RESUMO
The aim of this study was to evaluate the effects of different concentrations of BMP4 on activation, development and mRNA expression of GDF9, BMP15, PCNA, Bax and Bcl2 in cultured bovine follicles enclosed in ovarian tissues. Ovarian tissue fragments were cultured for 6 days in α-MEM+ alone or supplemented with different concentrations of BMP4 (10, 50 or 100 ng/ml). Classical histology was performed to analyze follicle growth and morphology, while real-time PCR was used to analyze mRNA levels in fresh and cultured tissues. After 6 days, the culture of ovarian tissue in α-MEM+ alone or supplemented with 10, 50 or 100 ng/ml BMP4 promoted follicular activation. The different concentrations of BMP4 maintained the percentage of normal follicles similar to results of the control. The presence of 100 ng/ml BMP-4 in culture medium increased oocyte and follicular diameters of primary and secondary follicles when compared with those follicles from uncultured control or cultured in α-MEM+ alone (P < 0.05). The tissues cultured in the presence of increasing concentrations of BMP4 had an increase in mRNA expression of the tested genes, but despite this the differences were not statistically significant. In conclusion, 100 ng/ml BMP4 promotes an increase in diameters of follicles and oocytes of primary and secondary follicles after 6 days of in vitro culture.
Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Ovário/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Proteína Morfogenética Óssea 15/genética , Bovinos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 9 de Diferenciação de Crescimento/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
The aim of this review was to evaluate the importance of the real-time PCR (qRT-PCR) as a technique for mRNA expression analysis in different tissues. Real-time PCR is widely used for quantification of mRNA levels and is a fundamental tool for basic research, molecular medicine and biotechnology.Genes of references are expressed in a wide variety of tissues and cells with minimal variations in their expression levels, and thus are used to normalize data of mRNA quantification. Software programs, such as geNorm, BestKeeper and NormFinder, have been developed to perform the normalization of data, which help to choose the most stable reference gene. Several genes, such as GAPDH, β-actin, β-tubulin, PGK, UBQ, RPL-19 and 18S rRNA have been suggested as standards in PCR studies, but these genes can have variation in their expression in different tissues, reinforcing the idea that there is no ideal reference gene.