Human sperm plasma membranes possess alpha-D-mannosidase activity but no galactosyltransferase activity

Previous studies from this laboratory and others have identified several enzymes on the surface of mammalian spermatozoa. Some of these enzymes, namely a galactosyltransferase and a novel alpha-D-mannosidase, are believed to play a ligand-like role in recognizing and binding to the complementary moiety(ies) present on zona pellucida glycoconjugates. However, little or no information is available about the occurrence of these enzymes in human spermatozoa. In the present report, we show that a very small amount of the total galactosyltransferase activity present in human semen is associated with spermatozoa. Moreover, our failure to find a significant amount of the enzyme on sperm plasma membranes suggests that the enzyme is not associated with the sperm surface. Therefore, it is unlikely that galactosyltransferase in humans has the same ligand-like role in zona binding that is demonstrated in mouse sperm. In contrast, nearly 5% of alpha-D-mannosidase activity was repeatedly found in the salt-washed plasma membrane fraction. The recovery and enrichment of the alpha-D-mannosidase was nearly one-half that observed for adenylate cyclase and nearly one-third that for phosphodiesterase I, the two sperm plasma membrane marker enzymes. The differential enrichment and recovery of the sperm surface alpha-D-mannosidase is consistant with our previous studies in rat spermatozoa, and suggests that alpha-D-mannosidase may be localized on morphologically distinct region(s) of the sperm plasma membranes. The properties of human sperm surface alpha-D-mannosidase are quite similar to those reported by us for rat sperm plasma membrane mannosidase, but quite different from human sperm acid alpha-D-mannosidase. In addition, whereas anti-rat epididymal alpha-D-mannosidase antibody (IgG-fraction) cross-reacted with the human sperm acid alpha-D-mannosidase, no cross-reactivity was observed with the sperm surface mannosidase. A small amount of fucosyltransferase (less than 1% of the enzyme originally present on spermatozoa) was found in the salt-washed plasma membrane, but the enrichment of the enzyme was only one-tenth of that observed for adenylate cyclase. The potential ligand-like role of human sperm surface alpha-D-mannosidase and other sperm surface enzymes during fertilization is discussed.     

Novel alpha-D-mannosidase of rat sperm plasma membranes: characterization and potential role in sperm-egg interactions

During the course of a study of glycoprotein processing mannosidases in the rat epididymis, we have made an intriguing discovery regarding the presence of a novel alpha-D-mannosidase on the rat sperm plasma membranes. Unlike the sperm acrosomal "acid" mannosidase which has a pH optimum of 4.4, the newly discovered alpha-D-mannosidase has a pH optimum of 6.2, and 6.5 when assayed in sperm plasma membranes and intact spermatozoa, respectively. In addition, the two enzymes show different substrate specificity. The acrosomal alpha-D-mannosidase is active mainly towards synthetic substrate, p-nitrophenyl alpha-D-mannopyranoside, whereas the sperm plasma membrane alpha-D-mannosidase shows activity mainly towards mannose-containing oligosaccharides. Evidence is presented which suggest that the sperm plasma membrane alpha-D-mannosidase is different from several processing mannosidases previously characterized from the rat liver. The newly discovered alpha-D-mannosidase appears to be an intrinsic plasma membrane component, since washing of the purified membranes with buffered 0.4 M NaCl did not release the enzyme in soluble form. The enzyme requires nonionic detergent (Triton X-100) for complete solubilization. The enzyme is activated by Co2+ and Mn2+. However, Cu2+ and Zn2+ are potent inhibitors of the sperm plasma membrane alpha-D-mannosidase. At a concentration of 0.1 mM, these divalent cations caused nearly complete inactivation of the sperm enzyme. In addition methyl-alpha-D-mannoside, methyl-alpha-D-glucoside, mannose, 2-deoxy-D-glucose, and D-mannosamine are inhibitors of the sperm surface alpha-D-mannosidase. The physiological role of the newly discovered enzyme is not yet known. Several published reports in three species, including the rat, suggest that the sperm surface alpha-D-mannosidase may have a role in binding to mannose-containing saccharides presumably present on the zona pellucida.     

Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro

In previous reports from this laboratory, we identified the presence of a novel alpha-D-mannosidase on the surface of rat, mouse, hamster, and human spermatozoa [J Cell Biol 1989; 109:1257-1267 and Biol Reprod 1990; 42:843-858]. Since it has been suggested that mannosyl residues on the egg zona pellucida may be important for sperm-egg binding, studies were undertaken to examine the potential role of the sperm alpha-D-mannosidase during fertilization. Incubation of mouse spermatozoa in the presence of increasing concentrations of the inhibitory sugars, alpha-methyl mannoside, alpha-methyl glucoside, D-mannose, or D-mannitol, resulted in a dose-dependent decrease in the number of spermatozoa bound per egg without a deleterious effect on sperm motility or on the sperm acrosome, and a dose-dependent inhibition of the sperm mannosidase activity. Galactose, however had no effect on sperm-egg binding or on sperm mannosidase activity. Two nucleotide sugars (UDP-GlcNAc and UDP-gal) were also tested and shown to reduce sperm-egg binding but with only a minimal effect on sperm mannosidase activity. In additional studies, spermatozoa incubated in the presence of a mannose-containing oligosaccharide exhibited a dramatic reduction in sperm-egg binding that correlated with a similar inhibition of sperm mannosidase activity. The oligosaccharide substrate did not affect sperm motility or the sperm acrosome. These studies suggest that the sperm alpha-D-mannosidase may play an important role during fertilization.     

Purification and biological characterization of N-acetyl beta-D glucosaminidase from Bufo arenarum spermatozoa

Fertilization in Bufo arenarum requires the sperm to penetrate the egg envelopes. The incubation of isolated vitelline envelopes with sperm induces the acrosome reaction, releasing proteases and glycosidases to the media. In the present work N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, and alpha-D-glucosidase activities are measured in spermatozoa. N-acetyl-beta-D-glucosaminidase is the major sperm glycosidase activity assayed. However, N-acetyl-beta-D-galactosamine show competitive inhibitory effect. The glycosidase pH optimum is 3.5 being inhibited at pHs higher than 7.5. In our study, N-acetyl-beta-D-glucosaminidase is the only glycosidase that in vitro binds to vitelline envelopes in conditions that resemble natural fertilization media. The isolation of the active enzyme will allow studies of its role in fertilization. The enzyme has been purified in a two-step procedure. After native gel electrophoresis, the activity-stained band was cut out and the eluted enzyme was finally subjected to ConA-sepharose chromatography. In SDS-PAGE, the denatured enzyme migrates as a single band with a molecular mass of 45 kDa. Furthermore, analysis by size-exclusion on HPLC showed a peak of activity at around 45 kDa. Preliminary localization studies showed higher relative activity in the acrosomal content. In addition, 10% of the N-acetyl-beta-D-glucosaminidase activity was associated with the reacted sperm. By in vitro fertilization assay, it was observed that the inhibition of the enzyme results in the inhibition of fertilization. This last study shows that N-acetyl-beta-D-glucosaminidase plays an important role in toad fertilization.     

Glycosidases in the plasma membrane of Ceratitis capitata spermatozoa

Fruit flies in the family Tephritidae are rated among the world’s most destructive agricultural pests. The Mediterranean fruit fly Ceratitis capitata is emerging as a model organism to study the fertilization in Insects. Three integral proteins with glycosidase activity are present in the plasma membrane of spermatozoa. The glycosidases have been purified and characterized. We have demonstrated the presence of three enzymes, a ??-N-acetylhexosaminidase, an ??-mannosidase and an ??-l-fucosidase. The molecular mass of the native enzymes estimated by gel filtration was 160 kDa for ??-N-acetylhexosaminidase, 310 kDa for ??-mannosidase and 140 kDa for ??-l-fucosidase. SDS-PAGE showed that ??-N-acetylhexosaminidase is a dimer of a single protein of 73 kDa, ??-mannosidase consists of six subunits with different molecular weights and ??-l-fucosidase is a dimer made up by two different monomers. Characterization of the purified enzymes included glycosylation pattern, pI, optimal pH, substrate preference, kinetic properties and thermal stability. Soluble forms similar to the sperm associated glycosidases are present. Polyclonal antibodies raised against synthetic peptides designed from the predicted products of the Drosophila melanogaster genes encoding ??-N-acetylhexosaminidase and ??-l-fucosidase were used. Immunofluorescence labelling of spermatozoa showed that the enzymes are present in the sperm plasma membrane overlying the acrosome and the tail. This work represents the first report on the characterization in C. capitata of sperm proteins that are potentially involved in primary gamete recognition.     

Effect of a fertilization-promoting peptide on the fertilizing ability and glycosidase activity in vitro of frozen-thawed spermatozoa in the pig

This study has evaluated the effect of fertilization-promoting peptide (FPP) on the fertilizing ability and glycosidase activity in vitro of frozen-thawed boar spermatozoa. Use of chlortetracycline (CTC) fluorescence analysis, as well as various glycosidase analyses and the oocyte penetration test showed that FPP can promote the fertilizing ability and glycosidase activity of frozen-thawed spermatozoa in vitro. There were significantly (P < 0.05) more acrosome-reacted and penetrated in medium with 100 nM FPP than with 0, 50, 200 or 400 nM. The beta-N-acetylglucosaminidase (beta-GlcNAcase) activity was at least two-fold higher than other glycosidase regardless of FPP concentrations. In the same glycosidase, there were no differences in medium with different concentrations of FPP. The percentages of spermatozoa that reached acrosome reaction were affected by different periods (0, 1, 2, 3 or 4 h) of spermatozoa preincubation and were higher in medium with than without FPP. Penetration rates were decreased with preincubation periods of spermatozoa when oocytes were inseminated with spermatozoa preincubated in medium with and without FPP for the different periods. These rates were higher in spermatozoa preincubated with that than without FPP and had a tendency to increase as time of culture periods when the sperm-oocyte were cultured for 4, 8, 12, 16, 20 or 24 h. The activities of alpha-fucosidase, alpha-mannosidase, beta-galactosidase and beta-GlcNAcase were higher in medium with that than without FPP regardless of periods of sperm preincubation and sperm-oocyte culture. These results suggest that FPP may have a positive role in promoting sperm function and glycosidase activity in the pig.     

An alpha-L-fucosidase potentially involved in fertilization is present on Drosophila spermatozoa surface

Drosophila is emerging as a model organism to investigate egg fertilization in insects and the possible conservation of molecular mechanisms of gamete interactions demonstrated in higher organisms. This study shows that the spermatozoa of several species of Drosophila belonging to the melanogaster group have a plasma membrane associated alpha-L-fucosidase with features in common with alpha-L-fucosidases from sperm of other animals, including mammals. The enzyme has been purified and completely characterized in D. ananassae, because of its stability in this species. The sperm alpha-L-fucosidase is an integral protein terminally mannosylated, with the catalytic site oriented toward the extracellular space. It has a M(r) of 256 kDa and a multimeric structure made up by subunits of 48 and 55 kDa. Enzyme characterization included kinetic properties, pI, optimal pH, and thermal stability. A soluble form of the enzyme similar to the sperm associated alpha-L-fucosidase is secreted by the seminal vesicles. Synthetic peptides designed from the deduced product of the D. melanogaster gene encoding an alpha-L-fucosidase were used to raise a specific polyclonal antibody. Immunofluorescence labeling of spermatozoa showed that the enzyme is present in the sperm plasma membrane overlying the acrosome and the tail. Lectin cytochemistry analysis of the egg surface indicated that alpha-L-fucose terminal residues are present on the chorion with a strongly polarized localization on the micropyle. The alpha-L-fucosidase of Drosophila sperm plasma membrane appears to be potentially involved in gamete recognition by interacting with its glycoside ligands present on the egg surface at the site of sperm entry.     

Sperm plasma membrane breakdown during Drosophila fertilization requires sneaky, an acrosomal membrane protein

Fertilization typically involves membrane fusion between sperm and eggs. In Drosophila, however, sperm enter eggs with membranes intact. Consequently, sperm plasma membrane breakdown (PMBD) and subsequent events of sperm activation occur in the egg cytoplasm. We previously proposed that mutations in the sneaky (snky) gene result in male sterility due to failure in PMBD. Here we support this proposal by demonstrating persistence of a plasma membrane protein around the head of snky sperm after entry into the egg. We further show that snky is expressed in testes and encodes a predicted integral membrane protein with multiple transmembrane domains, a DC-STAMP-like domain, and a variant RING finger. Using a transgene that expresses an active Snky-Green fluorescent protein fusion (Snky-GFP), we show that the protein is localized to the acrosome, a membrane-bound vesicle located at the apical tip of sperm. Snky-GFP also allowed us to follow the fate of the protein and the acrosome during fertilization. In many animals, the acrosome is a secretory vesicle with exocytosis essential for sperm penetration through the egg coats. Surprisingly, we find that the Drosophila acrosome is a paternally inherited structure. We provide evidence that the acrosome induces changes in sperm plasma membrane, exclusive of exocytosis and through the action of the acrosomal membrane protein Snky. Existence of testis-expressed Snky-like genes in many animals, including humans, suggests conserved protein function. We relate the characteristics of Drosophila Snky, acrosome function and sperm PMBD to membrane fusion events that occur in other systems.     

Characterization of the pharmacological-sensitivity profile of neoglycoprotein-induced acrosome reaction in mouse spermatozoa.

Mammalian spermatozoa undergo the acrosome reaction (AR) in response to the interaction of a carbohydrate-recognizing molecule(s) on the sperm plasma membrane (sperm surface receptor) and its complementary glycan (ligand) moiety(ies) on the zona pellucida (ZP). Previously, we demonstrated that a hexose (mannose) or two amino sugars (glucosaminyl or galactosaminyl residues) when covalently conjugated to a protein backbone (neoglycoproteins) mimicked the mouse ZP3 glycoprotein and induced the AR in capacitated mouse spermatozoa (Loeser and Tulsiani, Biol Reprod 1999; 60:94-101). To elucidate the mechanism underlying sperm-neoglycoprotein interaction and the induction of the AR, we have examined the effect of several AR blockers on neoglycoprotein-induced AR. Our data demonstrate that two known L-type Ca(2+) channel blockers prevented the induction of the AR by three neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA). The fact that the L-type Ca(2+) channel blockers (verapamil, diltiazem) had no inhibitory effect on sperm surface galactosyltransferase or alpha-D-mannosidase, two carbohydrate-recognizing enzymes thought to be sperm surface receptors, suggests that the reagents block the AR by a mechanism other than binding to the active site of the enzymes.     

suREJ3, a polycystin-1 protein, is cleaved at the GPS domain and localizes to the acrosomal region of sea urchin sperm.

The sea urchin sperm acrosome reaction (AR) is a prerequisite for sperm-egg fusion. This report identifies sea urchin sperm receptor for egg jelly-3 (suREJ3) as a new member of the polycystin-1 family (the protein mutated in autosomal dominant polycystic kidney disease). suREJ3 is a multidomain, 2,681-amino acid, heavily glycosylated orphan receptor with 11 putative transmembrane segments (TMS) that localize to the plasma membrane covering the sperm acrosomal vesicle. Like the latrophilins and other members of the secretin family of G-protein-coupled receptors, suREJ3 is cleaved at the consensus GPS (G-protein-coupled receptor proteolytic site) domain. Antibodies to the extracellular 1,455-residue NH(2)-terminal portion identify a band at 250 kDa that shifts in electrophoretic mobility to 180 kDa upon glycosidase digestion. Antibodies to the 1,226-residue COOH-terminal portion identify a band at 150 kDa that shifts to 140 kDa after glycosidase treatment. Antibodies to both portions of suREJ3 localize exclusively to the plasma membrane over the acrosomal vesicle. Immunoprecipitation shows that both portions of suREJ3 are associated in detergent extracts. This is the first report showing that a polycystin family member is cleaved at the GPS domain. Localization of suREJ3 to the acrosomal region provides the first suggestion for the role of a polycystin-1 protein (components of nonselective cation channels) in a specific cellular process.     

Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa

Previous studies from our laboratory have demonstrated the presence of two integral proteins with glycosidase activity in the plasma membrane of Drosophila melanogaster spermatozoa and we have suggested that these enzymes might have a role in sperm-egg binding. In this study the glycosidases have been purified and characterized. We have evidenced the presence of three distinct enzymes, two beta-N-acetylhexosaminidase isoforms, named HEX 1 and HEX 2, and an alpha-mannosidase. The molecular size of the native enzymes estimated by gel filtration was 158 kDa for beta-hexosaminidases and 317 kDa for alpha-mannosidase. SDS-PAGE showed that HEX 1 and HEX 2 are dimers formed by subunits with different molecular sizes, whereas alpha-mannosidase consists of three subunits with different molecular weights. All the enzymes are terminally glycosylated. Characterization of the purified enzymes included their 4-methylumbelliferyl-substrate preferences, kinetic properties, inhibitor constants and thermal stability. On the basis of substrate specificity, kinetics and the results of inhibition studies, beta-hexosaminidases appear to differ from each other. HEX 1 and HEX 2 are similar to mammalian isoenzyme A and isoenzyme B, respectively.These findings represent the first report on the characterization of sperm proteins that are potentially involved in interactions with the egg in Insects.     

Glycosidases are present on the surface of Drosophila melanogaster spermatozoa

We investigated the presence of enzymes on the surface of Drosophila melanogaster spermatozoa that might bind to the carbohydrate residues of the egg shell. Spectrophotometric and fluorimetric studies were used on whole spermatozoa to assay galactosyltransferase and glycosidase activities. No galactosyltransferase is present on the sperm surface, whereas two glycosidases, β-N-acetylglucosaminidase (GlcNAc′ase) and α-mannosidase (Man′ase), have been evidenced. They have an optimal pH of 6–6.5 and 4, respectively. The same glycosidases were detected as soluble forms probably secreted by the seminal vesicle epithelium. We suggest that these enzymes might be involved in the recognition of α-mannose and β-N-acetylglucosamine residues present on the egg shell at the site of sperm entry. Mol. Reprod. Dev. 48:276–281, 1997. © 1997 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.     

Assessment of different sperm functional tests in golden‐headed lion tamarins (Leontopithecus chrysomelas)

The golden‐headed lion tamarin (Leontopithecus chrysomelas) is an endangered species endemic to Brazil's Atlantic Forest, a shrinking biodiversity hotspot. As in other Neotropical primates, its semen characteristics and freezability are poorly studied. Hence, reproductive technologies for callitrichids would greatly benefit from reliable methods of semen analysis. In a bid to promote reproductive research in tamarins, we validated simple and inexpensive sperm function tests that can be used to monitor sperm‐egg binding, plasma membrane and acrosome integrity, mitochondrial activity, and DNA fragmentation. Ejaculates from adult males were individually diluted and divided into control and damage‐induced aliquots, and then samples comprising assorted amounts of damaged spermatozoa were examined by organelle‐specific tests. Our findings showed that sperm‐binding in chicken egg perivitelline membrane (EPM) positively correlated with the number of spermatozoa injured by snap‐freezing. Eosin‐nigrosin (EN) and propidium iodide readings were correlated with each other, and both provided robust measurements of plasma membrane integrity. A high correlation between expected and measured amounts of acrosome‐intact spermatozoa was found using Fast Green‐Rose Bengal (FG‐RB), Coomassie Blue (CB), and FITC‐PSA stains, and all three methods exhibited comparable results. Likewise, different percentages of UV‐irradiated spermatozoa were accurately assessed for DNA integrity by Toluidine Blue (TB) and sperm chromatin dispersion (SCD) tests. Comparisons between 3,3′‐diaminobenzidine (DAB) and JC‐1 stains also indicated the reliability of the former assay to ascertain gradual increases in spermatozoa with greater mitochondrial function. These data confirmed that different parts of the tamarin spermatozoa can be simply and consistently evaluated by EPM, EN, FG‐RB, CB, TB, and DAB protocols.     

Induction of the acrosome reaction in the spermatozoa of the goat by secretions of the male accessory glands and milk

The balancing effects of bulbourethral gland secretion (BUS) and of seminal vesicle secretion (SVS) on goat semen quality were previously demonstrated. In the present study, electron microscope observations revealed a high frequency of spermatozoa with a reacted acrosome among spermatozoa from cauda epididymis exposed to BUS in the presence of milk. This frequency was significantly reduced when SVS had been added either before or after BUS. No reacted acrosome was observed in the absence of milk. All mount spermatozoa were incubated with milk or SVS or BUS or combinations of the three materials labeled with colloidal gold. SVS attached specifically on the plasma membrane covering the anterior part of the acrosome, whereas BUS spread all over the sperm head. Milk attached on the anterior half of the sperm head only when BUS was present in the sperm environment. It is concluded that BUS plays an active role in the induction of the acrosome reaction in the presence of milk and that SVS counteracts this role.     

Changes in rat sperm membrane glycosidase activities and carbohydrate and protein contents associated with epididymal transit

Rat spermatozoa were recovered from the caput, corpus, and cauda epididymides and assayed for glycosidase activity, total nonamino (neutral) carbohydrate, and protein content. The activities of beta-glucosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and beta-N-acetylgalactosaminidase were fluorometrically assayed in spermatozoa and membrane-enriched fractions. Except for beta-glucosidase, the activities of the glycosidases based on protein content were greatest in whole sperm and membrane-enriched fractions obtained from the cauda epididymides. Based on sperm concentration, however, glycosidase activities increased proceeding from the caput to the corpus epididymides, then declined from the corpus to the cauda epididymides. Analyses of nonamino carbohydrate and protein content based on sperm number indicated regional trends similar to those of glycosidase activity. Total nonamino carbohydrate and protein content were highest in corpus sperm, and lowest in cauda sperm. These data indicate major quantitative changes in cell surface carbohydrate as spermatozoa traverse the epididymis. A positive correlation for the membrane-enriched fraction between increasing glycosidase activity and decreasing carbohydrate and protein content suggests that glycosidases may play a significant role in modifying the spermatozoon surface during epididymal transit and maturation.     

Acrosomal enzymes of human spermatozoa before and after in vitro capacitation

The effect of in vitro capacitation (events that occur before the acrosome reaction) on the acrosomal enzymes of human spermatozoa was determined. Capacitation of human spermatozoa was assessed by their ability to penetrate denuded hamster oocytes. The activities of a number of enzymes commonly associated with the sperm acrosome, including nonzymogen acrosin, proacrosin, inhibitor-bound acrosin, hyaluronidase, acid phosphatase, beta-glucuronidase, beta-glucosidase, beta-N-acetylglucosaminidase, beta-galactosidase and beta-N-acetylgalactosaminidase were assessed. With the exception of acid phosphatase, no alteration in enzyme activity occurred after 4 h of incubating the spermatozoa under capacitation conditions although gamete fusion took place. The acid phosphatase levels decreased twofold, presumably due to the loss of seminal (prostatic acid phosphatase that loosely adheres to spermatozoa. After 8 h of capacitation, a large decrease in sperm enzyme levels took place only in the case of hyaluronidase, although small decreases were also noted in total acrosin, proacrosin and inhibited acrosin. No new electrophoretically migrating forms of acrosin were observed. Decreases in total acrosin and proacrosin, but not in inhibited acrosin, also occurred when spermatozoa were incubated under noncapacitating conditions for 8 h, indicating that capacitation may specifically cause the release of some acrosin inhibitor from human spermatozoa. It is concluded that, with the possible exception of hyaluronidase, the in vitro capacitation of human spermatozoa does not cause a major change in its acrosomal enzyme content so that these hydrolases are fully present before the acrosome reaction takes place during gamete fusion. Serum albumin appears to protect against the loss of some of these enzymes since the activity of several glycosidases was significantly reduced when the spermatozoa were incubated for 8 h in human serum albumin-free medium.     

The Interactions of Sea Urchin Gametes During Fertilization

The interactions between sea urchin spermatozoa and ova duringfertilization usually exhibit a high degree of species specificity.Under natural conditions and reasonable gamete concentrations,most interspecific inseminations fail to yield zygotes. Macromoleculeson the external surfaces of the apposing gametes must surelybe responsible for successful gamete recognition, adhesion andfusion. Species specific recognition between surface componentsof sperm and egg could occur during at least three events comprisingthe fertilization process. The first event is the interactionof the sperm plasma membrane with the egg jelly coat. This inducesthe sperm acrosome reaction resulting in the exocytosis of the"bindin" -containing acrosome granule and also the extrusionof the acrosome process from the anterior tip of the sperm.The second event is the adhesion of the bindin-coated acrosomeprocess to glycoprotein "bindin receptors" on the external surfaceof the egg vitelline layer. The third event is the penetrationof the vitelline layer and the fusion of sperm and egg plasmamembranes. With the isolations of the component of egg jellywhich induces the acrosome reaction, sperm bindin from the acrosomevesicle and the egg surface bindin receptor from the vitellinelayer, there is hope of discovering the molecular basis of thismost interesting intercellular interaction which results inthe activation of embryonic development.     

Sperm-egg interaction in the painted frog (Discoglossus pictus): An ultrastructural study

The ultrastructure of sperm changes and penetration in the egg was studied in the anuran Discoglossus pictus, whose sperm have an acrosome cap with a typical tip, the apical rod. The first stage of the sperm apical rod and acrosome reaction (AR) consists in vesiculation between the plasma membrane and the outer acrosome membrane. The two components of the acrosome cap are released in sequence. The innermost component (component B) is dispersed first. The next acrosome change is the dispersal of the outermost acrosome content (component A). At 30 sec postinsemination, when the loss of component B is first observed, holes are seen in the innermost jelly coat (J1), surrounding the penetrating sperm. Therefore, this acrosome constituent might be related to penetration through the innermost egg investments. At 1 min postinsemination, during sperm penetration into the egg, a halo of finely granular material is observed around the inner acrosome membrane of the spermatozoon, suggesting a role for component A at this stage of penetration. Gamete-binding and fusion take place between D1 (the egg-specific site for sperm interaction) and the perpendicularly oriented sperm. Spermatozoa visualized at their initial interaction (15 sec postinsemination) with the oolemma are undergoing vesiculation. The first interaction is likely to occur between the D1 glycocalyx and the plasma membrane of the hybrid vesicles surrounding the apical rod. As fusion is observed between the internal acrosome membrane and the oolemma, it can be postulated that gametic interaction might be followed by fusion of the latter with the apical rod internal membrane that extends posteriorly into the inner acrosome membrane. Insemination of the outermost jelly layer (J3) dissected out of the egg, and observations of the ultrastructural changes of spermatozoa in this coat, indicate that J3 rather than the vitelline coat (VC) induces the AR. Interestingly, at the late postinsemination stage, VC fibrils are seen crosslinking the inner acrosome membrane. The role of this binding is here discussed. Mol. Reprod. Dev. 47:323–333, 1997. © 1997 Wiley-Liss, Inc.     

A third sea urchin sperm receptor for egg jelly module protein, suREJ2, concentrates in the plasma membrane over the sperm mitochondrion

Sea urchin spermatozoa are model cells for studying signal transduction events underlying flagellar motility and the acrosome reaction. We previously described the sea urchin sperm receptor for egg jelly 1 (suREJ1) which consists of 1450 amino acids, has one transmembrane segment and binds to the fucose sulfate polymer of egg jelly to induce the sperm acrosome reaction. We also cloned suREJ3 which consists of 2681 amino acids and has 11 putative transmembrane segments. Both these proteins localize to the plasma membrane over the acrosomal vesicle. While cloning suREJ1, we found suREJ2, which consists of 1472 amino acids, has two transmembrane segments and is present in the entire sperm plasma membrane, but is concentrated over the sperm mitochondrion. Experimental evidence suggests that, unlike suREJ1 and suREJ3, suREJ2 does not project extracellularly from the plasma membrane, but is an intracellular plasma membrane protein. All three sea urchin sperm REJ proteins possess a protein module of > 900 amino acids, termed 'the REJ module', that is shared by the human autosomal dominant polycystic kidney disease protein, polycystin-1, and PKDREJ, a testis-specific protein in mammals whose function is unknown. In the present study, we describe the sequence, domain structure and localization of suREJ2 and speculate on its possible function.