不同发育时期蒙古马睾丸组织中GH和IGF-Ⅰ及其受体的表达
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摘要
为进一步揭示蒙古马发育与生殖调控的分子机制提供试验数据和理论依据,研究不同发育年龄蒙古马睾丸组织中生长激素(Growth hormone,GH)、生长激素受体(Growth hormone receptor,GHR)、胰岛素样生长因子Ⅰ(Insulin-like growth factorsⅠ,IGF-Ⅰ)和胰岛素样生长因子Ⅰ受体(Insulin-like growth factorⅠreceptor,IGF-ⅠR)表达的发育性变化模式,以性成熟前期(5匹)、性成熟期(5匹)、成年期(5匹)的蒙古公马为研究对象,采集其睾丸组织,利用实时荧光定量PCR法(RT-qPCR)对不同发育年龄蒙古马睾丸组织中的GH、GHR、IGF-Ⅰ和IGF-ⅠR基因的表达差异进行分析,采用免疫组织化学方法对性成熟前期和性成熟期蒙古马睾丸组织中的GH和IGF-Ⅰ蛋白质进行定位。RT-qPCR试验结果表明蒙古马睾丸组织中上述4个基因的表达量从性成熟前期到性成熟期显著增加,成年期下降。免疫组织化学结果表明GH和IGF-Ⅰ免疫标记定位于精母细胞、精原细胞、精子细胞和睾丸间质细胞,其在相关细胞中的信号从性成熟前期到性成熟期逐渐增强。研究发现睾丸组织中GH、GHR、IGF-Ⅰ和IGF-ⅠR的mRNA协同表达对蒙古马睾丸发育和性成熟有重要调节作用,GH和IGF-Ⅰ蛋白可能通过自分泌/旁分泌的方式调节蒙古马的精子形成和睾丸间质细胞的功能。
To provide basis for further unraveling the mechanisms of development and reproduction regulation of Mongolian horse,the developmental pattern of growth hormone(GH),growth hormone receptor(GHR),insulin-like growth factors Ⅰ(IGF-Ⅰ)and insulin-like growth factorⅠ receptor(IGF-ⅠR)expression in testicular tissue of Mongolian horse were assessed at both gene and protein level.Fifteen Mongolian horses at the early maturation(n=5),sexual maturity period(n=5)and adulthood(n=5)were selected as experimental animals in this study.The expression profile of GH,GHR,IGF-Ⅰ and IGF-ⅠR genes in testicular tissue of Mongolian horses at the early maturation,sexual maturity period and adulthood were evaluated by SYBR Green Ⅱ-based RT-qPCR assay.The existence and location of GH and IGF-Ⅰ proteins in testicular tissue of Mongolian horses at the early maturation and sexual maturity period were identified by immunohistochemical method.The RT-qPCR assay demonstrated that the mRNA expression level of the four genes was all gradually increased from the early maturation to sexual maturity period and was subsequently decreased at adulthood.Immunohistochemical analysis showed that the germ cells and Leydig cells were found to be positive for GH and IGF-Ⅰ proteins expression;the expression of GH and IGF-Ⅰ proteins wasgradually enhanced in both of the cells from the early maturation to sexual maturity period.In conclusion,the coexpression of GH,GHR,IGF-Ⅰ and IGF-ⅠR mRNA displayed an important regulating effect on the development and sexual maturation of Mongolian horses,and the GH and IGF-Ⅰ proteins might regulate the spermatogenesis and Leydig cells function in an autocrine/paracrine manner.
To provide basis for further unraveling the mechanisms of development and reproduction regulation of Mongolian horse,the developmental pattern of growth hormone(GH),growth hormone receptor(GHR),insulin-like growth factors Ⅰ(IGF-Ⅰ)and insulin-like growth factorⅠ receptor(IGF-ⅠR)expression in testicular tissue of Mongolian horse were assessed at both gene and protein level.Fifteen Mongolian horses at the early maturation(n=5),sexual maturity period(n=5)and adulthood(n=5)were selected as experimental animals in this study.The expression profile of GH,GHR,IGF-Ⅰ and IGF-ⅠR genes in testicular tissue of Mongolian horses at the early maturation,sexual maturity period and adulthood were evaluated by SYBR Green Ⅱ-based RT-qPCR assay.The existence and location of GH and IGF-Ⅰ proteins in testicular tissue of Mongolian horses at the early maturation and sexual maturity period were identified by immunohistochemical method.The RT-qPCR assay demonstrated that the mRNA expression level of the four genes was all gradually increased from the early maturation to sexual maturity period and was subsequently decreased at adulthood.Immunohistochemical analysis showed that the germ cells and Leydig cells were found to be positive for GH and IGF-Ⅰ proteins expression;the expression of GH and IGF-Ⅰ proteins wasgradually enhanced in both of the cells from the early maturation to sexual maturity period.In conclusion,the coexpression of GH,GHR,IGF-Ⅰ and IGF-ⅠR mRNA displayed an important regulating effect on the development and sexual maturation of Mongolian horses,and the GH and IGF-Ⅰ proteins might regulate the spermatogenesis and Leydig cells function in an autocrine/paracrine manner.
引文
[1] Clemmons A J,Thompson D L Jr,Johnson L.Local initiation of spermatogenesis in the horse[J].Biology of Reproduction,1995,52(6):1258-1267
[2] Hull K L, Harvey S.Growth hormone:A reproductive endocrine-paracrineregulator?[J]. Reviewsof Reproduction,2000,5(3):175-182
[3] Hull K L, Harvey S. Growth hormone:Roles in male reproduction[J].Endocrine,2000,13(3):243-250
[4] Hull K L,Harvey S.GH as a co-gonadotropin:The relevance of correlative changes in GH secretion and reproductive state[J].Journal of Endocrinology,2002,172(1):1-19
[5] Childs G V. Growth hormone cells as co-gonadotropes:Partners in the regulation of the reproductive system[J].Trends in Endocrinology&Metabolism,2000,11(5):168-175
[6] Gravance C G,Breier B H,Vickers M H,Casey P J.Impaired sperm characteristics in postpubertal growth-hormone-deficient dwarf(dw/dw)rats[J].Animal Reproduction Science,1997,49(1):71-76
[7] Bartlett J M S,Charlton H M,Robinson I C A F,Nieschlag E.Pubertal development and testicular function in the male growth hormone-deficient rat[J].Journal of Endocrinology,1990,126(2):193-201
[8] Spiteri-Grech J,Nieschlag E.The role of growth hormone and insulin-like growth factorⅠin the regulation of male reproductive function[J].Hormone Research in Paediatrics,1992,38(S1):22-27
[9] Zheng J X,Liu Z Z,Yang N.Deficiency of growth hormone receptor does not affect male reproduction in dwarf chickens[J].Poultry Science,2007,86(1):112-117
[10] Hess M F,Roser J F.A comparison of the effects of equine luteinizing hormone(eLH),equine growth hormone(eGH)and human recombinant insulin-like growth factor(hrIGF-I)on steroid production in cultured equine Leydig cells during sexual maturation[J].Animal Reproduction Science,2005,89(1-4):7-19
[11] Aurich J E,Kranski S,Parvizi N,Aurich C.Somatostatin treatment affects testicular function in stallions[J].Theriogenology,2003,60(1):163-174.
[12] Ilkbahar N, Wu K, Thordarson G, Talamantes F.Expression and distribution of messenger ribonucleic acids for growth hormone(GH)receptor and GH-binding protein in mice during pregnancy[J].Endocrinology,1995,136(2):386-392
[13] Lobie P E,Breipohl W,García Aragón J,Watesr M J.Cellular localization of the growth hormone receptor/binding protein in the male and female reproductive systems[J].Endocrinology,1990,126(4):2214-2221
[14] Picard F,Postel-Vinay M C.Hypophysectomy and growth hormone receptors in liver membranes of male rats[J].Endocrinology,1984,114(4):1328-1333
[15] Chandrashekar V,Bartke A,Coschigano K T,Kopchick J J.Pituitary and testicular function in growth hormone receptor gene knockout mice[J].Endocrinology,1999,140(3):1082-1088
[16]李发弟,夏东,陈杰,赵茹茜.二花脸猪和大白猪睾丸生长激素受体基因表达的发育性变化[J].南京农业大学学报,2006,29(2):131-133Li F D,Xia D,Chen J,Zhao R Q.Developmental changes of growth hormone receptor gene expression in testicular of Erhualian and Large White pigs[J].Journal of Nanjing Agricultural University,2006,29(2):131-133(in Chinese)
[17] Breier H B,Vickers H M,Gravance G C,Casey P J.Growth hormone(GH)therapy markedly increases the motility of spermatozoa and the concentration of insulin-like growth factor-Ⅰin seminal vesicle fluid in the male GHdeficient dwarf rat[J].Endocrinology,1996,137(9):4061-4064
[18] Champion Z J,Vickers M H,Gravance C G,Breier B H,Casey P J.Growth hormone or insulin-like growth factor-Ⅰextends longevity of equine spermatozoa in vitro[J].Theriogenology,2002,57(7):1793-1800
[19] Vickers M H,Casey P J,Champion Z J,Gravance C G,Breier B H.IGF-Ⅰtreatment increases motility and improves morphology of immature spermatozoa in the GHdeficient dwarf(dw/dw)rat[J].Growth Hormone&IGF Research,1999,9(4):236-240
[20] Vannelli B G,Barni T,Orlando C,Natali A,Serio M,Balboni G C.Insulin-like growth factor-Ⅰ(IGF-Ⅰ)and IGF-Ⅰreceptor in human testis:An immunohistochemical study[J].Fertility and Sterility,1988,49(4):666-669
[21] Hansson H A,Billig H,Isgaard J.Insulin-like growth factorⅠin the developing and mature rat testis:Immunohistochemical aspects[J].Biology of Reproduction,1989,40(6):1321-1328
[22] Baker J,Hardy P M,Zhou J,Bondy C,Lupu F,BellvéR A,Efstratiadis A.Effects of an Igf1gene null mutation on mouse reproduction[J].Molecular Endocrinology,1996,10(7):903-1018.
[23] Yoon M J,Roser J F.A synergistic effect of insulin-like growth factor(IGF-Ⅰ)on equine luteinizing hormone(eLH)-induced testosterone production from cultured Leydig cells of horses[J].Animal Reproduction Science,2011,126(3-4):195-199
[24] Yoon M J,Berger T,Roser J F.Localization of insulin-like growth factor-Ⅰ(IGF-Ⅰ)and IGF-Ⅰreceptor(IGF-ⅠR)in equine testes[J].Reproduction in Domestic Animals,2011,46(2):221-228
[25] Yagci A,Zik B.Immunohistochemical localization of insulinlike growth factor-Ⅰreceptor(IGF-ⅠR)in the developing and maturerattestes[J]. Anatomia Histologia Embryologia,2006,35(5):305-309
[26]李发弟.生长轴激素在猪睾丸表达的发育性变化及其对睾酮分泌的影响[D].兰州:甘肃农业大学,2003Li F D.Developmental changes of growth-related genes expression in porcine testis and effect of rpGH on testosterone secretion by porcine Leydig cell in vitro[D].Lanzhou:Gansu Agricultural University,2003(in Chinese)
[27] Richards R G,Klotz D M,Walker M P,Diaugustine R P.Mammary gland branching morphogenesis is diminished in mice with a deficiency of insulin-like growth factor-Ⅰ(IGF-Ⅰ),but not in mice with a liver-specific deletion of IGF-Ⅰ[J].Endocrinology,2004,145(7):3106-3110
[28] Luna M,Martínez-Moreno C G,Ahumada-Solórzano M S,Harvey S,Carranza M,Arámburo C.Extrapituitary growth hormone in the chicken reproductive system[J].General and Comparative Endocrinology,2014,203:60-68
[29] Müller L,Kowalewski M P,Reichler I M,Kollár E,Balogh O.Different expression of leptin and IGF1in the adult and prepubertal testis in dogs[J].Reproduction in Domestic Animals,2017,52(S2):187-192
[30] Ariyaratne H B S,Chamindrani Mendis-Handagama S M L.Changes in the testis interstitium of sprague dawley rats from birth to sexual maturity1[J].Biology of Reproduction,2000,62(3):680-690
[31] Fu Z,Kubo T,Noguchi T,Kato H.Developmental changes in the mRNA levels of IGF-Ⅰand its related genes in the reproductive organs of Japanese quail(Coturnix coturnix japonica)[J].Growth Hormone&IGF Research,2001,11(1):24-33
[32] Mauras N,Rogol A D,Haymond M W,Veldhuis J D.Sex steroids,growth hormone,insulin-like growth factor-1:neuroendocrine and metabolic regulation in puberty[J].Hormone Research,1996,45(1/2):74-80
[33] Hess M F,Roser J F.The effects of age,season and fertility status on plasma and intratesticular insulin-like growth factorⅠconcentration in stallions[J].Theriogenology,2001,56(5):723-733
[34] Kanzaki M,Morris P L.Growth hormone regulates steroidogenic acute regulatory protein expression and steroidogenesis in Leydig cell progenitors[J].Endocrinology,1999,140(4):1681-1686
[35] Ewton D Z,Falen S L,Florini J R.The typeⅡinsulin-like growth factor(IGF)receptor has low affinity for IGF-I analogs:Pleiotypic actions of IGFs on myoblasts are apparently mediated by the type I receptor[J].Endocrinology,1987,120(1):115-123
[36]陈红,宋学茹,岳天孚.生长激素对生殖功能调节的研究进展[J].国际生殖健康/计划生育杂志,2009,28(1):27-30Chen H,Song X R,Yue T F.Research progress of growth hormone on the regulation of reproductive function[J].Journal of International Reproductive Health and Family Planning,2009,28(1):27-30(in Chinese)
[37] Yao J F,Zuo H Y,Gao J,Wang M M,Wang D,Li X D.The effects of IGF-1 on mouse spermatogenesis using an organ culture method[J].Biochemical&Biophysical Research Communications,2017,491(3):840-847
[38] Yoon M J,Roser J F.Insulin-like growth factor-Ⅰ(IGF-Ⅰ)protects cultured equine Leydig cells from undergoing apoptosis[J].Animal Reproduction Science,2010,122(3/4):353-358
[39] Martínez-Moreno C G,Palma L,Carranza M,Harvey S,Arámburo C,Luna M.Cellular and intracellular distribution of growth hormone in the adult chicken testis[J].General and Comparative Endocrinology,2011,172(3):344-357
[40] Yoon M, JiangJ, ChungKH, RoserJF.Immunolocalization of insulin-like growth factor-Ⅰ(IGF-Ⅰ)and its receptors(IGF-ⅠR)in the equine epididymis[J].Journal of Reproduction&Development,2015,61(1):30-34
[2] Hull K L, Harvey S.Growth hormone:A reproductive endocrine-paracrineregulator?[J]. Reviewsof Reproduction,2000,5(3):175-182
[3] Hull K L, Harvey S. Growth hormone:Roles in male reproduction[J].Endocrine,2000,13(3):243-250
[4] Hull K L,Harvey S.GH as a co-gonadotropin:The relevance of correlative changes in GH secretion and reproductive state[J].Journal of Endocrinology,2002,172(1):1-19
[5] Childs G V. Growth hormone cells as co-gonadotropes:Partners in the regulation of the reproductive system[J].Trends in Endocrinology&Metabolism,2000,11(5):168-175
[6] Gravance C G,Breier B H,Vickers M H,Casey P J.Impaired sperm characteristics in postpubertal growth-hormone-deficient dwarf(dw/dw)rats[J].Animal Reproduction Science,1997,49(1):71-76
[7] Bartlett J M S,Charlton H M,Robinson I C A F,Nieschlag E.Pubertal development and testicular function in the male growth hormone-deficient rat[J].Journal of Endocrinology,1990,126(2):193-201
[8] Spiteri-Grech J,Nieschlag E.The role of growth hormone and insulin-like growth factorⅠin the regulation of male reproductive function[J].Hormone Research in Paediatrics,1992,38(S1):22-27
[9] Zheng J X,Liu Z Z,Yang N.Deficiency of growth hormone receptor does not affect male reproduction in dwarf chickens[J].Poultry Science,2007,86(1):112-117
[10] Hess M F,Roser J F.A comparison of the effects of equine luteinizing hormone(eLH),equine growth hormone(eGH)and human recombinant insulin-like growth factor(hrIGF-I)on steroid production in cultured equine Leydig cells during sexual maturation[J].Animal Reproduction Science,2005,89(1-4):7-19
[11] Aurich J E,Kranski S,Parvizi N,Aurich C.Somatostatin treatment affects testicular function in stallions[J].Theriogenology,2003,60(1):163-174.
[12] Ilkbahar N, Wu K, Thordarson G, Talamantes F.Expression and distribution of messenger ribonucleic acids for growth hormone(GH)receptor and GH-binding protein in mice during pregnancy[J].Endocrinology,1995,136(2):386-392
[13] Lobie P E,Breipohl W,García Aragón J,Watesr M J.Cellular localization of the growth hormone receptor/binding protein in the male and female reproductive systems[J].Endocrinology,1990,126(4):2214-2221
[14] Picard F,Postel-Vinay M C.Hypophysectomy and growth hormone receptors in liver membranes of male rats[J].Endocrinology,1984,114(4):1328-1333
[15] Chandrashekar V,Bartke A,Coschigano K T,Kopchick J J.Pituitary and testicular function in growth hormone receptor gene knockout mice[J].Endocrinology,1999,140(3):1082-1088
[16]李发弟,夏东,陈杰,赵茹茜.二花脸猪和大白猪睾丸生长激素受体基因表达的发育性变化[J].南京农业大学学报,2006,29(2):131-133Li F D,Xia D,Chen J,Zhao R Q.Developmental changes of growth hormone receptor gene expression in testicular of Erhualian and Large White pigs[J].Journal of Nanjing Agricultural University,2006,29(2):131-133(in Chinese)
[17] Breier H B,Vickers H M,Gravance G C,Casey P J.Growth hormone(GH)therapy markedly increases the motility of spermatozoa and the concentration of insulin-like growth factor-Ⅰin seminal vesicle fluid in the male GHdeficient dwarf rat[J].Endocrinology,1996,137(9):4061-4064
[18] Champion Z J,Vickers M H,Gravance C G,Breier B H,Casey P J.Growth hormone or insulin-like growth factor-Ⅰextends longevity of equine spermatozoa in vitro[J].Theriogenology,2002,57(7):1793-1800
[19] Vickers M H,Casey P J,Champion Z J,Gravance C G,Breier B H.IGF-Ⅰtreatment increases motility and improves morphology of immature spermatozoa in the GHdeficient dwarf(dw/dw)rat[J].Growth Hormone&IGF Research,1999,9(4):236-240
[20] Vannelli B G,Barni T,Orlando C,Natali A,Serio M,Balboni G C.Insulin-like growth factor-Ⅰ(IGF-Ⅰ)and IGF-Ⅰreceptor in human testis:An immunohistochemical study[J].Fertility and Sterility,1988,49(4):666-669
[21] Hansson H A,Billig H,Isgaard J.Insulin-like growth factorⅠin the developing and mature rat testis:Immunohistochemical aspects[J].Biology of Reproduction,1989,40(6):1321-1328
[22] Baker J,Hardy P M,Zhou J,Bondy C,Lupu F,BellvéR A,Efstratiadis A.Effects of an Igf1gene null mutation on mouse reproduction[J].Molecular Endocrinology,1996,10(7):903-1018.
[23] Yoon M J,Roser J F.A synergistic effect of insulin-like growth factor(IGF-Ⅰ)on equine luteinizing hormone(eLH)-induced testosterone production from cultured Leydig cells of horses[J].Animal Reproduction Science,2011,126(3-4):195-199
[24] Yoon M J,Berger T,Roser J F.Localization of insulin-like growth factor-Ⅰ(IGF-Ⅰ)and IGF-Ⅰreceptor(IGF-ⅠR)in equine testes[J].Reproduction in Domestic Animals,2011,46(2):221-228
[25] Yagci A,Zik B.Immunohistochemical localization of insulinlike growth factor-Ⅰreceptor(IGF-ⅠR)in the developing and maturerattestes[J]. Anatomia Histologia Embryologia,2006,35(5):305-309
[26]李发弟.生长轴激素在猪睾丸表达的发育性变化及其对睾酮分泌的影响[D].兰州:甘肃农业大学,2003Li F D.Developmental changes of growth-related genes expression in porcine testis and effect of rpGH on testosterone secretion by porcine Leydig cell in vitro[D].Lanzhou:Gansu Agricultural University,2003(in Chinese)
[27] Richards R G,Klotz D M,Walker M P,Diaugustine R P.Mammary gland branching morphogenesis is diminished in mice with a deficiency of insulin-like growth factor-Ⅰ(IGF-Ⅰ),but not in mice with a liver-specific deletion of IGF-Ⅰ[J].Endocrinology,2004,145(7):3106-3110
[28] Luna M,Martínez-Moreno C G,Ahumada-Solórzano M S,Harvey S,Carranza M,Arámburo C.Extrapituitary growth hormone in the chicken reproductive system[J].General and Comparative Endocrinology,2014,203:60-68
[29] Müller L,Kowalewski M P,Reichler I M,Kollár E,Balogh O.Different expression of leptin and IGF1in the adult and prepubertal testis in dogs[J].Reproduction in Domestic Animals,2017,52(S2):187-192
[30] Ariyaratne H B S,Chamindrani Mendis-Handagama S M L.Changes in the testis interstitium of sprague dawley rats from birth to sexual maturity1[J].Biology of Reproduction,2000,62(3):680-690
[31] Fu Z,Kubo T,Noguchi T,Kato H.Developmental changes in the mRNA levels of IGF-Ⅰand its related genes in the reproductive organs of Japanese quail(Coturnix coturnix japonica)[J].Growth Hormone&IGF Research,2001,11(1):24-33
[32] Mauras N,Rogol A D,Haymond M W,Veldhuis J D.Sex steroids,growth hormone,insulin-like growth factor-1:neuroendocrine and metabolic regulation in puberty[J].Hormone Research,1996,45(1/2):74-80
[33] Hess M F,Roser J F.The effects of age,season and fertility status on plasma and intratesticular insulin-like growth factorⅠconcentration in stallions[J].Theriogenology,2001,56(5):723-733
[34] Kanzaki M,Morris P L.Growth hormone regulates steroidogenic acute regulatory protein expression and steroidogenesis in Leydig cell progenitors[J].Endocrinology,1999,140(4):1681-1686
[35] Ewton D Z,Falen S L,Florini J R.The typeⅡinsulin-like growth factor(IGF)receptor has low affinity for IGF-I analogs:Pleiotypic actions of IGFs on myoblasts are apparently mediated by the type I receptor[J].Endocrinology,1987,120(1):115-123
[36]陈红,宋学茹,岳天孚.生长激素对生殖功能调节的研究进展[J].国际生殖健康/计划生育杂志,2009,28(1):27-30Chen H,Song X R,Yue T F.Research progress of growth hormone on the regulation of reproductive function[J].Journal of International Reproductive Health and Family Planning,2009,28(1):27-30(in Chinese)
[37] Yao J F,Zuo H Y,Gao J,Wang M M,Wang D,Li X D.The effects of IGF-1 on mouse spermatogenesis using an organ culture method[J].Biochemical&Biophysical Research Communications,2017,491(3):840-847
[38] Yoon M J,Roser J F.Insulin-like growth factor-Ⅰ(IGF-Ⅰ)protects cultured equine Leydig cells from undergoing apoptosis[J].Animal Reproduction Science,2010,122(3/4):353-358
[39] Martínez-Moreno C G,Palma L,Carranza M,Harvey S,Arámburo C,Luna M.Cellular and intracellular distribution of growth hormone in the adult chicken testis[J].General and Comparative Endocrinology,2011,172(3):344-357
[40] Yoon M, JiangJ, ChungKH, RoserJF.Immunolocalization of insulin-like growth factor-Ⅰ(IGF-Ⅰ)and its receptors(IGF-ⅠR)in the equine epididymis[J].Journal of Reproduction&Development,2015,61(1):30-34