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

摘要/Abstract

摘要：

旨在探索骨形态发生蛋白4(bone morphogenetic protein 4, BMP4)对绵羊卵巢颗粒细胞中间隙连接蛋白基因(gap junction protein alpha 1, GJA1)表达的影响及其分子调控机制。本研究利用廊坊市屠宰场收集的2~4岁健康绵羊卵巢分离颗粒细胞，采用免疫荧光染色技术定位GJA1在颗粒细胞中的分布。将细胞随机分为4组，分别添加0、10、50和100 ng·mL^-1浓度的重组BMP4蛋白，每组3个重复，培养24 h，利用CCK-8法评估细胞活性，RT-qPCR和Western blot研究BMP4对GJA1的mRNA和蛋白表达水平的影响。为探究BMP4调控GJA1表达的潜在机制，将细胞随机分为3组，每组3个重复，除对照组外分别添加10 μmol·L^-1 BMP I型受体抑制剂(Dorsomorphin)和干扰小RNA敲除SMAD家族蛋白4(SMAD family member 4, SMAD4)，均添加100 ng·mL^-1的BMP4处理细胞24 h，RT-qPCR检测GJA1和SMAD4表达量，Western blot分析测定GJA1和SMAD4表达水平以及SMAD1/5/8的磷酸化水平，最后利用划痕染料示踪试验检测绵羊卵巢颗粒细胞之间的间隙连接活性。结果显示，BMP4显著抑制了绵羊卵巢颗粒细胞中GJA1表达和间隙连接活性(P < 0.05)，此抑制效应在添加Dorsomorphin和敲除SMAD4后显著减弱(P < 0.05)，同时，BMP4处理显著增加了SMAD1/5/8的磷酸化水平(P < 0.05)。综上，BMP4通过SMAD1/5/8-SMAD4信号转导调控GJA1表达进而影响颗粒细胞间隙连接活性，本结果增加了对绵羊BMP/SMAD通路调控颗粒细胞间隙连接活性的了解，为改进体外卵泡成熟方法和高繁母羊的分子育种提供了基础。

关键词: 绵羊, 卵巢颗粒细胞, BMP4, 间隙连接, GJA1, SMAD4

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

中图分类号:

S826.3

何雨, 王翔宇, 狄冉, 储明星, 梁琛. 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[J]. 畜牧兽医学报, 2025, 56(2): 679-688.

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.

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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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