转化生长因子β信号通路对卵泡刺激素调节卵巢颗粒细胞功能的影响

doi:10.16098/j.issn.0529-1356.2021.01.019

摘要/Abstract

摘要：

目的探讨大鼠卵巢颗粒细胞中卵泡刺激素（FSH）和转化生长因子β（TGF-β）信号通路之间的相互作用。方法取21 d SD大鼠卵泡分离的颗粒细胞原代培养，实验分为对照组、FSH处理组和转化生长因子β Ⅱ型受体(TGF-β RⅡ）中和组。通过免疫细胞化学和 Western blotting定位和检测TGF-β RⅡ以及增殖细胞核抗原（PCNA）的表达，通过流式细胞术(FCM)评估细胞增殖、细胞周期变化和凋亡细胞的百分比，并通过ELISA测定颗粒细胞分泌的雌二醇(E2)水平。结果当颗粒细胞的TGF-β信号通路受到抑制时，FSH促进颗粒细胞PCNA的表达，增加增殖指数，调节细胞周期以及抑制颗粒细胞凋亡的功能明显减弱(P<0.05）；FSH刺激颗粒细胞雌激素分泌的作用未明显受到TGF-β信号通路抑制的影响（P>0.05）。结论 FSH对卵巢颗粒细胞的调节作用部分受TGF-β信号通路的影响。

关键词: 颗粒细胞培养, 卵泡刺激素, 转化生长因子β, 免疫组织化学, 免疫印迹法, 大鼠

Abstract:

Objective To explore the interaction between follicle stimulating hormone (FSH) and transforming growth factor Beta (TGF-β) signaling pathway in rat ovarian granulosa cells. Methods The granulosa cells isolated from the follicles of 21 days SD rats. The experiments were divided into three groups: control group, FSH treated group and transforming growth factor beta receptor Ⅱ (TGF-β RⅡ) neutralizated group. Immunocytochemistry (ICC) and Western blotting were then used to locate and detect the expression level of TGF-β RⅡ and proliferating cell nuclear antigen (PCNA). The proliferation index (PI), cell cycle and percentage of apoptotic cells were assessed by flow cytometry (FCM), and the level of estradiol (E2) was determined by ELISA. Results FSH increased the expression of PCNA and PI, changed the cell cycle and inhibited apoptosis of GCs, and these actions were reduced significantly when TGF-βsignaling pathway was inhibited(P<0.05); FSH stimulated the secretion of E2, and the increase in E2 levels was not regulated by inhibition of the TGF-β signaling pathway(P>0.05). Conclusion The effects of FSH on ovarian granulosa cells are partly affected by the TGF-β signaling pathway.

Key words: Granulosa cells culture, Follicle stimulating hormone, Transforming growth factor β, Immunohistochemistry, Western blotting, Rat

中图分类号:

R322

王雨桐卫晓红葛玲杨云风徐健. 转化生长因子β信号通路对卵泡刺激素调节卵巢颗粒细胞功能的影响[J]. 解剖学报, 2021, 52(1): 118-123.

WANG Yu-tong WEI Xiao-hong GE Ling YANG Yun-feng XU Jian. Transforming growth factor β signaling pathway regulating the function of follicle stimulating hormone in ovarian granulosa cells[J]. Acta Anatomica Sinica, 2021, 52(1): 118-123.

参考文献

［1］ Ocon-Grove OM, Poole DH, Johnson AL. Bone morphogenetic protein 6 promotes FSH receptor and anti-Müllerian hormone mRNA expression in granulosa cells from hen prehierarchal follicles ［J］. Reproduction (Cambridge, England), 2012, 143(6): 825-833.

［2］ Mcgee EA, Oakley JI, Xu J. Transforming growth factor β (TGF-β) and activin stimulate nuclear translocation of Smad proteins in granulosa cells ［J］. Fertil Steril, 2001, 76(3): S38.

［3］ Gueripel X, Benahmed M, Gougeon A. Sequential gonadotropin treatment of immature mice leads to amplification of transforming growth factor beta action, via upregulation of receptor-type 1, Smad 2 and 4, and downregulation of Smad 6 ［J］. Biol Reprod, 2004, 70(3): 640-648.

［4］ Wei XH, Qi LH, Xu J, et al. The effects of FSH on the phosphorylation of Smad2/Smad3 protein in rat ovarian granulosa cells［J］.Chinese Journal of Anatomy, 2008, 31(2): 141-144.（in Chinese）

卫晓红, 祁丽花, 徐健，等. 卵泡刺激素调节大鼠卵巢颗粒细胞中Smad2/Smad3蛋白的表达及磷酸化［J］. 解剖学杂志, 2008, 31(2): 141-144.

［5］ Dong JX, Wei XH, Xu J, et al. Establishment of an in vitro blocking model of transforming growth factor beta signal transduction pathway in ovarian granulosa cells ［J］.Chinese Journal of Anatomy, 2008, 31(1):128-129.（in Chinese）

董静霞,卫晓红,徐健，等. 体外阻断卵巢颗粒细胞转化生长因子β信号转导通路模型的建立［J］. 解剖学杂志, 2008, 31(1):128-129.

［6］ Kossowska-Tomaszczuk K, De Geyter C, De Geyter M, et al. The multipotency of luteinizing granulosa cells collected from mature ovarian follicles ［J］. Stem cells (Dayton, Ohio), 2009, 27(1): 210-219.

［7］ Shen ChY, Dong JX, Xu J. Smad3 promotes the autophagy of granulosa cells in rats［J］. Acta Anatomica Sinica, 2014, 45(1): 109-113.（in Chinese）

申春艳, 董静霞, 徐健. Smad3促进大鼠卵巢颗粒细胞自噬［J］. 解剖学报, 2014, 45(1): 109-113.

［8］ Visser JA, Schipper Ⅰ, Laven JSE, et al. Anti-Müllerian hormone: an ovarian reserve marker in primary ovarian insufficiency ［J］. Nat Rev Endocrinol, 2012, 8(6): 331-341.

［9］ Pigny P, Merlen E, Robert Y, et al. Elevated serum level of anti-mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest ［J］. J Clin Endocrinol Metab, 2003, 88(12): 5957-5962.

［10］ Chang HM, Qiao J, Leung PCK. Oocyte-somatic cell interactions in the human ovary-novel role of bone morphogenetic proteins and growth differentiation factors ［J］. Hum Reprod Update, 2016, 23(1): 1-18.

［11］ Zafeiriou S, Loutradis D, Michalas S. The role of gonadotropins in follicular development and their use in ovulation induction protocols for assisted reproduction ［J］. Eur J Contracept Reprod Health Care, 2000, 5(2): 157-167.

［12］ Liu YC, Marraccino RL, Keng PC, et al. Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle ［J］. Biochemistry, 1989, 28(7): 2967-2974.

［13］ Sun XF, Sun XH, Cheng SF, et al. Interaction of the transforming growth factor-β and Notch signaling pathways in the regulation of granulosa cell proliferation ［J］. Reprod Fertil Dev, 2016, 28(12): 1873-1881.

［14］ Liu Y, Chen X, Xue X, et al. Effects of Smad3 on the proliferation and steroidogenesis in human ovarian luteinized granulosa cells ［J］. IUBMB Life, 2014, 66(6): 424-437.

［15］ Wang D, Di X, Wang J, et al. Increased formation of follicular antrum in aquaporin-8-deficient mice is due to defective proliferation and migration, and not steroidogenesis of granulosa cells ［J］. Front Physiol, 2018, 9:1193.

［16］ Tomic D, Miller KP, Kenny HA, et al. Ovarian follicle development requires Smad3 ［J］. Mol Endocrinol, 2004, 18(9): 2224-2240.

［17］ Gong X, Mcgee EA. Smad3 is required for normal follicular follicle-stimulating hormone responsiveness in the mouse ［J］. Biol Reprod, 2009, 81(4): 730-738.

［18］ Shen M, Jiang Y, Guan Z, et al. Protective mechanism of FSH against oxidative damage in mouse ovarian granulosa cells by repressing autophagy ［J］. Autophagy, 2017, 13(8): 1364-1385.

［19］ Boone DL, Tsang BK. Identification and localization of deoxyribonuclease Ⅰ in the rat ovary ［J］. Biol Reprod, 1997, 57(4): 813-821.

［20］ Li Y, Jin Y, Liu Y, et al. SMAD3 regulates the diverse functions of rat granulosa cells relating to the FSHR/PKA signaling pathway ［J］. Reproduction (Cambridge, England), 2013, 146(2): 169-179.

［21］ Bernard DJ, Tran S. Mechanisms of activin-stimulated FSH synthesis: the story of a pig and a FOX ［J］. Biol Reprod, 2013, 88(3): 78.

［22］ Bendell JJ, Dorrington J. Estradiol-17 beta stimulates DNA synthesis in rat granulosa cells: action mediated by transforming growth factor-beta ［J］. Endocrinology, 1991, 128(5): 2663-2665.

[1]	洪晓敏李三强崔钦奕郑润月杨孟利罗仁利李前辉 . 酒精性肝纤维化核因子κB信号通路与性别的差异 [J]. 解剖学报, 2024, 55(1): 55-61.
[2]	谢惠兰方芳林毅. Chir99021调控Wnt/β-catenin 信号通路抑制大鼠牙髓干细胞成骨诱导分化的机制[J]. 解剖学报, 2024, 55(1): 67-72.
[3]	魏茜茜李超红赵晨露唐家萍刘玉珍. 大鼠颈上神经节中Ⅰ组代谢型谷氨酸受体表达及慢性间歇性低氧对其的影响 [J]. 解剖学报, 2024, 55(1): 3-9.
[4]	袁蕾杨智伟杨惠刘政海何洁万炜. 三七总皂苷抑制小鼠星形胶质细胞活化缓解完全弗氏佐剂所致炎性痛 [J]. 解剖学报, 2024, 55(1): 25-31.
[5]	马婷婷陈建红刘爱翠李海宁. 抑制lncRNA TUG1下调核苷酸结合寡聚结构域样受体蛋白1炎症小体在延缓阿尔茨海默病进展的作用 [J]. 解剖学报, 2024, 55(1): 32-42.
[6]	邹成功冯浩陈兵唐辉邵川孙谋杨荣何家全. 创伤性颅脑损伤中神经功能障碍与认知功能损伤的动态变化 [J]. 解剖学报, 2024, 55(1): 43-48.
[7]	吕海燕沈西雅赵富生朱梅. 松茸多糖对 1-甲基-4-苯基-吡啶离子诱导 PC12 细胞损伤的影响 [J]. 解剖学报, 2024, 55(1): 49-54.
[8]	郑丽平刘霞杜晓华吴亚娟刘珊珊 . 脑源性神经营养因子在牦牛与黄牛端脑不同区域的表达与分布 [J]. 解剖学报, 2024, 55(1): 10-16.
[9]	苏芃王兴仪梁景岩熊天庆. 一种低密度及高纯度胎鼠原代海马神经元培养方法的优化及鉴定[J]. 解剖学报, 2024, 55(1): 113-119.
[10]	郝永明郭磊周程辉裴汉军李莉赵哲 . 改良腹主动脉缩窄法建立心肌肥厚模型[J]. 解剖学报, 2024, 55(1): 120-124.
[11]	许亚平余悦欣陈金福王柯柯郭志坤 . 高龄大鼠心室肌间质弹性纤维和胶原纤维的结构分布特征及其机制 [J]. 解剖学报, 2023, 54(6): 716-721.
[12]	刘丽平朱梦妮徐慧 . 白细胞介素6对脂多糖诱导所致类子痫前期大鼠有核红细胞的影响 [J]. 解剖学报, 2023, 54(6): 722-729.
[13]	赵卫明李晓余国营王改平常翠芳 . 丝氨酸肽酶抑制因子Kazal型1对肝癌细胞增殖的影响及其机制 [J]. 解剖学报, 2023, 54(6): 695-702.
[14]	王文娟丁雨桐苏昊任捷孙艳荣王涵菲陆嘉莉张林倩白钰秦丽华. 低雌激素状态下大鼠海马及纹状体Nogo-A的表达变化[J]. 解剖学报, 2023, 54(6): 620-627.
[15]	张琴杨俊霞蒋青松. 福辛普利对糖尿病视网膜病变小鼠血管紧张素转化酶2和血管内皮生长因子的影响[J]. 解剖学报, 2023, 54(6): 628-634.