| It is important for male antifertility to find effective, safe and reversible contraceptives. Besides developing new effective constituents, conbined usage of contraceptives having different pharmacologic action is a promising alternative. Studing for many years, we have found that combined regimen of low-dose gossypol and steroid hormones has reversible antifertility action with low side effects and the combined regimen has faster effect than that of low dose gossypol or steroid hormones alone, although these results demonstrate that low-dose gossypol and steroid hormones have synergistic effect on antiferlity, the sites of synergistic effect remained unknown. Our previous study also showed combined regimen can induce spermatocyte appostosis, however, the changes of other sperm cells in seminiferous epithelium are not clear, especially the change of spermatogonia and the molecular mechanism for it.Adult male rats were divided into four groups randomly, group GH:rats were fed orally with Gossypol acetic acid (GA,12.5mg/kg·d) and Desogestrel (DSG, 0.125mg/kg·d)/Ethinylestradiol (E,0.025mg/kg·d)/Testosterone undecanoate (TU, 100mg/kg·d); group G:a single does of GA (12.5mg/kg·d) was given; group H: DSG(0.125mg/kg·d)/E(0.025mg/kg·d)/TU(100mg/kg-d) were administered; group C:rats only received vehicle(1% methyl cellulose). The testis and epididymis were removed at 4、6 and 8 week for detecting changes of spermatogenesis, sperm motility, numbers of sperm cells on seminiferous epithelium and stereological parameters of testicular tissue. The results were analyzed using method of statistical interaction analysis for finding the synergistic effect sites. Among the changes of sperm cell number of seminiferous epithelium, the quantitative change spermatogonia (including spermatogonial stem cell) is key point for identifying whether the spamatogonia are influenced by the combined regiman. The expressions of spermatoginial stem cell niche related molecues, including glial cell line-derived neurotrophic factor (GDNF), Ets related molecule (ERM) and stem cell factor(SCF), were also detected by immunohistochamical method, Real-time PCR and Western blot in order to find the effector molecules of the combined regiman. Our resultes show that combined low-dose gossypol and steroid hormones can supress spermatogenesis, the production and motility of epididymal mature sperm were obviously reduced. Low-dose gossypol and steroid hormones did not paly synergistic effects on spermatogenesis supression, changes of tesicular stereological parameters and umbers of germ cell on seminiferous epithelium, while they had synergistic effect on reduction of number and motility of mature sperm in epididymis. Afeter combined regiman, the numbers of non-B type spermatogonia(including spermatogial stem cell, A and In spermatogonia), stageⅧ-ⅩⅣpachytene and diplontene spermatocyte and round spermatid were significant decrease, the numbers of B type spermatogonian, preleptotene, leptotene and zygotene spermatocyte and stageⅠ-Ⅶpachytene spamatocyte were not obviously changed. Among the spermatogonial stem cell niche related molecules, the expression of GDNF was reduced and ERM and SCF were not changed.In conclusion, low-dose gossypol and steroid hormones have own action sites, stereoid hormones mainly paly spermatogenesis supression role at testis, while gossypol acts on mature sperm and epididymis, and they have synergistic effect on reduction of number and motility of mature sperm in epididymis. The non-B type spermatogonia (including spermatogial stem cell, A and In spermatogonia) are influenced by the combined regiman. Spermatogonial stem cell niche is changed through downregulating GDNF by the steroid horments in the combined regimen. The self renewal of spermatogonial stem cell may be slowed. While the unchanged expression of SCF suggests the differetation of spermatogonial stem cell is normal. |
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