- Title
- Investigating the profile of miRNAs in the mammalian male reproductive tract
- Creator
- Reilly, Jackson Nicholas
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2018
- Description
- Masters Research - Master of Philosophy (MPhil)
- Description
- Approximately 20 % of the human population is affected by infertility, leading to an increasing concern regarding the reproductive health of our species. In around half these cases, a male factor is implicated and as a result, many research groups are actively exploring the causes of male infertility and the development of therapeutic interventions to alleviate this infertility. After leaving the testis, nascent sperm gain their potential for functional competence as they progressively transit the epididymis, a long and convoluted tubule that connects the testes to the vas deferens. This accessory organ of the male reproductive tract is characterised by segment specific microenvironments that result from differential protein secretion by the epithelium of the tubule. Recently, it has been shown that an additional tier of regulation involving non-proteincoding RNAs (ncRNAs), such as the microRNA (miRNA) small RNAs, is also highly influential in creating the dynamic intraluminal environment of the epididymis. There is also emerging interest in the contribution that these species of epididymal small RNA (sRNA) have in transgenerational inheritance owing to their potential to be transferred to maturing spermatozoa within the lumen of the duct. Thus, in recognition of the potential importance of epididymal sRNA, the aims of this project were to investigate the profile of miRNAs differentially expressed throughout the mouse epididymis, with a particular focus on identifying novel and miRNAs generated within this organ. The results of this study revealed that mouse epididymal epithelial cells are characterised by a cohort of 218 miRNAs. Interestingly, these populations were relatively stable, with only a small portion of these molecules (15 %) undergoing the significant changes expected of candidates involved in regulating differential gene expression along the length of the tubule. A number of these miRNAs were identified as playing regulatory roles in pathways well documented to influence epididymal physiology, including 12 and 10 miRNAs mapping to androgen regulation and endocytotic pathways, respectively. An impressive 295 miRNA species were identified within the spermatozoa sourced from differing epididymal segments. In marked contrast to epithelial cells, the miRNA population harboured by epididymal spermatozoa was found to be far more variable, with pronounced changed in both the number and abundance of miRNAs in sperm being observed as these cells progress through the epididymis. Among the miRNAs enriched in caudal sperm are a cohort of 28 molecules that have been experimentally confirmed to target the genes encoding several members of the TGFβ signalling pathway, which has been documented in the modulation of the female reproductive tract prior to fertilization. Further studies revealed that epididymosomes, small exosome-like vesicles produced by the epididymal epithelium, are replete with 358 miRNAs, ~48 % of which were characterised by significant changes in accumulation between proximal and distal ends of the tract. Additionally, the first empirical evidence to suggest that epididymosomes may transfer their payload to sperm after co-incubation in vitro has been provided. Analysis of the presence of novel miRNAs (Nov-miRs) in the mouse epididymis resulted in the identification of 22 putative candidates, mapping to > 6,200 reads. Of these, five were selected for further validation and target identification, resulting in the documentation of 19 key biological processes potentially regulated by these molecules. Three of the five Nov-miRs chosen for validation were confirmed to be present in sperm via RT-qPCR. The ongoing characterisation of these Nov-miRs and the role they play in regulation of epididymal physiology will form the basis of future work in the Nixon laboratory.
- Subject
- sperm; epididymis; miRNA; reproductive; regulation; NGS; PCR
- Identifier
- http://hdl.handle.net/1959.13/1383982
- Identifier
- uon:32001
- Rights
- Copyright 2018 Jackson Nicholas Reilly
- Language
- eng
- Full Text
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