Isolation and partial characterization of the plasma membrane of the sea urchin egg

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.     

Diacylglycerol induces fusion of nuclear envelope membrane precursor vesicles

Purified membrane vesicles isolated from sea urchin eggs form nuclear envelopes around sperm nuclei following GTP hydrolysis in the presence of cytosol. A low density subfraction of these vesicles (MV1), highly enriched in phosphatidylinositol (PtdIns), is required for nuclear envelope formation. Membrane fusion of MV1 with a second fraction that contributes most of the nuclear envelope can be initiated without GTP by an exogenous bacterial PtdIns-specific phospholipase C (PI-PLC) which hydrolyzes PtdIns to form diacylglycerides and inositol 1-phosphate. This PI-PLC hydrolyzes a subset of sea urchin membrane vesicle PtdIns into diglycerides enriched in long chain, polyunsaturated species as revealed by a novel liquid chromatography-mass spectrometry analysis. Large unilammelar vesicles (LUVs) enriched in PtdIns can substitute for MV1 in PI-PLC induced nuclear envelope formation. Moreover, MV1 prehydrolyzed with PI-PLC and washed to remove inositols leads to spontaneous nuclear envelope formation with MV2 without further PI-PLC treatment. LUVs enriched in diacylglycerol mimic prehydrolyzed MV1. These results indicate that production of membrane-destabilizing diglycerides in membranes enriched in PtdIns may facilitate membrane fusion in a natural membrane system and suggest that MV1, which binds only to two places on the sperm nucleus, may initiate fusion locally.     

Interaction of the sperm adhesive protein, bindin, with phospholipid vesicles. I. Specific association of bindin with gel-phase phospholipid vesicles

Bindin is a 30,000-mol-wt protein of sea urchin sperm that is responsible for the specific adhesion of the sperm acrosomal process to the vitelline layer covering the egg plasma membrane during fertilization. Sulfated glycoconjugates are believed to be the egg surface receptors for bindin, but the mechanism by which bindin associates with the sperm acrosomal membrane is unknown. Here I report that bindin specifically associates with phospholipid vesicles in vitro. Interaction of the bindin polypeptide with liposomes was found to cause an increase in the density of the liposomes and induce the aggregation of the vesicles. A novel property of this association of bindin with membranes was that it required phospholipids in a gel phase. The interaction of bindin with liposomes was greatly reduced at temperatures above the phase transition temperature. The interaction of bindin with gel-phase vesicles appeared to be reversible, since the aggregated vesicles dissaggregated as the temperature was raised above the phase transition temperature. Association of bindin with the bilayer did not alter the accessibility of the polypeptide to cleavage by trypsin, which suggests that most of the polypeptide chain remains exposed at the surface of the membrane.     

Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein

We have identified an integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD that cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) which contributes the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. We suggest that p56 is a sea urchin LBR homologue that targets membranes to chromatin and later anchors the membrane to the lamina.     

Response of isolated sperm plasma membranes from sea urchin to egg jelly

The acrosome reaction in sea urchin sperm is induced by a glycoprotein jelly surrounding the egg and is accompanied by changes in ion permeability of sperm plasma membrane. In an attempt to learn what membrane components are involved in the response to jelly, we have begun to reassemble sperm membrane components into artificial membranes and assay for permeability changes mimicking those that occur in sperm. Jelly in sea water at concentrations that induce the acrosome reaction did not significantly change 45Ca2+ uptake of sonicated unilamellar vesicles made with soybean lipid only (ratio jelly:control uptake = 1.08 +/- 0.36 SD, n = 21). Experiments with pure lipid planar bilayers made with soybean lipid or a lipid extract from sperm and held at various voltages, also did not reveal substantial permeability changes at comparable jelly concentrations. Thus, jelly by itself does not change the conductance of a pure lipid bilayer. In contrast, significant (P----0.0005, t test for two sample means) 45Ca2+ uptake was observed with vesicles made by cosonicating soybean phospholipids and Strongylocentrotus purpuratus sperm membranes isolated by the method of Cross, N. L. [1983, J. Cell Sci. 59, 13-25] (ratio jelly: control uptake = 1.51 +/- 0.75, n = 20, 16 positive out of 20 experiments). The calcium uptake response of the mixed vesicles was also species-specific: it did not occur with jelly from Arbacia punctulata (ratio Arbacia jelly: control = 1.18 +/- 0.51; ratio Strongylocentrotus jelly: control = 1.71 +/- 0.97, n = 10; P----0.025, paired t statistic). Vesicles made with soybean lipid and an octyl glucoside extract of sperm membranes also responded to jelly with increased 45Ca2+ uptake. Our results indicate that we have the starting conditions to isolate and characterize the sperm membrane components that participate in the egg jelly induced permeability changes.     

Single-channel activity of bilayers derived from sea urchin sperm plasma membranes at the tip of a patch-clamp electrode

Changes in the plasma membrane permeability of echinoderm sperm play a fundamental role in the acrosome reaction. During the reaction there is an increase in intracellular Ca2+ and Na+ and an efflux of H+ and K+. We have formed bilayers at the tip of patch pipets from a mixture of lipid vesicles and sea urchin sperm plasma membranes (12-50 microgram protein/ml). We observed three types of K+ channels (conductances: 22, 46, and 82 pS), two of which are partially blocked by TEA, and one Cl- channel (148 pS). The presence of K+ channels in sperm plasma membranes is consistent with the inhibition by TEA of the acrosome reaction in whole sperm and the membrane potential change that occurs during the reaction.     

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.     

Multiple GTP-binding proteins in sea urchin sperm: Evidence for Gs and small G-proteins

Sea urchin sperm plasma membranes isolated from heads and flagella were used to examine the presence of Gs (stimulatory guanine nucleotide-binding regulatory protein) and small G-proteins. Flagellar plasma membranes incubated with [³²P]NAD and cholera toxin (CTX) displayed radiolabeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX-catalyzed [³²P]ADP-ribosylation in conjunction with immunoprecipitation with anti-Gs, followed by electrophoresis and autoradiography, revealed one band of 48 kDa. Head plasma membranes, in contrast, did not show substrates for ADP-ribosylation by CTX. In flagellar and head plasma membranes pertussis toxin (PTX) ADP-ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP-ribosylation by PTX was higher in flagellar than in head membranes. Small G-proteins were investigated by [³²P]GTP-blotting. Both head and flagellar plasma membranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [α-³²P]GTP-binding in a concentration-dependent manner. A monoclonal antibody against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G-proteins, including Ras . Gs, Gi and Ras are enriched in flagellar membranes while the other small G-proteins do not display a preferential distribution along the sea urchin sperm plasma membrane. The role of these G-proteins in sea urchin sperm is presently under investigation.     

Rearrangements of sea urchin egg cytoplasmic membrane domains at fertilization

Fertilization in the sea urchin is accompanied by rapid reorganization of the egg endoplasmic reticulum (ER). ER-derived vesicles contribute to one of three classes of membranes used in assembling the male pronuclear envelope in vitro. We provide here biochemical evidence for the rearrangement of sea urchin egg cytoplasmic membrane domains at fertilization up to the first mitosis, with respect to two nuclear envelope markers, lamin B and lamin B receptor (LBR), using purified vesicles prepared from homogenates fractionated by floatation on sucrose gradients. In unfertilized eggs, immunoprecipitation data indicate that most of lamin B and LBR are localized in the same vesicles but do not interact. By 3 min post-fertilization, both proteins are more widely distributed across the gradients and by 12 min most of lamin B and LBR are localized in vesicles of different densities. This partitioning is maintained throughout S phase. At mitosis, most lamin B and LBR remain in distinct vesicles, while a small proportion of lamin B and LBR, likely derived from the disassembled nuclear envelope, associate in a minor subset of vesicles. The results illustrate a dynamic reorganization of egg cytoplasmic membranes at fertilization, and the establishment of distinct membrane domains enriched in specific nuclear envelope markers during the first cell cycle of sea urchin development. Additionally, we demonstrate that male pro-nuclear membrane assembly occurs only when both cytosol and membranes originate from fertilized but not unfertilized eggs, suggesting that fertilization-induced membrane rearrangements contribute to the ability of the egg to assemble the male pronuclear envelope.     

Sea urchin sperm cation-selective channels directly modulated by cAMP

Components of the sea urchin outer egg jelly layer such as speract drastically change second messenger levels and membrane permeability in sperm. Ion channels are deeply involved in the sperm-egg dialogue in sea urchin and other species. Yet, due to the small size of sperm, studies of ion channels and their modulation by second messengers in sperm are scarce. In this report we offer the first direct evidence that cation-selective channels upwardly regulated by cAMP operate in sea urchin sperm. Due to their poor selectivity among monovalent cations, channel activation in seawater could contribute to sperm membrane repolarization during the speract response.     

Morphology of gametes in sea urchins from Peter The Great Bay,Sea of Japan

The fine structure of the gametes in six sea urchin species of the Sea of Japan was studied. The sperm in Strongylocentrotus nudus, S. intermedius, Echinocardium cordatum, Scaphechinus mirabilis, Sc. griseus and Echinarachnius parma are species-specific. The conical head and symmetrically disposed ring-shape mitochondrion are common to regular sea urchin sperm cells. S. nudus is characterized by the bulb-shaped head of the sperm; S. intermedius, by a bullet-shaped one. The sperm spearhead and small amount of post-acrosome material are common to irregular sea urchins; the sperm width: length ratio varies for different species, with the highest for Sc. mirabilis. The sperm of Sc. griseus is characterized by two lipid drops in the middle part of sperm. Asymmetrical mitochondrion disposal is usual for E. parma. Actin filaments are found in the postacrosome material in the sperm of heart-shaped sea urchins. The differences in the fine structure of sperm in cosmopolitan species Ech. cordatum inhabiting the Sea of Japan and coastal areas of the Northeast Atlantic may bear record to the complex existence of species Ech. cordatum. The fine structure of sperm is unique for each of the studied families, Strongylocentrotidae, Scutellidae, and Loveniidae. The eggs of all the species are characterized by vitelline and jelly-like membranes. The vitelline membrane is formed by cytoplasm protrusions; the area between them is filled with fibrillar material. The jelly-like membrane is formed by fibrillar material associated with apical parts of microvilli of the vitelline membrane. The irregular sea urchins Sc. griseus, Sc. mirabilis and E. parma are characterized by chromatophores situated in the jelly-like membrane, with the highest abundance in Sc. mirabilis.     

Remodeling the sperm nucleus into a male pronucleus at fertilization

After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male pronucleus. We have investigated the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin-binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed.     

Fluorescence staining of living cells with fluorescamine

Summary The interaction of fluorescamine with living plant and animal cells was investigated to determine which subcellular structures and molecular species might react with the dye and to assess its effects on cell viability and function.Plasma and nuclear membranes ofXenopus erythrocytes, mitochondria of sea urchin sperm, growing apices of Timothy root hairs, and various organelles ofNitella andEuglena were labelled as judged by fluorescence microscopy. Cytoplasmic fluorescence was particulate inNitella and easily displaced by moderate centrifugal fields in sea urchin eggs. Chloroplasts and nuclei isolated from cells labelledin vivo exhibited fluorescamine dependent fluorescence.Reaction seemed to have little or no effect on cell viability (Euglena) photoautotrophic growth (Euglena), cell motility (sperm), fertilizability (sperm or egg), embryonic development (sea urchin), or cytoplasmic streaming (Nitella, Timothy).Quantitative fluorometric analysis of thein vivo reactants in sperm indicated a reaction preference for phospholipid over protein compared to control cells dissociated in SDS prior to labelling. The bulk of labelled lipid was phosphatidylethanolamine.These results suggest that fluorescamine is a true vital dye which can label the cell surface as well as penetrate deeply within cells to label a variety of organelles. The distribution of fluorescence and results of chemical analysis suggest thatin vivo the dye may preferentially react with membrane.     

Sea urchin sperm head plasma membranes: characteristics and egg jelly induced Ca2+ and Na+ uptake

Sea urchin sperm respond to egg factors with changes in the ionic permeability of their plasma membrane. It has been previously shown that plasma membranes isolated preferentially from sea urchin sperm flagella respond to egg jelly increasing their Ca2+ and Na+ uptake (Darszon et al. (1984) Eur. J. Biochem. 144, 515-522). However, the egg jelly induced acrosome reaction occurs in the sperm head, and there is evidence for an heterogeneous distribution of plasma membrane components within the various regions of this cell. We here report a method for purifying sperm head membranes using positively charged beads according to Jacobson (1977) Biochim. Biophys. Acta 471, 331-335). Under the transmission electron microscope these membranes appeared homogeneous and apparently free of internal membranes. The yield of the preparation was 0.9% of the total protein in the sperm homogenate. The preparation contained less than 5% of the mitochondrial marker cytochrome oxidase, and 10% of the total DNA/mg protein. Surface labeling with 125I indicated a 2.5-3-fold enrichment in specific activity of the head membranes with respect to whole sperm. The SDS band pattern and the lipid composition of this preparation were different from those of isolated flagellar membranes. Phosphatidylcholine was higher in the head membranes, while phosphatidylserine and phosphatidylethanolamine were lower. The head membranes displayed a 1.7-2.3-fold higher Ca2+-ATPase activity and a 2.5-fold lower Na+/K+-ATPase activity, than the flagellar membranes. These results are consistent with a heterogeneous distribution of membrane components along the sea urchin sperm plasma membranes. Isolated head membranes sonicated in the presence of soybean phospholipid liposomes responded to egg jelly with a species-specific increase in Ca2+ and Na+ uptake. As in whole sperm, Ca2+ uptake was inhibited by the Ca2+ channel blocker nisoldipine. A close analog of this compound, [3H]nitrendipine, binds with high affinity to head membranes in a saturable, reversible manner, showing a Kd and Bmax of 31 nM and 5.3 pmol/mg protein, respectively.     

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratas sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation.     

Isolation of intact sperm asters from fertilized sea urchin eggs

We have developed a procedure for isolating intact sperm asters in quantity from fertilized sea urchin eggs. This procedure is based on detergent-extraction methods developed previously for the bulk isolation of mitotic apparatuses. Using this protocol it is possible to isolate sperm asters as soon as they appear in the fertilized egg or at any subsequent point in their brief existence.     

Alteration of sea urchin embryo cell surface properties by mycostatin, a sterol binding antibiotic

Sea urchin embryos incubated in sea water containing mycostatin (MST), a polyene antibiotic, dissociate into single cells. Reaggregation of dissociated sea urchin embryo cells, and uptake of labeled precursors by these cells are also greatly inhibited although O₂ consumption is only slightly affected by this compound. It is known that mycostatin binds primarily to membrane sterols and affects only cells containing membrane sterols. Sea urchin cell membranes contain sterols. The effects of mycostatin on cell adhesion, reaggregation, and permeability seen in this study may be a result of an interaction with cell membrane sterols or sterol-associated molecules.     

Glycoprotein synthesis in developing sea urchin embryos

Surface membrane glycoproteins have been postulated in many mammalian cells to be involved in external surface membrane functions such as cell adhesion, cell-cell recognition, and cell movement. In developing echinoderm embryos, cell adhesion, recognition, and movement of individual cell types have been attributed to differences in the external surface membranes of these cells. Results reported here suggest that the three cell types of 16-cell sea urchin embryos have a mechanism that could establish differences in the carbohydrate portion of glycoproteins located in the external surface membrane. The results demonstrate 1) that glycoproteins are synthesized during early sea urchin development and 2) that slightly different rates of glycoprotein synthesis exist for the three types of blastomeres from 16-cell sea urchin embryos.     

A reevaluation of the fertilizin hypothesis of sperm agglutination and the description of a novel form of sperm adhesion

The classical isoagglutination of sea urchin sperm by egg jelly is not an agglutination of cells, as proposed by the fertilizin-antifertilizin hypothesis. Sperm motility is required to obtain the isoagglutination of Strongylocentrotus purpuratus sperm, and the sperm do not adhere to each other in the isoagglutination clusters, which cannot be fixed for microscopy and which disperse rapidly into individual cells when sperm motility is inhibited. These observations suggest that isoagglutination is the swarming of freely moving sperm to a common focus and is quite distinct from the agglutination of sperm by known crosslinking agents (antibodies or lectins).A previously unrecognized form of sperm agglutination is described which follows induction of an acrosome reaction by egg jelly, ammonia, or the ionophore A23187 in a suspension of sea urchin or sand dollar sperm. The sperm form rosettes of up to 100 cells in which the newly extended acrosomal processes adhere to each other. Rosettes can form containing sperm of different species, in which the acrosomal processes adhere without species preference.As observed by transmission electron microscopy, the acrosomal process of Lytechinus pictus sperm consists of an acrosomal tubule covered by a sheath of extracellular material. Rosette formation results from attachment between the extracellular materials of adjacent sperm.Less frequently, the acrosomal process of one sperm adheres to the midpiece of another by fusion of the acrosomal tubule and midpiece plasma membranes.     

A tale of two cells: endocannabinoid-signaling regulates functions of neurons and sperm

Sea urchin and human sperm contain receptors for neurotransmitters and psychoactive drugs, including cannabinoid receptors (CNRs). Anandamide, arachidonoylethanolamide (AEA), is a lipid-signal molecule that is an endogenous agonist for CNRs. AEA is enyzmatically released from membrane phospholipids when neurons are stimulated. Retrograde AEA signals from depolarized postsynaptic neurons inhibit neurotransmitter release at synapses in mammalian brain. Analogous processes regulate sperm functions during fertilization in sea urchins. AEA and (-)delta9tetrahydrocannabinol [(-)delta9THC], the major psychoactive constituent of marijuana, inhibit fertilization by blocking acrosomal exocytosis/acrosome reactions (AR) stimulated by egg jelly. The acrosome is a Golgi-derived secretory granule in sperm analogous to synaptic vesicles in neurons. AEA and (-)delta9THC do not block ionophore-induced AR, suggesting that they inhibit AR by modulating signal transduction event(s) before opening of ion channels. Unfertilized sea urchin eggs have enzymes required to release AEA from membrane phospholipids. These results indicate that sea urchin eggs may release AEA after activation by the fertilizing sperm. Released AEA may then react with CNRs in nearby sperm to block AR, thereby helping to prevent polyspermy. AEA is present in human seminal plasma, midcycle oviductal fluid, and follicular fluid. Sperm are sequentially exposed to these fluids as they move from the vagina to the site of fertilization in the oviduct. R-methanandamide (AM-356), a metabolically stable AEA analog, and (-)delta9THC modulate capacitation and fertilizing potential of human sperm in vitro. These findings suggest that AEA signaling directly affects sperm functions required for fertilization and provide additional evidence for common signaling processes in neurons and sperm.