Zhang Yunxia, Wu Qiqian, Bai Furong, Hu Yanqin, Xu Bufang, Tang Yujie, Wu Jingwen
Department of Histoembryology, Genetics and Developmental Biology, Shanghai Jiao Tong University School of Medicine, Building 5, Room 506 280 South Chongqing Road, Huangpu District, Shanghai, 200025, China.
Shanghai Key Laboratory of Reproductive Medicine, Shanghai, 200025, China.
J Ovarian Res. 2025 Apr 9;18(1):75. doi: 10.1186/s13048-025-01651-0.
Follicle development is a complicated biological process that produces mature oocytes, and requires nutrients, growth factors, and steroids produced by ovarian granulosa cells (GCs). High fork head box J2 (FOXJ2) expression might negatively regulate ovarian function; however, the mechanism is unclear. This study aimed to investigate the effect and mechanism of FOXJ2 overexpression in GCs on regulating follicle development and fertility.
A GC-specific conditional Foxj2 knock-in mouse model (Amh-cre; Foxj2 mouse) was generated. Reproductive phenotypes were compared between Amh-cre; Foxj2 and control mice using fertility evaluation, oocyte collection, estrus cycle analysis, hormone evaluation, and ovarian follicle assessment. Then, RNA sequencing and bioinformatic analyses were used to detect the altered transcriptome of GCs collected from the Amh-cre; Foxj2 and wild-type mice. Western blotting, transmission electron microscopy, immunofluorescence staining, and flow cytometry were used to explore apoptosis and mitochondrial calcium homeostasis. Furthermore, Chromatin immunoprecipitation-PCR and dual-luciferase reporter assays were used to detect the target gene of FOXJ2. Moreover, short hairpin RNA interference was performed on primary GCs and human ovarian granulosa-like tumor (KGN) cells to explore the relationship between FOXJ2 and its target gene in apoptosis and mitochondrial calcium overload.
FOXJ2 overexpression in GCs led to reduced fertility, hormonal abnormalities, and follicle atresia, starting at the initiation of sexual maturity, resulting in a premature ovarian insufficiency (POI)-like phenotype. Increased apoptosis and mitochondrial calcium overload were detected in the GCs of Amh-cre; Foxj2 mice. Mcu (encoding a mitochondrial calcium uniporter) was observed to be upregulated in the GCs of the Amh-cre; Foxj2 mice and was a direct target of FOXJ2. Moreover, Mcu knockdown restored mitochondrial calcium homeostasis and reduced the apoptosis in the GCs of the Amh-cre; Foxj2 mice and in KGN cells transfected with FOXJ2-overexpression lentivirus.
卵泡发育是一个复杂的生物学过程，可产生成熟的卵母细胞，并且需要卵巢颗粒细胞（GCs）产生的营养物质、生长因子和类固醇。叉头框J2（FOXJ2）高表达可能对卵巢功能产生负调控作用；然而，其机制尚不清楚。本研究旨在探讨GCs中FOXJ2过表达对卵泡发育和生育能力的影响及其机制。
构建了GC特异性条件性Foxj2基因敲入小鼠模型（Amh-cre; Foxj2小鼠）。通过生育力评估、卵母细胞采集、发情周期分析、激素评估和卵巢卵泡评估，比较了Amh-cre; Foxj2小鼠和对照小鼠的生殖表型。然后，利用RNA测序和生物信息学分析检测从Amh-cre; Foxj2小鼠和野生型小鼠收集的GCs的转录组变化。采用蛋白质免疫印迹法、透射电子显微镜、免疫荧光染色和流式细胞术探讨细胞凋亡和线粒体钙稳态。此外，采用染色质免疫沉淀-PCR和双荧光素酶报告基因检测法检测FOXJ2的靶基因。此外，对原代GCs和人卵巢颗粒细胞瘤（KGN）细胞进行短发夹RNA干扰，以探讨FOXJ2与其靶基因在细胞凋亡和线粒体钙超载中的关系。
GCs中FOXJ2过表达导致从性成熟开始生育力降低、激素异常和卵泡闭锁，导致类似卵巢早衰（POI）的表型。在Amh-cre; Foxj2小鼠的GCs中检测到细胞凋亡增加和线粒体钙超载。观察到Mcu（编码线粒体钙单向转运体）在Amh-cre; Foxj2小鼠的GCs中上调，并且是FOXJ2的直接靶标。此外，Mcu敲低恢复了线粒体钙稳态，并减少了Amh-cre; Foxj2小鼠的GCs和用FOXJ2过表达慢病毒转染的KGN细胞中的细胞凋亡。
