Zhou Y, Zhao H, Shuai L, She J, Diao R, Wang L
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.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Nov 20;42(11):1611-1617. doi: 10.12122/j.issn.1673-4254.2022.11.04.
To investigate the role of tacrolimus-binding protein 38 (FKBP38) in follicle development and the mechanism by which gene deletion causes premature ovarian insufficiency (POI).
The Cre-loxp system was used to construct oocyte-specific 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 knockout mice and non-transgenic littermate control mice were counted with HE staining under a microscope for analyzing the effect of 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.
The oocyte-specific 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 ( < 0.05), demonstrating POI-like changes. Compared with the control mice, oocyte-specific 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 ( < 0.05).
FKBP38 plays an important role in follicle development, and gene deletion in mice causes POI possibly by activating the mTOR signaling pathway and inducing granulosa cell apoptosis.
探讨钙调神经磷酸酶结合蛋白38（FKBP38）在卵泡发育中的作用以及基因缺失导致卵巢早衰（POI）的机制。
利用Cre-loxp系统构建卵母细胞特异性敲除转基因小鼠。采用聚合酶链反应（PCR）鉴定转基因小鼠的基因型，并验证卵母细胞中FKBP38的表达。通过苏木精-伊红（HE）染色在显微镜下计数敲除小鼠和非转基因同窝对照小鼠的原始卵泡、初级卵泡、次级卵泡和窦状卵泡数量，以分析基因缺失对卵泡发育的影响。通过繁殖实验和酶联免疫吸附测定（ELISA）确定小鼠的生育能力和血清性激素水平，以评估卵巢功能。采用末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）检测小鼠卵巢颗粒细胞凋亡情况。检测雷帕霉素靶蛋白（mTOR）信号通路磷酸化核糖体S6（PS6）下游靶蛋白的活性，并采用免疫荧光测定法测定BCL-2和BAX蛋白的表达，以评估小鼠卵母细胞的发育情况。
成功构建了卵母细胞特异性敲除转基因小鼠模型，该模型显示生育能力下降、性激素水平紊乱，卵巢中的原始卵泡、初级卵泡和次级卵泡显著减少（P<0.05），呈现出类似POI的变化。与对照小鼠相比，卵母细胞特异性敲除导致mTOR信号通路激活，颗粒细胞凋亡显著增加，并且通过增加卵母细胞中BAX的表达和降低BCL-2的表达，明显提高了BAX/BCL-2比值（P<0.05）。
FKBP38在卵泡发育中起重要作用，小鼠基因缺失可能通过激活mTOR信号通路和诱导颗粒细胞凋亡导致POI。
