The existing knowledge of the involvement of vinculin (VCL) in the control of ovarian cell functions is insufficient. To understand the role of VCL in the control of basic porcine ovarian granulosa cell functions, we decreased VCL activity by small interfering RNA (VCL siRNA). The expression of VCL, accumulation of VCL protein, cell viability, proliferation (accumulation of PCNA and cyclin B1), proportion of proliferative active cells, apoptosis (accumulation of bax, caspase 3, p53, antiapoptotic marker bcl2, and bax/bcl-2 ratio), DNA fragmentation, and release of steroid hormones and IGF-I were analyzed by RT‒qPCR, Trypan blue exclusion test, quantitative immunocytochemistry, XTT assay, TUNEL assay, and ELISA. The suppression of VCL activity inhibited cell viability, the accumulation of the proliferation-related proteins PCNA and cyclin B1, the antiapoptotic protein bcl2, and the proportion of proliferative active cells. Moreover, VCL siRNA inhibited the release of progesterone, estradiol, and IGF-1. VCL siRNA increased the proportion of the proapoptotic proteins bax, caspase 3, p53, the proportion of DNA fragmented cells, and stimulated testosterone release. Taken together, the present study is the first evidence that inhibition of VCL suppresses porcine granulosa cell functions. Moreover, the results suggest that VCL can be a potent physiological stimulator of ovarian functions.

中文翻译：

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抑制纽蛋白活性对猪卵巢细胞有不良影响

关于纽蛋白（VCL）参与卵巢细胞功能控制的现有知识还不够。为了了解 VCL 在控制基本猪卵巢颗粒细胞功能中的作用，我们通过小干扰 RNA (VCL siRNA) 降低了 VCL 活性。VCL表达、VCL蛋白积累、细胞活力、增殖（PCNA和cyclin B1积累）、增殖活性细胞比例、凋亡（bax、caspase 3、p53、抗凋亡标志物bcl2、bax/bcl-2积累）通过 RT-qPCR、台盼蓝排除试验、定量免疫细胞化学、XTT 测定、TUNEL 测定和 ELISA 分析 DNA 碎片以及类固醇激素和 IGF-I 的释放。VCL活性的抑制会抑制细胞活力、增殖相关蛋白PCNA和细胞周期蛋白B1、抗凋亡蛋白bcl2的积累以及增殖活跃细胞的比例。此外，VCL siRNA 抑制孕酮、雌二醇和 IGF-1 的释放。VCL siRNA增加了促凋亡蛋白bax、caspase 3、p53的比例、DNA碎片细胞的比例，并刺激睾酮释放。综上所述，本研究首次证明抑制 VCL 会抑制猪颗粒细胞功能。此外，结果表明 VCL 可以成为卵巢功能的有效生理刺激剂。