牦牛和黄牛PGRMC1基因的克隆及在繁殖相关组织中的表达比较研究
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摘要
【目的】孕激素受体膜组分1 (PGRMC1)在动物繁殖活动中有重要调节作用,本研究旨在探讨牦牛PGRMC1基因的序列及其在生殖轴的组织表达特性。【方法】试验采集5头牦牛和5头黄牛的下丘脑、脑垂体、卵巢、输卵管和子宫,以GenBank上已发布的普通牛PGRMC1基因序列设计引物,采用RT-PCR方法克隆牦牛和黄牛的PGRMC1基因,并使用相关的生物信息学软件对克隆所得序列进行分析;采用qRT-PCR法检测该基因在牦牛和黄牛不同组织中的表达情况。【结果】结果得到牦牛和黄牛PGRMC1基因cDNA序列(GenBank登录号:MF803753和MF803754),编码区(CDS)全长都为585 bp,编码194个氨基酸。其编码蛋白中含有1个Cyt-b5保守结构域。qRT-PCR结果表明:PGRMC1基因在牦牛和黄牛下丘脑、脑垂体、卵巢、输卵管和子宫都有表达,牦牛和黄牛的垂体中的PGRMC1的表达差异显著(P<0.05),但在其他组织品种间差异不显著;牦牛PGRMC1在卵巢和垂体的表达量显著高于其他组织(P<0.05)。【结论】本研究提示PGRMC1基因可能在牦牛卵泡发育、发情、排卵等繁殖机能调控中发挥重要作用。
[Purpose]Progesterone receptor membrane component 1(PGRMC1) plays important roles in regulation of animal reproduction, therefore, we aimed to study the sequence and expression characteristics of PGRMC1 gene in yak.[Method]Hypothalami, pituitaries, ovaries, oviducts and uteri were collected from 5 female yaks and 5 cows. The primers were designed by using mRNA sequence of Bos taurus PGRMC1 gene published in GenBank. The PGRMC1 gene was cloned by RTPCR and the sequences were analyzed by using related bioinformatics software. The expression levels of the gene in different tissues were detected by qRT-PCR.[ Result] The cDNA sequences of PGRMC1 gene(GenBank accession No.: MF803753 and MF803754) were cloned for yak and cattle,and the coding area(CDS) was 585 bp in length, encoding 194 amino acids. The encoding protein contained a Cyt-b5 conserved domain. The results of qRT-PCR analysis showed that PGRMC1 gene was expressed in the hypothalamus, pituitary, ovary, oviduct and uterus of yak and cattle. The expression of PGRMC1 in the pituitary was significantly different(P<0.05) between yak and cattle, but there was no difference in other tissues between these two species. The expression levels of PGRMC1 in ovary and pituitary of yak were significantly higher than that in other tissues of yaks(P<0.05).[Conclusion]The result indicated that PGRMC1 gene may play an important role in the yak reproduction of follicular development, estrus and ovulation.
[Purpose]Progesterone receptor membrane component 1(PGRMC1) plays important roles in regulation of animal reproduction, therefore, we aimed to study the sequence and expression characteristics of PGRMC1 gene in yak.[Method]Hypothalami, pituitaries, ovaries, oviducts and uteri were collected from 5 female yaks and 5 cows. The primers were designed by using mRNA sequence of Bos taurus PGRMC1 gene published in GenBank. The PGRMC1 gene was cloned by RTPCR and the sequences were analyzed by using related bioinformatics software. The expression levels of the gene in different tissues were detected by qRT-PCR.[ Result] The cDNA sequences of PGRMC1 gene(GenBank accession No.: MF803753 and MF803754) were cloned for yak and cattle,and the coding area(CDS) was 585 bp in length, encoding 194 amino acids. The encoding protein contained a Cyt-b5 conserved domain. The results of qRT-PCR analysis showed that PGRMC1 gene was expressed in the hypothalamus, pituitary, ovary, oviduct and uterus of yak and cattle. The expression of PGRMC1 in the pituitary was significantly different(P<0.05) between yak and cattle, but there was no difference in other tissues between these two species. The expression levels of PGRMC1 in ovary and pituitary of yak were significantly higher than that in other tissues of yaks(P<0.05).[Conclusion]The result indicated that PGRMC1 gene may play an important role in the yak reproduction of follicular development, estrus and ovulation.
引文
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[12]高磊,沈敏,甘尚权,等.绵羊PGRMC1蛋白的cDNA克隆、序列分析及组织表达[J].农业生物技术学报,2016,24(1):52.DOI:10.3969/j.issn.1674-7968.2016.01.007.
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[15]SAMBROOK J,FRITSCH E F,MANIATIS T.Molecular cloning:a laboratory manual[M].Cold Spring Harbor Laboratory Press,1989.
[16]CRUDDEN G,CHITTI R E,CRAVEN R J.Hpr6(heme-1 domain protein)regulates the susceptibility of cancer cells to chemotherapeutic drugs[J].The Journal of Pharmacology and Experimental Therapeutics,2006,316:448.
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[18]MIN L,STRUSHKEVICH NV,HARNASTAI I N,et al.Molecular identification of adrenal inner zone antigen as a heme-binding protein[J].The FEBS journal,2005,272(22):5832.DOI:10.1111/j.1742-4658.2005.04977.x.
[19]INTLEKOFER K A,PETERSEN S L.Distribution of mRNAs encoding classical progestin receptor,progesterone membrane components 1 and 2,serpine mRNAbinding protein 1,and progestin and ADIPOQ receptor family members 7 and 8 in rat forebrain[J].Neuroscience,2011,172:55.DOI:10.1016/j.neuroscience.2010.10.051.
[20]PELUSO J J,PRU J K.Non-canonical progesterone signaling in granulosa cell function[J].Reproduction,2014,147(5):R169.DOI:10.1530/REP-13-0582.
[21]SAINT-DIZIER M,SANDRA O,PLOYART S,et al.Expression of nuclear progesterone receptor and progesterone receptor membranecomponents 1 and 2 in the oviduct of cyclic and pregnant cows during the post-ovulation period[J].Reproductive Biology and Endocrinology,2012,10:76.DOI:10.1186/1477-7827-10-76.
[2]TERZAGHI L,TESSARO I,RAUCCI F,et al.PGRMC1 participates in late events of bovine granulose cells mitosis and oocyte meiosis[J].Cell cycle,2016,15(15):2019.DOI:10.1080/15384101.2016.1192731.
[3]PELUSO J J,ROMAK J,LIU X.Progesterone receptor membrane component-1(PGRMC1)is the mediator of progesterone’s antiapoptotic action in spontaneously immortalized granulosa cells as revealed by PGRMC1small interfering ribonucleic acid treatment and functional analysis of PGRMC1 mutations[J].Endocrinology,2008,149(2):534.DOI:10.1210/en.2007-1050.
[4]SCHUSTER J,KARLSSON T,KARLSTR?M P O,et al.Down-regulation of progesterone receptor membrane component 1(PGRMC1)in peripheral nucleated blood cells associated with premature ovarian failure(POF)and polycystic ovary syndrome(PCOS)[J].Reproductive Biology and Endocrinology,2010,8:58.DOI:10.1186/1477-7827-8-58.
[5]MANSOURI M R,SCHUSTER J,BADHAI J,et al.Alterations in the expression,structure and function of progesterone receptor membrane component-1(PGRMC1)in premature ovarian failure[J].Human Molecular Genetics,2008,17(23):3776.DOI:10.1093/hmg/ddn274.
[6]ELASSAR A,LIU X,SCRANTON V,et al.The relationship between follicle development and progesterone receptor membrane component-1 expression in women undergoing in vitro fertilization[J].Fertility and Sterility,2012,97(3):572.DOI:10.1016/j.fertnstert.2011.12.026.
[7]ENGMANN L,LOSEL R,WEHLING M,et al.Progesterone regulation of human granulosa/luteal cell viability by an RU486-independent mechanism[J].The Journal of Clinical Endocrinology and Metabolism,2006,91(12):4962.DOI:10.1210/jc.2006-1128.
[8]PELUSO J J,PAPPALARDO A,LOSEL R,et al.Progesterone membrane receptor component 1 expression in the immature rat ovary and its role in mediating progesterone’s antiapoptotic action[J].Endocrinology,2006,147(6):3133.DOI:10.1210/en.2006-0114.
[9]BISHOP C V,SATTERWHITE S,XU L,et al.Microarray analysis of the primate luteal transcriptome during chorionic gonadotrophin administration simulating early pregnancy[J].Molecular Human Reproduction,2012,18(4):216.DOI:10.1093/molehr/gar073.
[10]KOWALIK M K,REKAWIECKI R,KOTWICA J.Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy[J].Theriogenology,2014,82(8):1086.DOI:10.1016/j.theriogenology.2014.07.021.
[11]LUCIANO A M,CORBANI D,LODDE V,et al.Expression of progesterone receptor membrane component-1 in bovine reproductive system during estrous cycle[J].European Journal of Histochemistry,2011,55(3):e27.DOI:10.4081/ejh.2011.e27.
[12]高磊,沈敏,甘尚权,等.绵羊PGRMC1蛋白的cDNA克隆、序列分析及组织表达[J].农业生物技术学报,2016,24(1):52.DOI:10.3969/j.issn.1674-7968.2016.01.007.
[13]PETERSEN S L,OTTEM E N,CARPENTER C D.Direct and indirect regulation of gonadotropin-releasing hormone neurons by estradiol[J].Biology of Reproduction,2003,69(6):1771.DOI:10.1095/biolreprod.103.019745.
[14]ZI X D.Reproduction in female yaks(Bos grunniens)and opportunities for improvement[J].Theriogenology,2003,59(5/6):1303.DOI:10.1016/S0093-691X(02)01172-X.
[15]SAMBROOK J,FRITSCH E F,MANIATIS T.Molecular cloning:a laboratory manual[M].Cold Spring Harbor Laboratory Press,1989.
[16]CRUDDEN G,CHITTI R E,CRAVEN R J.Hpr6(heme-1 domain protein)regulates the susceptibility of cancer cells to chemotherapeutic drugs[J].The Journal of Pharmacology and Experimental Therapeutics,2006,316:448.
[17]NEBERT D W,RUSSELL D W.Clinical importance of the cytochromes P450[J].Lancet,2002,360(9340):1155.DOI:10.1016/S0140-6736(02)11203-7.
[18]MIN L,STRUSHKEVICH NV,HARNASTAI I N,et al.Molecular identification of adrenal inner zone antigen as a heme-binding protein[J].The FEBS journal,2005,272(22):5832.DOI:10.1111/j.1742-4658.2005.04977.x.
[19]INTLEKOFER K A,PETERSEN S L.Distribution of mRNAs encoding classical progestin receptor,progesterone membrane components 1 and 2,serpine mRNAbinding protein 1,and progestin and ADIPOQ receptor family members 7 and 8 in rat forebrain[J].Neuroscience,2011,172:55.DOI:10.1016/j.neuroscience.2010.10.051.
[20]PELUSO J J,PRU J K.Non-canonical progesterone signaling in granulosa cell function[J].Reproduction,2014,147(5):R169.DOI:10.1530/REP-13-0582.
[21]SAINT-DIZIER M,SANDRA O,PLOYART S,et al.Expression of nuclear progesterone receptor and progesterone receptor membranecomponents 1 and 2 in the oviduct of cyclic and pregnant cows during the post-ovulation period[J].Reproductive Biology and Endocrinology,2012,10:76.DOI:10.1186/1477-7827-10-76.