Expressão dos fatores de crescimento fibroblástico 17 e 18 (FGF-17 e FGF-18) em folículos antrais bovinos
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Data
2008-06-30
Autores
Machado, Mariana Fernandes [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Fatores de crescimento fibroblástico têm sido descritos como importantes reguladores locais do desenvolvimento folicular. Recentemente, foi relatada a expressão do FGF-8 e seus receptores, FGFR-3c e -4, em folículos antrais bovinos. O FGF-17 e o FGF-18 pertencem à subfamília do FGF-8 e ativam eficientemente o FGFR-3c e FGFR-4. Objetivando identificar outros FGFs capazes de ativar estes receptores, investigou-se (a) os padrões de expressão do FGF-17 e FGF-18 em folículos antrais bovinos; (b) a regulação da expressão do RNAm do FGF-17 pelo FSH e IGF-1 em células da granulosa; (c) a expressão do RNAm do FGF-17 em oócitos imaturos e maturados in vivo e in vitro, e (d) a expressão das proteínas FGF-17 e FGF-18 em folículos antrais bovinos. Amostras de células da teca e células da granulosa foram obtidas de ovários de vacas de abatedouro e o RNA total destas células foi extraído. As concentrações de estradiol (E2) e progesterona (P4) no fluido folicular foram mensuradas por Radioimunoensaio (RIA) e os folículos agrupados de acordo com a razão E2:P4 intrafoliculares (atrésicos; <0,01, transicionais; 1-0,01 e saudáveis; >1). “Pools” contendo 20 oócitos foram obtidos pela remoção das células do cumulus de complexos cumulus-oócito (CCOs) e o RNA total extraído. A expressão do FGF-17 e do FGF-18 foi investigada por RT-PCR em tempo real e normalizada pela expressão dos controles endógenos GAPDH, CYC-A ou Histona (H2a). Células da granulosa de folículos de 4 a 8 mm foram cultivadas em meio livre de soro fetal e tratadas com FSH (0; 0,1; 1; 10 ou 100 ng/mL) ou IGF-1 (0; 5; 10; 50; 100 ng/mL) para avaliar os efeitos dos tratamentos na expressão do RNAm do FGF-17. A presença das respectivas proteínas foi investigada por imunohistoquímica. Os dados foram analisados por ANOVA ou testes não-paramétricos quando a distribuição não foi normal.A expressão do RNAm...
Fibroblast growth factors have been described as important local regulators of follicle development. FGF-8 and its receptors, FGFR-3c and FGFR-4, have already been reported in bovine antral follicle. Aiming to identify other FGFs capable to activate this receptors, we assessed (a) the spatial and temporal patterns of FGF-17 and FGF-18 gene expression in bovine antral follicles; (b) FGF-17 mRNA regulation by FSH and IGF-1 in cultured granulosa cells ; (c) FGF-17 mRNA expression in immature oocytes and in vivo and in vitro matured oocytes; and (d) FGF-17 and FGF-18 protein expression in bovine antral follicles. Theca and granulosa cells samples were obtained from abattoir ovaries for total RNA extraction. Estradiol (E2) and progesterone (P4) concentrations were measured by RIA in the follicular fluid. Follicles were grouped according to health status based on the E2:P4 ratio (atretic: <0.01; transitional: 0.01-1; healthy: >1). Pools containing 20 oocytes were obtained by removing cumulus cells of cumulus-oocyte complexes (COCs) and total RNA was extracted. FGF-17 and FGF-18 mRNA expression was examined by semiquantitative real time RT-PCR using GAPDH, CYC-A or histone H2a as controls. Granulosa cells from follicles (4-8 mm) were cultured in serum-free medium supplemented with FSH (0, 0.1, 1, 10, 100 ng/mL) or IGF-1 (0, 5, 10, 50, 100 ng/mL) to assess effects of treatments on FGF-17 mRNA expression. The presence of FGF-17 and FGF-18 proteins was investigated by immunohistochemistry. Data were analized by ANOVA or non-parametric analysis when not normally distributed. FGF-17 mRNA expression was detected predominantly in granulosa cells but also in theca cells, and was higher in atretic follicles. FGF-17 mRNA was also detected in immature oocytes, was reduced in in vivo matured oocytes and absent in in vitro matured oocytes. FSH and IGF-1 inhibited FGF-17 mRNA expression in cultured granulosa... (Complete abstract click electronic access below)
Fibroblast growth factors have been described as important local regulators of follicle development. FGF-8 and its receptors, FGFR-3c and FGFR-4, have already been reported in bovine antral follicle. Aiming to identify other FGFs capable to activate this receptors, we assessed (a) the spatial and temporal patterns of FGF-17 and FGF-18 gene expression in bovine antral follicles; (b) FGF-17 mRNA regulation by FSH and IGF-1 in cultured granulosa cells ; (c) FGF-17 mRNA expression in immature oocytes and in vivo and in vitro matured oocytes; and (d) FGF-17 and FGF-18 protein expression in bovine antral follicles. Theca and granulosa cells samples were obtained from abattoir ovaries for total RNA extraction. Estradiol (E2) and progesterone (P4) concentrations were measured by RIA in the follicular fluid. Follicles were grouped according to health status based on the E2:P4 ratio (atretic: <0.01; transitional: 0.01-1; healthy: >1). Pools containing 20 oocytes were obtained by removing cumulus cells of cumulus-oocyte complexes (COCs) and total RNA was extracted. FGF-17 and FGF-18 mRNA expression was examined by semiquantitative real time RT-PCR using GAPDH, CYC-A or histone H2a as controls. Granulosa cells from follicles (4-8 mm) were cultured in serum-free medium supplemented with FSH (0, 0.1, 1, 10, 100 ng/mL) or IGF-1 (0, 5, 10, 50, 100 ng/mL) to assess effects of treatments on FGF-17 mRNA expression. The presence of FGF-17 and FGF-18 proteins was investigated by immunohistochemistry. Data were analized by ANOVA or non-parametric analysis when not normally distributed. FGF-17 mRNA expression was detected predominantly in granulosa cells but also in theca cells, and was higher in atretic follicles. FGF-17 mRNA was also detected in immature oocytes, was reduced in in vivo matured oocytes and absent in in vitro matured oocytes. FSH and IGF-1 inhibited FGF-17 mRNA expression in cultured granulosa... (Complete abstract click electronic access below)
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Palavras-chave
Bovino - Reprodução, Bovine
Como citar
MACHADO, Mariana Fernandes. Expressão dos fatores de crescimento fibroblástico 17 e 18 (FGF-17 e FGF-18) em folículos antrais bovinos. 2008. 63 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Medicina Veterinária e Zootecnia, 2008.