Molecular cloning of equine 17β-hydroxysteroid dehydrogenase type 1 and its downregulation during follicular luteinization in vivo

The type 1 form of 17 beta-hydroxysteroid dehydrogenase (17 beta HSD1) was the first isoform to be identified and is capable of converting estrone to 17 beta-estradiol. This study was aimed at characterizing the molecular structure of the equine 17 beta HSD1 gene and cDNA, as well as its molecular regulation during human chorionic gonadotropin (hCG)-induced follicular luteinization/ovulation in vivo. The equine 17 beta HSD1 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 17 beta HSD1 transcript in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment. Results demonstrated the presence of high 17 beta HSD1 mRNA expression prior to hCG treatment with a marked decrease observed 12 In after hCG (P < 0(.)05). Analyses on isolated preparations of granulosa and theca interna cells identified the granulosa cell layer as the site of 17 beta HSD1 transcript expression and downregulation (P < 0.05). A 1412 bp fragment of the equine 17 beta HSD1 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), NF kappa B, GATA, and Sp1 cis-elements are involved. Supershift assays using an antibody against the p65 subunit of NF kappa B led to the displacement of the binding nuclear proteins to the DNA probe, whereas the use of an anti-equine SIF-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 nuclearextracts isolated 30 h post-hCG were used, which paralleled the decrease in 17 beta HSD1 transcript after hCG treatment. Thus, this study is the first to report the gonadotropin-dependent downregulation of 17 beta HSD1 transcript expression in a monoovulatory species, the presence and regulation of protein/DNA interactions in the proximal region of the 17PHSD1 promoter during gonadotropin treatment, and the characterization of the primary structure of equine 17PHSD1 cDNA and gene.