减数分裂期雄性大菱鲆孕激素及受体基因的表达分析
|
摘要
为研究孕激素在大菱鲆(Scophthalmus maximus)精原细胞增殖到减数分裂过程中的作用,以6~22月龄的大菱鲆精巢为材料,通过组织学方法和定量amh、sycp3基因确定精原细胞增殖期和减数分裂期的大菱鲆精巢发育过程。通过酶联免疫吸附技术(ELISA)检测6~22月龄雄性大菱鲆血清中的孕酮(P4)和17α,20β,双羟孕酮(DHP)含量变化,利用q RT-PCR技术和原位杂交技术检测孕酮核受体(pgr)和膜受体(m PRα)在不同组织和不同发育时期的表达情况。结果表明孕激素在精原细胞增殖期高表达,开始出现初级精母细胞时降低,在精子细胞数量增加时表达量再次升高,在精子细胞变态形成精子时降低。pgr在减数分裂初期定位于精巢中的sertoli细胞,在精原细胞增殖至减数分裂期表达量逐渐增加,出现精子后显著降低;m PRα在精原细胞增殖和初级精母细胞增加时表达量都很低,在精子细胞不断增加时显著增加。推测在精原细胞增殖和减数分裂阶段孕激素可能主要通过调节pgr的表达量来促进精巢发育,而在减数分裂II期pgr和m PRα都发挥作用,在精子细胞变态成熟时,主要是m PRα发挥作用。
To explore the function of progestin during spermatogonial proliferation and meiosis,we used histological methods to investigate the gonadal development process of the male turbot(Scophthalmus maximus) 6 to 22 months of age.We detected the expression of amh and sycp3 via the real-time quantitative polymerase chain reaction(q RT-PCR).Using enzyme-linked immunosorbent(ELISA) technology,q RT-PCR,and in-situ hybridization,we detected the serum P4 and DHP levels and the expression patterns of the nuclear progesterone receptor(pgr) and membrane progestin receptor alpha(m PRa) during different development periods.The results reveal that the P4 and DHP levels were high during spermatogonial proliferation and meiosis Ⅱ.The pgr expression was high from spermatogonial proliferation to meiosis I,whereas the mPRα expression was high during meiosis Ⅱ and spermiogenesis.Our in-situ hybridization results indicate pgr m RNA to be predominantly located in the Sertoli cells that were in contact with the proliferating spermatogonia and primary spermatocyte and that the mPRα m RNA was undetectable.Taken together,our data indicate the pgr expression to be involved in mediating the progestin stimulation of spermatogonia proliferation and meiosis,whereas the m PRa plays an important role in meiosis Ⅱ and spermiogenesis.
To explore the function of progestin during spermatogonial proliferation and meiosis,we used histological methods to investigate the gonadal development process of the male turbot(Scophthalmus maximus) 6 to 22 months of age.We detected the expression of amh and sycp3 via the real-time quantitative polymerase chain reaction(q RT-PCR).Using enzyme-linked immunosorbent(ELISA) technology,q RT-PCR,and in-situ hybridization,we detected the serum P4 and DHP levels and the expression patterns of the nuclear progesterone receptor(pgr) and membrane progestin receptor alpha(m PRa) during different development periods.The results reveal that the P4 and DHP levels were high during spermatogonial proliferation and meiosis Ⅱ.The pgr expression was high from spermatogonial proliferation to meiosis I,whereas the mPRα expression was high during meiosis Ⅱ and spermiogenesis.Our in-situ hybridization results indicate pgr m RNA to be predominantly located in the Sertoli cells that were in contact with the proliferating spermatogonia and primary spermatocyte and that the mPRα m RNA was undetectable.Taken together,our data indicate the pgr expression to be involved in mediating the progestin stimulation of spermatogonia proliferation and meiosis,whereas the m PRa plays an important role in meiosis Ⅱ and spermiogenesis.
引文
[1]Lau E L,Lee M F,Chang C F,et al.Conserved sex-specific timing of meiotic initiation during sex differentiation in the protandrous black porgy Acanthopagrus schlegelii[J].Biol Reprod,2013,88(6):1-13.
[2]Scott A P,Sumpter J P,Stacey N,et al.The role of the maturation-inducing steroid,17,20beta-dihydroxypregn-4-en-3-one,in male fishes:a review[J].J Fish Biol,2010,76(1):183-224.
[3]Higuchi M,Miura C,Iwai T,et al.Trypsin regulates meiotic initiation in the Japanese eel(Anguilla japonica)by promoting the uptake of taurine into germ cells during spermatogenesis[J].Biol Reprod,2013,89(3):1-9,58.
[4]Liu G,Luo F,Song Q,et al.Blocking of progestin action disrupts spermatogenesis in Nile tilapia(Oreochromis niloticus)[J].J Mol Endocrinol,2014,53(1):57-70.
[5]Chen S X,Bogerd J,Schoonen E,et al.A progestin(17alpha,20beta-dihydroxy-4-pregnen-3-one)stimulates early stages of spermatogenesis in zebrafish[J].Gen Comp Endocrinol,2013,185:1-9.
[6]Chen S X,Bogerd J,García-López A,et al.Molecular cloning and functional characterization of a zebrafish nuclear progesterone receptor[J].Biol Reprod,2010,82(1):171-181.
[7]张瑞,阙华勇,张国范,等.海湾扇贝维甲酸受体_RXR_c DNA克隆与表达分析[J].海洋科学,2016,40(9):1-8.Zhang Rui,Que Huayong,Zhang Guofan,et al.Molecular cloning and expression of the retinoid X receptor gene in Argopecten irradians[J].Marine Sciences,2016,40(9):1-8.
[8]Salazar M.Progestin-mediated activation of MAPK and AKT in nuclear progesterone receptor negative breast epithelial cells:The role of membrane progesterone receptors[J].Gene,2016,591(1):6-13.
[9]郭佳伟,谭训刚,尤锋,等.大菱鲆周期蛋白依赖激酶基因cdk1、cdk6克隆及表达分析[J].海洋科学,2016,40(4):11-21.Guo Jiawei,Tan Xungang,You Feng,et al.Cloning and expression analysis of cdk1 and cdk6 in Scophthalmus maximus[J].Marine Sciences,2016,40(4):11-21.
[10]Hattori R S,Murai Y,Oura M,et al.A Y-linked anti-Mullerian hormone duplication takes over a critical role in sex determination[J].Proc Natl Acad Sci USA,2012,109(8):2955-2959.
[11]Li M,Sun Y,Zhao J,et al.A tandem duplicate of anti-mullerian hormone with a missense SNP on the Y chromosome is essential for male sex determination in nile tilapia,Oreochromis niloticus[J].PLo S Genet,2015,11(11):1-23.
[12]Liu S,Sun B,Liang Z,et al.Cloning and expression of anti-mullerian hormone gene in halfsmooth tongue-sole,Cynoglossus semilaevis[J].Journal of Fishery Sciences of China,2013,20(1):35-43.
[13]Yoshinaga N,Shiraishi E,Yamamoto T,et al.Sexually dimorphic expression of a teleost homologue of Mullerian inhibiting substance during gonadal sex differentiation in Japanese flounder,Paralichthys olivaceus[J].Biochemical and Biophysical Research Communications,2004,322(2):508-513.
[14]Shiraishi E,Yoshinaga N,Miura T,et al.Mullerian inhibiting substance is required for germ cell proliferation during early gonadal differentiation in medaka(Oryzias latipes)[J].Endocrinology,2008,149(4):1813-1819.
[15]Liang Y Q,Huang G,Ying G,et al.The effects of progesterone on transcriptional expression profiles of genes associated with hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes during the early development of zebrafish(Danio rerio)[J].Chemosphere,2015,128:199-206.
[16]Miura T,Higuchi M,Ozaki Y,et al.Progestin is an essential factor for the initiation of the meiosis in spermatogenetic cells of the eel[J].Proc Natl Acad Sci U S A,2006,103(19):7333-7338.
[17]Tubbs C,Tan W,Shi B,et al.Identification of 17,20beta,21-trihydroxy-4-pregnen-3-one(20beta-S)receptor binding and membrane progestin receptor alpha on southern flounder sperm(Paralichthys lethostigma)and their likely role in 20beta-S stimulation of sperm hypermotility[J].Gen Comp Endocrinol,2011,170(3):629-639.
[18]Chen S X,Jan B,Eva A,et al.Cloning,pharmacological characterization,and expression analysis of Atlantic salmon(Salmo salar L.)nuclear progesterone receptor[J].Reproduction,2011,141(4):491-500.
[19]Chen S X,Fernanda A,Eva A,et al.Cloning,pharmacological characterization and expression analysis of Atlantic cod(Gadus morhua,L.)nuclear progesterone receptor[J].Gen Comp Endocrinol,2012,179(1):71-77.
[20]Milla S,Terrien X,Sturm A,et al.Plasma 11-deoxycorticosterone(DOC)and mineralocorticoid receptor testicular expression during rainbow trout Oncorhynchus mykiss spermiation:implication with 17alpha,20betadihydroxyprogesterone on the milt fluidity?[J].Reprod Biol Endocrinol,2008,6:19.
[21]Hanna R N,Zhu Y.Expression of membrane progestin receptors in zebrafish(Danio rerio)oocytes,testis and pituitary[J].Gen Comp Endocrinol,2009,161(1):153-157.
[22]Tan W,Aizen J,Thomas P.Membrane progestin receptor alpha mediates progestin-induced sperm hypermotility and increased fertilization success in southern flounder(Paralichthys lethostigma)[J].Gen Comp Endocrinol,2014,200:18-26.
[2]Scott A P,Sumpter J P,Stacey N,et al.The role of the maturation-inducing steroid,17,20beta-dihydroxypregn-4-en-3-one,in male fishes:a review[J].J Fish Biol,2010,76(1):183-224.
[3]Higuchi M,Miura C,Iwai T,et al.Trypsin regulates meiotic initiation in the Japanese eel(Anguilla japonica)by promoting the uptake of taurine into germ cells during spermatogenesis[J].Biol Reprod,2013,89(3):1-9,58.
[4]Liu G,Luo F,Song Q,et al.Blocking of progestin action disrupts spermatogenesis in Nile tilapia(Oreochromis niloticus)[J].J Mol Endocrinol,2014,53(1):57-70.
[5]Chen S X,Bogerd J,Schoonen E,et al.A progestin(17alpha,20beta-dihydroxy-4-pregnen-3-one)stimulates early stages of spermatogenesis in zebrafish[J].Gen Comp Endocrinol,2013,185:1-9.
[6]Chen S X,Bogerd J,García-López A,et al.Molecular cloning and functional characterization of a zebrafish nuclear progesterone receptor[J].Biol Reprod,2010,82(1):171-181.
[7]张瑞,阙华勇,张国范,等.海湾扇贝维甲酸受体_RXR_c DNA克隆与表达分析[J].海洋科学,2016,40(9):1-8.Zhang Rui,Que Huayong,Zhang Guofan,et al.Molecular cloning and expression of the retinoid X receptor gene in Argopecten irradians[J].Marine Sciences,2016,40(9):1-8.
[8]Salazar M.Progestin-mediated activation of MAPK and AKT in nuclear progesterone receptor negative breast epithelial cells:The role of membrane progesterone receptors[J].Gene,2016,591(1):6-13.
[9]郭佳伟,谭训刚,尤锋,等.大菱鲆周期蛋白依赖激酶基因cdk1、cdk6克隆及表达分析[J].海洋科学,2016,40(4):11-21.Guo Jiawei,Tan Xungang,You Feng,et al.Cloning and expression analysis of cdk1 and cdk6 in Scophthalmus maximus[J].Marine Sciences,2016,40(4):11-21.
[10]Hattori R S,Murai Y,Oura M,et al.A Y-linked anti-Mullerian hormone duplication takes over a critical role in sex determination[J].Proc Natl Acad Sci USA,2012,109(8):2955-2959.
[11]Li M,Sun Y,Zhao J,et al.A tandem duplicate of anti-mullerian hormone with a missense SNP on the Y chromosome is essential for male sex determination in nile tilapia,Oreochromis niloticus[J].PLo S Genet,2015,11(11):1-23.
[12]Liu S,Sun B,Liang Z,et al.Cloning and expression of anti-mullerian hormone gene in halfsmooth tongue-sole,Cynoglossus semilaevis[J].Journal of Fishery Sciences of China,2013,20(1):35-43.
[13]Yoshinaga N,Shiraishi E,Yamamoto T,et al.Sexually dimorphic expression of a teleost homologue of Mullerian inhibiting substance during gonadal sex differentiation in Japanese flounder,Paralichthys olivaceus[J].Biochemical and Biophysical Research Communications,2004,322(2):508-513.
[14]Shiraishi E,Yoshinaga N,Miura T,et al.Mullerian inhibiting substance is required for germ cell proliferation during early gonadal differentiation in medaka(Oryzias latipes)[J].Endocrinology,2008,149(4):1813-1819.
[15]Liang Y Q,Huang G,Ying G,et al.The effects of progesterone on transcriptional expression profiles of genes associated with hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes during the early development of zebrafish(Danio rerio)[J].Chemosphere,2015,128:199-206.
[16]Miura T,Higuchi M,Ozaki Y,et al.Progestin is an essential factor for the initiation of the meiosis in spermatogenetic cells of the eel[J].Proc Natl Acad Sci U S A,2006,103(19):7333-7338.
[17]Tubbs C,Tan W,Shi B,et al.Identification of 17,20beta,21-trihydroxy-4-pregnen-3-one(20beta-S)receptor binding and membrane progestin receptor alpha on southern flounder sperm(Paralichthys lethostigma)and their likely role in 20beta-S stimulation of sperm hypermotility[J].Gen Comp Endocrinol,2011,170(3):629-639.
[18]Chen S X,Jan B,Eva A,et al.Cloning,pharmacological characterization,and expression analysis of Atlantic salmon(Salmo salar L.)nuclear progesterone receptor[J].Reproduction,2011,141(4):491-500.
[19]Chen S X,Fernanda A,Eva A,et al.Cloning,pharmacological characterization and expression analysis of Atlantic cod(Gadus morhua,L.)nuclear progesterone receptor[J].Gen Comp Endocrinol,2012,179(1):71-77.
[20]Milla S,Terrien X,Sturm A,et al.Plasma 11-deoxycorticosterone(DOC)and mineralocorticoid receptor testicular expression during rainbow trout Oncorhynchus mykiss spermiation:implication with 17alpha,20betadihydroxyprogesterone on the milt fluidity?[J].Reprod Biol Endocrinol,2008,6:19.
[21]Hanna R N,Zhu Y.Expression of membrane progestin receptors in zebrafish(Danio rerio)oocytes,testis and pituitary[J].Gen Comp Endocrinol,2009,161(1):153-157.
[22]Tan W,Aizen J,Thomas P.Membrane progestin receptor alpha mediates progestin-induced sperm hypermotility and increased fertilization success in southern flounder(Paralichthys lethostigma)[J].Gen Comp Endocrinol,2014,200:18-26.