Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo

Background PGF2 alpha 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 PGF2 alpha. EGR1 is involved in the transactivation of many genes, including TGF beta 1, which plays an important role during luteal regression. Methods The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGF beta 1 during PGF2 alpha induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2 alpha for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3 beta HSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2 alpha induced luteal regression via induction of TGF beta 1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. Result The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2 alpha treatment. Quantification of EGR1 and TGF beta 1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2 alpha induction. In order to validate the role of PGF2 alpha on stimulating the expression of TGF beta 1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2 alpha and it was observed that EGR1 KO did not modulate the PGF2 alpha induced expression of TGF beta 1. In PGF2 alpha treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2 alpha treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2 alpha treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2 alpha-treated wild type luteal cells. Conclusion These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2 alpha induced TGF beta 1 signaling for luteolysis.

Automated summary

This study aimed to better understand the role of EGR1 in transactivation of TGF beta 1 during PGF2 alpha induced luteal regression using buffalo luteal cells. The results showed that EGR1 and TGF beta 1 mRNA expression were significantly upregulated at 12 hours post PGF2 alpha induction, and knocking out EGR1 did not modulate the expression of TGF beta 1. Additionally, the Caspase 3 mRNA expression was significantly increased in EGR1 KO luteal cells treated with PGF2 alpha compared to wild type luteal cells.