Search Results

You are looking at 1 - 3 of 3 items for

  • Author: Kristy A Brown x
Clear All Modify Search
Free access

Evan R Simpson and Kristy A Brown

Obesity is now recognised to be an inflammatory condition in which dysregulated metabolism plays an integral role. Inflammatory mediators regulate aromatase expression in the human breast as one mechanism whereby they increase the risk of breast cancer, especially in women who are obese.

Free access

Kristy A Brown, Khampoune Sayasith, Nadine Bouchard, Jacques G Lussier and Jean Sirois

The type 1 form of 17β-hydroxysteroid dehydrogenase (17βHSD1) was the first isoform to be identified and is capable of converting estrone to 17β-estradiol. This study was aimed at characterizing the molecular structure of the equine 17βHSD1 gene and cDNA, as well as its molecular regulation during human chorionic gonadotropin (hCG)-induced follicular luteinization/ovulation in vivo. The equine 17βHSD1 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 17βHSD1 transcript in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment. Results demonstrated the presence of high 17βHSD1 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 17βHSD1 transcript expression and downregulation (P < 0.05). A 1412 bp fragment of the equine 17βHSD1 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), NFκB, GATA, and Sp1 cis-elements are involved. Supershift assays using an antibody against the p65 subunit of NFκB 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 17βHSD1 transcript after hCG treatment. Thus, this study is the first to report the gonadotropin-dependent downregulation of 17βHSD1 transcript expression in a monoovulatory species, the presence and regulation of protein/DNA interactions in the proximal region of the 17βHSD1 promoter during gonadotropin treatment, and the characterization of the primary structure of equine 17βHSD1 cDNA and gene.

Free access

Khampoune Sayasith, Kristy A Brown, Jacques G Lussier, Monique Doré and Jean Sirois

Early growth response factor-1 (EGR-1) is a transcription factor that is involved in the transactivation of several genes. The objective of this study was to characterize gonadotropin-dependent regulation of bovine EGR-1 in preovulatory follicles prior to ovulation. Bovine EGR-1 cDNA was obtained by RT-PCR, 5′- and 3′-RACE, its open reading frame composed of 1623 bp, and its coding region encodes a 540-amino acid protein that displays 62–93% identity to known mammalian homologs. The regulation of EGR-1 mRNA was studied in bovine preovulatory follicles which were isolated 0–24 h post-hCG using semiquantitative RT-PCR/Southern blot. Results revealed that the levels of EGR-1 mRNA were very low in follicles at 0 h, markedly increased at 6 h (P < 0.05) when compared to 0 h, and decreased between 12 and 24 h post-hCG. High levels of the EGR-1 mRNA were also observed in corpus luteum, uterus, kidney, pituitary, and spleen, moderate and low in other bovine tissues tested. Analyses performed on isolated preparations of granulosa and theca cells indicated that EGR-1 mRNA was regulated in both cell types, with a predominant expression in granulosa cells. Immunohistochemistry on sections of preovulatory follicles isolated before and after hCG confirmed its protein expression in granulosa cells, 24 h post-hCG. Studies of EGR-1 regulation in primary granulosa cells cultured with forskolin showed that levels of EGR-1 mRNA were low at 0 h, highly increased at 6 h post-forskolin (P < 0.05), and declined to steady state thereafter. Immunoblotting confirmed forskolin-induced EGR-1 protein in cultures. Interestingly, overexpression of EGR-1 increased the levels of mRNA for prostaglandin (PG) G/H synthase-2 (PGHS-2), PG E synthase (PGES), PG E2 receptor (EP2), LH receptor (LH-R), but not for cytochrome P450-side chain cleavage (P450scc), and cytochrome P450 aromatase (P450arom) in granulosa cultures. Thus, this study reports for the first time, a gonadotropin-dependent induction of follicular EGR-1 prior to ovulation in large monoovulatory species and its stimulating effect on the expression of genes known to be involved in prostaglandin biosynthesis pathway, thereby suggesting its potential involvement in the regulation of preovulatory events in cattle.