Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process

Steroidogenic factor-1 (SF-1, NR5A1a) is a member of the NR5A nuclear recep tor subfamily and has been implicated as a key transcriptional regulator of all ovarian steroidogenic genes in vitro. To establish links between the e xpression of SF-1 and that of the steroidogenic genes in vivo, the objectiv es of this study were to clone equine SF-I and examine the regulation of it s messenger RNA (mRNA) in follicular cells during human CG(hCG)-induced ovu lation. The equine SF-1 primary transcript was cloned by a combination of R T-PCR techniques. Results showed that the transcript was composed of a 5'-u ntranslated region (UTR) of 161 bp, an open reading frame (ORF) of 1386 bp that encodes a highly-conserved 461-amino acid protein, and a 3'-UTR of 518 bp. The cloning of SF-1 also led to the unexpected and serendipitous isola tion of the highly-related orphan nuclear receptor NR5A2, which was shown t o include a 5'-UTR of 243 bp, an ORF of 1488 bp, and a 3'-UTR of 1358 bp. T he NR5A2 ORF encodes a 495-amino acid protein that is 60% identical to SF-I , including 99%-similar DNA-binding domains. Northern blot analysis reveale d that SF-1 and NR5A2 were expressed in all major steroidogenic tissues, wi th the exception that NR5A2 was not present in the adrenal. Interestingly, NR5A2 was found to be, by far, the major NR5A subfamily member expressed in the preovulatory follicle and the corpus luteum. Using a semiquantitative RT-PCR/Southern blotting approach, the regulation of SF-1 and NR5A2 mRNAs i n. vivo was studied in equine follicular cells obtained from preovulatory f ollicles isolated between 0 and 39 h post hCG. Results showed that the thec a interna was the predominant site of SF-I mRNA expression in the follicle, and that hCG caused a significant decrease in SF-1 levels between 12-39 h in theca interna and between 24-39 h post hCG in granulosa cells (P < 0.05) . In contrast, the granulosa cell layer was the predominant, if not the sol e, site of NR5A2 mRNA expression in the follicle. Importantly, NR5A2 was mu ch more highly expressed in granulosa cells than SF-1. The administration o f hCG caused a significant decrease in NR5A2 transcripts in granulosa cells at 30, 36, and 39 h post hCG (P < 0.05). Thus, this study is the first to report the concomitant regulation of SF-1 in theca interna and granulosa ce lls throughout the ovulation/luteinization process, and to demonstrate the novel expression and hormonal regulation of NR5A2 in ovarian cells. Based o n the marked expression of NR5A2 in equine granulosa and luteal cells and o n mounting evidence of a functional redundancy between SF-1 and NR5A2 in ot her species, it is proposed that NR5A2 may play a key role in the regulatio n of gonadal steroidogenic gene expression.