A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida

Past studies have suggested that mouse sperm surface galactosyltransferase may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the zona pellucida. In this paper, we examined further the role of sperm surface galactosyltransferase in mouse fertilization. Two reagents that specifically perturb sperm surface galactosyltransferase activity both inhibit sperm-zona binding. The presence of the milk protein alpha- lactalbumin specifically modifies the substrate specificity of sperm galactosyltransferase away from GlcNAc and towards glucose and simultaneously inhibits sperm binding to the zona pellucida. Similarly, UDP-dialdehyde inhibits sperm binding to the zona pellucida and sperm surface galactosyl-transferase activity to identical degrees. Of five other sperm enzymes assayed, four are unaffected by UDP-dialdehyde, and one is affected only slightly. Covalent linkage of UDP-dialdehyde to sperm dramatically inhibits binding to eggs, while treatment of eggs with UDP-dialdehyde has no effect on sperm binding. Heat-solubilized or pronase-digested zona pellucida inhibit sperm-zona binding, and they can be glycosylated by sperm with UDP-galactose. Sperm are also able to glycosylate intact zona pellucida with UDP-galactose. Thus, solubilized and intact zona pellucida act as substrates for sperm surface GlcNAc:galactosyltransferases. Finally, pretreatment of eggs with beta- N-acetylglucosaminidase inhibits sperm binding by up to 86%, while under identical conditions, pretreatment with beta-galactosidase increases sperm binding by 55%. These studies, in conjunction with those of the preceding paper dealing with surface galactosyltransferase changes during capacitation, directly suggest that galactosyltransferase is at least one of the components necessary for sperm binding to the zona pellucida.     

Fertilization studies in the hamster : The role of cell-surface carbohydrates

The nature of complementary binding sites on the surfaces of hamster gametes has been analysed using mono- and oligosaccharides, glycoproteins and glycosidases in an in vitro system. The binding of capacitated spermatozoa to the zona pellucida was inhibited by several mono- and oligosaccharides related to fucose, galactose, and acetylated amino sugars, but not by unrelated sugars. Several glycoproteins with prosthetic carbohydrate groups rich in or terminated by galactose or N-acetylglucosamine residues were also potent inhibitors of fertilization. Of all the glucoproteins tested, two plasma glycoproteins, α₁-acid glycoproteins (orosomucoid) and fetuin were most effective. In their native form they were non-inhibitory but their desialylated (galactoseterminated) forms completely prevented the sperm-zona binding. Agalacto-orosomucoid with N-acetylglucosamine terminals also inhibited fertilization. The treatment of capacitated spermatozoa with α- -fucosidase, α- -galactosidase and β-N-acetylhexosaminidase, but not with other glycosidases, trypsin and arylsulphatase, resulted in the complete inhibition of fertilization. Inhibitory saccharides and glycosidases did not interfere with sperm motility and had no effect on sperm-oolemma fusion. The pretreatment of cumulus-free oocytes with these agents did not inhibit sperm zona pellucida binding either. These results provide evidence that sperm-zona pellucida binding is mediated by ligands on the sperm surface containing fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine residues.     

Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm

Background  

The functions of three zona glycoproteins, ZP1, ZP2 and ZP3 during the sperm-zona pellucida (ZP) interaction are now well established in mice. The expression of an additional zona glycoprotein, ZPB/4, in humans, led us to reconsider the classical mouse model of gamete interaction. We investigated the various functions of human ZP (hZP) during the interaction of spermatozoa with fertilised and unfertilised oocytes.     

Porcine sperm surface beta1,4galactosyltransferase binds to the zona pellucida but is not necessary or sufficient to mediate sperm-zona pellucida binding.

The binding of sperm to the zona pellucida is an integral part of the mammalian fertilization process, investigated most extensively in the mouse. Several sperm receptors for the murine zona pellucida have been studied (Snell WJ, White JM. 1996. Cell 85:629-637; Wassarman PM. 1999. Cell 96:175-183), but the most compelling evidence exists for beta-1,4-galactosyltransferase (GalTase). Considering that GalTase is present on the surface of porcine sperm (Larson JL, Miller DJ. 1997. Biol Reprod 57:442-453), we investigated the role of GalTase in porcine sperm-zona binding. Sperm surface GalTase catalyzed the addition of uridine diphosphate-[(3)H]galactose to the 55 kDa group of the porcine zona pellucida proteins implicated in sperm binding, demonstrating that GalTase binds the porcine zona. The functional importance of GalTase-zona pellucida binding was tested. Addition of uridine diphosphate galactose, a substrate that completes the GalTase enzymatic reaction and disrupts GalTase mediated adhesion, had no effect on binding of sperm to porcine oocytes. Furthermore, removal of the GalTase zona ligand by incubation of oocytes with N-acetylglucosaminidase had no effect on binding of sperm to oocytes. These results suggest that GalTase is not necessary for sperm to bind to the zona pellucida. Digestion of isolated porcine zona proteins with N-acetylglucosaminidase did not affect the biological activity of soluble porcine zona proteins in competitive sperm-zona binding assays, suggesting that GalTase alone is not sufficient to mediate sperm-zona attachment. From these results, it appears that, although GalTase is able to bind porcine zona proteins, its function in porcine sperm-zona binding is not necessary or sufficient for sperm-zona binding. This supports the contention that porcine sperm-zona binding requires redundant gamete receptors.     

Comparative immunoreactivity of mouse and hamster sperm proteins recognized by an anti-P26h hamster sperm protein

The binding of the spermatozoon to the zona pellucida is a species-specific phenomenon. We have previously shown that the binding of hamster sperm to the homologous zona pellucida involves a sperm 26-kDa glycoprotein, the P26h, originating in the epididymis. In order to establish to what extent this sperm protein is involved in the species-specific recognition of the egg's extracellular coat, we have compared the inhibitory properties of anti-P26h antibodies in a sperm-zona pellucida assay using hamster and mouse gametes. Anti-P26h IgGs inhibit, in a dose-dependent manner, gamete interactions in both species, although in a less efficient manner in the mouse than in the hamster. While anti-26kDa Fab fragments are as efficient as the intact IgG to inhibit hamster sperm-zona pellucida binding, they have no effect on mouse gamete interaction. ELISA, Western blot, and immunohistochemical experiments have been performed in order to characterize the mouse antigen(s) recognized by the anti-P26h antiserum. ELISA and Western blots showed that this antiserum recognized two proteins on mouse spermatozoa that are less reactive than the hamster P26h. These antigens are localized in the acrosomal region of epididymal spermatozoa of both species. These results indicate that the hamster P26H involved in zona pellucida interaction has certain unique epitopes, while others are common to the sperm of both species. © 1995 Wiley-Liss, Inc.     

Mannose-binding molecules of rat spermatozoa and sperm-egg interaction

We have previously reported the occurrence and partial characterisation of an alpha-D-mannosidase activity on plasma membranes of rat, mouse, hamster and human spermatozoa. A soluble isoform of the rat sperm surface mannosidase was purified and polyclonal antibody raised. Since several reports have suggested that mannosyl residues on the rat, mouse and human zona pellucida may be involved in sperm-zona binding, studies were undertaken to examine the receptor-like role of mannose-binding molecules on rat spermatozoa. Sprague-Dawley rats (25-30-days old) were superovulated and eggs collected from the oviduct were treated with 0.3% hyaluronidase to remove the cumulus cells. Spermatozoa, collected from the cauda epididymis were capacitated for 5 h at 37 degrees C in 5% CO2 in air. The sperm-zona binding assay was performed in the presence of increasing concentrations of several sugars as well as preimmune and immune (anti-mannosidase or anti-mannose binding protein) IgG. Data from these studies show that: (1) significantly fewer sperm bound per egg in the presence of competitive inhibitors of mannosidase; (2) among the sugars examined, D-mannose was the most potent inhibitor causing 70% reduction in the number of sperm bound per egg; (3) anti-mannosidase or anti-mannose binding protein (but not preimmune) IgG showed a dose-dependent reduction in the number of sperm bound per egg; (4) anti-mannosidase IgG (but not anti-mannose binding protein IgG) showed a dose-dependent inhibition of sperm surface mannosidase activity; (5) the competitive inhibitors of mannosidase or the immune IgG had no effect on sperm motility or the sperm acrosome reaction. These result suggest that mannose-binding molecule(s) such as alpha-D-mannosidase or mannose-binding protein on the spermatozoa may recognise mannosyl residues on zona pellucida, and play a receptor-like role in sperm-egg interaction in the rat.     

Aggregation of beta-1,4-galactosyltransferase on mouse sperm induces the acrosome reaction

beta-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, alpha-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-Gal-Tase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface.     

Reassessing the role of protein-carbohydrate complementarity during sperm-egg interactions in the mouse

Despite years of intense study by many investigators, it may appear that we have made little progress towards a molecular understanding of mammalian sperm binding to the egg zona pellucida. An abundance of evidence derived from in vitro assays suggests that sperm-zona pellucida binding is dependent upon sperm recognition of specific glycan moieties on the zona pellucida glycoproteins. However, there is considerable disagreement regarding the identity of the zona pellucida sugars thought to mediate sperm binding, as well as disagreement over the identity of the sperm receptors themselves. Moreover, results from in vivo gene-targeting strategies fail to support a role for many, if not all, of the sperm receptors and their zona pellucida ligands implicated from in vitro assays. Nevertheless, a retrospective view of the literature suggests that some common principles are emerging regarding the molecular basis of mammalian sperm-zona binding, both with respect to the nature of the components that mediate binding, as well as the involvement of distinct receptor-ligand interactions, that involve both protein- and carbohydrate-dependent mechanisms of binding.     

Defining oligosaccharide specificity for initial sperm-zona pellucida adhesion in the mouse

The identity of the sperm surface protein(s) responsible for sperm-zona pellucida binding in the mouse, as well as the characteristics of the oligosaccharide groups on zona pellucida glycoprotein 3 (ZP3) having ligand activity toward this receptor, remain controversial. Conflicting results from several groups have made interpretation of the current data difficult. By developing a quantitative binding assay to evaluate the molecular interactions between mammalian sperm and the zona pellucida during initial gamete interactions, we directly quantified sperm-ZP binding interactions at the molecular level for the first time. The ZP binding assay demonstrated that live, capacitated mouse sperm bind solubilized ¹²⁵I-labeled ZP glycoproteins in a concentration-dependent manner characterized by a rapid forward rate constant of 3.0 × 10⁷ M⁻¹ min⁻¹. Following the initial characterization, the binding assay was used to examine the roles of the sperm surface enzymes galactosyltransferase (GalTase) and fucosyltransferase (FucTase) in sperm-zone pellucida binding in the mouse. These data indicate that substrates for FucTase, but not for GalTase, inhibit sperm-ZP binding, in contrast to earlier reports in which GalTase substrates significantly inhibited sperm binding to intact ZPs. A model is presented which resolves conflicting results between assays using intact ZPs and the results obtained here using soluble ¹²⁵I-ZPs. Assuming a complex binding/recognition site, monosaccharides that could occupy part of the binding site would have a dramatic effect on sperm-ZP binding to the intact ZP, since they need only occupy the binding sites for a short time (∼ 100 msec) to disrupt binding. The current results suggest that the sperm ZP3 receptor binding site minimally recognizes the galβ1,3GlcNAc moiety also recognized by FucTases. The current data do not exclude the possibility that additional sugar residues form part of the ligand oligosaccharide group and are recognized by a yet-to-be-identified sperm surface protein which serves as the ZP3 receptor. © 1996 Wiley-Liss, Inc.     

Further characterization of the mouse sperm surface zona-binding site with galactosyltransferase activity

One of the mouse sperm surface binding sites for zona pellucida ligands exhibits galactosyltransferase (GT) enzyme activity. The present study was undertaken to ascertain whether the GT site behaves as a noncatalytic binding site in its physiological capacity, with no glycosylation of zona ligands, or whether glycosylation of zona ligands is an integral part of sperm-zona binding. The effects of Mn2+, the obligatory cation for GT catalysis, on enzyme activity and sperm-zona binding were examined. With uridine-5'-diphosphogalactose (UDPgal) as galactose donor, and N-acetylglucosamine (GlcNAc) as galactose acceptor, increasing concentrations of Mn2+ in the range of 0.1-10 mM increased GT enzyme activity, with half-maximal activation at 0.65 mM Mn2+ (Vmax = 20 pmol/hr/10(6) cells). In the presence of 0-2 mM Mn2+, sperm-zona binding was inhibited in a concentration-dependent manner; 50% inhibition occurred at 1.25 mM Mn2+. At this concentration, GT enzyme activity was at 65% Vmax. To determine the specificity of the GT site for glycoprotein terminal carbohydrate residues, spermatozoa were incubated with, asialo-ovine submaxillary mucin (N-acetylgalactosamine residues), asialo-, -alpha 1-acid glycoprotein (beta 1-4 galactose residues) ovalbumin (Ov; GlcNAc residues), and asialo-agalacto-/alpha 1-acid glycoprotein (AsAgAGP; GlcN-Ac residues). Only Ov and AsAgAGP acted as acceptors for galactose in the enzyme assay and inhibitors in the sperm-zona binding assay. The kinetics of the interaction of AsAgAGP with the GT site were determined: the Km was 3.6 mg/ml, with Vmax of 33 pmol/hr/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of hot-spot residues in protein-protein interactions by computational docking

Background  

The study of protein-protein interactions is becoming increasingly important for biotechnological and therapeutic reasons. We can define two major areas therein: the structural prediction of protein-protein binding mode, and the identification of the relevant residues for the interaction (so called 'hot-spots'). These hot-spot residues have high interest since they are considered one of the possible ways of disrupting a protein-protein interaction. Unfortunately, large-scale experimental measurement of residue contribution to the binding energy, based on alanine-scanning experiments, is costly and thus data is fairly limited. Recent computational approaches for hot-spot prediction have been reported, but they usually require the structure of the complex.     

Behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro

In this study we examined the behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro by assessing the effect of its pertinent monoclonal antibody mMC101. Experiments were designed to assess the effect of mMC101 on sperm-zona pellucida binding, the acrosome reaction, zona pellucida penetration, sperm-egg fusion, and fertilisation in vitro. mMC101 did not affect sperm motility or primary and secondary binding to the zona pellucida, but significantly inhibited fertilisation of zona-pellucida-intact oocytes in a dose-dependent manner. In the presence of mMC101 at 100 microg/ml concentration in TYH medium, none of the oocytes developed to pronuclear stage by 5 h after co-incubation of the gametes, but the pronucleus formation rate recovered to some extent (45.3%) after 8 h, indicating a delay of early embryonic development. mMC101 also delayed and significantly suppressed zona pellucida penetration by sperm. Acrin 3 dispersed and did not remain on completely acrosome-reacted sperm. Although mMC101 did not influence the zona-pellucida-induced acrosome reaction, it significantly inhibited fertilisation when acrosome-reacted sperm in the presence of mMC101 inseminated zona-pellucida-free oocytes. However, fertilisation remained unaffected when acrosome-reacted sperm in the absence of mMC101 inseminated zona-pellucida-free oocytes even in its presence. Thus, acrin 3 appears to facilitate zona pellucida penetration and is also likely to be involved in sperm-oocyte fusion by modifying the sperm plasma membrane during the acrosome reaction.     

Male mice deficient for germ-cell cyritestin are infertile

Cyritestin is a membrane-anchored sperm protein belonging to the ADAM (f1.gif" BORDER="0"> f2.gif" BORDER="0">isintegrin and f1.gif" BORDER="0"> f3.gif" BORDER="0">etalloprotease) family of proteins, which are proposed to be involved in cell-cell adhesion through binding to integrin receptors. Several lines of evidence support a role of cyritestin and other members of this protein family in the fusion of sperm and the egg plasma membrane. In an effort to elucidate the physiological function of cyritestin, we have disrupted its locus by homologous recombination. Male homozygous null mutants are infertile, even though spermatogenesis, mating, and migration of sperm from the uterus into the oviduct are normal. In vitro experiments showed that infertility is due to the inability of the cyritestin-deficient sperm to bind to the zona pellucida. However, after removal of the zona pellucida, sperm-egg membrane fusion monitored by the presence of pronuclei and generation of 2- and 4-cell embryos did not reveal any differences from the wild-type situation. These results demonstrate that cyritestin is crucial in the fertilization process at the level of the sperm-zona pellucida interaction.     

Binding of caput epididymal mouse sperm to the zona pellucida

Immature sperm from the caput epididymis are immotile and infertile. It is thought that caput epididymal sperm are infertile due to their immotility, as well as to an inability to bind to the zona pellucida, suggesting the absence of a functional receptor for the zona. However, the sperm receptor for the zona pellucida has been identified previously as the enzyme galactosyltransferase (GalTase) (L. C. Lopez et al. (1985) J. Cell Biol. 101, 1501-1510) and is present on the surface of caput as well as cauda epididymal sperm (N. F. Scully et al., (1987) Dev. Biol. 124, 111-124.). In this paper we examine this apparent conflict and show that immotile caput epididymal sperm are able to bind to the zona pellucida if they are first washed free of caput epididymal secretions, which contain factors that inhibit sperm-zona binding. Consistent with this finding are results that show that caput epididymal fluid is capable of inhibiting the binding of mature, cauda epididymal sperm to the zona pellucida. Caput epididymal fluid contains, among many other components, a soluble GalTase and an alpha-lactalbumin-like protein, both of which are capable of inhibiting mouse sperm-zona binding. Thus, caput epididymal sperm have the appropriate receptor, i.e., GalTase, for the zona pellucida, to which they can bind if removed from the inhibitory factors that mask their zona-binding ability.     

精子膜麦芽凝集素结合糖蛋白抗原某些特性的研究

应用自制的抗牛精子膜麦芽凝集素结合糖蛋白血清,对兔、人、小鼠和仓鼠精子进行了免疫细胞化学定位,结果各种动物精子均呈阳性反应,且以精子顶体区标记最强,与麦芽凝集素亲和细胞化学的标记结果相似。用抗血清处理地鼠精子,再与同种卵子进行体外结合试验,结果精子与卵于透明带的结合受到显著抑制、本实验的结果提示,牛精子膜麦芽凝集素结合糖蛋白抗原具有种间交叉反应性,并可能在精子与卵子透明带结合过程中具有重要作用。     

APIS: accurate prediction of hot spots in protein interfaces by combining protrusion index with solvent accessibility

Background  

It is well known that most of the binding free energy of protein interaction is contributed by a few key hot spot residues. These residues are crucial for understanding the function of proteins and studying their interactions. Experimental hot spots detection methods such as alanine scanning mutagenesis are not applicable on a large scale since they are time consuming and expensive. Therefore, reliable and efficient computational methods for identifying hot spots are greatly desired and urgently required.     

Polypeptide backbone derived from carboxyl terminal of mouse ZP3 inhibits sperm-zona binding

For mammalian organism, fertilization begins with species-specific recognition between sperm and egg, a process depending upon egg zona pellucida glycoproteins and putative sperm interacting protein(s). In mouse, zona pellucida glycoprotein ZP3 is believed to be the primary receptor for sperm and inducer of sperm acrosomal reaction, and its function has been attributed to the specific O-linked oligosaccharides attached to polypeptide backbone. While lots of reports have focused on the role of ZP3's oligosaccharides in fertilization, there are few concerning its polypeptide backbone. To investigate whether mZP3 polypeptide backbone is involved in sperm-egg recognition, three partially overlapping cDNA fragments, together covering entire mouse ZP3, were cloned, expressed and purified under denaturing condition. Although all three refolded proteins possess native conformation, only one derived from the carboxyl terminal showed inhibitory effect to the sperm-zona binding during in vitro fertilization. This phenomenon could not be explained by enhanced acrosomal exocytosis rate, in that the acrosomal reaction assay demonstrated its inability to induce the acrosomal reaction. Our results suggest that the carboxyl terminal of mZP3 polypeptide backbone interacts with sperm and such interaction plays a significant role in sperm-zona binding, ultimately successful fertilization.     

Aberrant distribution of ADAM3 in sperm from both angiotensin-converting enzyme (Ace)- and calmegin (Clgn)-deficient mice

Male mice deficient for the calmegin (Clgn) or the angiotensin-converting enzyme (Ace) gene show impaired sperm migration into the oviduct and loss of sperm-zona pellucida binding ability in vitro. Since CLGN is a molecular chaperone for membrane transport of target proteins and ACE is a membrane protein, we looked for ACE on the sperm membranes from Clgn-/- mice. ACE was present and showed normal activity, indicating that CLGN is not involved in transporting ACE to the sperm membranes. The ablation of the Adam2 and Adam3 genes generated animals whose sperm did not bind the zona pellucida, which led us to examine the presence of ADAM2 and ADAM3 in Clgn-/- and Ace-/- sperm. ADAM3 was absent from Clgn-/- sperm. In the Ace-/- mice, while ADAM2 was found normally in the sperm, ADAM3 disappeared from the Triton X-114 detergent-enriched phase after phase separation, which suggests that ACE is involved in distributing ADAM3 to a location where it can participate in sperm-zona pellucida binding. This diminished amount of ADAM3 in the Triton X-114 detergent-enriched phase may explain the inability of Clgn-/- and Ace-/- sperm to bind to the zona pellucida.