To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l^-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l^-1), whereas higher concentrations (>5 μmol l^-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca²⁺-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.
8-Bromo Cyclic Adenosine Monophosphate/pharmacology* ; Acrosome/metabolism* ; Acrosome Reaction/drug effects* ; Calcimycin/pharmacology* ; Cyclic AMP/metabolism* ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors* ; Exocytosis/drug effects* ; Guanine Nucleotide Exchange Factors/metabolism* ; Humans ; Male ; Protein Kinase Inhibitors/pharmacology* ; Signal Transduction/drug effects* ; Spermatozoa/metabolism* ; Thapsigargin/pharmacology*

Stress affects the male reproductive system and can cause sub-fertility or infertility. Although Phyllanthus emblica L. (PE) extract has been shown to have high antioxidant capacity and protective properties in damaged tissue, the preventive effects of PE extract on testicular function from stress-related impairment have never been demonstrated. This study aimed to investigate the effects of PE aqueous leaf extract on testicular impairment and protein marker changes in rats suffering from chronic stress. Adult male rats were divided into four groups: a control group, a chronic stress (CS) group, and two groups with CS that received different doses of PE extract (50 or 100 mg/kg body weight (BW)). In the treatment groups, the animals were given PE extract daily before stress induction for 42 consecutive days. Stress was induced through immobilization (4 h/d) followed by forced cold swimming (15 min/d). Sperm quality and the histology of the testes and caudal epididymis were examined, as were levels of serum corticosterone, testosterone, and malondialdehyde (MDA). The expressions of testicular steroidogenic acute regulatory (StAR) and tyrosine-phosphorylated proteins were investigated using immuno-Western blot analysis, as these proteins are assumed to play important roles in spermatogenesis and androgen synthesis. The results showed that PE (50 mg/kg BW) significantly increased sperm concentration and testosterone levels, while decreasing corticosterone levels, MDA levels, sperm head abnormalities, and acrosome-reacted sperm in CS rats. In addition, PE at both doses was found to diminish testicular histopathology in the CS rats. We also found that 50 mg/kg BW of PE significantly improved StAR protein expression and altered the intensities of some tyrosine-phosphorylated proteins in testis. We conclude that PE leaf extract at 50 mg/kg BW can prevent testicular damage in rats with CS.
Acrosome Reaction ; Animals ; Antioxidants/pharmacology* ; Corticosterone/blood* ; Epididymis/metabolism* ; Male ; Malondialdehyde/blood* ; Phosphoproteins/metabolism* ; Phosphorylation ; Phyllanthus emblica/chemistry* ; Plant Extracts/pharmacology* ; Plant Leaves/chemistry* ; Rats ; Rats, Sprague-Dawley ; Sperm Count ; Spermatogenesis/drug effects* ; Spermatozoa/drug effects* ; Stress, Physiological ; Testis/drug effects* ; Testosterone/blood* ; Tyrosine/chemistry*

OBJECTIVETo study the effects of nonylphenol and cadmium on acrosome reaction in vitro in mouse spermatozoa.

METHODSSperm were collected from the vas deferens of mice, capacitated in vitro and stimulated with A23187 at 30 micromol/L to induce acrosome reaction. Then the sperm suspension was treated with nonylphenol at 10, 20, 30, 60 and 100 micromol/L or cadmium at 500, 2500 and 5 000 micromol/L, and the control group treated with the carrier solvent. Acrosome reaction of the sperm was analyzed by FITC-PSA staining.

RESULTSCompared with the control group, nonylphenol significantly inhibited acrosome reaction at the concentration of > 60 micromol/L (P < 0.01), but not at < 30 micromol/L (P > 0.05), and the sperm survival rate was reduced with increased concentration of nonylphenol. However, cadmium exhibited no significant influence on either acrosome reaction (P > 0.05) or sperm survival rate at 500 - 5 000 micromol/L.

CONCLUSIONNonylphenol and cadmium affect the spermatogenesis of mice in different ways; the former directly inhibits sperm acrosome reaction, while the latter has no direct effect on it.

Acrosome ; drug effects ; Acrosome Reaction ; drug effects ; Animals ; Cadmium ; pharmacology ; In Vitro Techniques ; Male ; Mice ; Mice, Inbred C57BL ; Phenols ; pharmacology ; Spermatozoa ; drug effects

OBJECTIVE: To investigate the relationship between sperm acrosome reaction (AR) and the clinical pregnancy rate of intrauterine insemination (IUI). METHODS: We detected the sperm spontaneous AR rate and Ca2+ ionophore A23187-induced AR rate in 128 patients who accepted IUI treatment, collected their clinical data and analysed the relationship between sperm AR rate and clinical pregnancy rate of IUI. RESULTS: There was no statistical difference between the spontaneous AR rates in the pregnant group and the non-pregnant group (7.7% vs. 7.0%, P>0.05), but there was statistical difference between the induced AR rates(51.9 % vs. 43.5%, P<0.05). There was statistical difference in the clinical pregnancy rate among the 3 IUI groups divided by induced AR rate (≤20.0%, 20.1%-49.9%, and ≥50.0%; 4.8% vs. 12.5% vs. 18.6%, P<0.05). CONCLUSION The spontaneous AR rate has nothing to do with the clinical pregnancy rate of IUI, but the induced AR rate is associated with the clinical pregnancy rate of IUI.
Acrosome Reaction ; drug effects ; physiology ; Adult ; Calcimycin ; pharmacology ; Female ; Humans ; Infertility ; therapy ; Insemination, Artificial, Homologous ; methods ; Male ; Pregnancy ; Pregnancy Rate ; Spermatozoa ; physiology

Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO3-, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca2+ mobilization in sperm, which can be mimicked by the PLC inhibitor, U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.
Acrosome ; drug effects ; metabolism ; Acrosome Reaction ; physiology ; Adult ; Animals ; Calcium ; metabolism ; Fluorescent Antibody Technique, Indirect ; Humans ; Immune Sera ; pharmacology ; Male ; Mice ; Middle Aged ; Phosphoinositide Phospholipase C ; immunology ; metabolism ; Sperm Capacitation ; drug effects ; physiology ; Spermatozoa ; drug effects ; metabolism

In vitro supplementation with date seed oil (DSO) can protect spermatozoa against hydrogen peroxide (H2O2)-mediated damage and can improve sperm function, possibly owing to antioxidant properties. We tested the antioxidant effects of DSO on human sperm motility, sperm viability, reacted acrosome and lipid peroxidation assessed in vitro after H2O2-mediated oxidative damage in spermatozoa. Sixteen patients (mean age: 35 years; range: 25-45 years) referred to the Histology-Embryology Laboratory of the Medicine Faculty of Sfax for semen analysis after 12-24 months of sexual intercourse without conception were selected. After spermiogram, sperm selection by two-interface discontinuous Sill Select gradient was performed, and selected spermatozoa were used in four experimental assays: control; incubation with 100 microm H2O2; incubation with 0.1% DSO; and co-incubation with 0.1% DSO and 100 microm H2O2. Motility and viability were determined using World Health Organization criteria. Acrosome reaction and lipid peroxidation were assessed by staining with fluorescein isothiocyanate-Pisum sativum and spectrophotometric measurement of malondialdehyde, respectively. Results showed that incubation with H2O2 alone led to a significant increase in lipid peroxidation (57.83%, P<0.05) associated with a significant decrease in sperm motility, sperm viability (after 30 min and 24 h) and percentage of reacted acrosome (P<0.05). Date seed oil improved sperm motility after 24 h of incubation (P<0.05) and protected spermatozoa against the deleterious effects of H2O2 on motility, viability, acrosome reaction and lipid peroxidation. We conclude that supplementation with DSO may have a function in antioxidant protection against male infertility.
Acrosome Reaction ; drug effects ; Adult ; Arecaceae ; Cell Survival ; drug effects ; Humans ; Hydrogen Peroxide ; pharmacology ; In Vitro Techniques ; Lipid Peroxidation ; drug effects ; Male ; Middle Aged ; Oxidants ; pharmacology ; Oxidative Stress ; drug effects ; Plant Oils ; pharmacology ; Seeds ; Sperm Motility ; drug effects ; Spermatozoa ; cytology ; drug effects ; metabolism

OBJECTIVETo investigate the difference in the responsiveness of intracellular free Ca2+ concentration ([Ca2+]i) to progesterone in the spermatozoa of normal fertile men and patients with unexplained infertility.

METHODSNine normal fertile men and 10 patients with unexplained infertility were selected in this study. After swim-up separation of the motile fraction and 2-hour in vitro capacitation, the spermatozoa were loaded with the fluorescent calcium indicator Fluo-3/AM (8.85 micromol/L) for 40 minutes away from the light, and then the sperm suspension was mixed with equal amount of 20% gelatin to immobilize the spermatozoa. The basal intracellular free [Ca2+]i and that induced by 10 micromol/L progesterone in the individual sperm were assessed by laser scanning confocal microscopy.

RESULTSThe infertile patients had a significantly lower basal level of [Ca2+]i in the capacitated sperm than the fertile men (P < 0.01). The sperm from the normal controls responded to progesterone by exhibiting a rapid but transient rise in [Ca2+]i, with the peak level significantly higher than the basal level (P < 0.05), while those from the infertile patients by showing a slight increase, with no significant difference between the peak and basal levels (P > 0.05). Both the peak of the progesterone-induced [Ca2+]i and its increase amplitude expressed as the difference between the peak and basal levels were significantly higher in the normal fertile group than in the infertile patients (P < 0.01).

CONCLUSIONThe responsiveness of [Ca2+]i to progesterone is reduced in the spermatozoa of patients with unexplained infertility, which suggests a functional defect in the non-genomic sperm membrane progesterone receptor responsible for calcium influx.

Acrosome Reaction ; drug effects ; Adult ; Calcium ; analysis ; Case-Control Studies ; Female ; Humans ; Infertility, Male ; physiopathology ; Male ; Progesterone ; pharmacology ; Spermatozoa ; drug effects ; Young Adult

AIMTo determine the cellular distribution of secretory phospholipase A(2) (sPLA(2)) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes.

METHODSAcrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA(2) and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used.

RESULTSAlthough sPLA(2) was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA(2) was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA(2), disturbance of acrosome structure, and loss of vitality.

CONCLUSIONThe ability of spermatozoa to release secretory phospholipase A(2) is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA(2) can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA(2) in intact spermatozoa might be an additional parameter for evaluating sperm quality.

Acrosome ; drug effects ; physiology ; Acrosome Reaction ; drug effects ; Annexin A5 ; metabolism ; Anti-Bacterial Agents ; pharmacology ; Calcimycin ; pharmacology ; Ethidium ; Flow Cytometry ; Fluorescent Dyes ; Humans ; In Vitro Techniques ; Male ; Microscopy, Confocal ; Pancreatic Elastase ; metabolism ; Phosphatidylserines ; metabolism ; Phospholipases A2, Secretory ; metabolism ; Semen ; cytology ; drug effects ; Spermatozoa ; drug effects ; enzymology

AIMTo investigate the in vitro effects of insulin and leptin on human sperm motility, viability, acrosome reaction and nitric oxide (NO) production.

METHODSWashed human spermatozoa from normozoospermic donors were treated with insulin (10 microIU) and leptin (10 nmol). Insulin and leptin effects were blocked by inhibition of their intracellular effector, phosphotidylinositol 3-kinase (PI3K), by wortmannin (10 micromol) 30 min prior to insulin and leptin being given. Computer-assisted semen analysis was used to assess motility after 1, 2 and 3 h of incubation. Viability was assessed by fluorescence-activated cell sorting using propidium iodide as a fluorescent probe. Acrosome-reacted cells were observed under a fluorescent microscope using fluorescein-isothiocyanate-Pisum sativum agglutinin as a probe. NO was measured after treating the sperm with 4,5-diaminofluorescein-2/diacetate (DAF-2/DA) and analyzed by fluorescence-activated cell sorting.

RESULTSInsulin and leptin significantly increased total motility, progressive motility and acrosome reaction, as well as NO production.

CONCLUSIONThis study showed the in vitro beneficial effects of insulin and leptin on human sperm function. These hormones could play a role in enhancing the fertilization capacity of human spermatozoa.

Acrosome Reaction ; drug effects ; Cell Survival ; drug effects ; Flow Cytometry ; Humans ; Hypoglycemic Agents ; pharmacology ; In Vitro Techniques ; Insulin ; pharmacology ; Leptin ; pharmacology ; Male ; Nitric Oxide ; metabolism ; Sperm Motility ; drug effects ; Spermatozoa ; drug effects ; metabolism

AIMTo develop a method for assessing sperm function by measuring released acrosin activity during the acrosome reaction (AR).

METHODSHuman semen samples were obtained from 24 healthy donors with proven fertility after 3-7 days of sexual abstinence. After collection, samples were liquefied for 30 min at room temperature. Standard semen parameters were evaluated according to World Health Organization (WHO) criteria. Calcium ionophore A23187 and progesterone (P4) were used to stimulate the sperm to undergo AR. After treatment, sperm were incubated with the supravital dye Hoechst33258, fixed in a glutaraldehyde-phosphate-buffered saline solution, and the acrosomal status was determined by fluorescence microscopy with fluorescein isothiocyanate-labeled Pisum sativum agglutinin (FITC-PSA). The percentage of sperm undergoing AR (AR%) was compared to sperm acrosin activities as assessed by spectrocolorimetry. The correlation between AR% and acrosin activity was determined by statistical analysis.

RESULTSThe AR% and released acrosin activity were both markedly increased with A23187 and P4 stimulation. Sperm motility and viability were significantly higher after stimulation with P4 versus stimulation with A23187 (P < 0.001). There was a significant positive correlation between released acrosin activity and AR% determined by FITC-PSA staining (r=0.916, P < 0.001).

CONCLUSIONSpectrocolorimetric measurement of released acrosin activity might serve as a reasonable alternative method to evaluate AR.

Acrosin ; physiology ; Acrosome Reaction ; Adult ; China ; Humans ; Male ; Progesterone ; pharmacology ; Semen ; drug effects ; physiology ; Sperm Motility ; drug effects ; physiology