p53通过mTOR信号通路调节原始卵泡激活

p53 regulates primordial follicle activation through the mTOR signaling pathway

本文旨在探讨p53在原始卵泡激活中的作用及可能机制。首先检测生理状况下新生3、5、7、9天小鼠卵巢中p53mRNA和新生小鼠卵巢中p53的亚细胞定位情况,确定p53的表达模式。再选用新生2天和3天小鼠分别进行卵巢体外培养3天,分别添加p53抑制剂Pifithrin-μ(PFT-μ,5μmol/L)或等体积二甲基亚砜,苏木素染色观察并全卵巢计数,确定p53在原始卵泡激活中的功能,采用免疫组化染色检测细胞增殖情况,用免疫荧光染色、蛋白印迹、qPCR检测原始卵泡相关经典通路关键分子的转录水平和蛋白水平变化。最后,采用雷帕霉素(rapamycin,RAP)干预mTOR信号通路,设立对照组、RAP(1μmol/L)组、PFT-μ(5μmol/L)组、PFT-μ(5μmol/L)+RAP(1μmol/L)组,苏木素染色并全卵巢计数确定各级卵泡数目情况。结果显示:在生理情况下,p53 mRNA表达水平随着原始卵泡的激活而降低;p53在原始卵泡和生长卵泡的颗粒细胞和卵母细胞胞质中均有表达,在原始卵泡中的表达高于生长卵泡;抑制p53促进卵泡激活并减少原始卵泡库存;抑制p53可促进颗粒细胞和卵母细胞增殖;抑制p53后PI3K/AKT信号通路关键分子(AKT、PTEN、FOXO3a)的mRNA和蛋白表达无显著变化,mTOR信号通路的下游RPS6/p-RPS6表达显著上调;抑制p53的同时抑制mTOR可以逆转抑制p53导致的原始卵泡激活。上述结果表明p53可能通过mTOR信号通路抑制原始卵泡激活,维持原始卵泡库存。

This paper aimed to investigate the role and potential mechanism of p53 on primordial follicle activation.Firstly,the p53mRNA expression in the ovary of neonatal mice at 3,5,7 and 9 days post-partum(dpp)and the subcellular localization of p53 were detected to confirm the expression pattern of p53.Secondly,2 dpp and 3 dpp ovaries were cultured with p53 inhibitor Pifithrin-μ(PFT-μ,5μmol/L)or equal volume of dimethyl sulfoxide for 3 days.The function of p53 in primordial follicle activation was determined by hematoxylin staining and whole ovary follicle counting.The proliferation of cell was detected by immunohistochemistry.The relative mRNA levels and protein levels of the key molecules involved in the classical pathways associated with the growing follicles were examined by immunofluorescence staining,Western blot and real-time PCR,respectively.Finally,rapamycin(RAP)was used to intervene the mTOR signaling pathway,and ovaries were divided into four groups:Control,RAP(1μmol/L),PFT-μ(5μmol/L),PFT-μ(5μmol/L)+RAP(1μmol/L)groups.The number of follicles in each group was determined by hematoxylin staining and whole ovary follicle counting.The results showed that the expression of p53 mRNA was decreased with the activation of primordial follicles in physiological condition.p53 was expressed in granulosa cells and oocyte cytoplasm of the primordial follicles and growing follicles,and the expression of p53 in the primordial follicles was higher than that in the growing follicles.Inhibition of p53 promoted follicle activation and reduced the primordial follicle reserve.Inhibition of p53 promoted the proliferation of the granulosa cells and oocytes.The mRNA and protein expression levels of key molecules in the PI3K/AKT signaling pathway including AKT,PTEN,and FOXO3a were not significantly changed after PFT-μtreatment,while the expression of RPS6/p-RPS6,the downstream effectors of the mTOR signaling pathway,was upregulated.Inhibition of both p53 and mTOR blocked p53 inhibition-induced primordial follicle activation.Collectively,these findings suggest that p53 may inhibit primordial follicle activation through the mTOR signaling pathway to maintain the primordial follicle reserve.