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Faculté de Médecine Vétérinaire, Centre de Recherche en Reproduction Animale, Université de Montréal, 3200 Sicotte, Saint-Hyacinthe, Québec, Canada J2S 7C6

(Requests for offprints should be addressed to J Sirois; Email: jean.sirois{at}umontreal.ca)

The type 1 form of 17ß-hydroxysteroid dehydrogenase (17ßHSD1) was the first isoform to be identified and is capable of converting estrone to 17ß-estradiol. This study was aimed at characterizing the molecular structure of the equine 17ßHSD1 gene and cDNA, as well as its molecular regulation during human chorionic gonadotropin (hCG)-induced follicular luteinization/ovulation in vivo. The equine 17ßHSD1 gene was cloned from an equine genomic library and shown to have a conserved genomic structure composed of six exons. Its cDNA sequence was also identified and coded for a 308 amino acid protein, 72.1–74.5% homologous to other mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of the 17ßHSD1 transcript in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment. Results demonstrated the presence of high 17ßHSD1 mRNA expression prior to hCG treatment with a marked decrease observed 12 h after hCG (P < 0.05). Analyses on isolated preparations of granulosa and theca interna cells identified the granulosa cell layer as the site of 17ßHSD1 transcript expression and downregulation (P < 0.05). A 1412 bp fragment of the equine 17ßHSD1 proximal promoter was sequenced and shown to contain many putative transcription factor binding sites. Electromobility shift assays (EMSA) using a fragment of the proximal promoter (–230/–30) and nuclear extracts prepared from granulosa cells isolated prior to hCG (0 h post-hCG) revealed the presence of a major complex, and results from competition assays suggest that steroidogenic factor-1 (SF-1), NFB, GATA, and Sp1 cis-elements are involved. Supershift assays using an antibody against the p65 subunit of NFB led to the displacement of the binding nuclear proteins to the DNA probe, whereas the use of an anti-equine SF-1 antibody demonstrated the clear formation of a DNA–protein–antibody complex, confirming the potential role of these transcription factors in EMSA results. Interestingly, a notable decrease in DNA binding was observed when granulosa cell nuclear extracts isolated 30 h post-hCG were used, which paralleled the decrease in 17ßHSD1 transcript after hCG treatment. Thus, this study is the first to report the gonadotropin-dependent downregulation of 17ßHSD1 transcript expression in a monoovulatory species, the presence and regulation of protein/DNA interactions in the proximal region of the 17ßHSD1 promoter during gonadotropin treatment, and the characterization of the primary structure of equine 17ßHSD1 cDNA and gene.

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