title: Expression and regulation of regulator of G-protein signaling protein-2 (RGS2) in equine and bovine follicles prior to ovulation: molecular characterization of RGS2 transactivation in bovine granulosa cells.
abstract: The luteinizing hormone preovulatory surge stimulates several signal pathways essential for ovulation, and the regulator of G-protein signaling protein-2 (RGS2) is thought to be involved in this process. The objectives of this study were to characterize the regulation of RGS2 transcripts in equine and bovine follicles prior to ovulation and to determine its transcriptional control in bovine granulosa cells. To assess the regulation of equine RGS2 prior to ovulation, RT-PCR was performed using total RNA extracted from equine follicles collected at various times after human chorionic gonadotropin (hCG) injection. Results showed that RGS2 mRNA levels were very low at 0 h but markedly increased 12-39 h post-hCG (P < 0.05). In the bovine species, results revealed that RGS2 mRNA levels were low in small and dominant follicles and in ovulatory follicles obtained at 0 h, but markedly increased in ovulatory follicles 6-24 h post-hCG (P < 0.05). To study the molecular control of RGS2 expression, primary cultures of bovine granulosa cells were used. Stimulation with forskolin induced an up-regulation of RGS2 mRNA in vitro. Studies using 5'-deletion mutants identified a minimal region containing full-length basal and forskolin-inducible RGS2 promoter activities. Site-directed mutagenesis indicated that these activities were dependent on CRE and ETS1 cis-elements. Electrophoretic mobility shift assays confirmed the involvement of these elements and revealed their interactions with CREB1 and ETS1 proteins. Chromatin immunoprecipitation assays confirmed endogenous interactions of these proteins with the RGS2 promoter in granulosa cells. Forskolin-inducible RGS2 promoter activity and mRNA expression were markedly decreased by PKA and ERK1/2 inhibitors, and treatment with an antagonist of PGR (RU486) and inhibitors of PTGS2 (NS398) and EGFR (PD153035) blocked the forskolin-dependent RGS2 transcript expression, suggesting the importance of RGS2 in ovulation. Collectively, this study reports for the first time the gonadotropin-dependent up-regulation of RGS2 in equine and bovine preovulatory follicles and presents some of the regulatory controls involved in RGS2 gene expression in granulosa cells.
authors.affiliation: Centre de recherche en reproduction animale and the Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada k.sayasith@umontreal.ca.
authors.forename: Khampoun
authors.initials: K
authors.lastname: Sayasith
authors.affiliation: Centre de recherche en reproduction animale and the Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada.
authors.forename: Jean
authors.initials: J
authors.lastname: Sirois
authors.affiliation: Centre de recherche en reproduction animale and the Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada.
authors.forename: Jacques G
authors.initials: JG
authors.lastname: Lussier
mesh_terms: D000818:Animals; D002417:Cattle / Q000235:genetics* / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D005498:Follicular Phase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006062:Gonadotropins / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D006736:Horses / Q000235:genetics* / Q000502:physiology; D006080:Ovarian Follicle / Q000187:drug effects / Q000378:metabolism*; D010060:Ovulation / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D020710:RGS Proteins / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D006062:Gonadotropins; D020710:RGS Proteins
keywords: follicle; gene regulation; granulosa cells; ovulation; signaling pathways