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.

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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.