Next Generation Sequencing Analysis Reveals Segmental Patterns of microRNA Expression in Mouse Epididymal Epithelial Cells

Abstract

The functional maturation of mammalian spermatozoa is accomplished as the cells descend through the highly specialized microenvironment of the epididymis. This dynamic environment is, in turn, created by the combined secretory and absorptive activity of the surrounding epithelium and displays an extraordinary level of regionalization. Although the regulatory network responsible for spatial coordination of epididymal function remains unclear, recent evidence has highlighted a novel role for the RNA interference pathway. Indeed, as noncanonical regulators of gene expression, small noncoding RNAs have emerged as key elements of the circuitry involved in regulating epididymal function and hence sperm maturation. Herein we have employed next generation sequencing technology to profile the genome-wide miRNA signatures of mouse epididymal cells and characterize segmental patterns of expression. An impressive profile of some 370 miRNAs were detected in the mouse epididymis, with a subset of these specifically identified within the epithelial cells that line the tubule (218). A majority of the latter miRNAs (75%) were detected at equivalent levels along the entire length of the mouse epididymis. We did however identify a small cohort of miRNAs that displayed highly regionalized patterns of expression, including miR-204-5p and miR-196b-5p, which were down- and up-regulated by approximately 39- and 45-fold between the caput/caudal regions, respectively. In addition we identified 79 miRNAs (representing ~ 21% of all miRNAs) as displaying conserved expression within all regions of the mouse, rat and human epididymal tissue. These included 8/14 members of let-7 family of miRNAs that have been widely implicated in the control of androgen signaling and the repression of cell proliferation and oncogenic pathways. Overall these data provide novel insights into the sophistication of the miRNA network that regulates the function of the male reproductive tract.

Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome

“…For instance, miR-34c and miR-467e were found to be predominantly accumulated into epididymosomes in the cauda and caput epididymis, respectively. In contrast, the expression of miR-34c was some 2-fold higher in the epithelium of the caput epididymis versus that of the cauda, and in the case of miR-467e , we failed to detect this miRNA in the epithelium of any segment of the epididymis (Supplementary Table S3)26. We did however note close similarities in the trends of miRNA abundance within both epididymosomes and spermatozoa sampled from equivalent segments (Supplementary Table S3)22.…”

“…Further, many of the profiled miRNA were determined to be at considerably enriched levels compared to parent cells (the epididymal epithelial cells). Indeed, almost a third of the miRNAs detected in the epididymosome fraction were not present in our equivalent profiling of epididymal epithelial cell miRNAs26. While we cannot entirely discount the possibility that such differences may, in part, reflect either: (i) greater depth of sequence coverage achieved in our current analysis, (ii) profiling of a subset of epididymosomes originating from a non-surveyed epithelial cell population upstream of the caput epididymis, or (iii) contamination of our samples with vesicles released from ruptured cytoplasmic droplets; the data presented here does nevertheless accord with independent evidence that the epididymosome miRNA signature diverges from that of the epithelial cells from which they originate11.…”

Paternal environmental enrichment transgenerationally alters affective behavioral and neuroendocrine phenotypes

“…Alternatively, emerging evidence supports a direct influence of the environment on the epigenetic profile of spermatozoa. Environmental enrichment for 28 days, which accounts for the spermatogenic cycle of Mus musculus (Oakberg, 1956), could be altering the microRNA content of exosomes secreted by the epididymal epithelial cells which then fuse with the mature sperm in the caudal epididymis (Nixon et al, 2015a(Nixon et al, , 2015b. Environmental enrichment may be altering the dynamics of exosomal release and the microRNA content of these epididysomes, similar to its recently demonstrated impact on peripheral blood mononuclear cells (Pusic and Kraig, 2014;Pusic et al, 2016).…”

Sperm microRNA Content Is Altered in a Mouse Model of Male Obesity, but the Same Suite of microRNAs Are Not Altered in Offspring’s Sperm

“…Thus it remains a distinct possibility that the abundance of these microRNAs might be differentially acquired in obesity during epididymal transit, as the epididymis is not as protected from environmental insults as the testis. This altered microRNA transfer may well be enacted by epididymosomes [70] that may result from the effect of a father’s obesity, that then might also have altered microRNA content in epididymal epithelial cells, whose physiology is likely altered by systemic impacts of obesity [71]. …”