Study of triple methylation of H3K4 in bovine embryos in vivo produced through superovulation cryopreserved or not

The cryopreservation of in vivo produced embryos is well established and widely used, but the same is not observed with embryos in vitro produced. The study of epigenetics in such embryos should help to identify differences between in vivo and in vitro produced embryos that could explain the differences of their viability after cryopreservation. Methylation at lysine 4 of histone 3 (H3K4), in general, is related to the active state of chromatin, allowing access of the transcription machinery. Accordingly, the objective of this study was to evaluate the epigenetic modifications in bovine embryos produced in vivo, fresh or cryopreserved, by identifying the trimethylation of H3K4. Girolando cows were submitted to superovulation protocol (SOV) with FSH and embryo were collected seven days after artificial insemination by uterine flushing. Thirty-seven SOV procedures were performed 60 days apart with semen from the same bull. Only embryos of excellent quality, according to the IETS standards were used for immunofluorescence. Fresh embryos (n=15) were fixed in paraformaldehyde (3%) supplemented with sacarose (2%) and stored at -20°C for later analysis. The embryos (n=15) were cryopreserved by slow freezing method and after few days they were thawed and stored in paraformaldehyde (3%) supplemented with sacarose (2%). After blocking and permeabilization, the embryos were exposed to polyclonal antibody against trimethylated H3K4 (CellSignaling®) at a concentration of 1:1000, overnight at 4°C. They were then exposed to the secondary antibody (anti-Rabbit Alexa 647; Invitrogen A21245) at a concentration of 1:200, and then subjected to staining of the nucleus (SYBR Green, Molecular Probes, Goettingen, Germany, 1:500). Embryos were individually evaluated under immunofluorescence and confocal microscopy. The same pattern of staining was observed in all groups, indicating that embryos produced in vivo, fresh and cryopreserved present an intense and well distributed patter of H3K4 in the trimethylated form.