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

OBJECTIVEHeavy metals such as cadmium (Cd) are widely distributed in the environment as industrial pollutants and characterized by their ability to affect the male reproductive system. The objective of the present study was to test the effect of Cd in the concentration range from 10 to 1000 micromol/L, in vitro, on the membrane and DNA integrity, motility, and ability of sperm to undergo acrosomal exocytosis in Holstein bull spermatozoa.

METHODSBull semen samples were processed for sperm analyses using semen-diluting fluid, PBS. Membrane integrity of the processed bull sperm was evaluated by lipoperoxidation (LPO) test. Gelatin digestion test was performed to determine the ability of bull spermatozoa to undergo acrosomal exocytosis. Single cell gel electrophoresis (SCGE) assay was performed to detect the DNA strand breaks and alkali labile damages in the individual cell.

RESULTSWe found a significant increase in the lipoperoxidation (LPO) indicating the deleterious effect of Cd on the sperm membrane integrity. This effect was prominent at the concentration of 1000 micromol/L Cd. There was a negative correlation between LPO rate and the percentage of motile spermatozoa (r = -0.94, P < 0.001). The gelatin digestion test indicated that Cd caused a decline in the percentage of acrosomal exocytosis of bull spermatozoa. A reverse correlation was also found between LPO rate and the percentage of halos (r = -0.97, P < 0.001). Data obtained from the comet assay revealed that Cd was capable of inducing DNA breaks in the sperm nuclei. Almost 93% of DNA damages were double-stranded breaks. The correlation between LPO rate and the percentage of DNA breaks was found to be 0.95 (P < 0.001).

CONCLUSIONCollectively, Cd induced membrane impairments, lowered motility, DNA breaks and a decreased rate in the acrosome reaction of bull spermatozoa, leading to sperm dysfunction. Entering Cd in the male gonads and seminal plasma may exert deleterious effects on the animal sperm cells.

Acrosome Reaction ; Animals ; Cadmium ; toxicity ; Cattle ; DNA Breaks ; Lipid Peroxidation ; Male ; Semen ; drug effects ; Sperm Motility

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

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

OBJECTIVETo find a convenient and exact method for evaluating acrosome reaction in human spermatozoa.

METHODSThe semen of the normal male was mixed and then divided into 6 groups. Coomassie brilliant blue (CBB) staining, chlortetracycline (CTC) fluorescence staining and acid phosphatase (ACP) detection were used for morphological observation and data analysis of the acrosome status of the human sperm treated with or without progesterone.

RESULTSThere were obvious morphological differences between the acrosome-reaction and acrosome-intact spermatozoa in CBB staining and CTC fluorescence staining, and significant differences were observed between the experimental and control spermatozoa by the three methods (P < 0.05).

CONCLUSIONAll the three methods can be used to assess acrosome reaction in human spermatozoa, but Coomassie brilliant blue (CBB) staining is much more convenient and stable.

Acid Phosphatase ; Acrosome Reaction ; drug effects ; Cells, Cultured ; Chlortetracycline ; Humans ; Male ; Progesterone ; pharmacology ; Rosaniline Dyes ; Spermatozoa ; cytology ; Staining and Labeling ; methods

AIMTo investigate if the phytoestrogen, genistein, affects essential functions of cryopreserved bovine spermatozoa.

METHODSThe effect of genistein upon motility was assessed by computer-assisted motion analysis. Hemizona assay was performed to detect the ability of spermatozoa binding to the zona pellucida. The inducibility of the acrosome reaction using progesterone and ZP3-6 peptide was analysed by fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA)/Hoechst 33258 double staining. Capacitation after incubation with genistein was assessed by the chlortetracycline (CTC) assay. Immunoblots showed the pattern of protein tyrosine phosphorylation of cryopreserved bovine spermatozoa.

RESULTSImmunodetection of tyrosine-phosphorylated proteins showed that genistein did not affect tyrosine phosphorylation in cryopreserved bovine spermatozoa. However, genistein significantly reduced the progesterone- and ZP3-6 peptide-mediated induction of the acrosome reaction and led to a dose-dependent inhibition of sperm-zona pellucida binding; while sperm motility and capacitation were not affected by this phytoestrogen, as indicated by computer-assisted sperm motion analysis and the CTC assay, respectively.

CONCLUSIONOur results suggest that in cryopreserved bovine spermatozoa, genistein affects a protein tyrosine phosphorylation-independent signal transduction pathway that is involved in sperm capacitation, the acrosome reaction and sperm-zona pellucida binding.

Acrosome Reaction ; drug effects ; physiology ; Animals ; Cattle ; Enzyme Inhibitors ; pharmacology ; Genistein ; pharmacology ; Male ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Sperm Capacitation ; drug effects ; physiology ; Zona Pellucida ; drug effects ; physiology

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

OBJECTIVETo discuss the important role of actin polymerization in calcium ionophore A23187-induced human acrosome reaction and its mechanism.

METHODSEach spermatozoon specimen was divided into five groups, treated with A23187 3 micromol/L in Group A, Phalloidin 40 micromol/L and A23187 3 micromol/L in Group B, SLO 0.5 U/ml and A23187 3 micromol/L in Group C, SLO 0.5 U/ ml, Phalloidin 40 micromol/L and A23187 3 micromol/L in Group D, and nothing added in Grpup E. Then the percentage of the human acrosome reaction was assessed with Rhodamine-PSA (10 microg/ml).

RESULTSThe difference of the human spermatozoon acrosome reaction was significant (P < 0.01) among the 5 groups with or without SLO, Phalloidin and calcium ionophore A23187 but not between Groups A and B (P > 0.01).

CONCLUSIONPhalloidin does not work on the acrosome reaction of intact human spermatozoa, but in an SLO-permeabilized human spermatozoal model, it can obviously decrease the percentage of human spermatozoon acrosome reaction, which indicates that the polymerization of actin plays an important role in the course of human spermatozoon acrosome reaction, and mostly acts on the acrosome inside.

Acrosome Reaction ; drug effects ; Actins ; physiology ; Bacterial Proteins ; pharmacology ; Calcimycin ; pharmacology ; Cells, Cultured ; Humans ; Ionophores ; pharmacology ; Male ; Phalloidine ; pharmacology ; Spermatozoa ; drug effects ; physiology ; Streptolysins ; pharmacology

OBJECTIVETo observe the effect of Shouwu-Huanjing Recipe(SWHJR) medicated serum on human sperm motility and fertility in vitro.

METHODSHuman sperm was co-cultured with SWHJR medicated serum in vitro. Human sperm motility was evaluated by computer-assisted semen analysis(CASA). The acrosome reaction and the capability of penetrating zona-free hamster eggs were also observed.

RESULTSThe co-cultured SWHJR medicated serum significantly increased the sperm motion velocity(VAP, VCL, VSL) (P < 0.01), the amplitude of lateral head movement (ALH) and the beat frequency of flagellum(BCF), the density of progressive motility sperms (P < 0.05), the acrosome reaction rate(P < 0.001), the fertilization rate(FR) and the fertilization index (FI) in sperm penetration assay(SPA) test (P < 0.01). The stimulation of SWHJR medicated serum occurred in dose-dependent manner.

CONCLUSIONSWHJR can improve human sperm motility and fertility.

Acrosome Reaction ; Animals ; Cricetinae ; Fertility ; drug effects ; Humans ; Male ; Medicine, Chinese Traditional ; Mesocricetus ; Rats ; Rats, Sprague-Dawley ; Sperm Motility ; drug effects

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