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Epigenetic profile - triple methylation of H3K4 - bovine embryos produced in vivo with sexed semen: preliminary results

2014; Colégio Brasileiro de Patologia Animal (CBPA); Volume: 11; Issue: 3 Linguagem: Inglês

1984-3143

Mirela Brochado Souza-Cáceres, W. A. L. Silva, Christopher Júnior Tavares Cardoso, H. Kischel, Jair Sábio de Oliveira, Marcelo Marcondes Seneda, Luciana Simões Rafagnin Marinho, Ralf Poehland, F. A. M. Sterza,

Epigenetics and DNA Methylation

Methylation at lysine 4 of histone 3 (H3K4), in general, is related to the active state of chromatin, allowing the access of the transcription machinery (Mellor, J. Trends in Genetics, 22:320-329, 2006). Accordingly, the objective of this study was to evaluate the epigenetic modifications of bovine embryos produced in vivo with sexed semen through the identification of trimethylation of H3K4. Girolando Cows (n=10) underwent superovulation protocol (SOV) with FSH and embryo collection was performed seven days after artificial insemination by uterine flushing. Thirty-seven procedures were carried out at intervals of 60 days, and half of them were performed with sexed semen and the other half with conventional semen from the same bull. Only embryos of excellent quality, according to the IETS standards, were used for evaluation by immunofluorescence. For this conventional (n=15) and sexed (n=15) embryos were fixed in paraformaldehyde (3%) supplemented with sucrose (2%) and stored in the refrigerator for later analysis. After blocking and permeabilization, the embryos were exposed to polyclonal antibody against trimethylated H3K4 (CellSignaling ®) at a concentration of 1:100, overnight at 4 ° C. After, the embryos were exposed to the secondary antibody (anti-Rabbit Alexa 647; Invitrogen A21245) in a concentration of 1:200, and then subjected to nuclear staining (SYBR Green, Molecular Probes, Goettingen, Germany, 1:500). Embryos were individually evaluated under confocal microscopy. The same labeling pattern was observed in all groups, indicating that embryos produced in vivo with sexed semen have the same pattern of H3K4 trimethylation as those produced with conventional semen.