Replacement by Drosophila melanogaster Protamines and Mst77F of Histones during Chromatin Condensation in Late Spermatids and Role of Sesame in the Removal of These Proteins from the Male Pronucleus

Abstract

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 melanogaster. Here we characterize three genes in the fly genome, Mst35Ba, Mst35Bb, and Mst77F. The results indicate that Mst35Ba and Mst35Bb encode dProtA and dProtB, respectively. These are considerably larger than mammalian protamines, but, as in mammals, both protamines contain typical cysteine/arginine clusters. Mst77F encodes a linker histone-like protein showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying Drosophila lines show that these proteins become the important chromosomal protein components of elongating spermatids, and His2AvDGFP vanishes. Mst77F mutants [ms(3)nc3] are characterized by small round nuclei and are sterile as males. These data suggest the major features of chromatin condensation in Drosophila spermatogenesis correspond to those in mammals. 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-deficient females, the paternal pronucleus remains in a protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the sesame gene product is essential for removal of protamines while Mst77F removal is independent of Sesame.Chromatin reorganization of the complete genome leading to compaction is an essential feature during spermiogenesis (8,21,22,41). The switch from the nucleosomal to the condensed conformation in mammals is essential to get a more hydrodynamic sperm head and also may protect the genome from physical and chemical damage. As this process leads to an extremely condensed state of the haploid genome in the sperm, a reorganization of the paternal genome in the male pronucleus after fertilization and before zygote formation is essential (32).For mammals, it is known that the somatic set of histones are modified, as these are in part replaced by specific variants during meiotic prophase. After meiosis, histones are replaced by major transition proteins TP1 and TP2 (34) and subsequently by highly basic protamines to ensure the remodeling of chromatin to a typically highly condensed and transcriptionally silent state of mature sperm. These replacements leads to a shift from histone-based nucleosomal conformation to a radically different conformation, resembling stacked doughnut structures containing protamines as major chromatin condensing proteins and DNA. Some mammals have only one protamine gene (13), while mice and humans have two genes encoding two different protamines, both of which are essential for fertility and are haploinsufficient (11). Recently, HILS1 (spermatid-specific linker histone H1-like protein) was proposed to participate in chromatin remodeling in mouse and human spermiogenesis (23,49). The transition between histone rem...

Elimination of Y chromosome-bearing spermatids during spermiogenesis in an autosomal <i>sex-ratio</i> mutant of <i>Drosophila simulans</i>

“…sperm nuclei. These results were congruent with the expression and localization patterns in D. melanogaster documented by Jayaramaiah Raja and Renkawitz-Pohl (2005).…”

“…Although little is known about the molecular mechanism underlying nuclear elongation, this dynamic morphological change requires chromatin reorganization with histoneprotamine transition: nucleosomal histone is displaced by protamine, a sperm-specific chromatin protein. Recently, protamine genes were characterized in D. melanogaster and their expression pattern was described by Jayaramaiah Raja and Renkawitz-Pohl (2005). To analyze whether histone-protamine transition occurs in exf abnormal spermatid nuclei, we firstly generated D. simulans transgenic flies expressing GFP-tagged His2AvD and ProtamineB (constructed from the D. melanogaster His2AvD and ProtamineB genes respectively, see MATERIALS AND METHODS).…”

“…The transformation vector plasmid, which contained a His2AvD-GFP insert (GFP fusion construct for the D. melanogaster His2AvD gene) was provided by R. Saint (Clarkson and Saint, 1999), and that of ProtamineB-eGFP (GFP fusion construct for the D. melanogaster ProtamineB gene) was provided by R. Renkawitz-Pohl (Jayaramaiah Raja and Renkawitz-Pohl, 2005). As a helper plasmid, we used phsπ.…”

The testis-specific proteasome subunit Prosα6T of D. melanogaster is required for individualization and nuclear maturation during spermatogenesis

“…Some, if not all, of the somatic histones become replaced by the small, highly basic protamines, ProtA and ProtB (also known as Mst35Ba and Mst35Bb, respectivelyFlyBase), and a sperm-specific linker histone-related protein Mst77F, during the process of sperm nuclear elongation and condensation (Jayaramaiah Raja and Renkawitz-Pohl, 2005;Rathke et al, 2007). The timing of the histone-protamine transition overlaps the expression patterns of Pros␣3T and Pros␣6T, raising the possibility that the somatic histones are degraded by testisspecific proteasomes to set the stage for recruiting the protamines and Mst77F to the sperm chromosomes.…”

<em>Ex vivo</em> Culture of <em>Drosophila</em> Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

“…Targeting the H-P switch in these assays was made possible by using a fly strain that expresses the fusion genes histone2AvD-RFP and protamineB-eGFP 12,22 in the endogenous patterns, and the observation that the first spermatids in pupal testes pass through the H-P switch between 24 and 48 hr APF 12 .…”