Zona-free Eggs Research Articles

Pharmacological Inactivation of CatSper Blocks Sperm Fertilizing Ability Independently of the Capacitation Status of the Cells: Implications for Non-hormonal Contraception.

Cation channel of sperm (CatSper), the main sperm-specific Ca2+ channel, plays a key role in mammalian fertilization, and it is essential for male fertility, becoming an attractive target for contraception. Based on this, in the present work, we investigated the effects of CatSper inactivation on in vitro and in vivo sperm fertilizing ability and the mechanisms underlying such effects. Exposure of cauda epididymal mouse sperm to different concentrations (1–20 μM) of the potent CatSper inhibitor HC-056456 (HC) during in vitro capacitation showed no effects on sperm viability but significantly affected Ca2+ entry into the cells, progressive motility, protein tyrosine phosphorylation, induced acrosome reaction, and hyperactivation, as well as the sperm’s ability to in vitro fertilize cumulus oocyte complexes and zona-free eggs. Whereas the presence of HC during gamete coincubation did not affect in vitro fertilization, exposure of either non-capacitating or already capacitated sperm to HC prior to gamete coincubation severely reduced fertilization, indicating that sperm function is affected by HC when the cells are incubated with the drug before sperm–egg interaction. Of note, insemination of HC-treated sperm into the uterus significantly or completely reduced the percentage of oviductal fertilized eggs showing, for the first time, the effects of a CatSper inhibitor on in vivo fertilization. These observations, together with the finding that HC affects sperm fertilizing ability independently of the sperm capacitation status, provide further insights on how CatSper regulates sperm function and represent a solid proof of concept for developing a male/female non-hormonal contraceptive based on the pharmacological blockage of CatSper activity.

Cross-species fertilization: the hamster egg receptor, Juno, binds the human sperm ligand, Izumo1.

Fertilization is the culminating event in sexual reproduction and requires the recognition and fusion of the haploid sperm and egg to form a new diploid organism. Specificity in these recognition events is one reason why sperm and eggs from different species are not normally compatible. One notable exception is the unusual ability of zona-free eggs from the Syrian golden hamster (Mesocricetus auratus) to recognize and fuse with human sperm, a phenomenon that has been exploited to assess sperm quality in assisted fertility treatments. Following our recent finding that the interaction between the sperm and egg recognition receptors Izumo1 and Juno is essential for fertilization, we now demonstrate concordance between the ability of Izumo1 and Juno from different species to interact, and the ability of their isolated gametes to cross-fertilize each other in vitro. In particular, we show that Juno from the golden hamster can directly interact with human Izumo1. These data suggest that the interaction between Izumo1 and Juno plays an important role in cross-species gamete recognition, and may inform the development of improved prognostic tests that do not require the use of animals to guide the most appropriate fertility treatment for infertile couples.

Transplantation of zona-free cleavage stage embryo into an empty zona with successful blastulation, a case series

Transplantation of zona-free cleavage stage embryo into an empty zona with successful blastulation, a case series

Evaluation of Testicular Sperm CRISP2 as a Potential Target for Contraception

Cysteine-rich secretory protein 2 (CRISP2) is a testicular sperm protein proposed to be involved in fertilization. With the aim of examining the relevance of CRISP2 for fertility and its potential use as a target for contraception, in the present work, male and female rats were immunized with recombinant CRISP2 coupled to maltose-binding protein (MBP) and evaluated for their subsequent fertility. As controls, animals were injected with either MBP or recombinant CRISP1. Enzyme-linked immunosorbent assay of sera collected at different intervals after immunization indicated that CRISP2 immunization raised specific antibodies in both sexes, with levels that increased as a function of time. Western blot studies revealed that anti-CRISP2 sera were capable of recognizing CRISP2 in testicular, epididymal, and sperm extracts, whereas histological studies showed no evidence of autoimmune orchitis or epididymitis. Indirect immunofluorescence experiments revealed the ability of anti-CRISP2 sera to recognize the native sperm protein in fresh, capacitated, and ionophore-induced acrosome-reacted cells. Moreover, anti-CRISP2 sera significantly inhibited the sperm ability to penetrate zona-free eggs, confirming the role of CRISP2 in rat gamete fusion. In spite of the presence of circulating anti-CRISP2 antibodies capable of inhibiting the sperm fertilizing ability, mating studies revealed no effects of CRISP2 immunization on male or female fertility, in contrast to the significant inhibition observed in both sexes in animals injected with CRISP1. Together, these observations indicated the immunogenic properties of testicular CRISP2 but do not support CRISP2 as a target for immunocontraception or as a molecule responsible for generating autoimmune orchitis or immunoinfertility.

Cysteine-rich secretory proteins (CRISP) and their role in mammalian fertilization.

Epididymal protein CRISPI is a member of the CRISP (Cysteine-RIch Secretory proteins) family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2). In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-deficient mice exhibited normal fertility, CRISP1-defficient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zona-free eggs in vitro, confirming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization.

Na,K-ATPase alpha4 Isoform Is Critical for Sperm Motility and Fertility.

Active exchange of Na+ and K+ across the sperm plasma membrane is under the control of the Na,K-ATPase, an integral membrane enzyme, composed of catalytic alpha and glycosylated α subunits. Two molecular variants of the alpha subunit, the ubiquitous α1 and the sperm specific alpha4 coexist in the male gamete. These isoforms exhibit different biochemical properties; however, their function in sperm fertility is unknown. Here, we show that genetic deletion of alpha4 in mice causes complete male infertility. Sperm from these mice are unable to fertilize zona intact and zona free eggs in vitro. Sperm null in the alpha4 isoform show abnormal oocyte binding. Deletion of alpha4 produces severe reduction in sperm total, progressive and in a series of critical parameters of sperm motility. Moreover, alpha4 null sperm shows drastic reduction in the hyperactivation typical of sperm capacitation. In addition, absence of alpha4 causes a characteristic bend in the sperm flagellum, indicative of abnormal sperm ion regulation. Also, sperm devoid of alpha4 presents other alterations, including depolarization of the cell plasma membrane and increase in intracellular Na+ levels. Overall, this demonstrates the absolute requirement of alpha4 for sperm fertility and the inability of the alpha1 isoform to compensate for alpha4. Our findings reveal alpha4 as an attractive biomarker for male fertility and a novel target for male contraception. [Supported by NIH grants HD043044 and HD055763]. (poster)

Fertilizability of Mouse Eggs Treated with Mannosidase

Abstract: Using zona-free mouse eggs treated with mannosidase, the role of mannose on the surface of eggs in fertilization was examined. The fertilization rates of eggs treated with mannosidase at concentrations of 0.05, 0.1, 0.5 or 1.0 units/100 µl were 47.8, 40.4, 28.2 and 18.8%, respectively, which were all significantly lower than the 69.2% of control eggs with no mannosidase treatment. The binding of Canavalia ensiformis agglutinin (Con A), a lectin with an affinity to mannose, was strong on the surface of all control eggs not treated with mannosidase. However, the rate of eggs showing strong Con A-binding was significantly decreased among those treated with mannosidase at a concentration of 0.1 units/100 µl (44.0%). These findings suggest that mannose on the surface of the cytoplasmic membrane, in addition to zona pellucida, of eggs has a possible role in sperm recognition.

Epididymal Protein CRISP1 Plays Different Roles During the Fertilization Process

Rat epididymal CRISP1, the first described member of the evolutionarily conserved Cysteine-RIch Secretory Protein (CRISP) family, is expressed in the proximal regions of the epididymis and associates with the sperm during epididymal transit. Evidence indicates the existence of 2 populations of CRISP1 in spermatozoa: a major one, loosely bound, which is released during capacitation and, therefore, proposed as a decapacitating factor; and a minor one, strongly associated with spermatozoa that remains on the cells after capacitation and is proposed to participate in gamete interaction. Originally localized to the dorsal region of capacitated sperm, CRISP1 migrates to the equatorial segment with capacitation and acrosome reaction. Consistent with these localizations, in vitro fertilization experiments support the involvement of CRISP1 in the first step of sperm-zona pellucida (ZP) interaction and subsequent gamete fusion through its interaction with egg-complementary sites. The potential roles of CRISP1 in capacitation and fertilization have been further supported by the finding that capacitated spermatozoa from CRISP1 "knockout" animals exhibit low levels of protein tyrosine phosphorylation and have an impaired ability to fertilize zona-intact and zona-free eggs in vitro. Moreover, recent evidence from mutant spermatozoa reveals that CRISP1 mediates the stage of sperm binding to the ZP. Altogether, these observations support the view that CRISP1 is a multifunctional protein playing different roles during fertilization through its different associations with and localizations on spermatozoa. We believe these results contribute to a better understanding of the molecular mechanisms involved in both the fertilization process and the acquisition of sperm-fertilizing ability that occurs during epididymal maturation.

Cysteine-Rich Secretory Proteins (CRISP) and their role in mammalian fertilization

Epididymal protein CRISPI is a member of the CRISP (Cysteine-RIch Secretory proteins) family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2). In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-deficient mice exhibited normal fertility, CRISP1-defficient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zona-free eggs in vitro, confirming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization.

Accelerated modification of the zona pellucida is the primary cause of decreased fertilizability of oocytes in the 129 inbred mouse strain

We investigated whether the small litter size in the 129 inbred mouse strain results from a reduction in oocyte fertilizability. Sensitivity of the zona pellucida to α-chymotrypsin was examined for oocytes collected at 14 h (shortly after ovulation), 17 h, and 20 h after hCG injection. Passage of spermatozoa through the zona pellucida (using an in vitro fertilization (IVF) technique) and the density of cortical granules were examined for oocytes collected at 14 and 17 h after hCG injection. The capability of the oolemma to fuse with the sperm plasma membrane was also evaluated by IVF using zona-free eggs. The zona pellucida became markedly resistant to the enzyme 17 h after hCG injection. IVF rates significantly decreased at this time. In addition, there was a significant reduction in the density of cortical granules. When zona-free oocytes were inseminated, high fertilization rates were obtained at both 17 and 14 h after hCG injection. These results indicate that accelerated modification of the zona pellucida primarily causes a decreased fertilizability of oocytes in 129 mice, resulting in the low reproductive performance of this strain.

Role of sperm αvβ3 integrin in mouse fertilization

Oocyte integrins have been described as essential for fertilization. But this concept has been challenged by deletion experiments. Recently, we have shown that sperm integrin alpha6beta1 plays a determinant role in mouse gamete interaction. In this study, we demonstrate the presence of alphavbeta3 integrin by Western blot and immunofluorescence on the sperm membrane. Oocytes and/or sperm preincubations with anti-alphav or anti-beta3 antibodies were performed before in vitro fertilization on cumulus-intact and zona-free egg assays. We observed inhibitory effects on the fusion process mostly by means of sperm function. An antibody directed against vitronectin inhibited gametes fusion, whereas the presence of exogenous vitronectin increased its efficiency. We suggest that vitronectin (on multimeric forms) can play a first nonspecific link corresponding to loosely bound spermatozoa to oocyte and that this link could be mediated by means of oocyte proteoglycans or integrins, and sperm alphavbeta3 integrin.

Immunocontraceptive potential of the Ig‐like domain of Izumo

To investigate whether the Ig-like domain of sperm protein Izumo or the other part of the protein could be used as an immunocontraceptive antigen, three partially overlapping cDNA fragments (PA, PB, and PC), together covering entire mouse Izumo, were cloned, expressed, and purified. PB contains the whole Ig-like domain of mouse Izumo. The anti-PB antibody significantly inhibited the fusion of sperm with zona-free mouse eggs with no effect on sperm motility, while anti-PA and anti-PC antibodies virtually had no effect on sperm-egg fusion at the same concentration. Furthermore, in the presence of anti-PB antibody, the anti-sperm reactivity could be competitively inhibited by recombinant PB protein. The PB-specific antibody staining was restricted to the acrosome region in acrosome-reacted mouse spermatozoa by indirect immunofluorescence. Active immunization with the PB antigen sharply raised the antibody titers in mouse that were enough to cause a significant reduction in fertility compared to the PA and PC immunized groups. In conclusion, our data indicate that the Ig-like domain of Izumo plays an important role in the fertilization process, as verified by the dose-dependent reduction in fertilization rates in mouse IVF trials and mouse mating assay. These results indicate that the Ig-like domain of Izumo might be a new candidate for the development of a contraceptive vaccine.

Strict criteria for the evaluation of human sperm morphology and its impact on assisted reproduction.

Sperm morphology abnormalities can be identified in a large proportion of patients with failed and delayed fertilization when assessing this parameter with strict criteria. Statistical analyses of a large IVF population confirm that, in patients with severe teratozoospermia, fertilization rates can be enhanced in many cases by increasing insemination concentration. However, implantation rates are impaired, thereby affecting the ability of these patients to establish a viable pregnancy. Data from couples undergoing controlled ovarian stimulation/intrauterine insemination seem to support the use of strict morphology as a predictor of outcome in this setting, too. Although a descriptive parameter, sperm morphology assessed by strict criteria can be used as a biomarker of sperm dysfunction(s). These dysfunctions are multiple and include abnormalities of motion parameters, poorer capacity to bind to homologous zona pellucidae and penetrate zona-free eggs, and low incidence of spontaneous and induced acrosome reaction. At a cellular level, poor morphology is associated with a higher content of creatine kinase and impaired capacity to undergo appropriate changes in intracellular calcium concentration. Whether nuclear/DNA abnormalities are present still remains to be fully determined. A definitively objective evaluation of morphology based on these criteria may allow all centres specializing in assisted reproduction to analyse semen in a homogeneous fashion.

Evaluation of sperm function tests in samples utilized in IVF

OBJECTIVE: The laboratory evaluation of male infertility remains an essential area of research as almost one third of cases are classified as idiopathic. The predictive value of traditional semen parameters and sperm function tests to the outcome of IVF treatment remains controversial. The objective of this study was to evaluate the relationship between IVF outcomes and sperm function tests performed on an aliquot of the same sperm sample utilized for insemination in that cycle. DESIGN: Prospective analysis of IVF outcomes and sperm function tests. MATERIALS AND METHODS: Following Institutional Research Ethics Board approval, 36 couples with normozoospermia (WHO criteria) underwent conventional IVF treatment. An aliquot of the remaining semen sample utilized for insemination was immediately transferred to the Andrology Laboratory which had no knowledge of the IVF outcome. The samples were analyzed for strict sperm morphology, hemi-zona pellucida binding assay (HZA) and hamster zona-free egg penetration test (HEPT). Statistical evaluation was performed with SPSS 15.0 software and results expressed as mean ± SD. RESULTS: Clinical pregnancies were achieved in 9 patients (25%). In 5 patients (14%), no embryo transfer (ET) was performed due to failed fertilization (n=4) or abnormal embryo development (n=1). Patients were divided into groups based on IVF outcome (clinical pregnancy rate).Tabled 1Not pregnant (n=27)ParameterGroup I Pregnant (n=9)Group II ET (n=22)Group III No ET (n=5)Female age33.6 ± 3.333.6 ± 3.336.6 ± 3.3Male age34.8 ± 3.334.5 ± 4.439.2 ± 7.9Strict morphology (%)15.9 ± 6.812.7 ± 6.49.0 ± 4.8Oocytes retrieved (n)12.3 ± 7.110.9 ± 4.68.6 ± 5.6Oocytes fertilized (n)6.6 ± 5.76.7 ± 3.00.8 ± 0.8∗P < 0.05 between all groupsFertilization rate (%)50.6 ± 22.263.7 ± 22.922.9 ± 43.2∗P < 0.05 between all groupsEmbryo score200.7 ± 198.8185.1 ± 90.46.4 ± 8.7∗P < 0.05 between all groupsHEPT score (n)21.4 ± 5.18.7 ± 6.45.1 ± 5.8∗P < 0.05 between all groupsHZA (%)60.9 ± 23.442.9 ± 26.318.8 ± 19.0∗P < 0.05 between all groups∗ P < 0.05 between all groups Open table in a new tab CONCLUSIONS: This is the first study to evaluate sperm function tests on samples used concurrently for insemination in IVF. Results showed that more than a quarter of the patients had abnormal tests of sperm binding or sperm penetration. Most abnormal results were detected in the group of patients with no pregnancies and no ET. The data support the importance of sperm function testing in the presence of normal semen analysis to identify appropriate treatment options, minimize the cost of treatment and to improve pregnancy rates.

PGAP1 Knock-out Mice Show Otocephaly and Male Infertility

A palmitate linked to the inositol in glycosylphosphatidylinositol (GPI) is removed in the endoplasmic reticulum immediately after the conjugation of GPI with proteins in most cells. Previously, we identified PGAP1 (post GPI attachment to proteins 1) as a GPI inositoldeacylase that removes the palmitate from inositol. A defect in PGAP1 caused a delay in the transport of GPI-anchored proteins (GPI-APs) from the endoplasmic reticulum to the cell surface in Chinese hamster ovary cells, although the cell-surface expression of GPI-APs in the steady state was normal. Nevertheless, in most cells, GPI-APs undergo deacylation. To elucidate the biological significance of PGAP1 in vivo, we established PGAP1 knock-out mice. Most PGAP1 knock-out mice showed otocephaly, a developmental defect, and died right after birth. However, some survived with growth retardation. Male knock-out mice showed severely reduced fertility despite the capability of ejaculation. Their spermatozoa were normal in number, motility, and ability to ascend the uterus, but were unable to go into the oviduct. In vitro, PGAP1-deficient spermatozoa showed weak attachment to the zona pellucida and a severely diminished rate of fertilization. Therefore, an extra acyl chain in GPI anchors caused severe deleterious effects to development and sperm function.

Alpha6beta1 integrin expressed by sperm is determinant in mouse fertilization

BackgroundBased on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization.ResultsUsing Western blot analysis and immunofluorescence, we showed that the mouse sperm expresses the alpha6beta1 integrin. As for oocyte, binding of GoH3 anti-alpha6 antibody to sperm induces a specific inhibition of sperm fertilizing ability. Comparing zona-intact and zona-free eggs in fusion tests, we showed that the removal of the zona pellucida by acid treatment bypasses fertilizing oocyte alpha6beta1 integrin's function in the adhesion/fusion process.ConclusionThese findings show that alpha6beta1 integrin is expressed by both gametes and is functional in their membranes interaction. These results and previous reports, about fertilization of alpha6 or beta1 integrin subunits deleted oocytes by wild type sperm, suggest that the presence of alpha6beta1 integrin on one of the two gamete membranes can rescue the fertilization process. This hypothesis is further supported by the exchange of membrane fragments occurring between gametes prior to fusion that we recently reported.

Evidence for the Involvement of Testicular Protein CRISP2 in Mouse Sperm-Egg Fusion1

CRISP2, originally known as Tpx-1, is a cysteine-rich secretory protein specifically expressed in male haploid germ cells. Although likely to be involved in gamete interaction, evidence for a functional role of CRISP2 in fertilization still remains poor. In the present study, we used a mouse model to examine the subcellular localization of CRISP2 in sperm and its involvement in the different stages of fertilization. Results from indirect immunofluorescence and protein extraction experiments indicated that mouse CRISP2 is an intraacrosomal component that remains associated with sperm after capacitation and the acrosome reaction (AR). In vitro fertilization assays using zona pellucida-intact mouse eggs showed that an antibody against the protein significantly decreased the percentage of penetrated eggs, with a coincident accumulation of perivitelline sperm. The failure to inhibit zona pellucida penetration excludes a detrimental effect of the antibody on sperm motility or the AR, supporting a specific participation of CRISP2 at the sperm-egg fusion step. In agreement with this evidence, recombinant mouse CRISP2 (recCRISP2) specifically bound to the fusogenic area of mouse eggs, as previously reported for rat CRISP1, an epididymal protein involved in gamete fusion. In vitro competition investigations showed that incubation of mouse zona-free eggs with a fixed concentration of recCRISP2 and increasing amounts of rat CRISP1 reduced the binding of recCRISP2 to the egg, suggesting that the proteins interact with common complementary sites on the egg surface. Our findings indicate that testicular CRISP2, as observed for epididymal CRISP1, is involved in sperm-egg fusion through its binding to complementary sites on the egg surface, supporting the idea of functional cooperation between homologous molecules to ensure the success of fertilization.

Sperm defects in mice lacking a functional Niemann–Pick C1 protein

The Niemann-Pick C1 (NPC1) gene encodes for a multiple membrane spanning protein, which regulates the trafficking of low-density lipoprotein-mediated endocytosed cholesterol. Mutation of the human NPC1 gene causes Niemann-Pick type C (NPC) disease. The Npc1(NIH) mice, a model of human NPC disease, bear a spontaneous mutation of the Npc1 gene, and are infertile. In this study, we have performed sperm analysis to search for the cause of male infertility in the Npc1(NIH) mouse. The number of cauda sperms in Npc1(-/-) mice was decreased roughly three-and-half-fold of that in wild-type mice. The decreased sperm number in Npc1(-/-) mice is due, at least in part, to partial arrest of spermatogenesis in the testes, as revealed by histological analysis. Compared to wild-type sperm, Npc1(-/-) sperm displayed a high frequency of morphological abnormalities, including tailless heads and aberrant heads. In the in vitro fertilization (IVF) assay using cumulus-intact eggs, Npc1(-/-) sperm failed to produce two-cell embryos. In the IVF assay where zona-free eggs were used, Npc1(-/-) sperm bound normally but could not fuse with the egg. Further analysis indicated that Npc1(-/-) sperms are drastically impaired in the binding to the egg zona pellucida, only 14% of the level of wild-type sperm. Moreover, on Npc1(-/-) cauda sperm, one-third of the total cyritestin protein was not proteolytically processed, while fertilin beta was processed normally. Taken together, these results demonstrate that there are multiple defects in sperms from mice lacking a functional NPC1 protein, and these observed sperm defects may result in sterility.

CD9 controls the formation of clusters that contain tetraspanins and the integrin α6β1, which are involved in human and mouse gamete fusion

The process of gamete fusion has been largely studied in the mouse and has revealed the crucial role of the tetraspanin CD9. By contrast, human gamete fusion remains largely unknown. We now show that an anti-alpha 6 integrin mAb (GoH3) strongly inhibited human sperm-egg fusion in human zona-free eggs. Furthermore, a mAb directed against CD151, a tetraspanin known to associate with alpha 6 beta1, partially inhibited sperm-egg fusion. By contrast, the addition of an anti-CD9 mAb to zona free eggs had no effect. The integrin alpha 6 beta1, CD151 and CD9 tetraspanins were evenly distributed on human zona-intact oocytes. On zona-free eggs, the integrin alpha 6 beta1 and tetraspanin CD151 patched and co-localized but the tetraspanin CD9 remained unchanged. CD9 mAb prevented alpha 6 beta1 integrin clustering and gamete fusion when added prior to, but not after, zona removal. Antibody-mediated aggregation of integrin alpha 6 beta1 yielded patches that were bigger and more heterogeneous in mouse oocytes lacking CD9. Moreover, a strong labelling of alpha 6 beta1 could be observed at the sperm entry point. Altogether, these data show that CD9 controls the redistribution of some membrane proteins including the alpha 6 beta1 integrin into clusters that may be necessary for gamete fusion.

Delineation of the functional domains of the extracellular region of YWK-II Protein/APLP2 of sperm membrane

The sperm membrane protein, designated as YWK-II protein/APLP2, is a member of the amyloid precursor protein (APP) superfamily and is a type I transmembrane protein involved in fertilization. Here, the structure-function of the domains of YWK-II protein was examined. Five segments with overlapping ends encompassing the entire extracellular region of mouse YWK-II gene were prepared, cloned and separately expressed in E. coli. The recombinant YWK-II segments were fused with glutathione S-transferase (GST), purified and evaluated for their antifertility activities by measuring their capacity to block in vitro mouse sperm-egg interaction. The structural domain(s) involved in the fertilization process was identified. The polypeptide segment corresponding to position 22-207 of YWK-II-763 inhibited the early stage of fertilization when the spermatozoa interacted with zona-free eggs; whereas the polypeptide segment 201-395 (lacking 309-364) of YWK-II-763 blocked sperm-egg membrane fusion. The remaining three segments, 201-395, 389-574 and 517-704 (lacking 613-624) of YWK-II-763, did not influence the in vitro fertilization process. The present results suggest that segment 22-308 of YWK-II-763 participates in the binding and fusion of sperm and egg plasma membranes thereby promoting fertilization.