BMP4/SMAD4通过下调GJA1基因表达影响绵羊卵巢颗粒间隙连接活性

doi:10.11843/j.issn.0366-6964.2025.02.019

Abstract

Abstract:

This study aimed to explore the effect of bone morphogenetic protein 4 (BMP4) on the expression of gap junction protein alpha 1 (GJA1) in sheep ovarian granulosa cells and its molecular regulatory mechanism. Granulosa cells were isolated from the ovaries of healthy 2-4-year-old sheep obtained from a slaughterhouse in Langfang city. Immunofluorescence staining was used to localize the distribution of GJA1 in granulosa cells. The cells were randomly divided into 4 groups, each treated with recombinant BMP4 protein at concentrations of 0, 10, 50, and 100 ng·mL^-1, with 3 replicates per group, and cultured for 24 hours. Cell viability was assessed using the CCK-8 assay, while RT-qPCR and Western blot were employed to examine the effects of BMP4 on GJA1 mRNA and protein expression levels. To investigate the potential mechanism by which BMP4 regulates GJA1 expression, the cells were divided into 3 groups with 3 replicates per group. In addition to the control group, cells were treated with 10 μmol·L^-1 BMP type I receptor inhibitor (Dorsomorphin) and small interfering RNA to knock down SMAD family member 4 (SMAD4), all treated with 100 ng·mL^-1 BMP4 for 24 hours. RT-qPCR was used to measure the expression of GJA1 and SMAD4, and Western blot was used to assess the expression levels of GJA1 and SMAD4 as well as the phosphorylation levels of SMAD1/5/8. Finally, a scratch dye tracking assay was performed to assess the gap junction activity between sheep ovarian granulosa cells. The results showed that BMP4 significantly inhibited the expression of GJA1 and gap junction activity in sheep ovarian granulosa cells (P < 0.05). This inhibitory effect was significantly weakened after treatment with Dorsomorphin and SMAD4 knockdown (P < 0.05). Moreover, BMP4 treatment significantly increased the phosphorylation levels of SMAD1/5/8 (P < 0.05). In conclusion, BMP4 regulates GJA1 expression and subsequently affects the gap junction activity of granulosa cells through the SMAD1/5/8-SMAD4 signaling pathway. These findings enhance the understanding of how the BMP/SMAD pathway regulates gap junction activity in sheep granulosa cells, providing a foundation for improving in vitro follicle maturation methods and molecular breeding of high-fertility ewes.

Key words: sheep, ovarian granulosa cells, BMP4, gap junction, GJA1, SMAD4

CLC Number:

S826.3

HE Yu, WANG Xiangyu, DI Ran, CHU Mingxing, LIANG Chen. BMP4/SMAD4 Downregulates GJA1 Gene Expression to Affect the Gap Junctional Intercellular Communication Activity in Sheep Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 679-688.

Figures/Tables 8

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基因 Gene	GenBank登录号 GenBank ID	引物序列(5′→3′) Primer sequence	产物长度/bp Products size
GJA1	XM_004011159.5	F：GGAAAGTACCAAACAGCAGCAGAC R：GCAAGCCAAGTGATTGAACTCCTC	114
SMAD4	XM_060405186.1	F：CTTCAGCACCACCCGCCTATG R：ACACCAATACTCAGGAGCAGGATG	120
si-NC		F：CTTCAGCACCACCCGCCTATG R：ACACCAATACTCAGGAGCAGGATG
si-SMAD4		F：CTTCAGCACCACCCGCCTATG R：ACACCAATACTCAGGAGCAGGATG

si-RNA名称 Small interfering RNA	A液		B液
si-RNA名称 Small interfering RNA	DNA	OPTI	Lipofectamine 2000	OPTI
si-NC	8.0	242	10	240
si-SMAD4	8.0	242	10	240

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BMP4/SMAD4 Downregulates GJA1 Gene Expression to Affect the Gap Junctional Intercellular Communication Activity in Sheep Ovarian Granulosa Cells

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