A combined regimen of gossypol plus methyltestosterone and ethinylestradiol as a contraceptive induces germ cell apoptosis and expression of its related genes in rats.

Attempts to develop gossypol and steroidal hormones alone as a male contraceptive have been tested for many years; however, both caused undesirable side effects that have prevented their acceptance. In this study, we formulated a regimen of combined gossypol at a low dose of 12 mg/kg or a high dose of 50 mg/kg plus methyltestosterone 20 mg/kg and ethinylestradiol 100 g/kg daily (12 mg G+H and 50 mg G+H) administered for 6 weeks in adult rats. The possible roles of germ cell apoptosis and related genes expression were studied by techniques of TdT-mediated dUTP nick end-labeling (TUNEL), agarose gel electrophoresis of low-molecular-weight DNA, in situ hybridization and reverse transcription-polymerase chain reaction detection. Results showed that germ cell apoptosis and related genes expression were significantly induced after combined drug administration. The apoptosis index increased 3.86- and 9.65-fold in the 12-mg and 50-mg G+H-treated groups, respectively, as compared to the control group. DNA ladder formation on the agarose gel further validated the findings of TUNEL-stained apoptotic cells. The apoptosis-related genes fas mRNA expression levels increased 0.44- and 1.39-fold, bax mRNA 0.74- and 2.56-fold, caspase-3 mRNA 0.60- and 1.29-fold, and caspase-9 mRNA 2.50- and 4.08-fold, respectively, in the 12-mg and 50-mg G+H-treated groups vs. the control group. These results indicated that our drug regimen applied as a contraceptive could induce rat germ cell apoptosis. The apoptotic process involved fas system, bax and caspase family genes and the apoptotic extent and cell types were gossypol dose-dependent.

Combined administration of low-dose gossypol acetic acid with desogestrel/mini-dose ethinylestradiol/testosterone undecanoate as an oral contraceptive for men.

To evaluate the efficacy and feasibility of a new regimen of low-dose gossypol acetic acid (GA) combined with desogestrel/ethinylestradiol and testosterone undecanoate (DSG/E/TU) as a male contraceptive, adult male rats were fed orally with GA (12.5 mg/kg/day) and DSG (0.125 mg/kg)/E (0.025 mg/kg)/TU (100 mg/kg) daily for 8 weeks as loading dose until infertility, and a similar low dose of GA alone for infertility maintenance. Control animals were administered a single low dose of GA (12.5 mg/kg/day) or DSG (0.125 mg/kg)/E (0.025 mg/kg)/TU (100 mg/kg), and vehicle, respectively. Results demonstrated that the combined dosage regimen could damage epididymal sperm motility and density, and induce infertility within 8 weeks in rats; the infertility could be consistently sustained by giving single GA (12.5 mg/kg/day), and was reversible in about 8 weeks following withdrawal of gossypol. The regimen rendered treated male rats with spermiation failure within a period of 6-20 weeks of treatment. Also, the serum luteinizing hormone, follicle-stimulating hormone and testicular interstitial fluid testosterone levels showed a transient decrease at the end of 6 or 8 weeks, which returned to control levels after 8 weeks of recovery phase. No hypokalemia or other adverse effects in viscera were observed. These results provide a promising approach to using the new regimen for the development of an effective and reversible oral male contraceptive.

Cloning and characterization of a novel gene SRG-L expressed in late stages of mouse spermatogenic cells.

A cDNA expressed specifically in late stages of mouse spermatogenic cells during spermatogenesis was cloned by using overlapping RT-PCR and RACE. The cDNA contained an open reading frame (ORF) of 2625 base pairs that encoded an 874 amino acids protein. Comparison analysis of amino acid sequence showed 91% and 80% identity to a rat homologue XP-226242 and a monkey homologue BAB63115 respectively. The expression of the mRNA was only observed in pachytene spermatocytes, round, and elongating spermatids. We named this gene as SRG-L (spermatogenesis related gene expressed in the late stages of spermatogenic cells, GenBank accession No. AY352586). The tissue-specific analysis showed that the SRG-L was highly expressed in spleen and testis. The results suggested that SRG-L might play special roles during spermatogenesis, particularly related to meiosis and spermiogenesis. Analysis of the amino acid sequence showed there was a coiled-coil region near the N-terminal region and rich phosphorylation sites, suggesting SRG-L might function as a transmembrane protein mediating signal transduction. This study also demonstrated that gene cloning by RT-PCR was applicable and convenient when its homologous gene was known.

[Cloning of ESTs of spermatogenesis-related genes from early stage spermatogenic cells of mice using a modified DDRT-PCR method].

To avoid the shortcomings of radioactive pollution and high rate of false positives in DDRT-PCR method, the technique have been modified by replacement of radioactive reagents with fluorescent reagents followed by confirming the results using reverse RNA dot blot. The modified DDRT-PCR method was used in this study to clone spermatogenesis-related genes from early stage spermatogenic cells of mice. Primitive spermatogonia were isolated from 6-day-old mice and type B spermatogonia from 9-day-old mice. The purity of isolated cells was over 90%. Total RNA was extracted from the cells, fluorescent differential display technique was performed to screen the differentially expressed genes. Differences in expression of the screened fragments were identified by reverse RNA dot blot. 16 ESTs were selected for sequencing. The analysis results in GenBank/Blast database revealed that 7 of them were novel ESTs, and they were then registered in GenBank. All of them expressed stronger in B spermatogonia than in primitive spermatogonia. 3 of them were chosen to further identify their expression patterns by semi-quantitative RT-PCR. Compared with traditional differential display technique, the modified method used in this study can avoid radioactive pollution and eliminate false positive results. The present study suggests that the gene activation or up-regulation in B spermatogonia may be related to some specific process in the following steps of spermatogenesis.

[Characterization of expression of the centrosomal protein, Cenexin, in rat spermatogenesis].

The centrosomal protein, Cenexin, is a molecular marker of mature centriole. To elucidate the variability and function of mature centriole in spermatogenesis, the high titer polyclonal antibody against rat cenexin was obtained by immunizing mice with recombinant cenexin which was made up in this study. The expression of cenexin in rat spermatogenesis was carried out by semi-quantitative RT-PCR, immunofluorescence and Western Blot. The results demonstrated that the level of Cenexin mRNA was higher in spermatogonia and spermatocytes, then decreased in following stages, while Cenexin protein was located on one centriole from spermatogonia to spermatids, showing mature centriole existed in these stages. Cenexin protein was localized to the basal body of the flagellum in elongated spermatids and the stained signal disappeared in the most of epidydimal sperms. These results suggested that the expression pattern of cenexin in rat spermatogenesis might be related to the initiation of the flagella formation.

[Expression of rat protamine gene in MEL cells].

OBJECTIVE: To investigate the expression of rat protamine (RP) gene in MEL cells and the effect on cell growth. METHODS: Eukaryotic expression plasmid pCR-3.1-RP was constructed and transfected into MEL cells. The changes of cell growth rate, mitotic index and colony-forming rate in semi-solid medium were investigated. RESULTS: Transfected MEL cells showed lower growth rate, mitotic index and colony-forming rate. Volumes of cells were reduced and reduction of RNA transcription was observed. CONCLUSION: These results suggest that expression of RP in MEL cell may inhibit the cell growth and proliferation.