WNT2在绵羊卵泡颗粒细胞的表达及功能研究

doi:10.11843/j.issn.0366-6964.2020.01.009

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (1): 74-82.doi: 10.11843/j.issn.0366-6964.2020.01.009

WNT2在绵羊卵泡颗粒细胞的表达及功能研究

薛丽娜^1,2, 毕锡麟¹, 王锴¹, 党文庆¹, 韩琦¹, 罗惠娣², 曹宁贤³, 吕丽华^1*

1. 山西农业大学动物科技学院, 太谷 030801;
2. 山西省农业科学院畜牧兽医研究所, 太原 030032;
3. 山西省畜禽繁育工作站, 太原 030001

收稿日期:2019-06-14 出版日期:2020-01-23 发布日期:2020-01-17
通讯作者: 吕丽华,主要从事动物生殖生理和动物繁育生物技术方面的研究,E-mail:lihualvsxau@126.com
作者简介:薛丽娜(1986-),女,山西临汾人,硕士生,主要从事动物遗传育种与繁殖研究,E-mail:xln19860429@163.com
基金资助:
山西省国际科技合作项目（201603D421006）；山西省青年三晋学者专项；国家自然科学基金（31873002）；山西省科技创新团队资金（201705D131028-20）；山西省农业科学院优势课题组自选（YYS1717）

Expression and Function Analysis of WNT2 in Ovine Follicular Granulosa Cells

XUE Lina^1,2, BI Xilin¹, WANG Kai¹, DANG Wenqing¹, HAN Qi¹, LUO Huidi², CAO Ningxian³, Lü Lihua^1*

1. College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China;
2. Institute of Animal Husbandry and Veterinary, Shanxi Academy of Agricultural Sciences, Taiyuan 030032, China;
3. Livestock Breeding Station of Shanxi Province, Taiyuan 030001, China

Received:2019-06-14 Online:2020-01-23 Published:2020-01-17

摘要/Abstract

摘要： 旨在探究WNT2在绵羊卵泡颗粒细胞（GCs）中的表达及功能。本研究选取4~6月龄健康母羊20只，采集双侧卵巢，免疫组化技术检测WNT2蛋白在卵泡中的表达定位；qRT-PCR及Western blot技术检测其在不同发育阶段卵泡颗粒细胞中的表达差异；siRNA沉默GCs中的 Wnt2基因后，qRT-PCR技术检测Wnt2基因及参与经典WNT信号通路关键基因CTNNB1的相对表达量，并测定GCs凋亡情况。结果表明：1）WNT2蛋白在绵羊卵泡内膜细胞、颗粒细胞以及卵丘细胞内均有表达。2）qRT-PCR及Western blot结果基本一致，均表明Wnt2 mRNA及蛋白在不同发育阶段卵泡颗粒细胞表达差异显著（P<0.05），且在大卵泡颗粒细胞内表达量显著高于中卵泡颗粒细胞（P<0.05），中卵泡颗粒细胞内表达量显著高于小卵泡颗粒细胞（P<0.05）。3）基因沉默后，沉默组Wnt2和CTNNB1的表达量均显著低于无义序列siRNA组（NC组）以及空白对照组（P<0.05），而Wnt2基因沉默组细胞凋亡率显著高于其他两组（P<0.05）。综上表明，WNT2是通过WNT2/CTNNB1信号通路促进绵羊卵泡颗粒细胞生物学功能的。

关键词: 绵羊, 卵泡, 颗粒细胞, WNT2, Wnt/β-catenin信号通路

Abstract: This study aimed to investigate the expression and function of WNT2 in ovine follicular granulosa cells (GCs). In this study, both ovaries of 20 healthy ewes aged 4-6 months were collected and the expression of WNT2 protein in follicles was localized by immunohistochemistry; qRT-PCR and Western blot were used to detect the differences of its expression in follicular granulosa cells at different developmental stages; The Wnt2 expression was silenced in GCs, and the relative expression of Wnt2 gene and the key gene CTNNB1 involved in the classical WNT signaling pathway were analyzed using qRT-PCR technology, and the apoptosis of GCs was detected. The results showed that:1) WNT2 protein was expressed in theca interna cells, granulosa cells and cumulus cells of ovine. 2) The results of qRT-PCR and Western blot were basically consistent, the expression of Wnt2 mRNA and protein in granulosa cells with different sizes were significantly different (P<0.05), and the expression in large follicle granulosa cell was significantly higher than that in medium follicles (P<0.05), and the expression in medium follicle granulosa cell was significantly higher than that in small follicles (P<0.05). 3) After silenced Wnt2 gene, the expressions of Wnt2 and CTNNB1 in Wnt2 siRNA group were significantly lower than those in nonsense sequence siRNA group (NC group) and blank control group (P<0.05), while the percentage of apoptosis in Wnt2 siRNA group was significantly higher than those in the other two groups(P<0.05). In conclusion, WNT2 can improve the biological function of ovaries granulosa cells in sheep by the WNT2/CTNNB1 signal pathway.

Key words: sheep, follicle, granulosa cells, WNT2, Wnt/β-catenin signaling pathway

中图分类号:

S826.3

薛丽娜, 毕锡麟, 王锴, 党文庆, 韩琦, 罗惠娣, 曹宁贤, 吕丽华. WNT2在绵羊卵泡颗粒细胞的表达及功能研究[J]. 畜牧兽医学报, 2020, 51(1): 74-82.

XUE Lina, BI Xilin, WANG Kai, DANG Wenqing, HAN Qi, LUO Huidi, CAO Ningxian, Lü Lihua. Expression and Function Analysis of WNT2 in Ovine Follicular Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(1): 74-82.

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WNT2在绵羊卵泡颗粒细胞的表达及功能研究

Expression and Function Analysis of WNT2 in Ovine Follicular Granulosa Cells

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