Molecular cloning of equine 17beta-hydroxysteroid dehydrogenase type 1 and its downregulation during follicular luteinization in vivo.
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.
Publication Date: 2007-01-24 PubMed ID: 17242170DOI: 10.1677/jme.1.02097Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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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.
Cite This Article
APA
Brown KA, Sayasith K, Bouchard N, Lussier JG, Sirois J.
(2007).
Molecular cloning of equine 17beta-hydroxysteroid dehydrogenase type 1 and its downregulation during follicular luteinization in vivo.
J Mol Endocrinol, 38(1-2), 67-78.
https://doi.org/10.1677/jme.1.02097 Publication
Researcher Affiliations
- Faculté de Médecine Vétérinaire, Centre de Recherche en Reproduction Animale, Université de Montréal, 3200 Sicotte, Saint-Hyacinthe, Québec, Canada J2S 7C6.
MeSH Terms
- 17-Hydroxysteroid Dehydrogenases / biosynthesis
- 17-Hydroxysteroid Dehydrogenases / genetics
- Amino Acid Sequence
- Animals
- Base Sequence
- DNA-Binding Proteins / physiology
- Down-Regulation / physiology
- Female
- Horses / genetics
- Luteinization / physiology
- Molecular Sequence Data
- Ovarian Follicle / enzymology
- Promoter Regions, Genetic
Citations
This article has been cited 2 times.- Jones BL, Walker C, Azizi B, Tolbert L, Williams LD, Snell TW. Conservation of estrogen receptor function in invertebrate reproduction. BMC Evol Biol 2017 Mar 4;17(1):65.
- Viger RS, Guittot SM, Anttonen M, Wilson DB, Heikinheimo M. Role of the GATA family of transcription factors in endocrine development, function, and disease. Mol Endocrinol 2008 Apr;22(4):781-98.
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