Essential role of the nonreducing terminal alpha-mannosyl residues of the N-linked carbohydrate chain of bovine zona pellucida glycoproteins in sperm-egg binding

It has been proposed that mammalian sperm bind species-specifically to carbohydrate chains of zona pellucida glycoproteins at fertilization. Although the sperm ligand carbohydrate chains have been characterized in mice and pigs, the existence of the ligands of other mammals remains unclear. In order to explore the bovine sperm ligand, two in vitro competition assay methods were applied. As a result, a high-mannose-type carbohydrate chain, Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc, which is the major neutral chain in bovine egg zona glycoproteins, was shown to possess bovine sperm ligand activity. When nonreducing terminal alpha-mannosyl residues were eliminated from the zona glycoproteins by alpha-mannosidase digestion, the ligand activity was reduced, indicating that the alpha-mannosyl residues play an essential role in bovine sperm-egg binding. The number of sperm binding to eggs was reduced to about one-half after fertilization. The ligand-active high-mannose-type chain may be buried after fertilization, since its amount remains unchanged. Pretreatment of bovine sperm with the sperm ligand-carbohydrate chain significantly inhibited penetration of the sperm into oocyte and the male pronucleus formation. Thus, a correlation between the sperm ligand activity and in vitro fertilization rate was observed.     

Insemination,intrafollicular fertilization and development of the fertilization plug during gestation in Heterandria formosa (Poeciliidae)

The viviparous teleost Heterandria formosa is a remarkable species for its reproductive characters including: (a) the smallest oocyte in viviparous fish species; (b) a high level of matrotrophy with a complex placenta; and (c) the highest level of superfetation. Superfetation involves (d) the continuous development of oocytes and fertilization at the same time with embryos in gestation. The sequential fertilization of oocytes requires (e) storage of spermatozoa in the ovary. Among these characteristics, fertilization is of fundamental interest, specifically the intrafollicular fertilization of poeciliids, species that do not present micropyle, and the consequent formation of the fertilization plug, a structure developed at the periphery of the follicle where the entrance of spermatozoa occurs. Both processes intrafollicular fertilization and formation of the fertilization plug have been rarely described. There is only one study illustrating, the fertilization plug of H. formosa with a drawing. In the context of reproductive aspects of H. formosa, the goal of this study is to describe the morphology of the ovary during insemination, intrafollicular fertilization and development of the fertilization plug. After insemination, spermatozoa enter the ovary and occupy folds of the lamella near follicles of all stages of oogenesis, the delle, where the germinal epithelium establishes contact with the follicular epithelium. The results of the present study provide evidence that both epithelia open at the distal end of the delle, this morphological change allow that the spermatozoa to make contact with the zona pellucida of the oocyte. After fertilization, the delle becomes blocked by proliferation of cells of the germinal epithelium, to form the fertilization plug that persists throughout gestation. Abundant reticular fibers and blood vessels are seen around the fertilization plug. Persistence of the fertilization plug suggests that it could be the site where the juvenile will gain entrance to the ovarian lumen during birth.     

Difference of acrosomal serine protease system between mouse and other rodent sperm

A fraction of acrosomal proteins dispersed during calcium ionophore A23187‐induced acrosome reaction was prepared from cauda epididymal sperm of wild‐type and acrosin‐deficient mice, rat, and hamster. The acrosome‐reacted sperm were further extracted by Nonidet P‐40 to obtain the detergent‐soluble protein fraction. Activities of serine proteases in the two protein fractions were examined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the presence of gelatin. A mixture of 42‐ and 41‐kDa gelatin‐hydrolyzing proteases was found in both fractions of the wild‐type mouse sperm, whereas the acrosin‐deficient mouse sperm contained the active 42‐kDa protease and apparently lacked the activity of the 41‐kDa protease. However, exogenous bovine pancreatic trypsin compensated for the absence of acrosin in the protein fractions of the mutant mouse sperm; the gelatin‐hydrolyzing activity of the 41‐kDa protease appeared when the sperm proteins of the mutant mice were treated with pancreatic trypsin. Two‐dimensional polyacrylamide gel electrophoresis revealed that the 42‐ and 41‐kDa proteases were distinguished from acrosin by the isoelectric point and immunoreactivity with affinity‐purified antibody against an oligopeptide corresponding to the N‐terminal amino acid sequence of mouse proacrosin. Moreover, the gelatin‐hydrolyzing proteins corresponding to these two proteases were not detected in rat and hamster sperm, in spite of the treatment of the sperm extracts with pancreatic trypsin, and the total amount of gelatin‐hydrolyzing activities in mouse was much smaller than those in rat and hamster. These results may reflect the difference of the serine protease system for the sperm penetration through the egg zona pellucida between mouse and other rodent animals, possibly explaining why the acrosin‐deficient mouse sperm are capable of penetrating the zona pellucida. Dev. Genet. 25:115–122, 1999. © 1999 Wiley‐Liss, Inc.     

Interaction of boar spermatozoa with porcine oocytes: Increase in proteins with high affinity for the zona pellucida during epididymal transit

Methods for the investigation of cell-associated calcium and intracellular calcium were studied in washed ejaculated human spermatozoa. Experiments using ⁴⁵Ca²⁺ indicated that human spermatozoa were permeant to calcium and that a significant proportion of the cellassociated calcium (approximately 50%) was accumulated in the mitochondrion. This necessitated the use of alternative procedures to measure cytoplasmic free calcium. The ability of human spermatozoa to accumulate and de-esterify the intracellular fluorescent calcium indicator Quin-2 was established. Using this technique, the resting level of free intracellular calcium in human spermatozoa was found to be 146.0 ± 19.9 nM, and was significantly elevated upon addition of the divalent cation ionophore ionomycin. In further experiments designed to illustrate the applications of the Quin technique, data was obtained suggesting that the mechanisms controlling intracellular calcium in human spermatozoa are temperature dependent but do not involve voltage-sensitive calcium channels.     

Characterization of the functional domains of boar acrosin involved in nonenzymatic binding to homologous zona pellucida glycoproteins

During the first steps of the gamete interaction, the proacrosin/acrosin system seems to play a crucial role in the secondary binding, holding acrosome-reacted spermatozoa during their passage through the zona pellucida. To analyze the functional domains of acrosin, we decided to express recombinant boar acrosin proteins in bacteria and to study their binding capacities to zona pellucida glycoproteins (ZPGPs). The expressed proteins were immunodetected by Western blot with a polyclonal antiacrosin antibody. The recombinant truncated β-acrosin has a typical hyperbolic curve of a zymogen enzymatic activation. Three of the five recombinant forms (truncated β-acrosin, Ser/Ala²²²-truncated β-acrosin, and truncated β-acrosin “heavy chain”) had the ability to bind ZPGPs. The two shorter forms (the amino and carboxy termini of truncated β-acrosin) failed to bind. The catalytic site mutant (Ser/Ala²²²) of truncated β-acrosin does not differ from the recombinant truncated β-acrosin in its mechanism of interaction to ZPGPs, indicating that this secondary binding is done by a nonenzymatic process. Our results show that binding between acrosin and ZPGPs depends on the secondary and tertiary structures of acrosin and does not depend on an active catalytic site. Mol. Reprod. Dev. 49:426–434, 1998. © 1998 Wiley-Liss, Inc.     

Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.     

Relevance of glycosylation of human zona pellucida glycoproteins for their binding to capacitated human spermatozoa and subsequent induction of acrosomal exocytosis

To delineate the functional aspects of zona pellucida (ZP) glycoproteins during fertilization in human, in the present study, fluorochrome-conjugated Escherichia coli (E. coli)- and baculovirus-expressed recombinant human ZP glycoprotein-2 (ZP2), -3 (ZP3), and -4 (ZP4) were employed. In an immunofluorescence assay, capacitated human sperm exhibited binding of the baculovirus-expressed recombinant ZP3 as well as ZP4 to either acrosomal cap or equatorial region whereas acrosome-reacted sperm failed to show any binding to the acrosomal cap. Using double labeling experiments, simultaneous binding of ZP3 and ZP4 to the acrosomal cap was observed suggesting the possibility of different binding sites of these proteins on the sperm surface. No binding of ZP2 was observed to the capacitated sperm. However, acrosome-reacted sperm (20.00 +/- 1.93%) showed binding of ZP2 that was restricted to only equatorial region. Interestingly, E. coli-expressed recombinant human zona proteins also showed very similar binding profiles. Competitive inhibition studies with unlabeled recombinant human zona proteins revealed the specificity of the above binding characteristics. Binding characteristics have been further validated by an indirect immunofluorescence assay using native human heat solubilized isolated zona pellucida. Employing baculovirus-expressed recombinant ZP3 and ZP4 with reduced N-linked glycosylation and respective E. coli-expressed recombinant proteins, it was observed that glycosylation is required for induction of acrosomal exocytosis but its absence may not compromise on their binding ability. These studies have revealed the binding profile of individual human zona protein to spermatozoa and further strengthened the importance of glycosylation of zona proteins for acrosomal exocytosis in spermatozoa.     

Modifications of the lectin binding pattern in the rat zona pellucida after in vivo fertilization

The zona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte. It is involved in the sperm-egg adhesion phenomenon, induces the acrosome reaction, and participates in the late blockage to polyspermy. Thus, during the process of fertilization the cortical reaction is induced and the biochemical and biological properties of the ZP are modified. Some of these changes have been suggested to prevent the polyspermy. However, the mechanisms behind most of these changes are not well understood. Carbohydrate residues of the ZP glycoproteins have been shown to play a key role in the early step of fertilization. In the present study, the changes produced in the terminal oligosaccharide sequences of the rat ZP glycoproteins after in vivo fertilization were investigated by means of lectin-gold cytochemistry. A comparative quantitative analysis of the density of labeling in the ZP before and after fertilization was carried out by automatic counting of gold particles. The ZP of fertilized and unfertilized eggs were labeled by a battery of lectins including PNA, LFA, MAA, AAA, DSA, RCA I, and WGA. For all lectin studied in both fertilized and unfertilized eggs the labeling was preferentially located in the inner region of the ZP. After fertilization, binding of PNA, LFA, MAA, AAA, and DSA decreased in both inner and outer regions of the ZP. Labeling of RCA l-binding sites only decreased in the inner ZP, whereas reactivity to WGA was increased in the inner area of the ZP. Digestion of the thin-sections with neuraminidase prior to labeling with WGA resulted in a decrease of labeling for WGA binding sites. However, the labeling density of WGA binding sites was similar in both unfertilized and fertilized eggs upon treatment with neuraminidase. The present results demonstrate that the oligosaccharide chains contained in the rat ZP are modified after fertilization of the oocyte. Cortical granules of the oocytes might be involved in these modifications by two mechanisms: 1) by hydrolysis of terminal carbohydrate residues of ZP glycoproteins by specific glycosidases contained in the granules; and 2) by addition of new glycoproteins to the ZP after the exocytosis of the cortical granules (cortical reaction). © 1996 Wiley-Liss, Inc.     

Structure of the zona pellucida and cumulus oophorus in three species of native Australian rodents

The sperm head of many Australian hydromyine rodents has three curved hooks projecting from its anterior margin; the structure of the hooks has been characterized, but their function is unknown. In this study, we have investigated whether the hooks might have evolved to assist sperm penetration through more formidable egg vestments, particularly the zona pellucida. Cumulus-oocyte complexes were obtained from two species that possess a three-hooked sperm head (Pseudomys australis and P. nanus) and one species that does not (Notomys alexis) and examined by light and electron microscopy. After fixation in the presence of ruthenium red, the zona pellucida was found to consist of a fibrillar meshwork, but there were no interspecific structural differences. A corona radiata was absent, and the cumulus extracellular matrix was composed of filaments and electron-dense granules in each species. Measurements of the zona thickness in freshly ovulated, unfixed oocytes revealed that it was thinnest (7.8 μm) in P. australis. Which has a three-hooked sperm head, and thickest (11.4 μm) in N. alexis, the species in which the ventral hooks are absent. Hence, no correlation was found between the thickness of the zona pellucida or the structure of the cumulus-oocyte complex, and the presence of three hooks on the sperm head. We conclude, therefore, that it is unlikely that the evolution of the three-hooked sperm head is an adaptation for penetration of increased barriers around the oocyte.     

Isolation of antiSLIP1-reactive boar sperm P68/62 and its binding to mammalian zona pellucida

Single-step purification of boar sperm P68/62 that is cross-reactive with a polyclonal antibody against sulfolipidimmobilizing protein 1 (SLIP1) was achieved by chromatofocusing. This method is useful for obtaining P68/62 in quantity. The two proteins, P68 and P62, were antigenically related, since the antibody generated specifically against the 68-kDa band reacted with both the 68- and 62-kDa bands. Like rat testis SLIP1, purified boar sperm P68/62 bound to sulfogalactosylglycerolipid (SGG) and inhibited sperm-egg binding in a dose-dependent manner when added exogenously to sperm-egg coincubates. This inhibitory effect occurred at the level of the zona pellucida (ZP), and further studies showed that biotinylated boar sperm P68/62 bound to the ZP of unfertilized mouse eggs. Furthermore, biotinylated boar sperm P68/62 bound to isolated ZP of unfertilized eggs from other species, including pig, rat, cat, dog, and human, as well as to ZP of intact fertilized mouse eggs and preimplantation embryos of various developmental stages, although the degree of its binding to the ZP of intact eight-cell embryos, morulae, and blastocysts was much lower than that of fertilized eggs and two-cell embryos. These results suggest that P68/62 of capacitated sperm must act together with other sperm surface proteins/molecules that regulate zona binding specificity within homologous species and in unfertilized eggs. Together with our previous findings, we suggest that rather than being a true ZP receptor, sperm P68/62 may be involved in the initial step of sperm-ZP binding that is adhesive in nature. Mol. Reprod. Dev. 49:203–216, 1998 © 1998 Wiley-Liss, Inc.     

Participation of the nonreducing terminal beta-galactosyl residues of the neutral N-linked carbohydrate chains of porcine zona pellucida glycoproteins in sperm-egg binding

The zona pellucida (ZP) surrounding the mammalian oocyte is composed of three glycoprotein components (ZPA, ZPB, and ZPC). Mammalian sperm bind to carbohydrate chains of a ZP glycoprotein in the initial phase of fertilization. Sperm-ligand carbohydrate chains have been characterized in mouse, cow, and pig. In pigs, triantennary/tetraantennary neutral complex-type chains from ZPB/ZPC mixture possess stronger sperm-binding activity than those of biantennary chains (Kudo et al., 1998: Eur J Biochem 252:492-499). Most of these oligosaccharides have beta-galactosyl residues at the nonreducing ends. This study used two in vitro competition assays to investigate the participation of the nonreducing terminal beta-galactosyl residues of the ligand active chains in porcine sperm binding. The removal of the nonreducing terminal beta-galactosyl residues from either the ligand active carbohydrate chains or endo-beta-galactosidase-digested glycoproteins significantly reduced their inhibition of sperm-egg binding, indicating that the beta-galactosyl residues at the nonreducing ends are involved in porcine sperm-egg binding. A correlation between the sperm-binding activity and in vitro fertilization rate is also presented.     

Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida

Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm-zona attachments.     

Mechanism of sperm-binding inhibition by anti-zona antisera

Mouse zonae pellucidae contain receptors for sperm throughout their structure since spermatozoa will bind to both the inner and outer surfaces of isolated zona fragments. Antibodies raised against mechanically isolated mouse zonae pellucidae were only capable of suppressing sperm binding to the outer zona surface in association with the formation of a precipitate in this region. These results indicate that such antisera are not capable of interacting directly with the sperm receptors on the zona pellucida but rely upon the less efficient mechanism of steric hindrance to prevent sperm from gaining access to the sperm binding sites.     

猪精子凝集素在精卵结合中的作用机制

猪精子凝集素（ＢＳＬ）位于精子头部,既与精子蛋白结合,亦与透明带糖蛋白ＺＰ３结合。并且ＢＳＬ自身分子间亦会聚合。与ＺＰ３结合的片段亦结合于岩藻素－Ｓｅｐｈａｒｏｓｅ亲和柱,但不与胎球蛋白－Ｓｅｐｈａｒｏｓｅ柱结合。该片段具有较强的抑制精卵结合的活性。ＢＳＬ经ＮＢＳ修饰后,血凝活力大大下降,同时推动与精子结合的活性,但不影响它与ＺＰ３的结合。修饰后的ＢＳＬ亦显著抑制精卵结合。表明ＢＳＬ以两个不同的部     

Wheat germ agglutinin treatment of follicle cell-free mouse oocytes inhibits fertilization

Controversy exists whether treatment of follicle cell-free oocytes with wheat germ agglutinin (WGA) prevents fertilization. Lack of inhibition in one case has led to the suggestion that acrosin may not be a zona lysin. To re-examine the effect of the WGA, the zona pellucida of follicle cell-free mouse oocytes was made more resistant to proteinase digestion by treatment with 10 or 50 μg/ml WGA. Such WGA-treated oocytes showed decreased fertilizability when washed to remove excess WGA and incubated with capacitated spermatozoa. Oocyte cleavage was used as an end point, because a large number of spermatozoa adhered to the eggs after WGA treatment, making observation of sperm penetration and pronucleus formation unreliable. Resistance to proteinase digestion increased, and the fertilizability decreased with the higher amount of WGA. The action of WGA was most likely not mediated by a direct effect on sperm motility, sperm acrosin activity, sperm binding to the zona pellucida, or oocyte cleavage. WGA did not affect the acrosome reaction of guinea pig spermatozoa. These data show that WGA treatment of follicle cell-free mouse oocytes results in decreased fertilizability, possibly by rendering the zona pellucida more resistant to sperm proteinase digestion.     

Ultrastructural and histochemical changes in the murine zona pellucida during the final stages of oocyte maturation prior to ovulation

The ultrastructural morphology of the mouse zona pellucida was studied in preovulatory follicles from the ovaries of immature mice treated with exogenous gonadotrophins. The ovaries were fixed in the presence of cetylpyridinium chloride, which precipitates carbohydrates, so that their loss during fixation and processing is substantially reduced. The semi-thin araldite sections obtained from osmicated material were viewed by conventional light microscopy, while the ultra-thin sections were examined by transmission electron microscopy. A parallel series of semi-thin sections of non-osmicated ovaries was deresined and subsequently stained with periodic acid Schiff (PAS). The morphological appearance of the zona pellucida in preovulatory oocytes changed during the final stages of oocyte maturation. A close correlation was observed between the ultrastructural appearance of the zona pellucida and that observed following PAS staining during the period studied. Real differences were observed in the location, density, and distribution of glycoconjugates within the zona pellucida during the final stages of oocyte maturation prior to and immediately following germinal vesicle breakdown. Similar changes in the zona were observed in adult females autopsied during proestrus and oestrus. The changes in the density and distribution of glycoconjugates are likely to have important consequences for fertilization by affecting sperm-zona binding and sperm-egg interactions.     

Evidence for a binding site on the sperm plasma membrane which recognizes the murine zona pellucida: A binding site on the sperm plasma membrane

Murine cauda epididymal sperm preincubated in either a modified Krebs-Ringer or M 199 solution bind to cumulus-free, zona pellucida-intact eggs. Pretreatment of such eggs with an affinity purified preparation of the seminal inhibitor binding component (acceptor), isolated from epididymal sperm, reduces in a concentration dependent manner, the number of sperm that bind. Treatment of cauda sperm, preincubated in either of the above two media, with the seminal inhibitor, also reduces the number of sperm able to bind. Incubation of cauda sperm in the Krebs-Ringer solution for up to 4 h does not affect their ability to bind the seminal inhibitor. Omission of bovine serum albumin from the preincubation medium results in a significant reduction in sperm binding. These data are interpreted to mean that the seminal inhibitor acceptor sites on the sperm surface of incubated sperm function in the in vitro binding to the zona pellucida.     

Ultrastructural studies of the porcine zona pellucida during the solubilization process by Li-3,5-diiodosalicylate

Isolated porcine zonae pellucidae were investigated using transmission electron microscopy. It was found that the fine structure of the zona is not homogenous. The region near the oocyte consists of a more tightly packed micellar structure than the structure near the external surface. However, no clear boundary between the two structural features could be detected. The assumption that the molecular structure of the external and internal surface of the zona is not the same is substantiated by the finding that antibodies against the whole zona structure react apparently only with the more loosely bound structure on the external surface. This effect is attributed to differences in the spacial arrangements of the micellar structures rather than to differences in chemical composition. Solubilization of the zona results in a reorientation of the otherwise randomly interconnected structure. At lower Li-3,5-diiodosalicylate concentration (0.05 M) the fibrils seems to expand so that the individual fibers lie almost parallel to each other. At a higher Li-3,5-diiodosalicylate concentration (0.2 M) the individual micelles begin to break up from the zona surface, while at a still higher concentration (0.3 M) the rigid structure of the zona is completely solubilized. In the latter case no residual material could be detected in the sediment following high speed centrifugation of the mixture. These electron microscopic results correlated with the protein concentrations in the supernatant indicating that the maximal protein content (35 ng/zona) is obtained at 0.3 M or higher Li-3,5-diiodosalicylate concentrations.