Characterization of bovine early growth response factor-1 and its gonadotropin-dependent regulation in ovarian follicles prior to ovulation
Early Growth Response Protein 1
Early growth response factor-1 (EGR-1) is a transcription factor that is involved in the transactivation of several genes. The objective of this study was to characterize gonadotropin-dependent regulation of bovine EGR-1 in preovulatory follicles prior to ovulation. Bovine EGR-1 cDNA was obtained by RT-PCR, 5'- and 3'-RACE, its open reading frame composed of 1623 bp, and its coding region encodes a 540-amino acid protein that displays 62-93% identity to known mammalian homologs. The regulation of EGR-1 mRNA was studied in bovine preovulatory follicles which were isolated 0-24 h post-hCG using semiquantitative RT-PCR/Southern blot. Results revealed that the levels of EGR-1 mRNA were very low in follicles at 0 h, markedly increased at 6 h (P < 0.05) when compared to 0 h, and decreased between 12 and 24 h post-hCG. High levels of the EGR-1 mRNA were also observed in corpus luteum, uterus, kidney, pituitary, and spleen, moderate and low in other bovine tissues tested. Analyses performed on isolated preparations of granulosa and theca cells indicated that EGR-1 mRNA was regulated in both cell types, with a predominant expression in granulosa cells. Immunohistochemistry on sections of preovulatory follicles isolated before and after hCG confirmed its protein expression in granulosa cells, 24 h post-hCG. Studies of EGR-1 regulation in primary granulosa cells cultured with forskolin showed that levels of EGR-1 mRNA were low at 0 h, highly increased at 6 h post-forskolin (P < 0.05), and declined to steady state thereafter. Immunoblotting confirmed forskolin-induced EGR-1 protein in cultures. Interestingly, overexpression of EGR-1 increased the levels of mRNA for prostaglandin (PG) G/H synthase-2 (PGHS-2), PG E synthase (PGES), PG E2 receptor (EP2), LH receptor (LH-R), but not for cytochrome P450-side chain cleavage (P450scc), and cytochrome P450 aromatase (P450arom) in granulosa cultures. Thus, this study reports for the first time, a gonadotropin-dependent induction of follicular EGR-1 prior to ovulation in large monoovulatory species and its stimulating effect on the expression of genes known to be involved in prostaglandin biosynthesis pathway, thereby suggesting its potential involvement in the regulation of preovulatory events in cattle.