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

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 17␣-hydroxylase/C17-20 lyase (P45017␣) 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 5Ј-untranslated region (UTR), 1509 bp of open reading frame encoding a well conserved 503-amino acid protein, and 1058 bp of 3Ј-UTR. Its 5Ј-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 5Ј-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 ϭ day of ovulation). The results showed a biphasic regulation of P450AROM mRNA expression: levels were highest in follicles at 0 h post-hCG, decreased significantly during the ovulatory process at 12 and 24 h (P Ͻ 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 P45017␣ mRNA were high in follicles at 0 h, but significantly decreased after hCG treatment (P Ͻ 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 P45017␣ 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)

Regulation of transcripts encoding ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs-1) and progesterone receptor by human chorionic gonadotropin in equine preovulatory follicles

“…Despite these advantages, however, the information derived from rodents does not always serve as an accurate predictor of what occurs in large monoovulatory species. This has been demonstrated notably by a recent series of studies in equine preovulatory follicles, which showed that several genes involved in steroidogenesis and ovulation exhibit spatial and temporal expression patterns within the follicle that are different from what has been reported in rodents (Boerboom & Sirois 1998, Boerboom et al 1999, Kerban et al 1999a). The present study further emphasizes these differences by demonstrating distinct patterns of ovarian ADAMTS-1 and PR expression and regulation.…”

Cloning of equine prostaglandin dehydrogenase and its gonadotropin-dependent regulation in theca and mural granulosa cells of equine preovulatory follicles during the ovulatory process

“…However, the information obtained from animal species does not always reflect the spectrum of potential expression among species. For example, in mares, follicular development and ovulation show several distinct characteristics that are not observed elsewhere: the Characterization and regulation of equine PGDH gonadotropin rise in preovulatory follicles induces a unique and remarkable modification of the granulosa cell layer in follicles prior to ovulation, characterized by extensive synthesis and assembly of a viscoelastic extracellular matrix (ECM) within the granulosa cell layer ) and the spatial and temporal regulation of several genes involved in ovulation and steroidogenesis in preovulatory follicles differs from those observed in the ovary of other mammals (Sirois et al 1991, Boerboom et al 1999. This difference in the pattern of gene regulation is further supported by the present study.…”

“…Follicles were dissected into three cellular preparations referred to as the follicular wall (theca interna with attached granulosa cells), and isolated mural granulosa cells and thecal layers, as described (Sirois et al 1991). The relative purity of each cellular preparation is estimated to exceed 95% based on the selective expression of P450 17a-hydroxylase-C17-20 lyase (CYP17A1) and P450 aromatase (CYP19A1) mRNAs by theca interna and granulosa cells respectively (Boerboom et al 1999). Testicular tissues were obtained from the Hô pital de grandes animaux, Faculté de médecine vétérinaire (Université de Montréal) following a routine castration, whereas, other non-ovarian tissues were collected at a local slaughterhouse.…”

Molecular characterization of tumor necrosis α-induced protein 6 and its human chorionic gonadotropin-dependent induction in theca and mural granulosa cells of equine preovulatory follicles

“…Follicles were dissected into three cellular preparations referred to as the follicular wall (theca interna with attached granulosa cells), and isolated granulosa cells and thecal layers, as described (Sirois et al 1991). The relative purity of each cellular preparation is estimated to exceed 95% based on the selective expression of P450 17a-hydroxylase-C17-20 lyase (CYP17A1) and P450 aromatase (CYP19A1) mRNAs by theca interna and granulosa cells respectively (Boerboom et al 1999). Testicular tissues were obtained from the Large Animal Hospital of the Faculté de médecine vétérinaire (Université de Montréal) following a routine castration, whereas other non-ovarian tissues were collected at a local slaughterhouse.…”