Acrosome Reaction In Capacitated Spermatozoa Research Articles

The compound YK 3-237 promotes pig sperm capacitation-related events

Before fertilization of the oocyte, the spermatozoa must undergo through a series of biochemical changes in the female reproductive tract named sperm capacitation. Spermatozoa regulates its functions by post-translational modifications, being historically the most studied protein phosphorylation. In addition to phosphorylation, recently, protein acetylation has been described as an important molecular mechanism with regulatory roles in several reproductive processes. However, its role on the mammal’s sperm capacitation process remains unraveled. Sirtuins are a deacetylase protein family with 7 members that regulate protein acetylation. Here, we investigated the possible role of SIRT1 on pig sperm capacitation-related events by using YK 3-237, a commercial SIRT1 activator drug. SIRT1 is localized in the midpiece of pig spermatozoa. Protein tyrosine phosphorylation (focused at p32) is an event associated to pig sperm capacitation that increases when spermatozoa are in vitro capacitated in presence of YK 3-237. Eventually, YK 3-237 induces acrosome reaction in capacitated spermatozoa: YK 3-237 treatment tripled (3.40 ± 0.40 fold increase) the percentage of acrosome-reacted spermatozoa compared to the control. In addition, YK 3-237 induces sperm intracellular pH alkalinization and raises the intracellular calcium levels through a CatSper independent mechanism. YK 3-237 was not able to bypass sAC inhibition by LRE1. In summary, YK 3-237 promotes pig sperm capacitation by a mechanism upstream of sAC activation and independent of CatSper calcium channel.

Ceramide induces a multicomponent intracellular calcium increase triggering the acrosome secretion in human sperm

Exocytosis of spermatozoon's secretory vesicle, named acrosome reaction (AR), is a regulated event that plays a central role in fertilization. It is coupled to a complex calcium signaling. Ceramide is a multitasking lipid involved in exocytosis. Nevertheless, its effect on secretion is controversial and the underlying cellular and molecular mechanisms remain unknown. Human spermatozoa are useful to dissect the role of ceramide in secretion given that the gamete is not capable to undergo any trafficking mechanisms other than exocytosis. We report for the first time, the presence of sphingolipid metabolism enzymes such as neutral-sphingomyelinase and ceramide synthase in sperm. Ceramidases are also present and active. Both the addition of cell-permeable ceramide and the rise of the endogenous one, increase intracellular calcium acting as potent inducers of exocytosis. Ceramide triggers AR in capacitated spermatozoa and enhances the gamete response to progesterone. The lipid induces physiological ultrastructural changes in the acrosome and triggers an exocytosis-signaling cascade involving protein tyrosine phosphatase 1B and VAMP2. Real-time imaging showed an increment of calcium in the cytosol upon ceramide treatment either in the absence or in the presence of extracellular calcium. Pharmacological experiments demonstrate that at early stages the process involves ryanodine receptors, CatSper (calcium channel of sperm), and store-operated calcium channels.We set out the signaling sequence of events that connect ceramide to internal calcium mobilization and external calcium signals during secretion. These results allow the coordination of lipids and proteins in a pathway that accomplishes secretion. Our findings contribute to the understanding of ceramide's role in regulated exocytosis and fertilization.

Progesterone induces capacitation in human spermatozoa

Mammalian sperm acrosome reaction is a prerequisite for oocyte fertilization, and to date the mechanisms regulating this event are still unclear. Recent studies have demonstrated that human follicular fluid is able to induce an influx of extracellular calcium and acrosome reaction in capacitated spermatozoa. More recently these effects have been attributed to progesterone. The results of our study demonstrated that progesterone is able to trigger the capacitation, acrosome reaction, and fertilizability in human spermatozoa through a calcium mediated mechanism, suggesting a clinical use of this steroid in the treatment of spermatozoa for the different techniques of assisted fertilization.

Nitric oxide induces acrosome reaction in cryopreserved bovine spermatozoa

The aim of this work was to study the effect of nitric oxide on acrosome reaction (AR) and the participation of protein kinases and reactive oxygen species in the AR of cryopreserved bovine spermatozoa. Spermatozoa were capacitated in Tyrode's albumin lactate pyruvate medium with heparin (10 IU ml(-1)) and then incubated with different concentrations of sodium nitroprusside (SNP) (1-200 micromol l(-1)). Methylene blue and haemoglobin were used to confirm the role of nitric oxide as an inducer of the AR. The participation of protein kinase A (PKA) , protein kinase C (PKC) and protein tyrosine kinase was evaluated using specific inhibitors of these enzymes (H-89, 50 micromol l(-1); bisindolylmaleimide I, 0.1 micromol l(-1) and genistein, 3 micromol l(-1)). The role of hydrogen peroxide or superoxide anion was evaluated by incubation with catalase or superoxide dismutase respectively. AR percentages were determined by the fluorescence technique with chlortetracycline. The highest levels of AR were obtained in capacitated spermatozoa treated with 5-200 micromol l(-1) SNP (24.8 +/- 1.8%). The presence of PKA, PKC and protein tyrosine kinase inhibitors likewise decreased AR percentages. The addition of superoxide dismutase had no effect on the AR level but catalase completely blocked it. These results indicate that nitric oxide induces AR in capacitated spermatozoa involving hydrogen peroxide and the participation of PKA, PKC and protein tyrosine kinase as part of the signal transduction mechanism which lead to the AR in cryopreserved bovine spermatozoa.

Induction of the acrosome reaction in bull spermatozoa with plasmin.

Proteolytic enzymes appear to have an essential role in multiple phases of mammalian fertilization. Plasmin, the active enzyme of the plasminogen activation system that stimulates fibrinolysis and proteolysis has a less well-documented role in reproduction. The current study was conducted to investigate the effect of the active protease, plasmin, on the ability of bovine sperm to undergo the acrosome reaction. Aliquots of freshly ejaculated bull sperm were incubated in capacitating conditions with 10 microg ml-1 of heparin for 4 h. Every 2 h an aliquot of spermatozoa was exposed to lysophosphatidylcholine (100 microg ml-1) or 0, 0.1, 1, 10 and 100 mU of plasmin to induce the acrosome reaction in capacitated spermatozoa. Plasmin increased the percentage of live acrosome reacted sperm after 4 h of incubation in the capacitation medium. Viability was not affected by any of the treatments. This study provides new information on bovine acrosome reaction during in vitro incubation with plasmin and indicates that this protease may participate in the proteolytic events that accompany fertilization.

Capacitation and induction of the acrosome reaction in bull spermatozoa with norepinephrine.

Identification of norepinephrine (NE) within the microenvironment of the bovine oviduct suggests a potential role for catecholamines in the events surrounding fertilization. Previous studies have shown that the catecholamines capacitate and induce the acrosome reaction in spermatozoa from several species. The current project was undertaken to investigate the role of catecholamines in bovine sperm capacitation and the acrosome reaction. Freshly ejaculated bovine spermatozoa were incubated in NE (0-1000 ng/mL) and induced to acrosome-react with lysophosphatidylcholine (LPC). Additionally, spermatozoa capacitated with heparin were incubated with NE (0-1000 ng/mL) to assess its ability to induce the acrosome-reaction in capacitated spermatozoa. Concentrations of NE were chosen on the basis of physiological concentrations previously determined for bovine oviductal fluid. NE at concentrations of 10 and 20 ng/mL capacitated bovine spermatozoa after 2 hours of incubation. Additionally, spermatozoa incubated for 2 hours with heparin were induced to acrosome-react with 10 and 20 ng/mL NE. Interestingly, higher concentrations of NE inhibited both capacitation and the acrosome reaction. Incubating spermatozoa with dopamine or epinephrine did not result in capacitation or the acrosome reaction, suggesting that the action of NE was specific to that catecholamine. The ability of NE to capacitate or induce the acrosome reaction appears to be dependent on the presence of another membrane-destabilizing factor. Although adrenergic receptors have not been identified on spermatozoa from any species, the action of NE on spermatozoa may be a receptor-mediated event. This study suggests a possible function for oviductal catecholamines in sperm preparation prior to fertilization.

The cyclic GMP-specific phosphodiesterase inhibitor, sildenafil, stimulates human sperm motility and capacitation but not acrosome reaction.

Capacitation is the series of transformations that spermatozoa undergo in the female genital tract in order to bind to the zona pellucida, initiate the acrosome reaction, and fertilize an egg. Cyclic adenosine monophosphate (cAMP) plays an important role in this process and its levels are regulated by 2 key enzymes, adenylyl cyclase and cyclic nucleotide phosphodiesterase (PDE), the latter being involved in cAMP degradation. Evidence was provided for the involvement of PDE in sperm motility and capacitation. Of the 10 gene families of PDE that exist in mammalian tissues, the calcium-calmodulin-dependent (type 1) and the cAMP-specific (type 4) have been found in human spermatozoa. Using sildenafil, we investigated a highly potent cyclic guanosine monophosphate (cGMP)-specific PDE (type 5) inhibitor and whether this PDE is present in human spermatozoa and is involved in sperm functions. Sildenafil inhibited PDE activity of Percoll-washed spermatozoa with an IC50 of 97+/-3 and 33+/-3 microM when cAMP and cGMP, respectively, were used as substrates. Because the IC50 of sildenafil obtained for PDE type 5 is much lower (2 to 6 nM) than that obtained with sperm PDE, the data suggest that PDE type 5 represents only a small fraction of the whole PDE activity of spermatozoa. Sildenafil causes dose-dependent increases in sperm cAMP levels and capacitation, which are associated with an increase in the levels of tyrosine phosphorylation of 2 fibrous sheath proteins (p105/81). Sperm velocity, amplitude of lateral head displacement, and hyperactivation were increased at 30-180 minutes. Sildenafil did not trigger the acrosome reaction in capacitated spermatozoa. These results suggest that under our experimental conditions, sildenafil triggers human sperm motility and capacitation, probably via its inhibitory action on PDE activity other than type 5 with a resultant rise in cAMP levels.

Functional Sperm Parameters and Fertility of Bull Semen Extended in Biociphos‐Plus® and Triladyl®

ContentsTwenty ejaculates from five dairy AI‐bulls were used to compare, in a split‐sample experiment, the fertility [56 day‐non‐return‐rate (NRR) from more than 14000 AI) and sperm viability post‐thaw of semen diluted with an egg yolk‐ (Triladyl®) or soybean‐based (Biociphos‐Plus®) commercial extender. The in vitro evaluations were divided in two experiments. Experiment 1 (n = 20) included post‐thaw evaluations of motility (subjective and computerized), membrane integrity (CalceinAM/EthD‐1, SYBR‐14/PI, and osmotic resistance test; ORT), and capacitation status (CTC/EthD‐1). Experiment 2 (n = 10) included evaluations of the capacitation‐(CTC/EthD‐1) and acrosome status (FITC‐PSA/EthD‐1) during incubation with/without a challenge with solubilized zona pellucida proteins (SZP). No significant difference in the fertility (69.1 ± 0.8 versus 69.2 ± 0.8) results was found between the two extenders. In experiment 1, the computerized motility evaluations post‐thaw (CASA) showed higher values for Biociphos‐Plus® processed semen for the velocity patterns and lateral sperm head displacement. After 6 h at room temperature (20–22°C) all the CASA motility patterns were significantly higher for Biociphos‐Plus®. The proportion of spermatozoa with intact membranes assessed by CalceinAM was significantly higher in Biociphos‐Plus® (p < 0.001) compared to Triladyl®, but such difference was not seen when using SYBR‐14 or the ORT‐assay. When using the CTC/EthD‐1 assay, a lower proportion of acrosome reacted (AR) spermatozoa post‐thaw (p < 0.01) was found in Biociphos‐Plus® processed semen, as well as a tendency (p < 0.07) for a higher number of uncapacitated spermatozoa. In experiment 2, the proportion of uncapacitated spermatozoa was significantly higher for Biociphos‐Plus® when semen was incubated (38°C and 5% CO2) without SZP at both 0 (p < 0.001) and 30 min (p < 0.05). Concomitantly, Triladyl® showed a higher percentage of capacitated spermatozoa at 0 (p < 0.01), 30 (p < 0.05) and 120 min (p < 0.05). A higher (p < 0.05) incidence of AR‐spermatozoa was seen in Triladyl® at the beginning of the incubation with SZP. No significant difference between extenders was detected for the acrosome status by the FITC‐PSA‐assay. Incubation with SZP induced acrosome reaction of capacitated spermatozoa in both extenders, which was detected by CTC and FITC‐PSA assays. In conclusion, fertility was not affected by Biociphos‐Plus® when 15 × 106 of spermatozoa per AI dose were inseminated. The finding that higher frequencies of spermatozoa seemed more membrane stable post‐thaw, when frozen in Biociphos‐Plus®, might indicate that this extender better protects the sperm viability compared with Triladyl®.

Involvement of reactive oxygen species in human sperm arcosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates.

Although recent evidence indicated that the production of reactive oxygen species (ROS) by human spermatozoa may be involved in the regulation of capacitation, very little is known about the role of ROS in the acrosome reaction. To address this issue, Percoll-washed spermatozoa were incubated in Ham's F-10 medium in the absence (no capacitation) or presence (capacitation) of fetal cord serum ultrafiltrate (FCSu) or progesterone. The effects of the ROS scavengers, superoxide dismutase (SOD), and catalase were then tested on the acrosome reaction induced by lysophosphatidylcholine (LPC), A23187, and ultrafiltrates from follicular fluid (FFu) and FCSu, as well as on the protein tyrosine phosphorylation associated with this process. 2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA)-amplified chemiluminescence was used to determine the extracellular superoxide (O2.-) production from spermatozoa. The observations that both SOD and catalase reduced (in the case of LPC) or totally prevented (in the other cases) the acrosome reaction of capacitated spermatozoa and that hydrogen peroxide (H2O2) or ROS generated by the combination of xanthine and xanthine oxidase (O2.-, which dismutates to H2O2) triggered the acrosome reaction indicated the involvement of ROS in this process. In fact, capacitated spermatozoa in which the acrosome reaction was induced by LPC, A23187, and FFu produced more O2.- than noncapacitated spermatozoa treated with the same agents. A23187 and LPC had minor effects on protein tyrosine phosphorylation of noncapacitated spermatozoa. However, these inducers caused a decrease in tyrosine phosphorylation of Triton-soluble proteins (mainly those of 37, 42, and 47 kDa) from capacitated spermatozoa, a decrease more pronounced in the presence of SOD. On the other hand, there was a marked increase in tyrosine phosphorylation of few proteins (70 to 105 kDa) from the Triton-insoluble fraction, which was partly reversed by SOD (in the case of LPC and A23187) or catalase (in the case of A23187), or abolished in the presence of the two antioxidants (in the case of A23187). These data indicate that the acrosome reaction is associated with an extracellular O2.- generation by spermatozoa and that both O2.- and H2O2 may be involved in the regulation of this process. The mechanism by which these ROS act is unknown but may involve tyrosine phosphorylation of sperm proteins.

R-025. Effect of peritoneal fluid from fertile women and from infertile women with endometriosis or unexplained infertility on the acrosome reaction of capacitated spermatozoa

R-025. Effect of peritoneal fluid from fertile women and from infertile women with endometriosis or unexplained infertility on the acrosome reaction of capacitated spermatozoa

Atrial natriuretic peptide induces acrosomal exocytosis in bovine spermatozoa

The induction of acrosomal exocytosis in capacitated bull spermatozoa by atrial natriuretic peptide (ANP) was studied in vitro. ANP markedly stimulated acrosomal exocytosis in a calcium-dependent manner. Typically, ANP exerts its action via activation of the ANP receptor (ANPR-A), a particulate guanylyl cyclase-linked receptor, and subsequent formation of guanosine 3',5'-cyclic monophosphate (cGMP). We found that the ANP-induced acrosome reaction was inhibited by the competitive ANPR-A receptor antagonist-anantin, indicating a receptor-mediated effect. We could mimic the effect of ANP on the acrosome reaction by using 8-bromo-cGMP, suggesting that cGMP may serve as a signal transducer mediating the acrosome reaction. Indeed, the ANP-induced acrosome reaction was associated with elevation of cGMP levels. cGMP can also be formed by activation of the soluble form of guanylyl cyclase. Sodium nitroprusside (SNP) stimulated cGMP accumulation and acrosome reaction of capacitated spermatozoa. Thus ANP and the nitric oxide-releasing compound SNP, via activation of guanylyl cyclase (the former activating the particulate and the latter activating the soluble form of the enzyme), may play a significant role in the induction of the acrosome reaction.

Comparison of four staining methods for evaluating acrosome status and viability of ejaculated and cauda epididymal bull spermatozoa

Four staining procedures for simultaneously assessing viability and acrosomal integrity of bovine, ejaculated and cauda epididymal spermatozoa (CES) were evaluated. Fast green FCF/eosin B (FG/EB), eosin B-aniline blue (E-Ab), trypan blue/Giemsa (TB/G), and propidium iodide/Pisum sativum agglutinin (PI/PSA) were compared with naphthol yellow and erythrosin B (NY/EB) and E-Ab as standards for acrosomal integrity and viability, respectively. Both ejaculated and CES samples were incubated with heparin (10 μg/ml; 5 % CO 2/air; 39 °C) for 4 h. Lysophosphatidylcholine was added to spermatozoa to induce the acrosome reaction in capacitated spermatozoa. The sperm cells were then stained by the different methods and evaluated as either intact live, intact dead, acrosome-reacted live, or acrosome-reacted dead. The results indicated that FG/EB, E-Ab and PI/PSA stains provided an accurate assessment of both viability and acrosomal integrity for ejaculated spermatozoa. Although PI/PSA was a reliable method of determining viability and acrosomal integrity of ejaculated and cauda epididymal sperm cells, it was not suitable for use with spermatozoa capacitated with heparin due to sperm agglutination. The E-Ab stain, which was originally used to determine viability only, was also found to be suitable for determining acrosomal integrity of both ejaculated and CES, when differential interference contrast microscopy was used.

Characterization of two second messenger pathways and their interactions in eliciting the human sperm acrosome reaction.

The human sperm acrosome reaction (AR) occurs via the activation of at least two signal transduction pathways. The purpose of this investigation was to characterize two of the pathways, the protein kinase A (PKA) and C (PKC) pathways, and determine whether pathway "crosstalk" occurs between them in eliciting the AR in capacitated spermatozoa. Stimulators of each pathway were tested in a dose-dependent manner. ARmax, ED50, and delta ARmax (%ARmax-%ARcontrol) values were calculated. The PKA pathway stimulators forskolin and dibutyryl cyclic AMP (dbcAMP) induced an ARmax at 1.0 microM and 1.0 mM, respectively. The ED50 and delta ARmax values were: 0.01 microM and 17% for forskolin and 0.069 mM and 13% for dbcAMP. Two stimulator types of the PKC pathway were tested: synthetic diacylglycerols (DG) and a phorbol diester. 1,2-dioleoyl-sn-glycerol and 1,2-dioctanoyl-sn-glycerol, analogues of the PKC-activating second messenger DG, each induced an ARmax at 50 microM. The ED50 and delta AR max values were: 33 microM and 24% for 1,2-dioleoyl and 34.8 microM and 34% for 1,2-dioctanoyl. 4 beta-Phorbol-12,13-didecanoate, a PKC stimulator, induced an ARmax at 0.1 microM. The ED50 and delta ARmax were 0.021 microM and 26%. An inhibitor of each kinase was added at the end of the capacitation period and prior to stimulation by inducers at their ARmax dose. KT5720, a PKA inhibitor, caused a dose-dependent reduction of the forskolin and dbcAMP-induced AR. Calphostin C, a PKC inhibitor, prevented stimulation of the AR by 1,2-dioleoyl and 4 beta-phorbol-12,13-didecanoate. To investigate pathway "crosstalk," the following experiments were conducted: (1) stimulators of each pathway were combined and tested at the ARmax and ED50 concentrations for each; (2) spermatozoa were pretreated with a kinase inhibitor and then stimulated using an alternative pathway stimulator; and (3) a PKA or PKC inhibitor and a combination of PKA and PKC stimulators, at ED50 concentrations, were tested. The results for (1) indicate an additive AR response of ED50 concentrations but not for ARmax doses. The results for (2) demonstrate that a kinase inhibitor for one pathway prevents induction of the AR by a stimulator of the alternative pathway. Finally, the results for (3) show that a kinase inhibitor for one pathway prevents induction of the AR by the combined use of separate pathway stimulators. When taken collectively, the present results suggest a convergent mechanism of crosstalk between the PKA and PKC pathways leading to the human sperm AR.

Atrial natriuretic peptide (ANP) as a stimulus of the human acrosome reaction and a component of ovarian follicular fluid: correlation of follicular ANP content with in vitro fertilization outcome.

Atrial natriuretic peptides (ANPs) from several species induced the human acrosome reaction. The maximal response was highest for human ANP (18.6% above unstimulated or baseline values) and decreased progressively for peptides derived from animals lower on the phylogenetic scale. ANP concentrations required for a half-maximal effect in noncapacitated spermatozoa ranged from 0.07 to 0.38 nM. ANP induced the acrosome reaction in capacitated spermatozoa, but the concentration required was higher than in noncapacitated cells. The response in noncapacitated spermatozoa was independent of added extracellular Ca2+ and was completely inhibited by 1 microM LY83583 (inhibits particulate guanylate cyclase). However, 10 microM N omega-nitro-L-arginine (inhibits soluble guanylate cyclase) had no effect. ANP (80 pM) and 3 microM 1,2-dihexanoyl-sn-glycerol each induced a nearly half-maximal acrosome reaction. Added in combination, they produced no increased response, suggesting antagonism. Follicular fluid had variable levels of immunoreactive ANP. Average ANP content was nearly zero in samples that contained no oocyte at the time of aspiration but was higher (6.9 pM; 90% confidence limits = 1.67-28.72 pM) in follicular fluid containing oocytes that did not fertilize in vitro. Highest concentrations of ANP were present in follicular fluid containing oocytes that fertilized in vitro (72.8 pM; 90% confidence limits = 38.1-139.1 pM). These data suggest that noncapacitated spermatozoa can acrosome react without added extracellular Ca2+ in response to an extracellular ligand. Also, human spermatozoa appear to contain receptors for ANP similar to those found in other cell types. The ANP content of follicular fluid might partly explain the ability of follicular fluid to induce the acrosome reaction.

Head-specific mannose-ligand receptor expression in human spermatozoa is dependent on capacitation-associated membrane cholesterol loss.

D-Mannose binding lectins appear on the human sperm head following in-vitro incubation under capacitating conditions. Surface expression of lectin is dependent on reduction of the sterol content of sperm membranes. Mannose-specific lectin distribution over the head differs in acrosome-intact and acrosome-reacted spermatozoa. Sugar competition experiments show that mannose is the only saccharide competitive with D-mannosylated albumin binding and that free mannose induces an acrosome reaction in capacitated spermatozoa. A total of 183 men with normozoospermic semen parameters were screened for the ability of their spermatozoa to bind fluorescein isothiocyanate (FITC)-labelled mannosylated albumins, before and after incubation in vitro. The spermatozoa from 176 men 'responded' to incubation by exhibiting time-dependent increases in head-directed mannosylated albumin binding, accompanied by increases in the percentage of spermatozoa showing spontaneous acrosome loss. Motile spermatozoa from the remaining seven men failed to express mannose-lectin binding activity after 18 h of incubation and only low percentages of their spermatozoa showed spontaneous acrosome loss. These seven men were classified as 'non-responders'. The relative amounts of non-esterified cholesterol in the sperm membranes of the responder and non-responder males were analysed by gas-liquid chromatography. Responder spermatozoa showed decreases in free cholesterol content whereas non-responder spermatozoa exhibited either no decrease or increases in relative free cholesterol per cell. Fresh swim-up spermatozoa contain sub-plasma membrane stores of mannose lectins which are revealed by FITC-mannosylated albumin labelling before and after removal of the plasma membrane by vortexing. In contrast, the mannose-lectin binding activity of capacitated spermatozoa is entirely limited to the sperm surface. Western blots of proteins isolated from sperm plasma membranes after capacitation revealed two molecular species reactive with polyclonal antibodies against human macrophage mannose receptors. A model is proposed for the molecular mechanism whereby mannose lectins are transposed from sub-plasma membrane sites to an integral membrane position as a consequence of the loss of cholesterol from the sperm membrane.

Modulation of the human sperm acrosome reaction by effectors of the adenylate cyclase/cyclic AMP second-messenger pathway.

The acrosome reaction of spermatozoa appears to be analogous to various somatic cell exocytotic events which involve cascade reactions, i.e., transmission of an external signal across the cell membrane resulting in activation of an "amplifier" enzyme and the generation of a second messenger. Using a synchronous acrosome reaction system (De Jonge et al., J. Androl., 10:232-239, '89a), it was found that analogues of the second-messenger cAMP, dibutyryl cAMP (dbcAMP) and 8-bromo cAMP, stimulated the acrosome reaction of capacitated spermatozoa. Additionally, treatment of spermatozoa with either xanthine or non-xanthine phosphodiesterase inhibitors induced a significant (P less than 0.05) increase in the percent acrosome reaction after a period of capacitation in comparison to untreated controls. These results indicate that analogues of cAMP or inhibitors which prevent cAMP hydrolysis can induce the human sperm acrosome reaction. Subsequent experiments were conducted to test whether the amplifier enzyme in the cascade reaction, adenylate cyclase, has a role in the acrosome reaction. Forskolin, an adenylate cyclase stimulator, caused a significant (P less than 0.01) increase in the percent acrosome reaction in comparison to controls. Modulators of adenylate cyclase--adenosine, 2'-0-methyladenosine, and 2',3'-dideoxyadenosine--significantly (P less than 0.01) inhibited the forskolin-induced acrosome reaction. dbcAMP was able to overcome the inhibition by adenosine. Two inhibitors of protein kinase A, the Walsh inhibitor and H-8, caused a significant (P less than 0.01) inhibition of the dbcAMP-induced acrosome reaction. Finally, in the absence of extracellular calcium, dbcAMP induced a significant (P less than 0.01) increase in the acrosome reaction in contrast to A23187. These results suggest that: 1) a molecular mechanism for the human sperm acrosome reaction involves the cAMP second-messenger system; i.e., activation of adenylate cyclase, the amplifier enzyme that produces cAMP, production of cAMP as a second messenger, and activation of cAMP-dependent kinase A; and that 2) activation of adenylate cyclase occurs after calcium influx.

Effect of phorbol diesters, synthetic diacylglycerols, and a protein kinase C inhibitor on the human sperm acrosome reaction.

The acrosome reaction of spermatozoa may be analogous to various somatic cell exocytotic events that incorporate cascade reactions. One such cascade system involves the hydrolysis of a membrane-bound phospholipid; generation of the intracellular second messenger, diacylglycerol; and activation of protein kinase C, followed by the phosphorylation of a number of intracellular proteins. Stimulators of protein kinase C, phorbol diesters and synthetic diacylglycerols, were evaluated to determine if this system functions in the human sperm acrosome reaction. Phorbol 12-myristate 13-acetate and 4 beta-phorbol 12,13-didecanoate caused a significant (P less than 0.01) increase in the acrosome reaction of capacitated spermatozoa. Conversely, an inactive phorbol diester had no significant (P greater than 0.05) stimulatory effect on the acrosome reaction. The synthetic diacylglycerols, 1-oleoyl-2-acetyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, and 1,2-dioleoyl-sn-glycerol caused a significant (P less than 0.01) increase in the acrosome reaction of capacitated spermatozoa, and to a similar extent as the phorbol diesters. A nonactivating isomer of 1,2-dioleoyl-sn-glycerol, 1,3-diolein, had no significant (P greater than 0.05) stimulatory effect on the acrosome reaction. Protein kinase C activation is a diacylglycerol-dependent and Ca2(+)-dependent process, and stimulation of the acrosome reaction by 1,2-dioctanoyl-sn-glycerol required the presence of calcium ions in the capacitation medium. An inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), prevented the diacylglycerol-induced acrosome reaction (P less than 0.01). These results support the hypothesis that protein kinase C, via activation by the intracellular second messenger diacylglycerol, has a role in the human sperm acrosome reaction.

Participation of human epididymal sperm coating antigens in fertilization.

A polyclonal antiserum directed against human sperm coating proteins of epididymal origin (anti-KCl) was tested for its ability to alter sperm function. Spermatozoa from normal ejaculates were selected by swim-up separation and capacitated by overnight incubation at room temperature. Exposure of these cells to anti-KCl (0.39 mg protein/ml), prior to their use in the hamster ova penetration test, reduced the penetration of denuded oocytes by 65% (P less than 0.005). Significant inhibitions of lesser magnitude were observed at lower serum concentrations (to 0.098 mg/ml). In an effort to understand the mechanism of this inhibition, other sperm function parameters thought to be related to oocyte penetration were studied. The inhibitory effect was exerted without noticeable changes in sperm motility (determined by the percentage of motile cells and their linear velocity), and in the absence of major sperm agglutination. Anti-KCl did not inhibit the occurrence of spontaneous or induced (by human follicular fluid) acrosome reaction in capacitated spermatozoa. In contrast, exposure to anti-KCl reduced the ability of capacitated spermatozoa to bind tightly to the hamster oolemma. None of these effects were elicited by a control preparation obtained from pre-immune rabbit sera. Exposure of zona-free oocytes to the antiserum did not alter their penetrability by normal sperm. These results suggest that the antigens recognized by anti-KCl participate in some specific step of the sperm-ovum interaction.

Zona-induced acrosome reaction of hamster spermatozoa.

It is well established that the zonae pellucidae of mature unfertilized eggs have the ability to induce the acrosome reaction of capacitated spermatozoa. To determine if this capacity of the zona is species-specific, hamster spermatozoa were allowed to attach to the zonae of homologous and heterologous eggs and examined for the acrosome reaction. The zonae of eggs from six different species were tested and the zona of hamster egg was found to have the strongest capacity to induce the acrosome reaction of hamster spermatozoa, followed by human and rat zonae. The zonae and mouse, guinea pig, and domestic fowl eggs were incapable of inducing the acrosome reaction of hamster spermatozoa. The acrosome reaction-inducing ability of the hamster zona was found to increase during maturation in the ovary. The zona of mature unfertilized hamster eggs maintained their acrosome reaction-inducing ability even after aldehyde fixation or storage in a highly concentrated solution of ammonium sulfate.

Albumin is required to support the acrosome reaction but not capacitation in mouse spermatozoa in vitro.

Albumin was required specifically for penetration of the zona pellucida (less than 10% of eggs fertilized in the absence of albumin), but was not required for capacitation. A similar rate of capacitation was observed in the presence of albumin at concentrations ranging from 30 to 1 mg/ml, while a slightly slower rate was observed in the presence of 0.25 and 0.1 mg albumin/ml. In the absence of albumin, capacitation occurred at a rate which lagged behind that of the albumin-incubated counterparts by about 30 min; once capacitated, the addition of albumin promoted rapid sperm penetration. In albumin-free media (+/- the macromolecule PVA), sperm motility was frequently reduced, with fewer cells exhibiting hyperactivated motility, but improvements were observed after introduction of albumin. Acrosome loss was significantly lower in the absence of albumin, but within 5 min of its addition at concentrations ranging from 30 to 0.1 mg/ml to capacitated sperm suspensions, acrosome loss was stimulated and reached levels similar to those seen in control samples. Therefore, albumin can trigger the acrosome reaction in capacitated spermatozoa. It appears to act by assisting in the removal of a surface-associated inhibitory component, the presence of which stabilizes the sperm membranes and inhibits the acrosome reaction.