127 ABUNDANCE OF mRNA FOR HISTONE VARIANTS, HISTONE, AND DNA MODIFICATION ENZYMES IN BOVINE IN VIVO OOCYTES AND PRE-IMPLANTATION EMBRYOS

Abstract

During early embryogenesis, chromatin composition and structure undergo dramatic changes due to replacement of canonical histones by histone variants, post-translational modifications of histones, and changes in DNA methylation. These dynamics of chromatin play important roles in the regulation of gene expression and development of embryonic cells. Our goal here is to describe the above-mentioned changes using recently established transcriptome profiles of bovine in vivo-produced oocytes and pre-implantation embryos (Jiang et al. 2014 BMC Genomics, 15, 1). Ten multiparous Holstein cows were synchronized and superovulated. Artificial insemination was conducted at 12 and 24 h post-standing heat using semen from bulls of proven fertility. In vivo-matured oocytes and 2- to 16-cell stage embryos were collected at 30 h, and 2 to 4 days after oestrus by oviducal flushing. Early morulae, compact morulae, and blastocysts were collected by non-surgical uterine flushing on days 5, 6, and 7 after oestrus. Single-cell deep sequencing libraries were prepared from oocytes/embryos (2 samples/stage) using a SOLiD^TM Total RNA-seq Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions and sequenced on a 5500xl Genetic Analyzer. The reproducibility of the preparation and sequencing methods were indicated by high Pearson correlation efficiencies between the replicates. Sequencing reads were normalized to transcripts per million as final results after trimming and mapping of the reads. We found that 8, 8, 7, 13, 10, 2, and 2 out of the 14, 52, 22, 31, 23, 4, and 3 annotated histone variants, histone methyl-tranferases, histone demethylases, histone acetyl-tranferases, histone deacetylases, DNA methyl-transferases, and DNA demethylases, respectively, were highly abundant (mean transformed transcripts per kilobase million > 50) in at least one of the pre-implantation development stages studied. Among histone variants with high mRNA abundance, H1FOO, H3F3A, and H3F3B were highly stored in oocytes, whereas other variants such as H2AFJ, H2AFV, H2AFX, H2AFY, H2AFZ, and CENPA were largely transcribed after the embryonic genome activation. H3F3A and H3F3B, however, were maintained at relatively high levels throughout pre-implantation development. Additionally, the mRNA for histone acetyl-transferases, TADA2A and TADA1; histone deacetylase, HDAC1 and HDAC3; histone methyl-transferases, EED and PRMT5; histone demethylase, KDM1A, were more abundant than others. It was also found that oocytes stored a large amount of DNA methyl-transferase, DNMT1, which degraded gradually after fertilization. Overall, in vivo-produced oocytes and early embryos contained more mRNA for histone-modifying enzymes than those for DNA modification. Taken together, our results suggest that although there are widely recognised and dramatic changes in embryonic DNA methylation through both active and passive mechanisms, the pre-implantation embryos may be more engaged in modifying histones than DNA.