Highly purified spermatozoal RNA obtained by a novel method indicates an unusual 28S/18S rRNA ratio and suggests impaired ribosome assembly

Abstract

Human spermatozoal RNA features special characteristics such as a significantly reduced quantity within spermatozoa compared with somatic cells is described as being devoid of ribosomal RNAs and is difficult to isolate due to a massive excess of genomic DNA in the lysates. Using a novel two-round column-based protocol for human ejaculates delivering highly purified spermatozoal RNA, we uncovered a heterogeneous, but specific banding pattern in microelectrophoresis with 28S ribosomal RNA being indicative for the amount of round cell contamination. Ejaculates with different round cell quantities and density-purified spermatozoa revealed that 18S rRNA but not 28S rRNA is inherent to a pure spermatozoal fraction. Transmission electron microscopy showed monoribosomes and polyribosomes in spermatozoal cytoplasm, while immunohistochemical results suggest the presence of proteins from small and large ribosomal subunits in retained spermatozoal cytoplasm irrespective of 28S rRNA absence.

Human spermatozoal RNAs

“…It is now agreed that transcriptional and translational activities are present in the mitochondria of mature spermatozoa, but not in the cytoplasm (5,6). Mature spermatozoa had been thought to lack 28S and 18S rRNAs, the essential components of 80S cytoplasmic ribosomes to support translational machinery, however, 18S rRNA, but not the large subunit backbone 28S rRNA, was recently shown to be present in highly purified spermatozoal RNA after rigorous elimination of somatic cells (7). Transmission electron microscopy (TEM) showed a significant number of electron-dense ribosomal structures in the spermatozoal cytoplasm, but they were mostly irregularly dispersed and suggestive of monosomes (7).…”

Isolation of total ribonucleic acid from fresh and frozen-thawed boar semen and its relevance in transcriptome studies

“…In this study the amounts of total RNA isolated from boar spermatozoa differ from other animal species, mainly because of differences in the RNA extraction procedures, quantification methods, and other factors (Das et al, 2010;CappalloObermann et al, 2011;Parthipan et al, 2015). According to Cappallo-Obermann et al (2011), highly purified RNA comprises 18S rRNA, with the 28S/18S rRNA ratio approximately 0.1, suggesting the inherent characteristic of human spermatozoa. In the current study, the full-length 28S/18S rRNA ratio was 0, which has been suggested as a strong indicator of RNA integrity because of the possibility of rapid deterioration of rRNA (Georgiadis et al, 2015).…”

“…Several mRNA transcripts have been shown to be useful markers for sperm phenotypes, such as motility, viability, capacitation and chromatin condensation. There is also evidence to suggest that sperm-derived RNA contributes to fertilization and embryo development (Cappallo-Obermann et al, 2011;Card et al, 2013: Georgiadis et al, 2015.The main objective of this preliminary study was to isolate high-quality total RNA from raw fresh and frozen-thawed boar spermatozoa using a modified RNA extraction protocol.Ejaculates were collected from six Polish Large White (PLW) boars (n = 6) and were frozen, using a standard cryopreservation protocol (Fraser et al, 2008;. The frozen samples were stored in liquid nitrogen (-196 °C) for a week, prior to post-thaw semen analysis.…”

Sperm RNA as a Mediator of Genomic Plasticity

“…Using Bioanalyzer traces, our laboratory and others detected only smaller (shorter) RNAs in mature mammalian sperm [56,69,70] Nevertheless, a general rule of thumb for the isolation of pure populations of sperm RNA for downstream analyses (where even small amounts of contaminating somatic cell RNAs could be problematic) is that if the sperm RNA profile is essentially free of 28S and 18S rRNA, it is also free of nonsperm cell contaminants. The corollary of this assumption is that detection of the large rRNA subunits indicates a potential nonsperm cell contamination [71]. The question, therefore, of whether sperm has a translational capacity that cannot be ascribed to another contaminating cell type (including contaminating bacteria) remains open.…”