Carbohydrate involvement in sperm-egg fusion in mice

The potential involvement of cell-surface carbohydrates in sperm-egg fusion in mice was evaluated in this study. Zona-free mouse eggs were inseminated in the presence of a variety of simple saccharides to determine if certain sugars would act as competitive inhibitors of sperm-egg fusion. Of the sugars tested, L-fucose, galactose, and N-acetylglucosamine caused the greatest inhibition of sperm penetration levels relative to controls. A number of complex saccharides or glycoproteins with differing carbohydrate structures, including fucoidan, ascophyllan, ovomucoid, ovalbumin, fetuin, asialofetuin, and chondroitin sulfate, were also tested as competitive inhibitors of fusion. Only the L-fucose containing saccharides fucoidan and ascophyllan caused significant inhibition of fusion at concentrations of 0.05-1.0 mg/ml and 0.1-5.0 mg/ml, respectively. None of the other compounds tested had any inhibitory effect on fusion when tested at concentrations up to 5 mg/ml. The effect of the inhibitory saccharides was not due to the presence of residual zona material on the surface of the zona-free eggs, since zona-free eggs did not bind an 125I-labeled antibody directed against the ZP3 protein of the mouse zona pellucida. Pretreatment of either sperm or eggs with fucoidan (1 mg/ml) for 60 min prior to insemination caused only small decreases in sperm penetration levels, indicating that fucoidan exerted its major inhibitory effect on fusion only when present during insemination. Treatment of sperm, but not zona-free eggs, with fucosidase prior to insemination caused significant reductions in sperm penetration levels. Other glycosidic enzymes, including glucosidase, galactosidase, and N-acetylglucosaminidase, had no inhibitory effect on the sperm. These data suggest that an L-fucose component of the sperm surface is involved in sperm-egg fusion in the mouse.     

A preferential site for sperm-egg fusion in mammals

The tendency of mammalian sperm-egg fusion to occur at a site away from the first polar body was investigated in a homologous (mouse oocytes and mouse spermatozoa) and in a heterologous model (hamster oocytes and mouse spermatozoa). Following micromanipulation of the zona pellucida either in proximity to or opposite the first polar body, in vitro fertilization was performed and subsequent differences in sperm-egg interaction were evaluated. Since spermatozoa from random-bred mice do not readily penetrate intact zonae pellucidae in vitro, it is likely that zona penetration occurred through the artificial holes in both models. The creation of a gap in the zona pellucida opposite the first polar body was associated with levels of sperm fusion that were significantly higher than those resulting from manipulation near the first polar body. Spermatozoa were rarely found to penetrate the hole completely, and in general few spermatozoa were observed in the pervitelline space. The proximity between pronuclei following sperm penetration was correlated with the position of the incision with respect to the polar body. The findings suggest that breaching the zona pellucida for microsurgical fertilization should be performed away from the microvillus-free area of the oocyte.     

Processes controlling sperm-egg fusion

Sperm interaction with the egg envelopes triggers the acrosome reaction. Indeed, sperm-egg fusion is accomplished by the fusion of the acrosomal process (or of the exposed inner acrosomal membrane in mammals) with the egg plasma membrane. Fusion must be preceded by the establishment of molecular contact between the two membranes. It is suggested that, as in the case of artificial phospholipid membranes, the two major obstacles to the establishment of molecular contact are electrostatic repulsion and the hydration barrier. It is argued that morphology of the acrosome is such as to favour the overcoming of such barriers. By analogy with the conditions governing fusion of artificial phospholipid membranes and cell fusion, it is proposed that the following processes play a role in sperm-egg fusion. The large calcium uptake accompanying the acrosome reaction may help fusion either through the known effect of calcium on fusion of phospholipid membranes or by shielding the surface charges of the acrosomal process. Fusogenic proteins at the surface of the acrosomal process are likely to play a role in the fusion of the acrosomal process with the egg plasma membrane. The activation of phospholipases in conjunction with the acrosome reaction may also be instrumental in sperm-egg fusion through the transient production of lysophosphatides. Clearance or translocation of intramembraneous proteins in the egg plasma membrane at the site of contact with the acrosomal process may also be required for fusion. Lastly it is suggested that a translocation or a conformational change of some proteins of the egg plasma membrane, which is required for fusion, may be induced by the depolarization of the egg plasma membrane that follows molecular contact with the acrosomal process.     

The sperm-egg fusion in the nematode Parascaris equorum

Summary

The process of fertilization and the sperm storage in the female apparatus in Parascaris equorum is described in this paper. The sperm approaches the egg by means of pseudopodia containing bundles of microfilaments. The sperm and egg membranes fuse and the sperm penetrates progressively into the ovum. The egg and sperm plasma membranes and glycocalyces disappear at the point of fusion. At the end of fertilization, they are reformed at the egg's surface, while the egg and sperm chromatin begins to decondense. Spermatozoa are stored in the female apparatus prior to fertilization; here they come into contact with the epithelial cells of the spermatheca, protruding pseudopodia rich in microfilaments into the cellular body.     

The molecular players of sperm-egg fusion in mammals

Fertilization includes a series of cellular interactions culminating with the fusion of gamete membranes, creating a zygote. Two ADAM proteins present on sperm, fertilin beta and cyritestin, drew much attention. However, gene deletion in mice showed that fusion can happen in their absence. The presence of the integrin alpha6beta1 on egg, a putative fertilin beta receptor, is also dispensable. In contrast, sperm lacking Izumo, a molecule with a single Ig domain, are unable to fuse. On the egg side, a role for GPI-anchored molecules has been shown, and in mice lacking both tetraspanins CD9 and CD81 fertilization is completely blocked.     

Fertilization in a chiton: Acrosome-mediated sperm-egg fusion

Contrary to the widely accepted view that chiton sperm lack acrosomes and that fertilization in this group occurs via a micropyle, we demonstrate here that fertilization in Tonicella lineata occurs by acrosome-mediated sperm-egg fusion. The acrosome is a small vesicle containing two granules located at the tip of the sperm. The eggs have an elaborate hull (=chorion), which is formed into cupules that remain covered by follicle cells until maturity. When dissected ripe eggs were exposed to sperm in vitro, the sperm were attracted only to open cupules, inside which they swam through one of seven channels to the base where they penetrated the hull. The acrosome fired on contact with, or in, the hull, and during passage through it the apical granule was exhausted while the basal granule was exposed. If sperm contacted follicle cells between the cupules the acrosome did not react. The vitelline layer beneath the hull contains pores arranged in a regular pattern. Embedded in the base of each pore is an egg microvillus. Having penetrated the hull the sperm anterior filament located a pore and fused with the tip of the egg microvillus projecting into it. This created a membranous tube, through which the sperm nucleus was injected into the egg. The egg membrane appeared to be raised up into a small fertilization cone around the penetrating sperm, the vitelline layer became slightly elevated, and some cortical granules were released by exocytosis.     

哺乳动物精子与卵子质膜粘附和融合的分子基础

精子与卵子质膜粘附并发生融合是哺乳动物完成受精过程所必需的步骤。近年来,学者们以现代分子生物学理论为基础,对参与精卵质膜粘附、融合过程的分子进行了研究,特别是精子表面的去整合素金属蛋白酶基因家族(ADAM)和卵子表面的整合素蛋白。本文通过对精子表面的受精素仅、受精素β、cyritestin,卵子表面的α6β1、CD9等蛋白分子的研究,揭示了这些分子对粘附、融合的重要作用,为提高受精率提供了重要的依据。     

Evidence for the involvement of testicular protein CRISP2 in mouse sperm-egg fusion

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.     

ADAMs参与哺乳动物精-卵结合与融合

ADAMs家族是含多结构域的跨膜蛋白。睾丸特异的ADAMs,在精子发生与附睾精子转运过程中,经过蛋白水解成为成熟精子的分子形式,与精．卵质膜结合和融合有关。对于精-卵质膜相互作用,ADAMs去整合素域具有关键氨基酸残基和特殊模体。模拟ADAM2和ADAM3去整合素域的短肽能用于鉴别特异性卵子识别蛋白。精子ADAMs去整合素域与卵子膜蛋白整合素β1、α4／α9、α6和CD9相互作用,介导了精卵质膜的结合与融合。     

A model for sperm-egg binding and fusion based on ADAMs and integrins

Once a sperm meets an egg, several events must occur in order for fertilization to proceed. Sperm must bind to the zona pellucida, undergo the acrosome reaction, penetrate the zona pellucida and then bind to and fuse with the egg plasma membrane. Shortly thereafter, the egg must be activated for zygotic development. This review focuses on mammalian sperm-egg plasma membrane binding and fusion, and in particular on the roles of two families of cell-adhesion molecules, ADAMs and integrins, in this important union.     

Analysis of the role of egg integrins in sperm-egg binding and fusion

Sperm-egg fusion is believed to be mediated via specific molecular interactions. Integrin alpha6beta1 is a strong candidate for a sperm receptor on the egg plasma membrane. However, the ability of the egg integrin alpha6beta1 to interact with molecules on intact sperm has not yet been proven. In this report, possible involvement of integrin alpha6beta1 in sperm-egg interactions was examined by biochemical and immunocytochemical analyses. To identify egg molecules that specifically interact with sperm, we first incubated sperm with biotin-labeled egg surface proteins. Under this condition, solubilized proteins from eggs inhibited sperm-egg fusion. Western blot analysis under reducing conditions indicated that a major-labeled band of 135 kDa bound to sperm. An immunodepletion experiment using the anti-integrin alpha6 antibody GoH3 indicated that the 135 kDa egg surface molecule that bound to sperm was the integrin alpha6 subunit. To investigate the potential involvement of integrin alpha6beta1 in sperm-egg fusion, we next examined the localization of integrin alpha6 and beta1 subunits before and after fertilization by confocal laser microscopy. At an early stage of sperm-egg fusion, the integrin alpha6 and beta1 subunits were accumulated at the sperm binding site. The frequency of cluster formation was closely related to that of sperm-egg fusion, indicating that integrin receptors are accumulated by sperm destined for fusion. Taken together, these results strongly suggest that the integrin alpha6beta1 is involved in sperm-egg binding leading to fusion via direct association of the integrin alpha6 with sperm.     

Mammalian sperm-egg fusion: evidence that epididymal protein DE plays a role in mouse gamete fusion

Rat epididymal protein DE associates with the sperm surface during epididymal maturation and is a candidate molecule for mediating gamete membrane fusion in the rat. Here, we provide evidence supporting a role for DE in mouse sperm-egg fusion. Western blot studies indicated that the antibody against rat protein DE can recognize the mouse homologue in both epididymal tissue and sperm extracts. Indirect immunofluorescence studies using this antibody localized the protein on the dorsal region of the acrosome. Experiments in which zona-free mouse eggs were coincubated with mouse capacitated sperm in the presence of DE showed a significant and concentration-dependent inhibition in the percentage of penetrated eggs, with no effect on either the percentage of oocytes with bound sperm or the number of sperm bound per egg. Immunofluorescence experiments revealed specific DE-binding sites on the fusogenic region of mouse eggs. Because mouse sperm can penetrate zona-free rat eggs, the participation of DE in this interaction was also investigated. The presence of the protein during gamete coincubation produced a significant reduction in the percentage of penetrated eggs, without affecting the binding of sperm to the oolemma. These observations support the involvement of DE in an event subsequent to sperm-egg binding and leading to fusion in both homologous (mouse-mouse) and heterologous (mouse-rat) sperm-egg interaction. The lack of disintegrin domains in DE indicates that the protein interacts with its egg-binding sites through a novel mechanism that does not involve the reported disintegrin-integrin interaction.     

Mammalian sperm-egg fusion: the rat egg has complementary sites for a sperm protein that mediates gamete fusion.

Rat epididymal protein DE is localized on the fusogenic region of the acrosome-reacted spermatozoa and has a potential role in sperm-egg fusion. We investigated the presence of DE binding sites on the egg surface by co-incubating zona-free eggs and capacitated sperm in different concentrations of pure DE. Results indicate that DE produced a concentration-dependent decrease in egg penetration by sperm (fusion), with almost complete inhibition at 200 micrograms/ml. This inhibition was not due to an effect of DE on initial sperm binding to the egg membrane, since the presence of this protein did not affect the percentage of oocytes with bound sperm nor the number of bound sperm per egg. Those sperm that failed to penetrate the egg in the presence of DE became able to do so after transfer of the eggs to protein- and sperm-free medium, indicating a role for DE in an event subsequent to binding and leading to fusion. Indirect immunofluorescence using a polyclonal antibody against DE revealed a patchy labeling over the entire egg surface, with the exception of the area overlying the second metaphase spindle. This conclusion was supported by the disappearance of the DE-negative area on the fertilized egg. Zona-free eggs, incubated with DE at 4 degrees C or fixed before exposure to DE, displayed a uniform staining, suggesting that the patchy labeling resulted from aggregation of DE binding sites by the purified protein. The aggregation of these egg components may represent a necessary step of the fusion process. To our knowledge, this is the first study reporting the existence and localization of complementary sites to a specific sperm protein on the plasma membrane of the mammalian egg.     

Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342

When unfertilized sea urchin eggs are pretreated with the bisbenzimide DNA-specific fluorochrome Hoechst 33342, then washed and fertilized, a single sperm bound to the egg surface becomes intensely fluorescent. The location of the fluorescent sperm on the egg surface coincides exactly with the epicenter of the cortical reaction and the site at which the insemination cone subsequently appears. These observations, coupled with studies of eggs treated with quercetin to prevent fusion, as well as eggs made polyspermic by halothane exposure, indicate that the sperm acquires fluorescence as a consequence of fusion with the fluorochrome preloaded egg. Using a modification of this technique, we have found that cytoplasmic continuity between the sperm and egg is established at 4-8 sec after the onset of the sperm-induced conductance increase in the egg.     

Potassium dependence of sodium transport in frog skin

22Na+ and 42K+ fluxes across the basolateral membrane of the isolated epithelium of frog skin were investigated with regard to dependence on K+ in the basolateral solution. When K+ was removed from the basolateral solution (K+-free Ringer), there was a transient rise in short circuit current (Isc) that could be eliminated by pretreatment with ouabain. Concurrently, the apparent sodium efflux across the basolateral membrane (JNa*13) showed either no change or an immediate (1-2 min) small decrease (approximately equal to 10%) that was followed by a small transient increase. K+ fluxes showed either no change or a small decrease under these conditions. JNa*13 was partially ouabain sensitive during all of the above treatments. Furosemide partially inhibited both sodium and potassium flux after K+-free treatment. The pump, as defined by ouabain sensitivity of Na+ flux, continued to work even after 20 minutes of K+-free treatment. Pump activity may be maintained by potassium leaking from the cells that is recycled by the pump. However, the ouabain-sensitive transient rise in Isc after K+-free treatment cannot readily be explained by changes in either Na+ or K+ flux. A change in pump coupling ratio provides one explanation for these data.     

Proteolytic processing of a protein involved in sperm-egg fusion correlates with acquisition of fertilization competence

A protein located on the surface of guinea pig sperm (PH-30) has been implicated in the process of sperm-egg fusion (Primakoff, P., H. Hyatt, and J. Tredick-Kline. 1987. J. Cell Biol. 104:141-149). In this paper we have assessed basic biochemical properties of PH-30 and have analyzed the molecular forms of PH-30 present at different stages of sperm maturation. We show the following: (a) PH-30 is an integral membrane glycoprotein; (b) it is composed of two tightly associated and immunologically distinct subunits; (c) both subunits are made as larger precursors; (d) processing of the two subunits occurs at different developmental stages; (e) the final processing step occurs in the region of the epididymis where sperm become fertilization competent; (f) processing can be mimicked in vitro; (g) processing exposes at least two new epitopes on PH-30-one of the newly exposed epitopes is recognized by a fusion-inhibitory monoclonal antibody. These results are discussed in terms of the possible role of PH-30 in mediating fusion with the egg plasma membrane.     

Molecular models for murine sperm-egg binding

Murine sperm initiate fertilization by binding to the specialized extracellular matrix of mouse eggs, known as the zona pellucida. Over the past decade, powerful genetic, biophysical, and biochemical techniques have been employed to gain new insights into this interaction. Evidence from these studies does not support either of two major models for binding first proposed over two decades ago. Two more recently established models suggest that protein-protein interactions predominate during this initial stage of fertilization. Another model proposes that about 75-80% of the murine sperm bound to zona pellucida under well defined in vitro conditions is carbohydrate dependent, with the remaining sperm bound via protein-protein interactions. Mounting evidence suggests that the carbohydrate sequences coating the murine egg could be employed as specific immune recognition markers. Continued investigation of this system may resolve many of these controversial findings and reveal novel functions for murine zona pellucida glycoproteins.     

Observations of hamster sperm-egg fusion in freeze-fracture replicas including the use of filipin as a sterol marker

We have extended the observations of previous transmission electron microscopy studies of sperm-egg fusion to include those of freeze-fracture replicas showing sperm-egg interactions before, during, and following sperm head fusion with the egg membrane. Hamster eggs were incubated with hamster sperm under polyspermic conditions and were observed after a period of 5-30 minutes. After fixation, the eggs and sperm were exposed to filipin, which binds beta-OH-sterols to form visible complexes in freeze-fracture replicas. Filipin can act as a marker for egg plasma membrane wherein it is abundant, while filipin is relatively scarce in the acrosome-reacted hamster sperm membrane, found only in the plasma membrane of the equatorial segment. The earliest sperm-egg interactions are observed between the egg microvilli and the perforatorium and the equatorial segment of the sperm, and the initial fusion between egg and sperm occurs in the vicinity of the equatorial segment. At later stages of fusion involving the postacrosomal segment, a clear line of demarcation is observed between the filipin-rich egg membrane and the filipin-poor sperm postacrosomal segment, suggesting that filipin binding lipids from the egg intercalate into the sperm membrane following membrane fusion. The anterior segment of the sperm does not fuse with the egg but is instead incorporated into a cytoplasmic vesicle derived from both sperm and egg membranes. In this latter step, filipin-sterol complexes are not found in sperm-derived membranes suggesting that there may be barriers to the movement of filipin binding lipids from the egg into these sperm membranes.     

Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion

Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The mAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 micrograms/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75%. The average number of sperm fused per egg was also reduced by 75%. In contrast a control mAb, PH-1, preincubated with sperm at 400 micrograms/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two 125I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes. Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears. After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to approximately 49 kD and approximately 33 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44- and 60-kD bands. In the absence of detergent, the purified protein precipitates, suggesting that either 60 or 44 kD could be an integral membrane polypeptide.