Chromatin condensation during Drosophila spermiogenesis and decondensation after fertilization.

Chromatin condensation is a typical feature of sperm cells. During mammalian spermiogenesis, histones are first replaced by transition proteins and then by protamines, while little is known for Drosophila. Here I characterize three male specific genes in the fly genome, Mst35Ba, Mst35Bb and Mst77F. With eGFP fusion for these above mentioned proteins, I show here that Mst35Ba and Mst35Bb indeed encode for dProtA (Drosophila ProtamineA) and dProtB (Drosophila ProtamineB), respectively and show 94% identity to each other. Drosophila protamines are considerably larger than mammalian protamines, but, as in mammals, both protamines contain typical cysteine/arginine clusters. Ectopic expression of both dProtA and dProtB in the salivary gland cells localizes to the nucleus. Both the protamines binds to the polytene chromosomes without any specificity for the euchromatin or the heterochromatin reflecting the binding status of protamines in sperm nucleus. Unlike in mammals, Drosophila protamine genes are not haplo insufficient. Screening of Zuker mutant collection did not show any mutation in both protamine genes, which argues for functional redundancy. Mst77F encode a spermatid specific linker histone-like protein. The expression pattern of Mst77F overlaps the pattern of protamines as a chromatin component. Mst77F shows significant similarity to mammalian HILS1 protein. The ProtamineA-eGFP, ProtamineB-eGFP and Mst77F-eGFP carrying Drosophila lines show that these proteins become the important chromosomal protein components at the canoe stage in elongating spermatids and stays in mature sperm nucleus and His2AvDGFP vanishes at the canoe stage. Thus, transition from histone based nucleosomal configuration to protamine based chromatin configuration takes place at the canoe stage of spermatid development. Mst77F mutants (ms(3)nc3) are characterized by small round nuclei and are male sterile. These data suggests, the major feature of chromatin condensation in Drosophila spermatogenesis corrrespond to those in mammals. During the canoe stage of spermatid development, histone H2AvD is degraded. Here I show a novel function of UbcD1 an E2-ubiquitin conjugating enzyme that is required for H2AvD degradation. E3 ligase Cullin-1 and Cullin-3 both are expressed during Drosophila spermatogenesis. Cullin-1 is expressed in all early germ cells and localizes to the fusomes and degraded during meiosis. In the later stages, Cullin-1 is expressed again and localizes to the perinuclear space at the canoe stage of spermatid differentiation. Cullin-3 is expressed in the elongated spermatids. During early fertilization steps, the paternal pronucleus still contains protamines and Mst77F, but regains a nucleosomal conformation before zygote formation. In eggs laid by sesame mutant females, the paternal pronucleus remains in a protamine-based chromatin status, but Mst77F-eGFP is removed, suggesting that the sesame gene product (HIRA) is essential for removal of protamines while Mst77F removal is independent of Sesame.