Deep Sequencing of Spermatozoal Accessible Chromatin Reveals Complex Arrangements Underlying Spermatozoal Chromatin Packaging and Fertilization

Could structural aspects of sperm DNA packaging contribute to fertilization and early embryo development? Structural aspects of sperm DNA include complex patterns of repetitive-DNA and developmental gene packaging that could delineate a paternal impact on fertilization and early development. Genetics, epigenetics and transcriptomics of ejaculate human spermatozoa could have important effects on the establishment of embryonic chromatin architecture that may impact early fetal development. Spermatozoa are transcriptionally silent; however, a small fraction (∼5%-15%) of their chromatin escapes repackaging by protamines and remains in a more open nucleosomal conformation that may be poised for transcription. The sensitivity of this fraction to progressive salt/detergent disruption is likely assayed in sperm chromatin dispersion (SCD) or halo tests that are commonly used in assessments of sperm DNA fragmentation. Eleven healthy male donors and 9 women undergoing IVF treatment agreed to participate in this study. Eleven normozoospermic semen samples were donated by male donors, aged 25-39 years, who abstained from sexual activity for 3 days. Seven MII oocytes and 5 pronuclear zygotes were donated from 5 and 4 women, respectively, undergoing IVF treatment. The samples were collected within a time frame of 6 months. Female donors provided either MII oocytes or pronuclear zygotes. Sperm chromatin was processed by differential salt extraction tailored with endonuclease or micrococcal nuclease treatment and RNA isolated using established protocols. Oocytes and pronuclear zygotes were analyzed at the single cell level. DNA library construction employed the NEBNext Ultra approach (NEB) and RNA libraries the Ovation Single Cell (NuGEN) for Illumina. Bioinformatics analysis included FastQC, HISAT2, StringTie, GREAT, edgeR and other source-software. Public datasets were employed to further increase the statistical power. The aim of our approach was to dissect the accessible sperm chromatin fraction, identifying regions of great importance that could contribute towards the formation of the zygote and/or early embryonic development. Differing extraction methods revealed complementary accessible and resistant fractions in sperm, with accessible regions being significantly enriched for chromatin important in very early embryonic development. These chromatin fractions contained various genomic features including promoter sequences, CpG-islands and CTCF-binding sites that could attract transcription binding factors in the paternally-derived chromatin. Accessible sperm chromatin also contains high levels of specific DNA repeat classes of retroviral origin, known to impact early gene expression and gene expression programs. Accessible sperm chromatin was strongly associated with developmental and regulatory signatures with Bonferroni P values ranging between 2.07x10-4 and 1.03x10-20, potentially revealing a role for paternal chromatin in the initial start-up of early zygotic development. We further investigated paternally- and maternally- derived transcripts and identified mono-allelically transcribed genes present in the zygotes, that were entirely absent from all seven oocyte transcriptomes, suggesting a paternal origin, and potentially important roles following oocyte fertilization. Due to the restraints of obtaining human material, the study relied on a relatively small sample size. However, sequencing results showed quite homogeneous clustering in both DNA/RNA samples, with at least 5 biological replicates from at least 4 donors per group, potentially reducing the impact of low sample size. A better understanding of differentially accessible sperm chromatin and residual RNA, could help support the discovery of biomarkers that can be used to select more competent spermatozoa for IVF cycles, resulting in higher success rates in couples undergoing ART. not applicable