Distinct functions of Mst77F and protamines in nuclear shaping and chromatin condensation during Drosophila spermiogenesis

Abstract

Chromatin reorganisation is a major event towards the end of mammalian and Drosophila spermatogenesis. In Drosophila, we previously identified protamine A, protamine B and Mst77F as major chromatin components of the mature sperm. Here, an antibody against Mst77F reveals a dual expression pattern of Mst77F as a chromatin component and in association with microtubules during nuclear shaping. Spermatids of ms(3)nc3 (Mst77F¹) mutants show disturbed nuclear shaping, instability of perinuclear microtubules but no obvious chromatin condensation defects. Furthermore, we generated a deletion including both protamine genes (protΔ) and observed that in Drosophila, protamine genes are not haploinsufficient in contrast to those of mice and humans. Moreover, we show that in protΔ mutants, histone degradation, distribution of DNA breaks and Tpl^94D-eGFP and Mst77F expression proceed as in wild-type males. Surprisingly, in homozygous protΔ mutants, males are fertile and sperm are motile, while about 20% of sperm show abnormally shaped nuclei. The latter phenotype can be rescued by supplying protamine-eGFP but not by supplying Mst77F-eGFP. Finally, we demonstrate a 21% increase in X-ray-induced mutation rate of protΔ sperm. These data support the long-standing hypothesis that the switch from a histone- to protamine-based chromatin protects the paternal genome from mutagens.

Introduction

Spermatogenesis is a highly specialised process of cellular differentiation resulting in the formation of functional spermatozoa for successful reproduction. During later stages of spermatid differentiation, the initially round spermatids undergo extensive morphological changes to give rise to elongated spermatids. Nuclear transformation including chromatin reorganisation in these spermatids is of particular interest. In mammals and Drosophila, histones that form the nucleosomal subunits are replaced first by transition proteins and then by a basic set of small proteins called protamines. This replacement connected with nuclear shaping leads to a highly compact and slim sperm nucleus. The volume of the nucleus is reduced by 20-fold in the case of mammals (Braun, 2001) and 200-fold in Drosophila (Fuller, 1993).

In mice, protamine-1 and protamine-2 are haploinsufficient; the loss of one copy causes male sterility (Braun, 2001; Cho et al., 2001; Kimmins and Sassone-Corsi, 2005; Sassone-Corsi, 2002). Also in humans, reduction in protamine level results in infertility (Aoki et al., 2005; Carrell et al., 2007; Iguchi et al., 2006; Oliva, 2006; Torregrosa et al., 2006). There is evidence from the mouse that sperm with a haploid content of protamines can form a zygote after ICSI (intracytoplasmic sperm injection)-mediated fertilisation, but those embryos develop only up to morula or blastula stage (Cho et al., 2003).

In Drosophila, numerous histone modifications characterise the spermatocyte stage with its extended meiotic prophase and these modifications persist after meiosis. As in mammals, after meiosis histone H4 of Drosophila becomes hyperacetylated before degradation. Subsequently, histones are replaced by Tpl^94D with a transition-protein-like expression pattern at a stage characterised by numerous DNA breaks (Rathke et al., 2007).

Using eGFP fusion proteins we previously showed in Drosophila that protamine A (ProtA), protamine B (ProtB) and Mst77F replace histones during spermatogenesis (Jayaramaiah Raja and Renkawitz-Pohl, 2005). The corresponding genes were first described to encode male-specific transcripts that may encode histone H5 or protamine-like molecules (Russell and Kaiser, 1993). ProtA and ProtB are encoded by two closely related genes, Mst35Ba and Mst35Bb. Drosophila protamines share characteristic cysteines with mammalian protamines but are less rich in arginine. However, both are expressed during spermiogenesis and persist in the chromatin of mature sperm as do protamines of mammals. The ms(3)nc3 allele of Mst77F (Mst77F¹) was identified by non-complementation (nc) with β2 tubulin mutations (Fuller et al., 1989; Tweedie et al., 2009). ms(3)nc3 mutants are homozygous lethal due to an additional mutation. Males carrying this allele over a deletion (ms(3)nc3/Df(3L)ri-79c), however, are sterile, show abnormal nuclear shaping and often have tiny, dot-shaped spermatid nuclei (Jayaramaiah Raja and Renkawitz-Pohl, 2005). It has not been clear whether the abnormal form of the nuclei is a consequence of defective microtubule-dependent nuclear shaping or due to the defective chromatin condensation. Here, we show that Mst77F indeed has a dual subcellular localisation. On the one hand, Mst77F localises close to or with β2 tubulin in the perinuclear microtubules during nuclear shaping; on the other hand, Mst77F is a distinctly localised component of spermatid and sperm chromatin. We present evidence that the ms(3)nc3 mutation disturbs nuclear shaping but not chromatin condensation.

Besides Mst77F, ProtA and ProtB are also abundant in the sperm chromatin of Drosophila. It has been speculated that protamine-based chromatin organisation leads to a compact hydrodynamic sperm head and protects the paternal genome from physical and chemical damage (Braun, 2001; Oliva, 2006). As mammalian sperm with a complete loss of protamines do not exist, the evolutionary importance of the change from histones to protamines remains unclear. Therefore, we established and analysed loss-of-function mutants for protamine genes in Drosophila. We generated a deletion (protΔ) of the protamine genes, Mst35Ba and Mst35Bb and three adjacent reading frames, which do most likely not encode for chromosomal proteins. Here, we show that in protΔ mutants histone degradation, Tpl^94D-eGFP expression and removal, Mst77F synthesis, nuclear shaping and spermatid individualisation do not depend on protamine synthesis. Surprisingly, flies lacking both protamines are fertile but about 20% of late spermatid nuclei look abnormal. Furthermore, we demonstrate that introducing protB-eGFP rescues the abnormal spermatid nuclei phenotype of protΔ flies, while Mst77F-eGFP does not. This led us to suggest that these proteins cannot substitute for each other to compact the chromatin but have distinct functions. Finally, we demonstrated that sperm of protΔ flies show an increased sensitivity to X-rays, consistent with the long-standing hypothesis that protamines protect the paternal genome from damage.