Guinea Pig Sperm Research Articles

Identification and Localization of a Novel Cathepsin S-like Proteinase in Guinea Pig Spermatozoa

Guinea pig spermatozoa were found to contain a novel cysteine proteinase that most closely resembled lysosomal cathepsin S. The responsible enzyme was shown to have an acrosomal localization. Like the cathepsin S of lymphocyte-rich tissues, e.g., lymph nodes and spleen, the spermatozoal enzyme digested protein substrates such as hemoglobin and Azocoll optimally at about pH 3.5 in the presence of pepstatin. No appreciable action was manifested in the range of pH 5-6 on protein substrates nor on the 7-(4-methyl)coumarylamide (NMec) derivatives of peptides commonly employed for the fluorometric assay of cathepsins B (benzyloxycarbonyl (Z)-Arg-Arg-NMec), H (Arg-NMec), and L (Z-Phe-Arg-NMec). Like spleen cathepsin S, the sperm enzyme selectively hydrolyzed Z-Phe-Arg-NMec, but its activity was uncharacteristically limited to a pH range of 3.0-3.5, where it displayed a typical high sensitivity to sulfhydryl reagents (HgCl 2, mersalyl acid, iodoacetate), leupeptin, Z-Phe-Phe-CHN 2, and L- trans-epoxysuccinylleucylamido(3-methyl)butane. Gossypol (a male antifertility agent) was also inhibitory. Inhibition by the mercurial sulfhydryl reagents was completely reversible with dithiothreitol. Pepstatin, a potent inhibitor of aspartic proteinases, and serine proteinase inhibitors (phenylmethylsulfonyl fluoride, beazamidine) were ineffective. Other properties displayed by the sperm extract that were uniquely characteristic of cathepsin S included stability under alkaline conditions, and a greater rate of hydrolysis when the P 2-phenylalanine of the assay substrate was replaced by two aliphatic residues, as in Z-Leu-Leu-Arg-NMec.

A Role for the WH-30 Protein in Sperm-Sperm Adhesion during Rouleaux Formation in the Guinea Pig

Mammalian spermatozoa participate in specific cell adhesion phenomena during their development and functional lifespan; this includes interaction with Sertoli cells, the zona pellucida, and the oolemma. In some species such as the guinea pig, an additional sperm-sperm adhesion occurs during epididymal maturation which results in the formation of rouleaux in which the sperm heads are stacked one upon the other and the periacrosomal plasma membranes of adjacent sperm are linked by periodic crossbridges. In this study, we have used a monoclonal antibody to investigate the role of the WH-30 protein on the sperm surface in the formation of the junctional zones between adjacent guinea pig sperm in rouleaux. WH-30 monoclonal antibodies caused a dose- and time-dependent dissociation of rouleaux and an increase in the percentage of single, acrosome-intact sperm; there were no effects on sperm motility (maintained at 80-90%) or ultrastructure during the 120-min incubations. The maximal effect of about 80% single sperm was obtained with a 1:4 dilution of the WH-30 hybridoma supernatant or 5-50 μg/ml of purified WH-30 IgG. In contrast, incubation of sperm in AH-20 IgG, myeloma cell supernatants, or purified, nonspecific mouse IgG 1 had no effect on rouleaux. Treatment of sperm with a WH-30 Fab fragment resulted in almost complete dissociation of rouleaux without any observed effect on sperm motility or acrosomal status. Surface labeling of sperm followed by immunoprecipitation and SDS-PAGE revealed that the WH-30 antibody recognizes a single polypeptide of 43-45 kDa. Using immunofluorescence, the WH-30 protein was localized over the entire surface of the sperm head (wholehead pattern), and immunogold labeling showed that WH-30 is localized in the glycocalyx on both the dorsal and ventral surfaces of the periacrosomal and postacrosomal plasma membranes. These results indicate that the WH-30 protein on the sperm surface is a cell adhesion protein which is involved in the molecular interactions that maintain guinea pig sperm in rouleaux.

Proteases are not involved in the membrane fusion events of the lysolecithin-mediated guinea pig sperm acrosome reaction

The guinea pig sperm acrosome reaction is characterized by a complex temporal and structural pattern of membrane fusions. In this study, we have used specific protease inhibitors to determine if proteases regulate this pattern of membrane fusions during the lysolecithin-mediated guinea pig sperm acrosome reaction. Inhibitors were chosen so as to cover a wide range of different types of proteases, and all were used at the highest concentration that did not adversely affect sperm motility. Of the eight inhibitors tested, leupeptin, soya bean trypsin inhibitor (SBTI), p-aminobenzamidine (pAB) and nitrophenyl p'-guanidino benzoate (NPGB) inhibited completion of the acrosome reaction, while diethylenetriaminepentaacetic acid (DTPA), phosphoramidon, bestatin and pepstatin had no effect. Sperm that had been acrosome-reacted in the presence of each inhibitor were examined by transmission electron microscopy to assess whether the inhibitors altered the pattern of membrane fusions during the acrosome reaction. DTPA, phosphoramidon, bestatin and pepstatin had no effect on membrane fusion or matrix dispersal. Serine protease inhibitors such as leupeptin, SBTI, pAB and NPGB prevented complete dispersal of the acrosomal matrix and completion of the acrosome reaction, but did not alter the temporal sequence or structural pattern of membrane fusions. The undispersed matrix was present along the dorsal and ventral aspects of the apical segment and throughout the principal segment. We conclude that proteases are not involved in regulating the temporal and structural pattern of membrane fusions which occurs during the lysolecithin-mediated acrosome reaction of guinea pig sperm.

Purification, cloning, and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin, from Bothrops jararaca venom. Insights into the disintegrin gene family

A large hemorrhagin, jararhagin, has been cloned from a Bothrops jararaca venom gland cDNA expression library. The cDNA sequence predicts a 421-amino acid residue molecule with strong amino acid sequence homology and similar domain structure to HR1B, a high molecular weight hemorrhagic metalloprotease isolated from Trimeresurus flavoviridis (Habu) venom. Like HR1B, jararhagin contains enzyme, disintegrin, and cysteine-rich carboxyl-terminal regions. In the disintegrin region, the Arg-Gly-Asp sequence is replaced by Glu-Cys-Asp, as found in non-Arg-Gly-Asp disintegrin regions of HR1B and a guinea pig sperm fusion protein PH-30 beta. The cDNA sequence of jararhagin predicts a precursor protein (proprotein) with striking similarity to cryptic regions in precursors of the disintegrin peptides trigramin and rhodostomin. Comparison of jararhagin with disintegrin precursors highlights the modular arrangement of proprotein, metalloprotease, and disintegrin domains in the metalloprotease/disintegrin family and provides an insight into their biosynthesis and evolution.

F-actin in guinea pig spermatozoa: its role in calmodulin translocation during acrosome reaction.

The presence of actin has been determined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F-actin and a possible function for it been presented. In this work, actin distribution and F-actin were determined in uncapacitated, capacitated, and acrosomal-reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine-phalloidin. With the last probe we found filamentous actin in these cells. By both techniques, actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDS-PAGE and Western blots immunostained with monoclonal and polyclonal anti-actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa. Actin was also detected in preparations of Percoll-purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F-actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosomal region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility.

A mammalian epididymal protein with remarkable sequence similarity to snake venom haemorrhagic peptides.

Following spermatogenesis in the testis, mammalian spermatozoa pass into the epididymis, where they undergo changes which confer on them forward motility and the ability to recognize and penetrate the egg. Many of these maturation events involve androgen-regulated epididymal proteins which become associated with the sperm membrane, and/or effect changes to integral sperm membrane proteins. Here we report the sequence of an 89 kDa androgen-regulated protein from rat (Rattus norvegicus) and monkey (Macaca fascicularis) epididymis that is synthesized exclusively in the caput region and is localized on the apical surface of its principal epithelial cells. This protein shows remarkable similarity to a variety of proteases and disintegrins found in snake venoms and is similar to, but distinct from, the guinea-pig sperm surface PH-30 alpha/beta complex recently implicated in sperm-egg recognition and fusion.

Calcium transport and Ca ++-ATPase activity in spermatozoal plasma membrane vesicles of nifedipine-administered guinea pigs

Spermatozoal plasma membrane vesicles isolated from distal portion of the epididymis and vas deferens were found to contain Ca ++-activated ATPase and calcium transport activities. Nifedipine was administered at two different doses (1.0 and 2.5 mg/kg b.w./day) and the effect was observed for both short- (4 week) and long-term (12 week) period. The cellular ionic calcium content and Ca ++-ATPase activity were observed to be enhanced in the drug-treated animals. The recovery studies carried out after 4 and 6 weeks of withdrawal of the drug treatment exhibited partial to complete restoration of observed changes. The stimulatory rather than inhibitory effect of Nifedipine, a specific calcium channel blocker, on calcium uptake may suggest that voltage-sensitive calcium channels may be lacking in guinea pig spermatozoa. The stimulatory effect of the drug is speculated to be either by inhibition of Na +-Ca ++ antiporter or G-protein activated agonistic effect or probably due to altered physicochemical properties of the drug-treated sperm plasma membranes.

Effect of spermine on sperm capacitation of guinea pig in vitro.

Spermine (Sp) 10(-5) mM had vigorous activity of guinea pig spermatozoa, while it completely abolished sperm forward motility (SFM) at a concentration of 10(-3) mM. There appeared to be a dose relationship to inhibition to motility. 2-Difluoromethylornithine 10 mM antagonized the Sp-induced inhibition of SFM after 3 h of incubation. Capacitation of a guinea pig sperm was inhibited by Sp in a concentration-dependent manner. The majority of acrosome-reacted sperm did not display hyperactivated motility. Precapacitated sperm were able to undergo the acrosome reaction (AR) in the presence of Sp. Moreover, Sp-mediated inhibition of capacitation was a reversible process. Once sperm capacitation was completed, Sp no longer inhibited AR. Before capacitation, the content of Sp in spermatozoa was 4.5 +/- 0.5 micrograms/5 x 10(7) cells, whereas in case of capacitated spermatozoa it was significantly decreased (2.1 +/- 0.4 micrograms/5 x 10(7) cells). The penetration of spermatozoa into the zona-free hamster eggs in the presence of Sp was markedly decreased, but it did not affect the fertilizability of ova as compared to the control. These results suggest that Sp may be an inhibitory agent of sperm capacitation in guinea pig in vitro, and it may also be involved in the modulation of capacitation.

Albumin is Required for the Guinea Pig Sperm Capacitation but is not Essential for Acrosome Reaction and Fusion with Eggs

The importance of serum albumin in supporting guinea pig sperm capacitation, acrosome reaction, and fusion with eggs in vitro was studied by incubating the spermatozoa in albumin-free medium containing different synthetic polymers. Serum albumin was found to be an obligatory component in the incubating medium for the capacitation of guinea pig spermatozoa. Albumin in the medium is not essential for the acrosome reaction and fusion with the eggs, but these phenomena take place most efficiently in presence of albumin.

The effect of permeant and impermeant osmoticants on exocytosis in guinea pig sperm

Guinea pig sperm were suspended in calcium-containing medium supplemented with various concentrations of the tetrasaccharide, stachyose. At concentrations up to and including 0.6 M, stachyose was without effect on the A23187-induced acrosome reaction. At 1.0 M stachyose, greater than 97% of sperm retained their acrosome after exposure to A23187, as judged by light microscopy. Electron microscopy demonstrated, however, that exocytotic membrane fusion had occurred, although with substantial retention of the acrosomal matrix. Sperm incubated in 1.0 M stachyose solutions also underwent exocytotic membrane fusion in the absence of A23187 and external calcium. Sperm suspended in 0.175 M ammonium chloride solution progressively lost motility over 30 min, but without acrosomal swelling. By contrast, sperm in 0.19 M ammonium acetate underwent substantial swelling of the acrosome within 2-5 min. 70-80% of these sperm were able to exclude the vital dye propidium iodide with their acrosomes swollen. These sperm underwent acrosomal shrinkage if resuspended in normal medium within 5-10 min, and the majority (60-70%) recovered some motility. These sperm could undergo an A23187-induced acrosome reaction. Electron microscopy indicated that swelling in ammonium acetate solution solubilizes much of the acrosomal matrix and causes internal fusion between adjacent regions of the outer acrosomal membrane. There was no exocytotic membrane fusion in ammonium acetate solution, however. The evidence suggests that there is no stachyose osmolality for guinea pig sperm which will suppress the membrane fusion associated with exocytosis, and that sufficiently high osmolalities cause exocytotic membrane fusion in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical and biochemical characterization of guanine nucleotide-binding regulatory proteins in mammalian spermatozoa

Polyclonal antisera directed against conserved and subtype-specific peptide sequences of the α-subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to characterize the nature of mammalian sperm G proteins and to determine whether their localization was consistent with their proposed roles in mediating ZP3-induced acrosomal exocytosis. Mouse and guinea pig sperm exhibit positive immunofluorescence in the acrosomal region using an antiserum directed against a peptide region common to all α-subunits of G proteins (G α). The immunofluorescence disappears after sperm have undergone the acrosome reaction, suggesting that the immunoreactive material is associated with the plasma membrane/outer acrosomal membrane region overlying the acrosome. The presence of G proteins in this region is confirmed by the presence of a M r 41,000 substrate for pertussis toxin (PT)-catalyzed [ 32P]ADP-ribosylation in purified plasma membrane/outer acrosomal membrane hybrid vesicles obtained from acrosome-reacted guinea pig sperm. Immunoprecipitation and polyacrylamide gel electrophoresis of PT-catalyzed [ 32P]ADP-ribosylated protein(s) using anti-peptide antisera generated against sequences unique to G iα1, G iα2, and G iα3 confirm the existence of all three G i subtypes in mouse sperm extracts. Indirect immunofluorescence using an antiserum directed against a peptide region present in G zα, a PT-insensitive G protein, demonstrates positive immunoreactivity in the postacrosomal/lateral face region of the mouse sperm head. This immunoreactivity is retained during acrosomal exocytosis in response to solubilized ZP and then disappears subsequent to this exocytotic event. These data demonstrate that G i protein α-subunits are present in the acrosomal region of mammalian sperm, consistent with their postulated role in regulating ZP3-mediated acrosomal exocytosis, and that PT-insensitive G zα is found in a region of the sperm head distinct from that of the G iα subunits.

Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains

Proacrosin from guinea pig cauda epididymal sperm has a lower molecular weight compared with the testicular zymogen. In this study, we have examined the structural basis of this change and where the conversion in proacrosin molecular weight occurs during sperm maturation. Immunoblotting of trifluoromethanesulfonic acid-deglycosylated testicular and cauda epididymal sperm extracts with antibody to guinea pig testicular proacrosin demonstrated that the polypeptide backbones of proacrosins from the testis and cauda epididymal sperm had the same molecular weights (approximately 44,000). Keratanase, an endo-beta-galactosidase specific for lactosaminoglycans, partially digested testicular proacrosin but had no effect on proacrosin from cauda epididymal sperm. In extracts of testis, caput epididymis, and corpus epididymis analyzed by immunoblotting, anti-proacrosin recognized a major antigen with an apparent molecular weight (Mr) of 55,000, although a 50,000-Mr minor antigen began to appear in the corpus epididymis. By contrast, extracts of cauda epididymis, vas deferens, and cauda epididymal sperm had the 50,000 Mr protein as the only immunoreactive antigen. By enzymography following electrophoresis, the major bands of proteolytic activity in extracts of testis, caput epididymis, and corpus epididymis had 55,000 Mr. A band of protease activity with 55,000 Mr also appeared in extracts of the corpus epididymis. However, the most prominent bands of proteolytic activity in cauda epididymis, vas deferens, and cauda epididymal sperm had 50,000 Mr. In addition, two other major protease activities were detected with 32,000 and 34,000 Mr; the relationships of these proteases to proacrosin are unclear. From these results, we conclude that the oligosaccharides of proacrosin are altered during epididymal transit and that this modification occurs in the corpus epididymis.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose regulation of guinea-pig sperm motility

Washed guinea-pig spermatozoa from the vas deferens re-acquired progressive motility within 1-2 min of incubation in minimal culture medium containing pyruvate and lactate. When glucose was added, either at the beginning or after 15 min of incubation, the cells showed stimulated motility (increased straight-line velocity, linearity and beat-cross frequency, P less than 0.01). Re-acquisition of progressive motility was preceded by a significant (P less than 0.005) transient increase in sperm concentration of cyclic adenosine 5'-phosphate (cAMP) with or without glucose in the medium. Papaverine caused another large significant (P less than 0.001) increase in cAMP concentration; and 5.56mM glucose with papaverine caused a further stimulation in cAMP beyond that with papaverine alone (P less than 0.005). Although 0.05 or 5.56mM glucose plus alpha-chlorohydrin stimulated sperm motility, they did not further stimulate the increase in cAMP after 30 s of incubation. Thus, there was no apparent correlation between the glucose-stimulating effect on sperm motility and the enhancement of cAMP at 30 s. However, there was a close correlation between glucose-stimulated motility and enhancement of ATP (P less than 0.05) by glucose even under incubation conditions in which glucose caused the Crabtree effect (decrease in respiration rate).

A mechanism for differential release of acrosomal enzymes during the acrosome reaction

To study the organization of fertilization enzymes in the sperm acrosome, we isolated and characterized two physicochemically distinct acrosomal fractions of guinea-pig spermatozoa. A soluble fraction contained the 25,000-Mr acrosomal autoantigen, AA1, and most of the acrosomal hyaluronidase and dipeptidyl peptidase II activity. A particulate fraction, designated acrosomal matrix (AM), consisted of membraneless crescent-shaped structures, and contained most of the acrosomal proacrosin. The AM also contained a 28,000-Mr putative proacrosin-binding protein, and a very-high-Mr component which, on reduction, was dissociated into 48,000-Mr and 67,000-Mr subunits. Autoproteolytic dissolution of the AM correlated with proteolysis by acrosin of the 28,000-Mr and 48,000-Mr AM molecules. Components of both the AM and the soluble fraction were localized by immuno-electron microscopy to the electron-dense region of the guinea-pig sperm acrosome. We conclude that acrosomal molecules are segregated into soluble and matrix compartments. This segregation is a function of disulphide bonding and non-covalent interactions among the relatively few components of the AM. Association of acrosin with the AM may be the mechanism by which this enzyme's release from the spermatozoon during the acrosome reaction is delayed relative to the release of other acrosomal molecules.

Requirement of an extracellular energy substrate for the guinea pig sperm acrosome reaction induced by calcium ionophore

It is well established that calcium ionophore A 23187 induces acrosome reaction (AcR) of uncapacitated spermatozoa in the presence of extracellular Ca2+ ions. In the present study, we have investigated how extracellular energy substrates (glucose, pyruvate, and lactate) affect the ionophore-induced AcR of guinea pig spermatozoa. It was found that 0.3 microM concentration of A 23187 had the maximum effect to initiate AcR of guinea pig spermatozoa. Virtually no spermatozoa underwent their AcR when incubated in substrate-free modified Tyrode's medium containing 0.3 microM A 23187 and 2 mM Ca2+. At least one exogenous substrate is essential for the ionophore-induced AcR of spermatozoa. As for efficacy of the substrates, lactate was more effective than pyruvate and glucose. However, a better result was observed when lactate was added along with pyruvate. Malonate inhibited the ionophore-induced AcR but not the hyperactivated motility of spermatozoa. The mitochondrial electron transport chain blockers rotenone, antimycin, and oligomycin failed to inhibit AcR, although in the presence of these blockers spermatozoa were unable to show hyperactivated motility. These results suggest that the mitochondrial citric acid cycle, not the electron transport chain, is probably the energy source for ionophore-induced AcR of guinea pig spermatozoa.

Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism

The redistribution of membrane proteins on the surface of cells is a prevalent feature of differentiation in a variety of cells. In most cases the mechanism responsible for such redistribution is poorly understood. Two potential mechanisms for the redistribution of surface proteins are: (1) passive diffusion coupled with trapping, and (2) active translocation. We have studied the process of membrane protein redistribution for the PH-20 protein of guinea pig sperm, a surface protein required for sperm binding to the egg zona pellucida ( P. Primakoff, H. Hyatt, and D. G. Myles (1985). J. Cell Biol. 101, 2239–2244 ). PH-20 protein is localized to the posterior head plasma membrane of the mature sperm cell. Following the exocytotic acrosome reaction, PH-20 protein moves into the newly incorporated inner acrosomal membrane (IAM), placing it in a position favorable for a role in binding sperm to the egg zona pellucida ( D. G. Myles, and P. Primakoff (1984). J. Cell Biol. 99, 1634–1641 ). To analyze the mechanistic basis for this protein migration, we have used fluorescence microscopy and digital image processing to characterize PH-20 protein migration in individual cells. PH-20 protein was observed to move against a concentration gradient in the posterior head plasma membrane. This result argues strongly against a model of passive diffusion followed by trapping in the IAM, and instead suggests that an active process serves to concentrate PH-20 protein toward the boundary separating the posterior head and IAM regions. A transient gradient of PH-20 concentration observed in the IAM suggests that once PH-20 protein reaches the IAM, it is freely diffusing. Additionally, we observed that migration of PH-20 protein was calcium dependent.

Ultrastructural analysis of the acrosome reaction in a population of single guinea pig sperm

Cauda epididymal guinea pig spermatozoa are arranged in rouleaux, with the sperm heads stacked one on top of the other; the plasma membranes over the apical segment of the acrosomes of adjacent sperm are linked and form non-fusigenic "junctional" zones. A complex structural and temporal sequence of membrane fusions occurs during the acrosome reaction of guinea pig sperm in rouleaux. In this study, we have devised a procedure for dispersing the rouleaux and isolating a population of single, motile guinea pig sperm, and have investigated the ultrastructural features of the acrosome reaction in single sperm to determine if the pattern of membrane fusions is different from sperm in rouleaux. The rouleaux were dispersed using trypsin, and damaged cells were removed by passing the sperm suspension through a glass bead column; a population of 70-90% motile, acrosome-intact, single sperm was obtained. Sperm were then induced to undergo lysolecithin-mediated, "synchronous" acrosome reactions, and processed for transmission electron microscopy. The acrosome reaction involved a complex sequence of membrane fusions between the plasma membrane (PM) and outer acrosomal membrane (OAM). On the convex surface of the apical segment, sheets of hybrid membrane and parallel arrays of hybrid membrane tubules formed; filaments were associated with the luminal surface of the residual OAM in these regions. Hybrid membrane vesicles were produced on the concave surface of the apical segment, but fusion was delayed relative to the convex surface. In the principal segment, branching arrays of hybrid membrane tubules formed and later vesiculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular Distribution of Phospholipase A2 and Atpases During Capacitation and Acrosome Reaction in Guinea Pig Spermatozoa

The presence, distribution, and levels of phospholipase A2 and ATPases activities in those structures of the guinea pig spermatozoa that participate in the acrosome reaction were studied, both before and after capacitation, as well as during the acrosome reaction induced in vitro. Spermatozoa were collected from the cauda epididymis and incubated in the absence and presence of 1.15 mmol/L calcium, with and without the addition of 1 mumol/L A23187. Membrane fractions were recovered by vortexing and discontinuous sucrose density gradient centrifugation. Most of the Na+, K(+)-ATPase was recovered in the acrosome-free spermatozoa, but a clear, distinct presence of this enzyme was observed in the plasma membrane (25 against 101 nmoles Pi released per milligram of protein, respectively). The activity of this enzyme in the periacrosomal plasma and in the outer acrosomal membrane increased during calcium incubation. Ca2(+)-dependent ATPase was found in both membrane fractions, being higher in the periacrosomal plasma membrane. The addition of calcium induced a significant inhibition of this acrosomal ATPase, whereas the activity in the acrosome-free spermatozoa increased. The activity of phospholipase A2, under all experimental conditions, was found to be restricted to the soluble fraction.

CDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm-egg adhesion and the wide distribution of its gene among mammals.

Sperm binding to the egg zona pellucida in mammals is a cell-cell adhesion process that is generally species specific. The guinea pig sperm protein PH-20 has a required function in sperm adhesion to the zona pellucida of guinea pig eggs. PH-20 is located on both the sperm plasma membrane and acrosomal membrane. We report here the isolation and sequence of a full-length cDNA for PH-20 (available from EMBL/GenBank/DDBJ under accession number X56332). The derived amino acid sequence shows a mature protein of 468 amino acids containing six N-linked glycosylation sites and twelve cysteines, eight of which are tightly clustered near the COOH terminus. The sequence indicates PH-20 is a novel protein with no relationship to the mouse sperm adhesion protein galactosyl transferase and no significant homology with other known proteins. The two PH-20 populations, plasma membrane and acrosomal membrane, could arise because one form of PH-20 is encoded and differentially targeted at different spermatogenic stages. Alternatively, two different forms of PH-20 could be encoded. Our evidence thus far reveals only one sequence coding for PH-20: Southern blots of guinea pig genomic DNA indicated there is a single PH-20 gene, Northern blots showed a single size PH-20 message (approximately 2.2 kb), and no sequence variants were found among the sequenced cDNA clones. Cross-species Southern blots reveal the presence of a homologue of the PH-20 gene in mouse, rat, hamster, rabbit, bovine, monkey, and human genomic DNA, showing the PH-20 gene is conserved among mammals. Since genes for zona glycoproteins are also conserved among mammals, the general features of sperm and zona proteins involved in mammalian sperm-egg adhesion may have been evolutionarily maintained. Species specificity may result from limited changes in these molecules, either in their binding domains or in other regions that affect the ability of the binding domains to interact.

Studies on relationship between Na,K-ATPase activity and sperm capacitation in guinea pig.

In order to elucidate the biochemical mechanism of sperm capacitation, the relationship between plasmalemma Na,K-ATPase, capacitation and acrosome reaction was investigated. Plasmalemma of guinea pig spermatozoa was isolated by sucrose gradient centrifugation for the determination of Na,K-ATPase activity. As far as the authors are aware, the Na,K-ATPase activity in plasmalemma of the guinea pig has never been detected. By treating sperm plasmalemma with 0.05% DOC (deoxycholate), enzyme activity could be quantitatively measured. After spermatozoa were incubated in capacitation medium for 8 h, Na,K-ATPase activity was found to be decreased to 35.6% as compared with that before incubation. The spermatozoa incubated for 10.5 h in capacitation medium containing 1 and 5 mumol/L ouabain showed 46.5% and 64.4% acrosome reactions respectively, while the acrosome reaction of the control group was only 27.4%. The above results suggest that the decrease in the Na,K-ATPase activity of guinea pig spermatozoa may be a prerequisite for capacitation. Experimental results demonstrated for the first time that Na,K-ATPase activity exists in the sperm plasmalemma of the guinea pig. It was further found that the decrease of Na,K-ATPase activity of plasmalemma enhances sperm capacitation. It is suggested that sperm capacitation in the guinea pig is possibly induced by the decrease in plasmalemma Na,K-ATPase and, as a consequence, the intracellular Na+ is increased, which would benefit the exchange of Na+out/Ca++in and the onset of acrosome reaction.