Abstract: The experiment investigated the effects of melatonin (MT) on the proliferation, apoptosis, steroid secretion, and the expression of related genes in sheep ovarian granulostic cells, so as to elucidate the pathways in which MT affects sheep reproduction. Fresh ovaries of 20 healthy ewes of small-tailed Han sheep at one-year-old with similar body weight and same feeding conditions were collected. Granulosa cells from follicles of 2-3 mm were collected and randomly divided into 5 groups with 5 replicates in each group, and 0, 10^-9, 10^-8, 10^-7, and 10^-6 mol·L^-1 MT were added, respectively. After 48 h of culture, the proliferation and apoptosis of granulosa cells were detected by CCK8 and Annexin V-FITC, and the culture concentration in vitro was determined to be 10^-7 mol·L^-1. The cells were randomly divided into 2 groups with 3 replicates in each group. The culture medium and lysate supernatant of the experimental group (supplemented with 10^-7 mol·L^-1 MT) and the control group (not supplemented with MT) were collected. The concentrations of progesterone (P₄), estradiol (E₂), and cAMP were detected by ELISA. RT-qPCR and Western blot were used to analyze the effects of MT on the mRNA and protein expression of MT receptor genes, apoptosis-related genes, and steroid secretion-related genes in sheep granulosa cells. The results showed that the proliferative activity of granulosa cells was significantly increased (P<0.05) in experimental groups supplemented with MT, and the proliferative activity of granulosa cells in the 10^-7, 10^-6 and 10^-8 mol·L^-1 groups was significantly higher (P<0.05) than that in the 10^-9 mol·L^-1 group. The cell proliferation activity of the 10^-7 mol·L^-1 MT group was the highest (P<0.05), but there was no significant difference (P>0.05) between the 10^-7, 10^-6 and 10^-8 mol·L^-1 groups. The early and late apoptosis rates of MT granulosa cells added with 10^-7, 10^-6 and 10^-8 mol·L^-1 were significantly lower (P<0.01) than those in the 10^-9 mol·L^-1 and control groups, and the 10^-7 mol·L^-1 group had the lowest (P<0.05) late apoptosis rate. However, there was no significant difference (P>0.05) between the 10^-7 mol·L^-1 and 10^-6, 10^-8 mol·L^-1, while the 10^-9 mol·L^-1 had no significant effect (P>0.05) on cell apoptosis. The addition of 10^-7 mol·L^-1 MT significantly decreased (P<0.05) P₄ secretion of granulosa cells, but had no significant effect (P>0.05) on the levels of E₂ and cAMP. The addition of 10^-7 mol·L^-1MT significantly up-regulated (P<0.05) the mRNA and protein expression of MT1, as well as the mRNA expression of anti-apoptotic gene Bcl-2, and significantly down-regulated (P<0.05) the mRNA and protein expression of FSHR, StAR, CYP11A1, and 3β-HSD, as well as the expression of apoptosis genes BAX, Caspase-3 and BAX/Bcl-2 ratio (P<0.05). There was no significant effect (P>0.05) on the expression of the receptor genes MT2, LHR and CYP19A1. In conclusion, the addition of MT in vitro inhibited the apoptosis of sheep ovarian granulosa cells by up-regulating the expression of anti-apoptotic genes and down-regulating the expression of apoptotic genes. The MT binding to the MT1 receptor inhibited P₄ secretion by down-regulating FSHR, StAR, CYP11A1 and 3β-HSD expression.

Key words: melatonin, sheep, granulosa cells, proliferation, steroid hormone