Nucleosome preservation in mammalian sperm: an epigenetic program ensuring the healthy male reproduction

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Based on current scientific knowledge, the sperm epigenome, especially the nucleosomes that were excluded from protamine-exchange, may potentially direct certain processes in early development. Our previous work showed that sperm-specific nucleosomes are largely retained in distal intergenic regions and associated with centromere repeats and retrotransposons, particularly those known as long, interspersed nuclear elements-1 (LINE1s). Remarkably, promoters and introns of genes relevant for pre-implantation development (e.g., factors for RNA-processing, signal transduction, and mitochondrial function) are also nucleosome-enriched. We reasoned that aberrations with regard to nucleosome-preservation pattern and nucleosome-associated genomic loci in sperm might cause severe errors in post-fertilization processes, and hence, may lead to idiopathic male infertility. The proposed project will address some crucial, open questions regarding the function of sperm nucleosomes, mechanisms that promote their preservation, and the potential risk of nucleosome aberrations for male fertility. Accordingly, our aims are: 1) To analyze the epigenetic status (CpG-methylation, histone modifications) of previously identified nucleosome-enriched gene promoters in a considerable number of sperm samples from fertile men. This point is essential to determine whether epigenetic variability in nucleosome-preserved promoters exists in the fertile population, and hence, can be tolerated; 2) To analyze the mRNA expression of human and bovine orthologous genes with nucleosome-enriched promoters, in early bovine embryos. This point is essential in selecting evolutionarily conserved genes, which are paternally-premarked and are relevant for mammalian pre-implantation development; 3) To analyze the promoters of the candidate genes selected in Aim 2 to determine their epigenetic status in sperm from sub-fertile patients; 4) To identify genome-wide aberrations related to nucleosome-preservation patterns in sperm. The mono-nucleosomal DNA from sub-fertile patients will be deep sequenced and compared to our previous data generated in fertile men. Moreover, the sperm methylome will be compared between sperm samples from sub-fertile and fertile men; 5) To elucidate the mechanisms that allow LINE1s to retain nucleosomes. We will analyze LINE1 mRNA expression and LINE1 CpG-methylation in different stages of spermatogenesis. LINE1 methylation will be compared to CpG-methylation in other repetitive DNA elements; 6) Our preliminary study showed that nucleosome-to-protamine exchange in spermiogenesis was accompanied by DNA hydroxymethylation. To elucidate the link between DNA demethylation and nucleosome preservation, we will analyze the hydroxymethylomes in spermatocytes and spermatids.