Determination of Oocyte-Manipulation, Zygote-Manipulation, and Genome-Reprogramming Effects on the Transcriptomes of Bovine Blastocysts

Abstract

Somatic cell nuclear transfer (scNT) embryos suffer from damage caused by micro-operation (manipulation) and inefficient genome reprograming that hinder their normal development at different levels and in distinct ways. These two effects are inseparable in the nature of the scNT embryo, although methods to separately measure them are needed to improve scNT technology and evaluate incoming reprogramming tools. As an attempt to meet these demands, we made bovine sham nuclear-transfer (shNT) blastocysts, special embryos made with a standard nuclear-transfer procedure at the zygote stage, while retaining an intact genome. We compared their transcriptomes with those of other blastocysts derived by in-vitro fertilization (IVF) or scNT. Correlation analysis revealed a singularity of shNT blastocysts as they separately gathered from the others. Analysis of developmentally important genes revealed that, in shNTs, the stemness-associated differentially expressed genes (DEGs), including OCT4, were mostly underrepresented. Overrepresented epi-driver genes were largely associated with heterochromatin establishment and maintenance. By multilateral comparisons of their transcriptomes, we classified DEGs into three groups: 561 manipulation-associated DEGs (MADs) common to shNTs and scNTs, 764 donor genome-associated DEGs (DADs) specific to scNTs, and 1743 zygote manipulation-associated DEGs (zMADs) specific to shNTs. GO enrichment analysis generated various terms involving “cell-cell adhesion,” “translation,” and “transcription” for MADs and “cell differentiation” and “embryo implantation” for DADs. Because of the transcriptomic specificity of shNTs, we studied zMADs in detail. GO enrichment analysis with the 854 zMADs underrepresented in shNTs yielded terms related to protein and mitochondria homeostasis, while GO enrichment analysis of 889 shNT-high zMADs yielded terms related to endoplasmic reticulum stress and protein transport. We summarized the DEGs, which, with further investigation, may help improve our understanding of molecular events occurring in cloned embryos and our ability to control clonal reprogramming.

Demethylation and derepression of genomic retroelements in the skeletal muscles of aged mice

“…Genomic DNAs (gDNAs) were extracted from the tissue samples using DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's instruction. Genomic DNAs obtained from three mice of the same ages were pooled before sonication using Bioruptor Pico (Diagenode) and purified using with AMPure XP beads (Beckman) (Min, Park, & Kang, ). For preparation of MBD capture, 7 μl MBD‐Biotin proteins per sample from MethylMiner Methylated DNA Enrichment Kit (Thermo) was first incubated with 10 μl of precleared Dynabeads (Thermo) at RT for 1 hr.…”

Sperm‐borne proteins improve rabbit cloning efficiency via regulating embryonic cleavage and epigenetics

“…Bioinformatics analysis of the sperm proteome suggested that abnormalities in cytoskeleton and nuclear reprogramming might be related to the deletion of sperm proteins. Cloned embryos had higher reactive oxygen species (ROS) levels, higher endoplasmic reticulum stress, and embryo activation block [23–25]. Here, we also found many sperm proteins enriched in ROS‐related processes, enzyme activity regulation, unfolded protein binding, and ion binding, which suggests that the higher stress state of cloned embryos might be related to the absence of sperm proteins.…”

Retroelement Insertion in a CRISPR/Cas9 Editing Site in the Early Embryo Intensifies Genetic Mosaicism

“…The genetic nature of a mutation, such as whether it is in or out of frame or is a missense or a non-sense mutation, is not considered by the equation, although it is unambiguously useful to include such genetic aspects of an indel in the calculation to permit assessment of the severity of the mutation in relation to molecular and biochemical function. However, PCR and sequencing errors are not rare, and the error rate may increase greatly when samples containing limited amounts of genomic material, such as early embryos, are used (Min et al, 2017, 2018a). We indeed observed single base-pair insertions, deletions, and substitutions that randomly appeared among the amplicon reads, and we excluded these uncertain single-nucleotide variations (SNVs) from the ID calculation.…”

“…Successful mating was determined the following morning by detection of a vaginal plug. Mouse zygotes were transferred to M2 medium (Sigma) containing 0.1% (w/v) hyaluronidase to remove cumulus cells and cultured in M16 medium (Sigma) at 37°C, 5% CO 2 in air (Min et al, 2018a).…”