61 Expression Profiles and Functional Roles of H3.3 and HIRA in Bovine Early Embryos

Abstract

Early embryo death is one major reason for poor reproductive efficiency in dairy cows. In particular, ~20 to 50% of high-producing cows are subject to pregnancy loss during the first week of gestation, indicating the importance of embryonic development from fertilization to the blastocyst stage. To highlight this importance, multiple critical molecular and developmental events, including zygote reprogramming, maternal RNA decay, and embryonic genome activation, occur during bovine pre-implantation development. However, the molecular mechanisms of these events have yet to be defined. H3.3 is a histone H3 variant that encoded by 2 genes, namely, H3F3A and H3F3B. It is generally believed that H3.3 is closely related to active transcribed genes. Of interest, H3.3 required for establishing proper chromatin structure during mouse oogenesis. Immediately following fertilization, H3.3 is incorporated to parental chromatins and essential for blastocyst formation in mice. HIRA is a chaperone for H3.3 deposition and indispensable for zygote development. Previously, our results showed that H3.3 is needed for bovine early embryonic development. Herein, experiments were designed to determine the mechanisms of functional requirement of H3.3 in bovine early embryos. Slaughterhouse-derived cumulus–oocyte complexes (COC) were matured in vitro and IVF was performed. To knock down genes of interest, small interfering (si)RNAs were delivered into zygotes via microinjection. The qPCR results showed that H3F3A mRNA level is stable, whereas H3F3B and HIRA mRNA are dynamic during early embryonic development (4 replicates). The mRNA abundance of H3F3B is significantly higher than that of H3F3A (4 replicates; P < 0.05), which is also found in mouse and human. Immunostaining results revealed a stage-specific pattern for the localization of H3.3 in bovine early embryos, and the H3.3 signal was not different between paternal and maternal pronuclei in zygotes, which was different from the pattern in mice. The siRNA-mediated silencing of H3.3 dramatically reduces the expression of CTGF (a putative trophectoderm marker) in bovine blastocysts (3 replicates; P < 0.05). Furthermore, we found that the signal intensity of dimethylation of histone H3 lysine 36 (H3K36me2) and linker histone H1 decreases in H3.3-ablated embryos, which is similar to CHD1 knockdown (3 replicates; P < 0.05). However, no difference was found for the intensity of trimethylation of histone H3 lysine 4, dimethylation of histone H3 lysine 9 (H3K9me2) and splicing factor 3 B1 (SF3B1). We also found that HIRA deletion does not affect bovine early embryonic development. Taken together, the results described herein suggest that H3.3 is required for proper epigenetic modifications and H1 deposition during bovine early embryonic development.

This project was supported by National Natural Science Foundation of China grant (No. 31672416) and the Fundamental Research Funds for the Central Universities.