Why did the sperm cross the cumulus? To get to the oocyte. Functions of the sperm surface proteins PH-20 and fertilin in arriving at, and fusing with, the egg

The sperm surface has an active role in the events of fertilization. The definition of the sperm surface in both its composition and domain organization begins during spermatogenesis and continues until the moment of sperm-egg fusion. Alterations of the surface proceed as a result of internal programming and environmental cues from both the male and female reproductive tracts, including interactions with the egg itself. We have investigated the sperm surface to understand its domain organization and the ongoing changes in this organization as well as the role of specific surface proteins in fertilization. Much of our research has concentrated on two surface proteins: PH-20 and fertilin. PH-20 is a single-chain protein, anchored in the membrane via a glycosyl phosphatidylinositol (GPI) anchor. The N-terminal domain of the molecule has a hyaluronidase activity. The hyaluronidase activity of PH-20 on the sperm plasma membrane enables sperm to penetrate the layer of cumulus cells surrounding the oocyte. PH-20 has a second function, unrelated to its hyaluronidase activity, in the binding of acrosome-reacted sperm to the zona pellucida (secondary sperm-zona binding). The fertilin molecule is an alpha,beta heterodimer whose two subunits are closely related transmembrane proteins. Fertilin beta has a disintegrin domain that has high sequence homology with the snake disintegrins, a known class of soluble integrin ligands. The binding site of the beta disintegrin domain functions to bind sperm to the egg plasma membrane via a mechanism that leads to sperm-egg fusion. The precursor of fertilin alpha, made in the testis, has an active metalloprotease site that could function in spermatogenesis. This metalloprotease domain is removed by proteolytic processing in the testis. Mature fertilin alpha on sperm also has a hydrophobic, putative "fusion peptide" that may promote the process of lipid bilayer fusion between sperm and egg plasma membranes. Fertilin alpha and beta are the first identified members of a new gene family of transmembrane proteins, the ADAM family, so called because they contain A Disintegrin And Metalloprotease domain. Many distinct ADAMs have now been found in diverse tissues and species (Drosophila to human) and are proposed to have a variety of functions in development and the adult. In addition to fertilin, other ADAMs are also present on the sperm plasma membrane and may participate with fertilin in sperm-egg fusion.     

Analysis of the process of localization of fertilin to the sperm posterior head plasma membrane domain during sperm maturation in the epididymis

Fertilin is a heterodimeric (subunits alpha and beta) sperm plasma membrane protein. Both subunits belong to the ADAM protein family of surface proteins that contain a disintegrin and a metalloprotease domain. Fertilin functions in sperm-egg fusion by binding the sperm to the egg plasma membrane via a binding site in the disintegrin domain of fertilin beta. On testicular sperm of guinea pig, fertilin is distributed on the plasma membrane over the entire sperm head, but is found only on the posterior head once sperm have passed through the epididymis. This redistribution of fertilin to the posterior head can be partially mimicked in vitro if testicular sperm are briefly treated with trypsin. In this study we used immunofluorescence and digital image analysis to analyze how fertilin becomes restricted to the posterior head. We found that fertilin became restricted to the posterior head by migration of anterior head fertilin molecules into the posterior head domain. Comparison of immunofluorescence patterns and immunoblots of fertilin from seven regions of the epididymis showed a temporal correlation between the beginning of fertilin's migration to the posterior head and the proteolytic processing of the full-length fertilin beta precursor (the 85-kDa pro-beta form) to a 75-kDa intermediate, pro-beta*. Completion of the migration coincided with the further cleavage of pro-beta* to the 25- to 28-kDa mature form. Our data suggest that the cleavage of fertilin pro-beta to pro-beta* may initiate fertilin's migration into the posterior head domain and, after localization to that membrane domain, pro-beta* is cleaved to mature beta. We also report evidence that a common mechanism may be used to change the localization pattern of other sperm surface molecules. Other surface proteins were shown to become localized to either the posterior or the anterior head membrane domains on sperm at the same time fertilin became localized to the posterior head. These restrictions of surface protein localizations were also shown to immediately precede the development of the sperm's ability to swim and undergo the acrosome reaction, and thus redistribution of surface proteins may be necessary before sperm become functional.     

Fertilization defects in sperm from mice lacking fertilin beta

Fertilin, a member of the ADAM family, is found on the plasma membrane of mammalian sperm. Sperm from mice lacking fertilin beta were shown to be deficient in sperm-egg membrane adhesion, sperm-egg fusion, migration from the uterus into the oviduct, and binding to the egg zona pellucida. Egg activation was unaffected. The results are consistent with a direct role of fertilin in sperm-egg plasma membrane interaction. Fertilin could also have a direct role in sperm-zona binding or oviduct migration; alternatively, the effects on these functions could result from the absence of fertilin activity during spermatogenesis.     

Stability in extraversion and aspects of social support at midlife

Fusion of sperm and egg plasma membranes is an early and essential event at fertilization but it is not known if it plays a part in the signal transduction mechanism that leads to the oscillations in the cytoplasmic free Ca2+ concentration ([Ca2+]i) that accompany mammalian egg activation. We have used two independent fluorescence methods and confocal microscopy to show that cytoplasmic continuity of egg and sperm precedes the onset of the first [Ca2+]i increase in mouse eggs. The Ca2+ indicator dye Ca2+-green dextran was microinjected and its transfer from egg to sperm was monitored. We found that it occurred before, and without a requirement for, any detectable [Ca2+]i increase in the egg. In separate experiments [Ca2+]i changes were recorded in populations of eggs, using fura red, and the eggs fixed at various times after some of the eggs had shown a [Ca2+]i transient. Fusion of the sperm and egg was then assessed by Hoechst dye transfer. All eggs that showed a [Ca2+]i increase had a fused sperm but more than half of the eggs contained a sperm but had not undergone a [Ca2+]i increase. These data indicate that sperm-egg fusion precedes [Ca2+]i changes and we estimate that the elapsed time between sperm-egg fusion and the onset of the [Ca2+li oscillations is 1-3 minutes. Finally, sperm-egg fusion was prevented by using low pH medium which reversibly prevented [Ca2+]i oscillations in eggs that had been inseminated. This was not due to disruption of signalling mechanisms, since [Ca2+]i changes still occurred if low pH was applied after the onset of oscillations at fertilization. [Ca2+]i changes also occurred in eggs in low pH in response to the muscarinic agonist carbachol. These data are consistent with the idea that the [Ca2+]i signals that occur in mammalian eggs at fertilization are initiated by events that are closely coupled to the fusion of the sperm and egg membranes.     

Human ZP3 restores fertility in Zp3 null mice without affecting order-specific sperm binding

The mammalian zona pellucida surrounding ovulated eggs mediates sperm binding at fertilization, provides a postfertilization block to polyspermy, and facilitates passage of pre-implantation embryos down the oviduct. Although the three zona proteins (ZP1, ZP2, ZP3) are well conserved, mammalian fertilization is relatively specific and human sperm do not bind to the mouse zona pellucida. There are considerable in vitro data that ZP3 acts as a primary sperm adhesion molecule in mice and, by analogy, a similar role has been postulated for human ZP3. Genetically altered mice lacking ZP3 (Zp3(tm/tm)) do not form a zona pellucida and are infertile. To rescue this phenotype, transgenic mice expressing human ZP3 (67% identical to mouse ZP3) were produced and bred with Zp3(tm/tm) null mice. The resultant human ZP3 rescue females had chimeric zonae pellucidae composed of mouse ZP1, mouse ZP2 and human ZP3. Human ZP3 expressed in mouse oocytes had an apparent mass (64 kDa) indistinguishable from native human ZP3 and distinct from mouse ZP3 (83 kDa). Despite the presence of human ZP3, human sperm did not bind to the chimeric zona pellucida, and notwithstanding the absence of mouse ZP3, mouse sperm bound to ovulated eggs in vitro and fertility was restored in vivo. These data have implications regarding the molecular basis of mouse and human sperm binding to their respective zonae pellucidae.     

Xenopus laevis sperm receptor gp69/64 glycoprotein is a homolog of the mammalian sperm receptor ZP2

Little is known about sperm-binding proteins in the egg envelope of nonmammalian vertebrate species. We report here the molecular cloning and characterization of a recently identified sperm receptor (gp69/64) in the Xenopus laevis egg vitelline envelope. Our data indicate that the gp69 and gp64 glycoproteins are two glycoforms of the receptor and have the same number of N-linked oligosaccharide chains but differ in the extent of O-glycosylation. The amino acid sequence of the receptor is closely related to that of the mouse zona pellucida protein ZP2. Most of the sequence conservation, including a ZP domain, a potential furin cleavage site, and a putative transmembrane domain are located in the C-terminal half of the receptor. Proteolytic cleavage of the gp69/64 protein by a cortical granule protease during fertilization removes 27 amino acid residues from the N terminus of gp69/64 and results in loss of sperm binding to the activated eggs. Similarly, we find that treatment of eggs with type I collagenase removes 31 residues from the N terminus of gp69/64 and has the same effect on sperm binding. The isolated and purified N terminus-truncated receptor protein is inactive as an inhibitor of sperm-egg binding. Earlier studies on the effect of Pronase digestion on receptor activity suggest that this N-terminal peptide may contain an O-linked glycan that is involved in the binding process. Based on these results and the findings on the primary structure of the receptor, a pathway for the maturation and secretion of gp69/64, as well as its inactivation following fertilization, is proposed.     

Evidence for a polyspermy block at the level of sperm-egg plasma membrane fusion in Urechis caupo

The results of sperm binding experiments reveal no change in the sperm binding properties of the egg surface coat at fertilization of Urechis caupo eggs. When fertilized eggs are reinseminated, sperm continue to attach to the egg surface coat. The acrosomal tubules of supernumerary sperm are observed in the perivitelline space closely apposed to the egg membrane. Thus, the polyspermy block in Urechis eggs involves neither alteration of sperm binding sites nor inhibition of the acrosome reaction. Our results suggest that the block is at the level of sperm-egg membrane fusion.     

Modelling human sperm-egg interactions in vitro: signal transduction pathways regulating the acrosome reaction

Recent advances in characterizing sperm surface receptors and ion channels, when combined with the rapidly expanding knowledge of interactions among second messenger systems in somatic cells, permit formulation of a tentative molecular mechanism for the regulation of the human sperm acrosome reaction. As spermatozoa pass through the cumulus mass, progesterone binds to its sperm surface receptor, alkalinizes the sperm head cytosol and potentiates changes in intracellular ionized calcium. Primary binding of spermatozoa to egg involves receptors for mannosyl, N-acetylglucosaminyl and, possibly, fucosyl residues of the glycosylated zona protein, ZP3. These receptors aggregate on multivalent ligand binding, migrate to the equatorial region along an actin filament network formed between the plasma and acrosomal membranes during capacitation, and activate a G protein/protein kinase A/protein kinase C second messenger system and a secondary proteolysis signal. Binding of a receptor tyrosine kinase to ZP3 amino acid residues simultaneous with the sugar recognition event triggers tyrosine phosphorylation signalling. All signals combine to open a voltage-dependent calcium channel. The resulting elevated calcium signal depolymerizes the inter-membrane actin network and activates phospholipases, leading to an acrosome reaction.     

The fertilization potential provides a fast block to polyspermy in lamprey eggs

At fertilization, the membrane potential of the egg of the lamprey, Lampetra japonica, shifted rapidly from its resting value of -12 to +36 mV and gradually returned to about the same resting level (fertilization potential). The amplitude of depolarization was influenced by the external Cl- concentration and by an anion channel blocker, DIDS, indicating that the positive shift of membrane potential resulted from Cl- efflux. A similar change in membrane potential (activation potential) was observed when the unfertilized egg was pricked with a fine needle or treated with A23187 to induce parthenogenetic activation. Pricking at the animal pole region (predetermined site for sperm entry) resulted in the occurrence of an immediate activation potential and the initiation of cortical granule exocytosis. A time lag between the pricking and the occurrence of the activation potential was observed when the egg was pricked at a distance from the animal pole. In this instance, the activation potential was produced immediately before the propagating cortical granule exocytosis initiated at the pricked site reached the animal pole region. Sperm-egg fusion was blocked in eggs voltage-clamped at +20 to +40 mV and inseminated, whereas it took place in eggs clamped at -60 to 0 mV. However, most eggs clamped at +20 to +40 mV did activate, indicating that the voltage dependence of egg activation differs from that of sperm-egg fusion. Although eggs voltage-clamped at negative membrane potentials permitted multiple sperm to fuse with the egg plasma membrane, the nucleus of the fused sperm did not necessarily enter the ooplasm. We conclude that: (1) A fast electrical block against polyspermy operates in this species and is effective for about 160 sec of the onset of the positive shift; (2) the opening of Cl- channels is responsible for the potential change; (3) the channels are largely localized in the animal pole region; (4) during voltage clamp at positive potentials, eggs can be activated without sperm-egg fusion; and (5) during voltage clamp at negative potentials, sperm-egg fusion occurs, but sperm entry into the egg cytoplasm does not always proceed.     

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 I-binding sites only decreased in the inner ZP, whereas reactivity to WGA was increased 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).     

Modifications of carbohydrate residues and ZP2 and ZP3 glycoproteins in the mouse zona pellucida after fertilization

Oligosaccharide side chains of zona pellucida (ZP) glycoproteins play a key role in the sperm-egg interaction phenomena during fertilization. In the present study, modifications of the ZP glycoproteins during the fertilization process in the mouse were studied by the lectin-gold technique and immunocytochemistry in conjunction with quantitative analysis. Binding of PNA, RCA I, DSA, LFA, MAA, AAA, and anti-ZP2 and anti-ZP3 antibodies was observed throughout the ZP of both unfertilized and fertilized eggs. However, HPA and BSAIB4 labeling was found only in the inner region of the ZP. After neuraminidase treatment (Neu), HPA showed an affinity for the entire ZP. Labeling by LFA, WGA, MAA, PNA, BSAIB4, and AAA decreased in the ZP of fertilized eggs; however, there was an increase in the binding of RCA I. HPA and Neu-HPA increased only in the inner region of the ZP. Immunoreactivity to antibodies against ZP2 and ZP3 also decreased after fertilization. The present results demonstrate that 1) terminal carbohydrate residues contained in the ZP glycoproteins are modified after fertilization and 2) inner and outer regions of the ZP contain different oligosaccharide side chains.     

Structural properties of the putative fusion peptide of fertilin, a protein active in sperm-egg fusion, upon interaction with the lipid bilayer

We recently demonstrated that a peptide representing the putative fusion domain of fertilin, a surface membrane protein of sperm involved in sperm-egg fusion, induces fusion of large unilamellar vesicles containing negatively charged lipids [Martin, I., and Ruysschaert, J. M. (1997) FEBS Lett. 405, 351-355]. In the present work, we demonstrate that increasing the concentration in negatively charged lipids strongly enhances the binding of the fertilin fusion peptide to the membrane, suggesting that electrostatic attractions play a crucial role in the binding process. While no significant change of the secondary structure content is observed by increasing the amounts of negatively charged lipids in the bilayer, the orientation of the alpha-helix changes from a parallel to an oblique orientation in the membrane. This topological change is confirmed by amide II hydrogen/deuterium exchange measurements that monitor the accessibility of the peptide to the water medium. Differential scanning calorimetry data also suggest that the fertilin fusion peptide lowers the bilayer to hexagonal phase transition temperature of model membranes composed of mixtures of dipalmitoleoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylserine and therefore promotes negative curvature in lipid vesicles. A comparison of the biophysical properties and the membrane-perturbing activities of fertilin and of viral fusion peptides is discussed in terms of sperm-egg fusion and virus cell fusion.     

Surface localization of the sea urchin egg receptor for sperm

The sea urchin egg receptor for sperm is thought to be involved in species-specific sperm-egg interactions at the egg surface. Recent revisions in the deduced amino acid sequence of the cloned cDNAs indicate that the protein encoded does not possess the common structural hallmarks of a membrane protein. Thus, investigation of the localization and association of the protein with the egg surface is crucial. We describe and characterize a new monoclonal antibody raised against recombinant sperm receptor protein. This antibody, in conjunction with several polyclonal antibodies, was used to study the receptor protein in eggs. Immunoprecipitation studies indicated that the antibodies recognize the high Mr (ca. 350 K) sperm receptor protein which copurified with egg plasma membrane-vitelline layer complexes. The sperm receptor protein was solubilized only by detergents and not by treatments designed to solubilize peripherally associated or lipid-anchored membrane proteins, suggesting a tight association with the membrane fraction. Confocal immunofluorescence microscopy of live eggs indicated surface staining. Finally, lysylendoproteinase C treatment of live eggs resulted in a loss of the high Mr receptor protein epitopes, and the concomitant release of a 70-kDa proteolytic fragment, which correlated with a reduced ability of the eggs to be fertilized. Taken together, these data indicate that at least some fraction of the sperm receptor protein is present on the egg surface, a requisite locale for a sperm binding protein.     

Regulation of the polyspermy block in the mouse egg: maturation-dependent differences in cortical granule exocytosis and zona pellucida modifications induced by inositol 1,4,5-trisphosphate and an activator of protein kinase C

Germinal vesicle (GV)-intact fully grown mouse oocytes do not undergo cortical granule (CG) exocytosis in response to A23187 treatment, whereas metaphase II (MII)-arrested eggs do. This differential response may reflect the development of the ability of the egg to undergo CG exocytosis, which is responsible for the biochemical modification of the glycoprotein ZP2 in the zona pellucida. Accordingly, we compared in these two stages the ability of 12-O-tetradecanoyl phorbol 13-acetate (TPA) or inositol 1,4,5-trisphosphate (IP3) to promote CG exocytosis and/or the ZP2 to ZP2f conversion; these agents are known to stimulate early events of mouse egg activation. TPA (10 ng/ml) treatment for 60 and 120 min resulted in a 25% and 52% CG loss in GV-intact oocytes and a 38% and 76% loss in MII eggs, respectively; fertilization resulted in a CG loss of approximately 70-80%. Although a similar extent of ZP2 to ZP2f conversion was observed in oocytes and eggs after a 120-min TPA treatment (approximately 70-80%), a greater extent of conversion was observed in oocytes after a 60-min treatment (80% for oocytes, 50% for eggs). Microinjection of IP3 (final concentration 1 microM) into MII eggs resulted in an extent of ZP2 conversion similar to that observed following fertilization, whereas little conversion occurred in GV-intact oocytes similarly injected. These results indicate that a protein kinase C sensitivity develops prior to meiotic maturation, whereas responsiveness to IP3 develops after maturation has resumed. We propose that the regulatory mechanism involving an IP3-mediated calcium release is deficient in GV-stage oocytes.     

Evidence that Gq family G proteins do not function in mouse egg activation at fertilization

Embryonic development is initiated after the fertilizing sperm contacts the egg and triggers a process termed "egg activation," resulting in calcium release, cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. Heterotrimeric guanine nucleotide-binding proteins (G proteins) may be involved in mouse egg activation since inhibition of G protein beta gamma subunits partially inhibits sperm-induced cell cycle resumption. In addition, specific events of egg activation can be initiated in the absence of sperm by acetylcholine stimulation of mouse eggs overexpressing the human m1 muscarinic receptor, a G protein-coupled receptor. In somatic cell, G proteins in the Gq family couple ligand stimulation of the m1 muscarinic receptor to activation of phospholipase C, resulting in the production of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated release of intracellular calcium. Since IP3-mediated calcium release is involved in egg activation at fertilization, we have examined the role of Gq family G proteins in both sperm-independent (muscarinic receptor-mediated) and sperm-induced egg activation using a function-blocking antibody raised against the common C-terminal region of Gq and G11 proteins. We show that this antibody effectively inhibits Gq family G proteins in mouse eggs by demonstrating that the antibody inhibits egg activation in response to stimulation of the m1 muscarinic receptor. This same antibody, however, does not inhibit sperm-induced egg activation events. These results indicate that although activation of Gq family G proteins can result in egg activation in the mouse, it is unlikely that these proteins are used by the sperm to initiate egg activation at fertilization.     

Meiotic cells monitor the status of the interhomolog recombination complex

Cortical granule exocytosis is important for the block to polyspermy at fertilization in the eggs of most vertebrates and many invertebrates. Cortical granules are poised at the cell surface and exocytose in response to sperm stimulation. Following exocytosis, the cortical granule contents modify the extracellular environment of the egg, the major result of which is to block additional sperm binding. Here we show that proteins homologous to members of the SNARE hypothesis-a molecular model designed to explain the trafficking, docking, and exocytosis of vesicles in the secretory compartment-are present in eggs at the right time and place to be involved in the regulation of cortical granule exocytosis. Using polymerase chain reaction (PCR) screens we have found homologues of synaptobrevin/VAMP, syntaxin, synaptotagmin, and rab3. Antibodies generated to fusion proteins or to synthetic peptides encoded by the cloned cDNAs were used in an immunofluorescence assay to show that each of the cognate proteins are present in the cortex of the egg. A synaptobrevin/VAMP homologue appears to be specifically associated with the membrane of cortical granules before fertilization and, following cortical granule exocytosis, is incorporated into the plasma membrane of the zygote. A rab3 homologue is also associated with cortical granules specifically but, following fertilization, the protein reassociates with different, yet undefined, vesicles throughout the cytoplasm of the zygote. Homologues of synaptotagmin and syntaxin are also present at the egg cortex but, in contrast to rab3 and VAMP, appear to be associated with the plasma membrane. Following fertilization, syntaxin and tagmin remain associated with the plasma membrane and are more readily immunolabeled, presumably due to an increased accessibility of the antibodies to the target protein domains. We also show by immunoblotting experiments that the cognate proteins are of the sizes predicted for these homologues. These results suggest that at least some steps in the biology of cortical granules may be mediated by SNARE homologues, and this finding, along with the unique biology of cortical granules, should facilitate examination of specific events of the fertilization reaction and the mechanism of stimulus-dependent exocytosis.     

Perioperative outcomes of major hepatic resections under low central venous pressure anesthesia: blood loss, blood transfusion, and the risk of postoperative renal dysfunction

Fish sperm head plasma membranes have been demonstrated to contain syndecan (transmembrane heparan sulfate proteoglycan) immunofluorohistochemically and using immunoblot analysis and transglutaminase (TGase) by histochemistry. In order to examine the involvement of syndecan in fertilization, mature eggs were inseminated by direct mixing with untreated sperm or with sperm pretreated with an antiheparan sulfate (HS) antibody monoclonal (mAb), bovine serum albumin, human transferrin, or a TGase inhibitor (monodansylcadaverine, cystamine, and iodoacetamide). The fertilization rates of eggs inseminated with untreated and albumin-pretreated sperm were approximately 99.3% and 94.7%, respectively. Those of eggs pretreated with the anti-HS antibody and transferrin were 0% and 5.4%, respectively, whereas use of sperm pretreated with TGase inhibitors resulted in fertilization rates of approximately 13.2-17.8%. These results indicate that sperm head syndecan play an important role in fish sperm-egg contact and/or binding and that TGase inhibitors may reduce the fertilization rate by inhibiting sperm motility.     

Phorbol esters down-regulate alpha-fetoprotein gene expression without affecting growth in fetal hepatocytes in primary culture

The mammalian spermatozoon undergoes continuous modifications during spermatogenesis, maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are capable of binding the zona-intact egg and undergoing the acrosome reaction. The fertilization process is a net result of multiple molecular events which enable ejaculated spermatozoa to recognize and bind to the egg's extracellular coat, the zona pellucida (ZP). Sperm-egg interaction is a species-specific event which is initiated by the recognition and binding of complementary molecule(s) present on sperm plasma membrane (receptor) and the surface of the ZP (ligand). This is a carbohydrate-mediated event which initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be a prerequisite which enables the acrosome reacted spermatozoa to penetrate the ZP and fertilize the egg. This review focuses on the formation and contents of the sperm acrosome as well as the mechanisms underlying the induction of the acrosome reaction. Special emphasis has been laid on the synthesis, processing, substrate specificity, and mechanism of action of the acid glycohydrolases present within the acrosome. The hydrolytic action of glycohydrolases and proteases released at the site of sperm-zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important factors regulating the penetration of ZP. We have discussed the most recent studies which have attempted to explain signal transduction pathways leading to the acrosomal exocytosis.     

Protein-carbohydrate interactions during fertilization

Interaction between gametes during fertilization is at least in part regulated by carbohydrate moieties of the zona pellucida (ZP) and carbohydrate binding proteins of the sperm surface. This review focuses on the protein-carbohydrate interactions during the primary binding of the sperm to the ZP in different species. Synthesis, structure and composition of the ZP are summarized. The functional significance of carbohydrate residues of the ZP as sperm receptor is discussed. Sperm surface proteins known to have specific ZP and carbohydrate-binding sites including the mouse beta1, 4-galactosyltransferase and sp56, the rabbit protein Sp17, a human mannose-binding protein and several members of the spermadhesin family are presented.