the method of oocyte collection and maturation. Most work in the Evans lab on the membrane block to polyspermy had used ovulated eggs collected from gonadotropinprimed mice (McAvey et al., 2002; Gardner et al., 2007a, Gardner et al., 2007b, Wortzman-Show et al., 2007); however, more recent experiments used prophase I oocytes that were matured in vitro to metaphase II (Gerolstein, 2011). Panels A and B of Figure 5 highlight these differences in past experiments using ovulated and in vitro

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matured eggs, respectively (McAvey et al., 2002; Gerolstein, ScM thesis, 2011). In light of this discrepancy, we compared ovulated eggs to in vitro matured eggs in our standard assay of sperm incorporation over time (Fig. 3). The second part of the work presented here tested the hypothesis that the Src family kinase Fyn plays a role in membrane block establishment. As discussed above (Section D4), previous experiments involving Fyn kinase in oocytes used a knockout mouse model, pharmacological inhibition, and siRNA knockdown (Luo et al., 2009; McGinnis et al., 2009). Significantly, all three methods of Fyn kinase suppression resulted in a dramatic reduction in the prophase I oocyte’s ability to undergo meiotic maturation to metaphase II due to meiotic spindle disruption and chromosomal alignment defects (McGinnis et al., 2009). Since fertilization normally occurs during the metaphase II stage, any long-term experimental manipulation that has an effect on maturation makes it difficult to draw conclusions specific to the blocks to polyspermy. As a result, data from experiments using long-term depletion of Fyn kinase, such as gene deletion, can provide insight to the role of Fyn kinase prior to fertilization; however, the defects in the completion of meiosis are confounding factors in examining a defect specifically in the membrane block to polyspermy. Based on the limitations of chronic Fyn kinase depletion, a more precise way to investigate the potential function of Fyn kinase in membrane block establishment in metaphase II eggs, is use of short-term, acute suppression of Fyn kinase. This can be achieved through the use of a pharmacological inhibitor, which was preferred in these studies for several reasons. As already discussed, Fyn affects meiotic maturation, which would make if difficult to obtain unaffected metaphase II eggs (McGinnis et al., 2009). Second, Fyn knockout models of mice were

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shown to overexpress another SFK, Yes kinase, which may compensate for Fyn kinase suppression (Luo et al., 2009; McGinnis et al., 2009). Third, SKI-606 is highly specific for Src-family protein kinases (Luo et al., 2009). By using SKI-606 as an inhibitor, we can analyze wild-type eggs prior to exposure to sperm. The third part of the research in this thesis addressed the hypothesis that endocytosis plays a role in the membrane block to polyspermy. As noted above (Section E), internalization of surface proteins through endocytosis could be a way to change the functionality of a membrane. If certain egg receptors that support binding to sperm ligands are internalized and degraded as a result of endocytosis, the plasma membrane of the egg could be converted from a receptive to an unreceptive state. Coupled with the observations that compensatory endocytosis occurs with exocytosis of cortical granules (Smith et al., 2000), this model could link the membrane block back to the increased cytosolic calcium necessary for cortical granule exocytosis. In fact, previous work in the Evans laboratory has demonstrated that the timing of the membrane block establishment is at least partly dependent on calcium (Gardner et al., 2007). The possible role of endocytosis in the membrane block to polyspermy could link the membrane block to two other cellular processes that have been associated with the membrane block. Ubiquitination has been implicated in membrane trafficking events [Acconia et al., 2009)] and as noted above (Section D1), the deubiquitinating enzyme UCH-L1 has been implicated in preventing polyspermic fertilization (Sekiguchi et al., 2006; Gerolstein, ScM thesis, 2011). Another possibility is the link between the actin cytoskeleton and endocytotic processes. Previous work in the Evans laboratory investigated the link between actin and the membrane block to polyspermy, showing that

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ZP-free eggs treated with cytochalasin D showed increased extents of polyspermy, suggesting that the actin cytoskeleton may play a role in changing the receptivity of the egg plasma membrane to sperm (McAvey et al., 2002). (McAvey et al., 2002). The GTPase dynamin is critical to the membrane scission event associated with clathrinmediated endocytosis, and also has effects on actin dynamics through interactions with other proteins, some of which are localized to the plasma membrane (Anitei and Hoflack, 2012). Dynamin has also been reported to promote organization and elongation of Factin bundles by binding and displacing their capping protein (Anitei and Hoflack, 2012). The changes that occur in the actin cytoskeleton due to endocytosis could provide a mechanism for the establishment of the membrane block to polyspermy. Alternatively, the cause of polyspermy in cytochalasin D-treated eggs could be that these eggs are less able to undergo endocytosis. A role for actin has been shown for certain types of endocytosis (e.g., macropinocytosis and phagocytosis; Engqvist-Goldstein and Drubin, 2003), and may also play a role in clathrin-dependent endocytosis, depending on the cell type (Fujimoto et al., 2000).

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MATERIALS & METHODS Prophase I Oocyte Collection Prophase I, germinal vesicle-intact (GVI) oocytes were collected from 6-8 week old CF-1 female mice (Harlan, Indianapolis, IN). Mice were sacrificed using cervical dislocation. The ovaries were dissected out and placed in a watch glass containing Whitten’s medium (109.5 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 0.23 mM pyruvic acid, 4.8 mM lactic acid hemicalcium salt; Ca2+ concentration is 2.4 mM [Whitten, 1971]) supplemented with 7 mM NaHCO3, 15 mM HEPES, and 0.05% polyvinyl alcohol (referred to as WH/PVA). Media for collection and culture of prophase I oocytes contained 0.25 mM dibutyryl cAMP (dbcAMP, Sigma, St. Louis, MO) in order to maintain meiotic arrest in prophase I (Cho et al., 1974); this culture medium is referred to as WH/PVA/dbcAMP medium. Once ovaries were placed in ~1 ml of WH/PVA/dbcAMP medium, oocytes were obtained by piercing the ovaries with two 27½ gauge syringe needles to puncture the ovarian follicles and release oocytes. Upon visualizing intact cumulus-oocyte-complexes (COCs), oocytes were denuded of cumulus cells by aspirating through a thin bore glass pipette. Next, the GVI oocytes were transferred to polystyrene culture dishes with 30 µl drops of Whitten’s medium (109.5 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 0.23 mM pyruvic acid, 4.8 mM lactic acid hemicalcium salt; Ca 2+ concentration is 2.4 mM [Whitten, 1971]) with 22 mM NaHCO3 (referred to as Whitten’s bicarbonate (WB) medium)) with 0.05 % PVA (WB/PVA) and 0.25 mM dbcAMP (WB/PVA/dbcAMP medium) covered in mineral oil, which were cultured in an incubator at 37°C with 5% CO2 in air.

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In Vitro Maturation of Prophase I Oocytes to Metaphase II Eggs In order for oocytes to progress through meiotic maturation, the dbcAMP necessary to maintain meiotic arrest must be washed away (Cho et al., 1974). To ensure sufficient washing of dbcAMP, GVI oocytes were pipetted through six 60 µl drops of WH/PVA before overnight culture in a 150 µl drop of WH/PVA covered in mineral oil. Prophase I oocytes were matured by overnight culture (14-16 hours) in a 37°C, 5% CO2 incubator in WH/PVA medium that lacked the dbcAMP necessary to maintain meiotic arrest. Using a dissecting microscope, maturation to metaphase II (MII) was confirmed by observing the absence of the germinal vesicle and the emission of the first polar body. Superovulation of Mice to Obtain Metaphase II Eggs Ovulated metaphase II eggs were collected from mice induced to ovulate by administering a series of gonadotropin injections, as previously described (McAvey, et al., 2002). Mice were first injected with pregnant mare serum gonadotropin (PMSG, 10 IU) and 46-48 hours later with human chorionic gonadotropin (hCG, 7.5 IU). Mice were sacrificed 13-14 hours post hCG-injection by cervical dislocation. Oviducts were harvested from CF-1 female mice (Harlan) and placed in WH medium supplemented with 3 mg/ml BSA (Albumax I from Gibco-BRL, Gaithersburg, MD) and 0.25% Type IV-S hyaluronidase (Sigma) (referred to as HY medium) to facilitate cumulus cell removal. Oviducts were sheared using two 27½ gauge syringes to release cumulus-enclosed metaphase II eggs. To remove cumulus cells, clusters of cumulus-enclosed eggs were pipetted up and down in 50 µl HY drops using a wide bore pipette and then immediately transferred to WH/PVA to wash off HY media. Next, eggs were moved to 65 µl drops of WB medium covered in mineral oil and placed in a 37°C, 5% CO2 incubator

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Zona Pellucida Removal Most experiments used zona pellucida (ZP)-free eggs. ZP removal was achieved by pipetting eggs through 60 µl drops of acidic culture medium-compatible buffer (Acidic MEMCO; 10 mM HEPES, 1 mM NaH2PO4, 0.8 mM MgSO4, 5.4 mM KCl, 116.4 mM NaCl, pH 1.5). After visually confirming the absence of the ZP using a dissecting microscope, eggs were transferred to WB medium for a one hour recovery period in a 37°C, 5% CO2 incubator. Sperm Collection CD-1 retired breeders (Harlan) were sacrificed by cervical dislocation. Both caudae epididymides and the upper ~1 cm of the vas deferens were collected and placed in separate oil-covered 125 µl drops of WB medium supplemented with 15 mg/ml BSA (WB15) to support sperm capacitation (Visconti et al., 1995). The tissues were minced using dissecting scissors, and then sperm were allowed to “swim out” for 10 minutes in a 37°C, 5% CO2 incubator. Next, tissues and mineral oil were removed using forceps and a P-1000 pipettor, respectively. The 125 µl of sperm solution was then transferred to the bottom of a 12 x 75 mm polystyrene culture tube with 750 µl WB15. This tube was placed in a 37oC, 5% CO2 incubator for 45 minutes to allow the sperm to “swim up” in order to select for the most motile sperm. The top 220 µl was removed and transferred to a fresh 12 x 75 mm (5 ml) polystyrene culture tube. These sperm that had swum up were allowed to continue to undergo capacitation and allow for spontaneous acrosome exocytosis for an additional two hours in a 37°C, 5% CO2 incubator. Sperm were then counted using a hemacytometer and diluted to the desired concentration for in vitro fertilization, typically 100,000 sperm/ml.

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In Vitro Fertilization (IVF) of ZP-free MII Eggs After dilution of the swim-up preparation to the desired concentration for insemination, the sperm suspension was pipetted into slightly oblong 10 µl insemination drops in a 60 x 15 mm culture dish, and the drops were covered with mineral oil. To achieve a sperm-to-egg ratio of 100:1, 10 eggs were placed in each drop. Eggs were typically inseminated for 1.5 or 3 hours for a given experiment (see figure legends for details, Fig 3); by 1.5 hr post-insemination, a second polar body should be visible if fertilization occurred. For assessing sperm incorporation over time as a way to examine establishment of the membrane block to polyspermy, two different post-insemination times were examined (1.5 and 3 hr; Gardner and Evans, 2006). Fertilized eggs were washed through a series of three 60 µl drops of WB15 to remove loosely bound sperm prior to fixation. To stain ZP-free prophase I, MII eggs, and inseminated eggs with 4’6’-diamidino2-phenylindole (DAPI; Fig. 1), cells were fixed in a 30 µl drop of freshly made 4% paraformaldehyde in PBS, buffered to a pH of 7.4, for a period of 30-60 minutes in a humidified chamber. Next, eggs were quickly washed through a 30 µl drop of 1X PBS, then permeabilized in a 17 µl drop (to avoid excessive bubbles due to detergent) of 0.1% Triton X-100 in PBS for 5-15 minutes. The eggs were then placed in a 30 µl drop of indirect immunofluorescence (IIF) blocking solution (PBS containing 0.1 % BSA and 0.01 % Tween-20) for 5-60 minutes. Eggs were mounting on a glass slide in a 10 µl drop of Vectashield mounting medium (Vector Labs, Burlingame, CA) containing 1.5 µg/ml DAPI. A coverslip was placed over the drop and was supported by small dabs of Vaseline on the corners in order to prevent flattening of the eggs.

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Eggs were viewed on a Zeiss Axio Observe Z1 microscope with an ApoTome optical sectioning option and AxioCam MRm Rev3 camera was used (Carl Zeiss Inc; Jena, Germany). Wide-field imaging was used for viewing of maternal DNA and sperm fusion, while optical sectioning imaging with use of the ApoTome for structured illumination was used for taking pictures. Exposure times were determined based on auto-exposure recommendations and were optimized (typically lowered) to reduce any saturation present. For assessing sperm-egg fusion and the extent of polyspermy at the specified post-insemination timepoints, the average number of sperm fused per egg (± the standard error of the mean) for each experimental and control group, was calculated. Treatment of ZP-free Metaphase II Eggs with the Src Family Kinase inhibitor SKI-606 Prior to IVF, ZP-free metaphase II eggs were cultured for 1 hr in 30 µl drops of WB/15 containing either 25 µM SKI-606 (gift from Bill Kinsey's lab, Kansas University Medical Center; prepared from a stock of 10 mM in DMSO), or 0.25% DMSO as a solvent control. Eggs were then washed in three drops of drug-free WB/15 and inseminated without the presence of drug to avoid effects on the sperm based on previous experiments (Luo et al., 2009). Treatment of ZP-free Metaphase II Eggs with the dynamin inhibitor dynasore Prior to IVF, ZP-free metaphase II eggs were cultured for 1 hr in 30 µl drops of WB/15 containing either 40 or 60 µM dynasore (D-7693, Sigma; prepared from a stock of 200 mM in DMSO), or 0.2% DMSO as a solvent control. IVF was performed in the presence of inhibitor by diluting the swim-up preparation of sperm 100,000 sperm/ml in WB/15 containing 0.2% DMSO or 40 or 60 µM dynasore.

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Treatment of ZP-free Metaphase II Eggs with Monodansylcadaverine Prior to IVF, ZP-free metaphase II eggs were cultured for 45 min in 3- µl drops of WB/15 containing either 5 or 20 µM monodansylcadaverine (MDC; #30432, Sigma; prepared from a stock of 20 mM in DMSO), or 0.1% DMSO as a solvent control. IVF was performed in the presence of inhibitor by diluting the swim-up preparation of sperm 100,000 sperm/ml in WB/15 0.1% DMSO or containing 5 or 20 µM monodansylcadaverine. Reinsemination Assay – Challenging Zygotes with a Second Batch of Sperm The reinsemination assay was optimized from the procedure previously described (Gardner et al., 2007). Metaphase II eggs were inseminated sequentially with two batches of fluorescently labeled sperm using Alexa Fluor succinimidyl esters fluorescent dyes (Invitrogen; see figure legend for details, Fig. 4). These dyes were made as stocks by dissolving in DMSO at 10 mg/ml to a final stock concentration of 9.27 mM for Alexa Fluor 546 (red) and 15.5 mM for Alexa Fluor 488 (green), and then stored in 3 µl aliquots at -80°C. Sperm were collected as described above; however, after tissue and oil removal, either the green or red Alexa Fluor dye was added to the sperm solution to obtain a final working concentration of 0.2 mM. The sperm were cultured in the dye-containing medium for one hour in a 37oC, 5% CO2 incubator. Next, the ~125 µl solutions of labeled sperm were carefully transferred to the bottom of a 750 µl column of WB15 in a 12 x 75 mm polystyrene culture tube. This was further incubated for a period of 60 minutes, and then the top 220 µl, “swim-up” fraction, was transferred to a fresh 5 ml tube. Afterwards, sperm were allowed to continue capacitating for another 90 minutes.

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For the first insemination, using sperm labeled with the Alexa Fluor 546 (red) fluorescent dye, ZP-free eggs were inseminated for 30 minutes using a 50:1 to 100:1 sperm:egg ratio, for the 20 µM MDC pharmacological inhibitor or DMSO control, respectively. Zygotes were then cultured for 1.5 hours to allow for establishment of the membrane block to polyspermy. After this culture period, zygotes were challenged with a second batch of sperm labeled with the Alexa Fluor 488 (green) fluorescent dye for 60 minutes. During this insemination, a higher sperm to egg ratio of 200:1 was used. Next, the eggs were washed to remove loosely bound sperm followed by fixing and DAPI staining as described above. Statistical Analyses ANOVA with Tukey’s post-hoc testing were performed using Prism (GraphPad Software; La Jolla, CA); p < 0.05 was considered significant. Error bars in figures represent the standard error of the mean (S.E.M.). χ2 analyses were conducted using StatView 5.0 (SAS Institute, Cary, NC). A p-value < 0.05 was considered significant.

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RESULTS Establishment of the membrane block to polyspermy in in vitro matured compared to ovulated eggs Previous work has demonstrated that that sperm incorporation into ZP-free eggs (measured by the number of sperm fused per egg) over time will plateau, indicating the successful establishment of the membrane block to polyspermy (Wolf, 1978; Binor et al., 1982; Sengoku et al., 1995; Olds-Clarke 1999; McAvey et al., 2002). Surprisingly, recent unpublished work in the Evans laboratory investigated the membrane block to polyspermy using in vitro matured oocytes, which showed a less robust plateau in sperm incorporation over time (Gerolstein, ScM thesis, 2011). These observed differences are summarized in Figure 5 showing a robust plateau in control ovulated eggs in panel A (adapted from McAvey et al., 2002) and a less robust plateau in control in vitro matured eggs in panel B (adapted from Gerolstein, ScM thesis, 2011). This is possibly suggestive of a difference in membrane block establishment or membrane receptivity to sperm in ovulated eggs as compared to eggs matured to metaphase II in vitro. Experiments were undertaken here for a direct, side-by-side comparison of sperm incorporation over time between eggs collected by these two different methods (Fig. 5C). This was particularly useful since day-to-day variability when comparing results from IVF experiments is observed. While previous studies exhibited higher averages at both time points it would be difficult to make conclusions on differences when using in vitro matured vs. ovulated since experiments were not performed concurrently (Fig. 5A, McAvey et al., 2002; 5B, Gerolstein, ScM Thesis, 2011; 5C).

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Eggs were either collected from the oviducts of superovulated mice or matured from prophase I oocytes collected from mouse ovaries (IVM). ZP-free eggs were inseminated using a sperm-to-egg ratio of 100:1 for 1.5 hours and 3 hours, then fixed and assessed for the number of fused sperm per egg (Fig. 5C). Ovulated eggs had 1.18 ± 0.10 sperm fused per egg at 1.5 hr post-insemination, and 1.25 ± 0.09 sperm fused per egg at 3 hr post-insemination (mean ± S.E.M.); these values were not statistically significantly different (see also figure legend for details on statistical comparisons; Fig. 5). IVM eggs had 1.02 ± 0.11 sperm fused per egg at 1.5 hr post-insemination, and 1.58 ± 0.13 sperm fused per egg at 3 hr post-insemination; this value was statistically significantly different from the value for the 1.5 hr time point for IVM eggs (p < 0.05). These data suggest that differences may exist in membrane block establishment or other regulation of membrane receptivity to sperm between ovulated eggs and in vitro matured eggs. Examination of a putative role of Fyn-kinase in the membrane block to polyspermy As addressed in the Literature Review (Section D4), Src family kinases (SFK), particularly Fyn kinase, have been implicated as regulators of a variety of egg functions. Mouse eggs from Fyn-deficient mice or treated with the SFK inhibitor SKI-606 have disrupted actin cytoskeletal networks, and ZP-intact eggs treated with SKI-606 showed trends of increased polyspermy when inseminated (Luo et al., 2009). (Note: Eggs from Fyn-deficient mice did not have significant increased extents of polyspermy in these experiments (Luo et al., 2009), although Fyn-null eggs show a upregulation of the related SFK Yes (Luo et al., 2010), which may compensate for certain Fyn functions.) Since these experiments used ZP-intact eggs, it was unclear if prevention of polyspermy could be affected at the level of the membrane block. In this project, experiments using ZP-free

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eggs were performed to examine Fyn kinase as a potential player in the establishment of the membrane block to polyspermy. In vitro fertilization (IVF) assays were performed to investigate sperm incorporation (the number of sperm fused per egg) at 1.5 hours and 3 hours postinsemination (sperm incorporation over time assay, Fig. 3). Ovulated, ZP-free eggs were treated with 25 µM SKI-606, for one hour and washed prior to insemination (to avoid drug effects on sperm during the insemination, as was done in the previous work [Luo et al., 2009]). Previous work (Luo et al., 2009) performed dose-response studies, and 25 µM was found to be the optimal dose for a variety of effects on oocytes, and thus 25 µM was chosen for experiments here. As a solvent control, ZP-free eggs were treated for one hour in 0.25% DMSO. Following DMSO or SKI-606 treatment, both groups of eggs were inseminated with a sperm-to-egg ratio of 100:1 and assessed at 1.5 and 3 hours post insemination. Second polar body emission had not occurred in the majority of eggs in both the control and the experimental group by the three-hour time point. In almost all cases, meiosis had progressed to the anaphase II stage. DMSO-treated eggs had 1.64 ± 0.12 sperm fused per egg at 1.5 hr post-insemination, and 2.01 ± 0.12 sperm fused per egg at 3 hr post-insemination (mean ± S.E.M.; Fig. 6). The SKI-606-treated eggs had 2.33 ± 0.16 sperm fused per egg at 1.5 hr post-insemination, and 2.45 ± 0.15 sperm fused per egg at 3 hr post-insemination. The average numbers of sperm fused per egg for DMSO-treated eggs and SKI-606-treated eggs were statistically different at both time points (p >0.05; see also figure legend for details on statistical comparisons; Fig. 6). Additionally, the frequency distribution in Figure 7 show that there were similar percentages of eggs with 0-2 sperm fused in the DMSO- and SKI-606-treated groups, but

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that there were more eggs with 3 or more fused sperm in the SKI-606 treated eggs 45% (39/87) as compared to 30% (22/73) for DMSO-treated eggs (see also figure legend for details on statistical comparisons; Fig. 7). These data show that perturbation of Fyn kinase has a modest effect on the extent of polyspermy of ZP-free eggs, and lend support to the hypothesis that Fyn kinase could play a role in the membrane block to polyspermy. Examination of a putative role of endocytosis in the membrane block to polyspermy As discussed in the Literature Review (Section E), endocytosis is a way by which the egg membrane could be changed and remodeled, through endocytic internalization of surface proteins. This thesis research examined this possibility through in vitro fertilization (IVF) assays to investigate sperm incorporation in eggs treated with inhibitors of endocytosis (sperm incorporation over time assay, Fig. 3). The first series of experiments used a cell-permeable endocytosis inhibitor of dynamin, dynasore [Macia et al., 2002; Newton et al., 2006; Lowther et al., 2011]. Ovulated ZP-free eggs were treated with 40 or 60 µM dynasore prior to and during insemination, or, as a solvent control, with 0.2% DMSO. These doses of dynasore were chosen because these were the doses used in previous experiments using mouse oocytes (Lowther et al., 2011). (It should be noted that 80 µM was also tested in preliminary experiments, as this dose was shown to have the greatest effect on oocytes undergoing spontaneous exit from prophase I arrest (Lowther et al., 2011); however, with ZP-free metaphase II eggs, we observed an apparent toxic effect that resulted in cell death by the end of insemination.) After 1 hr pretreatment, drug-treated and control eggs were inseminated with a sperm-to-egg ratio of 100:1 for 1.5 or 3 hours post insemination, then fixed and assessed for the number of fused sperm per egg.

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DMSO-treated eggs had 1.51 ± 0.18 sperm fused per egg at 1.5 hr postinsemination and 1.34 ± 0.14 sperm fused per egg at 3 hr post-insemination (Fig. 8). Eggs treated with 40 µM dynasore had 1.60 ± 0.18 sperm fused per egg at 1.5 hr postinsemination, and 1.88 ± 0.22 sperm fused per egg at 3 hr post-insemination. Eggs treated with 60 µM dynasore had 1.56 ± 0.18 sperm fused per egg at 1.5 hr postinsemination, and 1.60 ± 0.15 sperm fused per egg at 3 hr post-insemination. None of these values were statistically significantly different (p > 0.05; see also figure legend for details on statistical comparisons; Fig. 8). The frequency distribution in Figure 9 showed roughly similar percentages of eggs with 0-2 sperm fused in all three groups (32/35 eggs [91%], DMSO; 35/49 eggs [71%], 40 µM dynasore; 38/45 eggs [84%], 60 µM dynasore). although, there were more eggs with 3 or more sperm in the eggs treated with 40 µM dynasore (14/49, 29%) as compared to eggs treated with DMSO (3/35, 9%) or 60 µM dynasore (7/45, 16%; see also figure legend for details on statistical comparisons; Fig. 9). The dynasore-treated eggs were the only eggs that had fused with 4 or more sperm (7/49 eggs, 40 µM dynasore; 3/45 eggs, 60 µM dyansore). This prompted us to test another inhibitor of endocytosis, monodansylcadaverine (MDC). This inhibitor had been used in studies of prophase I oocytes (Lowther et al., 2011). Moreover, a colleague had performed studies of MDC's effects in IVF of ZP-free eggs and reported to us that she observed an increased extent of polyspermy in eggs treated with MDC (control eggs had 1.10 ± 0.088 sperm fused per egg, eggs treated with 5 µM MDC had 1.94 ± 0.16 sperm fused/egg, and eggs treated with 20 µM MDC had 3.06 ± 0.24 sperm fused per egg; Carmen Williams, NIEHS, unpublished data). In experiments here, ovulated, ZP-free eggs were treated with MDC (5 or 20 µM), prior to

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and during insemination, or, as a solvent control, with 0.1% DMSO. After 45 min pretreatment, the eggs were inseminated with a sperm-to-egg ratio of 100:1, and then assessed at 1.5 hours post insemination and 3 hours post insemination. DMSO-treated eggs had 1.18 ± 0.09 sperm fused per egg at 1.5 hr postinsemination and 1.49 ± 0.11 sperm fused per egg at 3 hr post-insemination (Fig. 10). Eggs treated with 5 µM MDC had 1.77 ± 0.16 sperm fused per egg at 1.5 hr postinsemination, and 2.08 ± 0.14 sperm fused per egg at 3 hr post-insemination. Eggs treated with 20 µM MDC had 3.17 ± 0.24 sperm fused per egg at 1.5 hr postinsemination and 3.26 ± 0.19 sperm fused per egg at 3 hr post-insemination. The average numbers of sperm fused per egg for each of these experimental groups were statistically different at both time points (p < 0.05; see also figure legend for details on statistical comparisons; Fig. 10). The frequency distribution in Figure 11 shows similar percentages of eggs with 0-2 sperm fused in eggs treated with DMSO and with 5 µM MDC (88% and 72%, respectively), but there were more eggs with 3 or more sperm (21/75 eggs, 28%) in the eggs treated with 5 µM MDC than eggs treated with DMSO (8/68 eggs, 12%). The eggs treated with 20 µM MDC had a lower percentage of eggs with 0-2 sperm fused per egg (30/82 eggs, 37%), and higher percentage of eggs with 3 or more sperm fused per egg (52/62 eggs, 63%) as compared to the DMSO-treated eggs and eggs treated with 5 µM MDC (see also figure legend for details on statistical comparisons; Fig. 11). Since these experiments suggested that MDC treatment could be affecting the establishment of the membrane block to polyspermy, an additional experiment was performed. This was a reinsemination assay, in which zygotes were challenged with a second batch of sperm, to determine if the zygote membrane had converted to an

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unreceptive state or could still be penetrated by sperm (Fig. 4). DMSO- and MDCtreated ZP-free eggs were inseminated for 30 min to create zygotes; these zygotes were cultured for 1.5 hour, and then subjected to a second insemination. The first insemination (called IVF1) used sperm labeled with a red dye (Alexa Fluor 546; Fig. 12), and the second insemination (called IVF2) used sperm labeled with a green dye (Alexa Fluor 488; Fig. 12). If the membrane block to polyspermy is established, the green sperm from the second insemination should not be able to fuse with the zygote. Conversely, if the membrane block is disrupted, the zygote would remain receptive to green sperm (Gardner et al., 2007b). Preliminary IVF experiments were performed with unlabeled, red-labeled (Fig. 12A), and green-labeled sperm (Fig. 12B) at 100,000 sperm/ml or 200,000 sperm/ml. After a 1.5 hr insemination, the average numbers of sperm fused per egg (means ± S.E.M.) were the following: unlabeled sperm at 100,000 sperm/ml, 1.36 ± 0.12 (n=22 eggs); unlabeled sperm at 200,000 sperm/ml, 1.42 ± 0.14 (n=19 eggs); red-labeled sperm at 100,000 sperm/ml, 1.07 ± 0.08 (n=29 eggs); red-labeled sperm at 200,000 sperm/ml, 1.71 ± 0.16 (n=24 eggs), green-labeled sperm at 100,000 sperm/ml, 1.27 ± 0.13 (n=22 eggs), and green-labeled sperm at 200,000 sperm/ml, 1.47 ± 0.16 (n=19 eggs). This provided information for the specific design of the reinsemination experiment (see also Panel A of Fig. 13 for a schematic diagram of the experimental design). Fertilization conditions to create zygotes from the control DMSO-treated eggs were based on previous work in the Evans lab; these eggs were inseminated with a sperm-to-egg ratio of 100:1 for a period of 30 minutes (IVF1; Gardner et al., 2007b). Since monospermic zygotes are preferred for these studies and due to the higher average

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number of sperm fused per egg in the MDC-treated eggs in the experiments noted above (Figs. 10, 11), a sperm-to-egg ratio of 50:1 for 30 minutes was used, similar to what was done in past work when a treatment was known to increase the extent of polyspermy (Gardner et al., 2007b). IVF2 conditions were the same for both groups (sperm-to-egg ratio of 200:1 for 1 hr) as well as two controls, unfertilized eggs or unfertilized eggs treated with 20 µM MDC. Eggs that were inseminated with only the second batch of sperm (IVF2 only controls) confirmed that the green-labeled sperm successfully fertilized eggs, and provided a baseline level of the average sperm fused per egg (0 µM-treated eggs, 1.64 ± 0.11; eggs treated with 20 µM MDC, 3.24 ± 0.20; Fig. 13B). In analysis of all reinseminated eggs (i.e., those that were fertilized in IVF1 and then underwent IVF2), DMSO-treated eggs had 0.02 ± 0.02 green-labeled sperm fused per egg, and MDCtreated eggs had and 0.52 ± 0.13 green-labeled sperm fused per egg (Fig. 13C). Data were also analyzed focusing on only the zygotes that were monospermic from the first insemination (IVF1); in this subset of eggs, DMSO-treated eggs had an average of 0.02 ± 0.02 green-labeled sperm fused per egg, while the MDC-treated eggs had average of 0.59 ± 0.18 green-labeled sperm fused per egg (Fig. 13D). The subset of eggs that were polyspermic from IVF1 also were analyzed. There were only five polyspermic zygotes in the DMSO-treated group, and none of these eggs were fertilized by green-labeled sperm. The polyspermic zygotes in the MDC-treated group (n = 21 eggs) had an average of 0.43 ± 0.20 green-labeled sperm fused per egg (Fig. 13E). These data support the hypothesis that endocytosis plays a role in the membrane block to polyspermy, although caveats to this interpretation are brought up in the Discussion.

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DISCUSSION The membrane block to polyspermy provides a mechanism at the level of the egg plasma membrane to prevent polyspermic fertilization. While this block has been well characterized in non-mammalian species, it has yet to be determined what cellular processes render the mammalian egg capable of establishing the membrane block in response to a fertilizing sperm (Florman and Ducibella 2006). Better understanding the molecular mechanisms involved in this process may have implications for the treatment of infertility, prevention of recurrent pregnancy loss, and the development of molecular contraceptives. This thesis provides data that support the role of Fyn kinase and endocytosis in the establishment of the membrane block to polyspermy. The results from experiments involving the treatment of ovulated eggs with various pharmacological inhibitors (SKI-606, dynasore, and monodansylcadaverine) will be discussed in detail below. Selecting Ovulated Eggs Over In Vitro Matured Oocytes for Assessing the Membrane Block to Polyspermy This thesis research first examined the extent of polyspermy in ovulated and in vitro matured metaphase II eggs; the rationale for this was in vitro matured eggs have shown a less robust plateau in average of number of sperm fused per egg over time when compared to ovulated eggs (Gerolstein, ScM thesis, 2011; McAvey et al., 2002; Gardner et al., 2007b, Gardner et al., 2007a, Wortzman-Show et al., 2007). Significantly, the average number of sperm fused per egg can vary from experiment to experiment even when using similar IVF conditions. In our experiments, we examined sperm incorporation at two different post-insemination time points (i.e.,

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sperm incorporation over time, as diagramed in Fig. 3) in eggs that were either in vitro matured or ovulated, so that direct comparisons could be made. While the differences in the number of sperm fused per IVM egg as compared to ovulated eggs at the 1 hour and 3 hour time points were similar (i.e., not statistically significant), it is worth noting that the IVM eggs have a statistically significant increase in the average number of sperm fused per egg in the 3 hour time point as compared to the 1 hour time point (Fig. 5C, p 0.05.

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Figure 7:

 

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Figure 8: Sperm incorporation over time in eggs treated with the dynamin inhibitor, dynasore ZP-free eggs were incubated WB/15 medium supplemented with either 0.1% DMSO (solvent control), 40 µM dynasore, or 60 µM dynasore for one hour prior to insemination. Eggs were inseminated with a sperm: egg ratio of 100:1 (100,000 sperm/ml in a 10 µl drop containing 10 eggs) for 1.5 or 3 hours, then washed free of loosely bound sperm, fixed, and stained with DAPI to determine the number of sperm fused per egg. The x-axis shows the average number of sperm fused per egg (± S.E.M.) and the y-axis shows time in hours. Data were pooled from two experiments (DMSO 1.5 hr group, n=35 eggs; 40 µM dynasore 1.5 hr group, n=54 eggs; 60 µM dynasore 1.5 hr group, n=45 eggs; DMSO 3 hr group, n=35 eggs; 40 uM dynasore 3 hr group, n=49 eggs; 60 uM dynasore 3 hr group, n=45 eggs). No groups were statistically significant (p-values < 0.5) from each other as determined by ANOVA analysis combined with Tukey’s post-host testing.

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Figure 8:

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Figure 9: Analysis of the extent of sperm-egg fusion (average number of sperm fused per egg) at 3 hours post-insemination in control, 40 µM dynasore, and 60 µM dynasore treated eggs These histograms correspond to data shown in Figure 8, showing a different analysis from the eggs that were treated with dynasore (40 or 60 µM) or DMSO (solvent control), inseminated, and then fixed at 3 hours post-insemination (panel A, DMSO-treated controls, n=47 eggs; panel B, 40 µM dynasore-treated group, n=49 eggs, panel C, 60 µM dynasore-treated group, n=45 eggs; Panel D, composite). The graphs show frequency distributions of the percentage of eggs (y-axis) with the indicated number of fused sperm (x-axis). Numbers over the bars show the actual numbers of eggs for the corresponding percentage on the y-axis. Statistical analysis was performed by χ2 analysis; comparisons of the control versus both experimental groups, and a comparison of the 40 µM dynasoretreated to the 60 µM dynasore-treated produced p-values > 0.05. However, the 40 µM dynasore-treated group was not statistically significantly different from the DMSO group.

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Figure 9:  

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Figure 10: Sperm incorporation over time in eggs treated with the endocytosis inhibitor, monodansylcadaverine (MDC) ZP-free eggs were incubated WB/15 medium supplemented with either 0.25% DMSO (solvent control), 5 µM MDC, or 20 µM MDC for 45 minutes prior to insemination. Eggs were inseminated with a sperm: egg ratio of 100:1 (100,000 sperm/ml in a 10 µl drop containing 10 eggs) for 1.5 or 3 hours, then washed free of loosely bound sperm, fixed, and stained with DAPI to determine the number of sperm fused per egg. The y-axis shows the average number of sperm fused per egg (± S.E.M.) and the x-axis shows time in hours. Data were pooled from two experiments (DMSO 1.5 hr group, n=67 eggs; 5 µM MDC 1.5 hr group, n=64 eggs; 20 µM MDC 1.5 hr group, n=69 eggs; DMSO 3 hr group, n=68 eggs; 5 µM MDC 3 hr group, n=75 eggs; 20 µM MDC 3 hr group, n=82 eggs). Groups that were statistically different (p < 0.05) from each other as determined by ANOVA analysis combined with Tukey’s post-hoc testing are: DMSO control 90 min. group vs. 20 µM MDC 90 min. group, DMSO control 3 hr group vs. 5 µM MDC 3 hr group, DMSO control 3 hr group vs. 20 µM MDC 3 hr group, 5 µM MDC 90 min. group vs. 20 µM MDC 90 min group, 5 µM MDC 90 min. group vs. 20 µM MDC 3 hr group, 20 µM MDC 90 min. group vs. DMSO control 3 hr. group, 20 µM MDC 90 min. group vs. 5 µM MDC 3 hr. group, DMSO control 3 hr group vs. 20 µM MDC 3 hr. group, and 5 µM MDC 3 hr group vs. 20 µM MDC 3 hr. group. An asterisk (*) indicates a statistically significant difference when compared to the time-matched control. * Note that all 90 min. groups for a given time point are not statistically significant from all  3

hr. groups at the matching time point; however, both time points of the DMSO

groups are statistically different from the corresponding 20 µM MDC groups.

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Figure 10:

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Figure 11: Analysis of the extent of sperm-egg fusion (average number of sperm fused per egg) at 3 hours post-insemination in control, 5 µM MDC, and 20 µM MDC treated eggs These histograms correspond to data shown in Figure 10, showing a different analysis from the eggs that were treated with MDC (5 µM, 20 µM) or DMSO (solvent control), inseminated, and then fixed at 3 hours post-insemination (panel A, DMSO-treated controls, n = 68 eggs; panel B, 5 µM MDC-treated group, n = 75 eggs, panel C, 20 µM MDC-treated group, n = 82 eggs; Panel D, composite). The graphs show frequency distributions of the percentage of eggs (y-axis) with the indicated number of fused sperm (x-axis). Numbers over the bars show the actual numbers of eggs for the corresponding percentage on the y-axis. Statistical analysis was performed by χ2 analysis; comparisons of the control versus groups treated with the two doses of MDC, as well as a comparison of the 5 µM MDC-treated group to the 20 µM MDC-treated group produced a p-values < 0.05.

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Figure 11:

 

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Figure 12: Labeling sperm with Alexa Fluor succinimidyl esters fluorescent dyes These optically sectioned fluorescent images overlaid on DIC images show sperm that have been labeled using Alexa Fluor succinimidyl esters fluorescent dyes (Invitrogen). The sperm were cultured in dye-containing medium at a working concentration of 0.2 mM with either red (Alexa Fluor 546) or green (Alexa Fluor 488) dye. A. “Red,” Alexa Fluor 564-labeled sperm, used in IVF1. B. “Green,” Alexa Fluor 488-labeled sperm, used in IVF2.

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Figure 12:

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Figure 13: Reinsemination of eggs treated with the endocytosis inhibitor, monodansylcadaverine (MDC) Panel A shows the experimental design, which included four groups: two naïve, unfertilized controls (IVF2 only, 0 µM MDC and IVF2 only, 20 µM MDC) and two groups where zygotes (created from red-labeled sperm in IVF1) were exposed to a fresh batch of green-labeled sperm (IVF2; IVF1+2, 0 µM MDC and IVF1+2, 20 µM MDC). For the purposes of data analysis, zygotes exposed to a second batch of sperm were separated into three groups: all fertilized, monospermic, and polyspermic. Panel B shows average number of green sperm fused per egg from the second insemination in the IVF2 only (green-labeled sperm) that were cultured in the presence of 0.1% DMSO or 20 µM MDC. Panel C shows the average number of green sperm fused per egg in all zygotes that were generated from IVF1 and then inseminated in IVF2 and continued culturing in the presence of 0.1% DMSO or 20 µM MDC. Panel D shows the subset of eggs that were monospermic after IVF1. Panel E shows the average number of green sperm fused per egg in the subset of eggs that were polyspermic after IVF1. Data are based on one experiment with 52-78 eggs per group. Note that the y-axis in Panels C, D, E is different than the y-axis in Panel B. Error bars represent the S.E.M. Numbers over the bars show the numbers of eggs that were fused any green sperm in the second insemination, but do not reflect the number of eggs with more than one green sperm. Of the five MDC-treated zygotes that were polyspermic from IVF1 and were penetrated by green-labeled sperm in IVF2, 3 eggs had 1 green-labeled sperm fused, and 2 eggs had 3 green-labeled sperm. Statistical differences between the groups were calculated by ANOVA analysis combined with Tukey’s post-hoc and by χ2 analysis. In all three sets of data (i.e., Panels A, B and

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C), ANOVA with Tukey’s post-hoc test revealed that 0 µM MDC and 20 µM MDC each were statistically different (p < 0.05) from the IVF2, 0 µM group and the IVF2 20 µM MDC group. χ2 analysis revealed that the 0 µM MDC was statistically different than the 20 µM MDC (p < 0.05).

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Figure 13:

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REFERENCES Abbott, A. L., and T. Ducibella. "Calcium and the Control of Mammalian Cortical Granule Exocytosis." Frontiers in bioscience : a journal and virtual library 6 (2001): D792-806. Abbott, A. L., R. A. Fissore, and T. Ducibella. "Identification of a Translocation Deficiency in Cortical Granule Secretion in Preovulatory Mouse Oocytes." Biology of reproduction 65.6 (2001): 1640-7. Acconcia, F., S. Sigismund, and S. Polo. "Ubiquitin in Trafficking: The Network at Work." Experimental cell research 315.9 (2009): 1610-8. Albuz, F. K., et al. "Simulated Physiological Oocyte Maturation (SPOM): A Novel in Vitro Maturation System that Substantially Improves Embryo Yield and Pregnancy Outcomes." Human reproduction (Oxford, England) 25.12 (2010): 2999-3011. Anitei, M., and B. Hoflack. "Bridging Membrane and Cytoskeleton Dynamics in the Secretory and Endocytic Pathways." Nature cell biology 14.1 (2011): 11-9. Austin CR. The mammalian egg. Springfield, IL: Charles C. Thomas: (1961). Baarends, W. M., H. P. Roest, and J. A. Grootegoed. "The Ubiquitin System in Gametogenesis." Molecular and cellular endocrinology 151.1-2 (1999): 5-16.

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Backs, J., et al. "The Gamma Isoform of CaM Kinase II Controls Mouse Egg Activation by Regulating Cell Cycle Resumption." Proceedings of the National Academy of Sciences of the United States of America 107.1 (2010): 81-6. Baibakov, B., et al. "Sperm Binding to the Zona Pellucida is Not Sufficient to Induce Acrosome Exocytosis." Development (Cambridge, England) 134.5 (2007): 933-43. Barros, C., and R. Yanagimachi. "Induction of Zona Reaction in Golden Hamster Eggs by Cortical Granule Material." Nature 233.5317 (1971): 268-9. Bedford, S. J., et al. "Intracellular Calcium Oscillations and Activation in Horse Oocytes Injected with Stallion Sperm Extracts Or Spermatozoa." Reproduction (Cambridge, England) 126.4 (2003): 489-99. Bement, W. M., et al. "Evidence for Direct Membrane Retrieval Following Cortical Granule Exocytosis in Xenopus Oocytes and Eggs." The Journal of experimental zoology 286.7 (2000): 767-75. Bernardini, G., M. Ferraguti, and R. Stipani. "Fertilization Induces Endocytosis in Xenopus Eggs." Cell differentiation 21.4 (1987): 255-60. Binor, Z., and D. P. Wolf. "In-Vitro Maturation and Penetration of Mouse Primary Oocytes After Removal of the Zona Pellucida." Journal of reproduction and fertility 56.1 (1979): 309-14.

81  

Bleil, J. D., and P. M. Wassarman. "Mammalian Sperm-Egg Interaction: Identification of a Glycoprotein in Mouse Egg Zonae Pellucidae Possessing Receptor Activity for Sperm." Cell 20.3 (1980): 873-82. Boue, J., A. Bou, and P. Lazar. "Retrospective and Prospective Epidemiological Studies of 1500 Karyotyped Spontaneous Human Abortions." Teratology 12.1 (1975): 1126. Brunet, S., and B. Maro. "Cytoskeleton and Cell Cycle Control during Meiotic Maturation of the Mouse Oocyte: Integrating Time and Space." Reproduction (Cambridge, England) 130.6 (2005): 801-11. Burkart, A. D., et al. "Ovastacin, a Cortical Granule Protease, Cleaves ZP2 in the Zona Pellucida to Prevent Polyspermy." The Journal of cell biology 197.1 (2012): 37-44. Centers for Disease Control and Prevention. “Assisted Reproductive Technology Surveillance System.” June 2011. National ART Surveillance System. 26 May 2012. . Chang, H. Y., et al. "Calmodulin-Dependent Protein Kinase Gamma 3 (CamKIIgamma3) Mediates the Cell Cycle Resumption of Metaphase II Eggs in Mouse." Development (Cambridge, England) 136.24 (2009): 4077-81. Dalo, D.T., ScM thesis, Analysis of membrane topography and cortical remodeling in mouse eggs: insights into factors affecting gamete membrane interactions and the membrane block to polyspermy. (2007)

82  

Ducibella, T., et al. "Egg-to-Embryo Transition is Driven by Differential Responses to Ca(2+) Oscillation Number." Developmental biology 250.2 (2002): 280-91. Ducibella, T. "The Cortical Reaction and Development of Activation Competence in Mammalian Oocytes." Human reproduction update 2.1 (1996): 29-42. Engqvist-Goldstein, A. E., and D. G. Drubin. "Actin Assembly and Endocytosis: From Yeast to Mammals." Annual Review of Cell and Developmental Biology 19 (2003): 287-332. Evans, J.P., and Bailey, J.L. How does the sperm make its way to the egg and how does fertilization take place? - Capacitation, acrosome exocytosis, steps of sperm-egg interaction, egg activation. In The Handbook of Andrology, Eds. Bernard Robaire and Peter Chang. American Society of Andrology. (2010) Evans, J. P., R. M. Schultz, and G. S. Kopf. "Mouse Sperm-Egg Plasma Membrane Interactions: Analysis of Roles of Egg Integrins and the Mouse Sperm Homologue of PH-30 (Fertilin) Beta." Journal of cell science 108 ( Pt 10).Pt 10 (1995): 3267-78. European Society of Human Reproduction and Embryology. "Assisted Reproductive Technology Fact Sheet." June 2010. ESHRE. 26 May 2012. .

83  

Fisher, G. W., and L. I. Rebhun. "Sea Urchin Egg Cortical Granule Exocytosis is Followed by a Burst of Membrane Retrieval Via Uptake into Coated Vesicles." Developmental biology 99.2 (1983): 456-72. Florman HM, Ducibella T. “Fertilization in Mammals.” Knobil and Neill’s Physiology of Reproduction, Third Edition. Ed. Jimmy D. Neil. Elsevier: (2006). Fraser, L. R. "Strontium Supports Capacitation and the Acrosome Reaction in Mouse Sperm and Rapidly Activates Mouse Eggs." Gamete research 18.4 (1987): 363-74. Fujimoto, G., et al. "Calcium Concentration and Fertilization by Subzonal Insemination of a Single Spermatozoon in Mouse Oocytes." Molecular reproduction and development 38.1 (1994): 54-60. Fujimoto, L. M., et al. "Actin Assembly Plays a Variable, but Not Obligatory Role in Receptor-Mediated Endocytosis in Mammalian Cells." Traffic (Copenhagen, Denmark) 1.2 (2000): 161-71. Gadella, B. M., and J. P. Evans. "Membrane Fusions during Mammalian Fertilization." Advances in Experimental Medicine and Biology 713 (2011): 65-80. Gardner, A. J., and J. P. Evans. "Mammalian Membrane Block to Polyspermy: New Insights into how Mammalian Eggs Prevent Fertilisation by Multiple Sperm." Reproduction, fertility, and development 18.1-2 (2006): 53-61. (B)

84  

Gardner, A. J., et al. "CaMKII can Participate in but is Not Sufficient for the Establishment of the Membrane Block to Polyspermy in Mouse Eggs." Journal of cellular physiology 212.2 (2007): 275-80. (A) Gardner, A. J., C. J. Williams, and J. P. Evans. "Establishment of the Mammalian Membrane Block to Polyspermy: Evidence for Calcium-Dependent and Independent Regulation." Reproduction (Cambridge, England) 133.2 (2007): 38393. Gerolstein, A., ScM thesis, Analysis of the role of ubiquitin c-terminal hydrolase L1 (UCH-L1) in the prevention of polyspermic fertilization in mammalian eggs. (2011) Gilchrist, R. B. "Recent Insights into Oocyte-Follicle Cell Interactions Provide Opportunities for the Development of New Approaches to in Vitro Maturation." Reproduction, fertility, and development 23.1 (2011): 23-31. Golbus, M. Chromosome aberrations and mammalian reproduction. In Fertilization and embryonic Development In Vitro (JD Biggers and L. Mastroianni, Eds. Plenum Press, NY (1981): 1-25. Grant, S. G., et al. "Impaired Long-Term Potentiation, Spatial Learning, and Hippocampal Development in Fyn Mutant Mice." Science (New York, N.Y.) 258.5090 (1992): 1903-10.

85  

Guttmacher Institute. “Facts on Unintended Pregnancy in the United States.” January 2012. Guttmacher Institute. 26 May 2012. . Hassold, T., et al. "A Cytogenetic Study of 1000 Spontaneous Abortions." Annals of Human Genetics 44.Pt 2 (1980): 151-78. Hedrick, J. L., and D. M. Hardy. "Isolation of Extracellular Matrix Structures from Xenopus Laevis Oocytes, Eggs, and Embryos." Methods in cell biology 36 (1991): 231-47. Hinckley, M., et al. "The G-Protein-Coupled Receptors GPR3 and GPR12 are Involved in cAMP Signaling and Maintenance of Meiotic Arrest in Rodent Oocytes." Developmental biology 287.2 (2005): 249-61. Horvath, P. M., et al. "Mechanistic Studies of the Plasma Membrane Block to Polyspermy in Mouse Eggs." Molecular reproduction and development 34.1 (1993): 65-72. Hunter RHF. Fertilization in the pig and horse. In: Dunbar BS, O'Rand MG (eds.), A Comparative Overview of Mammalian Fertilization. New York, NY: Plenum Press: (1991): 329-349. Igusa, Y., S. Miyazaki, and N. Yamashita. "Periodic Hyperpolarizing Responses in Hamster and Mouse Eggs Fertilized with Mouse Sperm." The Journal of physiology 340 (1983): 633-47.

86  

Igusa, Y., and S. Miyazaki. "Effects of Altered Extracellular and Intracellular Calcium Concentration on Hyperpolarizing Responses of the Hamster Egg." The Journal of physiology 340 (1983): 611-32. Inoue, M., and D. P. Wolf. "Fertilization-Associated Changes in the Murine Zona Pellucida: A Time Sequence Study." Biology of reproduction 13.5 (1975): 546-51. Jacobs, P. A., et al. "The Origin of Human Triploids." Annals of Human Genetics 42.1 (1978): 49-57. Jaffe, L. A., A. P. Sharp, and D. P. Wolf. "Absence of an Electrical Polyspermy Block in the Mouse." Developmental biology 96.2 (1983): 317-23. Jaffe LA, Gould M. Polyspermy-preventing mechanisms. In “Biology of Fertilization: The fertilization Response of the Egg” (C. B. Metz and A. Monroy eds.) Academic Press, Orlando, FL. (1985): Vol. 3, pp. 223-250. Johnson, J., et al. "Calcium/calmodulin-Dependent Protein Kinase II and Calmodulin: Regulators of the Meiotic Spindle in Mouse Eggs." Developmental biology 204.2 (1998): 464-77. Kirchhausen, T., E. Macia, and H. E. Pelish. "Use of Dynasore, the Small Molecule Inhibitor of Dynamin, in the Regulation of Endocytosis." Methods in enzymology 438 (2008): 77-93.

87  

Kishimoto, T., et al. "Determinants of Endocytic Membrane Geometry, Stability, and Scission." Proceedings of the National Academy of Sciences of the United States of America 108.44 (2011): E979-88. Kline, D., and J. T. Kline. "Repetitive Calcium Transients and the Role of Calcium in Exocytosis and Cell Cycle Activation in the Mouse Egg." Developmental biology 149.1 (1992): 80-9. Kline, D., and J. T. Kline. "Thapsigargin Activates a Calcium Influx Pathway in the Unfertilized Mouse Egg and Suppresses Repetitive Calcium Transients in the Fertilized Egg." The Journal of biological chemistry 267.25 (1992): 17624-30. Kline, D., and J. Stewart-Savage. "The Timing of Cortical Granule Fusion, Content Dispersal, and Endocytosis during Fertilization of the Hamster Egg: An Electrophysiological and Histochemical Study." Developmental biology 162.1 (1994): 277-87. Knott, J. G., et al. "Calmodulin-Dependent Protein Kinase II Triggers Mouse Egg Activation and Embryo Development in the Absence of Ca2+ Oscillations." Developmental biology 296.2 (2006): 388-95. Knott, J. G., et al. "Transgenic RNA Interference Reveals Role for Mouse Sperm Phospholipase Czeta in Triggering Ca2+ Oscillations during Fertilization." Biology of reproduction 72.4 (2005): 992-6.

88  

Kryzak, C. ScM thesis, Sperm-egg interactions and establishment of the membrane block to polyspermy in prophase I mouse oocytes. (2008) Kurokawa, M., K. Sato, and R. A. Fissore. "Mammalian Fertilization: From Sperm Factor to Phospholipase Czeta." Biology of the cell / under the auspices of the European Cell Biology Organization 96.1 (2004): 37-45. Le Guen, P., et al. "Distribution and Role of Microfilaments during Early Events of Sheep Fertilization." Gamete research 22.4 (1989): 411-25. Levi, M., and R. Shalgi. "The Role of Fyn Kinase in the Release from Metaphase in Mammalian Oocytes." Molecular and cellular endocrinology 314.2 (2010): 228-33. Lewis WH, Wright ES. On the early development of the mouse egg. Carnegie Inst Contrib Embryol (1935): 25:113-143. Longo, F. J. "Effects of Cytochalasin B on Sperm--Egg Interactions." Developmental biology 67.2 (1978): 249-65. Lowther, K. M., V. O. Nikolaev, and L. M. Mehlmann. "Endocytosis in the Mouse Oocyte and its Contribution to cAMP Signaling during Meiotic Arrest." Reproduction (Cambridge, England) 141.6 (2011): 737-47. Luo, J., L. K. McGinnis, and W. H. Kinsey. "Fyn Kinase Activity is Required for Normal Organization and Functional Polarity of the Mouse Oocyte Cortex." Molecular reproduction and development 76.9 (2009): 819-31.

89  

Luo, J., L. K. McGinnis, and W. H. Kinsey. "Role of Fyn Kinase in Oocyte Developmental Potential." Reproduction, fertility, and development 22.6 (2010): 966-76. Macaluso, M., et al. "A Public Health Focus on Infertility Prevention, Detection, and Management." Fertility and sterility 93.1 (2010): 16.e1,16.10. Macia, E., et al. "Dynasore, a Cell-Permeable Inhibitor of Dynamin." Developmental cell 10.6 (2006): 839-50. Madgwick, S., M. Levasseur, and K. T. Jones. "Calmodulin-Dependent Protein Kinase II, and Not Protein Kinase C, is Sufficient for Triggering Cell-Cycle Resumption in Mammalian Eggs." Journal of cell science 118.Pt 17 (2005): 3849-59. Malcuit, C., M. Kurokawa, and R. A. Fissore. "Calcium Oscillations and Mammalian Egg Activation." Journal of cellular physiology 206.3 (2006): 565-73. Maleszewski, M., Y. Kimura, and R. Yanagimachi. "Sperm Membrane Incorporation into Oolemma Contributes to the Oolemma Block to Sperm Penetration: Evidence Based on Intracytoplasmic Sperm Injection Experiments in the Mouse." Molecular reproduction and development 44.2 (1996): 256-9. Maleszewski, M. "Decondensation of Mouse Sperm Chromatin in Cell-Free Extracts: A Micromethod." Molecular reproduction and development 27.3 (1990): 244-8. Maluchnik, D., and E. Borsuk. "Sperm Entry into Fertilised Mouse Eggs." Zygote (Cambridge, England) 2.2 (1994): 129-31.

90  

Marcello, M. R., et al. "Lack of Tyrosylprotein Sulfotransferase-2 Activity Results in Altered Sperm-Egg Interactions and Loss of ADAM3 and ADAM6 in Epididymal Sperm." The Journal of biological chemistry 286.15 (2011): 13060-70. Markoulaki, S., S. Matson, and T. Ducibella. "Fertilization Stimulates Long-Lasting Oscillations of CaMKII Activity in Mouse Eggs." Developmental biology 272.1 (2004): 15-25. Maro, B., et al. "Changes in Actin Distribution during Fertilization of the Mouse Egg." Journal of embryology and experimental morphology 81 (1984): 211-37. McAvey, B. A., et al. "Involvement of Calcium Signaling and the Actin Cytoskeleton in the Membrane Block to Polyspermy in Mouse Eggs." Biology of reproduction 67.4 (2002): 1342-52. McCulloh, D. H., C. E. Rexroad Jr, and H. Levitan. "Insemination of Rabbit Eggs is Associated with Slow Depolarization and Repetitive Diphasic Membrane Potentials." Developmental biology 95.2 (1983): 372-7. McFadden, D. E., and S. Langlois. "Parental and Meiotic Origin of Triploidy in the Embryonic and Fetal Periods." Clinical genetics 58.3 (2000): 192-200. McGinnis, L. K., W. H. Kinsey, and D. F. Albertini. "Functions of Fyn Kinase in the Completion of Meiosis in Mouse Oocytes." Developmental biology 327.2 (2009): 280-7.

91  

Mehlmann, L. M., et al. "The Gs-Linked Receptor GPR3 Maintains Meiotic Arrest in Mammalian Oocytes." Science (New York, N.Y.) 306.5703 (2004): 1947-50. Michelmann, H. W., A. Bonhoff, and L. Mettler. "Chromosome Analysis in Polyploid Human Embryos." Human reproduction (Oxford, England) 1.4 (1986): 243-6. Miyamoto, H., and T. Ishibashi. "The Role of Calcium Ions in Fertilization of Mouse and Rat Eggs in Vitro." Journal of reproduction and fertility 45.3 (1975): 523-6. Miyazaki, S., and Y. Igusa. "Fertilization Potential in Golden Hamster Eggs Consists of Recurring Hyperpolarizations." Nature 290.5808 (1981): 702-4. Newton, A. J., T. Kirchhausen, and V. N. Murthy. "Inhibition of Dynamin Completely Blocks Compensatory Synaptic Vesicle Endocytosis." Proceedings of the National Academy of Sciences of the United States of America 103.47 (2006): 17955-60. Odor, D. L., and R. J. Blandau. "The Frequency of Occurrence of Supernumerary Sperm in Rat Ova." The Anatomical Record 104.1 (1949): 1-9. Redkar, A. A., and P. J. Olds-Clarke. "An Improved Mouse Sperm-Oocyte Plasmalemma Binding Assay: Studies on Characteristics of Sperm Binding in Medium with Or without Glucose." Journal of andrology 20.4 (1999): 500-8. Runft, L. L., L. A. Jaffe, and L. M. Mehlmann. "Egg Activation at Fertilization: Where it all Begins." Developmental biology 245.2 (2002): 237-54.

92  

Sachdev, M., et al. "Oocyte Specific Oolemmal SAS1B Involved in Sperm Binding through Intra-Acrosomal SLLP1 during Fertilization." Developmental biology 363.1 (2012): 40-51. Sato, K. "Polyspermy-Preventing Mechanisms in Mouse Eggs Fertilized in Vitro." The Journal of experimental zoology 210.2 (1979): 353-9. Schatten, G., et al. "Latrunculin Inhibits the Microfilament-Mediated Processes during Fertilization, Cleavage and Early Development in Sea Urchins and Mice." Experimental cell research 166.1 (1986): 191-208. Schatten, H., et al. "Microtubule Assembly is Required for the Formation of the Pronuclei, Nuclear Lamin Acquisition, and DNA Synthesis during Mouse, but Not Sea Urchin, Fertilization." Gamete research 23.3 (1989): 309-22. Schlegel, R., et al. "Amantadine and Dansylcadaverine Inhibit Vesicular Stomatitis Virus Uptake and Receptor-Mediated Endocytosis of Alpha 2-Macroglobulin." Proceedings of the National Academy of Sciences of the United States of America 79.7 (1982): 2291-5. Schwarz, L. A., and G. N. Patrick. "Ubiquitin-Dependent Endocytosis, Trafficking and Turnover of Neuronal Membrane Proteins." Molecular and cellular neurosciences 49.3 (2012): 387-93.

93  

Sekiguchi, S., et al. "Localization of Ubiquitin C-Terminal Hydrolase L1 in Mouse Ova and its Function in the Plasma Membrane to Block Polyspermy." The American journal of pathology 169.5 (2006): 1722-9. Sengoku, K., et al. "Plasma Membrane Block to Polyspermy in Human Oocytes and Preimplantation Embryos." Journal of reproduction and fertility 105.1 (1995): 85-90. Shafi, R., et al. "The O-GlcNAc Transferase Gene Resides on the X Chromosome and is Essential for Embryonic Stem Cell Viability and Mouse Ontogeny." Proceedings of the National Academy of Sciences of the United States of America 97.11 (2000): 5735-9. Smith, R. M., et al. "Exocytotic Insertion of Calcium Channels Constrains Compensatory Endocytosis to Sites of Exocytosis." The Journal of cell biology 148.4 (2000): 75567. Smitz, J. E., J. G. Thompson, and R. B. Gilchrist. "The Promise of in Vitro Maturation in Assisted Reproduction and Fertility Preservation." Seminars in reproductive medicine 29.1 (2011): 24-37. Stein, P. L., et al. "Pp59fyn Mutant Mice Display Differential Signaling in Thymocytes and Peripheral T Cells." Cell 70.5 (1992): 741-50. Stein, P. L., H. Vogel, and P. Soriano. "Combined Deficiencies of Src, Fyn, and Yes Tyrosine Kinases in Mutant Mice." Genes & development 8.17 (1994): 1999-2007.

94  

Stewart-Savage, J., and B. D. Bavister. "A Cell Surface Block to Polyspermy Occurs in Golden Hamster Eggs." Developmental biology 128.1 (1988): 150-7. Suarez, S. S., and A. A. Pacey. "Sperm Transport in the Female Reproductive Tract." Human reproduction update 12.1 (2006): 23-37. Sun, Q. Y., et al. "Dynamic Events are Differently Mediated by Microfilaments, Microtubules, and Mitogen-Activated Protein Kinase during Porcine Oocyte Maturation and Fertilization in Vitro." Biology of reproduction 64.3 (2001): 879-89. Susor, A., et al. "Role of Ubiquitin C-Terminal Hydrolase-L1 in Antipolyspermy Defense of Mammalian Oocytes." Biology of reproduction 82.6 (2010): 1151-61. Sutovsky, P., et al. "Early Degradation of Paternal Mitochondria in Domestic Pig (Sus Scrofa) is Prevented by Selective Proteasomal Inhibitors Lactacystin and MG132." Biology of reproduction 68.5 (2003): 1793-800. Sutovsky, P., C. S. Navara, and G. Schatten. "Fate of the Sperm Mitochondria, and the Incorporation, Conversion, and Disassembly of the Sperm Tail Structures during Bovine Fertilization." Biology of reproduction 55.6 (1996): 1195-205. Sutovsky, P. "Ubiquitin-Dependent Proteolysis in Mammalian Spermatogenesis, Fertilization, and Sperm Quality Control: Killing Three Birds with One Stone." Microscopy research and technique 61.1 (2003): 88-102.

95  

Swann, K., et al. "PLCzeta(Zeta): A Sperm Protein that Triggers Ca2+ Oscillations and Egg Activation in Mammals." Seminars in cell & developmental biology 17.2 (2006): 264-73. Tatone, C., et al. "Possible Role for Ca(2+) Calmodulin-Dependent Protein Kinase II as an Effector of the Fertilization Ca(2+) Signal in Mouse Oocyte Activation." Molecular human reproduction 8.8 (2002): 750-7. Tatone, C., et al. "Biochemical and Biological Effects of KN-93, an Inhibitor of Calmodulin-Dependent Protein Kinase II, on the Initial Events of Mouse Egg Activation Induced by Ethanol." Journal of reproduction and fertility 115.1 (1999): 151-7. Terada, Y., C. Simerly, and G. Schatten. "Microfilament Stabilization by Jasplakinolide Arrests Oocyte Maturation, Cortical Granule Exocytosis, Sperm Incorporation Cone Resorption, and Cell-Cycle Progression, but Not DNA Replication, during Fertilization in Mice." Molecular reproduction and development 56.1 (2000): 89-98. Visconti, P. E., and H. M. Florman. "Mechanisms of Sperm-Egg Interactions: Between Sugars and Broken Bonds." Science signaling 3.142 (2010): pe35. Vjugina, U., and J. P. Evans. "New Insights into the Molecular Basis of Mammalian Sperm-Egg Membrane Interactions." Frontiers in bioscience : a journal and virtual library 13 (2008): 462-76.

96  

Wolf, D. E., and C. A. Ziomek. "Regionalization and Lateral Diffusion of Membrane Proteins in Unfertilized and Fertilized Mouse Eggs." The Journal of cell biology 96.6 (1983): 1786-90. Wolf, D. P. "On the Contents of the Cortical Granules from Xenopus Laevis Eggs." Developmental biology 38.1 (1974): 14-29. Wortzman, G. B., and J. P. Evans. "Membrane and Cortical Abnormalities in PostOvulatory Aged Eggs: Analysis of Fertilizability and Establishment of the Membrane Block to Polyspermy." Molecular human reproduction 11.1 (2005): 1-9. Wortzman-Show, G. B., et al. "Calcium and Sperm Components in the Establishment of the Membrane Block to Polyspermy: Studies of ICSI and Activation with Sperm Factor." Molecular human reproduction 13.8 (2007): 557-65. Xiong, S., et al. "Effects of Cumulus Cells Removal After 6 h Co-Incubation of Gametes on the Outcomes of Human IVF." Journal of assisted reproduction and genetics 28.12 (2011): 1205-11. Yanagimachi, R. "Calcium Requirement for Sperm-Egg Fusion in Mammals." Biology of reproduction 19.5 (1978): 949-58. Yanagimachi, R. "Intracytoplasmic Injection of Spermatozoa and Spermatogenic Cells: Its Biology and Applications in Humans and Animals." Reproductive biomedicine online 10.2 (2005): 247-88.

97  

Yoon, S. Y., and R. A. Fissore. "Release of Phospholipase C Zetaand [Ca2+]i Oscillation-Inducing Activity during Mammalian Fertilization." Reproduction (Cambridge, England) 134.5 (2007): 695-704. Zhu, G. Z., et al. "Testase 1 (ADAM 24) a Sperm Surface Metalloprotease is Required for Normal Fertility in Mice." Molecular reproduction and development 76.11 (2009): 1106-14. Zuccotti, M., R. Yanagimachi, and H. Yanagimachi. "The Ability of Hamster Oolemma to Fuse with Spermatozoa: Its Acquisition during Oogenesis and Loss After Fertilization." Development (Cambridge, England) 112.1 (1991): 143-52.

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CURRICULUM VITAE

BRENT TURNER 2810 E. Marshall St. #2 Richmond, VA 23223 540-761-2635 [email protected]

EDUCATION & HONORS Medical College of Virginia. M.D. Candidate, 2016.

The Johns Hopkins University Bloomberg School of Public Health. Sc.M., Reproductive & Cancer Biology, 2012. GPA: 3.87. Maternal & Child Health Certificate Population & Health Certificate Chair: Lesbian, Gay, Bisexual, & Transgender Society Teaching Assistant: Molecular Endocrinology Department Representative: Student Assembly Reproductive Division Journal Club

University of Mary Washington. B.S., Biochemistry, cum laude, 2009. GPA: 3.38. Ruby Y. Weinbrecht Award (Scholarship & Professionalism) Chair: Academic Affairs Council Executive Secretary: Association of Residence Halls President: Russell Hall

Kudan Institute of Language & Culture. Study Abroad, Tokyo, Japan, 2008.

PROFESSIONAL EXPERIENCE Research Assistant. Janice Evans, Ph.D., 2011-2012. Mammalian gamete collection and in vitro fertilization assays to examine molecular mechanisms behind the membrane block to polyspermy using fluorescent microscopy.

Scribe. Mary Washington & Stafford Hospital, 2006-2010. Triaged emergency room patients and performed real-time transcription of medical history during physician consultation.

Research Assistant. Deborah Zies, Ph.D., 2008-2009. Chromatin immunoprecipitation to investigate molecular mechanisms behind aldosterone’s regulation of blood pressure.

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BRENT TURNER 2810 E. Marshall St. #2 Richmond, VA 23223 540-761-2635 [email protected]

COMMUNITY SERVICE Instructor. Community Adolescent Sexuality Education (CASE), 2012. Lectured in Baltimore City Public Schools on relationships, reproductive anatomy, sex, pregnancy, contraception, and sexually transmitted infections.

HIV Counselor. Chase Brexton Health Services, 2010-2011. Provided HIV testing and counseling to high-risk populations in Baltimore City, Maryland.

Delegate. The United Nations Children’s Fund (UNICEF), 2004-2009. Fundraised for global immunization, iodine deficiency disorder, and maternal and newborn health.

Spanish Interpreter. Van L. Lewis, M.D., Carilion Clinic, 2006. Medical translator for interventional radiology specialist at Roanoke Memorial Hospital.

CONFERENCES & SYMPOSIA Medical Students for Choice Annual Meeting. Baltimore, MD. 2011.

Lesbian, Gay, Bisexual, & Transgender Leadership Institute. American Medical Student Association, Baltimore, MD, 2011.

Gordon Research Conference, Fertilization & Activation of Development. Holderness, NH, 2011.

Women’s Empowerment Symposium. American Medical Student Association, Baltimore, MD, 2011.

ABSTRACTS Christianson MS, Wilkinson A, Turner BC, Evans JP. Role of the deubiquitinating enzyme ubiquitin c-terminal hydrolase L1 (UCH-L1) in the prevention of polyspermic fertilization in mouse eggs. American Society of Reproductive Medicine Annual Meeting, San Diego, CA, October 20-24, 2012.

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