Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5-azacytidine and cultured in differentiation medium containing BMP2, BMP4 or follicular fluid

• Published in: Zygote (Cambridge) Vol. 25; no. 3; pp. 341 - 357
• Main Authors: Costa, José Jackson do Nascimento, de Souza, Glaucinete Borges, Bernardo, Joyla Maria Pires, Ribeiro, Regislane Pinto, Passos, José Renato de Souza, Bezerra, Francisco Taiã Gomes, Saraiva, Márcia Viviane Alves, Silva, José Roberto Viana
• Format: Journal Article
• Language: English
• Published: Cambridge, UK Cambridge University Press 01.06.2017
• Subjects: Animals; Antibiotics; Azacitidine - pharmacology; Azacytidine; Bone Morphogenetic Protein 2 - pharmacology; Bone Morphogenetic Protein 4 - pharmacology; Cattle; Cell culture; Cell Differentiation - drug effects; Cell Differentiation - genetics; Culture Media - chemistry; Culture Media - pharmacology; DEAD-box RNA Helicases - genetics; Differentiation; Efficiency; Female; Fibroblasts; Fibroblasts - cytology; Fibroblasts - drug effects; Fibroblasts - physiology; Follicular fluid; Follicular Fluid - physiology; Gene expression; Genetic engineering; Genetic Markers - genetics; Germ cells; Infertility; Laboratories; Markers; Meiosis; Mutation; Oct-4 protein; Oocytes; Penicillin; Physical characteristics; Pluripotency; Pluripotent Stem Cells - physiology; Skin; Skin - cytology; Skin - embryology; Stem cells; Studies; Viability; Zona Pellucida Glycoproteins - genetics; Fibroblasts; Follicular fluid; BMP; Gene expression; 5-Azacytidine
• ISSN: 0967-1994; 1469-8730
• DOI: 10.1017/S0967199417000211

This study aims to investigate the effect 5-azacytidine (5-Aza) during induction of pluripotency in bovine fibroblasts and to evaluate the effects of BMP2, BMP4 or follicular fluid in the differentiation of reprogrammed fibroblasts in primordial germ cells and oocytes. It also analysis the mRNA levels for OCT4, NANOG, REX, SOX2, VASA, DAZL, cKIT, SCP3, ZPA and GDF9 after culturing 5-Aza treated fibroblasts in the different tested medium. Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 μM of 5-Aza for 18 h, 36 h or 72 h. Then, the cells were cultured in DMEM/F12 supplemented with 10 ng/ml BMP2, 10 ng/ml BMP4 or 5% follicular fluid. After culture, morphological characteristics, viability and gene expression were evaluated by qPCR. Treatment of skin fibroblasts with 2.0 μM 5-Aza for 72 h significantly increased expression of mRNAs for SOX2, OCT4, NANOG and REX. The culture in medium supplemented with BMP2, BMP4 or follicular fluid for 7 or 14 days induced formation of oocyte-like cells, as well as the expression of markers for germ cells and oocyte. In conclusion, treatment of bovine skin-derived fibroblasts with 2.0 μM 5-Aza for 72 h induces the expression of pluripotency factors. Culturing these cells in differentiation medium supplemented with BMP2, BMP4 or follicular fluid induces morphological changes and promotes expression of markers for germ cells, meiosis and oocyte.