GENETIC DISSECTION OF SPERM INDIVIDUALIZATION IN DROSOPHILA-MELANOGASTER

The morphogenesis of spermatids generally takes place within a syncyti um, in which all spermatid nuclei descended from a primary spermatocyt e remain connected via an extensive network of cytoplasmic bridges. A late step in sperm maturation therefore requires the physical resoluti on of the syncytium, or cyst, into individual cells, a process sometim es referred to as sperm individualization. Despite the identification of specialized machinery involved in the individualization of Drosophi la spermatids (Tokuyasu, K. T., Peacock, W. J. and Hardy, R. W. (1972) Z. Zellforsch 124, 479-506), and of many Drosophila genes mutable to male-sterile phenotypes, little is known of the mechanisms by which th is extensive remodeling of the cyst is accomplished. Here, the identif ication of a major cytoskeletal component of the individualization com plex as actin is confirmed with a simple fluorescence assay. Using rho damine-phalloidin as a probe, the individualization complex is readily visualized forming around bundles of spermatid nuclei at one end of h ighly elongated cysts, then translocating along the length of the cyst s. The structure of the individualization complex in a male-sterile cl athrin heavy chain (Chc) mutant is observed to be reduced or disrupted relative to wild-type, consistent with the individualization-deficien t phenotype of this mutant. Using the fluorescence assay, a sampling o f male-sterile mutant phenotypes in which spermatogenesis proceeds to the assembly of highly elongated cysts distinguishes at least four dif ferent phenotypic classes: (1) mutations (nanking class) that block or significantly retard the assembly of the actin-based individualizatio n complex around the nuclear bundle, (2) mutations (dud class) in whic h the individualization complex assembles in/around the nuclear bundle , but fails to translocate down the cyst, (3) mutations (mulet class) that allow the assembly of a morphologically normal individualization complex around the nuclear bundle, but result in a breakdown in the co mplex after it begins to translocate down the cyst, and (4) mutations (purity of essence class) that allow the assembly of a motile but morp hologically altered or reduced individualization complex. Individualiz ation also fails in a number of mutants with altered nuclear shape, co nsistent with the hypothesis that spermatid nuclei provide a physical scaffolding for the assembly of the individualization complex. Genetic analysis suggests that a substantial number of additional loci with p henotypes distinguishable with this assay remain to be identified. The large proportion of male-sterile mutations resulting in a late block to spermatogenesis, in which highly elongated cysts fail to be individ ualized, suggest a substantial susceptibility of this process to a bro ad range of cellular perturbations. The massive reorganization of cyst cytoplasm required at individualization is expected to be a correspon dingly complex function requiring exquisite coordination of multiple c ytoplasmic functions, and may account for the previously noted high fr equency with which Drosophila genes are mutable to male-sterile phenot ypes.