The presence, role and clinical use of spermatozoal RNAs

Abstract

BACKGROUND Spermatozoa are highly differentiated, transcriptionally inert cells characterized by a compact nucleus with minimal cytoplasm. Nevertheless they contain a suite of unique RNAs that are delivered to oocyte upon fertilization. They are likely integrated as part of many different processes including genome recognition, consolidation-confrontation, early embryonic development and epigenetic transgenerational inherence. Spermatozoal RNAs also provide a window into the developmental history of each sperm thereby providing biomarkers of fertility and pregnancy outcome which are being intensely studied. METHODS Literature searches were performed to review the majority of spermatozoal RNA studies that described potential functions and clinical applications with emphasis on Next-Generation Sequencing. Human, mouse, bovine and stallion were compared as their distribution and composition of spermatozoal RNAs, using these techniques, have been described. RESULTS Comparisons highlighted the complexity of the population of spermatozoal RNAs that comprises rRNA, mRNA and both large and small non-coding RNAs. RNA-seq analysis has revealed that only a fraction of the larger RNAs retain their structure. While rRNAs are the most abundant and are highly fragmented, ensuring a translationally quiescent state, other RNAs including some mRNAs retain their functional potential, thereby increasing the opportunity for regulatory interactions. Abundant small non-coding RNAs retained in spermatozoa include miRNAs and piRNAs. Some, like miR-34c are essential to the early embryo development required for the first cellular division. Others like the piRNAs are likely part of the genomic dance of confrontation and consolidation. Other non-coding spermatozoal RNAs include transposable elements, annotated lnc-RNAs, intronic retained elements, exonic elements, chromatin-associated RNAs, small-nuclear ILF3/NF30 associated RNAs, quiescent RNAs, mse-tRNAs and YRNAs. Some non-coding RNAs are known to act as epigenetic modifiers, inducing histone modifications and DNA methylation, perhaps playing a role in transgenerational epigenetic inherence. Transcript profiling holds considerable potential for the discovery of fertility biomarkers for both agriculture and human medicine. Comparing the differential RNA profiles of infertile and fertile individuals as well as assessing species similarities, should resolve the regulatory pathways contributing to male factor infertility. CONCLUSIONS Dad delivers a complex population of RNAs to the oocyte at fertilization that likely influences fertilization, embryo development, the phenotype of the offspring and possibly future generations. Development is continuing on the use of spermatozoal RNA profiles as phenotypic markers of male factor status for use as clinical diagnostics of the father's contribution to the birth of a healthy child.

Keywords: embryogenesis; epigenetics modifiers; fertility biomarkers; spermatozoal RNA; transgenerational epigenetic inherence.

Figure 1

Composition of spermatozoal RNAs. The distribution of the various classes of RNAs as determined by RNA-seq is shown. The most abundant class is ribosomal RNAs followed by mitochondrial RNA (mitoRNAs), annotated coding transcripts, small non-coding RNAs (snc-RNAs), intronic retained elements, lnc-RNAs and Transcribed regions of Unknown Coding Potential (TUCP), short expressed regions, transposable elements and annotated non-coding RNAs, including snars, sno, pri-mir and RNU.

Figure 2

The degree of transcript fragmentation can influence the sequencing profile after Poly(A⁺) selection. (A) Intact RNA shows minimal 3′ bias, even with poly(A⁺) selection. However, transcripts which are biologically fragmented (i.e. prior to the typical fragmentation step of most RNA-seq protocols) show significant 3′ end profile bias as selection preferentially retains the 3′ poly(A⁺) containing ends. (B) For example, SPACA4, exhibits fairly even coverage across transcript length in testes, and marked 3′ bias in sperm.

Figure 3

Alternative polyadenlation of sperm transcripts. GIGYF2 encodes a protein that interacts with GRB10 and may be involved in the regulation of tyrosine kinase receptor signaling. The 3′UTR region of GIGYF2 gene is highlighted (upper panel). RNA-seq (lower panel) of this specific region exhibits a truncated 3′UTR in sperm (green). This contrasts with coverage extending over most of the UTR observed in testes (black). (See Supplementary data, Fig. S1 for more details.)

Figure 4

Alignment of short RNAs (18–24 nt) from human sperm sample AS062 to the LINE1 repeat. Some LINE1 elements in the genome act as active transposable elements encoding both an ORF2 endonuclease and reverse transcriptase as well as the RNA-binding protein p40 encoded by ORF1. Specific LINE1 fragments are abundant in the small RNA fraction (blue peaks). Some of these fragments are purine rich sequences (red boxes), which may, through the formation of triplex structures, promote expression of complete LINE1 elements in the fertilized oocyte.

Figure 5

Sperm intronic retained elements. The structure of DNAH1 is shown in the upper panel. The sequence reads obtained from sperm (green) and testes (black) RNA-seq within the highlighted region are shown (lower panel). The levels of specific intronic sperm RNAs are enhanced, while the coding regions of this transcript are absent in sperm. In equivalently sequenced testes samples, these intronic regions are underrepresented and resemble levels observed across the complete transcript (note y-axis). (See Supplementary data, Fig. S2 for more details.)

Figure 6

Unique sperm lnc-RNAs isoforms. A 30 kb region of chromosome 3 containing a series of putative lnc-RNAs as identified by the Human Body Map lincRNA UCSC track (Trapnell et al., 2010; Cabili et al., 2011) is shown in upper panel. Although low- level expression of a number of identified lnc-RNAs is evident across this region in testes, a single highly expressed two exon RNA is observed in sperm (lower panel). Many junction reads, as measured by RUM (Grant et al., 2011) (box), confirm that these two exons are part of a single spliced transcript, which was not previously identified as a unique lnc-RNA isoform. (See Supplementary data, Fig. S3 for more details.)

Figure 7

Exonic sperm element. An overview of the structure of ARFGEF1 is provided in the upper panel. In testes, significant coverage of the complete transcript is observed (Supplementary data, Fig. S4). In contrast, sperm show virtually no transcript within the length of the coding region. A ∼100 nt sperm-specific element is observed however within the 5′UTR (highlighted region), but is virtually absent in equivalently prepared testes samples. This unique sperm element may serve to accelerate degradation of its containing transcript. (See Supplementary data, Fig. S4 for more details.)

Figure 8

Enrichment of mse-tRNA fragments in human sperm samples. Short read alignment to specific tRNAs is highlighted in the dark green box. Significant enrichment of fragments corresponding to particular regions of each tRNA is observed. The corresponding region within each folded structure is marked in green. The upper panel highlights the enrichment of the 3′ fragment of tRNA58Leu. Lower panel, enrichment of the 5′ fragment of tRNA122-Ala.

Figure 9

The potential actions of spermatozoal RNAs during early embryo development. Spermatozoal RNAs are delivered to the oocyte acting during the first steps of embryogenesis. Some intact paternal mRNAs like INST1 could be translated by maternal machinery. On one hand, paternal mature miRNAs like mouse miR-34c are essential for the first cell division. On the other hand, primicroRNAs like 181c, can be processed and thus activated by maternal DICER to their mature miRNAs regulating transcript stability, whereas others may target promoters. Interestingly, some non-coding RNAs act through triplex structures and perhaps are transcriptional regulators. For example, homopurine fragments of LINE1 provided by spermatozoa induce LINE1 transcription during the first divisions of the zygote. It has also been proposed that piRNAs, miRNAs and other potential RNAs may be the pathway to confrontation and consolidation.