Molecular cloning of equine 17β-hydroxysteroid dehydrogenase type 1 and its downregulation during follicular luteinization in vivo
Bibliographic record
Abstract
The type 1 form of 17beta-hydroxysteroid dehydrogenase (17betaHSD1) was the first isoform to be identified and is capable of converting estrone to 17beta-estradiol. This study was aimed at characterizing the molecular structure of the equine 17betaHSD1 gene and cDNA, as well as its molecular regulation during human chorionic gonadotropin (hCG)-induced follicular luteinization/ovulation in vivo. The equine 17betaHSD1 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 x 1-74 x 5% homologous to other mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of the 17betaHSD1 transcript in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment. Results demonstrated the presence of high 17betaHSD1 mRNA expression prior to hCG treatment with a marked decrease observed 12 h after hCG (P<0 x 05). Analyses on isolated preparations of granulosa and theca interna cells identified the granulosa cell layer as the site of 17betaHSD1 transcript expression and downregulation (P<0 x 05). A 1412 bp fragment of the equine 17betaHSD1 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), NFkappaB, GATA, and Sp1 cis-elements are involved. Supershift assays using an antibody against the p65 subunit of NFkappaB 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 17betaHSD1 transcript after hCG treatment. Thus, this study is the first to report the gonadotropin-dependent downregulation of 17betaHSD1 transcript expression in a monoovulatory species, the presence and regulation of protein/DNA interactions in the proximal region of the 17betaHSD1 promoter during gonadotropin treatment, and the characterization of the primary structure of equine 17betaHSD1 cDNA and gene.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".