- Title
- The impact of environmental insult on mouse epididymal spermatozoa
- Creator
- Trigg, Natalie A.
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2021
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- The possibility of inheritance that deviates from canonical Mendelian rules (epigenetic / non-genetic inheritance) has been considered since the beginning of genetics research, however, only in recent years has experimental work provided the evidence needed to substantiate epigenetic inheritance. Indeed, models of epigenetic inheritance have now been established in species ranging from worms to mice. The putative ‘information carriers’ responsible for epigenetic inheritance, include DNA and histone modifications, chromatin modifications and, small non-coding RNAs (sncRNAs) and the modifications they harbour. Recently, several lines of evidence have supported sperm-borne sncRNA as an integral intergenerational signalling molecule following their delivery to the oocyte at the moment of fertilisation. It is also now apparent that the sperm sncRNA profile is dynamic and susceptible to modification during sperm maturation and in response to paternal environmental and lifestyle factors; each of which have potentially significant post-fertilisation consequences.While, the importance of the sperm sncRNA profile emerges, what remains less clear are the molecular mechanism(s) underpinning the response to environmental insult that leads to an altered sperm sncRNA profile. The studies in this thesis were designed to begin to bridge this important knowledge gap. Specifically, we aimed to investigate how paternal exposure to environmental factors influence the sperm sncRNA profile and the consequences of the delivery of an altered sncRNA profile to the oocyte (Chapter 2). The sncRNA profile of spermatozoa undergoes major remodelling during the sperm cells passage of the epididymis. Moreover, the balance of evidence has implicated the epididymis as a vulnerable site in which sperm acquire environmental signals, such as an altered sncRNA profile. Owing to the transcriptionally and translationally quiescent state of epididymal spermatozoa, modification to the sncRNA profile is facilitated by the complex luminal microenvironment. A key component of which are small membrane bound vesicles, termed epididymosomes. These vesicles are produced by the epididymal epithelial cells and deliver a diverse cargo of macromolecules, including sncRNA, to spermatozoa. Additionally, epididymosomes have recently been implicated in delivery of an altered sncRNA profile to sperm under paternal stress conditions. Hence, in examining the mechanism(s) driving sperm sncRNA changes, we focused on epididymosomes as mediators of this dialogue and more specifically, on the cognate receptor-ligand(s) that underpin epididymosome-sperm adhesion (Chapter 3). The data presented within this thesis confirms the acute sensitivity of the sperm sncRNA payload to the environmental toxicant, acrylamide, encountered during their post testicular development. We have traced the differential accumulation of miRNAs to coincide with sperm transit of the proximal (caput) epididymal segment. Indeed, we identified alterations in the epididymosomes secreted by the caput epididymis following environmental insult. In expanding this mechanistic investigation, we profiled the proteome of the caput epididymal epithelium and 10 revealed that acrylamide exposure alters the expression of a subset of proteins. In resolving a causal link, we identified the increased expression of seven transcription factors in the caput epithelium of acrylamide exposed mice, each of which have been implicated in the regulation of acrylamide-sensitive miRNAs. In identifying the consequences of an altered sperm sncRNA profile following acrylamide exposure our analysis revealed a subset of dysregulated genes in embryos fertilised by exposed sperm. This gene dysregulation was demonstrated to be in part driven by the sncRNA changes in the sperm and thus substantiate sperm-borne sncRNAs as important epigenetic messengers. Having confirmed an integral role for epididymosome mediated communication in facilitating a response to paternal environmental exposure (acrylamide), we next sought to further our understanding of the mechanistic basis underlying the interaction between epididymosomes and recipient sperm. In exploring the proteomic composition of epididymosomes, previous work from our laboratory identified a putative role for an epididymosome resident ligand, milk fat globule-EGF factor 8 (MFGE8), in the efficient transfer of cargo from epididymosomes to spermatozoa. MFGE8 has been implicated in the adhesion of extracellular vesicles (EVs) isolated from non-reproductive tissues to recipient cells and this finding indicated a conserved function for MFGE8 in EVs isolated from the epididymis. Therefore, the remaining studies herein focused on the role of MFGE8 as a key molecular ligand in this intercellular form of communication in the epididymis (Chapter 3). We utilised an immortalised caput epididymal (mECap18) cell line as a model and first confirmed its suitability with which to study the mechanism(s) of sperm-epididymosome interaction. Through additional inhibition studies and the ablation of MFGE8 functional domains, we identified MFGE8 as being of fundamental importance for efficient sperm-EV interaction. However, the failure to completely block sperm-EV interaction indicates redundancy in the EV tethering mechanisms and highlights the need for further research to gain a complete understanding of the cognate receptors and ligands that mediate this interaction. Taken together, the findings of this thesis contribute to our understanding of the sncRNA profile of mature mouse spermatozoa and its dynamic response to environmental insult. Importantly, these studies have advanced our knowledge of the molecular basis of epididymosome-sperm interactions, the importance of this intercellular communication in directing sperm sncRNA changes following stress, the mechanistic understanding of how paternal exposures affect remodelling of the sperm small RNA profile and the consequences of such changes for embryonic development.
- Subject
- mice; epididymal spermatozoa; sperm-borne sncRNA; environmental insult; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1476747
- Identifier
- uon:49853
- Rights
- Copyright 2021 Natalie A. Trigg
- Language
- eng
- Full Text
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