Systematic identification and characterization of long non-coding RNAs in mouse mature sperm

Abstract

Increasing studies have shown that mature spermatozoa contain many transcripts including mRNAs and miRNAs. However, the expression profile of long non-coding RNAs (lncRNAs) in mammalian sperm has not been systematically investigated. Here, we used highly purified RNA to investigate lncRNA expression profiles in mouse mature sperm by stranded-specific RNA-seq. We identified 20,907 known and 4,088 novel lncRNAs transcripts, and the existence of intact lncRNAs was confirmed by RT-PCR and fluorescence in situ hybridization on two representative lncRNAs. Compared to round spermatids, 1,794 upregulated and 165 downregulated lncRNAs and 4,435 upregulated and 3,920 downregulated mRNAs were identified in sperm. Based on the “Cis and Trans” RNA-RNA interaction principle, we found 14,259 targeted coding genes of differently expressed lncRNAs. In terms of Gene ontology (GO) analysis, differentially expressed lncRNAs targeted genes mainly related to nucleic acid metabolic, protein modification, chromatin and histone modification, heterocycle compound metabolic, sperm function, spermatogenesis and other processes. In contrast, differentially expressed transcripts of mRNAs were highly enriched for protein metabolic process and RNA metabolic, spermatogenesis, sperm motility, cell cycle, chromatin organization, heterocycle and aromatic compound metabolic processes. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the differentially expressed lncRNAs were involved in RNA transport, mRNA surveillance pathway, PI3K-Akt signaling pathway, AMPK signaling pathway, protein processing in endoplasmic reticulum. Metabolic pathways, mRNA surveillance pathway, AMPK signaling pathway, cell cycle, RNA transport splicesome and endocytosis incorporated with the differentially expressed mRNA. Furthermore, many lncRNAs were specifically expressed in testis/sperm, and 880 lncRNAs were conserved between human and mouse. In summary, this study provides a preliminary database valuable for identifying lncRNAs critical in the late stage of spermatogenesis or important for sperm function regulation, fertilization and early embryo development.

Intergenerational Effects of Alcohol: A Review of Paternal Preconception Ethanol Exposure Studies and Epigenetic Mechanisms in the Male Germline

“…In addition to chromatin, sperm contain RNA, though only ~1% of that carried by somatic cells (Zhang et al., ). Due to the largely arrested transcriptional state of fully mature sperm, it was long assumed that this limited amount of RNA in sperm was purely comprised of degraded transcripts from the earlier transcriptionally active stages of spermatogenesis in the testis.…”

Sperm and seminal plasma RNAs: what roles do they play beyond fertilization?

“…Sperm RNA isolation is a challenging process that requires careful consideration of the following points: (i) one spermatozoon contains an exceptionally low quantity of RNA, which ranges from 10-50 fg in human sperm (Goodrich et al 2013, Jodar et al 2013, approximately 100 fg in mice and rat sperm (Zhang et al 2017), 20 fg in stallion sperm (Das et al 2010), 20-30 fg in bull sperm (Selvaraju et al 2017) and 1-8 fg in boar sperm (Gòdia et al 2018b, Kasimanickam et al 2019; (ii) the ejaculate frequently contains non-sperm contaminant cells with ~100 times more RNA than sperm cells, which could mask the acquisition of accurate sperm RNA profiles by deep sequencing technologies and (iii) complete lysis of sperm is necessary to fully release the high proportion of RNAs embedded in the sperm nuclei (Johnson et al 2015). However, the elevated level of disulfide bridges between protamines, which are the main DNA packaging proteins of the sperm nucleus, hinders the complete release of nucleic acids from sperm (Oliva 2006, Goodrich et al 2013, Jodar & Oliva 2014.…”

Environmental toxicant induced epigenetic transgenerational inheritance of ovarian pathology and granulosa cell epigenome and transcriptome alterations: ancestral origins of polycystic ovarian syndrome and primary ovarian insufficiency

“…Long noncoding RNAs are hypothesized to maintain epigenetic memory by posttranscriptional regulation and to assist in regulating DNA methylation, chromatin remodeling, and histone modifications [19]. Small noncoding RNAs are known to regulate gene expression by affecting transcript stability and have been shown to be abundant in sperm with a single spermatozoon containing above 20,000 long and short noncoding RNAs [21,22]. Recently, concurrent alterations of DNA methylation, ncRNA, and histone alterations have been identified in sperm mediating the epigenetic transgenerational inheritance of pathology [23,24].…”