80 EFFECT OF BOVINE OVIDUCTAL FLUID ON DNA METHYLATION OF BOVINE BLASTOCYSTS PRODUCED IN VITRO

During the transit through the oviduct, the early embryo undergoes an epigenetic reprogramming of its genome, which induces changes in DNA methylation pattern. Given that epigenetic modifications are susceptible to environmental influence, the oviducal milieu may affects DNA methylation marks in the developing embryo. The aim of this study was to evaluate whether bovine oviducal fluid (OF) exerts an effect on methylation status of genomic regions at different time points of embryo development. In vitro-produced zygotes were cultured in SOF + 3 mg mL−1 BSA (control, C) or in SOF + 1.25% OF at 3 different time points: until 98 h post-insemination (hpi) (OF1–16: 1–16 cell), 52 hpi (OF1–8: 1–8 cell), or from 52 until 98 hpi (OF8–16: 8–16 cell). The OF used was acquired from Embryocloud (Murcia, Spain) from cow oviducts at the early luteal phase (Day 1–4). After, embryo culture took place in control medium up to Day 8. For all the groups, the speed of development was considered, and normal developing embryos that reached ≥6 cells at 52 hpi and ≥16 cells at 98 hpi were selected and separately cultured from slow developing embryos. Cleavage (52 hpi) and blastocyst yield (Day 7–8) were analysed by ANOVA (8 replicates). Expanding blastocysts (Day 7–8) from the normal developing groups were collected for bisulfite sequencing analysis. The DNA bisulfite conversion was performed with a MethylEdge Bisulfite Conversion System kit (Promega, WI, USA) in groups of 20 blastocysts obtained from 5 replicates. Methylation status was analysed on regions localised in 4 developmental important genes (MTERF2, ABCA7, OLFM1, and GMDS) and within 2 LINE L1 elements located on chromosomes 9 (L9) and 29 (L29). Methylation percentages (10 sequenced clones/group) were compared using statistical z-test. No significant differences were found on cleavage rate (C: 89.7 ± 1.0, OF1–16: 84.9 ± 1.7; OF1–8: 85.4 ± 1.9; OF8–16: 89.1 ± 1.9%) and blastocyst yield between normal developing embryos (C: 36.8 ± 5.3; OF1–16: 34.7 ± 3.7; OF1–8: 41.0 ± 3.8; OF8–16: 43.9 ± 5.1%). Blastocysts derived from all OF groups showed the CpG region of MTERF2 hypomethylated compared with C group (20.0, 26.2, and 32.9% v. 56.2%, respectively; P < 0.001). The CpG sequence of ABCA7 exhibited significant hypomethylation in embryos from OF1–16 group compared with OF1–8, OF8–16, and C groups (31.1 v. 56.8, 57.9, and 65.8%, respectively; P < 0.001). Although the methylation of the CpG region within OLFM1 did not differ between OF1–16 and C groups (24.1 v. 19.4%, respectively), embryos from OF1–8 group showed a highly methylated region (47.1%) compared with OF1–16 and C groups (P < 0.001). The CpG sequence on L9 showed a high methylation level in blastocysts derived from OF1–16 group compared with OF8–16 and C groups (36.4 v. 14.5 and 20.0%, respectively; P < 0.05). There were no differences in methylation marks between groups examined for CpG regions of GMDS and L29. These results indicated that embryos exhibit a temporal sensitivity to OF at early embryonic stages, which is reflected by DNA methylation changes of specific genes at blastocyst stage. This is the first report describing that OF could modify specific epigenetic marks of the bovine embryonic genome.