Mitochondria are highly dynamic organelles capable of significant reorganisation to meet cellular demands. Spermatogenesis – a complex developmental process that generates male gametes – is accompanied by substantial alterations in mitochondrial shape, quantity, and distribution. Previous studies have mainly focused on mitochondrial dynamics in post-meiotic spermatids, consequently little is known about mitochondrial dynamics during early spermatogenesis and its impact on sperm development. A potent example of this is the striking reorganization of the mitochondrial network into a large ball-like structure adjacent to the nucleus during the pre-meiotic spermatocyte stage in Drosophila melanogaster. This distinct cluster of mitochondria was initially observed in an electron microscopy study by Tates et al. in 1971 but has remained uncharacterized since then. Our lab termed this cluster of mitochondria the "mitoball”. The main objective of this thesis is to investigate the genetic basis of mitochondrial dynamics within the novel in vivo environment of the mitoball and explore its impact on sperm cell development and male fertility.

Our lab showed mitoballs are conserved among many insect species and are densely packed with other organelles. To explore the function of mitoball, I investigated the role of Milton, an adaptor protein involved in the microtubule-based transport of mitochondria. Through my research, I discovered that Milton collaborates with Mitochondrial Rho (Miro), a protein located on the mitochondrial outer membrane, and Khc, a microtubule motor, to facilitate the transportation of mitochondria along microtubules and form the mitoball. By generating homozygous viable milton mutants, I demonstrated that a 54-amino acid region in the C-terminus of Milton is essential for mitoball formation. I further observed that flies lacking mitoballs had swollen mitochondria in their spermatocytes, reduced ATP production, altered transcriptome, and compromised male fertility. These findings indicate that the subcellular distribution of mitochondria can modulate mitochondrial morphology and function during early spermatogenesis to impact male fertility.

To reveal other players regulating mitoball formation, I performed a forward genetic screen by feeding D. melanogaster with EMS, a mutagen that induces random mutations in the nuclear genome, and visualising mitoballs by confocal imaging. This screen identified 121 lines with abnormal mitochondrial and testis morphology. Focusing on nine EMS lines with mitoball defects, I performed whole genome sequencing and deletion mapping to identify the responsible genes that modulate mitochondrial dynamics in early spermatogenesis. I further validated the role of eight genes using RNAi knockdown and knockout mutants. This part of my research uncovered both previously known and novel regulators of mitochondrial dynamics.

Overall, this thesis investigates mitochondrial dynamics in Drosophila spermatogenesis within the context of the mitoball and establishes the essential role of Milton for mitoball formation. Since premeiotic clustering of mitochondria is observed in various insect species, regulators of mitoball formation could be utilised as targets for inducing male sterility in genetically modified insects, offering an environmentally friendly approach to pest control. Moreover, uncovering conserved genetic factors that influence mitochondrial dynamics during spermatogenesis holds the potential to address male fertility disorders in humans, as well as neurodegenerative and metabolic diseases associated with mitochondrial abnormalities.