Localization of intracellular calcium during the acrosome reaction in ram spermatozoa

Calcium was identified by a pyroantimonate-osmium fixation technique in ram spermatozoa undergoing a spontaneous acrosome reaction induced by incubation of diluted semen at 39°C. Intracellular calcium was only detected in diluted spermatozoa and increased in amount and distribution over 4 hr At 4 hr, the majority of the spermatozoa displayed ultrastructural evidence of an acrosome reaction. Calcium was initially evident on the outer acrosomal membrane in multiparticulate clusters, which were seen to be located on scalloped crests of acrosomal membrane as fusion developed; it was also located in the region of the acrosomal ridge beneath the outer acrosomal membrane. Vesiculation commenced just anterior to the equatorial segment and proceeded anteriorly. As vesiculation advanced, calcium particles became associated with the periphery of the vesicles attached in the region of the fusion between the two membranes, but were never seen inside the vesicles. The equatorial segment was not labelled until much later in the reaction, at which time calcium particles were also evident on the nuclear membrane; vesiculation of the equatorial segment was also noted at this time. Dense labelling of the postacrosomal dense lamina was seen in all incubated spermatozoa. At the anterior margin of this structure the labelling was seen to be in a “sawtooth” arrangement. The disposition of the calcium both temporally and spatially is discussed in relation to its possible mechanisms in bringing about membrane fusion. © 1995 Wiley-Liss, Inc.     

Control of membrane fusion during spermiogenesis and the acrosome reaction

Membrane fusion is important to reproduction because it occurs in several steps during the process of fertilization. Many events of intracellular trafficking occur during both spermiogenesis and oogenesis. The acrosome reaction, a key feature during mammalian fertilization, is a secretory event involving the specific fusion of the outer acrosomal membrane and the sperm plasma membrane overlaying the principal piece of the acrosome. Once the sperm has crossed the zona pellucida, the gametes fuse, but in the case of the sperm this process takes place through a specific membrane domain in the head, the equatorial segment. The cortical reaction, a process that prevents polyspermy, involves the exocytosis of the cortical granules to the extracellular milieu. In lower vertebrates, the formation of the zygotic nucleus involves the fusion (syngamia) of the male pronucleus with the female pronucleus. Other undiscovered membrane trafficking processes may also be relevant for the formation of the zygotic centrosome or other zygotic structures. In this review, we focus on the recent discovery of molecular machinery components involved in intracellular trafficking during mammalian spermiogenesis, notably related to acrosome biogenesis. We also extend our discussion to the molecular mechanism of membrane fusion during the acrosome reaction. The data available so far suggest that proteins participating in the intracellular trafficking events leading to the formation of the acrosome during mammalian spermiogenesis are also involved in controlling the acrosome reaction during fertilization.     

Cytochemical study of intracellular calcium in hamster spermatozoa during the acrosome reaction

Calcium was localized by a pyroantimonate technique in hamster spermatozoa during the acrosome reaction and pyroantimonate precipitates were observed in the anterior region of the acrosome. The calcium was also localized in the postacrosomal lamina of spermatozoa undergoing the acrosome reaction. Spermatozoa, incubated in capacitating medium containing verapamil, showed denser precipitates with an increase in concentration of this drug. Ionophore A23187 enhanced binding of calcium to the acrosomal region. The sodium channel inhibitor amiloride inhibited the acrosome reaction and the pyroantimonate precipitates were absent in these spermatozoa, whereas ionophore monensin enhanced the acrosome reaction. This suggests that the Na⁺/Ca⁺⁺ antiporter may be responsible for intracellular Ca⁺⁺ regulation during the acrosome reaction in hamster spermatozoa.     

Intracellular calcium in hamster spermatozoa in testis,epididymis and during the acrosome reaction

Hamster spermatozoa from the testis and cauda epididymis were examined for intracellular localization of calcium using a pyroantimonate technique. Calcium precipitates were found in spermatozoa from the testis but not in those from cauda epididymis. Spermatozoa from the epididymis which had been induced to undergo capacitation and the acrosome reaction in vitro, were also investigated. In these, the anterior region of the acrosome showed precipitates indicating calcium binding at specific sites, possibly prior to transport across the membranes. The additional presence of calcium precipitates in the postacrosomal region suggests that the appearance of free calcium in that region is an important step in the acrosome reaction.     

Some effects of chondroitin sulphate on the in-vitro acrosome reaction in ram spermatozoa

The effect of chondroitin sulphate on the incidence of the acrosome reaction in ram spermatozoa incubated in vitro was examined over a 24-h incubation period. Four concentrations of chondroitin sulphate (0, 1, 10 and 100 μg/ml) were used to supplement the incubating medium (Brackett's Defined Medium). The results indicate that 100 μg/ml chondroitin sulphate was effective in increasing the percentage of live, acrosome-reacted spermatozoa from 25% (no CS) to 39.9% after 24 h incubation. There was no difference in the percentage of live, acrosome-reacted spermatozoa between the other three concentrations. Chondroitin sulphate did not appear to affect the survival of ram spermatozoa over the incubation period.     

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.     

Induction of acrosomal reaction and calcium uptake in ram spermatozoa by ionophores

Ram spermatozoa incubated in the presence of Ca2+ and the Ca2+-ionophore A23187 undergo a process which is known as the acrosome reaction. This reaction is characterized by fusion of the outer acrosomal membrane and the overlying plasma membrane to form mixed vesicles which can be seen in the electron microscope. As a result, the trypsin-like acrosin is released from the cells to the medium. The occurrence of the acrosome reaction was determined by following acrosin activity in the medium. After 2 h of incubation of the cells in the presence of ionophore and Ca2+, the released acrosin activity is related to the ionophores according to the sequence: A23187 greater than monensin greater than valinomycin greater than FCCP = without ionophore. The study of Ca2+ uptake by the cells revealed that Ca2+ enters the cell prior to the release of acrosin. Monensin can induce Ca2+ uptake and acrosin release only when Na+ is present in the incubation medium. There is no increase in Ca2+ uptake with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). We suggest that the Na+/H+ exchange induced by monensin causes an increase in intracellular Na which is the driving force for the Ca2+ entry via a Ca2+/Na+ antiporter. Since monensin can induce an increase in Ca2+ uptake only in the presence of Na+, FCCP enhances Ca2+ uptake in the presence of valinomycin, and A23187 is a Ca2+/2H+ exchanger, we suggest that alkalization of the intracellular space is involved in the acrosome reaction. Calcium uptake in the presence of monensin is not affected by the uncoupler FCCP, a result which indicates that Ca2+ is not accumulated in the mitochondria. Incubation of cells for 3 h in the absence of Ca2+ or ionophore caused a 3-fold increase in the rate of acrosin release when monensin and Ca2+ were added together. There was no change in this rate when A23187 was used. We suggest that during the preincubation time (known as capacitation) the permeability of the plasma membrane to Ca2+ is enhanced. This study shows that acrosin release and Ca2+ uptake can be used as a quantitative asay for the determination of the acrosome reaction.     

Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae

The binding of mammalian spermatozoa to the egg's extracellular coat, the zona pellucida, is a complex process which culminates in species-specific penetration of the sperm to the egg plasma membrane. To investigate where on the spermatozoon's surface the zona binding sites are located, whole rabbit zonae were labeled with FITC, heat solubilized and used to observe the surface binding patterns on live spermatozoa. Before the acrosome reaction the zona binding sites are located either over the entire head as well as the middle piece or alternatively in patches along the apical ridge of the head. After the acrosome reaction there is a 29% loss of fluorescence and the zona binding sites are present in the posterior aspect of the acrosomal region, the anterior postacrosomal region and the middle piece. These results demonstrate the presence of zona binding sites after the acrosome reaction which would account for the sperm's ability to remain bound to the zona after the acrosome reaction. Further, we report for the first time that solubilized rabbit zonae pellucidae will induce the acrosome reaction in in vitro capacitated rabbit sperm whereas solubilized pig zonae pellucidae will not. Since rabbit sperm bind pig zonae, the induction and specificity of the physiological acrosome reaction must reside in the affinity of the binding rather than the binding itself.     

Evidence for the activation of phospholipases during acrosome reaction of human sperm elicited by calcium ionophore A23187

Incubation of washed human sperm with [3H]- or [14C]arachidonic acid allowed a major incorporation of the label into phospholipids, provided that the final concentration of the fatty acid did not exceed 20 microM. A further challenge with calcium ionophore A23187 of spermatozoa suspended in a calcium-containing medium led to phospholipid hydrolysis, which could account for 10-12% of total cell radioactivity. Degradation products were identified as free, unconverted arachidonic acid, occurring with some diacylglycerol. Phospholipid hydrolysis was significant after 15 min of incubation and became maximal after 120 min. It was found to be calcium dependent, diacylglycerol and free arachidonate production occurring maximally at 2 mM and 5 mM CaCl2, respectively. Phosphatidylcholine and phosphatidylinositol were the most significantly degraded phospholipids after 60 min of incubation. Similar incubations conducted with 32P-labeled sperm confirmed the selective hydrolysis of phosphatidylcholine and revealed an increase production of phosphatidic acid probably due to a phosphorylation of diacylglycerol. Under the same conditions, one third of the cells remained motile and electron microscopy revealed that acrosome reaction was completed in 40% of the cells and displayed an intermediary state in 40-50% of the spermatozoa. Furthermore, a good parallelism was observed between the extent of the acrosome reaction and the extent of phospholipid hydrolysis promoted by increasing concentrations of A23187. It is concluded that calcium entry into the cells activates both a phospholipase A2 and a phospholipase C, leading to the production of substances, like lysophospholipid, diacylglycerol or phosphatidic acid, which may or may not be involved in acrosome reaction.     

Surface mapping of binding of oviductin to the plasma membrane of golden hamster spermatozoa during in vitro capacitation and acrosome reaction

Oviductins are high-molecular-weight glycoproteins synthesized and secreted by nonciliated oviductal epithelial cells and have been shown to play a role in fertilization and early embryo development. The present study was carried out to examine the in vitro binding capacity of hamster oviductin to homologous sperm and to determine the sites of its localization in untreated, capacitated, and acrosome-reacted spermatozoa. Freshly prepared epididymal and capacitated sperm as well as acrosome-reacted sperm were incubated with oviductal fluid prepared from isolated hamster oviducts, fixed and then probed with a monoclonal antibody against hamster oviductin. Results obtained with pre-embedding immunolabeling experiments revealed binding of oviductin to the acrosomal cap and the apical aspect of the postacrosomal region. Immunolabeling of both regions appeared to be more intense in capacitated spermatozoa. Acrosome-reacted sperm showed an immunoreaction of moderate intensity over the postacrosomal region. The plasma membrane overlying the equatorial segment also exhibited a weak labeling. Quantitative analysis obtained with the surface replica technique indicated that oviductin had a higher binding affinity for the acrosomal cap than the postacrosomal region and that the binding of oviductin to the latter plasma membrane domain was enhanced during capacitation. Binding of oviductin to the postacrosomal region, however, was attenuated after acrosome reaction. Immunolabeling for oviductin was found to be the weakest over the equatorial segment regardless of the experimental conditions. The binding of hamster oviductin to specific membrane domains of the homologous sperm and the changes in its distribution during capacitation and acrosome reaction may be important for the function of hamster oviductin preceding and during fertilization.     

The effects of washing and protein supplementation on the acrosome reaction of ram spermatozoa in vitro

Ram spermatozoa were incubated for 12 h at a concentration of 1–5 × 10⁶ ml^?1 in a modified BWW medium with TRIS (pH 8.0 in air, 308 mOsm) and a variety of pre-treatments were examined. These included washing in hypertonic (360 mOsm) medium by centrifugation and also supplementing with purified bovine serum albumin (BSA), with fatty acid-depleted BSA and with 5% v/v heat-inactivated sheep serum. Motility was assessed by phase contrast microscopy, and the incidence of acrosome reactions among live spermatozoa was estimated from nigrosin-eosin live-dead smears. All treatments showed a steady decline in sperm motility with time, and progressive increases in the proportion of live, acrosome-reacted spermatozoa (% ARS). However, there were no significant differences between washing regimes except for the treatment with hypertonic medium., where % ARS was elevated significantly after 9 and 12 h incubation. No differences were seen in % ARS between the various protein supplementations, although serum promoted significantly better survival.     

Production,characterization, and use of ionophore-induced,calcium-dependent acrosome reaction in ram spermatozoa

A system has been developed for inducing a calcium-dependent acrosome reaction in ram spermatozoa in vitro using the calcium ionophore A23187. The resultant reaction is accompanied by release of the acrosomal enzymes hyaluronidase and acrosin, but there is no release of the cytoplasmic enzyme glucose 6-phosphate isomerase. In any given cell, the visible acrosome reaction apparently takes place rapidly, but there is a variable delay before the reaction occurs. Under optimum conditions, about 90% of treated spermatozoa show an acrosome reaction within one hour. Preincubation of the spermatozoa with the proteinase inhibitors p-amino-benzamidine or p-nitrophenylguanidinobenzoate allows two stages of the reaction to be distinguished ultrastructurally, a membrane fusion stage followed by a dispersal of the acrosomal matrix. In the presence of the inhibitors, the first stage is delayed but is completed within 1 hour, whereas the second remains largely incomplete. In the presence of calcium, ionophore concentrations which induce an acrosome reaction abolish sperm motility rapidly and completely. However, by adding serum albumin shortly after addition of ionophore, motility can be preserved while the acrosome reaction occurs as usual; the motility pattern observed under these conditions is of the “whip-lash” or “activated” type. Although the motile ionophore-treated spermatozoa were unsuccessful at penetrating normal mature sheep oocytes in vitro, they were able to penetrate zona-free oocytes, after which swelling and decondensation of the sperm head took place.     

Cross-linking a maturation-dependent ram sperm plasma membrane antigen induces the acrosome reaction

ESA152 is a highly hydrophobic 18 kDa sialoglycoprotein, which becomes expressed on ram sperm in the proximal cauda epididymis. ESA 152 is expressed on all regions of the sperm surface, most strongly on the posterior region of the head, most weakly on the anterior region of the head. In this paper, we show that induction of the acrosome reaction with Ca2+ ionophore causes ESA152 to be redistributed from the posterior to the anterior region of the head plasma membrane. Cross-linking ESA152 with bivalent antibody causes similar redistribution and induces the acrosome reaction. Induction of the acrosome reaction with ESA152 antibody requires Ca2+ but is insensitive to (10 ng/ml) pertussis toxin.     

Interaction of calcium and cholesterol sulphate induces membrane destabilization and fusion: implications for the acrosome reaction

Cholesterol sulphate is a potent stabilizer of membrane bilayer structure in both dielaidoylphosphatidylethanolamine and egg phosphatidylethanolamine model membranes, however, the addition of calcium abolishes this bilayer stabilization. Calcium also induces fusion and leakage of egg phosphatidylethanolamine large unilamellar vesicles containing cholesterol sulphate, but has no effect on fusion or leakage of egg phosphatidylcholine large unilamellar vesicles containing cholesterol sulphate. With egg phosphatidylethanoiamine liposomes, the initial rate, and extent of fusion, at constant calcium concentration, vary inversely with the mol percentage of cholesterol sulphate present in the vesicle membrane. The interaction of calcium and cholesterol sulphate, which causes membrane destabilization and fusion in phosphatidylethanolamine containing model systems, may play a role in the acrosome reaction in human sperm.     

Effects of taurine and hypotaurine supplementation and ionophore concentrations on post-thaw acrosome reaction of dog spermatozoa

The aim of this work was to study of the effect of the amino acids (AA) taurine (T) and hypotaurine (H) and of different calcium ionophore concentrations on the ability of capacitated frozen-thawed dog sperm to undergo the acrosome reaction (AR). Fifteen ejaculates grouped into five pools were used. Sperm was frozen at a concentration of 80 × 10⁶ sperm cells/mL in the Uppsala Equex extender (UE) supplemented with 25, 50 and 75 mM of either AA. The UE extender without T or H was used as control. After thawing, sperm was capacitated with Canine Capacitation Medium for 20 min. Sperm was then challenged with calcium ionophore A23178 at 0, 2.5 and 10 μM concentration and evaluated for integrity of plasma and acrosome membranes after 5, 15 and 30 min of incubation, utilizing PI/Fitc-PNA fluorescent staining and flow cytometry.Sperm cryopreserved in UE supplemented with 50 mM T (UE 50T) had higher AR rates than sperm cryopreserved with UE 75T, UE 25H and UE 50H, but AR rates were similar to semen frozen with the control extender. Challenges with 2.5 and 10 μM/L of calcium ionophore increased AR in frozen-thawed sperm incubated for 5, 15 and 30 min. The combination of calcium ionophore concentration and incubation time resulting in the highest AR rate was 10 μM and 15 min.     

The effect of heparin, caffeine and calcium ionophore A23187 on in vitro induction of the acrosome reaction in frozen-thawed bovine and caprine spermatozoa

The effect of heparin (5 IU), caffeine (5 mM) and calcium-ionophore A23187 (0.1 mM) on motility and in vitro induction of the acrosome reaction in glass wool filtered frozen-thawed bull and goat semen was studied. The motile spermatozoa fraction was obtained after glass wool filtration of frozen-thawed semen. The seminal plasma was removed from filtered semen by centrifugation, and the sperm pellet was resuspended in Sperm-TALP medium. Samples of treated and untreated control semen of both species were incubated at 37 degrees C. At 1, 15 and 30 min of incubation the proportions of progressively motile and acrosome-reacted spermatozoa were assessed. Trypan blue and Giemsa stain was used to differentiate live and dead spermatozoa having undergone acrosome reaction. Glass wool filtration enhanced the proportion of motile spermatozoa from 43% to 62% in the bovine and from 41% to 60% in the caprine. Whereas the effect of incubation with caffeine, heparin and calcium-ionophore on spermatozoan motility was negligible, the treatment of semen with calcium-ionophore resulted in a significantly improved percentage of live spermatozoa with true acrosome reaction at all stages of incubation, both in the bovine and the caprine.     

Relationship between the M42 antigen of mouse sperm and the acrosome reaction induced by ZP3

A murine monoclonal antibody, M42 mAb, directed against 200/220 Kd protein of mouse sperm, has been employed to study the molecular events of gamete interaction. We have reported previously that M42 mAb blocks mouse fertilization in a zona-dependent manner; the reagent specifically inhibits physiologically induced (zonae), but not pharmacologically induced (A23187), acrosome reactions in mouse sperm. Using solubilized mouse zonae pellucidae and purified ZP3, we demonstrate that M42 mAb inhibits acrosome reactions (ARs) induced by ZP3 to the same extent as those induced by total zonae. We have also studied AR inhibition using the fluorescent antibiotic chlortetracycline (CTC), which permits visualization of three different acrosomal patterns during the AR. In the presence of M42 IgG, greater than 70% of capacitated sperm treated with zonae are arrested in the acrosome-intact state (B-pattern), in contrast to the majority of sperm (60-70%) in the absence of M42 IgG, which progress through the intermediate phase (S-pattern) to the fully acrosome-reacted (AR-pattern) state. Incubation of sperm with zona proteins modified by incubating eggs with phorbol esters arrests sperm in the S-pattern (Y. Endo, R.M. Schultz, and G.S. Kopf, 1987, Dev. Biol. 119, 199-209). We show that once sperm have reached such a state, M42 mAb no longer exerts an inhibitory effect. The addition of unmodified ZP to S-pattern sperm permits the completion of the acrosome reaction. These results indicate that M42 mAb blocks an early step in the AR cascade and that M42 mAb is unable to prevent subsequent events of this cascade once it has been initiated.     

Factors affecting the acrosome reaction in human spermatozoa

Large pieces of human cumulus oophorus were exposed for 20-30 min to washed spermatozoa or to spermatozoa recovered after a swim-up procedure, and then fixed for electron microscopy. Spermatozoa of both populations penetrated deeply into the cumulus within that time, and none of 48 observed clearly had undergone an acrosome reaction (AR). As measured by fluorescence microscopy, an AR rate of 12% in spermatozoa obtained at 4 h following a swim-up increased to about 25% in samples incubated in culture dishes for approximately 20 h. However, this latter AR rate was no different in the presence or absence of a cumulus/oocyte complex, and was only moderately greater in 50% follicular fluid. Nor was it affected to any degree by the absence of calcium or by a low (26 degrees C) temperature, both of which are regulators of the physiological AR in other species. By contrast, a clear dose-related enhancement of the AR by the calcium ionophore A23187 was almost completely Ca2(+)-dependent. We conclude that the human cumulus oophorus does not rapidly induce an AR in spermatozoa capacitated in vitro and, unlike the situation in some other mammals, that washed human spermatozoa do not first require a period of capacitation in order to penetrate it. The results also point to the likelihood that ARs monitored in free-swimming human spermatozoa are for the most part spurious or artefactual, and they show that in-vitro AR rates in such populations do not parallel their fertilizing ability.     

Spatiotemporal characterization of intracellular Ca2+ rise during the acrosome reaction of mammalian spermatozoa induced by zona pellucida

The mammalian sperm acrosome reaction (AR) is an essential event prior to sperm-egg fusion at fertilization, and it is primarily dependent on an increase in intracellular Ca2+ concentration ([Ca2+]i). Spatiotemporal aspects of the [Ca2+]i increase during the AR induced by solubilized zona pellucida (ZP) in hamster spermatozoa were precisely investigated with a Ca2+ imaging technique using confocal laser scanning microscopy with two fluorescent Ca2+ indicators. A rapid rise in [Ca2+]i occurred immediately after the application of ZP solution through a micropipette. The rise was always initiated in the sperm head, even when the application was directed toward the tail. The elevated [Ca2+]i was little attenuated during measurement for 30-40 s. Acrosomal exocytosis was detected as a sudden decrease of fluorescence in the acrosomal vesicle approximately 20 s after the onset of the [Ca2+]i rise. High-resolution imaging revealed that the [Ca2+]i rise in the sperm head began at the region around the equatorial segment and spread over the posterior region of the head within 0.6 s, whereas Ca2+ concentration in the acrosomal vesicle appeared to be unaltered. The [Ca2+]i rise was completely abolished under Ca2+-free extracellular conditions, indicating that it is totally attributable to Ca2+ influx. Nifedipine, an inhibitor of L-type Ca2+ channels, did not affect the rising phase of the ZP-induced Ca2+ response, but accelerated the decline of the [Ca2+]i rise and inhibited acrosomal exocytosis. The present study provides implicative information about the spatial organization of functional molecules involved in the signal transduction in mammalian AR.