褪黑激素对绵羊卵巢颗粒细胞增殖、凋亡、类固醇激素分泌的影响

Effects of Melatonin on Proliferation,Apoptosis of Ovarian Granulosa Cells,and the Its Secretion of Steroid Hormones in of Sheep

旨在研究褪黑素(melatonin,MT)对绵羊卵巢颗粒细胞增殖、凋亡、类固醇激素分泌及相关基因表达的影响,以期解析MT影响绵羊繁殖的路径。本研究采集1岁龄体重相近饲养条件相同的20只健康小尾寒羊母羊新鲜卵巢,收集2~3 mm卵泡的颗粒细胞,随机分为5组,每组5个重复,分别添加0、10^(-9)、10^(-8)、10^(-7)、10^(-6)mol·L^(-1)MT,培养48 h后CCK8和Annexin V-FITC分别检测颗粒细胞的增殖和凋亡,确定体外培养浓度为10^(-7)mol·L^(-1),将细胞随机分为2组,每组3个重复,收集试验组(添加10^(-7)mol·L^(-1))和对照组(不添加)培养液及裂解液上清,ELISA检测孕酮(P_(4))、雌二醇(E_(2))和cAMP的含量,并利用RT-qPCR和Western blot分析MT对绵羊颗粒细胞MT受体基因、凋亡相关基因和类固醇分泌相关基因mRNA和蛋白表达的影响。结果表明,添加MT各试验组均显著提高了颗粒细胞增殖活力,其中10^(-7)、10^(-6)和10^(-8)mol·L^(-1)组颗粒细胞增殖活力显著高于10^(-9)mol·L^(-1)组(P<0.05),10^(-7)mol·L^(-1)细胞增殖活力最高,但与10^(-6)和10^(-8)mol·L^(-1)组间差异不显著(P>0.05)。添加10^(-7)、10^(-6)和10^(-8)mol·L^(-1)MT颗粒细胞早期凋亡和晚期凋亡率极显著低于10^(-9)mol·L^(-1)组和对照组(P<0.01),其中10^(-7)mol·L^(-1)组细胞晚期凋亡率最低,但与10^(-6)mol·L^(-1)和10^(-8)mol·L^(-1)间差异不显著(P>0.05),而10^(-9)mol·L^(-1)组对细胞凋亡没有显著影响(P>0.05)。添加10^(-7)mol·L^(-1)MT颗粒细胞P_(4)分泌显著降低(P<0.05),而对E_(2)和cAMP水平无显著影响(P>0.05)。添加10^(-7)mol·L^(-1)MT可显著上调MT受体基因MT 1的mRNA和蛋白表达水平及抗凋亡基因Bcl-2的mRNA表达(P<0.05),显著下调了FSHR、StAR、CYP11 A1和3β-HSD的mRNA和蛋白表达(P<0.05),以及凋亡基因BAX、Caspase-3的表达及BAX/Bcl-2比值(P<0.05),对受体基因MT2、LHR和CYP19 A1的表达无显著影响(P>0.05)。综上,绵羊卵巢颗粒细胞体外培养添加MT通过上调抗凋亡基因表达,下调凋亡基因表达,抑制绵羊卵巢颗粒细胞凋亡;MT与MT 1受体结合通过下调FSHR、StAR、CYP11A 1和3β-HSD表达,抑制了P_(4)的分泌。

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(P_(4)),estradiol(E_(2)),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)mol·L^(-1),10^(-6)mol·L^(-1),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)mol·L^(-1),10^(-6)mol·L^(-1),and 10^(-8)mol·L^(-1)groups.The early and late apoptosis rates of MT granulosa cells added with 10^(-7)mol·L^(-1),10^(-6)mol·L^(-1),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)mol·L^(-1)and,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)P_(4)secretion of granulosa cells,but had no significant effect(P>0.05)on the levels of E_(2)and cAMP.The addition of 10^(-7)mol·L^(-1)MT significantly up-regulated(P<0.05)the mRNA and protein expression of MT 1,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,CYP 11A1,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 difference effect(P>0.05)on the expression of the receptor genes MT2,LHR and CYP19 A1.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 MT 1 receptor inhibited P_(4)secretion by down-regulating FSHR,StAR,CYP 11A1 and 3β-HSD expression.