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**This study was supported by Natural Sciences and Engineering Research Council of Canada Grant OPG0171135. The nucleotide sequences reported in this paper have been submitted to GenBank, with accession numbers AF157626, AF168796, AF203911, and AF203913. (2000)

Steroidogenic factor-1 (SF-1, NR5A1a) is a member of the NR5A nuclear receptor subfamily and has been implicated as a key transcriptional regulator of all ovarian steroidogenic genes in vitro. To establish links between the expression of SF-1 and that of the steroidogenic genes in vivo, the objectives of this study were to clone equine SF-1 and examine the regulation of its messenger RNA (mRNA) in follicular cells during human CG (hCG)-induced ovulation. The equine SF-1 primary transcript was cloned by a combination of RT-PCR techniques. Results showed that the transcript was composed of a 5'-untranslated 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 isolation of the highly-related orphan nuclear receptor NR5A2, which was shown to include a 5'-UTR of 243 bp, an ORF of 1488 bp, and a 3'-UTR of 1358 bp. The NR5A2 ORF encodes a 495-amino acid protein that is 60% identical to SF-1, including 99%-similar DNA-binding domains. Northern blot analysis revealed that SF-1 and NR5A2 were expressed in all major steroidogenic tissues, with 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 in vivo was studied in equine follicular cells obtained from preovulatory follicles isolated between 0 and 39 h post hCG. Results showed that the theca interna was the predominant site of SF-1 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 sole, site of NR5A2 mRNA expression in the follicle. Importantly, NR5A2 was much more highly expressed in granulosa cells than SF-1. The administration of 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 cells throughout the ovulation/luteinization process, and to demonstrate the novel expression and hormonal regulation of NR5A2 in ovarian cells. Based on the marked expression of NR5A2 in equine granulosa and luteal cells and on mounting evidence of a functional redundancy between SF-1 and NR5A2 in other species, it is proposed that NR5A2 may play a key role in the regulation of gonadal steroidogenic gene expression.