Gamete Interaction Research Articles

Confounding of linkage disequilibrium patterns in large scale DNA based gene-gene interaction studies

BackgroundIn Genome-Wide Association Studies (GWAS), the concept of linkage disequilibrium is important as it allows identifying genetic markers that tag the actual causal variants. In Genome-Wide Association Interaction Studies (GWAIS), similar principles hold for pairs of causal variants. However, Linkage Disequilibrium (LD) may also interfere with the detection of genuine epistasis signals in that there may be complete confounding between Gametic Phase Disequilibrium (GPD) and interaction. GPD may involve unlinked genetic markers, even residing on different chromosomes. Often GPD is eliminated in GWAIS, via feature selection schemes or so-called pruning algorithms, to obtain unconfounded epistasis results. However, little is known about the optimal degree of GPD/LD-pruning that gives a balance between false positive control and sufficient power of epistasis detection statistics. Here, we focus on Model-Based Multifactor Dimensionality Reduction as one large-scale epistasis detection tool. Its performance has been thoroughly investigated in terms of false positive control and power, under a variety of scenarios involving different trait types and study designs, as well as error-free and noisy data, but never with respect to multicollinear SNPs.ResultsUsing real-life human LD patterns from a homogeneous subpopulation of British ancestry, we investigated the impact of LD-pruning on the statistical sensitivity of MB-MDR. We considered three different non-fully penetrant epistasis models with varying effect sizes. There is a clear advantage in pre-analysis pruning using sliding windows at r2 of 0.75 or lower, but using a threshold of 0.20 has a detrimental effect on the power to detect a functional interactive SNP pair (power < 25%). Signal sensitivity, directly using LD-block information to determine whether an epistasis signal is present or not, benefits from LD-pruning as well (average power across scenarios: 87%), but is largely hampered by functional loci residing at the boundaries of an LD-block.ConclusionsOur results confirm that LD patterns and the position of causal variants in LD blocks do have an impact on epistasis detection, and that pruning strategies and LD-blocks definitions combined need careful attention, if we wish to maximize the power of large-scale epistasis screenings.

S100 A9 is expressed and secreted by the oviduct epithelium, interacts with gametes and affects parameters of human sperm capacitation in vitro.

Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [35 S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [35 S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.

Ovarian stimulation induces high expression of interleukin-1β and disrupts the histological features of the fallopian tube

The objective of this study was to investigate whether there is an association between ovarian stimulation and interrupted gamete interaction in the rat fallopian tube in vivo. We evaluated the protein expression of interleukin-1s (IL-1s) via immunohistochemistry and assessed the key histological features of the ovaries, fallopian tubes, and uteri of the rats following treatment with mild (15 IU) and high (30 IU) doses of gonadotropins compared to an unstimulated control group. The serum concentration of estradiol was also determined by immunoassay. The results revealed that high levels of circulating estrogen were associated with higher expression of the IL-1s protein in the ovaries and fallopian tubes of highly stimulated rats than in those of the mildly stimulated and unstimulated control groups. In contrast, endometrial IL-1s immunoexpression was not affected by ovarian stimulation. The histological features of the fallopian tubes were also dramatically altered upon ovarian stimulation, reducing the efficiency of the fallopian tube. In conclusion, the resulting high circulating estrogen level disrupts the histological features of the fallopian tubes and can lead to higher expression of IL-1s, thus negatively affecting gamete interactions in vivo. These results provide useful information regarding the lower fertilization rate observed following ovarian stimulation.

Ovarian fluid and its impacts on spermatozoa performance in fish: A review

Factors such as gamete quality can profoundly affect fertility, but the spawning micro-environment that surrounds the spermatozoa and eggs during gamete contact has largely been neglected. In fishes, understanding these gametic interactions is crucial because each female creates a unique spawning environment by simultaneously expelling her distinct ovarian fluid (OF) along with an egg batch. In turn, OF has been shown to influence spermatozoa performance traits by modifying spermatozoa behaviors and fertilization outcomes. Here, we shed light on these gametic interactions by overviewing literature on OF and how it impacts spermatozoa performance traits. Fish OF is clear or has slight coloration and can constitute ≤10–30% of egg mass. Viscosity of the OF is ∼2- to 3-fold higher than water and its pH ranges 6.2 to 8.8. Osmolality of the OF is lower in freshwater (190–322 mOsmol/kg) than marine species (289–514 mOsmol/kg). Na+ (98.3–213.7 mmol/L) and Cl− (89.8–172.7 mmol/L) are predominant ions in OF, while K+ (1.7–19.3 mmol/L), Mg2+ (0.4–8.1 mmol/L), and Ca2+(0.5–9.7 mmol/L) ions are detected at lower concentrations. Protein levels can be high in OF and exhibit intra- and inter-species variation (54–826 mg/100 mL). Fish OF also contains a series of organic components and substances that enhance and/or attract sperm towards the vicinity of an egg. OF can also differentially impact sperm based on genetic relatedness of mates, male phenotype (i.e. alternative reproductive tactics), or geographic origin. To conclude, when testing further reproductive paradigms, we suggest a shift from classic spermatozoa activation medium (water only) to more natural spawning media, which encompass OF-spermatozoa interactions.

The Molecular Mechanisms of Gametic Incompatibility in Invertebrates.

Fertilization (gamete fusion followed by zygote formation) is a multistage process. Each stage is mediated by ligand-receptor recognition of gamete interaction molecules. This recognition includes the movement of sperm in the gradient of egg chemoattractants, destruction of the egg envelope by acrosomal proteins, etc. Gametic incompatibility is one of the mechanisms of reproductive isolation. It is based on species-specific molecular interactions that prevent heterospecific fertilization. Although gametic incompatibility may occur in any sexually reproducing organism, it has been studied only in a few model species. Gamete interactions in different taxa involve generally similar processes, but they often employ non-homologous molecules. Gamete recognition proteins evolve rapidly, like immunity proteins, and include many taxon-specific families. In fact, recently appeared proteins particularly contribute to reproductive isolation via gametic incompatibility. Thus, we can assume a multiple, independent origin of this type of reproductive isolation throughout animal evolution. Gametic incompatibility can be achieved at any fertilization stage and entails different consequences at different taxonomic levels and ranges, from complete incompatibility between closely related species to partial incompatibility between distantly related taxa.

Membrane Microdomains as Platform to Study Membrane-Associated Events During Oogenesis, Meiotic Maturation, and Fertilization in Xenopus laevis.

Studies on the egg plasma membrane-associated tyrosine kinase Src have shed light on the identity of the molecular machinery that is responsible for gamete interaction and possibly fusion in African clawed frog Xenopus laevis. Here we describe our protocol for identifying and analyzing molecular and cellular machinery that contributes to a variety of biological processes in the course of oogenesis, oocyte maturation, egg fertilization, and early embryogenesis in Xenopus. Our current special interest is to evaluate the hypothesis that the oocyte/egg membrane microdomain (MD)-associated uroplakin III-Src system is responsible for mediating sperm-egg membrane interaction/fusion signal to the oocyte/egg cytoplasm to initiate embryonic and zygotic development in this species. Therefore, this chapter contains a brief introduction to biology of oocytes and eggs in Xenopus and addresses the following questions: (1) What is oocyte/egg MD? (2) Why do we study oocyte/egg MD? (3) How to manipulate oocyte/egg MD? (4) What has been achieved by oocyte/egg MD studies? (5) What are the next steps in oocyte/egg MD studies?

Genetic Contributions to Ectopic Sperm Cell Migration in Caenorhabditis Nematodes.

Reproductive barriers involving gametic incompatibilities can act to enhance population divergence and promote the persistence of species boundaries. Observing gametic interactions in internal fertilizing organisms, however, presents a considerable practical challenge to characterizing mechanisms of such gametic isolation. Here we exploit the transparency of Caenorhabditis nematodes to investigate gametic isolation mediated by sperm that can migrate to ectopic locations, with this sperm invasion capable of inducing female sterility and premature death. As a step toward identifying genetic factors and mechanisms associated with female susceptibility to sperm invasion, we characterized a panel of 25 C. elegans genetic mutants to test for effects on the incidence and severity of sperm invasion in both conspecific and inter-species matings. We found genetic perturbations to contribute to distinct patterns of susceptibility that identify ovulation dynamics and sperm guidance cues as modulators of ectopic sperm migration incidence and severity. Genotypes confer distinctive phenotypic sensitivities to the sperm from conspecific C. elegans males vs. heterospecific C. nigoni males, implicating evolution of functional divergence in the history of these species for components of sperm-reproductive tract interactions. Sexually-antagonistic co-evolution within species that drives divergent trait and molecular evolution between species provides a working model to explain mismatched species-specific gametic interactions that promote or mitigate ectopic sperm migration.

The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants.

All flowering plants exhibit a unique type of sexual reproduction called 'double fertilization' in which each pollen tube-delivered sperm cell fuses with an egg and a central cell. Proteins that localize to the plasma membrane of gametes regulate one-to-one gamete pairing and fusion between male and female gametes for successful double fertilization. Here, we have identified a membrane protein from Lilium longiflorum generative cells using proteomic analysis and have found that the protein is an ortholog of Arabidopsis DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9)/DUO1-ACTIVATED UNKNOWN 2 (DAU2). The flowering plant DMP9 proteins analyzed in this study were predicted to have four transmembrane domains and be specifically expressed in both generative and sperm cells. Knockdown of DMP9 resulted in aborted seeds due to single fertilization of the central cell. Detailed imaging of DMP9-knockdown sperm cells during in vivo and semi-in vitro double fertilization revealed that DMP9 is involved in gamete interaction that leads to correct double fertilization.

Demographic consequences of reproductive interference in multi-species communities

BackgroundReproductive interference can mediate interference competition between species through sexual interactions that reduce the fitness of one species by another. Theory shows that the positive frequency-dependent effects of such costly errors in mate recognition can dictate species coexistence or exclusion even with countervailing resource competition differences between species. While usually framed in terms of pre-mating or post-zygotic costs, reproductive interference manifests between individual Caenorhabditis nematodes from negative interspecies gametic interactions: sperm cells from interspecies matings can migrate ectopically to induce female sterility and premature death. The potential for reproductive interference to exert population level effects on Caenorhabditis trait evolution and community structure, however, remains unknown.ResultsHere we test whether a species that is superior in individual-level reproductive interference (C. nigoni) can exact negative demographic effects on competitor species that are superior in resource competition (C. briggsae and C. elegans). We observe coexistence over six generations and find evidence of demographic reproductive interference even under conditions unfavorable to its influence. C. briggsae and C. elegans show distinct patterns of reproductive interference in competitive interactions with C. nigoni.ConclusionsThese results affirm that individual level negative effects of reproductive interference mediated by gamete interactions can ramify to population demography, with the potential to influence patterns of species coexistence separately from the effects of direct resource competition.

Matroclinic Haploidy and Gamete Interaction in Maize

The review analyzes the literature on the maize matroclinic haploids and genes that control the gamete interactions and membrane fusion of Arabidopsis and Zea mays plants.

Can the antral follicular count modulate the gene expression of bovine oviducts in Aberdeen Angus and Nelore heifers?

The number of visible ovarian antral follicles (antral follicle count—AFC) is repeatable in bovine individuals, but highly variable between animals, and with differences between Bos taurus and Bos indicus breeds. Several studies have tried to determine the correlation between AFC and increased fertility in cattle. While the impacts of AFC on embryo production, hormonal levels, and pregnancy rates have been described, the molecular effects of AFC on bovine oviducts have not yet been investigated. Here, the aim was to investigate the impact of breeds, such as Aberdeen Angus and Nelore heifer with high or low AFC, on abundance of transcripts and protein related to oviductal transport, sperm reservoir formation, monospermy control, and gamete interaction in the oviducts. In summary, the ovulation side was the major factor that affected transcript abundance on bovine oviducts. However, a discreet effect among AFC and cattle breeds was also observed. Based on this, we concluded and reinforced here that differential microenvironments between ipsilateral and contralateral oviducts have a major effect on modulating the transcripts related to oviductal transport, sperm reservoir formation, monospermy control, and gamete interaction. However, we cannot exclude that there is minimal effect of AFC or breed on regulation of some genes (such as AGTR1, ACE1, FUCA1, and VEGFA) in bovine oviducts.

LOSP: A putative marker of parasperm lineage in male reproductive system of the prosobranch mollusk Littorina obtusata.

Reproductive isolation is the key attribute of biological species and establishment of the reproductive barriers is an essential event for speciation. Among the mechanisms of reproductive isolation, gamete incompatibility due to the variability of gamete interaction proteins may drive fast divergence even in sympatry. However, the number of available models to study this phenomenon is limited. In case of internally fertilized invertebrates, models to study gamete incompatibility and sperm competition mechanisms are restricted to a single taxon: insects. Here, we propose a group of closely related Littorina species as a new model for such studies. Particularly since periwinkles are already thoroughly studied in terms of morphology, physiology, ecology, phylogeny, and ecological speciation. Earlier, we have identified the first species-specific Littorina sperm protein (LOSP) with no known conservative domains or homologies. LOSP is relatively abundant component of sperm extracts and might be involved in gamete incompatibility. Here, we characterize its definitive localization and mRNA expression pattern in the male reproductive system by immunocytochemistry and RNA in situ hybridization. We demonstrate that LOSP distribution is limited to the parasperm cells. Losp gene expression occurs only at the early stages of parasperm development. The protein is stored within granules of mature parasperm and, most likely, is released after ejaculation inside female reproductive system. Thus, LOSP is the only described molluscan paraspermal protein to date, and there is a possibility for LOSP to be involved in gamete incompatibility since heterospermy is a common phenomenon among Littorina.

40 years of bovine IVF in the new genomic selection context.

The development of a complex technology such as in vitro fertilization (IVF) requires years of experimentation, sometimes comparing several species to learn how to create the right in vitro environment for oocytes, spermatozoa and early embryos. At the same time, individual species characteristics such as gamete physiology and gamete interaction are recently evolved traits and must be analysed within the context of each species. In the last 40 years since the birth of Louise Brown, IVF techniques progressed and are now used in multiple domestic and non-domestic animal species around the world. This does not mean that the technology is completely matured or satisfactory; a number of problems remain to be solved and several procedures still need to be optimized. The development of IVF in cattle is particularly interesting since agriculture practices permitted the commercial development of the procedure and it is now used at a scale comparable to human IVF (millions of newborns). The genomic selection of young animals or even embryos combined with sexing and freezing technologies is driving a new era of IVF in the dairy sector. The time has come for a retrospective analysis of the success and pitfalls of the last 40 years of bovine IVF and for the description of the challenges to overcome in the years to come.

NFKB1 rs28362491 and pre-miRNA-146a rs2910164 SNPs on E-Cadherin expression in case of idiopathic oligospermia: A case-control study

A notable proportion of idiopathic male infertility cases is accompanied by oligozoospermia; and yet, the molecular mechanisms of fertilization problem underlying this defect are still unclear. Epithelial cadherin has been involved in several calcium-dependent cell-to-cell adhesion events; however, its participation in gamete interaction has also not been fully investigated. The aim was to investigate the changes in the expression of E-cadherin, based on the frequency of Single nucleotide polymorphisms in Nuclear Factor Kappa-B 1 and pre-mir-146a in oligospermic men. In this case-control study, semen and blood samples of 131 oligospermic men as the case group and 239 fertile healthy men as the control group were analyzed. Variants single nucleotide polymorphisms rs28362491 and rs2910164 were performed using polymerase chain reaction-restriction fragment length polymorphism method and E-cadherin expression were determined by immunoprecipitation studies. ins/ins genotype of rs28362491 was determined as a risk factor for idiopathic oligospermia by 1.73 times (p=0.0218), whereas no significant differences were found between the groups concerning pre-mir-146a rs2910164 polymorphism (p=0.2274 in case of GC genotype and p=0.9052 in case of GG genotype). Combined genotype analysis results did not show any notable differences between the multiple comparisons of 28362491-rs2910164 in oligospermic men and control groups. In addition, E-cadherin expression of oligospermic men with ins/ins genotype was significantly lower than patients with del/ins genotype (p=0.0221). E-cadherin expression level was low in oligospermic men with respect to the control group in presence of ins/ins genotype of NFKB1 gene. These results suggest that ins allele prevents binding of surface proteins to spermatozoa, leading to a low affinity of sperm-oocyte interaction in oligospermic men.

Postmating sexual conflict and female control over fertilization during gamete interaction.

Males and females rarely have identical evolutionary interests over reproduction, and when the fitness of both sexes is dependent upon paternity outcomes, sexual conflict over fertilization is inevitable. In internal fertilizers, the female tract is a formidable selective force on the number and integrity of sperm that reach the egg. Selection on sperm quality is intensified when females mate multiply and rival males are forced to compete for fertilizations. While male adaptations to sperm competition have been well documented (e.g., increased sperm fertilizing capacity), much less attention has been given to the evolutionary consequences of postmating sexual conflict for egg form and function. Specifically, increased sperm competitiveness can be detrimental by giving rise to an elevation in reproductive failure resulting from polyspermy. Spanning literature on both internal and external fertilizers, in this review I discuss how females respond to sperm competition via fertilization barriers that mediate sperm entry. These findings, which align directly with sexual conflict theory, indicate that females have greater control over fertilization than has previously been appreciated. I then consider the implications of gametic sexual conflict in relation to the development of reproductive isolation and speculate on potential mechanisms accounting for "egg defensiveness." Finally, I discuss the functional significance of egg defensiveness for both the sexes, and sperm selection for females.

Effects of the toxic benthic dinoflagellate Ostreopsis cf. ovata on fertilization and early development of the sea urchin Lytechinus variegatus

Blooms of the benthic dinoflagellate Ostreopsis cf. ovata have been recorded with increasing frequency, intensity and geographic distribution. This dinoflagellate produces potent toxins that may cause mortality of marine invertebrates. Adults of sea urchins are commonly affected by O. cf. ovata exposure with evidence of spines loss and high mortality during periods of high dinoflagellate abundances. Here, we report on the effects of the toxic dinoflagellate O. cf. ovata on fertilization and early development of the sea urchin Lytechinus variegatus, a key ecological herbivore. Lytechinus variegatus eggs and sperm were experimentally exposed to different concentrations of Ostreopsis cf. ovata (4, 40, 400, and 4000 cells ml−1) to test the hypothesis that fertilization success, embryonic and larval development of the sea urchin are negatively affected by the toxic dinoflagellate even at low abundances. Reduced fertilization, developmental failures, embryo and larval mortality, and occurrence of abnormal offspring were evident after exposure to O. cf. ovata. Fertilization decreased when gametes were exposed to high O. cf. ovata abundances (400 and 4000 cells ml−1), but just the exposure to the highest abundance significantly reduced fertilization success. Sea urchin early development was affected by O. cf. ovata in a dose-dependent way, high dinoflagellate abundances fully inhibited the early development of L. variegatus. Ostreopsis cf. ovata significantly increased the mortality of sea urchin eggs and embryos in the first hours of exposure (∼1–3 h), regardless of dinoflagellate abundance. Abundances of 400 and 4000 O. cf. ovata cells ml−1 induced significantly higher mortality on sea urchin initial stages in the first hours, and no egg or embryo was found in these treatments after 18 h of incubation. The early echinopluteus larva was only reached in the control and in treatments with low Ostreopsis cf. ovata abundances (4 and 40 cells ml−1). The exposure to O. cf. ovata led to significantly higher occurrence of skeletal anomalies in the early larva of L. variegatus. Interactions of sea urchin gametes and Ostreopsis cells may naturally occur in coastal areas due to the match between O. cf. ovata blooms and L. variegatus reproductive period. Reduced larval density and increased larval abnormalities were observed even at low abundances (4 and 40 cells ml−1) frequently found in tropical environments all year round. The chronic exposure to O. cf. ovata could significantly impact larval fitness, thus compromising recruitment success, and highlight the negative effects of benthic HABs on sea urchin populations and its possible broader ecological implications.

Fertilizing ability of gametes at different post-activation times and the sperm-oocyte ratio in the artificial reproduction of pikeperchSander lucioperca

The time period during which oocyte and spermatozoa retain their fertilizing ability after contacting with water was evaluated in pikeperch (Sander lucioperca). In addition, success of in vitro fertilization was examined regarding to the sperm-to-oocyte ratio (SOR). In the first trial, oocytes were placed in Petri dishes containing 5 ml of the hatchery water, to which freshly collected and pooled sperm were added to each sample at 0, 15, 30, 60, 90, 120, 150 and 180 s post oocyte activation. The oocytes retained their fertility for at least 30 s after contacting with water. The second trial tested the maximum time period during which spermatozoa retained fertilizability after contacting with water. Milt (50 μl) was collected from each male and added to 5 ml of water in Petri dishes. Thereafter, oocytes were added at 0, 5, 15, 30, 60 and 75 s post-sperm activation. Delays exceeding 10 s affected negatively the fertilization success. The third trial examined the optimum SOR; in which was found that 100 × 103 spermatozoa per oocyte were the minimum ratio to ensure fertilization rates above 70%. Overall, the data clarified some biological interactions of gametes in the artificial propagation of pikeperch.

Immunocontraceptive target repertoire defined by systematic identification of sperm membrane alloantigens in a single species.

Sperm competence in animal fertilization requires the collective activities of numerous sperm-specific proteins that are typically alloimmunogenic in females. Consequently, sperm membrane alloantigens are potential targets for contraceptives that act by blocking the proteins’ functions in gamete interactions. Here we used a targeted proteomics approach to identify the major alloantigens in swine sperm membranes and lipid rafts, and thereby systematically defined the repertoire of these sperm-specific proteins in a single species. Gilts with high alloantibody reactivity to proteins in sperm membranes or lipid rafts produced fewer offspring (73% decrease) than adjuvant-only or nonimmune control animals. Alloantisera recognized more than 20 potentially unique sperm membrane proteins and five sperm lipid raft proteins resolved on two-dimensional immunoblots with or without prior enrichment by anion exchange chromatography. Dominant sperm membrane alloantigens identified by mass spectrometry included the ADAMs fertilin α, fertilin ß, and cyritestin. Less abundant alloantigens included ATP synthase F1 β subunit, myo-inositol monophosphatase-1, and zymogen granule membrane glycoprotein-2. Immunodominant sperm lipid raft alloantigens included SAMP14, lymphocyte antigen 6K, and the epididymal sperm protein E12. Of the fifteen unique membrane alloantigens identified, eleven were known sperm-specific proteins with uncertain functions in fertilization, and four were not previously suspected to exist as sperm-specific isoforms. De novo sequences of tryptic peptides from sperm membrane alloantigen “M6” displayed no evident homology to known proteins, so is a newly discovered sperm-specific gene product in swine. We conclude that alloimmunizing gilts with sperm membranes or lipid rafts evokes formation of antibodies to a relatively small number of dominant alloantigens that include known and novel sperm-specific proteins with possible functions in fertilization and potential utility as targets for immunocontraception.

Effects of latrunculin A on the relocation of sperm IZUMO1 during gamete interaction in mouse.

The sperm protein IZUMO1 plays a central role in gamete fusion. In mouse sperm, IZUMO1 is enriched at the acrosomal cap before the acrosome reaction, and at the equatorial segment following this reaction; its relocation is dependent on filamentous actin. How actin polymerization affects IZUMO1 relocation during gamete interaction remains unknown. The present study addressed these processes using latrunculin A (LatA), an inhibitor of actin polymerization. We report that 25 µM LatA blocked actin polymerization in the capacitated sperm head, resulting in a marked decrease in sperm with relocated IZUMO1 during the A23187-induced acrosome reaction and cumulus layer penetration. Treated sperm also exhibited reduced zona pellucida penetration and fertilizing capacity. Interestingly, LatA-treated sperm present in the perivitelline space of eggs did not show impaired IZUMO1 relocation. Thus, IZUMO1 relocation represents one method by which eggs may select for or rescue sperm that are competent to undergo gamete adhesion/fusion. These data support the hypothesis that dynamic movement of IZUMO1 is essential for gamete fusion during mouse fertilization.

Novel species-specific glycoprotein on the surface of Mytilus edulis and M. trossulus eggs

Protein–protein interactions play a central role in the gamete attraction, binding, and fusion stages of gamete interactions and fertilization for broadcast spawning species, such as marine mussels in the Mytilus edulis species complex. Although assortative gamete interaction has been implicated in the level of reproductive isolation among the three species in this complex, the molecular basis of these interactions has not been elucidated. Using mass spectrometry peptide sequencing, cDNA sequencing, and bioinformatics approaches, we have investigated species-level variation in the proteins expressed on the surface of mussel eggs. We herein describe an extracellular protein, MESP-1, from the surface of the eggs of M. edulis and M. trossulus that has a unique domain structure when compared to protein structures that have heretofore been identified. Given variation in the size of MESP-1 predicted from cDNA sequences versus those estimated from SDS-PAGE gels, we conclude this protein is subject to significant species-specific post-translation modifications. Further, bioinformatic analysis of the novel structure of MESP-1 suggests that this protein may be an integral membrane protein involved in sperm–egg fusion, and/or released to the vitelline envelope.