Estimated Diversity of Messenger RNAs in Each Murine Spermatozoa and Their Potential Function During Early Zygotic Development1

Abstract

To study the diversity of mRNAs in murine spermatozoa and their potential function during zygotic development, total RNAs in murine spermatozoa were sequenced via RNA-Seq and analyzed through bioinformatics techniques. The delivery and translation of sperm-borne mRNA in fertilized oocyte were detected using RT-PCR (reverse transcription-polymerase chain reaction), Western blot, and immunofluorescence. A total of 35 288 825 reads matching 33 039 transcripts, including 27 310 coding transcripts, were obtained. Based on our analyses, we hypothesized that the transcripts with RPKM (reads per kilobase of exon model per million mapped reads) higher than six may exist in each sperm cell as consistently retained transcripts. There were 4885 consistent transcripts in each sperm, and the remainder were randomly retained. If the baseline RPKM increased, the remaining coding transcripts were more likely related to reproduction and development. The sperm-borne transcripts Wnt4 and Foxg1 were delivered into fertilized oocytes on fertilization. Furthermore, Wnt4 was translated into protein in zygotes, whereas Foxg1 was not translated. In conclusion, approximately 4885 mRNAs were present in each murine spermatozoon, and the spermatozoal mRNAs related to reproduction and development were more likely retained. The sperm-borne mRNA Wnt4 was delivered into the fertilized oocyte and translated, evidence of a paternal effect on zygotic development.

Sperm RNA as a Mediator of Genomic Plasticity

“…Distinct ontological descriptions persist, however, among the most abundant transcripts. A similar finding has been reported on murine sperm that also included rare evidence for the postfertilisation translation of some sperm RNAs in mouse zygotes [32]. Human sperm appear to have many RNAs with alternative polyadenylation (APA) sites compared with corresponding testis RNAs and, 3 truncation may be a normal consequence of APA and hence under active cellular control (rather than a passive consequence of RNA fragmentation).…”

“…The advantage of SAGE (like all DNA sequencing based strategies) is that it is not limited by the presence of specific probes on array platforms and so should provide more accurate descriptions of the relative abundance of mRNAs. The wider adoption of massively parallel RNA-seq in conjunction with better annotated genomes from domestic species has led to recent reports on the complexity of bovine [74], porcine [79], equine [80], and murine [2,32] sperm RNA. At the time of writing, the most comprehensive description of sperm RNA is from the human.…”

“…Upregulation of endogenous endonuclease activity in sperm has been reported elsewhere [30,31]; hence, an active RNAse activity during sperm maturation is likely. Nevertheless, the presence of small noncoding RNAs in the sperm of many species targeting a variety of gene sequences unrelated to spermatogenesis suggests that sperm RNA is more than just a residual relic of spermatogenesis [2,16,18,29,[32][33][34][35][36][37][38][39][40][41][42][43][44][45][46]. Several reports, the most recent of which focused on sperm capacitation, have provided evidence that sperm RNA can be translated de novo [47,48].…”

Comparative profiling of small RNAs of pig seminal plasma and ejaculated and epididymal sperm

“…The presence of coding RNAs in sperms of humans (Lambard et al 2004), pigs (Yang et al 2009), bovines (Gilbert et al 2007, Lalancette et al 2008, mice (Fang et al 2014) and stallions (Das et al 2013) is well established. Even though the functions of the majority of the sperm mRNAs remain enigmatic, it has been proposed that sperm mRNAs may play roles in the regulation of sperm morphology, motility and capacitation and that sperm transcriptional profiles might provide clinical markers for male fertility (Lambard et al 2004, Li et al 2007, Bansal et al 2015.…”

“…Similarly, sperm transcriptome studies have been initiated in bulls and have revealed the differences between the mRNA profiles of high-and low-fertility males (Lalancette et al 2008, Bissonnette et al 2009. Recently, the spermborne mRNA Wnt4 was found to be delivered into the fertilized oocyte and translated, which represents evidence of a paternal effect on zygotic development (Fang et al 2014); however, the functional significance of sperm mRNAs in the development of zygotes remains largely unknown.…”