- Title
- Functional characterization of dynamin in spermatozoa epididymal maturation and acrosomal exocytosis
- Creator
- Zhou, Wei
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2019
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Human infertility is now a major clinical problem affecting approximately one in six couples; with a male factor contributing to nearly 50% of these cases. Clinical analysis has shown that a majority of male infertile patients are still able to produce enough spermatozoa to achieve fertilization. However, for reasons that remain poorly defined, the functionality of these cells has become compromised. Improved understanding of how sperm acquire functional maturity would not only beneficial in terms of uncovering the causative basis of male gamete dysfunction, but also for the provision of urgently needed biomarkers of sperm quality to reliably predict the outcome of assisted reproductive technology treatments. In this context, it is generally accepted that spermatozoa released from the testes require additional phases of post-testicular development that occur during their transit through the epididymis and female reproductive tract before acquiring functional competence. Both biophysical and biochemical changes occur along this journey, eventually culminating in the ability of sperm to undergo an acrosome reaction and recognize the ocyte. Notably, due to spermatozoa being both transcriptionally and translationally silent, the acquisition of functional maturity is reliant on communication between the spermatozoa and the extrinsic factors that they encounter within the male and female reproductive tracts. Our recent work has shown that the dynamin family of enzymes may regulate several key steps in these communication pathways. The dynamin family comprises a group of large GTPases responsible for the regulation of membrane trafficking events such as endocytosis, exocytosis and intracellular trafficking. Such diverse functionality relies on the ability of dynamin to polymerize into a helix structure around the template lipid membrane, whereupon GTP hydrolysis drives the lengthwise constriction of the helix structure and leads to scission of the connection between the two membrane templates. Dynamin has three canonical isoforms, namely dynamin 1, dynamin 2 and dynamin 3. Although sharing over 80% sequence homology, recent studies have shown that each isoform may play distinct roles in regulating membrane trafficking events, depending on their localization and ability to interact with other protein targets. This is especially the case in male reproduction with our previously published studies having shown that dynamin 1 and 2 putatively regulate acrosomal exocytosis whilst dynamin 2 plays an essential role in regulating spermatogenesis in the mouse model. Herein, we have provided further evidence that dynamin is involved in sperm maturation through the regulation of epididymal epithelium secretion. Our detailed characterization of the three canonical dynamin isoforms have revealed that each are highly expressed during the early development phases of epididymal differentiation. Interestingly however, the widespread localization of these isoforms in the juvenile epididymis is replaced by segment and cell specific patterns coinciding with the arrival of testicular sperm into the tract. Notably, the expression of dynamin 2 in the Golgi apparatus of caput epithelial cells ideally positions the enzyme to regulate the classical merocrine pathway of protein secretion. This hypothesis was tested through the use of an in vitro caput epithelial cell line; i.e. mECap18 cells. Accordingly, pharmacological inhibition of dynamin selectively inhibited the secretion of a subset of proteins, such as CCT3 and CCT8, from the mECap18 cells. Having demonstrated that dynamin influences the secretion of epididymal proteins, we elected to explore if members of this family also participate in downstream communication between epididymal soma and sperm via the control of extracellular vesicle uptake. For this purpose, we elected to focus on epididymosomes, small membrane encapsulated vesicles that have been implicated in establishing the sperm proteomic and epigenetic landscape. Through the establishment of an in vitro co-culture model, we have documented the kinetics of epididymosome-mediated transfer of proteins to spermatozoa and identified the post-acrosomal sheath as the domain responsible for initial epididymosome – sperm adhesion. Such adhesion appears to be followed by the uptake of epididymosome cargo into the cell, a process that is reliant on both dynamin 1 and lipid rafts. In continuing our investigation of dynamin, we also elected to study the role of this family of mechanoenzymes in regulating the acrosome reaction in human spermatozoa. Based on previous data generated in a mouse model, we hypothesized that dynamin 1 and 2 play a conserved role in facilitating acrosomal exocytosis in human spermatozoa, and that this activity is linked to the phosphorylation status of the dynamin proteins. Consistent with this hypothesis, dynamin 1 and 2 were localized to the acrosomal domain of human spermatozoa and their pharmacological inhibition significantly compromised the ability of human spermatozoa to complete an acrosome reaction. This activity appears to be tied to the phosphorylation of dynamin, with our data identifying CDK1 as an important targeting kinase for dynamin 2. Accordingly, we recorded a significant loss of dynamin 2 expression in the acrosomal domain of poor quality human spermatozoa; a loss that was accompanied by a significant reduction in the ability of these cells to complete an acrosome reaction. Collectively these data support a conserved role for dynamin in regulating the acrosome reaction in both mouse and human spermatozoa. In summary, the data summarized in this thesis implicates dynamin as a key regulatory enzyme in both epididymal sperm maturation and downstream acrosomal exocytosis. In contrast to the overlapping role of the dynamin family members in somatic cells, our findings raise the prospect that different dynamin members fulfill distinct functions in the male reproductive system. Such distinctions raise the intriguing possibility of being able to target specific dynamin members for the purpose of male fertility regulation. Moreover, the functional conservation we observed between human and mouse models supports the utility of conditional knockout mouse models as an important tool with which to further dissect the role of dynamin in human male (in)fertility.
- Subject
- dynamin; epididymis; sperm maturation; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1401122
- Identifier
- uon:34873
- Rights
- Copyright 2019 Wei Zhou
- Language
- eng
- Full Text
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