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 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 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 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

AIMTo evaluate the key lesions in spermatogenesis suppressed partially by testosterone undecanoate (TU) treatment.

METHODSAdult male SD rats were treated with vehicle or TU (19 mg/kg) injection (i.m.) every 15 days for 130 days. The numbers of all types of cells (nuclei) in the seminiferous tubules and the interstitial tissue were estimated using a contemporary stereological tool, the optical disector.

RESULTSIn response to TU treatment, the numbers of non-type B spermatogonia, type B spermatogonia and late elongated spermatids per testis were reduced to 51 %, 66 % and 14 % of the controls, respectively. The conversion ratios from type B spermatogonia to early spermatocytes and pachytene spermatocytes were not significantly affected and the ratios to the later germ cell types fell to 51 % - 65 % of the controls. Less than 1.0 % of immature round spermatids were seen sloughing into the tubule lumen, 4.0 % of elongated spermatids retained in the seminiferous epithelium, and about half of the elongated spermatid nuclei appreciably malformed. Leydig cells were atrophied but their number and the peritubular myoid cell number per testis were unchanged.

CONCLUSIONDouble inhibition of spermatogenesis (i.e. inhibition at spermiation and spermatogonial conversion to type B spermatogonia), a scenario seen in the monkey and human following gonadotrophin withdrawal, was not sufficiently effective for a complete spermatogenic suppression in the rat after TU treatment, probably due to ineffective inhibition of the Leydig cell population and therefore the intra-testicular testosterone levels.

Animals ; Cell Nucleus ; drug effects ; ultrastructure ; Depression, Chemical ; Leydig Cells ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Sertoli Cells ; drug effects ; Sperm Count ; Spermatids ; drug effects ; Spermatogenesis ; drug effects ; Testosterone ; analogs & derivatives ; blood ; pharmacology

OBJECTIVETo explore the antifertility effect and safety of 30% ethanol retro-injection into the vas deferens of the rat.

METHODSThirty Sprague-Dawley male rats, 3 m of age and (200 +/- 20) g in weight, were equally randomized into an experimental group and a control group. The former received 30% ethanol (0.5 ml) and the latter 0.9% sodium chloride (0.5 ml), both retro-injected into the vas deferens. Pregnancy rates were obtained through pregnancy tests with 60 Sprague-Dawley female adult rats 1.5 m and 3 m after the injection. All the male rats were sacrificed three months later, and tests were done for the rates of sperm motility and deformity as well as for the apoptosis of spermatogenic cells with TUNEL.

RESULTSThe 1.5 m pregnancy rate was 0 and the 3 m sperm motility and pregnancy rates were (0.32 +/- 1.12)% and (0.58 +/- 1.27)%, significantly decreased (P < 0.05) as compared with those of the control group, which were (80.62 +/- 2.68)%, (70.68 +/- 1.62)% and (86.62 +/- 1.68)%, respectively. While the 3 m sperm deformity rate in the experimental group was (78.26 +/- 1.08)%, increased significantly (P < 0.05), and the apoptosis index (AI) of spermatogenic cells was (7.63 +/- 1.16)% as compared with (5.62 +/- 1.32)% of the control group, with no significant difference between the two groups (P > 0.05).

CONCLUSIONRetro-injection of 30% ethanol into the vas deferens of the rat produces significant antifertility effect on rats, but has no significant influence on their spermatogenic cells.

Animals ; Apoptosis ; Epididymis ; drug effects ; Ethanol ; administration & dosage ; pharmacology ; Female ; Male ; Pregnancy ; Pregnancy Rate ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sperm Motility ; drug effects ; Spermatids ; drug effects ; Testis ; cytology ; Vas Deferens ; drug effects

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 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

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

OBJECTIVETo investigate the effects of Flunarizine (Flu) on T-type calcium currents (ICaT) in spermatogenic cells.

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

RESULTSFlu of 0.1, 1, 10, 100 micromol/L inhibited ICaT in mouse spermatogenic cells significantly with the K50 value of 0.289 micromol/L (P < 0.05). With the holding potential at -90 mV and stimulating potential at -30 mV, the inhibition rates of Flu were (23.34 +/- 2.76)%, (46.04 +/- 3.52)%, (62.52 +/- 3.70)% and (73.52 +/- 3.12)%, respectively.

CONCLUSIONFlu has significant inhibitory effects on ICaT in mouse spermatogenic cells, with concentration dependence. Ca(v)3.2 is the main contributor to T-type Ca2+ currents in spermatogenic cells.

Animals ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, T-Type ; drug effects ; physiology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Flunarizine ; pharmacology ; Male ; Mice ; Mice, Inbred ICR ; Patch-Clamp Techniques ; Spermatids ; drug effects ; physiology