Influência das proteínas morfogenéticas ósseas 2 (BMP-2) e 4 (BMP-4) sobre o crescimento, expressão gênica e ultra estrutura de folículos bovinos cultivados in vitro

Abstract

The objectives of this study were: 1) to evaluate the effect of different concentrations of BMP-2 (10, 50 or 100 ng/mL) and BMP-4 (10, 50 or 100 ng/mL) on the activation, survival and growth of bovine preantral follicles included in ovarian tissue cultured in vitro for 6 days; 2) to evaluate the expression of messenger RNA for GDF-9, BMP-15, PCNA, Bax and Bcl2 in bovine preantral follicles after 6 days of in vitro culture in presence of BMP-4; 3) to evaluate the effect of different concentrations of BMP-2 (10, 50 or 100 ng/ml) on the in vitro development of isolated secondary follicles during 18 days of culture; 4) to evaluate the effect of BMP-2 (10 ng/mL) associated with FSH, as well as the expression of messenger RNAs for GDF-9, NLRP-5 and NPM-2 in secondary follicles cultured for 18 days and 5) to evaluate the effect of the interaction of both BMP-2 and 4, on the growth/viability of isolated bovine secondary follicles after 18 days of in vitro culture, 6) as well as quantification of levels of messenger RNAs for GDF-9, Cyclin B1, BMPR1A, BMPR1B, BMPRII, FSHR and SMAD1 before and after the culture of these follicles. For in situ culture, fragments of ovarian cortex were cultured in vitro for 6 days in α-MEM+ added with different concentrations of BMP2 or BMP4. Before and after culturing, the ovarian tissue fragments were fixed, analyzed by classical histology and transmission electron microscopy. The follicles were classified as primordial, primary and secondary, as well as in normal or atresic. In addition, oocyte and follicular diameters were evaluated. For follicular culture, follicles were microdissected and cultured by: a) 12 days in α-MEM+ with different concentrations of BMP-2 (10, 50 or 100 ng/mL); b) 18 days in α-MEM+ supplemented with BMP2 (10 ng/mL) associated or not to sequential FSH (50, 100 or 200 ng / mL) and c) 18 days in TCM199+ supplemented with BMP2 (10 ng/mL), BMP4 (100 ng/mL), or the interaction of both. After culturing, the follicles were evaluated morphologically and their diameters were measured. The RT-PCR technique was used to quantify the messenger RNAs. For in situ culture, the results showed that BMP2 and BMP4 do not influence the activation of primordial follicles in vitro. However, the addition of 100 ng/mL BMP4 increased the oocyte and follicular diameters of primary and secondary follicles included in ovarian tissue cultured in vitro for 6 days. BMP4 did not influence the expression of messenger RNAs to GDF-9, BMP-15, PCNA, Bax and Bcl2 after culturing. Regarding the culture of isolated secondary follicles, it was observed that BMP2 at 10 ng/mL, alone or associated with FSH, maintained the follicular ultrastructure after 12 days of culture. In addition, BMP2 (10 ng/mL) promoted follicle growth, antrum formation, maintained viability, and modulated GDF-9, NLRP-5 and NMP-2 expression of 10 follicles after 18 days of in vitro culture. BMP4 (100 ng/mL) promoted follicular growth, antrum formation and maintained follicular viability after in vitro culture. However, BMPs 2 and 4 together did not influence messenger RNA expression levels for GDF-9, Cyclin B1, BMPR1A, BMPR1B, BMPRII, FSHR and SMAD1. In conclusion, the addition of BMP2 (10 ng/ml) and BMP-4 (100 ng/ml) to the culture medium promoted growth and contributed to the maintenance of the ultrastructure of bovine preantral follicles after in vitro culture.