Since the mechanisms of spermatogenesis are highly conserved, insights into the causes of human male infertility, as well as insights into novel forms of male contraception, may come from studies on model organisms. Spermatogenesis in animals occurs in a syncytium, and individual spermatozoa are resolved from this syncytium during a post-meiotic step of spermatogenesis known as spermatid individualization. Individualization in Drosophila begins when a membrane-cytoskeleton complex known as the individualization complex (IC) assembles around the sperm heads and travels down the tails, squeezing out excess cytoplasm from between the sperm tails and reorganizes membrane around each of the spermatids as it travels. After individualization, each syncytial spermatid has been encased within its own plasma membrane and is thus transformed into an individual spermatozoon. ICs are, in part, composed of 64 F-actin-based investment cones, and each spermatid is individualized by one investment cone. While the investment cones of wild-type ICs travel in a coordinated ensemble, ICs from mulet mutant testes become severely discoordinated upon departure from the nuclei and fail to individualize, indicating that mulet is required for individualization. Since the mulet gene encodes Tubulin-Binding Cofactor E (TBCE)-like, a protein required for microtubule depolymerization, microtubule removal may be necessary for spermatid individualization. Indeed, a network of parallel inter-flagellar microtubules, normally removed from between the sperm tails prior to individualization, persists in mulet mutant testes. Thus, the removal of these microtubules appears to be necessary for the coordinated travel of the investment cones and spermatid individualization. The proposed work seeks to characterize the role of TBCE-like in Drosophila individualization. First, the mulet mutant phenotype will be fully characterized by fluorescence and confocal microscopy using known markers of individualization in order to determine if the observed individualization defect is caused by a defect in the IC itself or by the persistence of the inter-flagellar microtubule network. In order o determine where TBCE-like protein is required during spermatogenesis, in situ hybridization and immunofluorescence analyses will be employed to localize TBCE- like mRNA and protein, respectively. Rescue and RNAi experiments will also be conducted using the UAS/GAL4 system in order to distinguish between requirements for TBCE-like in the germline and the soma. Finally, the role of the inter-flagellar microtubule network in spermatid individualization will be elucidated using the UAS/GAL4 system to ectopically stabilize or depolymerize the network and examining the subsequent effects on spermatid individualization. The relationship between the investment cones and the microtubule network will also be examined using standard and spinning disk confocal microscopy. Ultimately, since TBCE- like is conserved in mammals and is expressed in mammalian testes, this work may uncover a novel aspect of individualization in flies and mammals, and thus pave the way for novel fertility treatments and contraception.
Failure to properly individualize, or shrink wrap, sperm into their own membranes is the most common cause of human male infertility, and thus a better understanding of the molecular mechanisms behind individualization can be useful for developing treatments for infertility and even developing novel means of contraception. Specifically, we are studying the role of one gene called mulet in Drosophila (fruit fly) individualization. Since sperm development is fundamentally similar in all animals, and since mulet is expressed in both human and fly testes, our work may uncover a fundamental aspect of the individualization process, common to flies and man, that may pave the way toward new infertility treatments and contraceptives.
