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The Partitioning of RNAs in sperm

ABSTRACT: A diverse pool of RNAs remain encapsulated within the transcriptionally and translationally silent spermatozoon. These transcripts persist within the male gamete despite the dramatic reduction in cellular volume achieved through expulsion of the cytoplasm and quite possibly the nucleoplasm. The precise location of RNAs retained within the sperm cell remains largely unknown. However, early evidence suggested that many are embedded within the nucleus (1). To discern the global pattern of transcript compartmentalization in sperm, total RNA was extracted from whole mouse spermatozoa and detergent demembranated nuclei fractionated through a sucrose gradient. Isolated RNAs were subjected to RNA-sequencing (RNA-seq) and their abundance used to infer localization. Transcripts enriched in the unfractionated cells were related to the production and function of mitochondria and surprisingly, exosomes. The absence of these extracellular vesicles associated RNAs within the inner-nuclear compartment was suggestive of an origin other than sperm. This contributes to the growing evidence for sperm-bound exosomes rich in RNA. In comparison, the majority of the remaining sperm RNAs were associated with the nucleus. This included the abundant fragmented ribosomal transcripts which likely persist between the nuclear envelope and the perinuclear theca. The spermatozoal inner-nuclear compartment was also enriched in repetitive transcribed sequences. This included LINE elements and simple repeat sequences both of which have been shown to contribute to chromatin structure in other cell types suggesting that they may serve parallel roles in the spermatozoon.

ORGANISM(S): Mus musculus

PROVIDER: GSE62874 | GEO | 2015/07/08

SECONDARY ACCESSION(S): PRJNA265872

REPOSITORIES: GEO

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Project description:It is now well established that mature mammalian spermatozoa carry a population of mRNA molecules, at least some of which are transferred to the oocyte at fertilisation. However, the function of the sperm transcriptome remains largely unclear. To shed light on the evolutionary conservation of this feature of sperm biology, we analysed highly purified populations of mature sperm from the fruitfly, Drosophila melanogaster. As with mammalian sperm, we found a consistently enriched population of mRNA molecules that are not likely to be derived from contaminating somatic cells or immature sperm. Using tagged transcripts for three of the spermatozoal mRNAs, we demonstrate that they are transferred to the oocyte at fertilisation and can be detected at least until the onset of zygotic gene expression. We find a remarkable conservation in the functional annotations associated with fly and human spermatozoal mRNAs, in particular a highly significant enrichment for transcripts encoding Ribosomal Proteins. The identification of a conserved set of spermatozoal transcripts opens the possibility of using the power of Drosophila genetics to address the function of this enigmatic class of molecules. RNA extracted from three biological replicates of purified sperm (Sperm rep1, Sperm rep2 and Sperm rep3) was used as a template for oligo-dT-primed reverse transcription, amplification, labelling of dye swapped technical replicates and hybridisation to long oligonucleotides microarrays. As a control, RNA from two biological replicates of dissected adult testis plus accessory glands (Testis_rep1, Testis_rep2) was amplified, labelled (dye-swap technical replicate) and hybridised to similar arrays. To help with the spot-finding of the arrays genomic DNA was co-hybridised in some cases (this genomic DNA data was excluded from further analysis). Genes with an intensity level below 200 in at least one channel across the Sperm or Testis set were removed (5579 transcripts present in all three sperm replicates, 5358 transcripts from the testis/accessory gland samples and 4295 transcripts common to both data sets). Then the quantile normalisation was independently applied to the Sperm replicate samples and Testis replicates.

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The Partitioning of RNAs in sperm

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