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.     

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

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

Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. II. Ultrastructural changes associated with inhibition of the acrosome reaction.

Pretreatment of Strongylocentrotus purpuratus sperm with delta 9-tetrahydrocannabinol (THC) prevents the triggering of the acrosome reaction by egg jelly. Examination of THC-treated sperm by transmission electron microscopy reveals that the membrane fusion reaction between the sperm plasma membrane and the acrosomal membrane is completely blocked. Electron-dense deposits are present in the subacrosomal fossa and in the centriolar fossa. The nuclear envelope is fragmented in close proximity to the electron-dense deposits. The electron-dense deposits are not bound by a limiting membrane, stain positively for lipid with thymol and farnesol, and disappear from THC-treated sperm that are extracted with chloroform:methanol (2:1) after glutaraldehyde fixation. The electron-dense deposits are lipid in nature and may be a hydrolytic product of the nuclear envelope. Electron-dense deposits are seen in sperm after 1-10 min treatment with 5-100 microM THC. The electron-dense deposits disappear after removal of THC from the sperm by washing, but the fragmented nuclear envelope in the subacrosomal fossa persists. Cannabidiol (CBD) and cannabinol (CBN) also inhibit the triggering of the acrosome reaction by egg jelly and produce ultrastructural changes in the sperm identical to those elicited by THC. Enhanced phospholipase activity stimulated by THC, CBD, and CBN may be the cause of the accumulation of lipid deposits in the sperm. Metabolites derived from this modification of membrane phospholipids may prevent triggering of the acrosome reaction by egg jelly and thereby inhibit fertilization.     

Wheat germ agglutinin blocks the acrosome reaction in Strongylocentrotus purpuratus sperm by binding a 210,000-mol-wt membrane protein

Wheat germ agglutinin (WGA) binds to the entire surface of Strongylocentrotus purpuratus sperm, and inhibits the egg jelly-induced acrosome reaction. The binding was found to be species dependent and was completely inhibited by 5 mM N-acetyl-D-glucosamine. Blockage of the acrosome reaction by WGA was bypassed by a combination of the ionophores A23187 and monensin, although neither ionophore was effective individually. These experiments suggest that WGA blocks both Ca2+ uptake and Na+/H+ exchange in these sperm, which was confirmed by direct measurements of 45Ca2+ uptake and H+ efflux. The target of WGA in S. purpuratus sperm appears to be a membrane glycoprotein of Mr = 210,000. Treatment of this protein with neuraminidase or endo-beta-N-acetylglucosaminidase F abolished WGA binding.     

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.     

Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction.

Jelly coat, a multicomponent extracellular matrix surrounding the sea urchin egg, induces the acrosome reaction in sperm. The jelly coats of the four species studied, Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus drobachiensis, and Lytechinus variegatus, were found to be very similar in chemical composition. A sialoprotein (approximately 20% of the mass of the jelly coat) and a fucose sulfate polysaccharide (approximately 80%) are the major macromolecular components of the jelly coat. The acrosome reaction inducing capacity resides solely in the fucose sulfate polysaccharide. Induction of the acrosome reaction ranges from highly species specific to nonspecific. Thus, A. punctulata and S. drobachiensis sperm are induced to undergo the acrosome reaction only with their homologous jelly coat, while S. purpuratus sperm react equally well with homologous or L. variegatus jelly coat, but not with A. punctulata jelly coat. L. variegatus sperm seem to be relatively nonspecific in response. Species-specific induction of the acrosome reaction resides solely in the fucose sulfate polysaccharide, suggesting that there must be structural differences in this polysaccharide in the various species. Therefore, in some species, fertilization appears to involve sperm-egg recognition at the level of the jelly coat as well as at the level of sperm-egg receptors.     

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.     

Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. I. Inhibition of the acrosome reaction induced by egg jelly.

delta 9-Tetrahydrocannabinol (THC) and two other major cannabinoids derived from marihuana--cannabidiol (CBD) and cannabinol (CBN)--inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of sperm (Schuel et al., 1987). Sperm fertility depends on their motility and on their ability to undergo the acrosome reaction upon encountering the egg's jelly coat. Pretreatment of S. purpuratus sperm with THC prevents triggering of the acrosome reaction by solubilized egg jelly in a dose (0.1-100 microM) and time (0-5 min)-dependent manner. Induction of the acrosome reaction is inhibited in 88.9 +/- 2.3% of sperm pretreated with 100 microM THC for 5 min, while motility of THC-treated sperm is not reduced compared to solvent (vehicle) and seawater-treated controls. The acrosome reaction is inhibited 50% by pretreatment with 6.6 microM THC for 5 min and with 100 microM THC after 20.8 sec. CBN and CBD at comparable concentrations inhibit the acrosome reaction by egg jelly in a manner similar to THC. THC does not inhibit the acrosome reaction artificially induced by ionomycin, which promotes Ca2+ influx, and nigericin, which promotes K+ efflux. THC partially inhibits (20-30%) the acrosome reaction induced by A23187, which promotes Ca2+ influx, and NH4OH, which raises the internal pH of the sperm. Addition of monensin, which promotes Na+ influx to egg jelly or to A23187, does not overcome the THC inhibition. Inhibition of the egg jelly-induced acrosome reaction by THC produces a corresponding reduction in the fertilizing capacity of the sperm. The adverse effects of THC on the acrosome reaction and sperm fertility are reversible.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Sperm–egg binding: Identification of a species-specific sperm receptor from eggs of strongylocentrotus purpuratus

We have attempted to identify a surface component of echinoderm eggs that is involved in the species-specific binding of sperm. Cell surface membranes from eggs of the sea urchins Strongylocentrotus purpuratus or Arbacia punctulata were radioiodinated, detergent-treated, and subjected to density-gradient centrifugation. In the presence of bindin, the complementary binding protein isolated from sperm, one component of the membranes sedimented to a different density. This membrane component bound-species specifically to sperm that had undergone the acrosome reaction. This binding led to an inhibition of the ability of treated sperm to fertilize eggs. Exhaustive proteolytic digestion of this receptor fraction yields a high molecular weight glycopeptide that can also bind to bindin. It therefore appears that this egg surface membrane fraction contains a functionally intact, species-specific receptor for sperm.     

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

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

Regulation of calcium content in bovine spermatozoa

Plasma membrane vesicles isolated from bovine epididymal and ejaculated spermatozoa have widely different capabilities for transporting Ca2+. Spermatozoa were ruptured by nitrogen cavitation, and the plasma membrane fraction was harvested after low speed and sucrose gradient centrifugation; purity was assessed by marker enzyme analyses, electron microscopy, and sedimentation properties. Plasma membrane vesicles isolated from epididymal sperm accumulate Ca2+ passively at a faster rate and to a greater extent than vesicles prepared from ejaculated sperm. Ca2+ transport across bovine sperm plasma membranes is an ATP-independent, Na+-dependent process that obligatorily exchanges intravesicular Na+ for external Ca2+. The rate of Na+/Ca2+ exchange is significantly lower in ejaculated sperm vesicles than in those of epididymal sperm. Bovine plasma membranes contain little or no Ca2+-dependent ATPase activity. It is suggested that, at the time of ejaculation, calcium flux into bovine sperm is prevented by the interaction of the plasma membrane with putative factors in seminal fluid that specifically interfere with Na+/Ca2+ exchange. We have isolated a protein from seminal plasma that prevents calcium accumulation by bovine epididymal sperm (Rufo, G. A., Jr., Singh, J. P., Babcock, D. F., and Lardy, H. A. (1982) J. Biol. Chem. 257, 4627-4632). A protein with properties resembling those of the seminal calcium transport inhibitor is found on the membrane vesicles from ejaculated sperm but not on membranes from epididymal sperm. We conclude that this protein binds strongly to the plasma membrane of bovine sperm and is responsible for preventing calcium uptake by ejaculated sperm.     

Evidence for the involvement of metalloendoproteases in the acrosome reaction in sea urchin sperm

An essential initial step in fertilization in the sea urchin Strongylocentrotus purpuratus is an intracellular membrane fusion event in the sperm known as the acrosome reaction. This Ca2+-dependent, exocytotic process involves fusion of the membrane of the acrosomal vesicle and the plasma membrane. Recently, metalloendoproteases requiring divalent metals have been implicated in several Ca2+-dependent membrane fusion events in other biological systems. In view of the suggested involvement of Zn2+ in the sea urchin sperm acrosome reaction (Clapper, D.L., Davis, J.A., Lamothe, P.J., Patton, C., and Epel, D. (1985) J. Cell Biol. 100, 1817-1824) and the fact that Zn2+ is a metal cofactor for metalloendoproteases, we investigated the potential role of this protease in the acrosome reaction. A soluble metalloendoprotease was demonstrated and characterized in sperm homogenates using the fluorogenic protease substrate succinyl-alanine-alanine-phenylalanine-4-aminomethylcoumarin. The protease was inhibited by the metal chelators EDTA and 1,10-phenanthroline, and activity of the inactive apoenzyme could be reconstituted with Zn2+. The metalloendoprotease substrate and inhibitors blocked the acrosome reaction induced either by egg jelly coat or by ionophore, but had no effect on the influx of Ca2+. These observations suggest that inhibition occurs at a step independent of Ca2+ entry. Overall, the results of this study provide strong indirect evidence that the acrosome reaction requires the action of metalloendoprotease.     

Studies on the specificity of sperm binding in echinoderm fertilization

Using gametes from the sea urchins Arbacia punctulata and Strongylocentrotus purpuratus, we have evaluated the role of the acrosome reaction and the sperm-egg binding process in the block to interspecific fertilization among echinoids. The results indicate that sperm preinduced to undergo the acrosomal reaction by two different methods still bind to homologous eggs in a species specific manner. These results, taken in conjunction with an earlier study on species specificity of jelly coat induction of the acrosomal reaction (SeGall and Lennarz 1978), indicate that both the acrosome reaction and the sperm binding process contribute to the species specificity of fertilization in S. purpuratus and A. punctulata.     

Stoichiometry of phosphate loss from sea urchin sperm guanylate cyclase during fertilization

Spermatozoa of the sea urchin Arbacia punctulata possess a phosphorylated guanylate cyclase as a major glycoprotein of the flagellar plasma membrane. When sperm cells contact the jelly layer surrounding the egg, the peptide "resact" binds the sperm cell surface and triggers the dephosphorylation of the cyclase. A large decrease in cyclase activity accompanies dephosphorylation. Before treatment of sperm cells with egg jelly the enzyme contains 17.95 +/- 1.24 moles phosphate per mole cyclase. After treatment of sperm cells with egg jelly this number decreases to 2.57 +/- 0.42. Based on a molecular weight of 137,250 for the peptide chain, approximately 15 phosphate groups are lost per molecule of guanylate cyclase at fertilization.     

Egg jelly triggers a calcium influx which inactivates and is inhibited by calmodulin antagonists in the sea urchin sperm

Sea urchin sperm must undergo the acrosome reaction to fertilize eggs. The natural inducer of this reaction is the most external coat of the egg, named 'jelly'. The ionic composition of the extracellular and intracellular media and the permeability properties of the sperm plasma membrane are fundamental in this reaction. As Ca2+ is required for the acrosome reaction to occur, its intracellular concentration ([Ca2+]i) was measured with fura-2. In 10 mM Ca2+, egg jelly induced the acrosome reaction and an increase in [Ca2+]i that lasted for several minutes. However, at 0.5 or 2 mM Ca2+, it became evident that the Ca2+-influx pathway activated by jelly opened only for a few seconds; this prevented both the full increase in [Ca2+]i and the acrosome reaction even after the concentration of Ca2+ was raised to 10 mM. In the presence of jelly, the time this permeability pathway remained open was inversely related to the extracellular concentration of Ca2+ ([ Ca2+]e). Using Bisoxonol (a permeant fluorescent membrane potential probe), it was found that the jelly-induced depolarization depended on [Ca2+]e and was proportional to the increase in [Ca2+]i. Since [Ca2+]i could affect the jelly-induced Ca2+ influx through calmodulin, two of its antagonists, trifluoperazine and W-7, were tested. Both compounds blocked the acrosome reaction by inhibiting the jelly-induced increase in [Ca2+]i. W-5 at the same concentration had no effect. The results suggest that one of the jelly-activated Ca2+-influx pathways, probably a channel, is the target of the calmodulin antagonists.     

Effect of temperature on the fluidity of boar sperm membranes

Fluidity was used to assess changes in molecular organization of boar spermatozoa plasma membranes from (1) the head and (2) the rest of the sperm body and acrosome as a consequence of temperature. The initial fluidity of the head membranes at 25 degrees C was less than that of the sperm body membranes (P less than 0.05). When held at 25 degrees C, the fluidity of the head membranes decreased for 105 +/- 8 min and then stabilized for the remainder of the 160-min incubation. Calcium (10 mM) caused a significantly greater decrease in fluidity. The fluidity of the sperm body membranes increased slightly over time in the absence of Ca2+, but decreased significantly with Ca2+. Cooling from 25 to 5 degrees C and subsequent heating to 40 degrees C (0.4 degrees C/min) caused marked alterations in the fluidity of each membrane. Cooling the head membranes prevented the fluidity increase seen at 25 degrees C, while reheating caused a dramatic decrease in fluidity. Fluidity of the head membranes was now unaffected by Ca2+. Lipid phase transitions, indicated by sharp break points in data curves, were detected at the onset of reheating (7 +/- 3 C) and at 23 +/- 4 degrees C during reheating. Fluidity of the sperm body membranes decreased slightly and in a linear fashion with Ca2+. Without Ca2+, the sperm body membranes showed an additional lipid phase shift at 31 +/- 5 degrees C, which led to a rapid fall in fluidity. These results suggest that the fluidity, and therefore the molecular structure, of sperm head and body membranes differ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphatidylcholine and phosphatidylinositol-specific phospholipases C of bull and rabbit spermatozoa.

Acrosomal reaction is an essential prerequisite to fertilization. The changes in lipid composition of sperm membranes cause fusion of the plasma and outer acrosomal membranes that results in the exocytosis of acrosomal contents. We report that both bull and rabbit spermatozoa contain a phosphatidylcholine-specific phospholipase C (PC-PLC) that hydrolyzes L-alpha-dipalmitoyl-(choline-methyl-14C-153.0 Ci/mmol and a phosphatidylinositol-specific phospholipase C (PI-PLC) that hydrolyzes L-alpha-(Myo-Inositol-2-3H (N)-5.2 Ci mmol. PI-PLC from bull sperm acrosome has been purified 568 x fold with a specific activity 6.25 +/- 0.6 nmol/min/mg protein, km 0.004 mM, and Vmax 12 nmol/min/mg protein. Both enzymes had optimum at pH 7.5. The activity of PC-PLC remained unaffected by varying concentrations of Ca2+, whereas PI-PLC activity was significantly increased. The bulk of PI-PLC was found to be associated with inner acrosomal membrane of bull and rabbit sperm, while PC-PLC was found in the outer acrosomal membranes in the bull sperm and the plasma membrane of the rabbit sperm. Both enzymes are compartmentalized in sperm cell.     

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

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

THE FERTILIZING CAPACITY OF SEA URCHIN SPERM RAPIDLY DECREASES AFTER INDUCTION OF THE ACROSOME REACTION*

When immotile, flagella-less sperm were added to acid-dejellied eggs of Strongylocentrotus purpuratus 11% of the eggs fertilized. Addition of soluble egg jelly increased the percentage fertilization to 90.5. Over 50% of the sperm exposed to egg jelly had undergone the acrosome reaction compared to only 3–5% in the absence of jelly. Egg jelly was added to flagella-less sperm to induce the acrosome reaction and dejellied eggs added at various times thereafter. The fertilizing capacity of the sperm decreased with first order kinetics with 50% loss by 23 sec after induction of the acrosome reaction. Intact, motile sperm bind to formaldehyde-fixed eggs with maximum binding occurring 40 sec after sperm addition. After 40 sec the sperm begin to detach from the fixed eggs and by 240 sec none remain attached. Sperm detachment from fixed eggs and loss of fertilizing capacity after the acrosome reaction show a close temporal correlation.