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

The research article discusses the investigation into the genetic and molecular structure of 17beta-hydroxysteroid dehydrogenase type 1 (17betaHSD1) in horses. This enzyme, which converts estrone to 17beta-estradiol, undergoes significant activity changes during the process of follicular luteinization, an essential component of ovulation.

Cloning and Characterizing Equine 17betaHSD1

• The researchers initially focused on characterizing the gene for equine 17betaHSD1. To do this, they successfully cloned the gene from an equine genomic library.
• Upon examination, the 17betaHSD1 gene was composed of six exons — a common characteristic among genes encoding proteins — and was conserved across mammalian species, similarities observed through gene comparisons with other mammals.
• They also isolated and identified the cDNA sequence of the 17betaHSD1 gene, which they found resulted in a protein consisting of 308 amino acids. This protein was shown to be 72-74.5% homologous to those in other mammals.

Role and Regulation of 17betaHSD1 during Follicular Luteinization

• To understand how the 17betaHSD1 gene is regulated during follicular luteinization, they isolated equine preovulatory follicles at intervals between 0 and 39 hours following injection with human chorionic gonadotropin (hCG), a hormone commonly used to trigger ovulation in animals.
• Using RT-PCR/Southern blot analyses, they found that expression of 17betaHSD1 mRNA was high prior to hCG treatment. However, 12 hours after treatment, there was a significant decrease.
• They identified that the regulation of this enzyme takes place in the granulosa cell layer of the ovarian follicle.

Investigations into the Promotor Sequence and Transcription Factors of 17betaHSD1

• The researchers sequenced a portion of the promoter sequence of the equine 17betaHSD1 gene and found it contained several areas that potentially bind transcription factors.
• Through Electromobility shift assays (EMSAs), they identified multiple components within the promoter that could bind to nuclear proteins, namely, NFkappaB, GATA, and Sp1 cis-elements.
• After additional analysis with specific antibodies, they confirmed the involvement of NFkappaB and SF-1 transcription factors in binding to the promoter region of the gene.
• Importantly, they observed the quantity of DNA binding decreased, parallel to the reduction in 17betaHSD1 gene expression, after hCG treatment.

In conclusion, this study unveils novel insights on gonadotropin-dependent downregulation of 17betaHSD1 expression during ovulation and the potential regulation of the gene promoter’s protein/DNA interactions. It also provides a thorough characterization of the gene structure of equine 17betaHSD1.