High temperatures cause several reproductive losses in cattle. Under in vitro conditions, heat shock decreases oocyte developmental competence and influences embryonic gene expression (Gendelman and Roth 2012 Anim. Reprod. Sci. 134, 125–134). This preliminary study aimed to evaluate whether heat shock during oocyte in vitro maturation (IVM) could have any further effect on chromatin remodelling of fertilized embryos at 4- and 8-cell stages, once such modifications are required for the gene activation in bovine embryos. We evaluated the distribution of heterochromatin 1 (HP1β) and of histone H3 trimethylated at lysine 9 (H3K9me3), both reportedly correlated with heterochromatin formation, in 4- and 8-cell stage embryos derived from control (C) and heat-shocked (HS) bovine oocytes. Immature cumulus-oocyte complexes (COC) collected from crossbred cows in Brazil were exposed for 12 h to 38.8°C (C group) or 41.0°C (HS group) followed by 12 h at 38.8°C, totalizing 24 h of IVM at 5% CO2 in air. Oocytes were in vitro fertilized (IVF) with non-sexed sperm and denuded zygotes were in vitro cultured in CR2aa medium at 38.8°C and 5% CO2, 5% O2 and 90% N2. Four- and 8-cell embryos at 44 h post-IVF were fixed in 4% paraformaldehyde and stained with anti-mouse HP1β and anti-rabbit H3K9me3 first antibodies. Immunofluorescence was evaluated by confocal microscopy (Zeiss LSM 700, MIMA platform, INRA) and 3D images processed by ZEN Lite software (Zeiss, Jena, Germany). Three different distribution patterns of fluorescence were identified based on morphological criteria: diffuse, little clusters, and big clusters. Proportions of embryos in every distribution pattern were compared between C and HS groups by Chi-squared test. No difference (P > 0.05) on cleavage rate was found between C and HS groups until 44 h post-fertilization. Embryos at the 4-cell stage from HS group displayed an increased (P < 0.01) proportion of nuclei with H3K9m3 big clusters (44%, n = 7/16 embryos), whereas embryos from C group displayed only few nuclei with this pattern (5%, n = 1/18). At the 8-cell stage, distribution of H3K9m3 was similar (P > 0.05) between C and HS groups. For HP1β, embryos at the 4-cell stage from HS group displayed an increased (P < 0.05) proportion of nuclei with little clusters (81%, n = 13/16 embryos), whereas embryos from C group had low proportion of nuclei with this same pattern (40%, n = 7/18). Mostly 4-cell stage embryos from C group presented the diffuse pattern (61%, n = 11/18 v.18%, n = 3/16 in the HS group; P < 0.05). At the 8-cell stage, some embryos from the C group (31%, n = 5/16) still showed nuclei with diffuse distribution of HP1β, whereas no nucleus with this pattern was found for the HS group. These preliminary data suggest that bovine embryos derived from heat-shocked oocytes can display precocious heterochromatin compaction, represented by the accumulation of H3K9me3 and HP1β at the 4-cell stage, compared with embryos derived from non-heat-shocked oocytes, which may affect embryonic genome activation with consequences for further gene expression.Research was supported by CNPq, FAPEMIG, FAPES and Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73).
Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos