Dual Regulation of Promoter II- and Promoter 1f-Derived Cytochrome P450 Aromatase Transcripts in Equine Granulosa Cells during Human Chorionic Gonadotropin-Induced Ovulation: A Novel Model for the Study of Aromatase Promoter Switching

0013-7227/99/$03.00/0 Endocrinology Copyright © 1999 by The Endocrine Society Vol. 140, No. 9 Printed in U.S.A. Dual Regulation of Promoter II- and Promoter 1fDerived Cytochrome P450 Aromatase Transcripts in Equine Granulosa Cells during Human Chorionic Gonadotropin-Induced Ovulation: A Novel Model for the Study of Aromatase Promoter Switching* DEREK BOERBOOM†, ABDURZAG KERBAN‡, AND JEAN SIROIS Centre de Recherche en Reproduction Animale and Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6 ABSTRACT Estradiol biosynthesis is a key biochemical trait of developing follicles. To study its regulation in equine follicles, the objectives of this study were to clone and determine the structure of equine cytochrome P450 aromatase (P450AROM), and characterize the regulation of P450AROM and P450 17a-hydroxylase/C17–20 lyase (P45017a) messenger RNAs (mRNAs) in vivo in equine preovulatory follicles isolated during hCGinduced ovulation. Two distinct P450AROM complementary DNAs (cDNAs) were isolated from an equine preovulatory follicle cDNA library. One clone was 2682 bp in length and included 115 bp of 59-untranslated region (UTR), 1509 bp of open reading frame encoding a well conserved 503-amino acid protein, and 1058 bp of 39-UTR. Its 59-most region represented the equine homolog of exon 1f, previously designated brain specific. The other cDNA clone encoded a truncated protein and contained a distinct 59-UTR characteristic of transcripts derived from promoter II, previously identified as the predominant ovarian mRNA. Northern blot analyses were performed using preovulatory follicles obtained during estrus between 0 –39 h after the administration of hCG and with corpora lutea isolated on day 8 of the estrous cycle (day 0 5 day of ovulation). The results showed a biphasic regulation of P450AROM mRNA expression: levels were highest in follicles at 0 h post-hCG, de- T HE AROMATASE cytochrome P450 (P450AROM), a product of the CYP19 gene, catalyzes the final ratelimiting step in the biosynthesis of estrogens from androgens (1– 4) and is expressed in the gonads and the brain of most vertebrate species (5– 8). However, a more extensive tissue distribution of the enzyme has been reported in humans, including expression in the placenta, adipose tissue, liver, and skin (5– 8). Placental expression of P450AROM has also been documented in cows (9, 10), pigs (11–13), and horses (9). A single CYP19 gene spanning more that 75 kb and containing nine coding exons (exons II–X) has been identified in Received December 1, 1998. Address all correspondence and requests for reprints to: Dr. Jean Sirois, Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Québec, Canada J2S 7C6. E-mail: siroisje@medvet. umontreal.ca. * This work 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 AF031520, AF031521, AF031893, and AF031894. † Supported by a Medical Research of Canada Doctoral Research Award. ‡ Supported by a fellowship from Al-Fateh University. creased significantly during the ovulatory 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 observed. Promoter II-derived transcripts accounted for the message present in follicles at 0 h post-hCG and in corpora lutea, whereas promoter 1f-derived mRNA was expressed exclusively during the ovulatory process (30 –39 h post-hCG). Levels of P45017a mRNA were high in follicles at 0 h, but significantly decreased after hCG treatment (P , 0.05), with lowest levels in follicles at 36 and 39 h post-hCG and in corpora lutea. Northern blots performed on isolated cellular preparations revealed that P450AROM and P45017a transcripts were localized exclusively in granulosa cells and theca interna, respectively. Equine aromatase promoters II and 1f were cloned from a genomic library, and putative transcription start sites were characterized by primer extension assays. Sequence analyses identified distinct potential regulatory elements in each promoter. Thus, this study identifies a novel aromatase promoter-switching phenomenon in equine granulosa cells during follicular luteinization and provides a new model in which aromatase promoter switching is induced in vivo. (Endocrinology 140: 4133– 4141, 1999) humans (5, 14, 15), but there is evidence for multiple distinct, but closely related, aromatase genes in pigs (13, 16, 17). The tissue-specific distribution of several aromatase transcripts has been linked in part to the use of different promoters (7, 9, 10, 12, 13, 18–21). In humans, these promoters direct the synthesis of distinct aromatase messenger RNA (mRNA) variants that differ only by their 59-noncoding termini. At least nine 59-untranslated first exons, and thus mRNAs variants, have been identified, including exons I.1, I.2, I.3, I.4, I.5, I.6, PII, 2a, and 1f (22). They are alternatively spliced into a common 59splice acceptor site found 38 bp upstream of the translation start site in exon 2 and generate transcripts that have distinct, but overlapping, tissue distribution (7). Increasing evidence suggests that switching of aromatase expression from one mRNA variant to another may be a key regulatory mechanism in several physiological and pathological processes. Distinct aromatase transcripts are expressed in early vs. midpregnancy in porcine placenta (12, 13, 23) as well as in fetal vs. adult human liver (24). Several studies have shown that a similar switch occurs in healthy vs. cancerous human breast adipose tissue (24 –29). It has been speculated that a switching mechanism may be involved in 4133 4134 AROMATASE PROMOTER SWITCHING IN EQUINE GRANULOSA CELLS the ovulation/luteinization process (24). However, results from the study of Jenkins et al. (30) did not support this concept, as only promoter II-derived aromatase transcripts were detected in human follicles and corpora lutea. One key function of P450AROM is to produce large amounts of estradiol in mammalian preovulatory follicles (31), and the obligatory role of estrogen synthesis in female reproduction was recently highlighted in mice by targeted disruption of the CYP19 gene (32). In contrast to that in other species, the molecular control of follicular steroidogenesis in mares has remained largely uncharacterized. Yet, the equine preovulatory follicle offers a good model for the study of ovarian gene expression because it has a relatively large size (40 –50 mm in diameter), and its development can be precisely monitored in vivo by ultrasound imaging (33, 34). Therefore, the objectives of this study were to clone and determine the primary structure of equine P4 (...truncated)