Academic Unit of Reproductive and Developmental Medicine, Department of Oncology and Metabolism, University of Sheffield, Sheffield, S10 2SF, United Kingdom.
Department of Obstetrics and Gynecology, McGill University Health Centre, Montreal, Canada.
Sci Rep. 2019 Apr 24;9(1):6513. doi: 10.1038/s41598-019-42878-4.
Primordial follicles, consisting of granulosa cell (GC)-enveloped oocytes are maintained in a state of developmental arrest until activated to grow. The mechanism that operates to maintain this arrested state in GCs is currently unknown. Here, we show the TGFβ-activated transcription factor SMAD3 is expressed in primordial GC nuclei alongside the cell cycle proteins, cyclin D2 (CCND2) and P27. Using neonatal C57/Bl6 mouse ovaries densely populated with primordial follicles, CCND2 protein co-localised and was detected in complex with P27 by immunofluorescence and co-immunoprecipitation, respectively. In the same tissue, SMAD3 co-precipitated with DNA sequences upstream of Ccnd2 and Myc transcription start sites implicating both as direct SMAD3 targets. In older ovaries follicle growth was associated with nuclear exclusion of SMAD3 and reduced P27 and CCND2 in GCs, alongside elevated Myc expression. Brief (2 H) exposure of neonatal ovaries to TGFβ1 (10 ng/ml) in vitro led to immediate dissociation of SMAD3 from the Ccnd2 and Myc promoters. This coincided with elevated Myc and phospho-S6, an indicator of mTOR signalling, followed by a small increase in mean primordial GC number after 48 H. These findings highlight a concentration-dependent role for TGFβ signalling in the maintenance and activation of primordial follicles, through SMAD-dependent and independent signalling pathways, respectively.
原始卵泡由被颗粒细胞（GC）包裹的卵母细胞组成，处于发育停滞状态，直到被激活生长。目前尚不清楚维持 GC 这种停滞状态的机制。在这里，我们发现 TGFβ 激活的转录因子 SMAD3 与细胞周期蛋白 D2（CCND2）和 P27 一起表达在原始 GC 核中。使用富含原始卵泡的新生 C57/Bl6 小鼠卵巢，CCND2 蛋白通过免疫荧光共定位，并且通过共免疫沉淀分别检测到与 P27 结合。在相同的组织中，SMAD3 与 Ccnd2 和 Myc 转录起始位点上游的 DNA 序列共沉淀，暗示两者都是 SMAD3 的直接靶标。在较老的卵巢中，卵泡生长与 GC 中 SMAD3 的核排斥以及 P27 和 CCND2 的减少以及 Myc 表达的升高有关。新生卵巢在体外短暂（2H）暴露于 TGFβ1（10ng/ml）会导致 SMAD3 立即从 Ccnd2 和 Myc 启动子上解离。这与 Myc 和磷酸化 S6 的升高同时发生，磷酸化 S6 是 mTOR 信号的指标，之后在 48H 后原始 GC 数量略有增加。这些发现强调了 TGFβ 信号在通过 SMAD 依赖和独立的信号通路分别维持和激活原始卵泡中的浓度依赖性作用。
