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Hippo信号通路主要分子与卵巢生殖干细胞相关因子在人和小鼠卵巢衰退过程中的表达相关

徐娇¹, 曹秀萍², 唐子娟³, 黄健⁴, 郑月慧^2,5, 李佳^4,5,*

¹南昌大学医学院第一临床医学院，南昌 330006；²南昌大学医学院基础医学院，南昌 330006；³南昌大学医学院护理学院，南昌 330006；⁴南昌大学医学院医学实验教学中心，南昌 330006；⁵南昌大学医学院江西省生殖生理与病理重点实验室，南昌 330006

摘要

本研究旨在探讨Hippo信号通路的主要分子与卵巢生殖干细胞(ovarian germline stem cell, OGSC)相关因子在人和小鼠衰老不同功能状态卵巢中表达的相关性。选择2月龄性成熟期(正常对照)、12月龄衰老期(生理性衰退) KM系小鼠卵巢，以及青年期(青春期后~35岁)、中年期(36~50岁)和绝经期(51~60岁)女性卵巢皮层样本；采用环磷酰胺/马利兰片(cyclophosphamide/busulfan, CY/BUS)腹腔注射方式构建小鼠病理性衰退卵巢模型，用HE染色法检测各级卵泡的变化，用免疫组织化学法和免疫荧光染色法检测Hippo信号分子与OGSC相关因子(MVH/OCT4)的定位和表达变化，用Western blot检测Hippo信号通路的主要分子和OGSC相关因子的蛋白表达水平。结果显示，在生理性和病理性衰退模型小鼠卵巢中已无正常卵泡，仅有一些闭锁卵泡存在；和正常对照小鼠相比，生理性和病理性衰退模型小鼠卵巢皮层Hippo信号通路的主要分子(pYAP1)和MVH/OCT4蛋白表达水平均显著降低，病理性衰退模型小鼠卵巢皮层pYAP1/YAP1比值升高。和青年期女性相比，中年期和绝经期女性卵巢表面上皮(ovarian surface epithelium, OSE)结构逐渐变得松散，皮层处细胞也逐渐减少；LATS2蛋白表达水平在青年期女性OSE最高，MST1蛋白表达水平在老年期女性OSE最低，而YAP1和pYAP1蛋白表达水平在中年期女性OSE最高；相比青年期女性，中年期和老年期OSE的pYAP1/YAP1比值显著降低，而二者之间无显著性差异。青年期女性OSE中MVH蛋白表达水平显著高于中年期和老年期。以上结果表明，Hippo信号通路的主要分子与OGSC相关因子的蛋白表达之间有相关性，提示Hippo信号通路可能调控OGSC相关因子表达，从而参与卵巢功能的生理性衰退和病理性衰退过程。

关键词： 功能状态卵巢; 卵巢衰退模型; 卵巢生殖干细胞; Hippo通路

分类号：Q291

Expression relationship of Hippo signaling molecules and ovarian germline stem cell markers in the ovarian aging process of women and mice

XU Jiao¹, CAO Xiu-Ping², TANG Zi-Juan³, HUANG Jian⁴, ZHENG Yue-Hui^2,5, LI Jia^4,5,*

¹The First Clinical Medical School, College of Medicine, Nanchang University, Nanchang 330006, China；²School of Basic Medical Sciences, College of Medicine, Nanchang University, Nanchang 330006, China；³Nursing School, College of Medicine, Nanchang University, Nanchang 330006, China；⁴Medical Laboratory Education Center, College of Medicine, Nanchang University, Nanchang 330006, China；⁵The Key Laboratory of Reproductive Physiology and Pathology of Jiangxi Province, College of Medicine, Nanchang University, Nanchang 330006, China

Abstract

The present study was aimed to investigate the expression relationship of Hippo signaling molecules and ovarian germline stem cell (OGSC) markers in the development schedule of OGSCs during ovarian aging in women and mice. The ovaries of 2-month-old mature (normal control) and 12-month-old (physiological ovarian aging) KM mice were sampled, and the ovarian cortex samples of young (postpuberty to 35 years old), middle age (36–50 years old) and menopausal period (51–60 years old) women were obtained with consent. The mice model of pathological ovarian aging was established by intraperitoneal injection of cyclophosphamide/busulfan (CY/BUS). HE staining was used to detect the changes of follicles at different stages, and the localization and expression changes of Hippo signaling molecules and OGSCs related factors (MVH/OCT4) were detected by immunohistochemistry and immunofluorescence staining. Western blot was used to detect the protein expression levels of the major molecules in the Hippo signaling pathway and OGSCs related factors. The results showed that there were not any normal follicles, but a few atresia follicles in the ovaries from physiological and pathological ovarian aging mice. Compared with the normal control mice, both the physiological and pathological ovarian aging mice showed decreased protein expression levels of the main Hippo signaling molecules (pYAP1) and MVH/OCT4; Whereas only the pathological ovarian aging mice showed increased ratio of pYAP1/YAP1. In comparison with the young women, the middle age and menopausal women showed looser structure of ovarian surface epithelium (OSE) and less ovarian cortical cells. The protein expression level of LATS2 in the OSE was the highest in young women, MST1 expression was the lowest in the menopausal period women, and the expression levels of YAP1 and pYAP1 were the highest in middle age women. Compared with the young women, the middle age and menopausal period women exhibited significantly decreased ratio of OSE pYAP1/YAP1, whereas there was no significant difference between them. The expression level of MVH protein in OSE from the young women was significantly higher than those of the middle age and menopausal period women. These results indicate that there is an expression relationship between the main molecules of Hippo signaling pathway and OGSCs related factors, which suggests that Hippo signaling pathway may regulate the expression levels of OGSCs related factors, thus participating in the process of physiological and pathological degeneration of ovarian.

Key words: functional ovarian; ovarian aging model; ovarian germline stem cells; Hippo signaling pathway

收稿日期：2018-06-20　　录用日期：2018-10-24

通讯作者：李佳　　E-mail: lijia4199@163.com

DOI: 10.13294/j.aps.2019.0034

引用本文：

徐娇, 曹秀萍, 唐子娟, 黄健, 郑月慧, 李佳. Hippo信号通路主要分子与卵巢生殖干细胞相关因子在人和小鼠卵巢衰退过程中的表达相关[J]. 生理学报 2019; 71 (3): 405-414.

XU Jiao, CAO Xiu-Ping, TANG Zi-Juan, HUANG Jian, ZHENG Yue-Hui, LI Jia. Expression relationship of Hippo signaling molecules and ovarian germline stem cell markers in the ovarian aging process of women and mice. Acta Physiol Sin 2019; 71 (3): 405-414 (in Chinese with English abstract).