A further study of lysolecithin−mediated acrosome rection of guinea pig spermatozoa

When guinea pig spermatozoa are preincubated for 1 hr in Ca²⁺?free medium containing a low concentration of lysolecithin (LC, 85 μg/ml) and then exposed to 2 mM Ca²⁺ by diluting the preincubation medium with an equal volume of LC?free, 4 mM Ca²⁺?containing medium, the majority of the spermatozoa undergo acrosome reaction promptly. On the other hand, when the preincubated spermatozoa are exposed to 2 mM Ca²⁺ without reducing the original concentration of LC in the medium, none of them undergo acrosome reaction. These spermatoza can acrosome?react if they are transferred to an LC?free medium. These results and those of some other experiments suggest that in the presistent presence of a high concentration of LC in the medium, exogenous Ca²⁺ essential for the acrosome reaction either does not penetrate the sperm plasma membrane or, if it does, it cannot alter the membrane for the acrosome reaction, at least under the experimental conditions employed. Freeze?fracture examination of the sperm plasma membrane has revealed that small areas or patches free of intramembranous paarticles (IMPs) appear in the membrance during sperm preincubation, and these IMP?free areas expand drastically in response to Ca²⁺ when the LC conccentration in the medium is reduced at the time Ca²⁺ is added to the medium. In contrast, IMP?free areas remain unchanged even after exposure of spermatozoa to Ca²⁺ if the concentration of LC remains at its original level of 85 μg/ml.     

The acrosome reaction of Strongylocentrotus purpuratus sperm. Ion requirements and movements

The acrosome reaction of sperm of the sea urchin, Strongylocentrotus purpuratus, is accompanied by ion movements. When the reaction is induced by the addition of egg jelly to sperm suspended in sea water, there is an acid release and an uptake (or exchange) of calcium ions. Verapamil and D600, drugs which block Ca²⁺ channels, inhibit induction of the acrosome reaction, acid release, and ⁴⁵Ca²⁺ uptake; this inhibition is reduced at higher concentrations of external Ca²⁺. Although acid release correlates temporally with extension of the acrosome filament, ⁴⁵Ca²⁺ uptake continues after the acrosome reaction has been completed. Neither the acrosome reaction nor acid release is inhibited by cyanide, azide, dinitrophenol (DNP), or carbonyl cyanide m-chlorophenylhydrazone (CCCP), whereas these metabolic inhibitors partially inhibit Ca²⁺ uptake. Tetraethylammonium (TEA) chloride, an inhibitor of delayed axonal potassium currents, inhibits the acrosome reaction. An increase in ⁸⁶Rb⁺ permeability accompanies the acrosome reaction, suggesting that movement of K⁺ is an important effector of the reaction. In support of this, the acrosome reaction may be triggered with nigericin, an ionophore that catalyzes the electrically neutral exchange of K⁺ and H⁺ across membranes. Induction of the acrosome reaction with nigericin can occur with either Na⁺ or K⁺ as the predominant external monovalent cation, while with jelly it requires external Na⁺. With nigericin, there is a delay in acid release, Ca²⁺ uptake, and filament extension, all of which follow a transient proton uptake. Taken together, these data suggest that triggering of the acrosome reaction involves linked permeability changes for monovalent and divalent ions.     

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.     

Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipids in the acrosome reaction

The effects of lipids on the survival, acrosome reaction, and fertilizing capacity of guinea pig spermatozoa were studied by incubating the spermatozoa in media containing various concentrations of the lipids. Lipids tested were: phosphatidyl-choline (PC), -ethanolamine (PE), -inositol (PI), -serine (PS), sphingomyelin (S), cholesterol (C), lysophosphatidyl-choline (LC), -ethanolamine (LE), -inositol (LI), -serine (LS), and glyceryl monooleate (M). When spermatozoa were incubated in a regular medium (containing 2 mM Ca²⁺) with M, the majority underwent the acrosome reaction within 1 hour. None of the other lipids were as effective as M, and some were totally ineffective under the same conditions. However, when spermatozoa were preincubated in Ca²⁺-free medium containing LC, LE, or LI, they gained the ability to undergo the acrosome reaction. One hour of preincubation in Ca²⁺-free medium with LC, LE, or LI was enough to render the vast majority of spermatozoa capable of undergoing the acrosome reaction in response to Ca²⁺. The optimum concentrations for LC, LE, and LI were approximately 85 μg/ml, 210 μg/ml, and 140 μg/ml, respectively. Spermatozoa that had undergone the acrosome reaction by pretreatment with LC, LE, or LI remained actively motile and were capable of fertilizing eggs. LS was totally ineffective in rendering the spermatozoa capable of undergoing the acrosome reaction, and in fact it inhibited the acrosome reaction by itself and also inhibited the LC-, LE-, or LI-mediated acrosome reaction. LS did not prevent acrosome-reacted spermatozoa from penetrating the zona pellucida, but did prevent sperm-egg fusion. Based on these findings, it is suggested that lysophospholipids are intricately involved in the sperm acrosome reaction and perhaps in sperm-egg fusion.     

High pH-induced acrosome reaction and Ca 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.     

Changes in guinea pig sperm intracellular sodium and potassium content during capacitation and treatment with monovalent ionophores

Guinea pig spermatozoa were collected from the caudae epididymides in various isotonic solutions and the intracellular sodium and potassium content was determined by atomic absorption spectroscopy. The sperm intracellular Na and K content was found to be influenced by large variations in the extracellular concentrations of these ions. Treatment of spermatozoa suspended in a saline-based solution with the monovalent ionophores monensin or nigericin caused an approximate 2-fold increase in the intracellular Na content and a 3–6 fold decrease in the intracellular K content. Incubation of the spermatozoa in a K⁺-free minimal culture medium (MCM-PL) at a pH of 7.6 or 8.3 for 2 hr caused an approximate 2-fold increase in the sperm intracellular Na content and a 5-fold decrease in the intracellular K content. The motile spermatozoa incubated for 2 hr at pH 7.6 showed less than 5% acrosome reactions, compared with 30–40% acrosome reactions after incubation at pH 8.3, in response to the addition of 5 mM Ca²⁺. Changes in the sperm intracellular elemental composition during culture in vitro, which may lead to an acrosome reaction, are discussed.     

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

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

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.     

A partial sequence of ionic changes associated with the acrosome reaction of Strongylocentrotus purpuratus

Relationships among several of the ion movements associated with the acrosome reaction of S. purpuratus were investigated. Egg jelly initiates ⁴⁵Ca²⁺ and ²²Na⁺ uptake, and K⁺ and H⁺ efflux. H⁺ efflux and ²²Na⁺ uptake occur with approximately equivalent stoichiometries as rapidly as the appearance of acrosomal rods, perhaps reflecting a linked process. Most K⁺ loss, as measured either by ⁴²K⁺ efflux or K⁺-ion-selective electrodes, occurs after the acrosome reaction is complete. Since an elevation of seawater K⁺ (from 10 to 15 mM) or the addition of 0.5 mM tetraethylammonium (TEA), an inhibitor of K⁺ channels, inhibits the acrosome reaction half-maximally, K⁺ movements or alterations of K⁺-dependent membrane potentials may regulate the triggering by jelly. Most, but not all, of the ⁴⁵Ca²⁺ influx is inhibited with a mixture of 10 μM FCCP, 1 mM CN^?, and 2 μg/ml oligomycin, suggesting that the mitochondria store most of the Ca²⁺. The extracellular Na⁺ concentration affects Ca²⁺ fluxes: sperm placed into 5 mM Na⁺ seawater have enhanced ⁴⁵Ca²⁺ uptake, but do not undergo the acrosome reaction, unless 30 mM Na⁺ is also added. Low Na⁺ concentrations lead to spontaneous triggering, by allowing for both Ca²⁺ influx and Na⁺-dependent H⁺ efflux. At least one early Ca²⁺ requirement precedes the Na⁺ and H⁺ movements, as inferred from attempts at reversing the inhibitors of jelly induction of the acrosome reaction. When sperm are incubated with jelly in the absence of Ca²⁺, then washed and incubated with jelly in the presence of Ca²⁺, the acrosome reaction is triggered only upon the second incubation. However, when sperm are mixed with jelly in the presence of the other inhibitors (verapamil, TEA, 5 mM Na⁺ seawater, low pH, or elevated K⁺), they are altered so that even upon subsequent washing, jelly-mediated triggering is no longer possible. This suggests the existence of an intermediate state in the reaction pathway, that follows an event for which Ca²⁺ is required, but that precedes the Na⁺ and H⁺ movements, which are inhibited by all inhibitors of the acrosome reaction. These data are used to develop a partial sequence of ionic changes associated with the triggering mechanism.     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.     

Role for Ca2+ channels in the signal transduction pathway leading to acrosomal exocytosis in human spermatozoa

Progesterone interaction with human spermatozoa promotes a rise in intracellular Ca²⁺ and can trigger acrosomal exocytosis in capacitated cells. We have used nifedipine, a 1,4-dihydropyridine Ca²⁺ channel antagonist, to investigate the possibility that Ca²⁺ channels play a role in the progesterone-stimulated exocytotic response. Cells were assessed biochemically for the generation of diacylglycerol (DAG) and microscopically for acrosome loss using chlortetracycline fluorescence. When motile cells were preincubated for 5 hr using culture conditions similar to those used for successful human in vitro fertilization, a short exposure to progesterone significantly stimulated DAG formation and acrosomal exocytosis. The addition of nifedipine (10 and 100 nM), either at time 0 or just prior to progesterone introduction, significantly inhibited both DAG formation and exocytosis, suggesting that Ca²⁺ channels are involved in the responses observed. Treatment of capacitated cells with a synthetic permeant DAG stimulated exocytosis irrespective of whether nifedipine was present, indicating that Ca²⁺ channels function prior to DAG generation. The possibility that an influx of Na⁺, as well as Ca²⁺, might be involved in the exocytotic pathway was investigated using the monovalent cation ionophores monensin and nigericin. Both significantly stimulated DAG generation and acrosome loss, but the prior inclusion of nifedipine significantly inhibited all responses. These results strongly suggest that the entry of Ca²⁺ through Ca²⁺ channels, with characteristics similar to those of L-type, voltage-sensitive Ca²⁺ channels found in cardiac and skeletal muscle, is a crucial step in the sequence of events leading to exocytosis in progesterone-stimulated human spermatozoa. An influx of Na⁺ also may play a role, but at a point prior to the opening of Ca²⁺ channels. © 1996 Wiley-Liss, Inc.     

Regulation of carrier-mediated and exocytotic release of [3H]GABA in rat brain synaptosomes

In this study we investigated the role of external monovalent cations, and of intracellular Ca²⁺ concentration ([Ca²⁺]i) in polarized and depolarized rat cerebral cortex synaptosomes on the release of [³H]--aminobutyric acid (³H-GABA). We found that potassium-depolarization, in the absence of Ca²⁺, of synaptosomes loaded with³H-GABA releases 7.4±2.1% of the accumulated neurotransmitter, provided that the external medium contains Na⁺, and an additional 19.0±2.5% is released upon adding 1.0 mM CaCl₂ to the exterior. The Ca²⁺-independent release component does not occur in a choline medium and it is only 3.4±0.8% of the³H-GABA accumulated in a Li⁺ medium, but both ions support the Ca²⁺-dependent release of³H-GABA (13.4±0.6% in choline and 15.4±1.5% in Li⁺), which suggests that the exocytotic release is independent of the external monovalent cation present, whereas the carrier-mediated release specifically requires Na⁺ outside. Furthermore, previous release of the cytosolic³H-GABA due to predepolarization in the absence of Ca²⁺ does not influence the amount of³H-GABA subsequently released by exocytosis due to Ca²⁺ addition (19.1±2.5% or 19.1±1.1%, respectively). In choline or Li⁺ medium, the value of the [Ca²⁺]i is raised by Na⁺/Ca²⁺ exchange to 663±75 nM or 782±54 nM, respectively, within three minutes after adding 1.0 mM Ca²⁺, in the absence of depolarization, and parallel release experiments show no release of³H-GABA in the choline medium, but a substantial release (7.1±2.1%) of³H-GABA occurs in the Li⁺ medium without depolarization. Subsequent K⁺-depolarization shows normal Ca²⁺-dependent release of³H-GABA in the choline medium (14.1±2.0%) but only 8.6±1.1% release in the Li⁺ medium, which suggests that raising the [Ca²⁺]i by Na⁺/Ca²⁺ exchange, without depolarization, supports some exocytotic release in Li⁺, but not in choline media. The role of [Ca²⁺]i and of membrane depolarization in the release process is discussed on the basis of the results obtained and other relevant observations which suggest that both Ca²⁺ and depolarization are essential for optimal exocytotic release of GABA.Special issue dedicated to Dr. Santiago Grisolia.     

Fluxes of Ca2+, Sr2+ and Mg2+ in synaptosomes.

Synaptosomes isolated from sheep brain cortex accumulate Ca²⁺, Sr²⁺ and Mg²⁺ when incubated in isosmotic sucrose media containing 5 mM of either of these cations. The maximal levels of cations retained per mg of protein are 100 nmol of Ca²⁺, 85 nmol of Mg²⁺ and 80 nmol of Sr²⁺. The loss of Ca²⁺ or Sr²⁺ from the preloaded synaptosomes is increased by monovalent cations in the following order: Na⁺> K⁺ > Li⁺> choline, whereas for the loss of Mg²⁺ this order is different: K⁺ > Na⁺ > Li ～ choline. The efflux of Ca²⁺ or Sr²⁺ induced by monovalent cations decreases as the temperature is lowered and it is nearly abolished at 0°C, whereas the efflux of Mg²⁺ is much less influenced by temperature. The results suggest that the mechanism of exchange of Ca²⁺ for Na⁺ in synaptosomes operates similarly for Sr²⁺, but not for Mg²⁺.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+-transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca²⁺ + Mg²⁺)-ATPase activity and in Ca²⁺ efflux at 37°C, depending on the side of the membrane at which the monovalent cations K⁺ and Na⁺ were placed. Under the conditions used, (Ca²⁺ + Mg²⁺)-ATPase activity and Ca²⁺ efflux was highest when K⁺ ($\text{35 ± 0.5}\text{mM (± S.E.)}$, mean of four experiments) was at the inside and Na⁺ (130 mM) at the outside of the ghost membrane.     

Monovalent ion and calcium ion fluxes in sarcoplasmic reticulum

Summary The ion permeability of sarcoplasmic reticulum vesicles from skeletal and heart muscle has been characterized by radioisotope flux, osmotic and membrane potential measurements, and by incorporating vesicles into planar phospholipid bilayers. The sarcoplasmic reticulum membrane is uniquely permeable to various biologically relevant monovalent ions. At least two and possibly three separate passive permeation systems for monovalent ions have been identified: 1) a K⁺, Na⁺ channel, 2) an anion channel, and 3) a H⁺ (OH^–) permeable pathway which may or may not be synonymous with the anion channel. A possible physiological function of these monovalent ion permeation systems is to permit rapid movement of K⁺, Na⁺, H⁺ and Cl across the membrane to counter electrogenic Ca²⁺ fluxes during Ca²⁺ release and uptake by sacroplasmic reticulum.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca²⁺ channels are important structural determinants for the passage of Ca²⁺ across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a_1S subunit of the skeletal L-type channel (Ca_v1.1) to lysine virtually eliminates passage of Ca²⁺ during step depolarizations. In this study, we examined the ability of this mutant Ca_v1.1 channel (SkEIIIK) to conduct inward Na⁺ current. When 150 mM Na⁺ was present as the sole monovalent cation in the bath solution, dysgenic (Ca_v1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na⁺. Ca²⁺ block of SkEIIIK-mediated Na⁺ current was revealed by the substantial enhancement of Na⁺ current amplitude after reduction of Ca²⁺ in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na⁺ currents through the mutant Ca_v1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na⁺ channel when Na⁺ is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca²⁺ permeability mediated by Ca_V channels on physiological processes that extend beyond channel gating and permeability.     

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.     

Regulation of spermatozoa motility in response to cations in Russian sturgeon Acipenser gueldenstaedtii

The aim of this study was to investigate the response of Russian sturgeon (Acipenser gueldenstaedtii) sperm to external cations (Na⁺, K⁺, Ca²⁺, and Mg²⁺) and their susceptibility on the induction of motility and swimming behavior. An in vitro spermatozoa motility assay was used by a computer-aided Motion-Analysis system. Sperm motility was inhibited by 60 mm NaCl (∼140 mOsm/kg) and 0.7 mm KCl solutions (∼ 21.4 mOsm/kg). The Ca²⁺ and Mg²⁺ ions were not able to inhibit spermatozoa motility. By contrast, Na⁺ within a limited concentration range (between 45 and 55 mm) was able to reverse the inhibitory effect of K⁺ at the critical concentration (0.7 mm). Ca²⁺ and Mg²⁺ were also able to reverse the K⁺-mediated spermatozoa motility restriction at concentrations starting at 0.01 and 0.1 mm, respectively. These results provide evidence for the role of K⁺ in suppressing spermatozoa motility, and suggest that Ca²⁺, Mg²⁺, and possibly Na⁺ trigger motility in Russian sturgeon sperm.     

Importance of sodium ion to the progesterone-initiated acrosome reaction in human sperm

Progesterone (P) has previously been shown to rapidly increase free intracellular calcium concentration ([Ca²⁻]_i), and subsequently to initiate the acrosome reaction (AR) in capacitated human sperm. The present study used cytochemical analysis of the AR, and spectrofluorometric determination of sperm [Ca²⁻]_i and intracellular pH (pH_i) in Na⁺-containing and Na⁺-deficient bicarbonate/CO₂-buffered media to investigate the role of Na⁺ in these P-initiated changes. We found that P failed to initiate the AR in Na⁺-deficient medium, and that the initial rise in [Ca²⁺]_i following P (1 μg/ml) stimulation was similar for both media; however, the [Ca²⁺]_i in the Na⁺-deficient medium regressed more rapidly and plateaued at a significantly lower [Ca²⁺]_i. Moreover, the differences in plateau [Ca²⁺]_i were directly related to the percentage of acrosome reactions, suggesting that the plateau phase is not due to [Ca²⁺]_i, but rather to the release of intracellular fura-2 into the medium during the AR. These [Ca²⁺]_i and AR results are in contrast to those reported previously by others for human sperm and suggest that a Na⁺-dependent mechanism is important in the P-initiated human sperm AR. Such a Na⁺ requirement may reflect the involvement of this ion in pH_i regulation, as capacitated sperm that were incubated in a Na⁺-deficient medium for ≥ 30 min displayed a significantly lower pH_i. © 1996 Wiley-Liss, Inc.     

Interaction of Na+ and Mg2+ with Ca2+ in pancreatic islets as visualized by chlorotetracycline fluorescence

The fluorescence of microdissected pancreatic islets of ob/ob-mice was studied by microscope photometry after incubation with 10 μM chlorotetracycline. In Krebs-Ringer bicarbonate buffer, excitation at 390 nm yielded peak emission at 530 nm, suggesting that chelated Ca²⁺ was the major source of fluorescence. In support of this interpretation, incubation in Ca²⁺-free buffer markedly decreased the fluorescence, whereas withdrawal of Mg²⁺ increased it. Raising the Mg²⁺ concentration to 15 mM suppressed the fluorescence. In the presence of Ca²⁺, the substitution of choline ions for Na⁺ increased the fluorescence considerably; in the absence of Ca²⁺, however, Na⁺ deficiency had only little effect. Control experiments showed that Na⁺ or choline ions had no effect on the fluorescence of Ca²⁺-chlorotetracycline in 70 or 90% methanol. In 90%, but not in 70%, methanol 15 mM Mg²⁺ slightly quenched the fluorescence from 2.5 mM Ca²⁺ and 10 μM chlorotetracycline. It is suggested that Na⁺, and perhaps Mg²⁺, tends to decrease the amount of membrane-bound Ca²⁺ in the pancreatic islets.