Heavy meromyosin-binding filaments in the mitotic apparatus of mammaliam cells

Guinea pig spermatozoa fail to fertilize eggs in Ca²⁺-free media primarily because of specific inhibition of the acrosome reaction and activation of the spermatozoa. In Ca²⁺-free media the spermatozoa undergo capacitation at the same rate as in Ca²⁺-containing media, but are arrested in the capacitated state. If Ca²⁺ is made available after the spermatozoa have reached the capacitated state, the spermatozoa immediately undergo the acrosome reaction and activation. The minimum concentration of Ca²⁺ necessary for the initiation of the acrosome reaction and activation is about 0.2 mM. Mg²⁺ cannot substitute for Ca²⁺ in initiating these processes. Possible mechanisms by which Ca²⁺ triggers the acrosome reaction and activation of guinea pig spermatozoa are discussed.     

Induction of the acrosome reaction in guinea pig spermatozoa by calmodulin antagonist W-7

The effect of the calmodulin antagonist W-7 on the capacitation and the acrosome reaction of guinea pig spermatozoa was examined. The characteristic features of the acrosome reaction induced by W-7 were the dependence on the composition and pH of the medium and on the presence of sodium bicarbonate. The most effective concentration of W-7 for inducing the acrosome reaction was approximately 5 μM, which is far less than the Kd for calmodulin. Moreover, W-7 enhanced the ability of spermatozoa to acquire capacitation in a Ca²⁺-free medium. The spermatozoa induced to undergo the acrosome reaction by W-7 were capable of penetrating the zona-free hamster eggs. W-5, which has a lower affinity for calmodulin than W-7, also induced the acrosome reaction in the same manner as W-7. These results suggest that the naphthalenesulfonamide derivatives W-7 and W-5 can induce the acrosome reaction in guinea pig spermatozoa via capacitation in a pH-dependent, Ca²⁺-calmodulin-independent manner.     

Promotion of capacitation of guinea pig spermatozoa by the membrane mobility agent,A2C,and inhibition by the disulfide-reducing agent,DTT

The membrane mobility agent A₂C accelerates the onset of the acrosome reaction of guinea pig spermatozoa by promoting capacitation. Spermatozoa incubated in a suspension of A₂C particles in Ca²⁺-free medium for one hour undergo a synchronous, rapid acrosome reaction upon the addition of Ca²⁺. These acrosome-reacted spermatozoa are capable of fertilization as assessed by their ability to penetrate (fuse with) zona-free hamster eggs. The disulfide-reducing agent, dithiothreitol (DTT) inhibits A₂C-mediated capacitation. It also blocks fertilization of zone-free eggs by acrosome-reacted spermatozoa by preventing attachment of the spermatozoa to the egg plasma membrane. The mode of A₂C action on spermatozoa is compared to that of A₂C-induced fusion in somatic cells. The similarity of the molecular events in the sperm membrane during capacitation and the acrosome reaction to these in other fusion events is pointed out. Inhibition of capacitation by DTT points to the importance of membrane and/or submembrane proteins and thiol groups in this process. Oxidation of sperm membrane SH groups may play an important role in in vivo capacitation.     

Signal transduction pathways that regulate sperm capacitation and the acrosome reaction

Ejaculated spermatozoa must undergo physiological priming as they traverse the female reproductive tract before they can bind to the egg’s extracellular coat, the zona pellucida (ZP), undergo the acrosome reaction, and fertilize the egg. The preparatory changes are the net result of a series of biochemical and functional modifications collectively referred to as capacitation. Accumulated evidence suggests that the event that initiates capacitation is the efflux of cholesterol from the sperm plasma membrane (PM). The efflux increases permeability and fluidity of the sperm PM and causes influx of Ca²⁺ ions that starts a signaling cascade and result in sperm capacitation. The binding of capacitated spermatozoa to ZP further elevates intrasperm Ca²⁺ and starts a new signaling cascade which open up Ca²⁺ channels in the sperm PM and outer acrosomal membrane (OAM) and cause the sperm to undergo acrosomal exocytosis. The hydrolytic action of the acrosomal enzymes released at the site of sperm-egg (zona) binding, along with the hyperactivated beat pattern of the bound spermatozoon, are important factors in directing the sperm to penetrate the ZP and fertilize the egg. The role of Ca²⁺-signaling in sperm capacitation and induction of the acrosome reaction (acrosomal exocytosis) has been of wide interest. However, the precise mechanism(s) of its action remains elusive. In this article, we intend to highlight data from this and other laboratories on Ca²⁺ signaling cascades that regulate sperm functions.     

Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro

After capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl₂ and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl₂ revealed that plasma membrane was removed only from the acrosomal region and remained predominately intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl₂ lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg²⁺, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM-PL) for 2 hours. Under these culture conditions the addition of Ca²⁺ (5 mM) to the capacitated spermatozoa induced approximately 78 ± 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 ± 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg²⁺, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2-deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.     

Thapsigargin stimulates acrosomal exocytosis in hamster spermatozoa

Thapsigargin (TG), a plant-derived sesquiterpene lactone, inhibits several isoforms of both the sarcoplasmic and endoplasmic reticulum Ca²⁺-ATPases. Thus, intracellular Ca²⁺ stores found in the endoplasmic reticulum can be released by this compound. The mammalian sperm acrosome reaction (AR) depends on influx of extracellular Ca²⁺. However, few reports have presented evidence for the involvement of putative Ca²⁺ stores and intracellular Ca²⁺ mobilization in the AR. Thus, we designed experiments to evaluate the effect of TG on the hamster sperm AR. Thapsigargin stimulated—in a dose-dependent manner—the AR of spermatozoa previously capacitated for at least 3 hr, not affecting sperm motility. A maximal stimulatory effect was apparent 3 min after addition of TG to spermatozoa previously capacitated for 4 hr and was dependent on external Ca²⁺ since ethyleneglycol-bis-(b-amino-ethyl ether) N,N′-tetra-acetic acid added 1 min before TG completely inhibited AR stimulation. The Ca²⁺ channel blockers diltiazem and nifedipine also abolished the TG-stimulatory effect when added to capacitated spermatozoa 10 min before the inhibitor. In addition, the trypsin inhibitors p-nitrophenyl-p′-guanidine-benzoate hydrochloride and benzamidine added to the sperm suspensions 10 min before TG inhibited by 70–80% the TG-induced AR. These results indicate that putative Ca²⁺ stores release may be involved in stimulation of extracellular Ca²⁺ influx required for the occurrence of the AR. In addition, a sperm trypsin-like protease may be part of the mechanism by which TG induces the hamster sperm AR. Mol. Reprod. Dev. 51:84–91, 1998. © 1998 Wiley-Liss, Inc.     

Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in the hamster

Using a semi-chemically defined medium, the requirement of extracellular Ca²⁺ for survival, capacitation, and acrosome reaction of spermatozoa as well as various stages of fertilization in the hamster was studied. A Ca²⁺-deficient environment is unfavorable for long-term survival of spermatozoa. Sperm capacitation may occur in Ca²⁺-deficient media, but not as efficiently as in normal media. The acrosome reaction definitely requires extracellular Ca²⁺. Other processes or phenomena that require extracellular Ca²⁺ are initiation and maintenance of hyperactivated motility of spermatozoa, penetration of acrosome-reacted spermatozoa into the zona pellucida, fusion of the spermatozoa with eggs, and the development of pronuclear eggs into two-cell embryos. Extracellular Ca²⁺ is apparently unnecessary for the attachment of spermatozoa to the zona and egg surfaces, decondensation of the sperm nucleus, and the development of sperm and egg pronuclei within the egg. These results were compared with data obtained in other species such as the sea urchin, mouse, rat and guinea pig.     

Effect of Ca2+ channel antagonists on the acrosome reaction of guinea pig and golden hamster spermatozoa

The effects of Ca²⁺ channel antagonists on the motility and acrosome reaction of guinea pig spermatozoa were examined by incubating the spermatozoa continuously in Ca²⁺-containing capacitating media with 10^?6 M to 10^?4 M antagonist. Antagonists tested were four voltage-gated Ca²⁺ channel antagonists (verapamil, nifedipine, nimodipine, and FR–34235) and two ligand-gated channel antagonists (NaNO₂ and Na-nitroprusside). None of these antagonists could block the acrosome reaction. Instead, three antagonists (verapamil, nimodipine, and FR-34235, each at 10^?4 M) accelerated the onset of the acrosome reaction with a subsequent decrease in sperm motility. Nifedipine and Na-nitroprusside at the same concentration caused a complete loss of sperm motility by 4 hr of incubation with no substantial effect on the rate of acrosome reaction. The detrimental effect of antagonists on the motility of spermatozoa appears to be due to a direct, Ca²⁺-independent, membrane-perturbing action of the reagents. The acrosome reaction was not inhibited when guinea pig spermatozoa were precapacitated in Ca²⁺-free medium (with a low concentration of lysolecithin) in the continuous presence of antagonists. An acceleration of the onset of the acrosome reaction by verapamil (10^?4 M) was also demonstrated in the golden hamster. These results may be interpreted as indicating that the entry of extracellular Ca²⁺ into spermatozoa, which triggers the acrosome reaction of guinea pig and hamster spermatozoa, is not mediated by Ca²⁺ channels. This is in marked contrast with the case reported in invertebrate spermatozoa. Possible mechanisms by which some of the antagonists stimulate the acrosome reaction and affect the motility of mammalian spermatozoa are discussed.     

Reactive oxygen species requirements for bovine sperm capacitation and acrosome reaction

Sperm capacitation is necessary for the fertilization of oocytes. During capacitation intracellular and membrane changes occur, that culminate with an exocytotic event called the acrosome reaction. The aim of this work was to study the participation of the superoxide anion (O2-.) and of hydrogen peroxide (H2O2) in the capacitation process and acrosome reaction in spermatozoa from cryopreserved bovine semen. Samples were capacitated with heparin or treated with the xanthine-xanthine oxidase-catalase system (X-XO-C) for the production of O2-. The percentage of capacitated spermatozoa was determined using the chlortetracycline (CTC) technique, by means of epifluorescence microscopy. Addition of X-XO-C to the incubation medium significantly induced capacitation (P < 0.05), but there were no differences with samples incubated with heparin. When the medium contained heparin or the X-XO-C, addition of superoxide dismutase (SOD, 0.5 mg/mL) significantly inhibited capacitation (P < 0.05). In samples treated with heparin and with diverse concentrations of H2O2 (10, 25, 50 and 250 microM) in the incubation medium, the percentage of capacitated spermatozoa was significantly reduced (P < 0.05); however, acrosome reaction was produced at concentrations of 10 and 25 microM H2O2. At concentrations greater than 25 microM H2O2 a deleterious effect was observed on sperm motility. From these results it may be inferred that O2-. is required in the capacitation process and that H2O2 may participate as an inductor of the acrosome reaction in spermatozoa from cryopreserved bovine semen.     

Cytochemical localization of membrane-bound Mg2+-dependent ATPase activity in guinea pig sperm head before and during the acrosome reaction

The distribution of ATPase activity in the heads of uncapacitated, capacitated, and acrosome-reacting guinea-pig spermatozoa was examined cytochemically using the Wachstein-Meisel's technique. In uncapacitated spermatozoa, the reaction products of the enzyme activity were localized on both the inner surface of the plasma membrane and the outer surface of the outer acrosomal membrane. The activity was Mg²⁺-dependent and inhibited by both Ca²⁺ and SH-blocking agents. This Mg²⁺-dependent ATPase activity was also demonstrated at the same sites in capacitated spermatozoa, whereas it was completely absent in acrosome-reacting spermatozoa. Although we did not determine the exact time of inactivation of the enzyme, it appeared to occur before the plasma membrane fused with the underlying outer acrosomal membrane. The abrupt loss of the Mg²⁺-dependent ATPase activity in the plasma and outer acrosomal membranes immediately before the onset of the acrosome reaction seems to suggest that inactivation of this enzyme by Ca²⁺ is one of the important biochemical events involved in the acrosome reaction.     

THE IMPORTANCE OF HYDROLYTIC ENZYMES TO AN EXOCYTOTIC EVENT, THE MAMMALIAN SPERM ACROSOME REACTION

The mammalian sperm acrosome reaction is a unique form of exocytosis, which includes the loss of the involved membranes. Other laboratories have suggested the involvement of hydrolytic enzymes in somatic cell exocytosis and membrane fusion, and in the invertebrate sperm acrosome reaction, but there is no general agreement on such an involvement. Although reference was made to such work in this review, the focus of the review was on the evidence (summarized below) that supports or fails to support the importance of certain hydrolytic enzymes to the mammalian sperm acrosome reaction. Because the events of capacitation, the prerequisite for the mammalian acrosome reaction, and of the acrosome reaction itself are not fully understood or identified, it is not yet always possible to determine whether the role of a particular enzyme is in a very late step of capacitation or part of the acrosome reaction. (1) The results of studies utilizing inhibitors of trypsin-like enzymes suggest that such an enzyme has a role in the membrane events of the golden hamster sperm acrosome reaction. The enzyme involved may be acrosin, but it is possible that some as yet unidentified trypsin-like enzyme on the sperm surface may play a role in addition to or instead of acrosin. Results obtained by others with guinea pig, ram and mouse spermatozoa suggest that a trypsin-like enzyme is not involved in the membrane events of the acrosome reaction, but only in the loss of acrosomal matrix. Such results, which conflict with those of the hamster study, may have been due to species differences or the presence of fusion-promoting phospholipase-A or lipids contaminating the incubation media components, and in one case to the possibly damaging effects of the high level of calcium ionophore used. The role of the trypsin-like enzyme in the membrane events of the hamster sperm acrosome reaction may be to activate a putative prophospholipase and/or to hydrolyse an outer acrosomal or plasma membrane protein, thus promoting fusion. A possible role of the enzyme in the vesiculation step rather than the fusion step of the acrosome reaction cannot be ruled out at present. (2) Experiments utilizing inhibitors of phospholipase-A₂, as well as the fusogenic lysophospholipid and cis-unsaturated fatty acid hydrolysis products that would result from such enzyme activity, suggests that a sperm phospholipase-A₂ is involved in the golden hamster sperm acrosome reaction. Inhibitor and LPC addition studies in guinea pig spermatozoa have led others to the same conclusion. The fact that partially purified serum albumin is important in so many capacitation media may be explained by its contamination with phospholipase-A and/or phospholipids. Serum albumin may also play a role, at least in part, by its removal of inhibitory products released by the action of phospholipase-A₂ in the membrane. The demonstration of phospholipase-A₂ activity associated with the acrosome reaction vesicles and/or the soluble component of the acrosome of hamster spermatozoa, and the fact that exogenous phospholipase A₂ can stimulate acrosome reactions in hamster and guinea pig spermatozoa, also support a role for the sperm enzyme. The actual site or the sites of the enzyme in the sperm head are not yet known. The enzyme may be on the plasma membrane as well as, or instead of, in the acrosomal membranes or matrix. A substrate for the phospholipase may be phosphatidylcholine produced by phospholipid methylation. It is possible that more than one type of ‘fusogen’ is released by phospholipase activity (LPC and/or cis-unsaturated fatty acids, which have different roles in membrane fusion and/or vesiculation. In addition to acting as a potential ‘fusogen’, arachidonic acid released by sperm phospholipase-A₂ probably serves as precursor for cyclo-oxygenase or lipoxygenase pathway metabolites, such as prostaglandins and HETES, which might also play a role in the acrosome reaction. Although much evidence points to a role for phospholipase-A₂, phospholipase-C found in spermatozoa could also have a role in the acrosome reaction, perhaps by stimulating events leading to calcium gating, as suggested for this enzyme in somatic secretory cells. (3) A Mg²⁺-ATPase H⁺-pump is present in the acrosome of the golden hamster spermatozoon. Inhibition of this pump by certain inhibitors of ATPases (but not by those that only inhibit mitochondrial function) leads to an acrosome reaction only in capacitated spermatozoa and only in the presence of external K⁺. The enzyme is also inhibited by low levels of calcium, and such inhibition, combined with increased outer membrane permeability to H⁺ and K⁺, and possibly plasma membrane permeability to H⁺ (perhaps by the formation of channels), may be part of capacitation and/or the acrosome reaction. The pH of the hamster sperm acrosome has been shown to become more alkaline during capacitation, and such a change may result in the activation of hydrolytic enzymes in the acrosome or perhaps in a change in membrane permeability to Ca²⁺. A similar Mg²⁺-ATPase has not been found in isolated boar sperm head membranes. However, that conflicting result could have been due to the use of noncapacitated boar spermatozoa for the preparation of the membranes or to protease modification of the boar sperm enzyme during assay. (4) Inhibition of Na⁺, K⁺-ATPase inhibits the acrosome reaction of golden hamster spermatozoa, and the activity of this enzyme increases relatively early during capacitation. A late influx of K⁺ is important for the acrosome reaction. However, this late influx may not be due to Na⁺, K⁺-ATPase, but instead may be due to a K⁺ permeability increase (possibly via newly formed channels) in the membranes during capacitation. It is suggested in this review that Na⁺, K⁺-ATPase has a role early in capacitation rather than directly in the acrosome reaction (although such a role cannot yet be completely ruled out). One possible role for the enzyme in capacitation might be to stimulate glycolysis (which appears to be essential for capacitation and/or the acrosome reaction of hamster and mouse spermatozoa). The function of the influx of K⁺ just before the acrosome reaction is probably to stimulate, directly or indirectly, the H⁺-efflux required for the increase in intraacrosomal pH occurring during capacitation. Direct stimulation of the acrosome reaction by a change in membrane potential resulting directly from K⁺-influx is not a likely explanation for the hamster results. However, the importance of an earlier membrane potential change, due to increased Na⁺, K⁺-ATPase during capacitation, and/or of later membrane potential changes resulting from the pH change, cannot be ruled out. Although K⁺ is required for the hamster acrosome reaction, other workers have reported that K+ inhibits guinea pig sperm capacitation. However, the experimental procedures used in the guinea pig sperm studies raise some questions about the interpretation of those inhibition results. (5) Ca²⁺-influx is known to be required for the acrosome reaction. Others have suggested that increased Ca²⁺-influx due to inhibition or stimulation of sperm membrane calcium transport ATPases are involved in the acrosome reaction. There is as yet no direct or indirect biochemical evidence that inhibition or stimulation of such enzymatic activity is involved in the acrosome reaction, and further studies are needed on those questions. (6) I suggest that the hydrolytic enzymes important to the hamster sperm acrosome reaction will also prove important for the acrosome reaction of all other eutherian mammals.     

RU486 suppresses progesterone-induced acrosome reaction in boar spermatozoa

The effects of progesterone on the acrosome reaction, as well as the effects of RU486 on the progesterone-induced acrosome reaction in capacitated boar spermatozoa, were investigated. Progesterone, a major steroid that is secreted by the cumulus cells of oocyte, clearly induced the acrosome reaction in a dose-dependent manner in capacitated boar spermatozoa, even though it failed to show similar effects in non-capacitated spermatozoa. RU486, a potent antiprogestin, significantly reduced the effects of progesterone on the progesterone-induced acrosome reaction; however, when treated alone, it showed no inhibitory effects on the acrosome reaction. The inhibitory effects of RU486 were also shown to be dose dependent. These results imply that in addition to the wellknown inducer of the acrosome reaction, zona pellucida, progesterone can also induce the acrosome reaction through its specific receptors on spermatozoa after the spermatozoa undergo capacitation.     

Ca2+-Regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis

We have used chlortetracycline (CTC) analysis to investigate mechanisms that may play important roles during bull sperm capacitation in a culture medium (containing glucose, heparin, and caffeine) known to promote capacitation and fertilization in vitro. In initial experiments employing the Ca²⁺ ionophore A23187, we identified three discrete CTC patterns so similar to those described for mouse and human sperm that we have employed the same nomenclature: “F,” characteristic of uncapacitated, acrosome-intact cells; “B,” characteristic of capacitated, acrosome-intact, cells; “AR,” characteristic of capacitated, acrosome-reacted cells. Over a 60-min period, A23187 stimulated significant increases in B and AR pattern cells, with concomitant decreases in F pattern cells, suggesting a very rapid transition from the uncapacitated to the capacitated state and then on to exocytosis. Without ionophore, significant changes in the proportions of F and B pattern cells were also observed, but the maximum responses required 4 hr; the proportion of AR cells was consistently ～ 15% throughout, indicating a low incidence of spontaneous acrosome loss. Analysis of cells in media with altered composition indicated that the inclusion of either heparin or caffeine significantly promoted capacitation to about the same extent, but together, heparin plus caffeine had an even more stimulatory effect. Despite this, none of these treatments triggered acrosome loss above the levels seen in media lacking these constituents. In the presence of caffeine, with or without heparin, the inclusion of glucose had little effect on responses, but in the presence of heparin there were fewer B cells. In the presence of either quercetin, a Ca-ATPase inhibitor used at 50–200 μM, or W-7, a calmodulin antagonist used at 5–125 μM, capacitation per se was accelerated, as evidenced by significant decreases in F and significant increases in B pattern cells; only the highest concentration of each caused significant increases in AR cells. In addition, 25 and 125 μM W-7 markedly stimulated motility, both quantitatively and qualitatively. Finally the Na⁺ ionophore monensin at 500 μM significantly accelerated both capacitation and acrosomal exocytosis. The addition of the dihydropyridine calcium channel blocker nifedipine at 10 nM, just prior to monensin, did not inhibit capacitation (F to B transition) but blocked acrosomal exocytosis (B to AR transition). We suggest that Ca²⁺ is required for functional changes in bull sperm, with a Ca²⁺-ATPase modulating intracellular Ca²⁺ during capacitation and calcium channels controlling the Ca²⁺ influx required for acrosomal exocytosis. © 1995 Wiley-Liss, Inc.     

Alteration of sperm thiol-disulfide status and capacitation in the guinea pig

Epididymal spermatozoa of the guinea pig were incubated under conditions known to promote a rapid synchronous capacitation in a large proportion of the spermatozoa (Ca²⁺-free medium with lysophosphatidylcholine, LC) or in Ca ²⁺-free medium without LC. To study the effects of altered thiol-disulfide status and content, incubations were conducted with reagents that maintain and increase thiol groups (DTT, GSH), maintain and increase disulfide groups (diamide, GSSG), or which irreversibly block thiol groups by alkylation (NEM). The permeable DTT inhibited LC-induced capacitation and at high concentrations diminished the percentage of acrosome reactions in capacitated spermatozoa. The permeable diamide exhibited a stimulatory effect upon capacitation. The largely impermeable GSH and GSSG exhibited effects similar to their respective permeable counterparts but their effects were moderate and required extremely high concentrations. The DTT inhibition of LC-induced capacitation was reversible by washing and a further 1 hr incubation. In this final incubation after removal of DTT by washing, LC was absent too so its stimulatory effect must have been accomplished prior to washing and in the presence of DTT. NEM-alkylation of the existing thiol population did not affect LC-induced capacitation but alkylation of the increased thiol population after prior DTT treatment was inhibitory of capacitation. These results suggest that the maintenance and/or formation of disulfide groups on enzymes or structural proteins may be a component of the capacitation process. In contrast, the formation and maintenance by alkylation of increased thiol groups but not the maintenance of existing thiol groups, is inhibitory of capacitation. The relevance of these findings to a role for a thiol-sensitive proteinase in capacitation is discussed.     

Capacitation and the acrosome reaction in squirrel monkey (Saimiri sciureus) spermatozoa evaluated by the chlortetracycline fluorescence assay

Capacitation and the acrosome reaction in squirrel monkey seminal spermatozoa diluted in Tyrode's medium (TALP) and TC-199 were monitored by a chlortetracycline (CTC) fluorescence assay. Four CTC patterns, similar to those found in human sperm, were readily characterized by fluorescent staining on the heads of the spermatozoa. The appearance of the capacitated (CP) pattern was dependent on the concentration of the bovine serum albumin. Acrosomal loss was observed in a maximum of 15% of the sperm in the populations studied here. Calcium ionophore A23187 (5 μM to 20μM) induced acrosomal loss in 60–70% of capacitated spermatozoa. However in freshly ejaculated sperm incubated under capacitating conditions or in spermatozoa incubated in Ca^{+ +}-free medium, A23187 failed to induce acrosomal loss. Furthermore, spermatozoa incubated in the presence of seminal plasma or spermatozoa obtained following a 1-hour “swim-up” procedure showed an identical timecourse of appearance of the CP pattern, indicating the lack of effect of seminal plasma on capacitation in the squirrel monkey.     

The role of Ca2+ and protein kinase C in the acrosome reaction of giant panda (Ailuropoda melanoleuca) spermatozoa

Fresh semen was collected from adult male giant pandas and the role of Ca2+, Ca2+ ionophore A23187 and protein kinase C (PKC) in sperm motility and acrosome reaction (AR) was assessed by lens culinaris agglutinin conjugated with fluorescein isothiocyanate (FITC-LCA) labeling and transmission electron microscopy. The AR in giant panda spermatozoa was characterized by vesiculation of the outer acrosomal membrane through its invagination. Both the sperm motility and the AR rate decreased significantly (p < 0.05) in Ca2+-free and low Ca2+ medium. The addition of 10 microM Ca2+ ionophore A23187 potently stimulated AR. After incubation for capacitation, the PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated AR in a dose-dependent manner and its effect could be overcome by the PKC inhibitor staurosporine. These results suggest that Ca2+ and PKC play an important role in the sperm acrosome reaction of the giant panda.     

Molecular modifications of MC31/CE9, a sperm surface molecule, during sperm capacitation and the acrosome reaction in the rat: is MC31/CE9 required for fertilization?

We examined the modification of the MC31 molecule during capacitation, the acrosome reaction, and studied its role in fertilization. These studies revealed that the molecular mass of MC31 in cauda spermatozoa was approximately 28,000-26,000 Dalton (28-26 kDa). A limited change in molecular mass was seen in capacitated spermatozoa. Treatment of sperm extracts with peptide-N-glycosidase (PN glycosidase) reduced the molecular mass of MC31 in both cauda and capacitated spermatozoa from 28-26 kDa to 23-20 kDa, suggesting that MC31 from both cauda and capacitated spermatozoa is glycosylated, and indicating that capacitation induces minor posttranslational modifications in the structure of the MC31 antigen. The MC31 antigen was redistributed from the midpiece of cauda epididymal spermatozoa to the head and equatorial segment after capacitation and acrosome reaction, respectively, when traced by indirect immunofluorescence under in vitro fertilization (IVF) conditions. Some spermatozoa did not stain for the MC31 antigen and might represent spermatozoa that have shed the antigen. IVF experiments conducted to assess the effect of an anti-MC31 monoclonal antibody (mMC31) revealed that this antibody significantly (P < 0.01) inhibited fertilization of cumulus-invested zona pellucida-intact and the zona pellucida-free oocytes in a dose-dependent manner. However, sperm-oolemma binding was not affected. These findings suggest the MC31 antigen facilitates sperm-oocyte interactions.     

Inhibition of the human sperm acrosome reaction by proteinase inhibitors

The analogue of the second messenger cAMP, dibutyryl cAMP (dbcAMP), was shown to induce the human sperm acrosome reaction to the same extent as calcium ionophore A23187, providing preliminary evidence for the involvement of the adenylate cydase system in the acrosome reaction (AR) of human spermatozoa. Using the human synchronous acrosome reaction system, proteinase inhibitors were tested for their effect on the dbcAMP-induced human sperm acrosome reaction. The proteinase inhibitor 4′-acctamidophenyl4-guanidinoben-zoate (AGB), an inhibitor of proacrosin activation and of acrosin, when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, significantly (P < 0.01) inhibited the acrosome reaction at final concentrations of 1 × 10^?4 M to 1 × 10^?6 M in comparison to dbcAMP treatment alone. At concentrations less than 1 × 10^?6 M, no significant inhibitory effect was seen. Similarly, para-aminobenzamidine (pAB), also an inhibitor of proacrosin activation and of acrosin, significantly (P < 0.01) inhibited the dbcAMP-induced acrosome reaction at final concentrations of 1 × 10-4 M to I × 10-6 M when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, in comparison to stimulation by dbcAMP alone. However, at concentrations less than 1 × 10^?6 M, no significant (P > 0.05) inhibitory effect was seen. These results indicate that a serine proteinase, most likely acrosin, has a role in the human sperm acrosome reaction and suggest that the enzyme functions after the involvement of the adenylate cyclase system.     

Effects of platelet activating factor on capacitation and acrosome reaction in mouse spermatozoa

Platelet activating factor (PAF) plays an important role in mammalian reproduction. The aim of this study was to investigate the effects of PAF on capacitation and acrosome reaction of mouse spermatozoa by chlortetracycline (CTC) fluorescence assay and coomassie blue staining. The percentage of capacitated mouse spermatozoa was increased (P < 0.05) by incubation with 50 ng/ml PAF for 20-120 min. The peak response occurred between 80 to 100 min of exposure to PAF. In contrast, the effects of PAF on acrosome reaction may be not receptor-mediated since lyso-PAF had the same effects. Ionophore A23187 stimulated an increase in acrosome-reacted spermatozoa of PAF-treated spermatozoa, but not of lyso-PAF-treated ones. These results suggest that PAF mainly acts on sperm capacitation.     

Immunolocalization of heat shock protein 70 (Hsp 70) in boar spermatozoa and its role during fertilization

The presence and cellular distribution of heat protein 70 (Hsp70) in ejaculated, capacitated, and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well-defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment. In reacted spermatozoa, Hsp70 seems to be not only relocalized but also translocated from the inner to the outer leaflet of the sperm plasma membrane, as a significant (P < 0.05) increase in the proportion of unfixed cells showing the fluorescent signal has been recorded. No differences in Hsp70 amount between fresh, capacitated, and reacted semen were observed by Western blot. The presence of anti-Hsp70 antibody in the fertilization medium significantly reduced, in a concentration-dependent manner, the fertilization rate of both zona-intact and zona-free oocytes. The overall data demonstrate that Hsp70 is present on boar sperm with a dynamic redistribution as the sperm undergoes capacitation and acrosome reaction and suggest an important role of this protein during porcine gamete interaction.