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

doi:10.11843/j.issn.0366-6964.2023.08.017

摘要/Abstract

摘要： 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^-6mol·L^-1MT，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^-7mol·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^-7mol·L^-1）和对照组（不添加）培养液及裂解液上清，ELISA检测孕酮（P₄）、雌二醇（E₂）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^-8mol·L^-1组颗粒细胞增殖活力显著高于10^-9mol·L^-1组（P<0.05），10^-7mol·L^-1细胞增殖活力最高，但与10^-6和10^-8mol·L^-1组间差异不显著（P>0.05）。添加10^-7、10^-6和10^-8mol·L^-1 MT颗粒细胞早期凋亡和晚期凋亡率极显著低于10^-9mol·L^-1组和对照组（P<0.01），其中10^-7mol·L^-1组细胞晚期凋亡率最低，但与10^-6mol·L^-1和10^-8mol·L^-1间差异不显著（P>0.05），而10^-9mol·L^-1组对细胞凋亡没有显著影响（P>0.05）。添加10^-7mol·L^-1 MT颗粒细胞P₄分泌显著降低（P<0.05），而对E₂和cAMP水平无显著影响（P>0.05）。添加10^-7mol·L^-1 MT可显著上调MT受体基因MT1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、CYP11A1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CYP19A1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MT1受体结合通过下调FSHR、StAR、CYP11A1和3β-HSD表达，抑制了P₄的分泌。

关键词: 褪黑素, 绵羊, 颗粒细胞, 增殖, 类固醇激素

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

中图分类号:

S826.3

贺名扬, 马钰静, 王泳, 杨若晨, 刘月琴, 张英杰, 段春辉. 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[J]. 畜牧兽医学报, 2023, 54(8): 3313-3324.

HE Mingyang, MA Yujing, WANG Yong, YANG Ruochen, LIU Yueqin, ZHANG Yingjie, DUAN Chunhui. Effects of Melatonin on Proliferation, Apoptosis of Ovarian Granulosa Cells, and Its Secretion of Steroid Hormones of Sheep[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3313-3324.

参考文献 39

[1]	SONG C, PENG W, YIN S N, et al.Melatonin improves age-induced fertility decline and attenuates ovarian mitochondrial oxidative stress in mice[J].Sci Rep, 2016, 6:35165.
[2]	冯肖艺, 杨柏高, 郝海生, 等.热应激导致奶牛胚胎质量下降的机制及解决措施[J].畜牧兽医学报, 2023, 54(3):868-876.FENG X Y, YANG B G, HAO H S, et al.Mechanism and solution of heat stress induced embryo quality decline in dairy cows[J].Acta Veterinaria et Zootechnica Sinica, 2023, 54(3):868-876.(in Chinese)
[3]	YANG L, ZHAO Z M, CUI M S, et al.Melatonin restores the developmental competence of heat stressed porcine oocytes, and alters the expression of genes related to oocyte maturation[J].Animals (Basel), 2021, 11(4):1086.
[4]	王梁, 刘国世, 史文清, 等.外源褪黑激素对梅花鹿超数排卵及体内促性腺激素的影响[C]//第二届中国鹿业发展大会论文集.长春:中国畜牧业协会, 2011:10.WANG L, LIU G S, SHI W Q, et al.Effects of exogenous melatonin on superovulation and gonadotrophin hormones in sika deer[C]//Proceedings of the second China Deer Industry Development Conference.Changchun:China Animal Agriculture Association, 2011:10.(in Chinese)
[5]	XIAO L F, HU J J, SONG L L, et al.Profile of melatonin and its receptors and synthesizing enzymes in cumulus-oocyte complexes of the developing sheep antral follicle-a potential estradiol-mediated mechanism[J].Reprod Biol Endocrinol, 2019, 17(1):1.
[6]	梁锦萍, 字向东.褪黑素对动物繁殖的调控作用机制研究进展[J].中国畜牧杂志, 2021, 57(5):53-58.LIANG J P, ZI X D.Advances in the regulation mechanism of melatonin on animal reproduction[J]. Chinese Journal of Animal Science, 2021, 57(5):53-58.(in Chinese)
[7]	ADRIAENS I, JACQUET P, CORTVRINDT R, et al.Melatonin has dose-dependent effects on folliculogenesis, oocyte maturation capacity and steroidogenesis[J].Toxicology, 2006, 228(2-3):333-343.
[8]	SOARES J M, MASANA M I, ERŞAHIN Ç, et al.Functional melatonin receptors in rat ovaries at various stages of the estrous cycle[J].J Pharmacol Exp Ther, 2003, 306(2):694-702.
[9]	白欣洁, 刘莎莎, 王功民, 等.褪黑素在雌性动物繁殖中的调控作用[J].中国兽医科学, 2020, 50(7):908-914.BAI X J, LIU S S, WANG G M, et al.The regulation of melatonin on female reproduction[J].Chinese Veterinary Science, 2020, 50(7):908-914.(in Chinese)
[10]	王雨曈.不同BMPR-IB基因型对绵羊初情期生殖激素及生长发育的影响[D].石河子:石河子大学, 2020.WANG Y T.Effects of different BMPR-IB genotypes on reproductive hormones, growth and development in puberty of sheep[D].Shihezi:Shihezi University, 2020.(in Chinese)
[11]	胡亚美, 宋湘容, 黄亮, 等.FGF21增强线粒体功能抑制猪卵巢颗粒细胞凋亡[J].畜牧兽医学报, 2023, 54(3):1034-1045.HU Y M, SONG X R, HUANG L, et al.FGF21 enhances mitochondrial function and inhibits apoptosis of porcine ovarian granulosa cells[J].Acta Veterinaria et Zootechnica Sinica, 2023, 54(3):1034-1045.(in Chinese)
[12]	ZHU Q, DING D, YANG H, et al.Melatonin protects mitochondrial function and inhibits oxidative damage against the decline of human oocytes development caused by prolonged cryopreservation[J].Cells, 2022, 11(24):4018.
[13]	张培培, 郝海生, 杜卫华, 等.OPU卵母细胞体外成熟体系的优化研究进展[J].畜牧兽医学报, 2023, 54(4):1359-1369.ZHANG P P, HAO H S, DU W H, et al.A review of optimization of in vitro maturation system of OPU oocytes[J].Acta Veterinaria et Zootechnica Sinica, 2023, 54(4):1359-1369.(in Chinese)
[14]	TAKETANI T, TAMURA H, TAKASAKI A, et al.Protective role of melatonin in progesterone production by human luteal cells[J].J Pineal Res, 2011, 51(2):207-213.
[15]	冯肖艺, 刘昱成, 王晶晶, 等.褪黑素在绵羊体外胚胎生产技术中的应用研究进展[J].中国畜牧杂志, 2023, 59(3):77-81.FENG X Y, LIU Y C, WANG J J, et al.Research progress on the application of melatonin to sheep in vitro embryo production technology[J].Chinese Journal of Animal Science, 2023, 59(3):77-81.(in Chinese)
[16]	LOREN P, SÁNCHEZ R, ARIAS M E, et al.Melatonin scavenger properties against oxidative and Nitrosative Stress:impact on gamete handling and in vitro embryo production in humans and other mammals[J].Int J Mol Sci, 2017, 18(6):1119.
[17]	张红春, 李勇, 张岩, 等.褪黑素在动物卵泡发育和闭锁中作用的研究进展[J].黑龙江畜牧兽医, 2021(7):38-43.ZHANG H C, LI Y, ZHANG Y, et al.Progress in the role of melatonin in animal follicular development and atresia[J]. Heilongjiang Animal Science and Veterinary Medicine, 2021(7):38-43.(in Chinese)
[18]	TIAN X Z, WANG F, ZHANG L, et al.Beneficial effects of melatonin on the in vitro maturation of sheep oocytes and its relation to melatonin receptors[J].Int J Mol Sci, 2017, 18(4):834.
[19]	HE Y M, DENG H H, SHI M H, et al.Melatonin modulates the functions of porcine granulosa cells via its membrane receptor MT2 in vitro[J].Anim Reprod Sci, 2016, 172:164-172.
[20]	SHI J X, LIU C Y, CHEN M Y, et al.The interference effects of bisphenol A on the synthesis of steroid hormones in human ovarian granulosa cells[J].Environ Toxicol, 2021, 36(4):665-674.
[21]	李宇, 段春辉, 宋志攀, 等.黄体期注射PGF2α对母羊血清生殖激素及相关细胞因子的影响[J].畜牧兽医学报, 2022, 53(6):1807-1818.LI Y, DUAN C H, SONG Z P, et al.Effects of PGF2α on Reproductive Hormones and Related Cytokines during Luteal Phase in Ewes[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(6):1807-1818.(in Chinese)
[22]	陈文俊, 盛文洁, 郭银华, 等.日节律紊乱对大鼠妊娠及子代的影响[J].生理学报, 2015, 67(5):521-526.CHEN W J, SHENG W J, GUO Y H, et al.The influence of interfered circadian rhythm on pregnancy and neonatal rats[J].Acta Physiologica Sinica, 2015, 67(5):521-526.(in Chinese)
[23]	RIAZ H, YOUSUF M R, LIANG A X, et al.Effect of melatonin on regulation of apoptosis and steroidogenesis in cultured buffalo granulosa cells[J].Anim Sci J, 2019, 90(4):473-480.
[24]	张良.褪黑素对放疗后大鼠卵巢功能的保护作用及其机制的研究[D].广州:南方医科大学, 2014.ZHANG L.The protective effects of melatonin on radiotherapy-induced ovarian damage in rats and its mechanism[D]. Guangzhou:Southern Medical University, 2014.(in Chinese)
[25]	VON GALL C, STEHLE J H, WEAVER D R.Mammalian melatonin receptors:molecular biology and signal transduction[J]. Cell Tissue Res, 2002, 309(1):151-162.
[26]	张红春.褪黑素对产蛋末期蛋鸡卵巢颗粒细胞的影响[D].郑州:河南农业大学, 2021.ZHANG H C.Effects of melatonin on ovary granulosa cells of laying hens in late laying period[D].Zhengzhou:Henan Agricultural University, 2021.(in Chinese)
[27]	赵淑琴.褪黑素介导的生物钟基因Cry对双峰驼雌二醇分泌的影响[D].兰州:甘肃农业大学, 2020.ZHAO S Q.Effect of melatonin-mediated circadian clock genes Cry on estradiol secretion of Bactrian camel[D].Lanzhou:Gansu Agricultural University, 2020.(in Chinese)
[28]	何珲, 姜冬梅, 康波.褪黑素调控动物繁殖功能的途径[J].中国生物化学与分子生物学报, 2014, 30(2):144-149.HE H, JIANG D M, KANG B.Regulation of melatonin in animal reproduction[J].Chinese Journal of Biochemistry and Molecular Biology, 2014, 30(2):144-149.(in Chinese)
[29]	WANG S J, LIU W J, WANG L K, et al.The role of melatonin receptor MTNR1A in the action of melatonin on bovine granulosa cells[J].Mol Reprod Dev, 2017, 84(11):1140-1154.
[30]	王建萍, 张克英.氧化应激对家禽卵泡闭锁的影响及机制[J].动物营养学报, 2021, 33(6):3001-3009.WANG J P, ZHANG K Y.Effects of oxidative stress on follicular atresia in poultry and its mechanism[J].Chinese Journal of Animal Nutrition, 2021, 33(6):3001-3009.(in Chinese)
[31]	阿依木古丽·阿不都热依木, 阿尔祖古丽·阿依丁, 王家敏, 等.大豆异黄酮对牦牛卵巢颗粒细胞增殖和凋亡的影响[J].中国农业科学, 2022, 55(8):1667-1675.ABUDUREYIMU·A, AYIDING·A, WANG J M, et al.Effects of soy Isoflavones on the proliferation and apoptosis of yak ovarian granulosa cells[J].Scientia Agricultura Sinica, 2022, 55(8):1667-1675.(in Chinese)
[32]	FU Y, HE C J, JI P Y, et al.Effects of melatonin on the proliferation and apoptosis of sheep granulosa cells under thermal stress[J].Int J Mol Sci, 2014, 15(11):21090-21104.
[33]	程华琴, 袁钰洁, 罗文, 等.褪黑素对牦牛黄体细胞生长及功能影响的研究[J].畜牧兽医学报, 2022, 53(2):459-469.CHENG H Q, YUAN Y J, LUO W, et al.Research on the effect of melatonin on the growth and function of yak luteal cells[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2):459-469.(in Chinese)
[34]	张金璧.类固醇激素合成酶及相关受体表达与猪卵泡闭锁过程的关系研究[D].南京:南京农业大学, 2010.ZHANG J B.Relationship between changes of steroidogenesis enzymes and related hormone Reseptors gene expression and follicular atresia of swine[D].Nanjing:Nanjing Agricultural University, 2010.(in Chinese)
[35]	MARSTERS P, KENDALL N R, CAMPBELL B K.Pre-translational regulation of luteinizing hormone receptor in follicular somatic cells of cattle[J].Anim Reprod Sci, 2015, 163:63-74.
[36]	杜婷, 任春娥, 韩海燕, 等.卵泡液中颗粒细胞FSHR的表达与体外受精-胚胎移植的相关研究[J].潍坊医学院学报, 2014, 36(3):217-219, 222.DU T, REN C E, HAN H Y, et al.Study of the relationship between the expression of FSH receptor on granulosa cells and in vitro fertilization and embryo transfer (IVF-ET)[J].Acta Academiae Medicinae Weifang, 2014, 36(3):217-219, 222.(in Chinese)
[37]	王磊, 何莉娜, 唐雪, 等.miR-495-3p对山羊卵巢颗粒细胞功能的影响[J].畜牧兽医学报, 2022, 53(2):436-446.WANG L, HE L N, TANG X, et al.Effects of miR-495-3p on ovarian granulosa cell functions in goat[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(2):436-446.(in Chinese)
[38]	贺亚媚.褪黑素对猪卵泡发育与闭锁的影响研究[D].杨凌:西北农林科技大学, 2017.HE Y M.Effects of melatonin on follicular development and atresia in the porcine[D].Yangling:Northwest A&F University, 2017.(in Chinese)
[39]	MATTI N, IRVING-RODGERS H F, HATZIRODOS N, et al.Differential expression of focimatrix and steroidogenic enzymes before size deviation during waves of follicular development in bovine ovarian follicles[J].Mol Cell Endocrinol, 2010, 321(2):207-214.