Diacylglycerol species as messengers and substrates for phosphatidylcholine re-synthesis during Ca2+-dependent exocytosis in boar spermatozoa

We have investigated pathways of lipid metabolism in spermatozoa and generation of various metabolites with potential messenger functions during exocytosis stimulated with A23187/Ca²⁺. Stimulation of boar spermatozoa resulted in a considerable rapid increase in saturated/unsaturated 1,2-diacylglycerol (1,2-SU-DAG) and, concomitantly, a substantial reduction in disaturated 1,2-diacylglycerol (1,2-DS-DAG), and in phosphatidylcholine (PC). These changes preceded the onset of exocytosis. Phosphatidic acid was sometimes generated in parallel, but usually rose later, suggesting that 1,2-SU-DAG may be formed directly by phospholipase C action. Lipid changes observed in stimulated spermatozoa that have been prelabelled with several lipid precursors ([¹⁴C]palmitic acid, [¹⁴C]glycerol, [¹⁴C]choline, or [¹⁴C]arachidonic acid) suggested the existence of a unique process involving the utilization of the high basal levels of 1,2-DS-DAG to form 1,2-SU-DAG, with the latter being subsequently employed to replenish the PC pool. An ensuing generation of lysoPC and arachidonic acid, which paralleled the occurrence of exocytosis, revealed that the newly synthesized PC was hydrolyzed by phospholipase A₂. The highest levels of 1,2-SU-DAG, minimum levels of 1,2-DS-DAG, and the regeneration of the PC pool were tightly coupled to the beginning of visible exocytosis. These results suggest that changes in these lipid metabolites may be fundamental processes during acrosomal exocytosis occurring in response to physiological agonists. Mol. Reprod. Dev. 48:95–105, 1997. © 1997 Wiley-Liss, Inc.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Extracellular localization of proteasomes in human sperm

The proteasome, a multienzymatic protease complex is present in human sperm. Here we present evidence indicating that the proteasome has an extracellular localization, on the plasma membrane of the sperm head. Motile sperm (>90%) in PBS were incubated with the proteasome inhibitors clasto-lactacystin beta-lactone or epoxomicin. Then, the substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC) was added and the enzyme activity evaluated in a spectrofluorometer. Other aliquots were resuspended in Tyrode's medium and incubated at different concentrations for various times with or without inhibitors in the presence of 0.4% azocasein. Hydrolysis of azocasein was evaluated at 440 nm. In addition, sperm membrane proteins were obtained incubating the sperm with Triton X-114 or with 0.5 M KCl plus Triton X-100 and removing insoluble material by centrifugation at 5,000g for 40 min. Proteasomal activity was evaluated with SLLVY-AMC and its presence corroborated by Western blotting. Formaldehyde fixed, unpermeabilized sperm were incubated with anti-proteasome monoclonal antibodies and evaluated using indirect immunofluorescence. The effect of proteasome inhibitors upon the progesterone-induced acrosome reaction was also evaluated. Results indicated that (a) whole, intact sperm were able to hydrolyze the proteasome substrates SLLVY-AMC and azocasein; this activity was inhibited by proteasome inhibitors; (b) proteasomal activity was detected in soluble sperm membrane protein preparations and Western blotting revealed the presence of the proteasome in these fractions; (c) indirect immunofluorescence revealed staining of the head region, particularly of the post acrosomal region; and (d) the proteasome plays an important role during the acrosome reaction.     

精子顶体反应期间磷脂酶A2的信号转导及其调节

磷脂酶A2是精子重要的脂解酶类,包括多种不同亚型。在精子顶体反应期间磷脂酶A2受天然激动剂卵透明带、孕酮和γ-氨基丁酸激活,引起胞外Ca2 内流,使磷脂水解为花生四烯酸和溶血磷脂酰胆碱,从而促进膜的融合即顶体反应。天然激动剂引起PLA2激活受Gi蛋白、甘油二酯、蛋白激酶A、蛋白激酶C、促分裂原蛋白激酶和活性氧等多条信号通路的调节,此外,磷脂酶A2与特异性磷酯酶C之间可以发生信号串话。研究PLA2的信号通路为了解受精机制、男性不育之病因和开发男性避孕药提供依据。     

Bimodal regulatory effect of melittin and phospholipase A2-activating protein on human type II secretory phospholipase A2

Melittin and phospholipase A2-activating protein (PLAP) are known as efficient activators of secretory phospholipase A2(sPLA2) types I, II, and III when phospholipid liposomes are used as substrate. The present study demonstrates that both peptides can either inhibit or activate sPLA2 depending on the peptide/phospholipid ratio when erythrocyte membranes serve as a biologically relevant substrate. Low concentrations of melittin and PLAP were observed to inhibit sPLA2-triggered release of fatty acids from erythrocyte membranes. The inhibition was reversed at melittin concentrations above 1 microM. PLAP-induced inhibition of sPLA2 persisted steadily throughout the used concentration range (0-150 nM). The two peptides induced a dose-dependent activation of sPLA2 at low concentrations, followed by inhibition when model membranes were used as substrate. This opposite modulatory effect on biological membranes and model membranes is discussed with respect to different mechanisms the interaction of the regulatory peptides with the enzyme molecules and the substrate vesicles.     

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.     

Induction of the acrosome reaction in guinea pig spermatozoa by calmodulin antagonist W-7

The effect of the calmodulin antagonist W-7 on the capacitation and the acrosome reaction of guinea pig spermatozoa was examined. The characteristic features of the acrosome reaction induced by W-7 were the dependence on the composition and pH of the medium and on the presence of sodium bicarbonate. The most effective concentration of W-7 for inducing the acrosome reaction was approximately 5 μM, which is far less than the Kd for calmodulin. Moreover, W-7 enhanced the ability of spermatozoa to acquire capacitation in a Ca²⁺-free medium. The spermatozoa induced to undergo the acrosome reaction by W-7 were capable of penetrating the zona-free hamster eggs. W-5, which has a lower affinity for calmodulin than W-7, also induced the acrosome reaction in the same manner as W-7. These results suggest that the naphthalenesulfonamide derivatives W-7 and W-5 can induce the acrosome reaction in guinea pig spermatozoa via capacitation in a pH-dependent, Ca²⁺-calmodulin-independent manner.     

Requirement of monovalent cations in the acrosome reaction of guinea pig spermatozoa

The majority of the spermatozoa precapacitated in Ca²⁺-free medium underwent the acrosome raction rapidly when they were transferred to Ca²⁺-containing medium. The presence of Na⁺ and Ca²⁺ in the medium was essential for the acrosome reaction. The vast majority of spermatozoa failed to undergo the reaction in Ca²⁺ medium lacking monovalent ions, although they remained motile. At the concentration of 140 mM, Na⁺, K⁺, Rb⁺, and Cs⁺ all supported the reaction at the maximum level, but at 50 mM the latter three ions were not as effective as Na⁺. Li⁺ was least effective in supporting the reaction. Virtually no acrosome reactions took place when precapacitated spermatozoa were first exposed to Na⁺ medium (no Ca²⁺) and then to Ca²⁺ medium (no Na⁺). On the other hand, a considerably higher proportion of spermatozoa acrosome reacted when they were exposed to these media in the reverse order. The most efficient acrosome reactions took place when the medium contained both a monovalent ion (Na⁺) and Ca²⁺ simultaneously. Possible mechanisms by which monovalent and divalent cations participate in the acrosome reaction are discussed.     

Enhancement of the acrosome reaction of hamster spermatozoa by the proteolytic enzymes,kallikrein, trypsin,and chymotrypsin

The involvement of a kallikrein−kinin system in the motility of mammalian spermatozoa has been suggested by several investigators. We found that incorporation of kallikrein (0.1–1.0) unit/ml) in the sperm incubation medium did not enhance the motility of hamster spermatozoa that were already active. However, this enzyme significantly increased the incidence of the acrosome reaction. Trypsin (1.8–18 units/ml) and chymotrypsin (0.34–3.4 units/ml) also increased the incidence of the acrosome reaction, and accelerated its onset. Kinins (bradykinin and kallidin) added to the medium in a wide concentration range (1 ng/ml to 1 mg/ml) had no marked effects on either the motility or the acrosome reaction. A kallikrein−kinin system is apparently not of primary importance at least for the acrosome reaction. The enhancement of the acrosome reaction by exogenous proteinases may be due in part to accelerated removal or alteration of the sperm surface coat (glycoprotein) by the enzyme peior to the acrosome reaction. Exogenous proteinases may also act synergistically with endogenous (acrosomal) proteinases (and other enzymes) in altering membrane proteins and dispersing the acrosome matrix during the course of teh acrosome reaction.     

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.     

Ethanol inhibits human and hamster sperm penetration of eggs

The effect of alcohol on the fertilizing ability of both human and hamster spermatozoa was examined by an in vitro fertilization assay using hamster ova. Spermatozoa were incubated in capacitating media for 3 hr (hamster sperm) and 4 hr (human sperm). Hamster ova were inseminated with preincubated sperm and were examined after 2 to 3 hr. Ethanol was added to the capacitating media at concentrations of 25, 50, 100, 200, and 400 mg%. Fertilization of zona-free hamster eggs by human spermatozoa was reduced from 49.6% in no alcohol to 16.7% in 400 mg% ethanol. Fertilization of hamster eggs by hamster sperm revealed a reduction from 63.6% to 33.7% in cumulus-intact eggs and from 65.8% to 10.8% in cumulus-free eggs in the presence of ethanol at 400 mg%. Hamster sperm acrosome reaction was reduced from 47% to 12%. When these hamster sperm with reduced acrosome reaction were placed with zona-free hamster eggs, the 100% fertilization rate was not reduced; however, the fertilization index, which reflects the number of swelling sperm heads per egg, was reduced from 8.5 to 1.8. This suggests that as little as 12% of the sperm with an acrosome reaction is sufficient to fertilize 100% of the zona-free eggs. If ethanol was added to the insemination media only, there was no inhibition of fertilization by human sperm or hamster sperm that had been previously capacitated in an ethanol-free media. Removal of the ethanol from the preincubated sperm produced fertilization at control levels; thus the inhibitory effect is reversible. These results indicate that ethanol may affect fertilization by an inhibition of the capacitation and/or acrosome reaction process.     

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space-filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A₂, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A₂ activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.     

Lipoprotein-associated phospholipase A2 decreases oxidized lipoprotein cellular association by human macrophages and hepatocytes

We investigated whether the presence of endogenous or exogenous lipoprotein-associated phospholipase A2 (Lp-PLA2) can modify the cellular association of oxidized low density lipoprotein (oxLDL) and oxidized lipoprotein(a) (oxLp(a)) by human monocyte-derived macrophages (MDM) and hepatocytes (HepG2). Purified recombinant Lp-PLA2 was used as a source of exogenous enzyme whereas Pefabloc (serine esterase inhibitor) was used to inhibit the endogenous Lp-PLA2 activity associated with isolated lipoproteins. Cellular association studies were performed with DiI-labeled oxLDL or oxLp(a) and human monocyte-derived macrophages and HepG2 cells. Active Lp-PLA2 decreased the cellular association of oxLDL and oxLp(a) in macrophages and HepG2 cells by approximately 30–40%, whereas the inactive enzyme did not significantly change oxidized lipoprotein cellular association by either cell type. OxLDL pretreated by Pefabloc increased oxLDL cellular association by MDM and HepG2 cells compared to untreated oxLDL. Therefore, unlike some lipases, Lp-PLA2 did not appear to have any catalytic independent function in oxLDL cellular association. To assess whether the reduced cellular association mediated by Lp-PLA2 was due to the hydrolysis of oxidized phosphatidylcholine (oxPC), we measured the concentration of lysophosphatidylcholine (lysoPC) in lipoprotein fractions after Lp-PLA2 treatment. LysoPC was increased by 20% (0.4 μM) and 87% (0.7 μM) by active Lp-PLA2 compared to inactive Lp-PLA2 for oxLDL and Lp(a), respectively. LysoPC at higher concentration dose-dependently increased the cellular association of oxLDL and oxLp(a) in MDM and HepG2 cells. We conclude that Lp-PLA2 mediates a decrease in oxidized lipoprotein cellular association in human macrophages and HepG2 cells by reducing the concentration of oxPC within these lipoproteins.     

Quantitative analysis of flagellar movement in hyperactivated and acrosome-reacted golden hamster spermatozoa

Caudal epididymal spermatozoa of golden hamsters were incubated in capacitation medium. Their movement patterns changed as they became hyperactivated and underwent the acrosome reaction. To understand the basic mechanism by which changes in movement pattern are brought about, digital image analysis was carried out on the flagellar movements recorded with a video system. The degree of flagellar bending increased with incubation time, especially in the proximal midpiece. The hyperactivated spermatozoa had remarkably asymmetrical flagellar waves of large amplitude because either the bends in the same direction as the hook of the head (referred as the "pro-hook bend") or the bends in the opposite direction to the hook of the head (referred as the "anti-hook bend") extremely increased their curvature; whereas, the acrosome-reacted spermatozoa had relatively symmetrical flagellar waves of large amplitude because both the pro- and anti-hook bends remarkably increased their curvature. Beat frequency significantly decreased while wavelength of flagellar waves increased after hyperactivation and further after the acrosome reaction. These results suggest that both extreme pro- and anti-hook bends are essential in the acrosome-reacted spermatozoa even though beat frequency decreased markedly.     

Effects of surfactants on the fertilizing capacity and acrosome reaction of sea urchin spermatozoa

The effects of seven surfactants on spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, were studied. All these surfactants induced the acrosome reaction and inhibited the fertilizing capacity of spermatozoa. There was a statistically significant correlation between the concentrations that induce the acrosome reaction and inhibit fertilization. The critical micelle concentrations (CMC) of surfactants in sea water were almost even and these values, which are inherent physical properties of surfactants, did not provide a direct measure of their inhibitory effect of fertilization. Among seven surfactants, p-menthanyl-phenol polyoxyethylene (8.8) ether (TS-88) with a characteristic hydrophobes was the most potent both in the induction of acrosome reaction and in the inhibition of fertilization. Various ethylene oxide adducts to p-menthanyl-phenol were also tested for the purpose of comparison. It is suggested that the effects of surfactants on sea urchin spermatozoa at low concentrations reflect their activity associated with the hydrophobic group inherent in each surfactant.     

Mouse lipocalin as an enhancer of spermatozoa motility

The 24p3 protein is a 25 kDa glycoprotein that is secreted into the uterine fluid during the proestrous phase of mice. We assessed the effects on spermatozoa motility and on the functions of mouse spermatozoa using the computer-assisted sperm analysis method, cytochemical staining and detection of the protein tyrosine phosphorylation pattern. Compared with the control cells, sperm motility was stimulated by the addition of 24p3 protein into the medium. Introducing 24p3 protein enhanced progressive motility but did not promote the appearance of hyperactivated movement. The presence of 24p3 protein in the medium did not allow the cells to undergo the capacitated protein tyrosine phosphorylation pattern and acrosome reaction. The tyrosine phosphorylation pattern shows phosphoproteins in the range of Mr 50000–106000 correlated with the sperm progressive motility after the addition of 24p3 protein into the medium. Using flow cytometry, we assessed the changes in the intracellular pH and measured the intracellular cAMP concentration with an immunodetection kit. The results indicated that the elevation in intracellular pH from 6.67 to 6.89, increase of intracellular cAMP accumulation, and protein tyrosine phosphorylation might be the factors in enhancement of sperm motility as the 24p3 protein bound to the spermatozoa. The 24p3 protein may have a role in regulating flagellar motility.     

Characterization of the human patatin-like phospholipase family

Several publications have described biological roles for human patatin-like phospholipases (PNPLAs) in the regulation of adipocyte differentiation. Here, we report on the characterization and expression profiling of 10 human PNPLAs. A variety of bioinformatics approaches were used to identify and characterize all PNPLAs encoded by the human genome. The genes described represent a divergent family, most with a highly conserved ortholog in several mammalian species. In silico characterization predicts that two of the genes function as integral membrane proteins and are regulated by cAMP/cGMP. A structurally guided protein alignment of the patatin-like domain identifies a number of conserved residues in all family members. Quantitative PCR was used to determine the expression profile of each family member. Affymetrix-based profiling of a human preadipocyte cell line identified several members that are differentially regulated during cell differentiation. Cumulative data suggest that patatin-like genes normally expressed at very low levels are induced in response to environmental signals. Given the observed conservation of the patatin fold and lipase motif in all human PNPLAs, a single nomenclature to describe the PNPLA family is proposed.     

The role of propeptide in the refolding of human group IB phospholipase A2

Phospholipase A2 (PLA2, EC 3.1.1.4) catalyzes the hy-drolysis of the 2-acyl ester bond of sn-3-phosphoglycerides,producing a free fatty acid and a lysophospholipid [1].Mammalian secretory phospholipase A2 is tentatively sug-gested to be involved in the in…     

Ultrastructural localization of proacrosin and acrosin in ram spermatozoa

Proacrosin and acrosin were localized immunocytochemically at the electron microscope level in ram spermatozoa undergoing an ionophore-induced acrosome reaction. Antigenicity was preserved after fixation with 0.5% w/v ethyl-(dimethylaminopropyl)-carbodimide, and an antibody preparation was used that reacted with all major forms of ram acrosin. All stages of the acrosome reaction could be observed in a single preparation. At the earliest stage, labeling was observed throughout the acrosomal contents, which were just beginning to disperse. As dispersal proceeded, labeling diminished, being associated only with visible remnants of the acrosomal matrix. By the time the acrosome had emptied, almost no labeling could be detected on the inner acrosomal membrane. The relationship between matrix dispersal and proacrosin activation was studied in isolated ram sperm heads. While proacrosin was prevented from activating, the acrosomal matrix remained compact; but as activation proceeded, the matrix decondensed and dispersed in close parallel. By the time proacrosin activation was complete, the acrosomal contents had almost entirely disappeared. We conclude that proacrosin is distributed throughout the acrosomal contents as an intrinsic constituent of the acrosomal matrix. During the acrosome reaction, proacrosin activation occurs, resulting directly in decondensation of the matrix. All the contents of the acrosome including acrosin disperse and, by the time the acrosome is empty and the acrosomal cap is lost, only occasional traces of acrosin remain on the inner acrosomal membrane. Since the acrosomal cap is normally lost during the earliest stages of zona penetration, acrosin's role in fertilization is unclear: it does not appear to be a zona lysin bound to the inner acrosomal membrane.