CTNNB1对猪卵巢颗粒细胞的调控

doi:10.3864/j.issn.0578-1752.2022.15.015

摘要/Abstract

摘要：

【目的】卵巢颗粒细胞的增殖和分化是原始卵泡生长启动的关键因素,卵巢颗粒细胞过度凋亡是卵泡闭锁的主要原因,因此卵巢颗粒细胞的功能对卵泡生长发育、排卵、激素分泌等至关重要。研究通过探究连环蛋白β1（catenin beta 1, CTNNB1）对猪卵泡发育过程中颗粒细胞的增殖、凋亡及类固醇激素分泌等功能的影响,为猪卵泡发育的分子调控机制研究提供参考。【方法】利用RNA抽提、实时定量PCR（Quantitative real time PCR, qRT-PCR）等方法,构建CTNNB1在母猪卵巢、肌肉、大脑等9个组织的表达谱;检测母猪性成熟过程中卵巢组织和小（≤3 mm）、中（3—6 mm）、大（≥6 mm）卵泡颗粒细胞中CTNNB1的表达情况。构建CTNNB1的真核表达载体及合成siRNA,转染至猪卵巢颗粒细胞,采用EdU、Annexin V- FITC/PI双染、ELISA等方法,检测CTNNB1对颗粒细胞增殖、凋亡、类固醇激素分泌的影响,以及对类固醇激素合成通路重要基因转录表达的影响。【结果】与肌肉、大脑等组织相比,CTNNB1在卵巢中mRNA表达水平最高。性成熟母猪卵巢中CTNNB1转录水平显著高于性成熟前和性成熟后的母猪;卵巢卵泡中CTNNB1转录和蛋白水平随卵泡发育明显上调;且卵巢颗粒细胞中CTNNB1转录和蛋白水平也随卵泡的发育逐渐上调。更重要的是,CTNNB1显著促进颗粒细胞增殖,抑制颗粒细胞凋亡;CTNNB1能够上调CYP1A1和HSD17B7的转录水平,下调CYP11A1、ESR1、ESR2、FSHR、LHR和NR5A1等类固醇激素合成相关基因的转录水平,进而促进颗粒细胞雌激素的分泌,抑制颗粒细胞雄激素和孕激素的分泌。【结论】本研究证实CTNNB1可能通过促进颗粒细胞增殖及雌激素的合成和分泌,抑制颗粒细胞凋亡及雄激素、孕激素的合成和分泌,进而促进卵泡的生长发育。

关键词: 猪, 卵巢, 卵泡, 颗粒细胞, CTNNB1

Abstract:

【Objective】The ovarian granulosa cells proliferation and differentiation are important to the initiation of primordial follicle growth. The excessive of ovarian granulosa cells apoptosis is one of the main reasons for follicular atresia. Therefore, the functions of ovarian granulosa cells are very important for follicular growth, development, ovulation, hormone secretion and so on. The objective of this study was to investigate the effects of CTNNB1 on the function of granulosa cells proliferation, apoptosis and steroid hormone secretion during porcine follicle development, so as to provide a reference for the molecular regulation mechanism of porcine follicular development. 【Method】In this study, the expressions of CTNNB1 in porcine ovary, muscle, brain and other 9 tissues were detected by RNA extraction and qRT-PCR. Then, qRT-PCR and Western blot were used to explore the expression of CTNNB1 in different stages of porcine ovarian tissue, as well as granulosa cells of small (≤3 mm), medium (3-6 mm) and large (≥6 mm) follicles. The eukaryotic expression vector and siRNA of CTNNB1 were constructed and transfected into porcine ovarian granulosa cells. The effects of CTNNB1 on the proliferation, apoptosis and steroid hormone secretion of granulosa cells were detected by EdU, annexin V-FITC/PI double staining assay, flow cytometry, and ELISA. Finally, qRT-PCR was also applied to explore the effect of CTNNB1 on the expression of genes related to steroid hormone synthesis. 【Result】The expression of CTNNB1 mRNA in ovary was higher than that in other tissues. The expression of CTNNB1 mRNA in ovaries of in-sexually-mature sows was significantly higher than that of pre-sexually-mature and post-sexually-mature sows. The expression of CTNNB1 in the follicles was gradually increased with the development of follicles, and the expression of CTNNB1 in granulosa cells of follicles was also increased with the development of follicles. In addition, the expression of CTNNB1 in granulosa cells as well as medium and large follicles was extremely higher than that in small follicles. CTNNB1 could promote the proliferation of granulosa cells and inhibit the apoptosis of granulosa cells. Moreover, CTNNB1 could also up-regulate the transcription level of CYP1A1 and HSD17B7, and down-regulate the transcription level of CYP11A1, ESR1, ESR2, FSHR, LHR and NR5A1, which were related to steroid hormone synthesis, thereby promoting the secretion of estrogen and suppressing the secretion of androgen and progesterone in granulosa cells. 【Conclusion】The above results confirmed that CTNNB1 regulated the function of granulosa cells by stimulating its proliferation and secretion of estrogen, and restraining its apoptosis and secretion of androgen and progesterone, thereby promoting the growth and development of follicles.

Key words: porcine, ovary, follicles, granulosa cells, CTNNB1

李丽莹,何颖婷,钟玉宜,周小枫,张豪,袁晓龙,李加琪,陈赞谋. CTNNB1对猪卵巢颗粒细胞的调控[J]. 中国农业科学, 2022, 55(15): 3050-3061.

LI LiYing,HE YingTing,ZHONG YuYi,ZHOU XiaoFeng,ZHANG Hao,YUAN XiaoLong,LI JiaQi,CHEN ZanMou. CTNNB1 Regulates the Function of Porcine Ovarian Granulosa Cells[J]. Scientia Agricultura Sinica, 2022, 55(15): 3050-3061.

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引物名称Name	引物序列Sequence (5′→3′)	产物长度Length (bp)
CTNNB1	F: GGGGTACCATGGCTACCCAAGCTGATTTG R: CGCTCGAGTTACAGGTCAGTATCAAACCAGGC	2346
CTNNB1-RT	F: GCTGTTCGCCTTCACTAC R: CTGATGAGCACGAACCAG	186
GAPDH-RT	F: TCGGAGTGAACGGATTTG R: TCACCCCATTTGATGTTGG	250
STARD1-RT	F: GCTTTTCCACTCTAGGGCGA R: AAGCTCCTGGCTGGATGAAC	184
CYP1A1-RT	F: TATCCTCCGTTACCTGCCCA R: TGCGCCCCTTCTCAAAGATT	119
CYP11A1-RT	F: CGCTCAGTCCTGGTCAAAGG R: TTCCAAGTTGCCGAGCTTCT	267
CYP17A1-RT	F: AAGCCAAGACGAACGCAGAAAG R: TAGATGGGGCACGATTGAAACC	228
CYP19A1-RT	F: TTCCTTGGCTGTACAGAAAGTATGA R: GGTGTCTGGTGCTGCAATTAG	221
HSD3B1-RT	F: ATCGTCCACTTGTTGCTGGA R: TGCTCTGGAGCTTAGAAAATTCC	103
HSD17B1-RT	F: GGGAGGCCTGATAGGTGAGT R: GGGCGGTAACTCACTGGAC	271
HSD17B7-RT	F: TGGACTTCACCTGTGCTTGG R: TGCTGACATCCACTTGCACA	116
ESR1-RT	F: ATGGCCATGGAATCTGCCAA R: CCCCTTTCATCATGCCCACT	241
ESR2-RT	F: GCCGACAAGGAACTGGTACA R: GAGCAAAGATGAGCTTGCCG	169
FSHR-RT	F: CGCGGTTGAACTGAGGTTTG R: TTGGGAAGGTTCTGGAAGGC	222
LHR-RT	F: ACATAACCACCGTACCAGCA R: GGAAGGCGTCATTGTGCATC	177
NR5A1-RT	F: CTGCTGGAGTGAGCTGCATGT R: ACTTGAGGCAGACGAACTCCT	218