Drosophila Protamine-Like Mst35Ba and Mst35Bb Are Required for Proper Sperm Nuclear Morphology but Are Dispensable for Male Fertility

During spermiogenesis, histones are massively replaced with protamines. A previous report showed that Drosophila males homozygous for a genomic deletion covering several genes including the protamine-like genes Mst35Ba/b are surprisingly fertile. Here, we have precisely deleted the Mst35B locus by homologous recombination, and we confirm the dispensability of Mst35Ba/b for fertility.

In most animal species, the mature sperm nucleus is characterized by an extreme level of DNA compaction achieved after the massive replacement of somatic-type histones with sperm-specific nuclear basic proteins (SNBPs) (Lewis et al. 2003;Miller et al. 2010;Ward 2010;Kanippayoor et al. 2013). In mammals, the bulk of sperm chromatin is organized with two small protamines (Protamine 1 and 2) highly enriched in arginine residues (Balhorn 2007). Drosophila comprises at least three SNBPs: two paralogous protamine-like proteins, Mst35Ba and Mst35Bb, which are conserved among drosophilids, and the HILS1related protein Mst77F (Russell and Kaiser 1993;Jayaramaiah Raja and Renkawitz-Pohl 2005;Alvi et al. 2013;Rathke et al. 2014). The almost identical Mst35Ba and Mst35Bb proteins are larger than mammalian protamines and are enriched in lysine residues. Mst35Ba/b proteins are incorporated in elongating spermatid nu-clei at the late canoe stage and remain associated with mature sperm nuclei until fertilization (Jayaramaiah Raja and Renkawitz-Pohl 2005). Although the functions of Drosophila SNBPs remain poorly understood, Rathke et al. (2010) reported the surprising observation that Drosophila males homozygous for a genomic deficiency covering the Mst35B locus were fertile. This result was indeed unexpected when considering for instance the haploinsufficiency of mouse protamine genes for male fertility (Cho et al. 2001). However, according to Flybase (Flybase.org), the deficiency generated by Rathke et al. (2010) (named protD) is a 73.6-kb deletion that not only uncovers Mst35Ba and Mst35Bb but also removes four additional protein encoding genes (CG42682, CG15279, CG4480, CG15278) as well as three noncoding RNAs (CR43805, CR45727, CR45302). Furthermore, all these genes and noncoding RNAs are expressed in the adult testis or accessory glands, with the exception of CG15279, and transcripts of three of these genes (CG33309, CG4480, and CG15278) were detected in early spermatids (Flybase; Rathke et al. 2010). Because the simultaneous deletion of these other genetic elements could potentially interfere with a detailed functional analysis of Mst35B genes, we generated a precise deletion of the Mst35B locus by homologous recombination using the "Ends-Out" targeting technique Golic 2003, 2004) ( Figure 1A). The resulting allele, named DMst35B, eliminates a 5-kb genomic DNA fragment that only contains the Mst35Ba and Mst35Bb genes. To validate the elimination of these genes in the new deletion allele, we raised an antiserum against a peptide common to Mst35Ba and Mst35Bb proteins ( Figure 1B). This antibody specifically stained late This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (http://creativecommons.org/licenses/ by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Supporting information is available online at Note 1. http://www.g3journal.org/ lookup/suppl/doi:10.1534/g3.114.012724/-/DC1 1 canoe stage spermatid nuclei of wild-type males but not those of DMst35B homozygous males ( Figure 1C). At later stages of spermiogenesis, the highly compacted chromatin of spermatids is no longer accessible to antibodies (Bonnefoy et al. 2007), thus explaining the absence of staining beyond the canoe stage in wild-type testes. In addition, another anti-Mst35B antibody raised against the whole Mst35Bb recombinant protein allowed us to confirm the absence of Mst35B proteins from DMst35B testicular protein extracts ( Figure 1D).
As expected, homozygous DMst35B males were fully viable (not shown) and at least partially fertile (see paragraphs to follow), thus confirming the dispensability of Mst35B proteins for male fertility. Spermiogenesis (the differentiation of postmeiotic spermatids) in mutant males nevertheless appeared severely disorganized, with many elongating spermatids showing abnormal nuclear morphology ( Figure  2, A2C). The spermiogenesis defects were similar in homozygous DMst35B and trans-heterozygous DMst35B/protD males, ruling out the possibility that the phenotypes associated with DMst35B were caused by a second-site mutation. In both allelic combinations, affected spermatid nuclei typically appeared bent compared with control spermatids, with the anterior tip of the nucleus sometimes folded into a hook-like structure (Figure 2, D and E). It is likely that the concentration of chromatin at one end of mutant spermatid nuclei observed by Rathke et al. (2010) actually correspond to folded nuclear extremities. A large proportion of mutant spermatids were scattered along the cysts instead of remaining tightly grouped in bundles of 64 nuclei, suggesting that they were progressively eliminated during the course of spermiogenesis (Figure 2, B and C). Accordingly, mutant males stored significantly less gametes in their seminal vesicles compared with control males ( Figure 3E). Interestingly, however, we did observe morphologically aberrant mature gametes stored in the seminal vesicles of homozygous DMst35B and DMst35B/protD males (Figure 3, A2C), in sharp contrast to previous observations (Rathke et al. 2010). A transgene expressing Mst35Ba-EGFP rescued the abnormal nuclear shaping of DMst35B spermatids, thus confirming that this phenotype is actually caused by the loss of Mst35B genes ( Figure 3D). However, a fraction of spermatids was still eliminated in rescued animals (Supporting Information, Figure S1), suggesting that the presence of a relatively large green fluorescent protein tag perturbs the functionality of the recombinant protein. Alternatively, both Mst35Ba and Mst35Bb proteins could be required for proper packaging of sperm DNA. In addition, we confirmed that a transgene expressing Mst77F-EGFP was normally incorporated into the chromatin of mutant gametes but failed to rescue the phenotype ( Figure 3B). Finally, using a specific antibody ( Figure S2), we also verified that the transition protein Tpl94D (Rathke et al. 2007) was normally incorporated in mutant spermatids at the histone-to-protamine transition (Figure 2, D and E), confirming that the nuclear defects in mutant spermatids appear after this stage.
Although the quantity and quality of gametes were affected by the loss of Mst35B genes, homozygous DMst35B and DMst35B/ protD males were nevertheless fertile, in agreement with the study by Rathke et al. (2010). In fact, the impact of DMst35B on male fertility was only revealed when mutant males were allowed to mate with a large excess of virgin females (1 for 10; Figure 3F) but not with a 1:1 sex ratio (not shown). In the presence of a large excess of females, the observed reduction of fertility is likely explained by the limiting amount of sperm produced by mutant males ( Figure 3E). Figure 1 Knocking-out Mst35Ba/b genes by homologous recombination. (A) Representation of the Mst35b locus before (wild-type; WT) and after (DMst35B, w+) "ends-out" targeting using the pW25 vector Golic, 2003, 2004; details are available upon request). The DMst35B allele was obtained after Cremediated excision of the white cassette. The tandem protamine genes Mst35Ba and Mst35Bb are represented with their coding regions in pale blue. The dark blue hexagons representing termination codons in the six reading frames and the Lox sites (black triangles) are from the pW25 ends-out targeting vector. Arrowheads indicate the position of the primer pair used in polymerase chain reaction analyses. (B) Sequence alignment of Mst35Ba and Mst35Bb proteins. The peptide used to generate the rabbit antipeptide "NGI" polyclonal antibody is highlighted in green and other identical residues are shaded in gray. (C) Immunofluorescence staining of WT or DMst35B spermatid nuclei with the antipeptide "NGI" antibody. The antibody stains WT spermatid nuclei in late canoe stage but not more advanced nuclei (asterisk), which are too compacted for antibody penetration (Bonnefoy et al. 2007). No staining was observed in mutant spermatid nuclei at the late canoe stage. Bar: 10 mm. (D) Western blot analysis of testicular protein extracts using a rabbit antibody directed against a recombinant full-length Mst35Bb protein. Mst35Ba/b-specific bands are detected in WT but not in DMst35B extracts. The anti-a-Tubulin antibody was used as loading control.
The organization of sperm chromatin in animals is poorly understood and most of our knowledge comes from studies on human or other mammalian species. Drosophila is an interesting, alternative model for the study of sperm chromatin at the functional level. The generation of a precise deletion allele of both protamine-like genes Mst35Ba/b provides an ideal tool for the functional study of Drosophila SNBPs. The fertility of DMst35B males reveals the extraordinary plasticity of the Drosophila sperm nucleus, which grossly maintains its architecture, motility and ability to fertilize eggs in the absence of what is considered a major component of its chromatin. It is likely that additional SNBPs compensate for the loss of the protamine-like proteins. In fact, we already know that the loss of Mst35Ba/b proteins does not perturb the incorporation of Mst77F in spermatid nuclei (this work and Rathke et al. 2010). Mst77F, which was originally identified in a genetic screen for b2 tubulin interactors (Fuller et al. 1989), is related to the mammalian spermatid-specific histone H1-like protein HILS1 (Iguchi et al. 2004;Yan et al. 2003). Interestingly, the D. melanogaster genome contains several recent copies of Mst77F on the Y chromosome, and eight of these Mst77Y genes are most likely functional (Russell and Kaiser 1993;Krsticevic et al. 2010). It has been proposed that Mst77F is essential for male fertility (Rathke et al. 2010), but this conclusion is based on the analysis of the antimorphic point mutation Mst77F 1 (see Krsticevic et al. 2010). Future work should aim at clarifying the nuclear function of Mst77F/Y proteins to determine if they can indeed maintain a sperm chromatin structure compatible with male fertility in the absence of Mst35B proteins.

ACKNOWLEDGMENTS
We thank Renate Renkawitz-Pohl and Cristina Rathke for fly stocks. This work was supported by research grants from the French ANR (ZygoPat-ANR-12-BSV6-0014), the Fondation ARC pour la Recherche sur le Cancer (SFI20121205765), and the NSF Frontiers in Integrative Biological Research (EF-0328363). S.T. is supported by a doctoral fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche. S.K. is supported by a fellowship from the Uehara Memorial Foundation. Millipore) in red. In control testes, groups of 64 spermatid nuclei are visible in each cyst. Needle-like shaped spermatid nuclei appear green after the removal of histones and the incorporation of Mst77F-EGFP. In DMst35B/DMst35B as in DMst35B/protD testes, many green fluorescent protein2 positive spermatid nuclei appear scattered along the length of the cyst (arrowheads), indicating that they are progressively eliminated. (D2E) Confocal images of spermatid nuclei from control (D) and protamine mutant testes (E) bearing a Mst77F-EGFP transgene. Testes were immunostained for Tpl94D, a transition protein facilitating the deposition of spermspecific nuclear basic proteins (Rathke et al. 2007). The anti-Tpl94D antibody was generated by rabbit immunization with full-length recombinant Tpl94D protein (details available upon request. See Figure S2). In both control and DMst35B mutant testes, spermatid nuclei successively incorporate Tpl94D and Mst77F-EGFP. However, mutant spermatids frequently exhibit a twisted shape compared to control nuclei. Scale bar: 10 mm LITERATURE CITED  (A2B) were acquired with a Z1 Imager microscope (Zeiss) and sperm nuclei were individually counted using the Image J software. A total of 9 control vesicles and 14 DMst35B mutant vesicles were counted and the results were statistically analyzed with a Mann-Whitney test. ÃÃÃ P , 0.001. (F) Reduced fertility of DMst35B males. 0-to 1-d-old males of the indicated genotypes were individually mated with 10 virgin w 1118 females. Embryo hatching rates were then determined as described in Orsi et al. (2010). Results were statistically analyzed with a Mann-Whitney U test. ns: nonsignificant. ÃÃ P , 0.01. ÃÃÃÃ P , 0.0001.