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Mol Cell Biol. 2005 July; 25(14): 6165–6177.
PMCID: PMC1168805

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


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 removal and its replacement by protamines in mice and humans is characterized by small 6- to 10-kDa transition proteins acting as a short-term chromosomal proteins (34). In mice, the transition proteins TP1 and TP2 are redundant in function. In fishes and birds, transition proteins are missing and protamines directly reorganize the chromatin. In annelids and echinoderms, the nucleosomal configuration is maintained in sperms(28, 48), while protamine-like proteins have been described for mussels (27). These protamine-like proteins lack the typical high cysteine content necessary for disulfide bridges (10). Therefore, a doughnut-type chromatin structure as in mammals is unlikely to occur in mussels. Lewis and Ausió (27) propose that the protamine-like proteins in mussels belong to the histone H1 family. The sperm chromatin of mussels contain core histones and thus a nucleosomal configuration, but histone H1 is replaced by protamine-like molecules which organize the higher order structure of the chromatin.

For Drosophila melanogaster, chromatin reorganization after meiosis has not been studied so far at the molecular level. At the light microscopic level, the Drosophila spermatid nucleus is initially round after meiosis and then is shaped to a thin needle-like structure with highly condensed chromatin, so that the volume of the nucleus is condensed over 200-fold (19). In mammals, the volume of the nucleus is reduced over 20-fold (8). In the mature sperms of Drosophila, core histones are not detectable by immunohistology (2). There is histochemical evidence for the presence of very basic proteins in sperm (14), but it still remains an open question whether histones are replaced by protamine-like basic proteins in Drosophila. The analysis of the Drosophila genome sequence (1, 9) revealed that the proteins encoded by two genes show similarity to mammalian protamines for which the male-specific transcripts Mst35Ba and Mst35Bb have been found (3) and proposed previously to encode protamine-like proteins (4). Another male specifically transcribed gene, Mst77F, is a distant relative of the histone H1/H5 (linker histone) family and has been proposed to play a role either as a transition protein or as a replacement protein for compaction of the Drosophila sperm chromatin (39). With enhanced green fluorescent protein (eGFP) fusion for these abovementioned proteins, we show here that Mst35Ba and Mst35Bb indeed encode protamines and Mst77F encodes a linker histone-like protein. The expression pattern of Mst77F overlaps the pattern of protamines as a chromatin component. Furthermore, we show that during fertilization, the removal of protamines from the male pronucleus requires the function of the maternal component, Sesame, but not for the removal of Mst77F. It has been shown that sesame mutants cause impairment of the entry of histones into the male pronucleus (30).


Fly strains and culture.

Flies were grown on standard medium at 25°C. Visible markers and chromosome balancers are as described in Flybase unless otherwise specified. w was used as the wild-type strain. Rb97D1, Df(2L)Exel8033, Df(3L)ri-79c, and PBac{PB}Pka-R1c06969 (45) stocks were obtained from Bloomington Stock Center; His2AvDGFP flies were provided by R. Saint (12); and ms(3)nc3 was obtained from M. T. Fuller (17). The sesame mutant stock ssm185b/FM7c was kindly provided by B. Loppin and P. Couble (29).

Constructs and P-element-mediated transformation.

Generally, gene control regions of genes expressed in the male germ line are relatively short in Drosophila melanogaster (7, 37, 40), and therefore we used only short upstream regions to establish protamine-eGFP fusion constructs. The construct protamineB-eGFP was made by PCR amplifying 2.1 kb of the genomic region using the primer pair A (at 665 bp upstream of the predicted 5′-untranslated region [UTR]) and B (for the predicted third exon of Mst35Bb) with internal restriction sites designed for KpnI and NcoI, respectively. The amplified DNA was restriction digested with KpnI and NcoI and cloned into pKS+eGFP in frame with eGFP. The clone was digested again with KpnI and NotI and subcloned into the germ line transformation vector pChabΔSal. protamineA-eGFP was constructed by PCR amplifying the genomic region 3.1kb using the primer pair C (at 510 bp upstream of the predicted 5′UTR) and D (for the predicted third exon of Mst35Ba) with internal restriction sites designed for KpnI and NcoI, respectively. The amplified DNA was restriction digested with internal EcoRI-NcoI and was cloned into pKS+eGFP in frame with eGFP. The clone was digested again with EcoRI-NotI and subcloned into the germ line transformation vector pChabΔSal. Mst77F-eGFP was constructed by PCR amplifying the genomic region 1.15 kb using the primer pair E (at 278 bp upstream of the predicted 5′UTR) and F (for the predicted second exon without stop for Mst77F) with internal restriction sites designed for EcoRI and XbaI, respectively. The amplified DNA was restriction digested with internal EcoRI-XbaI and was cloned into pKS+eGFP in frame with eGFP. The clone was digested again with EcoRI-NotI and subcloned into the germ line transformation vector pChabΔSal. The primer names (and sequences) are the following: A (5′-GGGGGGTACCGTCTACTCTCCCTGTGTGCGT-3′), B (5′-GCCGCCATGGTGCAAATCCGTCGGCGCTTGTGG-3′), C (5′-GGGGGTACCATTTTCATTGATTTGATACCATTCG-3′), D (5′-GCCGCCATGGATTGCTGGCAAATCCGTCGGC-3′), E (5′-GGGGAATTCCGCGTTACTCAGCTAGTCGGA-3′); F (5′-GGGTCTAGACATCGAGCACTTGGGCTTGGA-3′).

Fertility test.

Young adult males were placed individually with three wild-type virgin females in separate vials at 25°C, and vials were scored for offspring after a week.

Analysis of the embryonic phenotype.

Wild-type virgin females were mated with ProtamineB-eGFP and ProtamineA-eGFP males separately, ssm185b/ssm185b virgin females were mated with ProtamineB-eGFP-, ProtamineA-eGFP-, and Mst77f-eGFP-containing male flies separately, and the embryos were collected every 15 to 30 min after egg deposition and were stored at 4°C up to 2 h before fixation. Egg collection and fixation were done essentially as described previously (29). After fixation, the embryos were incubated for 1 h in a 2 mg/ml RNaseA solution at 37°C, rinsed with phosphate-buffered saline (PBS)-0.1% Triton X-100, stained with 5 μg/ml propidium iodide (PI) for 30 min, washed with PBS-0.1% Triton X-100 twice for 15 min each, and mounted with Fluoromount medium (Southern Biotech). Photomicrographs of PI-stained male and female pronuclei were made with a Leitz confocal microscope and processed with Adobe Photoshop 6.0.

In situ hybridization.

Whole-mount in situ hybridization was performed with digoxigenin (Dig)-labeled Mst35Ba cDNA probe and digoxigenin-labeled Mst35Bb cDNA probe using expressed sequence tag clones BG634974 and AI134395, respectively. Mst77F probe was generated by PCR with digoxigenin labeling using primers (E and F) mentioned above with Mst77F-eGFP as the template. In situ hybridization to the whole-mount adult testis was essentially done according to the protocol described by White-Cooper et al. (47) with some modification. Prehybridization, hybridization, and washes were all done at 45°C. Hybridized Dig-DNA probes were detected by alkaline phosphatase-conjugated anti-Dig antibody at a dilution of 1:2,000.


In Drosophila melanogaster, two putative protamine genes and Mst77F encode proteins of the sperm chromatin.

We characterized the genes encoding two predicted protamines of D. melanogaster, Drosophila ProtamineA (dProtA) and Drosophila ProtamineB (dProtB). With the predicted molecular mass and pI of 16.9 kDa and 10.63 for dProtA and 16.6 kDa and 10.24 for dProtB, respectively, they exhibit a very basic character. These genes, Mst35Ba and Mst35Bb, are present at cytological position 35B6 and 35B6-7, respectively, on the chromosome arm 2L. These two genes are arranged in tandem, and both consist of three exons (Fig. (Fig.1A).1A). The 5′UTR, coding region, and the 3′UTR of these genes are highly identical (data not shown), as they probably arose from a recent gene duplication. The encoded protamines show over 94% identity to each other (Fig. 2A and B; see also the supplemental data).

Molecular genetic analysis of Drosophila protamines and Mst77F genes. (A) Genomic organization of the two protamine genes. The map illustrates the position of Mst35Bb (thick black lines) and Mst35Ba (thick grey lines) exons and introns (thin lines). The ...
FIG. 2.
Sequence alignment of protamines and Mst77F. Black boxes show identity, gray boxes show similarity, and conserved cysteines are marked by asterisks. (A) Alignment of Drosophila ProtamineA and ProtamineB (dProtA and dProtB) with human Protamine-1 (hProt1) ...

A remarkable feature of protamines is their ability to form intermolecular disulfide bridges, which is reflected by the conserved cysteine residues within mammalian protamines (10). The dProtA and dProtB are of 146 amino acids (aa) and 144 aa, respectively, and thus longer than even the human and mouse Protamine-2, which are 102 aa and 107 aa, respectively (Fig. (Fig.2B).2B). Both Drosophila protamines contain 10 cysteines each and show significant similarity, particularly with respect to a high cysteine, lysine, and arginine content to mammalian protamines (Fig. 2A and B and Table Table1).1). Human and mouse Protamine-1 aligns to the N-terminal half of the Drosophila protamines (from aa positions 27 to 82), and four cysteine residues are conserved and regularly spaced (Fig. (Fig.2A,2A, asterisks). On the other hand, Protamine-2 of human and mouse show relatively high similarity to the C-terminal half of the Drosophila protamines, with four cysteines in this region which are conserved and regularly spaced (Fig. (Fig.2B,2B, asterisks), whereas one cysteine (Fig. (Fig.2B,2B, first asterisk) is shared with the mouse and human Protamine-1 (Fig. (Fig.2A,2A, last asterisk).

Chromatin condensing proteins of mouse, human, and fly

Mst77F is present at the cytological position 77F on the chromosome arm 3L and lies within the large intron of PKA-R1. Mst77F is also male specifically transcribed, and the encoded protein has been proposed to be a linker histone H1/H5 type, which could also play the role of a transition protein or a protamine (39). The predicted gene structure is as shown in Fig. Fig.1B.1B. We observed that the Mst77F protein shares a significant similarity to the HILS1 protein of mouse (Fig. (Fig.2C)2C) and human HILS1 (see the supplementary data), where the percentages of cysteine, lysine, and arginine are similar to that of mHILS1 and hHILS1 (Table (Table1).1). HILS1 protein has been recently described as a component of the mammalian sperm nucleus (49). Drosophila Mst77F encodes a protein of 215 aa with a molecular mass of 24.5 kDa and with a pI of 9.86. mHILS1 is of 170 aa and shows 39% similarity to Mst77F (Fig. (Fig.2C).2C). Mst77F contains 10 cystine residues as in Drosophila protamines (Table (Table1),1), and mHILS1 contains eight cystine residues, of which four residues are conserved (Fig. (Fig.2C,2C, asterisks).

As there are considerable differences between the mammalian protamines as well as between the mammalian HILS1 proteins and the presumptive Drosophila homologue Mst77F, additional experiments are essential to clarify if these proteins are indeed involved in the condensation of sperm chromatin.

Mst35Ba, Mst35Bb, and Mst77F are transcribed in the male germ line from the primary spermatocyte stage onward.

To check whether Mst35Ba, Mst35Bb, and Mst77F are expressed in the male germ line, whole-mount in situ hybridizations on testis of adult flies were performed. The results showed that indeed Mst35Ba (Fig. (Fig.1C,1C, panel 1), Mst35Bb (Fig. (Fig.1C,1C, panel 2), and Mst77F (Fig. (Fig.1C,1C, panel 4) transcripts are detectable in the cytoplasm from the primary spermatocyte stage onward, while stem cells, spermatogonia, and the testis sheath are free of transcript. As discussed below, these Mst35Ba (Fig. (Fig.1C,1C, panel 3), Mst35Bb, and Mst77F (Fig. (Fig.1C,1C, panel 6) mRNAs are translationally repressed until the elongated spermatid stage.

Loss of histones during nuclear shaping with the simultaneous accumulation of protamine A, protamine B, and Mst77F in the sperm head.

During spermiogenesis, the round nuclei dramatically change their shape from a spherical structure (Fig. (Fig.3A3A and and4F)4F) into a hooklike structure forming at the tip of the nucleus as a result of onset of nuclear shaping (Fig. (Fig.3A3A and and4G).4G). The nuclei start to flatten on one side and appear thereafter canoe or banana shaped, with the densely stained chromatin interspersed with lightly stained chromatin showing a “net like” appearance when stained with Hoechst as a result of onset of chromatin condensation process (Fig. (Fig.3A3A and and4H).4H). Furthermore, as the condensation proceeds, eventually the densely stained chromatin appears to be evenly distributed, and the nuclei cluster together (Fig. (Fig.3A3A and and4I)4I) and lead into a thin needle-shaped nucleus just prior to individualization (Fig. (Fig.3A3A and and4J).4J). Each sperm from the cluster individualizes synchronously with a fully condensed chromatin compacted in a thin needle-shaped nucleus. (Fig. (Fig.3A3A and and4K).4K). During this process the volume of the nucleus is reduced approximately 200-fold (19). To analyze the fate of histones during spermiogenesis, we made use of an existing His2AvDGFP fly line (12). His2AvDGFP fluorescence in these fly lines was retained in all the early sperm nuclei up to canoe stage, indicative of the presence of histone-based nucleosomal conformation, but during the canoe stage, the His2AvDGFP begins to fade and is no longer detectable in the needle-shaped nuclei of elongated spermatids (Fig. (Fig.3B,3B, from left to right).

FIG. 3.
Loss of histone His2AvDGFP and appearance of ProtamineB-eGFP, ProtamineA-eGFP, and Mst77F-eGFP during nuclear shaping and chromatin condensation. (A, C, E, and G) Hoechst counter staining for the chromatin (blue) visualizes the transformation from round ...
Defective spermatid nuclei in ms(3)nc3 and rescue of a mutant phenotype by the Mst77F-eGFP transgene. Testis squash preparation colors: Hoechst (red), Mst77F-eGFP (green), and Merge (yellow). (A) Wild-type (WT) Hoechst staining. Uncondensed early spermatid ...

To analyze whether core histones are replaced by dProtA, dProtB, and Mst77F, we established the transgenic lines bearing an eGFP fusion to dProtA, dProtB, and Mst77F (see Materials and Methods) by adding cytoplasmic eGFP in frame to the putative protamine coding regions. eGFP was fused at the C terminus since it is known from mammals that the protamines are processed at the N terminus (10). We chose 665 bp upstream of the predicted 5′UTR for Mst35Bb and 280 bp upstream of the predicted 5′UTR of Mst35Ba and the gene region and fused eGFP in frame with the last exon. The polyadenylation signal was supplied by the transformation vector pChabΔSal. Using these constructs we established several independent transgenic fly lines. These respective genes are transcribed much earlier in the primary spermatocytes (Fig. (Fig.1C,1C, panels 1, 2, and 4). The proteins are made after several days in the elongated spermatid stage (Fig. (Fig.1C,1C, panel 3 and panel 6, respectively). Thus, protamine mRNAs and Mst77F mRNAs are translationally repressed for several days. Mst77F-eGFP is also clearly seen transiently in the flagellum of the sperm bundles (Fig. (Fig.1C,1C, panel 6). These data reflect the pattern of expression and localization of these proteins in the Drosophila male germ line.

The pattern of these fusion proteins (green) were analyzed by testis squash preparation and counter staining with Hoechst for DNA (blue). All three fusion proteins, ProtamineA-eGFP, ProtamineB-eGFP, and Mst77F-eGFP, show the onset of these proteins at the canoe stage; the fluorescence became brighter in the later steps and stayed in the mature sperm nucleus (Fig. 3D, F, and H, respectively). The comparison of DNA distribution to eGFP fused to protamines and Mst77F distribution in the nucleus revealed that both protamines and Mst77F start to colocalize with DNA at the canoe stage of nuclear shaping (Fig. 3C to H). At this stage His2AvDGFP fluorescence begins to diminish (Fig. (Fig.3B).3B). Protamines and Mst77F persist in individualized sperms until after fertilization (see below).

We checked whether protamines indeed localize to chromatin. Ectopic expression of both ProtamineA-eGFP and ProtamineB-eGFP in the salivary gland cells under the control of a Sgs-GAL4 driver showed that ProtamineA-eGFP and ProtamineB-eGFP cover the whole chromosome (S. Jayaramaiah Raja and R. Renkawitz-Pohl, unpublished data).

As mentioned earlier, the role of the Mst77F gene product was not clear. Interestingly, the Mst77F-eGFP pattern was not restricted to the nucleus alone, but a clear Mst77F-eGFP expression also in the flagellum of the sperm bundles was observed from canoe stage onwards (Fig. (Fig.1C,1C, panel 6, and 4B and C). Just prior to individualization, Mst77F-eGFP was no longer seen in the sperm flagellum but still remained in the nucleus (Fig. (Fig.4B).4B). One or two additional copies of ProtamineA-eGFP, ProtamineB-eGFP, and Mst77F-eGFP do not disturb the normal process of spermatogenesis.

Mst77F is essential for male fertility.

We started to investigate whether all three basic proteins are essential for male fertility in flies. For mice (11) it was shown that mutation in Protamine-1 or Protamine-2 is haploinsufficient and causes male sterility. Analyzing a haploid situation for the Mst35Ba and Mst35Bb genes with the deficiency Df(2L)Exel8033/+ showed normal spermatogenesis (data not shown) and male fertility (Table (Table2).2). Deficiency Df(2L)Exel8033 is defined by the breakpoint 35B1;35B8, which deletes over 35 genes, including Mst35Ba and Mst35Bb. As this deficiency was not viable in a homozygous situation, it was not possible to check for complete removal of both Mst35Ba and Mst35Bb. So far, no mutants for these genes are available.

Fertility and phenotype

In the male sterile Zuker mutant collection (26, 46), 31 male sterile mutant lines were mapped to the region 35B2 to 35D7 using a large deficiency, Df(2L)A48 (D. L. Lindsley, personal communication). We screened these lines against a smaller deficiency, Df(2L)Exel8033 (breakpoint 35B1;35B8) and isolated several lines mapping to this region. Two mutant lines which were allelic to each other in this region showed chromatin condensation defects (with normal nuclear shaping) from the canoe stage onwards. Sequencing Mst35Ba and Mst35Bb from these homozygous mutants showed no mutation in both these genes (S. Jayaramaiah Raja and R. Renkawitz-Pohl, unpublished). It might be that these small reading frames of Mst35Ba and Mst35Bb were either not mutated in this large screen or, alternatively, dProtA and dProtB, which are 94% identical, may be functionally redundant so that the mutation in one is rescued by the other.

Besides the nuclear localization, Mst77F-eGFP also shows a transient pattern in the flagella of canoe-stage spermatids to a stage just before individualization (Fig. 4B and C). In order to elucidate the function of Mst77F we analyzed two existing mutants, ms(3)nc3 and PBac{PB}Pka-R1c06969. ms(3)nc3 is a second-site noncomplementation (nc) mutation that was isolated in an ethylmethanesulfonate screen to identify the interacting proteins involved in microtubule function in Drosophila (18). ms(3)nc3 fails to complement class I alleles at the β2 tubulin locus on the chromosome arm 3R and was mapped to 3L at 77E-77F (17). We tested ms(3)nc3 in trans-heterozygotic situations with Df(3L)ri-79c (77B7 to 77F1), and these males were sterile (Table (Table2).2). PBac{PB}Pka-R1c06969 is a Piggy bac P-element insertion (45) in the promoter region of Mst77F at 158 bp upstream of the presumptive transcription start site. PBac{PB}Pka-R1c06969/PBac{PB}Pka-R1c06969 homozygotes and PBac{PB}Pka-R1c06969/Df(3L)ri-79c trans-heterozygote males were viable and fully fertile (Table (Table2).2). However, PBac{PB}Pka-R1c06969/ms(3)nc3 trans-heterozygotes were sterile (Table (Table2).2). In situ hybridization showed that the level of transcript was greatly reduced in PBac{PB}Pka-R1c06969/PBac{PB}Pka-R1c06969 homozygous testis in comparison to the wild-type testis (Fig. (Fig.1C,1C, panels 5 and 4, respectively). This led us to infer that ms(3)nc3 is a mutation in Mst77F, and this mutation is dosage dependent. Sequencing Mst77F in ms(3)nc3/Df(3L)ri-79c trans-heterozygotes revealed a single missense mutation from T→A causing the substitution of threonine instead of serine at aa position 149 (Fig. (Fig.1B1B).

ms(3)nc3 was not investigated concerning nuclear condensation. In this respect, we analyzed ms(3)nc3/Df(3L)ri-79c and PBac{PB}Pka-R1c06969/ms(3)nc3 to exclude any further mutation on the ms(3)nc3-carrying chromosome that contribute to the phenotype. In an overview, nuclei of spermatids from these males are mainly scattered and are round or ellipsoid (Fig. (Fig.4D);4D); some spermatid nuclei begin to elongate but do so far less than wild-type spermatids (Fig. (Fig.4A).4A). In the wild-type situation, at higher magnification the chromatin distribution was observed very well, changing from a broad network to a highly condensed form (Fig. 4F to K). In spermatids of ms(3)nc3/Df(3L)ri-79c testis, the early spermatid nuclei appear to be normal (Fig. 4L and M). The accumulation of the chromatin in spots (net like) was also observed, though the nuclear shaping does not proceed (Fig. 4N and O). Later the nuclei are smaller and ellipsoid, but no further development was seen (Fig. 4P and Q). PBac{PB}Pka-R1c06969/ms(3)nc3 showed a slightly less severe phenotype (Fig. (Fig.4E)4E) compared to ms(3)nc3/Df(3L)ri-79c, with no individualized sperms leading to male sterility (Table (Table22).

Mst77F-eGFP rescues ms(3)nc3 mutants.

Transgenic flies carrying Mst77F-eGFP on the second chromosome were used to clarify if Mst77F-eGFP could rescue the phenotype caused by ms(3)nc3/Df(3L)ri-79c. In these males the Mst77F-eGFP transgene clearly could restore chromatin condensation. These males were fertile (Table (Table2),2), with the nuclei clustered like in the wild-type situation (see Fig. Fig.4R4R for an overview), and spermatids individualized to mature sperms (Fig. (Fig.4S)4S) with a few tiny-shape nuclei (Fig. (Fig.4R).4R). ms(3)nc3/+ males, though fertile, also showed in a few tiny nuclei in scattered (Fig. (Fig.4T)4T) and individualized sperms (Fig. (Fig.4U)4U) comparable to that of ms(3)nc3/Df(3L)ri-79c rescued by Mst77F-eGFP (Table (Table2).2). These results indicate that ms(3)nc3 is indeed a mutation in Mst77F, and incorporation of the mutant Mst77F protein might act as a poison to the complex.

ProtamineB-eGFP cannot replace Mst77F.

It was proposed that Mst77F mRNA might encode a protamine (39), which might be supported by the fact that the Mst77F protein contains many cysteines. Therefore, in order to check whether Mst77F might be functionally redundant to protamines, spermiogenesis was analyzed with one copy of ProtamineB-eGFP in an ms(3)nc3/Df(3L)ri-79c genetic background. Clearly, spermiogenesis was not improved in this situation (compare Fig. Fig.5A5A to to4D).4D). Thus, Mst77F cannot be replaced by increasing the amount of protamine B. Furthermore, we checked whether ProtamineB-eGFP was deposited on the chromatin in these mutants, and this clearly was the case (Fig. (Fig.5A).5A). The detailed comparison of nuclear stages of spermatids from wild-type and ms(3)nc3/Df(3L)ri-79c testis squash preparations revealed that ProtamineB-eGFP was made at identical stages as that of the wild type (compare Fig. 5J to M to D to G). When the sizes of the round-shaped uncondensed nuclei in Fig. Fig.5H5H and Fig. Fig.5M5M taken with the same 100× magnification are compared, it is clear that the nuclear size is reduced (Fig. (Fig.5M),5M), which is an indication of chromatin condensation and the removal of nucleoplasm, but we do not know if the chromatin condensation is complete with Mst77F mutant protein in ms(3)nc3.

FIG. 5.
Protamines in Mst77F mutant. Testis squash preparation is Hoechst (red). ProtamineB-eGFP is in green, and merge is in yellow. (A) ProtamineB-eGFP in ms(3)nc3/Df(3L)ri-79c males. Shown are uncondensed early spermatid nuclei undergoing nuclear shaping (arrow) ...

Protamine deposition is independent of nuclear shaping.

We know that Mst35Ba and Mst35Bb are transcribed during the early primary spermatocyte stage (Fig. (Fig.1C,1C, panels 1 and 2) and that the transcripts are translationally repressed until the postmeiotic stage, when the repression is relieved and the protein is made (Fig. (Fig.1C,1C, panel 3). Rb97D encodes an RNA binding protein which is associated with the ks-1 fertility locus on the Y chromosome and limited to premeiotic stages in the nucleus (20). As the protamine mRNAs are localized in cytoplasm of primary spermatocytes (Fig. (Fig.1C,1C, panels 1 and 2), it is unlikely that Rb97D binds to protamine mRNA. Mutation in Rb97D cause developmental arrest during spermiogenesis (24). The Rb97D1 mutant is of interest with respect to the question of whether nuclear shaping and protamine deposition to the chromatin are independent processes or not. Therefore, we analyzed the expression of eGFP-tagged version of ProtamineB in Rb97D1 mutants. In the wild-type situation, protamines just start to accumulate in the nuclei at the canoe stage, with a gradual increase in the later stages (Fig. 6A and B). In Rb97D1 mutants, DNA staining shows that as the nuclei start to elongate, some reach the canoe stage of nuclear shaping (Fig. (Fig.6C).6C). Nuclear shaping does not proceed further, but the chromatin condensation continues as large amounts of protamines were observed (Fig. (Fig.6D)6D) in contrast to the wild type (Fig. (Fig.6B).6B). From these observations we infer that synchronous advancement in nuclear shaping is not essential for protamine deposition but is an independent process.

FIG. 6.
Protamine deposition in Rb97D mutants. Squash preparation of testis with ProtamineB-eGFP in the Rb97D1 mutant. Testes are stained with Hoechst (blue) in panels A and C and ProtamineB-eGFP (green) in panels B and D. The ProtamineB-eGFP protein is made ...

ProtamineA and ProtamineB removal from the male pronucleus depends on the maternally supplied Sesame protein.

Protamines are the typical chromatin organizing proteins in the mature sperm. Before zygote formation, the chromatin of the male pronucleus has to be remodeled to obtain the nucleosomal conformation. We used transgenic flies with eGFP-tagged versions of protamines for the analysis of protamine removal from the male pronucleus.

In eggs laid by wild-type females, the needle-shaped male pronucleus is visible by eGFP-tagged ProtamineA (Fig. (Fig.7A).7A). No sign of ProtamineA-eGFP is visible in the first mitotic division after zygote formation (Fig. (Fig.7B).7B). A comparable result is found for ProtamineB-eGFP (Fig. 7C and D).

FIG. 7.
Loss of both protamines in the male pronucleus is dependent on the maternally encoded protein Sesame, while loss of Mst77F is independent of Sesame. Shown are propidium iodide (red), eGFP fusion proteins (green), and merge (yellow). (A to F) Wild-type ...

It is likely that maternally supplied proteins induce the chromatin reorganization of the male pronucleus. Indeed, a maternal effect mutant, sesame, has been described in which the male pronucleus is not able to form a zygote with the female pronucleus, probably caused by a defect in chromatin reorganization of the male pronucleus. In sesame mutants, the male pronucleus looses the needle-like shape and becomes spherical, like in the wild type, but the male pronucleus is not competent enough to fuse with the female pronucleus because maternal histones are not incorporated into the male pronucleus (30). This raises the question of whether the male pronucleus still remains in the protamine-based chromatin structure in sesame mutants. Homozygous virgin sesame mutant female flies were collected and mated to transgenic males carrying protamine-eGFP fusion genes separately. In eggs laid by homozygous sesame mutants, DNA staining visualizes the male pronucleus (m) and female pronucleus (f) dividing after fertilization with ProtamineA-eGFP sperm (Fig. (Fig.7G).7G). The male pronucleus transformed from its needle-like shape to a round shape but surprisingly retained ProtamineA-eGFP (Fig. (Fig.7H).7H). With ProtamineB-eGFP, essentially the same situation was observed, with the male pronucleus still containing ProtamineB-eGFP (Fig. (Fig.7J).7J). With these observations we conclude that Sesame function is required for removal of both dProtA and dProtB from the male pronucleus for the successful restoration of a nucleosomal chromatin configuration.

Removal of Mst77F from the male pronucleus is independent of Sesame.

In the wild-type situation Mst77F is seen in the nucleus of sperm until shortly after fertilization (Fig. (Fig.7E)7E) but is removed from the male pronucleus before zygote formation (Fig. (Fig.7F).7F). To check whether this process also depends on maternally supplied Sesame, we observed homozygous sesame mutant eggs fertilized with Mst77F-eGFP sperm. Interestingly, in Sesame mutant eggs, Mst77F-eGFP is removed from the male pronucleus. No zygote is formed, and the haploid female nucleus starts to divide (Fig. 7K and L). This clearly illustrated that Mst77F removal from the male pronucleus is independent of Sesame function.


Mst35B, Mst35Bb, and Mst77F are transcribed at primary spermatocyte stage and are translationally repressed until the elongated spermatid stage.

Drosophila protamine mRNAs are transcribed at the primary spermatocyte stage, whereas in mammals protamine mRNAs are synthesized at the round spermatid stage (42) and translationally repressed until the elongated stage, which is mediated by 3′UTR (50, 52, 53). The Drosophila ProtamineA-eGFP and ProtamineB-eGFP constructs do not contain the 3′UTR of the respective protamine genes. Nevertheless, the transgenic flies carrying these constructs still show repression of translation. So, in Drosophila, the region responsible for the translational repression is most likely in the 5′UTR. Deletion constructs of Mst35Bb and Mst77F 5′UTRs fused to the reporter lacZ showed the translation repression element is indeed present in the 5′UTR (data not shown). This holds true also for the mRNA of the Mst77F-eGFP fusion gene, as is the case for all mRNAs investigated concerning translational repression so far in male germ lines of Drosophila (37). In contrast to mammalian spermatogenesis (41), in Drosophila transcription ceases already with the entry into meiotic divisions (19). Since the protamines are made in the elongated spermatids, the transcriptional silencing in Drosophila spermatogenesis seems to be independent of protamines.

Comparison of mammalian and Drosophila protamines.

When primary amino acid sequences of Drosophila protamines are compared to mammalian protamines, it is quite evident that Drosophila protamines are relatively large. dProtA and dProtB are over 94% identical to each other. This could explain that both the protamines may be functionally redundant. Human and mouse Protamine-1 aligns with the N terminus of both Drosophila protamines (Fig. (Fig.1A),1A), and Protamine-2 aligns more to the C terminus (Fig. (Fig.1B).1B). It is possible that the Drosophila protamines undergo posttranslational cleavage at the N terminus, as is known for mammals (28). The cytoplasmic eGFP fused at the C terminus shows clear nuclear localization, indicating that the tagged protamine is functionally intact. Drosophila protamines each contain 10 cysteine residues at identical positions, while over 4 of 10 cysteines at the N terminus and the C terminus are conserved with human and mouse Protamine-1 and Protamine-2, respectively (Fig. 1A and B, asterisks). With nine cysteines, the content is highest in Protamine-1 of mice. Inter- or intradisulfide bridges can be formed between the cysteine-rich protamines to condense the DNA. For mice (11) it is shown that mutation in protamine-1 or protamine-2 is haploinsufficient and causes male sterility. We analyzed a haploid situation for the Mst35Ba and Mst35Bb genes with the deficiency Df(2L)Exel8033/+; these flies are fertile males (Table (Table2)2) and show normal spermatogenesis (data not shown). The large amount of identity that both dProtA and dProtB exhibit can contribute to the functional redundancy.

Histone displacement and incorporation of protamines and Mst77F during Drosophila spermiogenesis.

Chromatin reorganization is an essential feature during spermiogenesis. The functional significance of chromatin compaction during spermiogenesis is still unknown. The main explanation seems to be that compaction of the sperm nucleus is an essential factor for its mobility as well as for the penetration of sperm into the egg and genomic stability. In mammals, somatic histones are in part replaced by spermatid-specific variants during meiotic prophase (15), later 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 lead to a shift from histone-based nucleosomal conformation to a radically different conformation, resembling stacked doughnut structures containing major chromatin condensing proteins and DNA in the nucleus (8).

In Drosophila, so far no proteins have been identified that are involved in the packaging of the genome in the mature sperm nucleus. One observation, that Histone3.3 variant and the somatic H3 isoform in Drosophila are vanishing at the time of chromatin condensation, supports the view of histone displacement (2, 22), but it was still a question of whether it is the real absence of histones at this stage in Drosophila or whether the antibodies are not accessible to the mature sperm due to the tight packaging of the chromatin (22). To circumvent this problem, we chose the GFP fusion approach, made use of the existing His2AvDGFP (12), and generated Protamine-eGFP and Mst77F-eGFP fusion transgenic flies in order to analyze the situation in Drosophila. Our results clearly show that histone His2AvD is lost from the spermatid nuclei at the time of appearance of protamines and Mst77F during later stages of spermatid differentiation. The exact molecular mechanisms underlying the histone displacement, degradation, and incorporation of protamines onto the chromatin are poorly understood (8). For mammals, evidence has been obtained that histone H2A is ubiquitinated in mouse spermatids around the developmental time period when histones are removed from the chromatin (5, 6). The mammalian HR6B ubiquitin-conjugating enzyme is the homologue of yeast RAD6, and both can ubiquitinate histones in vitro (38). The mechanism of histone displacement and protamine incorporation is unknown during spermiogenesis in Drosophila so far. In flies as well as in mammals, many questions remain unanswered about these underlying mechanisms of chromatin remodeling during spermiogenesis which need to be addressed.

In Drosophila, mammalian HILS1-related protein Mst77F is coexpressed with protamines.

In mammals, transition proteins act as intermediates in the histone-to-protamine transition (34). In mice, the onset of HILS1 and transition proteins TP1 and TP2 (major forms) overlaps with the pattern of Protamine-1 and later with Protamine-2 but is no longer present in the mature sperm. It was recently shown that mice lacking both TP1 and TP2 show normal transcriptional repression, histone displacement, nuclear shaping, and protamine deposition but show the loss of genomic integrity with large numbers of DNA breaks leading to male sterility (51). In Drosophila, we now know that histones are displaced with synchronous accumulation of protamines and Mst77F. Mst77F, a distant relative of the histone H1/H5 (linker histone) family, has been proposed to play a role either as a transition protein or as a protamine for compaction of the Drosophila sperm chromatin (39). Mst77F shows highest similarity to HILS1 with respect to the cysteines and basic amino acid content (Fig. (Fig.2C2C and Table Table1)1) but not to mouse TP1, TP2, or H1t (Table (Table1).1). Moreover, our results show that the pattern of expression of Mst77F in the nucleus is similar to that of mHILS1 in the nucleus, with the exception that Mst77F is also transiently detected in the flagella and persists in mature sperm nuclei, unlike mHILS1. In mammalian mature sperm nuclei, it is only the protamines that are the chromatin condensing proteins which persist. This again raises the question of whether Mst77F could also play the role of protamines. However, one additional copy of dProtB (dProtA and dProtB showing 94% identity may be functionally redundant) does not rescue the ms(3)nc3 phenotype, indicating that the role of Mst77F may be completely or partially different from that of protamines in the nucleus. However, a null mutation for Mst77F is required to answer this question with respect to chromatin condensation. In ms(3)nc3 mutants, the chromatin condensation with the native protamines continues to take place. When we took a closer look at the deposition of ProtamineB-eGFP in ms(3)nc3/Df(3L)ri-79c trans-heterozygotes, it revealed that the condensed chromatin in the tid-shaped nuclei (Fig. (Fig.5M)5M) is concentrated at the two opposite ends, with a lightly stained chromatin spaced in the center. So the chromatin condensation takes place but may not be complete with the incorporation of the mutant Mst77F protein. The large amount of chromatin compaction or condensation seen in Drosophila mature sperm when compared to that of mouse and human sperm possibly could be the result of persistence of Mst77F in the mature sperm nuclei. It remains to be clarified whether the sperm nucleus contains further not yet properly annotated protamines.

Role of Mst77F in nuclear shaping.

ms(3)nc3 is a second-site noncomplementation (nc) mutation that was isolated in an ethylmethanesulfonate screen to identify interacting proteins involved in microtubule function in Drosophila (18). We have shown that ms(3)nc3 is a single missense mutation from a T→A transition, causing the substitution of threonine instead of serine at aa position 149 (Fig. (Fig.1B).1B). Mst77F shows a pattern of expression similar to protamines in the nucleus (Fig. (Fig.3H3H and and4B)4B) and was also seen in the flagella until the individualization stage (Fig. 4B and C). Since ms(3)nc3 fails to complement class I alleles at the β2 tubulin locus (17), it is possible that Mst77F has a dual role to play as a chromatin condensing protein in the nucleus and for the normal nuclear shaping. Nuclear shaping is a microtubule-based event (19). ms(3)nc3 leads to a tid-shaped nuclear phenotype, where the nucleus fails to shape into a needle-like nuclei. Similar defective nuclear shaping is seen with the few homozygous and heteroallelic combinations of class I alleles of β2 tubulin (16). The incorporation of the defective subunit encoded by ms(3)nc3 may interfere with the function of the resulting complex. These data suggest the involvement of an Mst77F (a linker histone variant) in the microtubule dynamics during the nuclear shaping. This again complements the role of sea urchin histone H1 in the stabilization of flagellar microtubules (35).

Sesame is essential to remove protamines from the male pronucleus but not for shape changes of the nucleus.

After the first steps in the fertilization process, the male gamete is still in the highly compact protamine-based chromatin structure. In a wild-type egg, the paternal pronucleus changes the shape from the needle-like to a spherical structure. Furthermore, the male pronucleus acquires a nucleosome-based structure before zygote formation and thus is transformed into a replication-competent male pronucleus. sesame is a maternal effect mutation in HIRA and had been mapped to 7C1 (31). In Drosophila, HIRA is expressed in the female germ line and a high level of HIRA mRNA is deposited in the egg (25). Human HIRA is shown to bind to histone H2B and H4 (33). The WD repeats present at the N-terminal part of HIRA could probably function as a part of a multiprotein complex (33). Xenopus HIRA proteins are also known in promoting chromatin assembly that is independent of DNA synthesis in vitro (36). Loppin et al. (29) analyzed the corresponding maternal effect mutant sesame, in which the sperm fertilizes the egg but no zygote is formed. Although the shape change of the nucleus to the spherical structure occurs in these mutants, maternal histones are not incorporated into the male pronucleus (30), which strengthens the function of HIRA in binding to the core histones. Here we show that both Drosophila protamines are not removed from the male pronucleus in sesame mutants. This lets us propose that the transport and incorporation of histones onto the chromatin in some manner is coupled to the removal of protamines in which HIRA could play an important role in the multiprotein complex required in this chromatin reconstitution process. We also show here that Mst77F removal from the male pronucleus in contrast to protamines is independent of HIRA.

During spermiogenesis, chromatin reorganization of the complete genome is an essential feature for male fertility. This process leads to an extremely condensed state of the haploid genome in the sperm and requires a reorganization of the paternal genome in the male pronucleus during fertilization and before zygote formation. With the characterization of the chromatin condensing proteins in Drosophila, it would be possible to gain more insight into the mechanisms of sperm chromatin reorganization during spermiogenesis and fertilization.

Supplementary Material

[Supplemental material]


We thank B. Loppin and P. Couble for the sesame mutants, M. T. Fuller for the ms(3)nc3 mutant, R. Saint for the His2AvDGFP fly strain, and C. Zuker, B. T. Wakimoto, and D. L. Lindsley for providing the male sterile mutant stocks. We thank Heike Sauer for excellent secretarial assistance and Ruth Hyland and Dominik Helmecke for excellent technical assistance. We thank Anton Grootegoed and Christoph Kirchner for critical reading of the manuscript.

This research was supported by the Deutsche Forschungsgemeinschaft within the European Graduate Program GRK 767 “Transcriptional Control in Developmental processes” and the Forschergruppe “Chromatin-mediated Biological Decisions” (RE 628/12-1) to R.R.-P.


 Supplemental material for this article may be found at


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