ABSTRACT
Spermiogenesis in Drosophila melanogaster is a highly conserved process and essential for male fertility. In this haploid phase of spermatogenesis, motile sperm are assembled from round cells, and flagella and needle-shaped nuclei with highly compacted genomes are formed. As transcription takes place mainly in spermatocytes and transcripts relevant for post-meiotic sperm development are translationally repressed for days, we comparatively analysed the proteome of larval testes (only germ cell stages before meiotic divisions), testes of 1-2-day-old pupae (germ cell stages before meiotic divisions, meiotic and early spermatid stages) and adult flies (germ cell stages before meiotic divisions, meiotic and early spermatid stages, late spermatids and sperm). We identified 6,171 proteins; 61 proteins were detected solely in one stage and are thus enriched, namely 34 in larval testes, 77 in pupal testes and 214 in adult testes. To substantiate our mass spectrometric data, we analysed the stage-specific synthesis and importance for male fertility of a number of uncharacterized proteins. For example, Mst84B (gene CG1988), a very basic cysteine- and lysine-rich nuclear protein and was present in the transition phase from a histone-based to a protamine-based chromatin structure. CG6332 encodes d-Theg, which is related to the mouse tHEG and human THEG proteins. Mutants of d-Theg were sterile due to the lack of sperm in the seminal vesicles. Our catalogue of proteins of the different stages of testis development in D. melanogaster will pave the road for future analyses of spermatogenesis.
Subject(s)
Drosophila Proteins/genetics , Infertility, Male/genetics , Proteome/genetics , Testis/metabolism , Animals , Drosophila Proteins/metabolism , Drosophila melanogaster , Male , Proteome/metabolism , Spermatogenesis/genetics , Testis/growth & developmentABSTRACT
In the course of spermatogenesis, germ cells undergo dramatic morphological changes that affect almost all cellular components. Therefore, it is impossible to study the process of spermatogenesis in its entirety without detailed morphological analyses. Here, we describe a method to visualize chromatin dynamics in differentiating Drosophila male germ cells using immunofluorescence staining. In addition, we demonstrate how to treat Drosophila sperm before immunofluorescence staining to help reveal epitopes in the highly condensed sperm chromatin that otherwise may be inaccessible to antibodies.
Subject(s)
Chromatin/metabolism , Drosophila/physiology , Spermatogenesis/genetics , Animals , Chromatin/immunology , Drosophila/genetics , Epitopes , Female , Fluorescent Antibody Technique/instrumentation , Fluorescent Antibody Technique/methods , Male , Spermatozoa/cytology , Spermatozoa/physiologyABSTRACT
The formation of motile spermatozoa involves the highly conserved formation of protamine-rich, tightly packed chromatin. However, genetic loss of protamine function in Drosophila and mice does not lead to significant decompaction of sperm chromatin. This indicates that other proteins act redundantly or together with protamines. Here, we identify Prtl99C as a Drosophila sperm chromatin-associated protein that is essential for male fertility. Whereas the loss of protamines results in modest elongation of sperm nuclei, knockdown of Prtl99C has a much stronger effect on sperm nuclei. Loss of protamines and Prtl99C indicates an additive effect of these proteins on chromatin compaction, in agreement with independent loading of these factors into sperm chromatin. These data reveal that at least three chromatin-binding proteins act together in chromatin reorganization to compact the paternal chromatin.
