CTNNB1对猪卵巢颗粒细胞的调控

doi:10.3864/j.issn.0578-1752.2022.15.015

Abstract

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

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.

Figures/Tables 7

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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

CTNNB1 Regulates the Function of Porcine Ovarian Granulosa Cells

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