Previous research has shown that embryos conceived by IVF show a sexual dimorphic phenotype and different metabolite expression into adulthood. Interestingly, female embryos have a higher apoptotic rate and are more vulnerable to environmental stressors. Our prior studies have examined DNA methylation changes in male and female embryos; however, limited knowledge is available with regard to chromatin accessibility in male and female embryos. The objective of this study was to assess if genome-wide chromatin accessibility changes occur in the inner cell mass (ICM) of the male v. female mouse embryo. For this experiment we utilised expanded blastocysts (CF-1 females×B6D2F1 males) that were obtained by IVF or flushed out of the uterus (FB, control). We performed IVF using KSOM with amino acids and 5% O2. The ICM were isolated from trophectoderm cells utilising immunosurgery and complement. The chromatin status was assessed using an assay for transposase-accessible chromatin status using a sequencing (ATAC seq) protocol modified from Wu et al. (2016 Nature 524, 652-657, DOI: 10.1038/nature18606). Briefly, cells were lysed and nuclei incubated with the Tn5 transposome and tagmentation buffer (Nextera, Illumina Inc., San Diego, CA, USA). After tagmentation, PCR amplification (15 cycles), mitochondrial depletion, and library purification were performed. The sex of the embryo was determined based on MSY (male sex region on the Y chromosome) and total Y chromosome aligning reads. Samples were then divided into 2 groups, male (n=20) and female (n=25), and sequencing was completed using Illumina HiSEqn 2500 (Illumina Inc.). Sequencing was completed using Illumina HiSEqn 2500. MACS2 (https://github.com/taoliu/MACS/) was used for peak identification from the sequencing results and DESEqn 2 (https://www.r-project.org/) was used for statistical analysis and false discovery rate<0.1 was considered significant. GREAT analysis (http://great.stanford.edu/public/html/) was used to identify the top biological processes. Overall, 17,136 regions were identified as more open in males and 19,460 regions were identified as more open in females. Interestingly, of these identified regions, only 28 were considered significant (25 increased and 3 decreased in males). The majority of these regions were located on chromosome Y and most of the regions that were increased in male correlated with gene Gm10352, a gene with an unknown function. While some pathway changes were expected, like the ones involved in sex determination, male gonadal development, and male sexual characteristics, others included histone demethylation, regulation of chromatin silencing, negative regulation of DNA replication, and antigen processing and presentation of peptide antigen. Our data are the first to analyse genome-wide chromatin accessibility changes in the ICM of male and female mouse embryos. Importantly, these data indicate that chromatin differences are already present in the ICM of male and female embryos. Further analysis will provide insights into epigenetic and sexual dimorphic changes present in embryos.
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Feb 28, 2024
Michael Wleklinski + 6
Open Access
