南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (11): 1611-1617.doi: 10.12122/j.issn.1673-4254.2022.11.04

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Fkbp38基因缺失导致小鼠早发性卵巢功能不全：基于激活mTOR通路并诱导细胞凋亡

周玉霞，赵茴茴，帅 领，佘加杰，刁瑞英，汪丽萍   

  1. 南方医科大学南方医院妇产科，广东 广州 510515；深圳市第二人民医院生殖医学科，广东 深圳 518035
  • 出版日期:2022-11-20 发布日期:2022-11-30

Fkbp38 deletion induces premature ovarian insufficiency in mice by activating mTOR signaling and inducing granulosa cell apoptosis

ZHOU Yuxia, ZHAO Huihui, SHUAI Ling, SHE Jiajie, DIAO Ruiying, WANG Liping   

  1. Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; Department of Reproductive Medicine, Shenzhen Second People's Hospital, Shenzhen 518035, China
  • Online:2022-11-20 Published:2022-11-30

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摘要： 目的 探讨他克莫司结合蛋白38（Fkbp38）在卵泡发育中的作用及其缺失导致早发性卵巢功能不全（POI）的机制。方法 采用Cre-loxp系统构建卵母细胞特异性敲除Fkbp38转基因小鼠，并应用PCR鉴定小鼠基因型，然后在蛋白水平上验证Fkbp38在卵母细胞中的敲除效果。通过HE染色在显微镜下计数敲除小鼠与同窝对照小鼠卵巢原始卵泡、初级卵泡、次级卵泡及窦卵泡数量分析卵母细胞缺失Fkbp38基因对小鼠卵泡发育的影响。通过繁殖实验及ELISA实验检测小鼠繁殖能力及血清性激素水平，明确卵母细胞敲除Fkbp38基因对卵巢功能的影响。TUNEL法检测敲除小鼠与同窝对照小鼠卵巢颗粒细胞凋亡情况。检测雷帕霉素靶蛋白（mTOR）信号通路下游靶蛋白磷酸化核糖体S6（PS6）活性验证Fkbp38基因对mTOR信号通路的影响。IF检测凋亡相关蛋白B细胞淋巴瘤/白血病-2（Bcl-2）及Bcl2-Associated X（BAX）表达明确Fkbp38基因敲除对卵母细胞发育的影响。结果 卵母细胞特异性敲除Fkbp38转基因小鼠模型构建成功。敲除小鼠较对照小鼠表现出生育力下降，性激素水平紊乱，卵巢内原始卵泡、初级卵泡和次级卵泡数均显著减少（P<0.05），引起POI样改变。卵母细胞特异性敲除Fkbp38基因后，mTOR信号通路激活，颗粒细胞凋亡增加，卵母细胞内BAX蛋白表达增强，Bcl-2蛋白表达减弱，BAX/Bcl-2蛋白比值显著升高（P<0.05）。结论 Fkbp38在卵泡发育中发挥重要作用，其缺失致POI发生的机制可能是通过激活mTOR信号通路诱导细胞凋亡而引起的。

关键词: 早发性卵巢功能不全；卵母细胞；Fkbp38；mTOR信号通路；细胞凋亡

Abstract: Objective To investigate the role of tacrolimus-binding protein 38 (FKBP38) in follicle development and the mechanism by which Fkbp38 gene deletion causes premature ovarian insufficiency (POI). Methods The Cre-loxp system was used to construct oocyte-specific Fkbp38 knockout transgenic mice. The genotype of the transgenic mice was identified using PCR, and the expression of FKBP38 in the oocytes was verified. The numbers of primordial follicles, primary follicles, secondary follicles and antral follicles in Fkbp38 knockout mice and non-transgenic littermate control mice were counted with HE staining under a microscope for analyzing the effect of Fkbp38 deletion on follicular development. The fertility and serum sex hormone levels of the mice were determined by reproduction experiments and ELISA to assess ovarian function. Ovarian granulosa cell apoptosis of the mice was assessed using TUNEL assay. The activity of the downstream target protein of phosphorylated ribosomal S6 (PS6) of mTOR signaling pathway was detected, and the expressions of BCL-2 and BAX proteins were determined using immunofluorescence assay for assessing oocyte development in the mice. Results The oocyte-specific Fkbp38 knockout transgenic mouse model was successfully constructed, which showed decreased fertility, disordered sex hormone levels, and significantly reduced primordial follicles, primary follicles and secondary follicles in the ovary (P<0.05), demonstrating POI-like changes. Compared with the control mice, oocyte- specific Fkbp38 knockout caused activation of the mTOR signaling pathway, significantly increased apoptosis of the granulosa cells, and obviously increased the BAX/BCL-2 ratio by increasing BAX expression and reducing BCL-2 expression in the oocytes (P<0.05). Conclusion FKBP38 plays an important role in follicle development, and Fkbp38 gene deletion in mice causes POI possibly by activating the mTOR signaling pathway and inducing granulosa cell apoptosis.

Key words: premature ovarian insufficiency; oocytes; Fkbp38; mTOR signaling pathway; apoptosis