Discovery and phenotyping of the determinants and receptors of post-fertilization sperm mitophagy
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This thesis is concerned with two important aspects of sperm physiology -- mitochondrial health prior to fertilization and mitochondrial inheritance after fertilization. The factors and mechanisms that play a role in maternal mitochondrial inheritance have been explored over the decades. It is known that there is mitochondrial DNA within the oocytes' mitochondria as well as in the mitochondrial sheath of the fertilizing spermatozoa. In most mammalian species, sperm mitochondrial DNA is degraded after fertilization, preventing it from being inherited. This process is known as sperm mitophagy. Post-fertilization sperm mitophagy is a developmental mechanism which ensures the inheritance of mitochondria from the maternal lineage in a variety of taxa. Inheritance of paternal, sperm-contributed mitochondrial genes is seldom observed in mammals due to post-fertilization targeting and degradation of sperm mitochondria. Post-fertilization sperm mitophagy is controlled by an interplay of autophagy and the ubiquitin proteasome system (UPS). In this system, the ubiquitin-binding pro-autophagic receptors such as SQSTM1 and the proteasome-interacting ubiquitinated protein dislocase VCP, contribute to sperm mitophagy. To identify additional proteins involved in this mitophagic process, we have developed a novel porcine cell-free system that recapitulates the early post-fertilization mitophagic events and interactions which take place between sperm organelles and oocyte proteins during fertilization. In the context of fertilization events, a multi-pronged proteomic and sperm phenotyping approach is used to further understand the function and distribution of 185 candidate sperm mitophagy substrates and determinants identified by proteomic analysis of boar spermatozoa exposed to porcine oocyte extracts. This approach allows for the implication of oocyte proteins in the processes of mitophagy as well as the observation of oocyte proteins translocating to sperm mitochondrial sheath, and other sperm head and tail structures, during sperm-extract coincubation. From the identified 185 candidate sperm mitophagy substrates, five have been selected for further investigation in this paper. These proteins of interest are being further studied and validated by using cell imaging, proteomics, in vitro fertilization (IVF) protocols and the aforementioned, porcine cell-free system, to compare the localization patterns of these selected proteins in the sperm mitochondria before and after targeting for mitophagy. Results from this study will validate the porcine cell-free system as a rare tool for the exploration of early fertilization events. A successful fertilization partly depends on the spermatozoa and how efficiently they are able to fertilize an egg. To effectively fertilize an egg the spermatozoa, need to have efficient and progressive motility. Over time, as semen is stored, they generate an abundance of reactive oxygen species (ROS). The increase in ROS production can lead to impaired spermatozoa function such as: loss of motility and mitochondrial activity, as well as DNA damage. It has been found that adding certain additives into semen extender during storage may help retain the spermatozoa quality over time. An additive being explored in this study with regard to sperm mitochondrial health is a plant-derived antioxidant known as gallocatechin, identified by collaborative studies involving our laboratory and colleagues at the University of Pretoria, South Africa. Different concentrations of gallocatechin have been added to boar semen extenders over the period of 10 days to determine which concentrations aid in preserving sperm motility and progressiveness. In all, this study explores gallocatechin as a promising means to extend semen quality over time. As well as advancing the understanding of mitochondrial inheritance and shedding light on the origins of certain mitochondrial diseases arising from the failure of post fertilization sperm mitophagy, causing paternal mtDNA leakage and heteroplasmy.
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M.S.