Optimization of real time RT-PCR system for the quantitative estimation of CatSper1 mRNA levels in human and mouse mature spermatozoa / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish and optimize a real time RT-PCR system for determining the transcript levels of CatSper1 in human and mouse mature spermatozoa containing microamount of RNA.</p><p><b>METHODS</b>Total RNA of human and mouse mature spermatozoa was isolated by using TRIzol reagent and reversely transcribed to complementary DNA respectively. Primers for real time RT-PCR were designed in the homologous area of the human and mouse CatSper1 mRNAs. Human sperm complementary DNA was used as the template to the optimize the conditions for SYBR Green I real time RT-PCR, including annealing temperature, Mg2+ concentration, fluorescence measurement temperature and the ratio between forward and reverse primers. The standard curve was constructed with serial dilutions of complementary DNA from human sperm to ascertain the amplification efficiency of SYBR Green I real time PCR and to quantitate the CatSper1 mRNA levels in the human and mouse mature spermatozoa.</p><p><b>RESULTS</b>The optimal conditions for real time RT-PCR, that is, annealing temperature, Mg2+ concentration and the ratio between forward and reverse primers were 63 degrees C, 3.0 mmol/L and 1:1 respectively. The fluorescence measurement temperature was 88 degrees C. The standard curves were Y = -3.402 log (X) + 25.99 and Y = -3.409 log(X) + 24.09 in the human sperm cDNA and mouse sperm cDNA as the template, with amplification efficiency of 96.8% and 96.5% respectively. The R2 value (an indicator of the quality of the fit of the standard curve to the standard data points plotted) of both standard curves was 0.998. The CatSper1 mRNA levels in the human and mouse mature spermatozoa could be determined according to the standard curve.</p><p><b>CONCLUSION</b>The general RT-PCR system, by adding SYBR Green I and optimizing its conditions, could be used to quantitate the mRNA levels in both human and mouse mature spermatozoa.</p>

Peroxidative damage induced by cumene hydroperoxide in testis and epididymis of rats in vivo / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish an oxidative stress model induced by cumene hydroperoxide (cHP) in testis and epididymis of rats in vivo, and to understand the peroxidative damage of oxidative stress in testis, epididymal sperm and its propensity to induce nuclear DNA damage during spermatogenesis and sperm maturation in vivo.</p><p><b>METHODS</b>An organic hydroperoxide, cHP, 70% aqueous, diluted by 0.9% NaCl, was employed as model prooxidant. Ninety-day-old male Wistar rats were divided into a control and three cHP groups, and were administered intraperitoneally 0, 1/10, 1/6 and 1/4 LD50 cHP per day respectively at a dose of 2 ml/kg, for 7 consecutive days and were observed for any toxic symptoms and mortality. Twenty-four hours after the last dose, rats were sacrificed and induction of oxidative stress was ascertained by monitoring the degree of lipid peroxidation expressed as nano molar of malondialdehyde (MDA) in testicular homogenate and epididymal sperm. Nuclear DNA damage in testes and epididymal sperms was determined by comet assay. Motility of caudal sperms was counted and the morphology of testes and epididymis was observed under light microscope.</p><p><b>RESULTS</b>Rats of cHP administered groups were less vigorous than those of the control, but there were not death of rats during treatment. 1/10 LD50 per day for 7 consecutive days resulted in only a marginal increase in testicular MDA levels. However, 1/6 and 1/ 4 LD50 per day for 7 days of cHP administered to adult rats induced marked oxidative stress in testis and epididymal sperms as evidenced by a marked increase in MDA or nuclear DNA damage in testis and caput sperms, as well as significant decreases both in the body weight-and motility of caudal sperms. While the nuclear DNA damage caput sperms of 1/6 and 1/4 LD50 cHP administered rats increased significantly, nuclear DNA damage in caudal sperms showed no treatment related alterations.</p><p><b>CONCLUSION</b>Oxidative stress in testis and epididymal sperms can be safely induced by applying multiple doses of cHP (1/6 and 1/4 LD50 per day for seven consecutive days). DNA damage caused by cHP induced oxidative stress may occurred mainly in testes.</p>

The expression and significance of CATSPER1 in human testis and ejaculated spermatozoa / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To investigate the distribution of cation channel of sperm 1 (CATSPER1) protein and the presence of CATSPER1 mRNA in human testis and ejaculated spermatozoa. The influence of anti-human CATSPER1 antibody upon human sperm motility was used to evaluate the function of human CATSPER1 and to estimate its possible use as a target for immunocontraception.</p><p><b>METHODS</b>Human ejaculated sperm from normozoospermic donors (n = 12) and liquid nitrogen frozen human testis were used for the study of mRNA and protein expression of CATSPER1 by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Spermatozoa from normozoospermic donors (n = 12) were individually processed using a swim-up procedure and were then incubated with CATSPER1 antibody at final concentrations of 20, 4 and 0.8 microg/mL. After 1, 2 and 6 h incubation, progressive motility and fast progressive motility were measured by means of computer-assisted semen analysis.</p><p><b>RESULTS</b>CATSPER1 transcript was detected in both human testis and each human ejaculated semen sample. CATSPER1 protein expressed in the membrane of spermatid and was localized in the principal piece of the sperm tail. The application of CATSPER1 antibody at all concentrations significantly inhibited both progressive motility and fast progressive motility after 1, 2 and 6 h incubation, and significant dose-dependent changes were observed.</p><p><b>CONCLUSION</b>CATSPER1 is meiotically and post-meiotically expressed in human testis tissue. CATSPER1 mRNA in human ejaculated spermatozoa could be a more feasible target for study and infertility screening than testis biopsy. In addition, our results suggest that human CATSPER1 could be a possible target for immunocontraception.</p>

Effect of ornidazole on sperm in rats and its mechanism of action / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the reductive effect of ornidazole on sperm motility in rats and its mechanism of action.</p><p><b>METHODS</b>Twenty rats were randomly divided into three groups, a low dosage group (LD group, n = 5), a high dosage group (HD group, n = 8) and a normal control group (n = 7). Ornidazole (200 mg/kg, 400 mg/kg) was given to the LD and HD groups, and 0.5% carboxymethylcellulose sodium (CMC) administered to the normal control, all for 20 consecutive days. Immediately after, sperm density, motility and the morphological changes of the testis and epidiclymis were measured, and the concentrations of lactate dehydrogenase (LDH), alpha-glycosidase, malondialdehyde (MDA) and fructose in the testis and epididymis tissues were monitored.</p><p><b>RESULTS</b>Compared with the normal control, there were no obvious changes in sperm density (P > 0.05), but a significant decrease in sperm motility in the LD and HD groups (P < 0.01), and the concentration of LDH obviously declined (P < 0.01) while that of MDA distinctly increased in the HD group (P < 0.05).</p><p><b>CONCLUSION</b>Spermatogenic cells could be damaged by the increase of inhibiting MDA, while sperm motility could be decreased by inhibiting energetic transferase or non-protein substance in the epididymis. This might be one of the mechanisms of ornidazole on weak sperm models in rats.</p>