Genome-Wide Distribution of Nascent Transcripts in Sperm DNA, Products of a Late Wave of General Transcription

Abstract

Mature spermatozoa contain a whole repertoire of the various classes of cellular RNAs, both coding and non-coding. It was hypothesized that after fertilization they might impact development, a claim supported by experimental evidence in various systems. Despite the current increasing interest in the transgenerational maintenance of epigenetic traits and their possible determination by RNAs, little remains known about conservation in sperm and across generations and the specificities and mechanisms involved in transgenerational maintenance. We identified two distinct fractions of RNAs in mature mouse sperm, one readily extracted in the aqueous phase of the classical TRIzol procedure and a distinct fraction hybridized with homologous DNA in DNA-RNA complexes recovered from the interface, purified after DNase hydrolysis and analyzed by RNA-seq methodology. This DNA-associated RNA (D RNA) was found to represent as much as half of the cell contents in differentiated sperm, in which a major part of the cytoplasmic material has been discarded. Stable complexes were purified free of proteins and identified as hybrids (R-loops) on the basis of their sensitivity to RNase H hydrolysis. Further analysis by RNA-seq identified transcripts from all the coding and non-coding regions of the genome, thus revealing an extensive wave of transcription, prior to or concomitant with the terminal compaction of the chromatin.

DNA-RNA Hybrid (R-Loop): From a Unified Picture of the Mammalian Telomere to the Genome-Wide Profile

“…In the testes, several studies have already reported genome-wide transcripts, and it is also assumed that from the last stage of elongated spermatids, with the elimination of the majority of cytoplasmic RNAs, a number of transcripts remain in the sperm [ 38 ]. We have recently reported profiles of DRNA-seq mouse sperm transcripts from all coding and non-coding regions of the genome [ 12 ]. Unlike in the analysis of previous individual transcripts [ 12 ], here, we performed peak finding in order to associate the location of the peaks and to compare maps in terms of their genomic characteristics.…”

“…We have recently reported profiles of DRNA-seq mouse sperm transcripts from all coding and non-coding regions of the genome [ 12 ]. Unlike in the analysis of previous individual transcripts [ 12 ], here, we performed peak finding in order to associate the location of the peaks and to compare maps in terms of their genomic characteristics.…”

“…Mouse spermatozoa were collected from the cauda epididymis and ejaculated human sperm kindly provided by the Fertility Clinics of the University. In both cases, the standard protocol was already reported previously by Kianmehr et al [ 12 ], briefly: motile spermatozoa were recovered and washed MEM buffer (1 mM Na pyruvate, 0.5 mM EDTA, 50 U/mL penicillin, 50 mg/mL streptomycin, and 0.1% BSA) by centrifugation at 3000 rpm. Sperm pellets were suspended in phosphate-buffered saline (PBS) and centrifuged again.…”

DNA-RNA hybrid (R-loop): from a unified picture of the mammalian telomere to the genome-wide

“…In both cases, standard protocol is already reported previously and Kianmehr & al. 24 summarized: motile spermatozoa were recovered and washed MEM buffer (1 mM Na pyruvate, 0.5 mM EDTA, 50 U/ml penicillin, 50 mg/ml streptomycin, and 0.1% BSA) by centrifugation at 3000 rpm. Sperm pellets were resuspended in phosphate-buffered saline (PBS) and centrifuged again.…”

“…In the testes, several studies are already reported genome-wide transcripts and it is also assumed that from last stage of elongated spermatids, with elimination of the majority of cytoplasmic RNAs, genome-wide transcripts remain in the sperm. We have recently reported profiles of DRNA-seq mouse sperm transcripts from all coding and non-coding regions of the genome 24 . Contrary to the analysis of the previous individual transcripts, we have here carried out a peak annotation in order to associate the location of peaks/region.…”