OBJECTIVETo search for an optimal protocol and freezing conditions for the cryopreservation of microamount round spermatids of the mouse.

METHODSWe compared the survival rates of frozen-thawed microamount round spermatids of the mouse achieved by vitrification or standard slow freezing with different concentrations of glycerol (5, 7, or 9%) and different lengths of equilibrium time (0, 15, 30, 45, or 60 min).

RESULTSUnder the conditions of 7% glycerol and 30 min equilibrium, both vitrification and standard slow freezing achieved high survival rates of spermatids, and the former obtained an even higher rate than the latter ([72.9 ± 15.4]% vs [58.2 ± 17.7]%, P < 0.05).

CONCLUSIONA high rate of frozen-thawed microamount round spermatids of the mouse can be achieved by vitrification under the conditions of 7% glycerol and 30 min equilibrium.

Animals ; Cell Survival ; Cryopreservation ; methods ; Cryoprotective Agents ; administration & dosage ; pharmacology ; Glycerol ; administration & dosage ; pharmacology ; Male ; Mice ; Spermatids ; Time Factors ; Vitrification ; drug effects

OBJECTIVETo search for an optimal activation protocol by comparing the chemical activation effects of single-activator and combined activation protocols on mouse oocytes following injection of round spermatids (ROSI) from spermatogenic cells cultured in vitro.

METHODSUsing different concentrations of ethanol, ionomycin (Ion), calcium ionophore A23187 (CIA), strontium chloride (SrCl2), cycloheximide (CHX), and 6-dimethylaminopurine (6-DMAP) , we activated post-ROSI oocytes for different times, and activated them by combined protocols at optimal concentrations and action times according to different activation channels. We compared the activation effects of single-activator and combined activation protocols by comparing the rates of fertilization, cleavages, and morulas and blastocysts.

RESULTSWith a single activator, the optimal protocols of different activators were as follows: 7% ethanol for 6 min, 5 micromol/L CIA for 5 min, 5 micromol/L Ion for 5 min, 2 mmol/L 6-DMAP for 2 h, 10 mmol/L SrCl2 for 1.5 h, and 10 microg/ml CHX for 1.5 h, among which 10 mmol/L SrCl2 for 1.5 h achieved the highest rate of morulas and blastocysts, significantly better than CHX (P < 0.05) but with no remarkable difference from other activators. The ethanol + 6-DMAP group showed a significantly higher rate of morulas and blastocysts (29.63%) than all other combined activation groups and single-activator groups except SrCl2 (P < 0.05), and it also exhibited higher rates of normal fertilization, cleavages and morula than the SrCl2 group, but with no significant difference.

CONCLUSIONThe single-activator 10 mmol/L SrCl2 for 1.5 h and the combined activation of 7% ethanol for 6 min + 2 mmol/L 6-DMAP for 2 h are the optimal protocols for chemical activation of mouse oocytes following ROSI, and the combined activation of ethanol + 6-DMAP is even superior to the single-activator protocol.

Animals ; Cycloheximide ; pharmacology ; Female ; Fertilization in Vitro ; Ionomycin ; pharmacology ; Male ; Mice ; Mice, Inbred Strains ; Oocytes ; cytology ; drug effects ; Spermatids ; cytology ; drug effects

The dynamic polar polymers actin filaments and microtubules are usually employed to provide the structural basis for establishing cell polarity in most eukaryotic cells. Radially round and immotile spermatids from nematodes contain almost no actin or tubulin, but still have the ability to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein (MSP) during spermiogenesis (sperm activation). However, the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly understood. Here we show that Ca(2+) oscillations induced by the Ca(2+) release from intracellular Ca(2+) store through inositol (1,4,5)-trisphosphate receptor are required for Ascaris suum sperm activation. The chelation of cytosolic Ca(2+) suppresses the generation of a functional pseudopod, and this suppression can be relieved by introducing exogenous Ca(2+) into sperm cells. Ca(2+) promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly. On the other hand, Ca(2+) promotes MSP disassembly by activating Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase calcineurin. In addition, Ca(2+)/camodulin activity is required for the fusion of sperm-specifi c membranous organelle with the plasma membrane, a regulated exocytosis required for sperm motility. Thus, Ca(2+) plays multifunctional roles during sperm activation in Ascaris suum.
Animals ; Ascaris suum ; metabolism ; Calcineurin ; metabolism ; Calcium ; metabolism ; Calmodulin ; metabolism ; Cytoskeleton ; metabolism ; Cytosol ; metabolism ; Egtazic Acid ; analogs & derivatives ; pharmacology ; Helminth Proteins ; metabolism ; Inositol 1,4,5-Trisphosphate Receptors ; metabolism ; Male ; Membrane Potential, Mitochondrial ; physiology ; Mitochondria ; metabolism ; Pseudopodia ; metabolism ; Signal Transduction ; Sperm Motility ; Spermatids ; drug effects ; physiology ; Spermatogenesis ; Type C Phospholipases ; metabolism

OBJECTIVES: Bisphenol A diglycidyl ether (BADGE) is a liquid compound obtained by condensation of two molecules of epichlorohydrin with one molecule of bisphenol A. General and reproductive toxicity with BADGE has been reported higher than 1000 mg/kg/day. This study was performed to show the effects of acute exposure to BADGE below 1000 mg/kg/day on the testis in adult male rats. METHODS: BADGE was administered by gastric lavage in a single dose of 500, 750, 1000, and 2000 mg/kg/day in 8-week old male SPF Sprague-Dawley rats. The right testis was processed for light microscopic analysis. The left testis was homogenized and spermatids were counted to determine the daily sperm production and daily abnormal sperm production. The sperm count, sperm motility, and incidence of abnormal sperm were estimated in the epididymis. In testicular sections, the seminiferous tubules were observed for qualitative changes. The progression of spermatogenesis was arbitrarily classified as full-matured, maturing, and immature. The specimen slide was observed at 3 points and 10 seminiferous tubules were evaluated at each point. RESULTS: The male rats exposed to single oral dose of BADGE at 750, 1000, and 2000 mg/kg/day were significantly increased the number of immature and maturing sperm on the testis. There were no significant differences with respect to sperm head count, sperm motility, and sperm abnormality in the BADGE treatment groups. CONCLUSIONS: These results suggest that single oral exposure of BADGE 750 mg/kg/day can affect adult male testis development.
Animals ; Dose-Response Relationship, Drug ; Epoxy Compounds/*toxicity ; Male ; Rats ; Rats, Sprague-Dawley ; Semen Analysis ; Spermatids/drug effects ; Spermatogenesis/drug effects ; Testis/*drug effects/metabolism

OBJECTIVETo investigate the effect of 17 beta-estradiol (E2) on T-type calcium currents (ICaT) in spermatogenic cells.

METHODSCa2+ currents were obtained in acutely dissociated mouse spermatogenic cells by the whole-cell patch clamp technique and the effects of E2 on ICaT were observed.

RESULTSE2 at the concentrations of 1, 10 and 100 micromol/L significantly inhibited ICaT in the mouse spermatogenic cells, with the KC50 value of 8.89 micromol/L and the inhibition rates of (13.48 +/- 1.86) %, (28.98 +/- 2.70) and (52.93 +/- 3.42)%, respectively (n = 6, P < 0.05). E2 of 100 micromol/L significantly changed the activation and inactivation of ICaT: the half activation potential (Va 1/2) and the activation steepness factor (Ka) from ( -48.94 +/- 0.22) mV and (5.19 +/- 0.19) mV to (-54.34 +/- 1.02) mV and (6.02 +/- 0.84) mV ( n = 5, P < 0.05) , and the half inactivation potential (Vi 1/2) and the inactivation steepness factor (Ki) from (-56.51 +/- 0.13) mV and (3.36 +/- 0.11) mV to (-61.78 +/- 0.50) mV and (4.25 +/- 0.37) mV, respectively (n = 5, P < 0.05).

CONCLUSIONE2 has a significant inhibitory effect on ICaT in mouse spermatogenic cells in a con-centration-dependent manner.

Animals ; Calcium Channels, T-Type ; physiology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Estradiol ; pharmacology ; Male ; Membrane Potentials ; drug effects ; Mice ; Patch-Clamp Techniques ; Spermatids ; cytology ; drug effects ; physiology

OBJECTIVETo investigate the possible involvement of calmodulin in mouse sperm capacitation.

METHODSCalmodulin antagonists W7 at the concentrations of 50, 100 and 200 micromol/L and calmidazolium (CZ) at the concentrations of 10, 20 and 30 micromol/ L, were coincubated with mouse sperm for 2 hours, respectively. The percentage of pattern B sperm was measured by chlorotetracycline staining. Then the sperm were coincubated with 100 micromol/L W7 or 10 micromol/L calmidazolium (CZ) before acrosome reaction was induced by 5 micromol/L progesterone and evaluated by the same method.

RESULTSAfter the treatment of the sperm with different concentrations of CZ or W7, the percentages of pattern B sperm decreased in a dose-dependent manner, significantly different from the control (P < 0.05). There was a statistic difference in the rate of acrosome reaction between the experiment and the control group (P < 0.01).

CONCLUSIONCalmodulin is a key protein involved in mouse sperm capacitation.

Animals ; Calmodulin ; antagonists & inhibitors ; Enzyme Inhibitors ; pharmacology ; Imidazoles ; pharmacology ; Male ; Mice ; Mice, Inbred ICR ; Sperm Capacitation ; drug effects ; Sperm Count ; Sperm Motility ; drug effects ; Spermatids ; cytology ; drug effects ; physiology

AIMTo understand the biological functions of the ectoplasmic specializations between Sertoli cells and maturing spermatids in seminiferous epithelia.

METHODSIn order to disrupt the function of the ectoplasmic specializations, nectin-2, which is expressed at the specialization, was neutralized with anti-nectin-2 antibody micro-injected into the lumen of the mouse seminiferous tubule. Anti-nectin-3 antibody was also micro-injected into the lumen in order to neutralize nectin-3, which is expressed at the specialization.

RESULTSThe actin filaments at the specialization disappeared, and exfoliation of maturing spermatids was observed by electron microscopy.

CONCLUSIONNectin-2 was neutralized by anti-nectin-2 antibody and nectin-3 was neutralized by anti-nectin-3 antibody, respectively. Inactivated nectin-2 and nectin-3 disrupted the nectin-afadin-actin system, and finally the actin filaments disappeared. As a result, the specialization lost the holding function and detachment of spermatids was observed. One of the functions of the specialization seems to be to hold maturing spermatids until spermiation.

Actins ; metabolism ; Animals ; Antibodies ; immunology ; pharmacology ; Cell Adhesion Molecules ; immunology ; metabolism ; Cell Communication ; drug effects ; physiology ; Intercellular Junctions ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Microfilament Proteins ; metabolism ; Microscopy, Confocal ; Nectins ; Seminiferous Epithelium ; cytology ; drug effects ; metabolism ; Sertoli Cells ; cytology ; drug effects ; metabolism ; Spermatids ; cytology ; drug effects ; metabolism

OBJECTIVE: To investigate the reproductive system impairment induced by cocaine in adult male rats and the possible underlying mechanism. METHODS: Thirty adult male rats were randomly divided into experimental and control groups, with 15 rats in each group. Rats of the experimental group were injected cocaine hydrochloride (15 mg/kg body weight) subcutaneously daily for four weeks. The weight of body and testis, as well as the level of serum hormone of the rats were examined. In addition, the apoptosis rate of testicular tissue by TUNEL and the expression of Fas gene in testicular tissue were examined by immunohistochemistry. RESULTS: Compared with the control, the weight of testis in the cocaine exposed group decreased significantly (P<0.05), and the serum testosterone level decreased significantly (P<0.05). Moreover, both the apoptosis rate and the expression of Fas gene increased in the testicular tissue of rats in the cocaine exposed group in comparison to the control group (P<0.05). The apoptosis rate was significantly correlated with the expression of Fas gene (r=0.9012, P<0.05). CONCLUSION Cocaine may cause reproductive system injury in adult male rats, and Fas-mediated apoptosis may be one of the functional mechanisms involved in the reproductive system injuried by cocaine.
Adaptor Proteins, Signal Transducing/metabolism* ; Animals ; Apoptosis/drug effects* ; Cocaine/adverse effects* ; Forensic Toxicology ; Male ; Molecular Chaperones ; Nuclear Proteins/metabolism* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatids/pathology* ; Substance-Related Disorders ; Testis/pathology* ; Testosterone/blood*

OBJECTIVETo investigate the effect of nifedipine of therapeutic dosage on the plasma membrane functional integrity and osmosensitive calcium influx in human sperm in vitro.

METHODSSperm samples were aseptically obtained from 10 healthy fertile men by masturbation and prepared by swim-up technique to produce a spermatozoal solution of high motility. The solution was then incubated with nifedipine of 20, 100 and 20 x 10(3) ng/ml respectively at 37 degrees C in vitro. The hypo-osmotic swelling (HOS) test was done to assess the sperm function. Intracellular calcium concentration was measured by fluorescent probe fura-2/AM before and after sperm medium dilution in distilled water.

RESULTSThe 20 x 103 ng/ml group showed significantly lower HOS scores than the control (P < 0.01). The 20, 100 and 20 x 10(3) ng/ml groups all showed significantly lower Ca2+ fluorescence D-value than the control (P < 0.01).

CONCLUSIONNifedipine can modify plasma membrane functional integrity and inhibit osmosensitive calcium influx in human sperm and affect male fertility in vitro in therapeutic dose.

Adult ; Calcium ; metabolism ; Cell Membrane ; Humans ; In Vitro Techniques ; Male ; Nifedipine ; pharmacology ; Osmotic Pressure ; Spermatids ; drug effects ; metabolism ; physiology

OBJECTIVETo preliminarily study the effect of prepubertal exposure of male SD (Sprague-Dawley) rats to diethylstilbestrol (DES) on the apoptosis of spermatogenic cells after sexual maturation and its mechanism.

METHODSThirty 21-day-old male SD rats were randomly divided into 4 experimental groups, DES 0.01, 0.1, 1.0 and 10.0 microg/(kg x d) and 1 control group. The experimental groups were injected (s.c.) with different doses of DES (dissolved in corn oil) during prepuberty [from postnatal day (PND) 22 to PND 35] and the control group with medium only. The apoptosis and related proteins Bcl-2 and Bax expressions of testicular spermatogenic cells were studied with TUNEL and immunohistochemistry after the rats sexual maturation (at PND 64).

RESULTSCompared with the control group, the apoptosis of testicular spermatogenic cells in the DES 0.01 microg/kg group had no difference, but significantly increased in the DES 0.1, 1.0 and 10.0 microg/kg groups and the apoptosis increased with the increase of DES dose. In the control and DES 0.01 microg/kg groups, Bax protein expressed weakly but Bcl-2 protein strongly in spermatogenic cells. With the increase of DES exposure, Bax protein expression in spermatogenic cells increased but Bcl-2 protein expression decreased.

CONCLUSIONPrepubertal exposure of SD rats to inappropriate dose of DES can make the apoptosis of spermatogenic cells increase after sexual maturation. Bax and Bcl-2 proteins participate in the apoptotic course caused by prepubertal DES exposure.

Animals ; Apoptosis ; drug effects ; Diethylstilbestrol ; toxicity ; Dose-Response Relationship, Drug ; Male ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sexual Maturation ; Spermatids ; drug effects ; metabolism ; bcl-2-Associated X Protein ; biosynthesis