Dual regulation of promoter II- and promoter 1f-derived cytochrome P450 aromatase transcripts equine granulosa cells during human chorionic gonadotropin-induced ovulation: A novel model for the study of aromatase promoter switching

Estradiol biosynthesis is a key biochemical trait of developing follicles. To study its regulation in equine follicles, the objectives of this study w ere to clone and determine the structure of equine cytochrome P450 aromatas e (P450AROM), and characterize the regulation of P450AROM and P450 17 alpha -hydroxylase/C17-20 lyase (P45017 alpha) messenger RNAs (mRNAs) in vivo in equine preovulatory follicles isolated during hCG-induced ovulation. Two di stinct P450AROM complementary DNAs (cDNAs) were isolated from an equine pre ovulatory follicle cDNA library. One clone was 2682 bp in length and includ ed 115 bp of 5'-untranslated region (UTR), 1509 bp of open reading frame en coding a well conserved 503-amino acid protein, and 1058 bp of 3'-UTR. Its 5'-most region represented the equine homolog of exon If, previously design ated brain specific. The other cDNA clone encoded a truncated protein and c ontained a distinct 5'-UTR characteristic of transcripts derived hom promot er II, previously identified as the predominant ovarian mRNA. Northern blot analyses were performed using preovulatory follicles obtained during estru s between 0-39 h after the administration of hCG and with corpora lutea iso lated on day 8 of the estrous cycle (day 0 = day of ovulation). The results showed a biphasic regulation of P450AROM mRNA expression: levels were high est in follicles at 0 h post-hCG, de-creased significantly during the ovula tory process at 12 and 24 h (P < 0.05), and increased again between 30-39 h post-hCG and in corpora lutea. When oligonucleotides specific for P450AROM mRNA variants were used as probes, a novel switching phenomenon was observ ed. Promoter II-derived transcripts accounted for the message present in fo llicles at 0 h post-hCG and in corpora lutea, whereas promoter II-derived m RNA was expressed exclusively during the ovulatory process (30-39 h post-hC G). Levels of P45017 alpha mRNA were high in follicles at 0 h, but signific antly decreased after hCG treatment (P < 0.05), with lowest levels in folli cles at 36 and 39 h post-hCG and in corpora lutea. Northern blots performed on isolated cellular preparations revealed that P450AROM and P45017a trans cripts were localized exclusively in granulosa cells and theca interna, res pectively. Equine aromatase promoters II and II were cloned from a genomic Library, and putative transcription start sites were characterized by prime r extension assays. Sequence analyses identified distinct potential regulat ory elements in each promoter. Thus, this study identifies a novel aromatas e promoter-switching phenomenon in equine granulosa cells during follicular luteinization and provides a new model in which aromatase promoter switchi ng is induced in vivo.