Physiology & Climatology Division, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 243122, India.
Animal Physiology, ICAR-National Research Centre on Pig, Guwahati, Assam, India.
Biol Res. 2021 Mar 12;54(1):9. doi: 10.1186/s40659-021-00333-7.
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.
PGF2α 对于黄体溶解至关重要，黄体溶解反过来又会减少孕激素的产生。早期生长反应 (EGR) 蛋白是一种 Cys2-His2 型锌指转录因子，与细胞增殖、存活和凋亡密切相关。在黄体溶解剂量的 PGF2α 作用后，EGR1 迅速升高。EGR1 参与许多基因的反式激活，包括 TGFβ1，其在黄体溶解过程中发挥重要作用。
本研究旨在探讨 EGR1 在 PGF2α 诱导的黄体溶解过程中反式激活 TGFβ1 中的作用，在水牛黄体细胞中进行。用不同剂量的 PGF2α 处理处于中期黄体期的水牛黄体细胞不同时间。分析孕激素生物合成途径（3βHSD、CYP11A1 和 StAR）；Caspase 3；AKT 编码酶的相对 mRNA 表达，以确认黄体溶解事件的发生。为了确定 EGR1 是否通过诱导 TGFβ1 表达参与 PGF2α 诱导的黄体溶解，我们使用 CRISPR/Cas9 敲除了 EGR1 基因。
本实验确定了黄体细胞中 EGR1 蛋白表达是否对 PGF2α 处理有反应。定量分析表明，PGF2α 诱导后 12 小时，水牛黄体细胞中 EGR1 和 TGFβ1 mRNA 显著上调。为了验证 PGF2α 通过 EGR1 依赖性机制刺激 TGFβ1 表达的作用，我们敲除了 EGR1。用 PGF2α 刺激 EGR1 缺失的黄体细胞，观察到 EGR1 KO 不能调节 PGF2α 诱导的 TGFβ1 表达。与用 PGF2α 处理的野生型黄体细胞维持 12 小时相比，用 PGF2α 处理的 EGR1 KO 黄体细胞中 Caspase 3 的 mRNA 表达显著增加。我们还研究了 EGR1 对甾体生成的影响。用 PGF2α 处理的 EGR1 KO 黄体细胞与用 PGF2α 处理的野生型黄体细胞相比，孕激素浓度或 StAR mRNA 表达均无明显差异。
这些结果表明，EGR1 信号不是调节 PGF2α 诱导的黄体溶解中 TGFβ1 信号的唯一因素。
