Evidence suggesting the importance of fatty acids and the fatty acid moieties of sperm membrane phospholipids in the acrosome reaction of guinea pig spermatozoa

When guinea pig spermatozoa were preincubated 1 hr in Ca2+-free medium containing dilysocardiolipin (100--125 micrograms/ml) then exposed to Ca2+, the majority underwent an immediate acrosome reaction. Monolysocardiolipin was much less effective and the native cardiolipin was totally ineffective. Some fatty acids added to the medium could also render the spermatozoa capable of undergoing their acrosome reactions, arachidonic acid in methyl ester form being most potent. It is known that sperm membrane contains phospholipase A which cleaves membrane phospholipids into lysophospholipids and fatty acids. Most lysophospholipids are known to be potent acrosome reaction-promoting agents. As some forms of fatty acids are also potent acrosome reaction-promoting agents, both products of membrane phospholipid hydrolysis by phospholipase A (i.e., both fatty acids and lysophospholipids) may work synergistically to effect the conversion of stable sperm membranes to a fusion competent state capable of engaging in the acrosome reaction.     

The laminin-induced acrosome reaction in human sperm is mediated by Src kinases and the proteasome

The aim of this work was to determine whether laminin (Ln), an extracellular matrix protein, induces the intracellular events that may be involved in producing the acrosome reaction in human sperm. To this end, we evaluated the effect of Ln on tyrosine phosphorylation, intracellular calcium concentration, proteasome activity, and phosphorylation in human sperm. Aliquots of highly motile sperm selected with a Percoll gradient, were incubated with different concentrations of Ln (0-20 μg/ml) for different periods (0-18 h). The percentage of viable acrosome-reacted sperm was evaluated using fluorescein isothiocyanate-labeled Pisum sativum agglutinin and Hoechst 33258 DNA dye. Tyrosine phosphorylation was evaluated by Western blot analysis. The chymotrypsin-like activity of the proteasome was evaluated with a fluorogenic peptide, and intracellular calcium concentration was measured with fura-2. The results indicate that Ln stimulated the acrosome reaction of human sperm in a dose-dependent manner. This increase was drastically inhibited in the presence of herbimycin A, SU6656, and epoxomicin. In addition, Ln increased proteasome activity and phosphorylation; both events were inhibited by herbimycin A and SU6656. Finally, Ln induced an increase in intracellular calcium concentration, which was inhibited by SU6656 and epoxomicin. These results suggest that Ln is able to induce the acrosome reaction. This effect may be mediated by Src kinase and the proteasome, with the consequent induction of a calcium influx.     

Aster Formation in Sea Urchin Eggs Induced by the injection of Enzyme-Treated Sperm Centrioles

To determine the responsible components of isolated sperm centrioles for the aster induction in sea urchin eggs, the sperm centriolar fraction was treated with various enzymes and was injected into the unfertilized eggs, then the aster formation in first division was observed after fertilization.
Treatment with 1 μg/ml or higher concentration of trypsin inhibited the centriolar activity for aster induction, whereas the treatment with 50 μg/ml of DNase 1, 80 μg/ml of RNase A, 40 μg/ml of RNase T1, or 0.1 μg/ml of trypsin had no inhibitory effect to induce asters. Injection of 0.5 μg/ml of RNase A or 1 mUg/ml of RNase T1 into the egg caused the detention of mitosis at the streak stage. To examine the temperature effect for aster induction, the centriolar fraction was pre-treated with boiling temperature, and it was found that the fraction became incapable to induce any aster.
Results obtained suggest that the effective components of the sperm centriolar fraction to induce asters in the fertilized sea urchin eggs are the proteins but not the nucleic acids. The aster inducing activity is destroyed by heat treatment.     

Zona pellucida-induced acrosome reaction in boar sperm

Induction of the acrosome reaction in boar sperm by the zona pellucida (ZP) was investigated. A modified cytochemical staining method for measuring the acrosome reaction in boar sperm gave equivalent results to those obtained with transmission electron microscopy. Isolated heat-solubilized ZP effectively induced the acrosome reaction in boar sperm at a concentration of 25 micrograms/ml. Electrophoretically purified ZP components were also tested for acrosome reaction-inducing activity; both the 55,000 and 90,000 components of the ZP were effective. The carbohydrate moiety of the 55,000 component was necessary for activity because the polypeptides derived by chemical deglycosylation of the two glycoproteins did not induce the acrosome reaction.     

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.     

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

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

Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the vitelline envelope during fertilization of the sea urchin Pseudocentrotus depressus

The roles of sperm proteasomes in fertilization were investigated in the sea urchin Pseudocentrotus depressus. Two proteasome inhibitors, MG-132 and MG-115, inhibited fertilization at 100 microM, whereas chymostatin and leupeptin showed no inhibition. Among three proteasome substrates, Z-Leu-Leu-Glu-MCA showed the strongest inhibition toward fertilization. MG-132 inhibited the egg-jelly-induced, but not ionomycin-induced, acrosome reaction. In addition, MG-132, but not E-64-d, inhibited fertilization of dejellied eggs by acrosome-reacted sperm. MG-132 showed no significant inhibition toward the binding of reacted sperm to the vitelline layer. Proteasomes were detected by Western blotting in the acrosomal contents, which are partially released upon exocytosis. We also found that the inhibition pattern of the caspase-like activity of the proteasome in the acrosomal contents by chymostatin and proteasome inhibitors coincided well with their inhibitory abilities toward fertilization. Furthermore, the vitelline layer of unfertilized eggs appears to be ubiquitinated as revealed by immunocytochemistry and Western blotting. Extracellular ATP, required for the degradation of ubiquitinated proteins by the proteasome, was also necessary for fertilization. These results indicate that the sperm proteasome plays a key role not only in the acrosome reaction but also in sperm penetration through the vitelline envelope, most probably as a lysin, during sea urchin fertilization.     

Role of the sperm proteasome during fertilization and gamete interaction in the mouse

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.     

Rabbit spermatozoa undergo an acrosome reaction in the presence of glycosaminoglycans

Rabbit-ejaculated spermatozoa were incubated in a chemically defined medium containing comercially available glycosaminoglycans (GAGs) at concentrations ranging from 0.1 to 100 μg/ml. Sperm were stained and examined for the degree of acrosome reaction and viability after 9 h of incubation. There were significant dose and treatment effects of the induction of the acrosome reaction. Viability did not differ significantly betweendoses or treatment. Heparin enhanced the acrosome reaction between concentrations of 0.1 to 1.0 μg/ml, whereas higher levels depressed the percentage of sperm undergoing the acrosome reaction. Seminal plasma added to sperm cultures depressed the stimulatory effect of GAGs. Treatment of chondroitin-4-sulfate with chondro-4-sulfatase prohibited the stimulatory effect. It is concluded that GAGs, components of the female reproductive tract, may promote the acrosome reaction so that successful fertilization can occur.     

Sperm phospholipid methyltransferase activity during preparation for exocytosis

The present report describes experiments to evaluate phospholipid methyltransferase activity in golden hamster spermatozoa incubated under different conditions. Washed cauda epididymal sperm were incubated with taurine, in the presence or absence of epinephrine. At various times, the sperm were separated, and phospholipid methyltransferase activity measured. Also, at each time, aliquots of the sperm suspension were assayed for motility, and acrosome reactions. Some sperm incubated in the presence of taurine and epinephrine were capacitated by 3·5 h, because about 40 per cent of them can undergo the acrosome reaction 10 min after addition of the fusogen lysophosphatidylcholine. In epinephrine-free incubations the fusogen failed to stimulated acrosome reactions. On the other hand, epinephrine stimulated by twofold phospholipid methyltransferase activity from ‘0 time’ incubated sperm, in comparison to that observed in taurine-treated cells. Enzyme activities from both taurine or epinephrine plus taurine-treated cells decreased as the incubation time of the sperm suspension increased. Kinetic properties of the sperm phospholipid methyltransferase acvity were modified by the presence of taurine and epinephrine when S-adenosylmethionine was used as the substrate. These results suggest that refined molecular events occur in the sperm cell during the acquisition of fertilizing ability.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

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.     

Does Phospholipase A2 Participate in the Acrosome Reaction of Sea Urchin Sperm?: A Pharmacological Study

We examined whether phospholipase A₂ (PLA₂) is involved in the initiation of the acrosome reaction of sperm of the sea urchin, Strongylocentrotus intermedius , using inhibitors and an activator of this enzyme. Quinacrine and p-bromophenacyl bromide (PBPB) inhibited the egg jelly-induced acrosome reaction at 100 μM, but not the ionomycin-induced one. Depression of egg jelly-induced increase of intracellular free Ca²⁺concentration ([Ca²⁺]_i) by these reagents was expected and examined using fura 2. Quinacrine interfered with the flourescence of fura 2, but PBPB was found to depress at concentrations which could inhibit the acrosome reaction. Furthermore, melittin, which is known to stimulate PLA², caused a [Ca²⁺]_i increase and a formation of acrosomal process-like structure on sperm head. These results suggest that PLA₂ participates in the early step(s) of the acrosome reaction of sea urchin sperm.     

Sperm head membrane reorganisation during capacitation

The sperm cell has a characteristic polarized morphology and its surface is also highly differentiated into different membrane domains. Junctional protein ring structures seal the surface of the mid-piece from the head and the tail respectively and probably prevent random diffusion of membrane molecules over the protein rings. Despite the absence of such lateral diffusion-preventing structures, the sperm head surface is also highly heterogeneous. Furthermore, lipid and membrane protein ordering is subjected to changes when sperm become capacitated. The forces that maintain the lateral polarity of membrane molecules over the sperm surface, as well as those that cause their dynamic redistribution, are only poorly understood. Nevertheless, it is known that each of the sperm head surface regions has specific roles to allow sperm to fertilize the oocyte: a specific region is devoted to zona pellucida binding, a larger area of the sperm head surface is involved in the acrosome reaction (intracellular fusion), while yet another region is involved in egg plasma membrane binding and fertilization fusion (intercellular membrane fusion). All three events occur in the area of the sperm head where the plasma membrane covers the acrosome. Recently, lipid ordered microdomains (lipid rafts) were discovered in membranes of many biological specimens including sperm. In this review, we cover the latest insights about sperm lipid raft research and discuss how sperm lipid raft dynamics may relate to sperm-zona binding and the zona-induced acrosome reaction.     

Interactions of seminal plasma and glycosaminoglycans on acrosome reactions in bovine spermatozoa in vitro

Previous reports indicate that glycosaminoglycans (GAGs) would enhance the occurrence of acrosome reactions in sperm in vitro, but continuous exposure of those sperm to seminal plasma prevented a significant incidence of acrosome reactions. This study was designed to evaluate the interaction of GAGs and seminal plasma to promote acrosome reactions in bull sperm in vitro. Epididymal sperm required 22 hr to exhibit acrosome reactions in response to GAGs whereas only 9 hr were needed to achieve the same effect with washed ejaculated sperm. Exposure of epididymal sperm to seminal plasma for 20 min shortened the time for induction of the acrosome reaction to 9 hr. Scatchard analyses of displacement data suggested an alteration in the binding affinity of ³H-heparin to epididymal sperm membrane following the short-term exposure to seminal plasma. High doses (250 and 500 μg/ml) of heparin, heparan sulfate, and chondroitin-4-sulfate were without effect, but doses <100 μg/ml were stimulatory in terms of enhancing acrosome reactions. Compositional studies with seminal plasma revealed a total GAG content of 1.6 mg/ml, proportioned as 61.6% chondroitin sulfates, 17.6% heparin-like material, 0.3% hyaluronic acid, and 20.5% undetermined GAG. It is proposed that seminal plasma can alter the ability of sperm to respond to GAGs, and the high concentrations of GAGs endogenous to seminal plasma may prevent premature initiation of the membrane perturbations necessary for the acrosome reaction.     

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.     

Proteasome (multicatalytic proteinase) of sea urchin sperm and its possible participation in the acrosome reaction

The egg jelly-induced acrosome reaction of the sea urchin, Strongylocentrotus intermedius, was inhibited by succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA), but not by Suc-Ala-Ala-Pro-Phe-MCA. The proteases with hydrolytic activity toward the former were purified from sperm extract by DEAE-Sephacel and hydroxylapatite chromatographies, Sephacryl S-300 gel filtration, and heparin-Sepharose CL-6B chromatography. Two types of protease were separated, and the molecular weights were estimated to be 65 and 700 kDa, respectively, by gel filtration. The former was accompanied by hydrolytic activity toward Suc-Ala-Ala-Pro-Phe-MCA, which was not hydrolyzed by the latter. Polyacrylamide gel electrophoresis of 700 kDa protease gave a single protein band under nondenaturing conditions and at least eight bands in the range of 22-33 kDa in the presence of sodium dodecyl sulfate (SDS). The substrate specificity and the inhibitor sensitivity of 700 kDa protease indicate that it contains two types of the activity, one is chymotrypsin-type and the other trypsin-type. The former activity was enhanced by poly-L-lysine or SDS. These properties of 700 kDa protease are similar to those of proteasomes (multicatalytic proteinases) isolated from various eukaryotic sources. We had previously shown that inhibitors of chymotrypsin-like proteases inhibit the increase of intracellular Ca2+ concentration by egg jelly, resulting in the inhibition of the acrosome reaction of St. intermedius (Matsumura and Aketa, Gamete Res 23:255-266, 1989). Bringing these findings together, we suggest that the chymotrypsin-like activity of sperm proteasome participates in the onset of the acrosome reaction of St. intermedius.     

Differential sensitivity of progesterone- and zona pellucida-induced acrosome reactions to pertussis toxin

Pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins) have previously been shown to mediate the zona pellucida-induced acrosome reaction in mammalian sperm. In this study we compared the inhibitory effect of pertussis toxin on the zona-induced acrosome reaction in human spermatozoa with that on the reaction induced by progesterone, another physiological acrosome reaction-promoting stimulus associated with the ovulated oocyte. Up to the concentration of 1 μg/ml, pertussis toxin did not produce any direct effects on the acrosome reaction frequency nor did it influence sperm movement and viability. However, preincubation of spermatozoa with the toxin at a concentration of 100 ng/ml completely abolished the increase in the acrosome reaction frequency upon subsequent exposure to solubilized zona pellucida material. In contrast, the same treatment did not impair the ability of spermatozoa to initiate the acrosome reaction in response to progesterone. Moreover, the preincubation with pertussis toxin did not modify the changes in the intracellular concentration of calcium ions occurring after progesterone addition. These data suggest that different physiological stimuli may utilize different signal transduction pathways to induce the human sperm acrosome reaction. © 1993 Wiley-Liss, Inc.     

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.     

Sustained High Protein-tyrosine Phosphatase 1B Activity in the Sperm of Obese Males Impairs the Sperm Acrosome Reaction

Evidence of a causal link between male obesity and subfertility or infertility has been demonstrated previously. However, the mechanism underlying this link is incompletely understood. Here, we report that sustained high protein-tyrosine phosphatase 1B (PTP1B) activity in sperm of obese donors plays an essential role in coupling male obesity and subfertility or infertility. First, PTP1B level and activity were significantly higher in sperm from ob/ob mice than in wild-type littermates. High PTP1B level and activity in sperm was also observed in obese patients compared with non-obese donors. The enhanced sperm PTP1B level and activity in ob/ob mice and obese patients correlated with a defect of the sperm acrosome reaction (AR). Second, treating sperm from male ob/ob mice or obese men with a specific PTP1B inhibitor largely restored the sperm AR. Finally, blockade of sperm AR by enhanced PTP1B activity in male ob/ob mice or obese men was due to prolonged dephosphorylation of N-ethylmaleimide-sensitive factor by PTP1B, leading to the inability to reassemble the trans-SNARE complexes, which is a critical step in sperm acrosomal exocytosis. In summary, our study demonstrates for the first time that a sustained high PTP1B level or activity in the sperm of obese donors causes a defect of sperm AR and that PTP1B is a novel potential therapeutic target for male infertility treatment.