The Limulus sperm motility-initiating peptide initiates acrosome reactions in sea water lacking potassium

During fertilization in Limulus, the spermatozoa first attach to the egg and then undergo an acrosomal reaction. In this reaction, the acrosomal vesicle exocytoses, and a long, preformed acrosomal filament is extruded (and subsequently penetrates the egg chorion). The egg surface component that triggers the acrosome reaction has not yet been solubilized; therefore, previous studies have examined either spontaneous acrosome reactions or acrosome reactions that were triggered by eggs (or insoluble egg fragments), elevated extracellular Ca2+, or Ca2+ ionophores. In this study, we report a new method for initiating acrosome reactions in Limulus sperm. When the Limulus sperm motility-initiating peptide (SMI) is added to sperm in K+-free sea water, greater than 90% acrosome reactions are initiated within 5 min. However, less than 5% acrosome reactions occur either in K+-free sea water lacking SMI or when SMI is added to sperm in either normal sea water or K+- and Ca2+-free sea water. Experiments with K+ ionophores (nigericin and valinomycin), a K+ channel blocking agent (tetraethyl ammonium), an Na+ ionophore (monensin), and reagents that increase the intracellular pH (monensin, nigericin, and NH4Cl) indicate that changes in intracellular K+, Na+, or H+ do not mediate SMI-initiated acrosome reactions. The K+/Ca2+ ratio determines whether or not SMI will initiate acrosome reactions, with greater than 50% acrosome reactions being initiated when this ratio is below 0.3. In that K+ movement does not appear to be the critical event, possibly the K+/Ca2+ ratio either determines the rate of Ca2+ entry or controls the conformation of sperm surface molecules to allow SMI to initiate acrosome reactions in low K+.     

Bicarbonate- and calcium-dependent induction of rapid guinea pig sperm acrosome reactions by monovalent ionophores

The monovalent cationic ionophores monensin and nigericin stimulated rapid guinea pig sperm acrosome reactions in the presence of extracellular Na+, Ca2+ and bicarbonate (HCO3-/CO2). Extracellular K+ (mM concentrations), in contrast, was not required for the stimulatory effect of the ionophores. The effect of HCO3-/CO2 is concentration, pH and temperature dependent, with maximal responses obtained with 50 microM monensin or 25 microM nigericin at a concentration of 30 mM HCO3-, 2.5% CO2 and pH 7.8 at 25 degrees C. At a constant HCO3- concentration (30 mM), monensin stimulated acrosome reactions within the pH range 7.5-7.8, whereas a higher or lower pH did not support acrosome reactions at 25 degrees C. At constant extracellular pH (7.8), monensin stimulated acrosome reactions in the presence of 30 mM HCO3-, whereas higher and lower concentrations did not support acrosome reactions at 25 degrees C. The permeant anions pyruvate and lactate were essential to maintain sperm motility when treated with monensin under these conditions. NH4Cl, sodium acetate and 4,41-diisothiocyano-2, 21-disulfonic acid stibene (DIDS; 25 microM), an anion transport inhibitor, blocked the ability of monensin to stimulate acrosome reactions. Verapamil (100 microM), a putative Ca2+ transport antagonist, in contrast, did not prevent the monensin-induced acrosome reactions. Physiological concentrations of Na+ were needed for monensin to stimulate acrosome reactions, but high concentrations of Mg2+ prevented the monensin stimulation. The Ca2+ ionophore A23187 (75 nM) also required physiological concentrations of Na+ for the rapid induction of maximal acrosome reactions at an elevated pH (8.3) but did not require the presence of extracellular HCO3-. These studies suggest that a monovalent ionophore-induced rise in sperm intracellular Na+ concentrations is a pre-Ca2+ entry event, that stimulates an endogenous Ca2+/Na+ exchange that allows a Ca2+ influx which in turn induces the acrosome reaction. The possible regulatory role of the sperm intracellular pH and Na+, K+-ATPase during the capacitation process under physiological conditions is discussed.     

Regulation of the starfish sperm acrosome reaction by cGMP, pH, cAMP and Ca2+

In the starfish, Asterias amurensis, three components in the jelly coat of eggs, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to induce the acrosome reaction (AR). Molecular recognition between the sperm surface molecules and the egg jelly molecules must underlie signal transduction events triggering the AR. Asterosap is a sperm-activating molecule, which stimulates rapid synthesis of intracellular cGMP, pH and Ca(2+). This transient elevation of Ca(2+) level is caused by a K(+)-dependent Na(+)/Ca(2+) exchanger, and the increase of intracellular pH is sufficient for ARIS to induce the AR. The concerted action of ARIS and asterosap could induce elevate intracellular cAMP levels in starfish sperm and the sustained increase in [Ca(2+)], which is essential for the AR. The signaling pathway induced by these factors seems to be synergistically regulated to trigger the AR in starfish sperm.     

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.     

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.     

VLA-6 integrin distribution and calcium signalling in capacitated boar sperm.

Previous investigations showed that VLA-6 integrin present on boar sperm membrane can induce acrosome reaction upon exposure to laminin accumulated in expanded cumuli (Mattioli et al., 1998. To further investigate this novel sperm egg-recognition system, the authors studied the distribution of VLA-6 integrin on the membrane of boar sperm throughout capacitation and following acrosome reaction, and analyzed intracellular Ca(2+) changes occurring in spermatozoa exposed to laminin. Immunofluorescent localisation of VLA-6 revealed a low proportion (nearly 22%) of positive cells in freshly ejaculated sperm, with integrin mainly concentrated in clustered spots. After 3 hr incubation most of the spermatozoa showed integrin molecules on the membrane, with three different labeling patterns: fluorescence localised on the edge of the acrosome (58.2 +/- 14.2% of the cells); fluorescence uniformly spread over the whole sperm head (5.0 +/- 1.9%) and finally fluorescence concentrated in clustered spots (7.6 +/- 5.6%), as recorded in freshly ejaculated sperm. Twenty-nine percent of cells did not show any distinct fluorescence. Following acrosome reaction sperm with fluorescence on the acrosomal region virtually disappeared and the proportion of unstained cells rose from 29.2 +/- 9.2 to 69.0 +/- 10.1%. Electron microscopy demonstrated that VLA-6 integrin was exclusively located on the sperm membrane of intact spermatozoa. Confocal analysis showed that laminin triggers distinct Ca(2+) raises, and that sperm exposed and kept in the presence of laminin fully retained their ability to rise intracellular Ca(2+) in response to zona pellucida proteins. These data indicate that boar sperm accumulate VLA-6 integrin on the membrane and concentrate it on the acrosomal region as capacitation progresses. Probably due to this compartmentalisation, sperm exposed to laminin experience a Ca(2+) raise that originates in the anterior sperm head where it is more adequate for the induction of acrosome reaction. Mol. Reprod. Dev. 59:322-329, 2001.     

Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction

The effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS-2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell-conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO₂ in air. Viability and the occurrence of true acrosome reactions were assessed using a triple-stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS-2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS-2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS-2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley-Liss, Inc.     

Involvement of zinc in the regulation of pHi, motility, and acrosome reactions in sea urchin sperm

When sperm of Strongylocentrotus purpuratus or Lytechinus pictus are diluted into seawater, motility is initiated; and when exposed to egg jelly, an acrosome reaction is induced. In the presence of a variety of structurally different metal chelators (0.1-1 mM EDTA, EGTA, phenanthroline, dipyridyl, cysteine, or dithiothreitol), motility initiation is delayed and the acrosome reaction is inhibited. Of the metals detected in the sperm of these two species, very low levels of Zn+2 (0.1 microM free Zn+2) uniquely prevent this chelator inhibition. L. pictus sperm concentrate 65Zn+2 from seawater, and EDTA removes 50% of the accumulated 65Zn+2 by 5 min. Since both sperm motility and acrosome reactions are in part regulated by intracellular pH (pHi), the effect of chelators on the sperm pHi was examined by using the fluorescent pH sensitive probe, 9-aminoacridine, EDTA depresses sperm pHi in both species, and 0.1 microM free Zn+2 reverses this pHi depression. When sperm are diluted into media that contain chelators, both NH4Cl and monensin (a Na+/H+ ionophore) increase the sperm pHi and reverse the chelator inhibition of sperm motility and acrosome reactions. The results of this study are consistent with the involvement of a trace metal (probably zinc) in the pHi regulation of sea urchin sperm and indicate a likely mechanism for the previously observed effects of chelators on sperm motility and acrosome reactions.     

Hamster sperm Na+, K+-adenosine triphosphatase: increased activity during capacitation in vitro and its relationship to cyclic nucleotides

These in vitro studies of golden hamster sperm were undertaken to determine whether: Na+, K+-adenosine triphosphatase (ATPase) activity is required for capacitation; Na+, K+-ATPase activity is altered during capacitation; and cyclic nucleotides can control this enzyme activity. Hamster sperm were incubated in a medium in which capacitation occurred in an asynchronous manner and in which acrosome reactions began to occur after approximately 3.5 h of incubation. Inhibition of the hamster sperm acrosome reaction by the Na+, K+-ATPase inhibitor ouabain (1 microM) added at Time (T) = 2 or T = 3 h could be fully reversed by the addition of the ionophore nigericin (0.1 microM) at T = 3.5 h. However, when ouabain was added at T = 0 or T = 1 h, similar nigericin addition could not completely reverse the inhibition. Na+, K+-ATPase activity of hamster sperm increased by 2 h of incubation (compared to that measured initially after 15 min) and this activity remained elevated at 3.5 h. Addition of either monobutyryl cyclic adenosine 3':5'-monophosphate ( BtcAMP ) (12.9 microM) or monobutyryl cyclic guanosine monophosphate ( BtcGMP ) (10.5 microM), or the phosphodiesterase inhibitor SQ20009 (10 microM) at 2 h produced a stimulation of acrosome reactions at 4 and 5 h. However, while BtcGMP and SQ 20009 also induced a further increase in Na+, K+-ATPase activity measured at 3.5 h, BtcAMP had no effect. Intracellular cAMP and cGMP levels measured showed cAMP increased by 2 h and remained elevated when measured at 3.5 h, while cGMP could not be consistently detected at 15 min, 2 h or 3.5 h. However, assays of high numbers of uncapacitated sperm did detect a low level of cGMP. These results suggest that Na+, K+-ATPase activity increases in and is essential for early capacitation [and thereby eventually for the acrosome reaction (AR)] of hamster sperm and that the increase in Na+, K+-ATPase activity occurring during capacitation is probably mediated by intracellular cGMP but not cAMP, although both cyclic nucleotides stimulate the hamster sperm AR.     

High pH-induced acrosome reaction and Ca2+ uptake in sea urchin sperm suspended in Na+-free seawater

The egg jelly-induced acrosome reaction of sea urchin sperm requires the presence of Ca2+ and Na+ in seawater at its normal pH 8. Sperm suspended in seawater at pH 9 undergo the acrosome reaction in the absence of jelly. We have attempted to understand the role of external Na+ in this reaction. Sperm were suspended in Na+-free seawater and the percentage of acrosome reaction and the amount of Ca2+ uptake were determined as a function of external pH. High pH (9.0) in Na+-free medium without jelly triggered a high percentage (above 65%) of sperm acrosome reactions and a two to fourfold increase in Ca2+ uptake. Both the percentage of acrosome reactions and the amount of Ca2+ uptake were similar to those induced by either jelly or pH 9 in Na+-containing seawater. On the other hand, the absence of Na+ in seawater inhibits jelly from inducing Ca2+ uptake and acrosome reactions at pH 8.0 and even at pH 8.5. These results indicate that the Na+ requirement for the acrosome reaction induced by jelly is lost when triggering is by high pH. In contrast, Ca2+ was strictly required since sperm did not react in Ca2+-free seawater at pH 9. We also found that like the jelly-induced acrosome reaction the high-pH-induced acrosome reaction and Ca2+ uptake in complete and Na+-free seawater were inhibited by D600. This finding suggests that the same transport system for Ca2+ uptake associated with the acrosome reaction operates at both triggering conditions, i.e., jelly or pH 9. Although D600 is not now considered a specific blocker, its effect has suggested the involvement of Ca2+ channels in the acrosome reaction. This proposal is supported by our results with nisoldipine, a highly specific inhibitor of calcium channels. The drug inhibited both the sperm acrosome reaction and Ca2+ uptake induced by jelly or pH 9 in complete seawater.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Observations on the acrosome reaction of human sperm in vitro

Human sperm were incubated in vitro in serum or the defined medium TMPA and were periodically assessed for acrosome reactions using two new methods of assay. The first method, FITC-RCA labeling, was previously shown to be valid for estimating the percentage of normal acrosome reactions of human sperm. The second method, a triple staining technique, is shown in this study to give results comparable to those obtained with FITC-RCA labeling. The percentage of acrosome-reacted sperm was determined at 0, 2.5, 5, and 7 hr of incubation. In both media, some sperm had reacted by 2.5 hr; a maximum percentage of reactions occurred between 5 and 7 hr. The maximum percentage never exceeded 20–25%, which represents only one-third of the live sperm, ie, those potentially able to undergo normal acrosome reactions. It will be important in future studies to determine if this low-peak percentage is due to the fact that: (1) Commonly used culture media are suboptimal or (2) only about 25% of the sperm in a human ejaculate are capable of undergoing normal acrosome reactions.     

猪精子体外获能前后离子成分变化

应用扫描电镜和镜射电镜能谱技术,为猪精子获能前后质膜表面和内部的离子成分进行了研究,结果表明,猪精子获能后质膜表面的Na~+和Al~(3+)升高,而Cl~-和Ca~(2+)降低;精子顶体内Na~+和Cl~-降低,Ca~(2+)、K~+和Fe~(2+)升高;中段线粒体内的Na~+、Ca~(2+)和Fe~(2+)升高,而K~+和Cl~-降低。文章分析了精子获能后顶体内Na~+、Cl~-、K~+、Ca~(2+)和Fe~(2+)变化的浓度比和摩尔比。     

The regulation of acrosomal exocytosis. I. Sperm capacitation is required for the induction of acrosome reactions by the bovine zona pellucida in vitro

The regulation of acrosomal exocytosis in capacitated bovine spermatozoa by soluble extracts of zonae pellucidae was examined. Kinetic studies demonstrated that zonae pellucidae stimulated synchronous acrosome reactions. The t1/2 of this process was 5-10 min and response was maximal at 20 min. The apparent initial rate of exocytosis in sperm populations was dependent upon the concentration of zona pellucida protein, with an ED50 and a maximally effective dosage of 20 and 50 ng protein/microliter, respectively. Zonae pellucidae caused up to a 48-fold increase in the apparent initial rate and a 3- to 4-fold stimulation in the net occurrence of exocytosis. In contrast, solubilized zonae pellucidae did not induce acrosome reactions in uncapacitated sperm. The development of a capacitated state, as assayed by the ability of sperm to fertilize eggs in vitro, was compared to the expression of zona pellucida-regulated acrosome reactions in a series of kinetic experiments. Both activities were manifest with similar kinetics and displayed identical dependencies toward stimulatory and inhibitory agents in vitro. It is concluded that capacitation is an essential prerequisite for the induction of acrosomal exocytosis in bovine sperm by the zona pellucida.     

Fusion of hamster and pig zona-free eggs stimulated by boar and guinea pig sperm at fertilization in vitro

In the course of in vitro fertilization of zona-free hamster and pig eggs by boar and guinea-pig spermatozoa it was observed that homologous and heterologous eggs fused together, forming cell hybrids between two or more cells. The fusogenic activity was attributed to spermatozoa and this was the hypothesis tested. The fusogenic activity (coinciding with sperm penetration activity) was dependent on the duration of sperm preincubation, which may be regarded as capacitation in vitro. Fusion occurred only after 3 hr of sperm preincubation and a narrow optimum was detected at 4–4.5 hr. Fusion of eggs was also dependent on sperm concentration. A relatively high proportion of fusions was observed at a sperm concentration of 4.0 × 10⁴ per ml and an optimum was attained at a concentration of 5.0 × 10⁵ per ml. The first fusions were observed at 90 min after semination. After 3 hr more than a half of the eggs reacted, and by 20 hr of incubation 80% of ova were fused. The fusability of eggs was tested and found to occur at 14 hr after ovulation. The fusion process was also studied using transmission electron microscopy. It is supposed that the process of egg fusion may be caused either by a similar mechanism to sperm-egg fusion, or by products released during the sperm acrosome reaction.     

Interactions of seminal plasma and glycosaminoglycans on acrosome reactions in bovine spermatozoa in vitro

Previous reports indicate that glycosaminoglycans (GAGs) would enhance the occurrence of acrosome reactions in sperm in vitro, but continuous exposure of those sperm to seminal plasma prevented a significant incidence of acrosome reactions. This study was designed to evaluate the interaction of GAGs and seminal plasma to promote acrosome reactions in bull sperm in vitro. Epididymal sperm required 22 hr to exhibit acrosome reactions in response to GAGs whereas only 9 hr were needed to achieve the same effect with washed ejaculated sperm. Exposure of epididymal sperm to seminal plasma for 20 min shortened the time for induction of the acrosome reaction to 9 hr. Scatchard analyses of displacement data suggested an alteration in the binding affinity of ³H-heparin to epididymal sperm membrane following the short-term exposure to seminal plasma. High doses (250 and 500 μg/ml) of heparin, heparan sulfate, and chondroitin-4-sulfate were without effect, but doses <100 μg/ml were stimulatory in terms of enhancing acrosome reactions. Compositional studies with seminal plasma revealed a total GAG content of 1.6 mg/ml, proportioned as 61.6% chondroitin sulfates, 17.6% heparin-like material, 0.3% hyaluronic acid, and 20.5% undetermined GAG. It is proposed that seminal plasma can alter the ability of sperm to respond to GAGs, and the high concentrations of GAGs endogenous to seminal plasma may prevent premature initiation of the membrane perturbations necessary for the acrosome reaction.     

Internal pH can regulate Ca2+ uptake and the acrosome reaction in sea urchin sperm

The egg jelly-induced acrosome reaction of sea urchin sperm is accompanied by intracellular alkalinization and Ca2+ entry. We have previously shown that in the absence of egg jelly, NH4Cl, which increases intracellular pH (pHi), induces Ca2+ uptake and the acrosome reaction in sperm of the sea urchin, Strongylocentrotus purpuratus. Here we show that at a constant concentration of NH4Cl (20 mM) in seawater, sperm react less as external pH is lowered from the normal 8 to 7.25. The pH dependence of the NH4Cl response is not very sensitive to temperatures between 12 and 17 degrees C. NH4Cl (15-50 mM) stimulates Ca2+ uptake and acrosome reactions in sperm suspended in Na+-free seawater, a condition known to inhibit the inductive effect of jelly. Jelly does not further stimulate Ca2+ uptake of sperm preincubated in NH4Cl, indicating that once the permeability to Ca2+ is increased by raising the pHi, the jelly has no further effect. We have used the membrane potential-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide to follow the membrane potential change that occurs when NH4Cl is added. Depolarization (25 mV) is associated with the acrosome reaction when either the natural inducer, egg jelly, or NH4Cl is added to sperm. Response to both inducers is inhibited under conditions known to abolish the acrosome reaction, i.e., low-pH seawater and nisoldipine. These results indicate that the NH4Cl-induced depolarization that accompanies the reaction is probably due to the opening of channels that allow Ca2+ to enter the cell and not to the depolarization by NH4+ ions. High-K+ seawater, which depolarizes sperm, and tetraethylammonium, a K+ channel blocker, inhibit the jelly-induced depolarization and the acrosome reaction, but do not inhibit NH4Cl-induced changes. It has already been shown that nigericin promotes Ca2+ entry and the acrosome reaction in sea urchin sperm. We found that the action of this ionophore depends on the pH of normal seawater. In the absence of external Na+ (replaced by choline), nigericin does not induce the reaction and does not stimulate Ca2+ uptake.     

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.     

Complex combined steroid mix of the female tract modulates human sperm

Human spermatozoa interact with a complex biochemical environment in the female reproductive tract en route to the site of fertilisation. Ovarian follicular fluid contributes to this complex milieu and is known to contain steroids such as progesterone, whose effects on sperm physiology have been widely characterised. We have previously reported that progesterone stimulates intracellular calcium concentration ([Ca²⁺]_i) signalling and acrosome reaction in human spermatozoa. To characterise the effects of the unified complete follicular fluid steroid hormone complement on human spermatozoa, a comprehensive, data-based, ‘physiological standard’ steroid hormone balance of follicular fluid (shFF) was created from individual constituents. shFF induced a rapid biphasic [Ca²⁺]_i elevation in human spermatozoa. Using population fluorimetry, we compared [Ca²⁺]_i signal amplitude in cells exposed to serial applications of shFF (6 steps from 10^-5X up to 1X shFF) with responses to the equivalent progesterone component alone (6 steps from 135 pM - 13.5μM). Threshold for the response to shFF was right-shifted (≈10-fold) compared to progesterone alone, but the maximum response to shFF was greatly enhanced. An acrosome reaction assay was used to assess functional effects of shFF-induced sperm calcium signalling. shFF as well as progesterone-treated spermatozoa showed a significant increase in % acrosome reaction (P < 0.01). All of this evidence suggests the modulation of progesterone-mediated responses by other follicular fluid steroids.     

Direct contact is required between serum albumin and hamster spermatozoa for capacitation in vitro

A dialysis unit was used to test whether direct physical contact between serum albumin and hamster spermatozoa is required for capacitation and/or the acrosome reaction. Sperm and bovine serum albumin (BSA) were incubated cither together (direct incubation) or separated by a dialysis membrane (indirect incubation). Sperm viability was supported with “sperm motility factors” (hypotaurine and epinephrine) and polyvinylalcohol (PVA). Spermatozoa became capacitated and underwent acrosome reactions when directly incubated in medium containing BSA (TALP-PVA), but did not undergo acrosome reactions when indirectly incubated with BSA (medium TLP-PVA). When sperm were first incubated for 4 hr indirectly with BSA, followed by 4 hr direct incubation with BSA, capacitation did not occur during indirect incubation. These findings indicate that an “intimate association” is necessary between serum albumin and spermatozoa to support capacitation under in vitro incubation conditions. The data are consistent with the concept of direct transfer of compounds from sperm to albumin and/or vice versa during sperm capacitation.