Glucose induces a Na(+),K(+)-ATPase-dependent transient hyperpolarization in human sperm. I. Induction of changes in plasma membrane potential by the proton ionophore CCCP

When human sperm was incubated in medium deprived of glucose, glucose restoration caused a transient hyperpolarization of the plasma membrane. This hyperpolarization was also induced by fructose but not by 2-deoxyglucose, a substrate that cannot be metabolized. The hyperpolarization was inhibited by NaF, a glycolysis inhibitor, but not by mitochondrial inhibitors (cyanide, rotenone and antimycin), suggesting that it depended on glycolysis. Furthermore, the hyperpolarization was still induced in medium containing a high concentration of KCl and was insensitive to the K(+) channel blocker TEA and the Cl(-) channel blocker niflumic acid, but it was blocked by ouabain. This suggested that upon glucose addition, there was an increase in the concentration of ATP, that in turns increased the Na(+),K(+)-ATPase activity. Since this pump is electrogenic (2K(+)/3Na(+)) the plasma membrane hyperpolarized. On the other hand, CCCP, a proton ionophore, inhibited the hyperpolarization induced by glucose. When CCCP was added to glucose-treated hyperpolarized sperm, it caused a depolarization that triggered a Ca(2+) influx sensitive to nickel, an inhibitor of voltage-dependent calcium channels. Moreover, CCCP caused hyperpolarization in sperm incubated in medium without calcium, a known condition that depolarizes sperm. This indicated that CCCP induced proton permeability in the plasma membrane that was able to change the membrane potential to a value corresponding to the E(H) and that was also able to clamp it, so that it prevented the hyperpolarization induced by glucose.     

Anion channels in the sea urchin sperm plasma membrane

Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent-solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion-selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO₃^? > CNS^? > Br^? > CI^?. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4′-diisothiocyano-2,2′ -stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS. © 1993 Wiley-Liss, Inc.     

Ion channels in boar sperm plasma membranes: characterization of a cation selective channel.

Plasma membranes isolated from cauda epididymal and ejaculated boar sperm were inserted into planar lipid bilayers and examined for the presence of ion channels. Channel fusion was frequently observed; the most prominent was a nonselective cation channel which conducted K, Na, Cs, Ca, and Ba. Channel opening did not show a strict dependence on voltage but was partially blocked by verapamil, nitrendipine, and ruthenium red. A channel with these characteristics was observed when plasma membranes were isolated by high-pressure nitrogen cavitation (650 psi, 78% sperm head plasma membranes) or at very low nitrogen pressures (50 psi, 90% sperm head plasma membranes), suggesting that this channel may be present in the plasma membrane overlying the sperm head.     

Activation of sperm motility in striped bass via a cAMP-independent pathway

The objective of the present study was to identify the effect of osmolality, ions (K+, H+, Ca2+, Mg2+) and cAMP on the initiation of sperm motility in striped bass (Morone saxatilis). Striped bass spermatozoa remained motile in solutions isotonic to seminal plasma (350 mOsm/kg) until osmolality reached 600 mOsm/kg. K+ (0-100 mM) had no effect ( p>0.05 ) on sperm motility, and sperm displayed a high percentage of motility over a wide range of pH (6.0-8.5). Sperm motility could be initiated in Ca2+-free solutions. In contrast, sperm motility was inhibited (P<0.01) by solutions containing > or =10 mM Ca2+, and sperm could not be reactivated by a Ca2+-free solution. This Ca2+ inhibition was not affected by verapamil, a Ca2+ channel blocker. However, if sperm motility was first initiated in a Ca2+-free solution, the addition of Ca2+ solutions, up to 80 mM, failed to inhibit sperm motility, suggesting that Ca2+ inhibited the initiation of motility, but had no control of motile spermatozoa. Mg2+ solutions had similar inhibitory effects on sperm motility as Ca2+ solutions. Therefore, initiation of motility in striped bass sperm may be related to voltage-gated channels across the cell's plasma membrane. Membrane permeable cAMP did not initiate motility of quiescent, intact striped bass spermatozoa, and motility of demembranated sperm could be activated in the absence of cAMP.     

Regional differentiation of the sea urchin sperm plasma membrane

In order to study the molecular basis for the functional localization and behavioral control of sperm, we have partially characterized plasma membranes prepared from isolated head and tail fractions. These membranes have similar amounts of the Na+ pump (as reflected by (Na+,K+)-ATPase activity), whereas they differ in protein composition, binding sites for Ca2+ channel antagonists, and in the localization of enzymes of cyclic nucleotide metabolism. The Ca2+ channel antagonist D600 (and related phenylalkylamines) binds to plasma membrane preparations from sperm heads and tails with much higher affinity than do the dihydropyridine antagonists. This binding is inhibited greatly by certain monovalent (but not divalent) ions, especially Na+, Tris+, glycine ethyl ester+, and methylamine+.K+,Li+, and choline+ are less effective. In media of ionic composition resembling seawater, sperm tail membranes exhibit 6.5-fold more binding sites for D600 than do membranes from sperm head. cGMP phosphodiesterase and adenylate cyclase are also enriched in plasma membranes from the tail. Thus, the highly polarized sperm cell exhibits a regional differentiation of plasma membrane proteins implicated in behavioral control.     

Estradiol inhibits the effects of extracellular ATP in human sperm by a non genomic mechanism of action

Steroid hormones, beside their classical genomic mechanism of action, exert rapid, non genomic effects in different cell types. These effects are mediated by still poorly characterized plasma membrane receptors that appear to be distinct from the classic intracellular receptors. In the present study we evaluated the non genomic effects of estradiol (17βE₂) in human sperm and its effects on sperm stimulation by extracellular ATP, a potent activator of sperm acrosome reaction. In human sperm 17βE₂ induced a rapid increase of intracellular calcium (Ca²⁺) concentrations dependent on an influx of Ca²⁺ from the extracellular medium. The monitoring of the plasma membrane potential variations induced by 17βE₂ showed that this steroid induces a rapid plasma membrane hyperpolarization that was dependent on the presence of Ca²⁺ in the extracellular medium since it was absent in Ca²⁺ free-medium. When sperm were pre-incubated in the presence of the K⁺ channel inhibitor tetra-ethylammonium, the 17βE₂ induced plasma membrane hyperpolarization was blunted suggesting the involvement of K⁺ channels in the hyperpolarizing effects of 17βE₂. Extracellular ATP induced a rapid plasma membrane depolarization followed by acrosome reaction. Sperm pre-incubation with 17βE₂ inhibited the effects of extracellular ATP on sperm plasma membrane potential variations and acrosome reaction. The effects of 17βE₂ were specific since its inactive steroisomer 17αE₂ was inactive. Furthermore the effects of 17βE₂ were not inhibited by tamoxifen, an antagonist of the classic 17βE₂ intracellular receptor.     

Roles for Potassium and Calcium Channels in the Initiation of Sperm Motility in Rainbow Trout.

It is well known that the motility of spermatozoa in rainbow trout is suppressed by K⁺. We showed here that although trout sperm are completely immotile in medium containing 5 mM K⁺, motility was initiated by the subsequent addition of several mM Ca²⁺, suggesting that both K⁺and Ca²⁺are related to the process of the initiation of sperm motility. It was further found that K⁺channel blockers tetraethylammonium, nonyltriethylammonium, Ba²⁺and Cs⁺, as well as the Ca²⁺channel blocker verapamil, inhibited the initiation of sperm motility at doses at which these reagents inhibit chnnel-related functions in other cells. However, Na⁺channel blocker, tetrodotoxin and anion channel blocker 4, 4-diisothiocyatatostilbene-2, 2'-disulfonic acid inhibited the motility only at extremely high doses. These results suggest that transport of K⁺and Ca²⁺through ion channels at the plasma membrane of spermatozoa is the first event that triggers the initiation of sperm motility in rainbow trout.     

Polycystin-2 associates with the polycystin-1 homolog, suREJ3, and localizes to the acrosomal region of sea urchin spermatozoa

Polycystin-2, the protein mutated in type 2 autosomal dominant polycystic kidney disease, is an integral transmembrane protein with nonselective cation channel activity. Here we report on the sea urchin sperm homolog of polycystin-2 (suPC2). Like other polycystin-2 family members, suPC2 is a six-pass transmembrane protein containing C-terminal cytoplasmic EF hand and coiled-coil domains. The protein localizes exclusively to the plasma membrane over the sperm acrosomal vesicle. This localization coincides with the previously reported localization of the sea urchin PC1 homolog, suREJ3. Co-immunoprecipitation shows that suPC2 and suREJ3 are associated in the membrane. The location of suPC2 suggests that it may function as a cation channel mediating the sperm acrosome reaction. The low cation selectivity of PC2 channels would explain data indicating that Na(+) and Ca(2+) may enter sea urchin sperm through the same channel during the acrosome reaction.     

The Sperm‐surface glycoprotein,SGP, is necessary for fertilization in the frog,Xenopus laevis

To identify a molecule involved in sperm‐egg plasma membrane binding at fertilization, a monoclonal antibody against a sperm‐surface glycoprotein (SGP) was obtained by immunizing mice with a sperm membrane fraction of the frog, Xenopus laevis, followed by screening of the culture supernatants based on their inhibitory activity against fertilization. The fertilization of both jellied and denuded eggs was effectively inhibited by pretreatment of sperm with intact anti‐SGP antibody as well as its Fab fragment, indicating that the antibody recognizes a molecule on the sperm's surface that is necessary for fertilization. On Western blots, the anti‐SGP antibody recognized large molecules, with molecular masses of 65–150 kDa and minor smaller molecules with masses of 20–28 kDa in the sperm membrane vesicles. SGP was distributed over nearly the entire surface of the sperm, probably as an integral membrane protein in close association with microfilaments. More membrane vesicles containing SGP bound to the surface were found in the animal hemisphere compared with the vegetal hemisphere in unfertilized eggs, but the vesicle‐binding was not observed in fertilized eggs. These results indicate that SGP mediates sperm‐egg membrane binding and is responsible for the establishment of fertilization in Xenopus.     

The enhancing effects of anion channel blockers on sperm activation by bicarbonate.

We found that anion channel blockers such as phosphotungstate and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) enhanced HCO3(-)-induced activation on porcine epididymal sperm. In the presence of these compounds, HCO3- increased the motility, respiration rate and especially the cAMP content of the sperm to a greater extent than did HCO3- alone. The enhancing effects were not observed in the absence of HCO3-, but were evident when the concentration of HCO3- was low. These compounds did not significantly alter the intracellular pH and did inhibit the adenylate cyclase activity of the sperm plasma membrane. When these compounds were added to sperm homogenate with ATP, the cAMP formed was reduced compared to the control. In addition, these compounds inhibited both the SO4(2-) influx and efflux of the sperm. From these results, we conclude that the anion channel blockers tested principally inhibit the efflux of endogenous HCO3- derived from metabolic CO2, so that HCO3- accumulates intracellularly and stimulates the adenylate cyclase of the sperm.     

Hyaluronic acid and the cumulus extracellular matrix induce increases in intracellular calcium in macaque sperm via the plasma membrane protein PH-20.

The hyaluronic acid (HA)-rich extracellular matrix (ECM) of the cumulus oophorus is known to facilitate fertilization. It has been suggested that HA may enhance fertilisation in a number of species, and in macaque sperm, HA has been shown to increase the number of acrosome reactions that follow sperm binding to the zona pellucida. In this study, we investigated the effects of HA on intracellular Ca2+ in capacitated cynomolgus macaque sperm. Fluorometry studies using the intracellular Ca2+ indicator Fluo-3 showed that addition of 100 micrograms/ml of HA induced a rapid increase in intracellular Ca2+. This Ca2+ increase (approximately 2-3 times above basal levels) was inhibited by preincubation of sperm with Fab fragments of anti-recombinant PH-20 IgG. The frequency of acrosome reactions in sperm exposed to HA was not above control levels. A synthetic gel was prepared with similar viscosity to the cumulus and with HA trapped in its matrix. Video imaging of individual sperm was used to demonstrate that capacitated sperm swimming into the HA gel had increased intracellular Ca2+ levels. Preincubation of sperm with Fab fragments of anti-PH-20 IgG inhibited the increased intracellular Ca2+ levels induced by the HA gel. Sperm in control gel (no HA) did not show increased intracellular Ca2+, while sperm in gel containing anti-PH-20 IgG showed increased Ca2+ (positive control). Sperm loaded with Fluo-3 were allowed to interact with cynomolgus macaque cumulus masses, and sperm within the cumulus ECM clearly showed increased intracellular Ca2+ that was inhibited when sperm were preincubated in anti-PH-20 Fab. Fluorescein isothiocyanate (FITC)-HA was found to bind to sperm over the acrosomal region, corresponding to PH-20 localisation, and this binding could be inhibited by preincubation of sperm with anti-PH-20 fragments. The results of this study show that HA increases intracellular Ca2+ in macaque sperm through interaction with plasma membrane PH-20. We propose that HA binding to plasma membrane PH-20 induces an aggregation of receptors that in turn results in intracellular signalling. As a result, sperm have higher basal CA2+ levels and are more responsive to induction of the acrosome reaction after binding to the zona pellucida.     

The retention of 125I-labeled plasma membrane proteins in bovine spermatozoa cryopreserved in egg-yolk citrate extender

Cryopreservation of bovine sperm in egg-yolk citrate extender (EYC) usually maintains fertility. Since plasma membrane proteins are important for the fertilizing potential of sperm, the possible loss of membrane proteins from sperm subjected to cryopreservation in EYC was evaluated. Sperm were washed and labeled with ¹²⁵I without significantly reducing motility. Radiolabeled sperm were a) held for 2 hr at 22°C in N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES)-buffered saline containing 1% polyvinyl alcohol, b) cooled to 5 °C in glycerol-free EYC and held for 3 hr, or c) frozen-thawed in EYC containing 7% glycerol. Sperm were solubilized and proteins were separated by electrophoresis under denaturing conditions. Freeze-thawing dislodged most egg-yolk proteins from the spermatozoal plasma membrane that were bound to and retained by sperm that only were cooled to 5 °C. Autoradiography resolved 11-18 bands of ¹²⁵I polypeptides. There was no difference (P > 0.05) in the amount of ¹²⁵I protein retained by frozenthawed and cooled sperm. However, the radioactivity in two polypeptide bands (MW = 105 K and 24.2 K) was less (P < 0.05) for sperm held at 22 °C in HEPES-buffered saline. Thus, holding sperm in buffered saline at 22 °C resulted in a greater loss of ¹²⁵I proteins from the plasma membrane than did cryopreservation of sperm in EYC. Cryopreservation did not induce greater loss of ¹²⁵I proteins from the plasma membrane than simply cooling sperm to 5 °C in EYC.     

Extragenomic actions of progesterone in human sperm and progesterone metabolites in human platelets.

Progesterone rapidly increased intracellular free calcium ([Ca2+]i) in human sperm, removal of extracellular Ca2+ prevented the increase in [Ca2+]i. The Ca2+ influx was not blocked by the T-type Ca2+ channel blocker mibefradil. However T-type calcium channels do appear to be present in human sperm because the neoglycoprotein mannose-albumin, an inducer of the acrosome reaction, was able to promote Ca2+ influx, which was blocked by mibefradil and more potently inhibited by Ni2+ than Cd2+. The receptor for progesterone that promotes the Ca2+ influx was located on the plasma membrane using FITC-progesterone-albumin. It is concluded that progesterone stimulates Ca2+ influx in human sperm via a unique Ca2+ channel possibly similar to a store-operated channel (SOC) or a receptor-operated channel (ROC). We have found that progesterone metabolites, such as pregnanolone and pregnanediol, promote a rapid rise in [Ca2+]i and aggregation in human platelets, similar to that observed with thrombin. The increase in [Ca2+]i was prevented when extracellular Ca2+ was removed or by the SOC inhibitor SKF-96365. The phospholipase C inhibitor U-73122 also prevented the increase in [Ca2+]i, suggesting that these metabolites interact with a cell surface receptor on the platelet to activate phospholipase C to produce inositol-P3, which mobilizes intracellular Ca2+, thereby activating the SOC in the plasma membrane. Progesterone and estradiol conjugated to albumin, also produced a rapid increase in [Ca2+]i, which was prevented by Ca2+ removal from the medium or when SKF-96365 or U-73122 were added. It is proposed that human platelets possess cell surface receptors for steroids.     

Isolation and partial purification of plasma membrane from porcine oocytes

Egg plasma membrane (EPM) was isolated in comparatively large amounts from porcine slaughterhouse ovaries. Ovaries were minced, and the oocyte containing fluid was filtered to retrieve zona pellucidae–intact oocytes. The oocytes were homogenized and filtered again to remove zona pellucidae. The egg filtrate was subjected to differential centrifugation to remove membrane bound organelles and the remaining plasma membrane containing material was pelleted by ultracentrifugation. Plasma membranes were further separated from cellular material by sucrose density gradient centrifugation and were collected from portions of the gradient that correspond to the densities of plasma membrane. The purity of isolated plasma membranes was assessed by membrane marker enzyme analysis and transmission electron microscopy. Activities of the plasma membrane marker enzymes 5' nucleotidase and alkaline phosphatase increased from nondetectable levels in the egg filtrate to relatively high levels in the plasma membrane preparation. Marker enzymes for mitochondrial and lysosomal membranes fell from detectable levels in the egg filtrate to levels that were at the lower limits of the assays to detect in the final preparation. Evidence provided by binding of biotin-labeled EPM to capacitated sperm suggests that the isolated EPM retains its biological activity. The procedure presented here represents a novel method of isolating procine egg plasma membranes for further study involving sperm–egg interaction. © 1994 Wiley-Liss, Inc.     

Analysis of a sperm surface molecule that binds to a vitelline envelope component of Xenopus laevis eggs

To analyze sperm surface molecules involved in sperm–egg envelope binding in Xenopus laevis, heat‐solubilized vitelline envelope (VE) dot blotted onto a polyvinylidene difluoride (PVDF) sheet was incubated with a detergent extract of sperm plasma membrane (SP‐ML). The membrane components bound to the VE were detected using an antibody library against sperm plasma membrane components, and a hybridoma clone producing a monoclonal antibody (mAb) 16A2A7 was identified. This mAb was used in a Far Western blotting experiment in which VE was separated by electrophoresis, and then transferred to a PVDF strip that was incubated with SP‐ML. It was found that SP‐ML binds to the VE component gp37 (Xenopus homolog of mammalian ZP1). The antigens reactive to mAb 16A2A7 showed apparent molecular weights of 65–130 and 20–30 kDa, and were distributed relatively evenly over the entire sperm surface. Periodate oxidation revealed that both the pertinent epitope on the sperm surface and the ligands of VE gp37 were sugar moieties. VE gp37 was exposed on the VE surface, and the mAb 16A2A7 dose‐dependently inhibited sperm binding to VE. The sperm membrane molecules reactive with mAb 16A2A7 also reacted with mAb 2A3D9, which is known to recognize the glycoprotein SGP in the sperm plasma membrane and is involved in interactions with the egg plasma membrane, indicating that the sperm membrane glycoprotein has a bifunctional role in Xenopus fertilization. Mol. Reprod. Dev. 77: 728–735, 2010. © 2010 Wiley‐Liss, Inc.     

THE FUCUS (PHAEOPHYCEAE) SPERM RECEPTOR FOR EGGS. I. DEVELOPMENT AND CHARACTERISTICS OF A BINDING ASSAY1

To explore the molecular basis of egg-sperm recognition in the brown alga , Fucus serratus L., we developed an in vitro binding assay involving egg plasma membrane vesicles (PMVs) and proteins contained in a KCl extract of sperm. Binding between the two components was measured using biotinylated PMVs followed by the addition of streptavidin conjugated to alkaline phosphatase and the appropriate substrate. Biotinylation did not affect the ability of egg PMVs to inhibit fertilization in a species-preferential manner. Binding of labeled egg PMVs to the sperm KCl extract was saturable and competable with unlabeled PMVs but was not species-specific. Protease treatment of the KCl extract abolished binding, whereas periodate had no effect, suggesting that sperm protein rather than carbohydrate was involved. Preincubation of the sperm extract with sulfated polysaccharides (e.g. fucoidan and ascophyllan) inhibited binding of egg plasma membranes. Sulfation seems to be important for this effect since desulfated fucoidan was far less effective at blocking binding. Polysaccharides which inhibited binding also inhibited fertilization. Overall, the results indicate that at least some aspects of binding between Fucus sperm and eggs are mediated by a protein(s) derived from sperm which recognizes sulfated glycoconjugates on the egg plasma membrane .     

Effect of verapamil and sulfhydryl reagents on calcium transport in bovine spermatozoa

Calcium uptake by intact bovine epididymal spermatozoa is not affected by low concentrations (up to 0.75 mM) of the calcium transport blocker verapamil. Under these conditions, calcium transport into sperm mitochondria is highly inhibited. At higher verapamil concentrations (1.0, 1.5 mM), calcium transport into intact sperm is also inhibited, and this inhibition cannot be relieved by disrupting the plasma membrane with filipin. Calcium uptake into intact sperm is highly inhibited by mersalyl and this inhibitory effect can be completely relieved when the plasma membrane is disrupted by filipin. This effect of mersalyl is not dependent on the presence of phosphate in the incubation medium. Phosphate itself, up to 2 mM, enhances calcium uptake into the cells; this effect decreases at higher concentrations and is depressed 57% at 10 mM phosphate. This inhibitory effect of high phosphate concentration can be blocked by mersalyl. It is suggested that the calcium carrier itself and not a phosphate carrier of the plasma membrane is inhibited by mersalyl. It is possible that there is a symporter for calcium and phosphate in the plasma membrane of bovine spermatozoa.     

Identification of homologous binding proteins in porcine and bovine gametes

The purpose of this study was to determine if sperm and oocyte proteins that mediate plasma membrane interaction during mammalian fertilization are conserved among porcine and bovine gametes. We examined homologous and heterologous sperm and zona-free oocyte interactions to determine the extent of cross-reactivity between the gametes of these two ungulate species. First, the numbers of ejaculated porcine and bovine sperm bound to the oocyte plasma membrane of intact porcine and bovine oocytes were determined in vitro. There was no significant difference between the number of porcine or bovine sperm that bound to porcine or bovine oocytes (P > 0.25). Second, individual porcine and bovine sperm plasma membrane proteins were identified by binding of homologous or heterologous oocyte plasma membrane to whole sperm plasma membrane on Western ligand blots. The relative amount of labeled oocyte plasma membrane bound to individual sperm plasma membrane proteins was analyzed by laser densitometry. Eight porcine sperm plasma membrane proteins and seven bovine sperm plasma membrane proteins were bound by both porcine and bovine oocyte plasma membrane. A significantly greater relative amount of porcine oocyte plasma membrane than bovine oocyte plasma membrane was bound to the 14- and 10-kD porcine sperm plasma membrane proteins (P < 0.001 and P < 0.01, respectively). A 27-kD bovine sperm plasma membrane protein bound proportionally more bovine oocyte plasma membrane probe than porcine oocyte plasma membrane probe (P < 0.04). These results are consistent with conservation of similar receptor ligand interactions at the gamete plasma membrane among porcine and bovine gametes.     

Importance of mammalian sperm metalloendoprotease activity during the acrosome reaction to subsequent sperm-egg fusion: inhibitor studies with human sperm and zona-free hamster eggs.

We have previously shown that each of the metalloendoprotease (MEP) inhibitors phosphoramidon, diethylenetriaminepentaacetic acid, and carbobenzoxy-L-phenylalanine, when present only during the human sperm acrosome reaction (AR), will not inhibit the AR or sperm motility but will decrease the number of sperm that penetrate zona-free hamster eggs. The present study was designed to investigate whether this inhibition of penetration is due to an effect on sperm binding to the egg plasma membrane and/or to an effect on the actual membrane fusion event. In these studies we used ionomycin to initiate the AR and assayed binding in a Ca(2+)-free medium and fusion in Ca(2+)-containing medium in the same experiment. Eggs were loaded with the fluorescent dye Hoechst 33342, and the appearance of fluorescence in a sperm head indicated that fusion had occurred. The three MEP inhibitors reduced binding only slightly but inhibited the actual fusion step by 50-60% (determined with an equation that corrected for any inhibition of fusion due to inhibition of binding). MEP inhibitors present only during gamete interactions had little or no effect on fusion. We also found that phosphoramidon-inhibitable MEP activity was released during the ionomycin-initiated AR. Incubation of AR supernatant containing MEP activity with previously acrosome-reacted, phosphoramidon-treated sperm resulted in a large reversal of the phosphoramidon-inhibitory effect on sperm-egg fusion. These results support the hypothesis that the acrosomal phosphoramidon-inhibitable MEP released during the AR acts directly or indirectly during that event to increase the fusibility of the sperm plasma membrane region required for subsequent sperm-egg fusion.