Genetic dissection of sperm individualization in Drosophila melanogaster

ABSTRACT The morphogenesis of spermatids generally takes place within a syncytium, in which all spermatid nuclei descended from a primary spermatocyte remain connected via an extensive network of cytoplasmic bridges. A late step in sperm maturation therefore requires the physical resolution of the syncytium, or cyst, into individual cells, a process sometimes referred to as sperm individualization. Despite the identification of specialized machinery involved in the individualization of Drosophila 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 this extensive remodeling of the cyst is accomplished. Here, the identification of a major cytoskeletal component of the individualization complex as actin is confirmed with a simple fluorescence assay. Using rhodamine-phalloidin as a probe, the individualization complex is readily visualized forming around bundles of spermatid nuclei at one end of highly elongated cysts, then translocating along the length of the cysts. The structure of the individualization complex in a male-sterile clathrin heavy chain (Chc) mutant is observed to be reduced or disrupted relative to wild-type, consistent with the individualization-deficient phenotype of this mutant. Using the fluorescence assay, a sampling of male-sterile mutant phenotypes in which spermatogenesis proceeds to the assembly of highly elongated cysts distinguishes at least four different phenotypic classes: (1) mutations (nanking class) that block or significantly retard the assembly of the actin-based individualization complex around the nuclear bundle, (2) mutations (dud class) in which 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 complex after it begins to translocate down the cyst, and (4) mutations (purity of essence class) that allow the assembly of a motile but morphologically altered or reduced individualization complex. Individualization also fails in a number of mutants with altered nuclear shape, consistent 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 phenotypes 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 individualized, suggest a substantial susceptibility of this process to a broad range of cellular perturbations. The massive reorganization of cyst cytoplasm required at individualization is expected to be a correspondingly complex function requiring exquisite coordination of multiple cytoplasmic functions, and may account for the previously noted high frequency with which Drosophila genes are mutable to male-sterile phenotypes.