Methylxanthines stimulate calcium transport and inhibit cyclic nucleotide phosphodiesterases in abalone sperm

Experiments were designed to determine the mechanism by which methylxanthines elevate abalone sperm cAMP concentrations and induce the acrosome reaction (AR). Theophylline or, more effectively, 1-methyl-3-isobutylxanthine (MIX) inhibit the cyclic nucleotide phosphodiesterase activities of abalone sperm homogenates. 45Ca2+ uptake by sperm is also stimulated by theophylline, and more effectively by MIX, and this stimulatory effect is blocked by KCN. Verapamil, a compound known to antagonize Ca2+ conductance, has no effect on the Ca2+ or MIX-induced cAMP elevation at concentrations up to 200 microM. However, verapamil reduces the sperm cAMP elevation caused by the addition of Ca2+ plus MIX. This inhibition is not complete, even at 200 microM verapamil. The AR induced by Ca2+ plus MIX is completely inhibited by 200 microM verapamil. The data suggest that these methylxanthines elevate abalone sperm cyclic nucleotide concentrations by inhibiting cyclic nucleotide phosphodiesterase activities. Furthermore, since sperm cAMP metabolism is modulated by Ca2+ flux, methylxanthines also appear to elevate abalone sperm cAMP concentrations by their effects on Ca2+ transport. The Ca2+-induced cAMP elevation occurs through a verapamil-insensitive mechanism, whereas the potentiation by MIX of the Ca2+ effect to elevate cAMP occurs through both verapamil-insensitive and -sensitive mechanisms. The methylxanthine-induced AR is mediated by a primary effect on Ca2+ transport and occurs through a verapamil-sensitive mechanism. Cyclic AMP may play a role in the methylxanthine-induced AR, but does not appear to act as the primary mediator of this exocytotic event.     

Characterization of basal and methylxanthine-stimulated Ca2+ transport in abalone spermatozoa

Methylxanthines, such as 1-methyl-3-isobutylxanthine (MIX) and theophylline, stimulate abalone sperm 45Ca2+ uptake in a time- and concentration-dependent manner. MIX is the most potent compound tested, and the ability of these compounds to alter 45Ca2+ uptake resides with methyl or isobutyl substitution of the xanthine nucleus at multiple sites. Methylxanthine-stimulated 45Ca2+ uptake does not occur as a secondary consequence of cyclic nucleotide phosphodiesterase inhibition, and added cyclic nucleotides are also without effect. The dramatic elevation of intracellular cAMP concentrations and induction of the acrosome reaction of sperm incubated with methylxanthines in the presence of Ca2+ is mediated by a primary effect of methylxanthines on Ca2+ transport. Basal 45Ca2+ uptake occurs through a verapamil-insensitive site that obeys the properties of a simple diffusion-mediated process. MIX-stimulated 45Ca2+ uptake occurs through a carrier-mediated transport site that has low affinity for Ca2+ (Km = 19.9 mM) and is verapamil sensitive. 45Ca2+ uptake through both basal and MIX-stimulated sites is enhanced by low extracellular Na+ concentrations (less than or equal to 15 mM) and is not affected by either extracellular Mg2+ or K+ X 45Ca2+ uptake through both sites is pH sensitive, but this sensitivity is different for each site. These data suggest that methylxanthines can affect sperm function via primary effects on Ca2+ transport, which occur through a specific carrier-mediated site(s). It is possible that many of the previously described effects of methylxanthines on sperm function are mediated via such changes in Ca2+ conductance.     

A depolarization can trigger Ca2+ uptake and the acrosome reaction when preceded by a hyperpolarization in L. pictus sea urchin sperm.

The acrosome reaction (AR) is an exocytotic event that allows sperm to recognize and fuse with the egg. In the sea urchin sperm this reaction is triggered by the outer investment of the egg, the jelly, which induces ionic movements leading to increases in intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi), a K(+)-dependent transient hyperpolarization which may involve K+ channels, and a depolarization which depends on external Ca2+. The present paper explores the role of the hyperpolarization in the triggering of the acrosome reaction. The artificial hyperpolarization of Lytechinus pictus sperm with valinomycin in K(+)-free seawater raised the pHi, caused a small increase in 45Ca2+ uptake, and triggered some AR. When the cells were depolarized with KCl (30 mM) 40-60 sec after the induced hyperpolarization, the pHi decreased and there was a significant increase in 45Ca2+ uptake, [Ca2+]i, and the AR. This waiting time was necessary in order to allow the pHi change required for the AR to occur. Thus, the jelly-induced hyperpolarization may lead to the intracellular alkalinization required to trigger the AR, and, on its own or via pHi, may regulate Ca2+ transport systems involved in this process. Because of the key role played by K+ in the triggering of the AR, the presence and characteristics of ion channels in L. pictus isolated sperm plasma membranes are being explored. Planar lipid bilayers into which these membranes were incorporated by fusion displayed 85 pS single channel transitions which were cation selective.     

Potassium ion influx and Na+,K+-ATPase activity are required for the hamster sperm acrosome reaction

The role of a K+ ion influx and Na+,K+-ATPase activity in the hamster sperm acrosome reaction (AR) was examined, using a range of concentrations of K+,K+ ionophores and a Na+,K+-ATPase inhibitor. Washed epididymal hamster sperm, capacitated in vitro in an artificial medium containing 2 mM Ca2+, 147 mM Na+, and 3, 6, 12, 18, or 24 mM K+, began undergoing the AR after 3 h of incubation. Sperm incubated in low K+ (0.9 mM) failed to undergo the AR even after 5 h of incubation. Sperm in 0.9 mM K+ could be induced to undergo the AR when either K+ (12 mM) alone or K+ (12 mM) with 0.1 microM nigericin was added after 3.5 h of incubation. The addition of K+ alone stimulated the AR in 30 min, whereas nigericin plus K+ stimulated the AR 15 min after addition. Neither nigericin added alone (0.9 mM K+) nor nigericin plus 12 mM K+ added to a low Ca2+ (0.35 mM) system resulted in acrosome reactions. Valinomycin (1 nM) did not stimulate the AR when added together with K+ (3-24 mM) to sperm incubated in 0.9 mM K+ for 3.5 h but markedly decreased sperm motility. Micromolar levels of ouabain blocked the AR when added between t = 0--3 h to sperm incubated with 3-24 mM K+. Inhibition of AR by the addition of 1 microM ouabain to sperm incubated with 3 mM K+ was completely reversed by the addition of 0.1 microM nigericin at t = 3.5 h. These results suggest that Na+,K+-ATPase activity and the resulting K+ influx are important for the mammalian sperm AR. Some similarities between requirements for the hamster sperm AR and secretory granule exocytosis are discussed.     

Correlation of increased intraacrosomal pH with the hamster sperm acrosome reaction

This study describes investigations of the importance of intraacrosomal pH in the hamster sperm acrosome reaction (AR). Washed cauda epididymal sperm were capacitated in vitro in a medium containing 2 mM Ca2+, 144 mM Na+, and 3 mM K+. Such sperm underwent a significant increase in the number of AR within 10 min after the addition of the Mg2+-ATPase (adenosine triphosphatase) inhibitors DCCD (20 microM) or NBD-Cl (10 microM) or the proton ionophore FCCP (6 micrograms/ml) at 3.5 hr of incubation or after addition of HN4Cl (3 mM) at 4 hr of incubation. Addition of the mitochondrial electron transport inhibitor rotenone (2.5 microM) at 3.5 hr or of NaCl (3 mM) or KCl (3 mM) at 4 hr did not stimulate AR over control levels, suggesting that the stimulation of AR by the other compounds was not directly due to depletion of acrosomal adenosine triphosphate (ATP) or alteration of the acrosomal transmembrane potential. The AR also was not stimulated by either DCCD or FCCP added prior to 3 hr of incubation of sperm, whereas both compounds were increasingly effective at stimulating AR with increasing length of preincubation of sperm before the addition of the test compounds. The intraacrosomal pH of sperm incubated in low [K+] (0.6-0.9 mM) for 3.5 hr rose by at least one pH unit (as measured with the fluorescent dye 9-aminoacridine) within 15-30 min after raising extracellular [K+] to 4.2-4.5 mM. The pH rise occurred even in the presence of the Ca2+-chelator EGTA (2 mM). Either FCCP (8 micrograms/ml) or DCCD (20 microM), but not rotenone (2.5 microM), plus K+ (3.6 mM), raised the intraacrosomal pH of sperm incubated for 3 hr in low [K+] within 10 min after addition. No pH rise occurred in the absence of additional K+. These results demonstrate that the intraacrosomal pH of the hamster sperm becomes more alkaline in a process not requiring high concentrations of external Ca2+, but requiring K+. The results of this and previous studies lead us to suggest here that the intraacrosomal pH rise may be mediated via a change in K+ and H+ permeability of sperm head membranes, which allows K+ influx and H+ efflux, and via inhibition of an acrosomal Mg2+-ATPase proton pump. We propose that the permeability changes and the consequent alkalinization of the acrosomal interior are important steps in late capacitation and/or the mammalian AR.     

The role of Ca-2+ and cyclic adenosine 3':5'-monophosphate in insulin release induced in vitro by the divalent cation ionophore A23187.

Insulin release from isolated perifused pancreatic islets was stimulated by the divalent ionophore A23187 in the absence of exogenous glucose. In addition, A23187 produced a 2-fold elevation of cyclic adenosine 3':5'-monophosphate (cAMP) levels in isolated perifused islets. The elevation of cAMP levels coincided with peak insulin release. Ionophore-induced insulin release was unaffected by pretreatment of the islets with theophylline (5 mM). Stimulation of insulin release produced by the ionophore occurred either in the presence or absence of extracellular Ca-2+; however, cAMP accumulation required the presence of extracellular Ca-2+. The ionophore (10 muM) had no effect on adenylate cyclase activity of homogenates of isolated islets. The results of this study are interpreted as indicating that intracellular Ca-2+ has an essential role in the insulin releasing mechanism, whereas the cAMP system has a modulatory effect on this process.     

Effect of methylxanthines on motility and fertility of frozen-thawed goat semen

We studied the effects of 2 methylxanthines (caffeine and theophylline) at different concentrations on goat sperm motility and live spermatozoa and on the percentage of acrosomal damage and fertility. Altogether, 144 semen samples collected from 12 bucks (3 each from Black Bengal and Beetal, and 6 from cross-breds) were diluted in TRIS extender, divided into 5 equal fractions; then caffeine and theophylline were added at 2 concentrations (2 and 5 mM) in different fractions. These samples were frozen in liquid nitrogen vapor, thawed at 37 degrees C for 15 sec, and evaluated for motility and other semen attributes. Addition of caffeine and theophylline had a stimulatory effect on goat spermatozoa. It was further observed that the effect of these agents was concentration-dependent, with 2 mM caffeine and 5 mM theophylline yielding the best results in respect to the percentage of motility in all 3 breeds of goats tested. Among the two methylxanthines used, caffeine was found to be the more effective in Improving motility than theophylline. There was no significant effect on the percentages of live spermatozoa and acrosomal damage due to the addition of these 2 methylxanthines to the extender. Fertility rates with Tris + 2 mM caffeine (60.20 %) and with Tris + 5 mM theophylline (58.88 %) extended semen were apparently higher than those with the Tris-diluted semen (50.0 %), although these differences were not significant.     

Characterization of a calcium-modulated adenylate cyclase from abalone spermatozoa

Abalone spermatozoa contain a particulate adenylate cyclase that displays maximal catalytic activity when Mn2+ is present as a metal cofactor in excess of ATP. Unlike other sperm adenylate cyclases, the abalone enzyme displays a high Mg2+-supported catalytic activity (Mg2+/Mn2+ activity ratio = 0.8). Kinetics analyses demonstrate that the enzyme contains both a MgATP catalytic site and a separate Mg2+ regulatory site. Mg2+-supported enzyme activity, however, is not stimulated by guanine nucleotides, NaF, cholera toxin, forskolin, or a variety of hormones. The enzyme from unfractionated sperm homogenates is inhibited by added Ca2+ in a concentration-dependent manner, when EGTA is not present in the assay. Methylxanthines, such as 1-methyl-3-isobutylxanthine and theophylline, also inhibit enzyme activity in a concentration-dependent manner through a noncompetitive mechanism. On the other hand, when intact cells are preincubated with Ca2+ prior to breakage and assayed for enzyme activity, Ca2+ stimulates enzyme activity at low concentrations. Enzyme activity of intact sperm preincubated with methylxanthines, in either the absence or presence of added Ca2+, is also stimulated. This effect is expressed via an effect on the velocity of the enzyme. A-23187 has similar stimulatory effects on the enzyme under these conditions. These data provide further support for the role of Ca2+ conductance in modulating sperm adenylate cyclase activity. The abalone sperm enzyme also appears to have regulatory properties that are unique among other sperm types.     

Induction of the acrosome reaction in guinea pig spermatozoa by cGMP analogues

The effect of cyclic nucleotide analgoues upon the immediate induction of the guinea pig acrosome reaction (AR) was studied. Dibutyryl (dB) CGMP and 8-bromo-cGMP, when added to sperm suspensions after varying periods of preincubation in glucose-free BWW medium (NaCl 94.59 mM, KCl 4.7 mM, CaCl2 1.71 mM, KH2PO4 1.19 mM, MgSO4 1.19 mM, NaHCO3 25.07 mM, pyruvate 0.25 mM, lactate 21.58 mM, and bovine seru albumen 1 g/liter), induced the AR in a large proportion of spermatozoa relative to controls. The proportion of ARs induced upon the addition of dB cGMP or 8-bromo-cGMP (10mM) at 1 h was equivalent to that obtained after a 5-h incubation in glucose-free BWW alone. The effect of cGMP analogues was concentration dependent over the tested range of 2-12 mM (less than 1-20%). The simultaneous addition if imidazole (10 mM), a cAMP phosphodiesterase stimulator, potentiated the effect (imidazole + 12 mM 8-bromo-cGMP: 73%). cAMP analogues were without effect. The presence of extracellular Ca++ was required, and it is suggested that a rise in the cGMP/CAMP ratio triggers Ca++ influx and the AR.     

Effect of Spermine-NONOate on acrosome reaction and associated protein tyrosine phosphorylation in Murrah buffalo (Bubalus bubalis) spermatozoa

The present study was conducted to know the role of Nitric Oxide (NO) on the acrosome reaction (AR) in Murrah buffalo (Bubalus bubalis) spermatozoa. Ejaculated buffalo spermatozoa were washed, suspended in sp-TALP media containing 6 mg BSA/mL and cell concentration was adjusted to 50×10(6) cells/mL. The cells were incubated for 6h in the absence or presence of heparin (10 μg/mL) to induce capacitation. Fully capacitated spermatozoa were incubated in presence of 100 μg/mL Lysophosphatidyl choline (LPC, T1) or 100 μM Spermine-NONOate (T2) or 100 mM L-NAME (T3) or 100 μM Spermine-NONOate+100 mM L-NAME (T4) or 1 mM db-cAMP + 0.1 mM IBMX (T5) or 100μM H-89 (T6) or 100 μM Spermine-NONOate+100 μM H-89 (T7) in combination to induce acrosome reaction. The extent of AR was assessed by dual-staining of spermatozoa with trypan blue/Giemsa stain. AR-associated tyrosine-phosphorylated proteins were detected by SDS-PAGE followed by immunoblotting using monoclonal anti-phosphotyrosine antibody. Significant (P<0.05) number of spermatozoa were acrosome reacted in Spermine-NONOate (T2) treated cells but it was significantly (P<0.05) lower than LPC (T1) induced AR. Addition of Spermine-NONOate + L-NAME (T4) resulted in non significant (P>0.05) decrease in acrosome reaction. On addition of H-89 + Spermine-NONOate (T7) to sperm culture medium, resulted in significant (P<0.05) decrease in the percent acrosome reaction. Conversely, addition of db-cAMP+IBMX (T5, cAMP analogue) resulted in the significantly (P<0.05) higher number of acrosome reacted spermatozoa. Pattern of sperm protein tyrosine phosphorylation was also different in NO induced acrosome reaction compared to that of LPC. The present study concluded that nitric oxide is involved in acrosome reaction of buffalo spermatozoa by causing the tyrosine phosphorylation of proteins mainly p17 and p20 and through activation of cAMP/PKA pathway.     

Calcium requirement and increased association with bovine sperm during capacitation by heparin

The requirement for external Ca+2 during capacitation of ejaculated bovine sperm with heparin and changes in sperm-associated 45Ca+2 during capacitation were investigated in vitro. Sperm capacitation was evaluated by ability to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine. The percentage of sperm which were capacitated during a 4 h incubation with heparin increased exponentially with increased exposure time to 2 mM Ca+2. When sperm were incubated with or without heparin in the presence of 45CaCl2, there was no difference in the amount of 45Ca+2 associated with sperm initially or at 1 h of incubation. Incubation with heparin resulted in a greater amount of sperm-associated 45Ca+2 at 2, 3, and 4 h as compared to sperm incubated without heparin. The amount of 45Ca+2 associated with sperm during capacitation was unaffected by washing with 2 mM EGTA-5 mM LaCl3. Glucose (5 mM) inhibited the effects of heparin on sperm-associated 45Ca+2 and on capacitation. The inhibitory effects of glucose could be overridden by 8-bromo-cAMP. The results suggest that the requirement for external Ca+2 during capacitation with heparin may be related to an increased association of external Ca+2 with sperm.     

Maitotoxin potently promotes Ca2+ influx in mouse spermatogenic cells and sperm, and induces the acrosome reaction

Maitotoxin (MTX), a potent marine toxin, activates Ca2+ entry via nonselective cation channels in a wide variety of cells. The identity of the channels involved in MTX action remains unknown. In mammalian sperm, Ca2+ entry through store-operated channels regulates a number of physiological events including the acrosome reaction (AR). Here we report that MTX produced an increase in the intracellular concentration of Ca2+ ([Ca2+]i) in spermatogenic cells that depended on extracellular Ca2+. Ni2+ and SKF96365 diminished the MTX-activated Ca2+ uptake, at concentrations they inhibit store-operated channels, and in a similar manner as they inhibit the Ca2+ influx activated following depletion of intracellular stores by thapsigargin (Tpg). In addition, MTX significantly increased [Ca2+]i in single mature sperm and effectively induced the AR with a half-maximal concentration (ED50) of approximately 1.1 nM. Notably, SKF96365 similarly inhibited the MTX-induced increase in sperm [Ca2+]i and the AR triggered by the toxin, Tpg and zona pellucida. These results suggest that putative MTX-activated channels may be involved in the Ca2+ influx required for mouse sperm AR.     

The role of anion channels in the mechanism of acrosome reaction in bull spermatozoa.

The involvement of anion channels in the mechanism of the acrosome reaction (AR) was investigated. The AR was induced by Ca2+ or by addition of the Ca2+ ionophore A23187. The occurrence of AR was determined by following the release of acrosin from the cells. In order to investigate the role of anion channels in the AR, several anion-channel inhibitors were tested, mainly DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). Other blockers, like SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), furosemide, probenecid and pyridoxal 5-phosphate, were also tested. We found that DIDS binds covalently to sperm plasma membrane in a time- and concentration-dependent manner. Maximal binding occurs after 2 h with 0.3 mM DIDS. DIDS and SITS inhibit AR in a concentration-dependent manner. The IC50 of DIDS and SITS in the presence of A23187 is 0.15 and 0.22 mM, respectively. Tributyltin chloride (TBTC), an Cl-/OH- exchanger, partially overcomes DIDS inhibition of the AR. HCO3- is required for a maximal acrosin release and Ca(2+)-uptake, in the presence or absence of A23187. It is known that HCO3- activates adenylate cyclase and therefore, increases the intracellular level of cAMP. The inhibition of the AR by DIDS decreases from 95 to 50% when (dibutyryl cyclic AMP (dbcAMP) was added, i.e., HCO3- is no longer required while elevating the level of cAMP in an alternative way. Moreover, we show that the stimulatory effect of HCO3- on Ca(2+)-uptake is completely inhibited by DIDS. We conclude that DIDS inhibits AR by blocking anion channels, including those that transport HCO3- into the cell.     

Calcium requirement for the inhibition by theophylline of histamine release from mast cells

When added to Ca2+-free Hanks' solution, Ca2+ (0.1-2.5 mM) had no significant effect on antigen-induced histamine release from rat mast cells, but Sr2+ (1.0-3.0 mM) dose-dependently increased the release. Ba2+ (1.0 and 2.0 mM) also enhanced the release. Ca2+ and Ba2+ inhibited compound 40/80-induced histamine release, in a dose-dependent manner. In ordinary Hanks' medium, theophylline and 3-isobutyl-1-methylxanthine (IBMX) dose-dependently inhibited the antigen-induced histamine release but these drugs were ineffective in Ca2+-free medium. Theophylline (1.0 mM) also inhibited compound 48/80-induced histamine release in the presence but not absence of Ca2+. There was an optimal Ca2+ concentration for the theophylline effect. Sr2+ but not Ba2+ could substitute for Ca2+ in supporting the theophylline effect. Theophylline (1.0 mM) and IBMX (1.0 mM) increased mast cell cyclic AMP levels both in the presence and absence of Ca2+. These results suggest that Ca2+ is required in the interaction of theophylline and specific sites on mast cells or in the mast cell response to theophylline which probably does not involve the cyclic AMP increase and is linked to the inhibition of histamine release.     

Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.     

Calcium-induced acrosomal exocytosis requires cAMP acting through a protein kinase A-independent, Epac-mediated pathway

Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca2+ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca2+ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyryl-cAMP and the Epac-selective 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca2+. This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca2+, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-alpha-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca2+ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca2+ in exocytosis.     

Minimum and maximum extracellular Ca2+ requirements during mouse sperm capacitation and fertilization in vitro

The minimum and maximum extracellular Ca2+ concentrations required to promote capacitation, the acrosome reaction, hyperactivated motility, zona penetration and gamete fusion in the mouse have been established. The traces of free calcium in Ca2+-deficient medium were shown not to enhance capacitation since the inclusion of EGTA to chelate free ions during a 120 min preincubation failed to alter the kinetics of capacitation from those observed in the absence of EGTA; 1 h after addition of 1.80 mM-Ca2+, both suspensions were highly fertile. Complete capacitation, when suspensions were immediately functional upon the addition of 1.80 mM-Ca2+, required the presence of greater than or equal to 90 microM-Ca2. Considerably higher concentrations were required to initiate optimal sperm responses: acrosome reaction, 900 microM; gamete fusion, 900 microM; hyperactivated motility, 1.80 mM; zona penetration, 1.80 mM. None of these changes was effected when Ca2+ was less than 450 microM. The responses to elevated Ca2+ were dependent on the length of incubation, being initially positive and then negative. A short (30 min) exposure to 3.40 mM-Ca2+ (x 2 the standard) accelerated capacitation, as evidenced by significantly increased acrosome loss, precocious expression of hyperactivated motility and enhanced fertilizing ability when Ca2+ was reduced to 1.80 mM. However, extended (120 min) preincubation irreversibly damaged sperm function. In the presence of 7.20 mM-Ca2+ (x 4), fertilizing ability was inhibited at both 30 and 120 min, despite a high incidence of acrosome loss. The primary deleterious effect appeared to be on motility which was judged to be more erratic than in 1.80 mM-Ca2+, possibly due to elevated intracellular Ca2+. Because of the considerable difference in threshold Ca2+ concentrations, it is now possible to dissociate the Ca2+-dependent events of capacitation from those of the acrosome reaction and motility changes.     

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.     

Evidence for tight coupling of phospholipase activation and Ca2+ influx during acrosome reaction of golden hamster spermatozoa

1. Phospholipases have been proposed to play a key role in sperm acrosome reaction. To examine the activation mechanism of phospholipases and subsequently sperm fertilizing capacity. Ca2+ fluxes and phospholipid turnover (breakdown and synthesis) were investigated in golden hamster spermatozoa during acrosome reaction. 2. Upon exposure of the spermatozoa to 1.7 mM Ca2+, a net uptake by the cells occurred in two distinguishable phases. 3. Depletion of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at a time that an initial Ca2+ uptake was observed to reach almost steady-state, prevented the secondary Ca2+ uptake and acrosome reaction. 4. The time course of an initial Ca2+ uptake seemed to precede that of the acrosome reaction. 5. Incubation of the spermatozoa with Ca2+ in the presence of [3H]glycerol induced a rapid increase in labeling of phosphatidic acid, a key intermediate of phosphinositide turnover initiated by the action of phospholipase C, which appeared to parallel the time course of a first phase of Ca2+. 6. Phospholipase A2 activation, detected by lysophospholipid formation, slightly delayed the initial events of first Ca2+ uptake and phosphatidic acid production. 7. It is concluded that first Ca2+ entry into the cells, associated with phosphatidic acid production, activates a phospholipase A2, leading to the production of substances, like lysophospholipids and fatty acids, which may contribute to acrosome reaction.