Expressão de marcadores de células germinativas e de oócitos em fibroblastos bovinos tratados com 5-aza-citidina e em células-tronco adultas cultivadas in vitro na presença de BMP-2, BMP-4 ou fluido folicular

Abstract

This study aimed to investigate the effect of 5-Aza-cytidine during induction of pluripotency, and evaluate the effects of culture in medium containing BMP-2, BMP-4 or follicular fluid in the differentiation of fibroblasts in primordias germ cells (CGP) and oocytes (stage 1). Furthermore, isolation of stem cells from the bovine ovarian epithelium and evaluate the effects of BMP-2, BMP-4 or follicular fluid on the differentiation of these stem cells into structures similar to oocytes (stage 2). In phase 1, fibroblasts were treated with 0.5, 1.0 or 2.0 μM of 5-Aza for 18, 36 or 72 h. Morphology cell viability and gene expression (OCT-4, NANOG, SOX2 and REX), were assessed, for the selection of the concentration/time more efficient. The fibroblasts were then cultured in medium supplemented with 10 ng/mL BMP-2 or 10 ng/mL BMP-4 or 5% bovine follicular fluid by 7 or 14 days. Subsequently, evaluation of the morphology and cell viability was taken, and gene expression (VASA, DAZL, c-Kit, SCP3, ZPA and GDF-9). For phase 2, the stem cells of ovarian surface were isolated, expanded, grown in differentiation medium containing 50 ng/mL BMP-2 or 50 ng/mL BMP-4, or BMP-2+BMP-4 or 5% bovine follicular fluid for 14 days. Morphological characteristics, cell viability and expression of alkaline phosphatase and gene expression (VASA, DAZL, C-KIT, SCP3, ZPA and GDF-9), were evaluated. The gene expression results were analyzed using ANOVA followed by Kruskal-Wallis test (P <0.05). In stage 1, the culture with 2.0 μM of 5-Aza for 72 h caused changes in morphology and cell proliferation rate, and significantly increased the expression of pluripotency factors. The culture in medium containing BMP-2, BMP-4 or follicular fluid for 7 or 14 days, altered cellular morphology, and expression of specific genes for stem cells and oocytes. In stage 2, the ovarian stem cells expressed pluripotent genes, and after culture, this cells showed morphologic characteristics similar to PGC and oocytes, including the expression of alkaline phosphatase, and the expression of specific genes for PCG and oocytes. In conclusion, the present study describes the possibility of conversion of the skin fibroblasts and stem cells from ovarian surface of the bovine, in oocyte-like cells, similar oocyte cells, through the cell reprogramming process associated with the supplementation of BMP-2, BMP- 4 and follicular fluid.