促卵泡素对体外培养牛卵泡类固醇合成相关基因表达的影响

doi:10.16431/j.cnki.1671-7236.2019.11.019

摘要/Abstract

摘要： 本研究旨在探究促卵泡素（follicle stimulating hormone，FSH）处理对体外培养的牛有腔卵泡颗粒细胞和膜细胞类固醇激素合成相关基因表达的影响。采集牛卵巢表面直径9~11 mm的有腔卵泡，用含不同浓度FSH的DMEM/F12体外培养牛有腔卵泡24 h。提取卵泡颗粒细胞、膜细胞RNA并反转录成cDNA，利用实时荧光定量PCR检测卵泡颗粒细胞、膜细胞中类固醇激素合成酶基因（CYP11A1、3β-HSD、CYP17A1、CYP19A1、17β-HSD）和促性腺激素受体基因（FSHR、LHR）的表达水平。结果显示，FSH处理上调了颗粒细胞中CYP11A1、3β-HSD和CYP19A1基因表达，其中，25 ng/mL FSH处理极显著上调了CYP11A1基因表达（P<0.01），10 ng/mL FSH处理显著上调了3β-HSD基因表达（P<0.05），50 ng/mL FSH处理显著上调了CYP19A1基因表达（P<0.05）；50 ng/mL FSH处理显著或极显著上调了膜细胞中CYP11A1、3β-HSD和CYP17A1基因表达（P<0.05；P<0.01），但在10和25 ng/mL FSH处理组中CYP11A1、3β-HSD和CYP17A1基因表达显著或极显著下调（P<0.05；P<0.01）。对FSHR、LHR基因研究结果显示，不同浓度FSH处理对颗粒细胞中FSHR、LHR基因的表达均无显著影响（P>0.05），只有25和50 ng/mL FSH处理显著或极显著上调了膜细胞中LHR基因表达水平（P<0.05；P<0.01），且不同处理组之间膜细胞中CYP11A1、3β-HSD和CYP17A1基因的表达变化与LHR基因表达变化趋势较一致。结果表明，FSH处理可提高牛有腔卵泡颗粒细胞中CYP11A1、3β-HSD和CYP17A1基因的表达，膜细胞中CYP11A1、3β-HSD和CYP17A1基因对LH的刺激更敏感，FSH可能通过影响LHR基因的表达来调节膜细胞中类固醇合成酶基因的表达。

关键词: 牛; 促卵泡素(FSH); 有腔卵泡; 类固醇合成相关基因; 体外试验

Abstract: The purpose of this study was to investigate the effects of FSH treatment on steroidogenesis genes expression in granulosa cells and theca cells of cultured bovine antral follicles in vitro.Cavernous follicles 9-11 mm in diameter on the surface of bovine ovaries were collected and cultured in vitro with DMEM/F12 containing different concentrations of FSH for 24 hours.The total RNA of granulosa cells and theca cells were extracted,and reverse transcribed into cDNA,the expression of steroidogenesis genes (CYP11A1,3β-HSD,CYP17A1,CYP19A1 and 17β-HSD) and gonadotropin receptor genes (FSHR and LHR) in follicular granulosa cells and theca cells were detected by Real-time quantitative PCR.The results in granulosa cells showed that FSH up-regulated the expression of CYP11A1,3β-HSD and CYP19A1 genes,25 ng/mL FSH treatment extremely significantly increased the expression of CYP11A1 gene (P<0.01),10 ng/mL FSH treatment significantly increased the expression of 3β-HSD gene (P<0.05),50 ng/mL FSH treatment significantly increased the expression of CYP19A1 gene (P<0.05).The results in theca cells showed that 50 ng/mL FSH treatment significantly or extremely significantly increased the expression of CYP11A1,3β-HSD and CYP17A1 genes (P<0.05;P<0.01),but 10 and 25 ng/mL FSH treatment significantly or extremely significantly decreased the expression of CYP11A1,3β-HSD and CYP17A1 genes (P<0.05;P<0.01).The results showed that FSH treatment had no significant effect on the expression of FSHR and LHR genes in granulosa cells (P>0.05),only 25 and 50 ng/mL FSH treatments significantly or extremely significantly increased the expression of LHR gene in theca cells (P<0.05;P<0.01).The expression of CYP11A1,3β-HSD and CYP17A1 genes in theca cells among the different treatment groups showed the same trend as that of LHR gene.The results indicated that FSH treatment could increase the expression of CYP11A1,3β-HSD and CYP17A1 genes in granulosa cells of bovine antral follicular,the expression of CYP11A1,3β-HSD and CYP17A1 genes in theca cells were more sensitive to the stimulation of LH.FSH might regulate the expression of steroidogenesis genes in theca cells by affecting the expression of LHR gene.

Key words: bovine; FSH; antral follicles; steroidogenesis genes; in vitro test

中图分类号:

S814.8

王洋洋, 许慧韬, 赵善江, 庞云渭, 郝海生, 邹惠影, 朱化彬. 促卵泡素对体外培养牛卵泡类固醇合成相关基因表达的影响[J]. 中国畜牧兽医, 2019, 46(11): 3299-3306.

WANG Yangyang, XU Huitao, ZHAO Shanjiang, PANG Yunwei, HAO Haisheng, ZOU Huiying, ZHU Huabin. Effects of FSH on Genes Expression Related to Steroidogenesis in Bovine Follicles Cultured in vitro[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(11): 3299-3306.

参考文献

[1] GARCIA G A,TRIBULO A,YAPURA J,et al.Lengthened superstimulatory treatment in cattle:Evidence for rescue of follicles within a wave rather than continuous recruitment of new follicles[J].Theriogenology,2015,84(3):467-476.
[2] SINGH J,DOMINGUEZ M,JAISWAL R,et al.A simple ultrasound test to predict the superstimulatory response in cattle[J].Theriogenology,2004,62(1-2):227-243.
[3] BARUSELLI P S,DE SA M F,MARTINS C M,et al.Superovulation and embryo transfer in Bos indicus cattle[J].Theriogenology,2006,65(1):77-88.
[4] KELLER D S,TEEPKER G.Effect of variability in response to superovulation on donor cow selection differentials in nucleus breeding schemes[J].Journal of Dairy Science,1990,73(2):549-554.
[5] FAN Q R,HENDRICKSON W A.Structure of human follicle-stimulating hormone in complex with its receptor[J].Nature,2005,433(7023):269-277.
[6] SHIMIZU T.Molecular and cellular mechanisms for the regulation of ovarian follicular function in cows[J].Journal of Reproduction and Development,2016,62(4):323-329.
[7] RODRIGUEZ K F,COUSE J F,JAYES F L,et al.Insufficient luteinizing hormone-induced intracellular signaling disrupts ovulation in preovulatory follicles lacking estrogen receptor-β[J].Endocrinology,2010,151(6):2826-2834.
[8] FORTUNE J E,YANG M Y,MURUVI W.The earliest stages of follicular development:Follicle formation and activation[J].Society of Reproduction and Fertility Supplement,2010,67(1):203-216.
[9] 王和建,张莉,余道伦,等.BMP4诱导的Smad9在小鼠卵泡发育中的作用及其机制的初步研究[J].中国畜牧兽医,2018,45(10):2902-2909. WANG H J,ZHANG L,YU D L,et al.Preliminary study on the role of BMP4-induced Smad9 in mouse follicular development and its mechanism[J].China Animal Husbandry & Veterinary Medicine,2018,45(10):2902-2909.(in Chinese)
[10] 陈超,李俊,华丽萍,等.RNA干扰水牛MTPN基因对颗粒细胞增殖、凋亡及激素分泌的影响[J].中国畜牧兽医,2019,46(3):800-807. CHEN C,LI J,HUA L P,et al.Effect of RNA interference buffalo MTPN gene on proliferation,apoptosis of granulosa cells and hormone secretion[J].China Animal Husbandry & Veterinary Medicine,2019,46(3):800-807.(in Chinese)
[11] MILLER W L.Steroid hormone synthesis in mitochondria[J].Molecular and Cellular Endocrinology,2013,379(1-2):62-73.
[12] SUMMERS A F,POHLMEIER W E,SARGENT K M,et al.Altered theca and cumulus oocyte complex gene expression,follicular arrest and reduced fertility in cows with dominant follicle follicular fluid androgen excess[J].PLoS One,2014,9(10):e110683.
[13] MILLER W L,AUCHUS R J.The molecular biology,biochemistry,and physiology of human steroidogenesis and its disorders[J].Endocrine Reviews,2011,32(1):81-151.
[14] HEDIN L,RODGERS R J,SIMPSON E R,et al.Changes in content of cytochrome P45017α,cytochrome P450scc,and 3-hydroxy-3-methylglutaryl CoA reductase in developing rat ovarian follicles and corpora lutea:Correlation with theca cell steroidogenesis[J].Biology of Reproduction,1987,37(1):211-223.
[15] LI H,CHEN Y,YAN L Y,et al.Increased expression of P450scc and CYP17 in development of endogenous hyperandrogenism in a rat model of PCOS[J].Endocrine,2013,43(1):184-190.
[16] KWINTKIEWICZ J,CAI Z,STOCCO C.Follicle-stimulating hormone-induced activation of Gata4 contributes in the up-regulation of Cyp19 expression in rat granulosa cells[J].Molecular Endocrinology,2007,21(4):933-947.
[17] CONLEY A J,BIRD I M.The role of cytochrome P45017 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase in the integration of gonadal and adrenal steroidogenesis via the delta 5 and delta 4 pathways of steroidogenesis in mammals[J].Biology of Reproduction,1997,56(4):789-799.
[18] ADASHI E Y,HSUEH A J.Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells[J].Journal of Biological Chemistry,1982,257(11):6077-6083.
[19] 关洪斌,李庆章.卵泡刺激素研究进展[J].东北农业大学学报,2002,33(3):306-311. GUAN H B,LI Q Z.The study advance of follicle stimulating hormone[J].Journal of Northeast Agricultural University,2002,33(3):306-311.(in Chinese)
[20] RASOLOMBOAHANGINJATOVO H S,CHORFI Y,DUPRAS R,et al.Effect of superovulation on uterine and serum biochemical parameters and its potential association with transferable embryos in Holstein dairy cows[J].Journal of Veterinary Science,2014,15(2):273-281.
[21] GARVERICK H A,BAXTER G,GONG J,et al.Regulation of expression of ovarian mRNA encoding steroidogenic enzymes and gonadotrophin receptors by FSH and GH in hypogonadotrophic cattle[J].Reproduction,2002,123(5):651-661.
[22] SHARMA G T,MAJUMDAR A C.Control of follicular steroidogenesis in early-and late-luteal phase goat ovaries[J].Small Ruminant Research,1999,34(2):111-117.
[23] HILLIER S G.Current concepts of the roles of follicle stimulating hormone and luteinizing hormone in folliculogenesis[J].Human Reproduction,1994,9(2):188-191.
[24] DUARTE A,PODEROSO C,COOKE M,et al.Mitochondrial fusion is essential for steroid biosynthesis[J].PLoS One,2012,7(9):e45829.
[25] ZIRILLI L,ROCHIRA V,DIAZZI C,et al.Human models of aromatase deficiency[J].Journal of Steroid Biochemistry and Molecular Biology,2008,109(3-5):212-218.
[26] YU G M,HAQ I U,KHAN S H,et al.Actin filaments are necessary for FSH-induced CYP19A1 transcription in bovine granulosa cells in vitro[J].Pakistan Veterinary Journal,2015,35(1):53-57.
[27] FITZPATRICK S L,RICHARDS J S.Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells[J].Endocrinology,1991,129(3):1452-1462.
[28] MANUEL SILVA J,PRICE C A.Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450 aromatase and cholesterol side-chain cleavage in bovine granulosa cells in vitro[J].Biology of Reproduction,2000,62(1):186-191.
[29] JOHNSON S K,DAILEY R A,INSKEEP E K,et al.Effect of peripheral concentrations of progesterone on follicular growth and fertility in ewes[J].Domestic Animal Endocrinology,1996,13(1):69-79.
[30] HATLER T B,HAYES S H,RAY D L,et al.Effect of subluteal concentrations of progesterone on luteinizing hormone and ovulation in lactating dairy cows[J].The Veterinary Journal,2008,177(3):360-368.
[31] SALONIEMI T,JOKELA H,STRAUSS L,et al.The diversity of sex steroid action:Novel functions of hydroxysteroid (17beta) dehydrogenases as revealed by genetically modified mouse models[J].Journal of Endocrinology,2012,212(1):27-40.
[32] HARTMANN R W,MARCHAIS-OBERWINKLER S,HENN C,et al.17beta-hydroxysteroid dehydrogenases (17beta-HSDs) as therapeutic targets:Protein structures,functions,and recent progress in inhibitor development[J].Journal of Steroid Biochemistry and Molecular Biology,2011,125(1-2):66-82.
[33] BOEHMER A L,BRINKMANN A O,SANDKUIJL L A,et al.17beta-hydroxysteroid dehydrogenase-3 deficiency:Diagnosis,phenotypic variability,population genetics,and worldwide distribution of ancient and de novo mutations[J].Journal of Clinical Endocrinology and Metabolism,1999,84(12):4713-4721.
[34] BARROS C M,SATRAPA R A,CASTILHO A C,et al.Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity,oocyte competence and embryo development in cattle[J].Reproduction,Fertility and Development,2012,25(1):17-25.
[35] NAKAO K,KISHI H,IMAI F,et al.TNF-alpha suppressed FSH-induced LH receptor expression through transcriptional regulation in rat granulosa cells[J].Endocrinology,2015,156(9):3192-3202.
[36] FA S,POGRMIC-MAJKIC K,SAMARDZIJA D,et al.Involvement of ERK1/2 signaling pathway in atrazine action on FSH-stimulated LHR and CYP19A1 expression in rat granulosa cells[J].Toxicology and Applied Pharmacology,2013,270(1):1-8.
[37] LUO W,WILTBANK M C.Distinct regulation by steroids of messenger RNAs for FSHR and CYP19A1 in bovine granulosa cells[J].Biology of Reproduction,2006,75(2):217-225.
[38] 谭萍.大鼠颗粒细胞Inhibin A抑制FSHR表达抑制FSH的作用[D].重庆:西南大学,2008. TAN P.Inhibin A inhibits FSH action by suppressing its receptor expression in cultured rat granulosa cells[D].Chongqing:Southwest University,2008.(in Chinese)
[39] LU C,YANG W,CHEN M,et al.Inhibin A inhibits follicle-stimulating hormone (FSH) action by suppressing its receptor expression in cultured rat granulosa cells[J].Biology of Reproduction,2009,298(1-2):48-56.
[40] NOGUEIRA M F,BURATINI J J,PRICE C A,et al.Expression of LH receptor mRNA splice variants in bovine granulosa cells:Changes with follicle size and regulation by FSH in vitro[J].Molecular Reproduction and Development,2007,74(6):680-686.
[41] ALLEN J J,HERRICK S L,FORTUNE J E.Regulation of steroidogenesis in fetal bovine ovaries:Differential effects of LH and FSH[J].Journal of Molecular Endocrinology,2016,57(4):275-286.
[42] SALHAB M,TOSCA L,CABAU C,et al.Kinetics of gene expression and signaling in bovine cumulus cells throughout IVM in different mediums in relation to oocyte developmental competence,cumulus apoptosis and progesterone secretion[J].Theriogenology,2011,75(1):90-104.