| Melatonin is a kind of indoles hormone secreted by pineal gland and controlled by illumination intensity. The secretion levels of melatonin is a positive correlation with illumination intensity. In north of china, autumn and winter are the breeding season for dairy goat, during these time, the secretion of melatonin reaches peak and the sperm quality of male goat is good. In our research, we collect the dairy goat testis of breeding and non-breeding season. The HE staining of tissue slice shows the spermatogenesis is more active and the expression level of self-renewal and proliferation relative markers are higher in breeding season. Immunohistochemical staining show that the spermatogenic cells of spermatogenesis all express the melatonin receptor and the expression level is higher in breeding season. The above results illustrate male goat is a seasonal breeder too, and melatonin may participate in the formation of seasonal reproduction phenomenon.Many studies show that the reproduction regulation of mammal depends on the hypothalamic-pituitary-gonadal axis and melatonin plays an important role in the regulation. Melatonin can act on hypothalamic, pituitary and germ cells to regulate the hormone secrete, spermatogenesis and oogenesis. Spermatogonial stem cells(SSCs) located at the base membrane of seminiferous tubule in testis, it has the ability of self-renewal and differentiate to sperm, it is the initiating cell of spermatogenesis. To determine whether melatonin can regulate spermatogenesis through SSCs, we culture goat SSCs in vitro and add various concentrations melatonin in the medium and get the following results.1. We separate dairy goat testis cells using Ⅳ collagenase and trypsin, and the undifferentiated SSCs were enriched using differential attachment technique. Then we get SSCs clone after cultured the SSCs using SSC medium, the clone shows CD49 f, GFRa1, PGP9.5 positive and AP positive, meanwhile, the clone express the SSCs marker PLZF in mRNA and protein level. This shows that the culture system can be applied to the dairy goat SSCs culture in vitro.2. The cell viability and proliferation relative marker analysis show that melatonin can promote the proliferation of dairy goat SSCs, and the clone size also can demonstrate the results. Meanwhile, we find that melatonin can promote the expression of SOX9 and GDNF, which are the markers of Sertoli cell. To test whether melatonin has effect on SSCs differentiation, we analys the mRNA expression level of SSCs differentiation markers Stra8, Dazl, Kit and Sycp3, the results show melatonin can inhibit the differentiation of dairy goat SSCs. The CCK-8, MTT and Brdu staining results show melatonin has none effect on the proliferation of mGSCs-I-SB, a goat SSCs line in our laboratory.3. The Elisa assay demonstrate that Sertoli cell can produce GDNF to maintain the proliferation and self-renewal when culturing goat SSCs. We also find the GDNF concentration is higher in melatonin added medium, melatonin can stimulate the sertoli cell produce GDNF. Western blotting show that GDNF activate the phosphorylation of AKT and ERK1/2 to maintain the self-renewal and proliferation. And the phosphorylation level of melatonin added medium is higher, which further show melatonin can affect the dairy goat SSCs through GDNF pathway.Based on the study of effect of melatonin on dairy goat SSCs in vitro, we hypothesis that in the breeding season, pineal can produce more melatonin and stimulate sertoli cell produce more GDNF to replenish the spermatogonia stem cell pool. Spermatogenesis is regulated by several hormones, melatonin in combination with these hormones form the season dependence reproduction. |
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