Abstract
The small RNA (sRNA) landscape of mammalian spermatozoa is considerably altered as these gametic cells migrate through the segment specific microenvironments of the epididymis. More specifically, the microRNA (miRNA) species of sRNA dominates the sRNA landscape of spermatozoa of the proximal caput segment of the epididymis. However, in sperm cells sourced from the distal cauda epididymal segment, the transfer RNA (tRNA)-derived RNA fragment (tRF) sRNA species is the most abundant. Here we show that the 5′ halves of fifteen mature tRNAs were used as processing substrates for the production of a specific subpopulation of tRF sRNAs, 30 to 33 nucleotides (30–33-nt) in length. A quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) approach was used to experimentally validate the sRNA sequencing identified trend of enriched abundance of this specific 30–33-nt tRF subpopulation in cauda spermatozoa. The length, and exclusive alignment of the cauda spermatozoa enriched tRF subpopulation to the 5′ half of each processed tRNA precursor, identified ANGIOGENIN (ANG) as the endonuclease likely responsible for tRF production in the mouse epididymis: a prediction confirmed via immunoblotting assessment of ANG abundance in spermatozoa sourced from the caput, corpus and cauda epididymal segments. When taken together with our previous profiling of miRNA and Piwi-interacting RNA (piRNA) sRNA abundance in spermatozoa sourced from the three segments of physiologically normal mouse epididymides, the tRF profile reported here adds greater depth of coverage to the global sRNA landscape of the mouse epididymis; a roadmap constructed to assist with the future molecular characterization of sRNA-directed responses to a wide range of imposed environmental stressors.
Highlights
In mammals, spermatozoa must undergo sequential phases of mitosis, meiosis and cytological differentiation, before they are rendered morphologically mature [1]
The small RNA accumulation profile of mouse spermatozoa is dramatically altered during their epididymal transit Assessment of the distribution of sRNA molecules 19–36nt in length across spermatozoa sourced from the proximal caput, to distal cauda segment of the mouse epididymis, revealed strikingly different abundance profiles (Fig. 1a)
We have previously demonstrated that in spermatozoa sourced from the caput, corpus and cauda segments of the mouse epididymis, Piwi-interacting RNA (piRNA) accumulation is enriched in cauda sperm [23]
Summary
Spermatozoa must undergo sequential phases of mitosis, meiosis and cytological differentiation, before they are rendered morphologically mature [1]. The drastically altered morphology of mature spermatozoa renders these cells silent, both at the transcriptional and translational level. Their functional maturation in the epididymis must be driven extrinsically: a process made possible by the secretive and absorptive activity of the epithelium that lines the lumen of the tract [6]. Historical research has focused heavily on the characterization of the complex interplay between the secretory and absorptive activity of the soma of each epididymal segment, and the maturing spermatozoa descending the tract [7]. Much of this research attention was aimed at documenting the complex segment-specific protein complement secreted into the epididymis lumen in an attempt to identify the molecules that direct the functional maturation of spermatozoa [8, 9]. Considering that the recipient spermatozoa are rendered transcriptionally and translationally silent prior to entry into the tract, the communicative role occupied by epididymosomes takes on further significance
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