PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFβ1, which plays an important role during luteal regression. The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFβ1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3βHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFβ1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFβ1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFβ1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFβ1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFβ1 signaling for luteolysis.

中文翻译：

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利用CRISPR识别水牛黄体中EGR1在前列腺素F2α诱导的黄体退化中的功能作用

PGF2α对于诱导黄体消退是必不可少的，而黄体消退又会减少孕激素的产生。早期生长反应（EGR）蛋白是Cys2-His2型锌指转录因子，与细胞增殖，存活和凋亡密切相关。黄体溶解剂量的PGF2α后观察到EGR1迅速升高。EGR1参与包括TGFβ1在内的许多基因的反式激活，而TGFβ1在黄体退化中起重要作用。当前的研究是在水牛黄体细胞中进行的，目的是更好地了解EGR1在PGF2α诱导的黄体退化过程中在TGFβ1反式激活中的作用。培养水牛中期黄体的黄体细胞，并用不同剂量的PGF2α处理不同的持续时间。孕激素生物合成途径（3βHSD，CYP11A1和StAR）内编码酶的mRNA的相对表达；半胱天冬酶3; 分析AKT以确认溶血事件的发生。为了确定EGR1是否通过诱导TGFβ1表达而参与PGF2α诱导的黄体退化，我们使用CRISPR / Cas9敲除了EGR1基因。本实验确定黄体细胞中EGR1蛋白表达是否对PGF2α处理有反应。在PGF2α诱导后12 h，水牛黄体细胞中EGR1和TGFβ1mRNA的定量显示明显上调。为了验证PGF2α在通过EGR1依赖性机制刺激TGFβ1表达中的作用，我们敲除了EGR1。PGF2α刺激了EGR1消融的黄体细胞，并且观察到EGR1 KO没有调节PGF2α诱导的TGFβ1的表达。在PGF2α处理的EGR1 KO黄体细胞中，与维持12 h的PGF2α处理的野生型黄体细胞相比，Caspase 3的mRNA表达显着增加。我们还研究了EGR1对类固醇生成的影响。用PGF2α处理的EGR1 KO黄体细胞与经PGF2α处理的野生型黄体细胞的孕酮浓度或StAR mRNA表达无明显差异。这些结果表明，EGR1信号不是在PGF2α诱导的黄体溶解TGFβ1信号的调节中起作用的唯一因素。与维持12小时的PGF2α处理的野生型黄体细胞相比，Caspase 3的mRNA表达显着增加。我们还研究了EGR1对类固醇生成的影响。用PGF2α处理的EGR1 KO黄体细胞与经PGF2α处理的野生型黄体细胞的孕酮浓度或StAR mRNA表达均无显着差异。这些结果表明，EGR1信号不是在PGF2α诱导的黄体溶解TGFβ1信号的调节中起作用的唯一因素。与维持12 h的PGF2α处理的野生型黄体细胞相比，Caspase 3的mRNA表达显着增加。我们还研究了EGR1对类固醇生成的影响。用PGF2α处理的EGR1 KO黄体细胞与经PGF2α处理的野生型黄体细胞的孕酮浓度或StAR mRNA表达均无显着差异。这些结果表明，EGR1信号不是在PGF2α诱导的黄体溶解TGFβ1信号的调节中起作用的唯一因素。