TGF-β1及其受体在性成熟前后藏绵羊睾丸的表达比较
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摘要
旨在比较研究转化生长因子-β1 (transforming growth factor-β1, TGF-β1)及其受体在性成熟前后藏绵羊睾丸组织的表达及分布特征,探索其对藏绵羊睾丸发育及生殖机能的调节作用。采用睾丸摘除手术收集1.0、1.5、2.0岁藏绵羊睾丸,应用免疫印迹、免疫组织化学SP法及IPP图像分析研究TGF-β1及其受体(transforming growth factor-βreceptor I, TGF-βR I)的表达及分布特征。结果表明:1)TGF-β1在藏绵羊睾丸组织中弱表达,性成熟前后差异不显著(P>0.05);1.5岁藏绵羊睾丸组织中TGF-βRⅠ表达均显著高于1.0及2.0岁(P<0.05)。2)各年龄组TGF-β1主要表达于睾丸间质细胞(Leydig)及小血管内皮细胞,1.5岁藏绵羊在各级生精细胞及支持细胞(Sertoli)亦有中等阳性表达,其他年龄组在生精上皮弱表达;TGF-βRⅠ在各年龄组生精上皮均有表达,1.0岁时在Leydig细胞无明显表达,1.5岁时强表达于圆形精子、Leydig细胞及淋巴管内皮细胞,2.0岁时在Leydig细胞及毛细淋巴管亦见阳性表达,但明显弱于1.5岁。3)各年龄组TGF-βRⅠ表达明显强于TGF-β1,TGF-β1表达组间差异不显著(P>0.05);TGF-βRⅠ在1.5岁时较1.0及2.0岁表达差异显著(P<0.05)。综上表明,高原地区藏绵羊睾丸组织TGF-β1和TGF-βRⅠ的阳性表达随着性成熟出现先上升后下降的变化趋势,提示性成熟前后TGF-β1及其受体通过调节Sertoli细胞在精子生成过程中发挥作用;各年龄段藏绵羊睾丸组织TGF-βRⅠ及TGF-β1阳性表达以Leydig细胞变化最为明显,为进一步分析TGF-β1/TGF-βRⅠ信号通路随着性成熟发育参与调节Leydig细胞生物合成提供研究基础。
This experiment was conducted to compare the distribution characteristics of TGF-β1 and its receptors(TGF-βRⅠ) in Tibetan sheep testis before and after sexual mature and attempt to explore their relationships with testicular spermatogenesis in sexual mature. The testis of 1.0-, 1.5-, 2.0-year-old Tibetan sheep were prepared by testicular removal surgery for describe the distribution density and the location of TGF-β1 and TGF-βRⅠ in the testis tissues using the Western blot, immunohistochemical SP combined with IPP(Image-Pro Plus) statistics methods. The results suggested that:1) The reactions of TGF-β1 antibody were weakly and there were no significant difference in Tibetan sheep testis before and after sexual mature(P>0.05);The expression of TGF-βR Ⅰ in the 1.5-year-old sheep testis tissue were significantly higher than 1.0-and 2.0-year-old(P<0.05). 2)The TGF-β1 was mainly present in Leydig cells and endothelial cells of small vascular at different ages, there were medium positive expression of TGF-β1 in Sertoli cells and spermatocytes at 1.5-year-old Tibetan sheep but weakly expressed in seminiferous epithelium at other 2 groups. The TGF-βRⅠwere expressed in seminiferous epithelium at 3 ages. In the 1.0-year-old sheep testis, the expression of TGF-βRⅠwas weakly expressed in Leydig cells, and at the age of 1.5, its expression in the round spermatid, Leydig cells and lymphatic endothelial cells were strongly positive, also there were immunolocalized in Leydig cells and capillary lymph vessel at 2-year-old, basically the expression of TGF-βRⅠin the 1.5-year-old sheep testis was more stronger than that of other 2 groups.3)The positive signal statistics indicated that the average absorbance of TGF-βRⅠ expression was significantly higher in the testis than TGF-β1,and the expression of TGF-β1 were not different in the testis of the Tibetan sheep before and after sexual mature(P> 0.05),the TGF-βRⅠwas expressed significantly higher in testis tissue of 1.5-year-old Tibetan sheep than that of 1.0-and 2.0-year-old sheep(P<0.05). In conclusion, these results demonstrate that the expression of TGF-β1 and TGF-βRⅠin Tibetan sheep testis appered with rise trend first and then fall with age in sexual mature, their expression in Sertoli cells had correlaton with the regulation of spermatogenesis; and the mainly changes in Leydig cells serve as a meaningful index for the further research of TGF-β1/TGF-βRⅠpathways involved in its steroid biosynthesis and basic insights into testicular development.
This experiment was conducted to compare the distribution characteristics of TGF-β1 and its receptors(TGF-βRⅠ) in Tibetan sheep testis before and after sexual mature and attempt to explore their relationships with testicular spermatogenesis in sexual mature. The testis of 1.0-, 1.5-, 2.0-year-old Tibetan sheep were prepared by testicular removal surgery for describe the distribution density and the location of TGF-β1 and TGF-βRⅠ in the testis tissues using the Western blot, immunohistochemical SP combined with IPP(Image-Pro Plus) statistics methods. The results suggested that:1) The reactions of TGF-β1 antibody were weakly and there were no significant difference in Tibetan sheep testis before and after sexual mature(P>0.05);The expression of TGF-βR Ⅰ in the 1.5-year-old sheep testis tissue were significantly higher than 1.0-and 2.0-year-old(P<0.05). 2)The TGF-β1 was mainly present in Leydig cells and endothelial cells of small vascular at different ages, there were medium positive expression of TGF-β1 in Sertoli cells and spermatocytes at 1.5-year-old Tibetan sheep but weakly expressed in seminiferous epithelium at other 2 groups. The TGF-βRⅠwere expressed in seminiferous epithelium at 3 ages. In the 1.0-year-old sheep testis, the expression of TGF-βRⅠwas weakly expressed in Leydig cells, and at the age of 1.5, its expression in the round spermatid, Leydig cells and lymphatic endothelial cells were strongly positive, also there were immunolocalized in Leydig cells and capillary lymph vessel at 2-year-old, basically the expression of TGF-βRⅠin the 1.5-year-old sheep testis was more stronger than that of other 2 groups.3)The positive signal statistics indicated that the average absorbance of TGF-βRⅠ expression was significantly higher in the testis than TGF-β1,and the expression of TGF-β1 were not different in the testis of the Tibetan sheep before and after sexual mature(P> 0.05),the TGF-βRⅠwas expressed significantly higher in testis tissue of 1.5-year-old Tibetan sheep than that of 1.0-and 2.0-year-old sheep(P<0.05). In conclusion, these results demonstrate that the expression of TGF-β1 and TGF-βRⅠin Tibetan sheep testis appered with rise trend first and then fall with age in sexual mature, their expression in Sertoli cells had correlaton with the regulation of spermatogenesis; and the mainly changes in Leydig cells serve as a meaningful index for the further research of TGF-β1/TGF-βRⅠpathways involved in its steroid biosynthesis and basic insights into testicular development.
引文
[1] MILES D C,WAKELING S I,STRINGER J M,et al.Signaling through the TGF beta-activin receptors ALK4/5/7 regulates testis formation and male germ cell development[J].PLoS One,2013,8(1):e54606.
[2] GONZALEZ C R,GONZALEZ B,RULLI S B,et al.TGF-β1 system in Leydig cells.Part I:effect of hCG and progesterone[J].J Reprod Dev,2010,56(4):389-395.
[3] MONSIVAIS D,MATZUK M M,PANGAS S A.The TGF-β family in the reproductive tract[J].Cold Spring Harb Perspect Biol,2017,9(10):a022251.
[4] 侯武刚,张远强,赵洁,等.转化生长因子β及其受体在小鼠精子发生过程中的表达[J].解剖学报,2006,37(2):226-231.HOU W G,ZHANG Y Q,ZHAO J,et al The expression of transforming growth factor β and their receptors in mouse spermatogenesis[J].Acta Anatomica Sinica,2006,37(2):226-231.(in Chinese)
[5] CHAN K H,GALUSKA S P,KUDIPUDI P K,et al.Signaling by TGF-betas in tubule cultures of adult rat testis[J].Am J Transl Res,2017,9(3):1173-1182.
[6] 闫永平,董常生,贺俊平,等.TGF-β1在羊驼睾丸的表达与定位[J].畜牧兽医学报,2008,39(1):97-102.YAN Y P,DONG C S,HE J P,et al.Expression and localization of transforming growth factor-β1 in alpaca testis[J].Acta Veterinaria et Zootechnica Sinica,2008,39(1):97-102.(in Chinese)
[7] MADRID E,REYES J G,HERNáNDEZ B,et al.Effect of normobaric hypoxia on the testis in a murine model[J].Andrologia,2013,45(5):332-338.
[8] 卢玮,黄磊,何惠华,等.TGF-β1在低氧诱导肺动脉内皮细胞向平滑肌样细胞转分化中的作用[J].华中科技大学学报:医学版,2006,35(5):561-565.LU W,HUANG L,HE H H.The role of transforming growth factor β1 in hypoxia inducing transdifferentiation of pulmonary artery endothelial cells to smooth muscle-like cells[J].Acta Medicinae Universitatis Scientiae et Technologiae Huazhong,2006,35(5):561-565.(in Chinese)
[9] 陈国娟.牦牛睾丸局部调节因子增龄性与季节性变化的表达研究[D].兰州:甘肃农业大学,2015.CHEN G J.The expression study of the local regulation factor in yak testis associated with age and season[D].Lanzhou:Gansu Agricultural University,2015.(in Chinese)
[10] 袁莉刚,张勇,李聪,等.高原地区藏绵羊与小尾寒羊睾丸细胞外基质组织分布特征比较[J].解剖学报,2017,48(2):179-186.YUAN L G,ZHANG Y,LI C,et al.Comparison of distribution characteristics of extracellular matrix components in the testis of the Tibetan sheep and the small-tail Han sheep from plateau[J].Acta Anatomica Sinica,2017,48(2):179-186.(in Chinese)
[11] GONZALEZ C R,MATZKIN M E,FRUNGIERI M B,et al.Expression of the TGF-beta1 system in human testicular pathologies[J].Reprod Biol Endocrinol,2010,8(1):148.
[12] TEERDS K J,DORRINGTON J H.Localization of transforming growth factor β1 and β2 during testicular development in the rat[J].Biol Reprod,1993,48(1):40-45.
[13] MULLANEY B P,SKINNER M K.Transforming growth factor-β(β1,β2,and β3) gene expression and action during pubertal development of the seminiferous tubule:potential role at the onset of spermatogenesis[J].Mol Endocrinol,1993,7(1):67-76.
[14] DAMESTOY A,PERRARD M H,VIGIER M,et al.Transforming growth factor beta-1 decreases the yield of the second meiotic division of rat pachytene spermatocytes in vitro[J].Reprod Biol Endocrinol,2005,3(1):22.
[15] CAUSSANEL V,TABONE E,HENDRICK J C,et al.Cellular Distribution of transforming growth factor betas 1,2,and 3 and their types Ⅰ and II receptors during postnatal development and spermatogenesis in the boar testis[J].Biol Reprod,1997,56(2):357-367.
[16] ZHANG Y Q,HE X Z,ZHANG J S,et al.Stage-specific localization of transforming growth factor β1 and β3 and their receptors during spermatogenesis in men[J].Asian J Androl,2004,6(2):105-109.
[17] OLASO R,PAIRAULT C,BOULOGNE B,et al.Transforming growth factor β1 and β2 reduce the number of gonocytes by increasing apoptosis[J].Endocrinology,1998,139(2):733-740.
[18] PARK E,SONG C H,PARK J I,et al.Transforming growth factor-β1 signaling represses testicular steroidogenesis through cross-talk with orphan nuclear receptor Nur77[J].PLoS One,2014,9(8):e104812.
[19] ?ZGüDEN-AKKO? C G,?ZER A.Immunohistochemical localization of transforming growth factor β1 and β2 in mouse testes during postnatal development[J].Biotech Histochem,2012,87(2):154-159.
[20] FAWCETT D W,NEAVES W B,FLORES M N.Comparative observations on Intertubular lymphatics and the organization of the interstitial tissue of the mammalian testis[J].Biol Reprod,1973,9(5):500-532.
[21] ITOH M,LI X Q,YANO A,et al.Patterns of efferent lymphatics of the mouse testis[J].J Androl,1998,19(4):466-472.
[22] IRIGOYEN M,ANSó E,SALVO E,et al.TGFβ-induced protein mediates lymphatic endothelial cell adhesion to the extracellular matrix under low oxygen conditions[J].Cell Mol Life Sci,2008,65(14):2244-2255.
[23] 白瑞,赵鹂,李栋,等.生长分化因子9及其受体在成年狗睾丸中的表达与定位[J].畜牧兽医学报,2014,45(4):566-571.BAI R,ZHAO L,LI D,et al.Expression and location of growth differentiation factor 9 (GDF9) and its receptor in adult dog testis[J].Acta Veterinaria et Zootechnica Sinica,2014,45(4):566-571.(in Chinese)
[24] TANG K,WANG L,JIN Y,et al.GDF9 affects the development and tight junction functions of immature bovine Sertoli cells[J].Reprod Domest Anim,2017,52(4):640-648.
[25] 张炜.TGF-β/Activin信号通路在干细胞定向分化过程中的作用[D].济南:山东师范大学,2007.ZHANG W.The role of TGF-β/Activin in the differentiation of hESCs[D].Ji’nan:Shandong Normal University,2007.(in Chinese)
[26] YANG Q,MA B,QIAO H,et al.TGFB1 represses the expression of SF1 and LRH1 to inhibit E2 production in rat LCs[J].Reproduction,2017,153(5):621-629.
[27] GUO Q Y,GAO Z Z,ZHAO L,et al.Expression of growth differentiation factor 9 (GDF9),ALK5,and claudin-11 in adult alpaca testis[J].Acta Histochem,2013,115(1):16-21.
[28] XU M Y,PANG Q Q,XU S Q,et al.Hypoxia-inducible factor-1α activates transforming growth factor-β1/Smad signaling and increases collagen deposition in dermal fibroblasts[J].Oncotarget,2018,9(3):3188-3197.
[2] GONZALEZ C R,GONZALEZ B,RULLI S B,et al.TGF-β1 system in Leydig cells.Part I:effect of hCG and progesterone[J].J Reprod Dev,2010,56(4):389-395.
[3] MONSIVAIS D,MATZUK M M,PANGAS S A.The TGF-β family in the reproductive tract[J].Cold Spring Harb Perspect Biol,2017,9(10):a022251.
[4] 侯武刚,张远强,赵洁,等.转化生长因子β及其受体在小鼠精子发生过程中的表达[J].解剖学报,2006,37(2):226-231.HOU W G,ZHANG Y Q,ZHAO J,et al The expression of transforming growth factor β and their receptors in mouse spermatogenesis[J].Acta Anatomica Sinica,2006,37(2):226-231.(in Chinese)
[5] CHAN K H,GALUSKA S P,KUDIPUDI P K,et al.Signaling by TGF-betas in tubule cultures of adult rat testis[J].Am J Transl Res,2017,9(3):1173-1182.
[6] 闫永平,董常生,贺俊平,等.TGF-β1在羊驼睾丸的表达与定位[J].畜牧兽医学报,2008,39(1):97-102.YAN Y P,DONG C S,HE J P,et al.Expression and localization of transforming growth factor-β1 in alpaca testis[J].Acta Veterinaria et Zootechnica Sinica,2008,39(1):97-102.(in Chinese)
[7] MADRID E,REYES J G,HERNáNDEZ B,et al.Effect of normobaric hypoxia on the testis in a murine model[J].Andrologia,2013,45(5):332-338.
[8] 卢玮,黄磊,何惠华,等.TGF-β1在低氧诱导肺动脉内皮细胞向平滑肌样细胞转分化中的作用[J].华中科技大学学报:医学版,2006,35(5):561-565.LU W,HUANG L,HE H H.The role of transforming growth factor β1 in hypoxia inducing transdifferentiation of pulmonary artery endothelial cells to smooth muscle-like cells[J].Acta Medicinae Universitatis Scientiae et Technologiae Huazhong,2006,35(5):561-565.(in Chinese)
[9] 陈国娟.牦牛睾丸局部调节因子增龄性与季节性变化的表达研究[D].兰州:甘肃农业大学,2015.CHEN G J.The expression study of the local regulation factor in yak testis associated with age and season[D].Lanzhou:Gansu Agricultural University,2015.(in Chinese)
[10] 袁莉刚,张勇,李聪,等.高原地区藏绵羊与小尾寒羊睾丸细胞外基质组织分布特征比较[J].解剖学报,2017,48(2):179-186.YUAN L G,ZHANG Y,LI C,et al.Comparison of distribution characteristics of extracellular matrix components in the testis of the Tibetan sheep and the small-tail Han sheep from plateau[J].Acta Anatomica Sinica,2017,48(2):179-186.(in Chinese)
[11] GONZALEZ C R,MATZKIN M E,FRUNGIERI M B,et al.Expression of the TGF-beta1 system in human testicular pathologies[J].Reprod Biol Endocrinol,2010,8(1):148.
[12] TEERDS K J,DORRINGTON J H.Localization of transforming growth factor β1 and β2 during testicular development in the rat[J].Biol Reprod,1993,48(1):40-45.
[13] MULLANEY B P,SKINNER M K.Transforming growth factor-β(β1,β2,and β3) gene expression and action during pubertal development of the seminiferous tubule:potential role at the onset of spermatogenesis[J].Mol Endocrinol,1993,7(1):67-76.
[14] DAMESTOY A,PERRARD M H,VIGIER M,et al.Transforming growth factor beta-1 decreases the yield of the second meiotic division of rat pachytene spermatocytes in vitro[J].Reprod Biol Endocrinol,2005,3(1):22.
[15] CAUSSANEL V,TABONE E,HENDRICK J C,et al.Cellular Distribution of transforming growth factor betas 1,2,and 3 and their types Ⅰ and II receptors during postnatal development and spermatogenesis in the boar testis[J].Biol Reprod,1997,56(2):357-367.
[16] ZHANG Y Q,HE X Z,ZHANG J S,et al.Stage-specific localization of transforming growth factor β1 and β3 and their receptors during spermatogenesis in men[J].Asian J Androl,2004,6(2):105-109.
[17] OLASO R,PAIRAULT C,BOULOGNE B,et al.Transforming growth factor β1 and β2 reduce the number of gonocytes by increasing apoptosis[J].Endocrinology,1998,139(2):733-740.
[18] PARK E,SONG C H,PARK J I,et al.Transforming growth factor-β1 signaling represses testicular steroidogenesis through cross-talk with orphan nuclear receptor Nur77[J].PLoS One,2014,9(8):e104812.
[19] ?ZGüDEN-AKKO? C G,?ZER A.Immunohistochemical localization of transforming growth factor β1 and β2 in mouse testes during postnatal development[J].Biotech Histochem,2012,87(2):154-159.
[20] FAWCETT D W,NEAVES W B,FLORES M N.Comparative observations on Intertubular lymphatics and the organization of the interstitial tissue of the mammalian testis[J].Biol Reprod,1973,9(5):500-532.
[21] ITOH M,LI X Q,YANO A,et al.Patterns of efferent lymphatics of the mouse testis[J].J Androl,1998,19(4):466-472.
[22] IRIGOYEN M,ANSó E,SALVO E,et al.TGFβ-induced protein mediates lymphatic endothelial cell adhesion to the extracellular matrix under low oxygen conditions[J].Cell Mol Life Sci,2008,65(14):2244-2255.
[23] 白瑞,赵鹂,李栋,等.生长分化因子9及其受体在成年狗睾丸中的表达与定位[J].畜牧兽医学报,2014,45(4):566-571.BAI R,ZHAO L,LI D,et al.Expression and location of growth differentiation factor 9 (GDF9) and its receptor in adult dog testis[J].Acta Veterinaria et Zootechnica Sinica,2014,45(4):566-571.(in Chinese)
[24] TANG K,WANG L,JIN Y,et al.GDF9 affects the development and tight junction functions of immature bovine Sertoli cells[J].Reprod Domest Anim,2017,52(4):640-648.
[25] 张炜.TGF-β/Activin信号通路在干细胞定向分化过程中的作用[D].济南:山东师范大学,2007.ZHANG W.The role of TGF-β/Activin in the differentiation of hESCs[D].Ji’nan:Shandong Normal University,2007.(in Chinese)
[26] YANG Q,MA B,QIAO H,et al.TGFB1 represses the expression of SF1 and LRH1 to inhibit E2 production in rat LCs[J].Reproduction,2017,153(5):621-629.
[27] GUO Q Y,GAO Z Z,ZHAO L,et al.Expression of growth differentiation factor 9 (GDF9),ALK5,and claudin-11 in adult alpaca testis[J].Acta Histochem,2013,115(1):16-21.
[28] XU M Y,PANG Q Q,XU S Q,et al.Hypoxia-inducible factor-1α activates transforming growth factor-β1/Smad signaling and increases collagen deposition in dermal fibroblasts[J].Oncotarget,2018,9(3):3188-3197.