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Cell Biology International (2010) 34, 991–996 (Printed in Great Britain)
The role of Drosophila hyperplastic discs gene in spermatogenesis
Julia A Pertceva†, Natalia V Dorogova*, Elena U Bolobolova*, Olga O Nerusheva*, Svetlana A Fedorova* and Leonid V Omelyanchuk†‡1
*Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia, †Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia, and ‡Novosibirsk State University, Novosibirsk 630090, Russia
In Drosophila, the ubiquitin ligase Hyd (hyperplastic disc) is required for regulation of cell proliferation during development [Martin et al. (1977) Dev Biol 55, 213–232; Mansfield et al. (1994) Dev Biol 165, 507–526]. Earlier, we demonstrated that the Drosophila tumour suppressor Merlin participates not only in imaginal discs proliferation control, but also performs a separate Nebenkern structural function in Drosophila spermatogenesis [Dorogova et al. (2008) BMC Cell Biol 9, 1. Here, we show that the hyd mutants also have spermatogenesis defects: chromosome condensation and attachment to the spindle, centrosome behaviour and cytokinesis in meiosis. The process of spermatid elongation was also greatly affected: nuclei were scattered along the cyst and had an abnormal shape, Nebenkern–axoneme angular relation and attachment was distorted, axonemes themselves lost correct structure. Since Hyd and pAbp protein families share a common PABC [poly(A)-binding protein C-terminal] protein domain, we also studied spermatogenesis in pAbp homozygotes and found defects in cytokinesis and spermatid elongation. However, our study of hyd and pAbp genetic interaction revealed only the phenotype of defective nuclei shape at the final stage of spermatid differentiation. So, the PABC domain is unlikely to be responsible for meiotic defects. Thus, our data document that, in addition to the tumour suppressor Merlin, another tumour suppressor, Hyd, also has a function in spermatogenesis. Key words: Hyd, pAbp, spermatogenesis, meiosis, spermatid, tumour suppressor Abbreviations: GFP, green fluorescent protein, Hyd, hyperplastic disc, PABC, poly(A)-binding protein C-terminal 1To whom correspondence should be addressed (email ome@mcb.nsc.ru). 1. Introduction In Drosophila, Hyd (hyperplastic disc) is required for the regulation of cell proliferation during development (Martin et al., 1977; Mansfield et al., 1994), with mutations in the hyd gene resulting in developmental abnormalities that include adult sterility caused by germ cell defects (Callaghan et al., 1998). A later study showed that in eye imaginal disc, Hyd function differs from what is expected for conventional tumour suppressor behaviour (Lee et al., 2002). It happens that in eye disc, homozygous hyd mutant clones induce non-autonomous overproliferation of nearby tissue directed by ectopic expression of Hh morphogene within the clones mediated by Hh regulator Ci. However, in the posterior compartment of the wing disc, this is not the case, and homozygous hyd mutation induces cell autonomous clone overgrowth by a separate mechanism (Lee et al., 2002). The tumour suppressor nature of Hyd protein was supported by the studies in mammals (Callaghan et al., 1998; Clancy et al., 2003; Fuja et al., 2004). Structurally, Hyd protein family ligases contain an ubiquitin-associated domain at their N-termini, two nuclear localization signals, a zinc-finger-like UBR domain involved in recognition of type 1 N-terminal regions (Tasaki et al., 2005), a domain highly homologous with the PABC [poly(A)-binding protein C-terminal] domain and a HECT domain at their extreme C-termini. While the HECT domain is a common feature of E3 ubiquitin ligases, the presence of PABC in Hyd is unique. PABC domains are commonly found in PABP proteins, whose function is related to mRNA translation. A number of studies document that the function of PABC is clearly different from the function of ubiquitin ligases (Khaleghpour et al., 2001; Kozlov et al., 2001; Roy et al., 2002; Uchida et al., 2002). Our evolutionary study showed that the PABC domain was transferred to the Hyd protein at the time when primitive metazoa emerged (Omelyanchuk et al., 2009a). The study of tumour suppressor genes in Drosophila melanogaster showed that such genes are active in a variety of tissues, but that only one, or at most two, cell types become malignantly transformed by any particular mutation (Gateff, 1994). This means that tumour suppressor proteins may have different functions in different tissues. Earlier, we demonstrated that Drosophila tumour suppressor Merlin participates not only in imaginal discs proliferation control, but also performs a separate Nebenkern structural function in Drosophila spermatogenesis (Dorogova et al., 2008). Here, we asked similar question for the Drosophila tumour suppressor Hyd. 2. Materials and methods 2.1. Fly stocks Flies were maintained at 25°C in standard cornmeal yeast–agar medium. This and our other experimental procedures used, followed internationally recognized guidelines. We used Bloomington stocks: kniri−1 hyd15 e1/TM3, Sb1 (3718) and y1 w1118; PBac{3HPy+}hydC017/TM3, Sb1 Ser1 (16256) as the source of hyd alleles. In addition, we used an allele hydhs1 obtained from P. Bryant (University of California). Following Bloomington, stocks were used as a source of pAbp alleles: w1118; P{EP}pAbpEP310 (17261), y1 w67c23; P{EPgy2}pAbpEY11561 (20684), y1 w67c23; P{lacW}pAbpk10109/CyO (10970). GFP (green fluorescent protein)-tag PTT insertion (37-2) for the pAbp (CG5119) gene was kindly provided by A. Debec (Université Pierre et Marie Curie, Observatoire Océanologique, Villefranche-sur-mer, France) and was balanced by the following way: w1118; P{w+mC = PTT−GA}pAbp/CyO, y+. 2.2. Antibody staining The procedures of testis preparation and mounting was described earlier (Dorogova et al., 2008). We used a mouse anti-γ-tubulin antibody (1:500 dilution; Sigma Chemicals) and a rabbit anti-α-tubulin antibody (1:100 dilution; Sigma Chemicals). After slide washing with 0.1 M PBS three times, a secondary antibody conjugate [Alexa 488-conjugated anti-mouse IgG (1:400 dilution) and a FITC-conjugated anti-rabbit antibody IgG (1:100 dilution)] was added for 2 h at room temperature. In some experiments, nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (1.5 μg/ml). 2.3. Visualization of mitochondria using MitoTracker Red Dissected testes were fixed in a drop of 0.25% glutaraldehyde in 100 mM sodium cacodylate buffer, pH 7.4, for 1 min. Then, testes were additionally fixed in 1.85% formaldehyde in 0.1 M PBS, pH 7.4, for 2 min. Preparations were washed once in 0.1 M PBS containing 0.5% Triton X-100 and then also washed twice in 0.1 M PBS. The stock 10 mM MitoTracker Red solution was diluted 1000 times in 0.1 M PBS and then was used to stain the testes for 5 min at room temperature. Preparations were then washed in 0.1 M PBS with 4 mM Pipes (pH 7.2) for 20–30 min, stained by DAPI and mounted as described in the work of Dorogova et al. (2008). 2.4. Electron microscopy The procedure coincides with that already described in the work of Dorogova et al. (2008). 2.5. Insertion site GFP-tag for the pAbp gene gives an opportunity to visualize the localization of the protein in the cell. To determine the location of artificial GFP exon within the pAbp gene, we extracted genomic DNA from w1118; P{w+mC = PTT−GA}pAbp/CyO, y+ flies, cut it with Kzo 9I restrictase, ligated it into circular form and amplified DNA fragments with internal PTT primers CCTTTCACTCGCACTTATT and GTGAGACAGCGATATGATTGT. The resulting DNA fragment was amplified with the same primers and sequenced with the TATCGCTGTCTCACTCAG primer. Sequence analysis showed that the PTT element was inserted into 2R chromosome arm at 14029525 bp of chromosome contig AE013599.4. In other words, protein-tag P-element is located in the second intron of pAbp gene at the position 1942 when counting from the gene transcription start. 3. Results 3.1. pAbp phenotype and localization Flies carrying hetero-allelic combinations of three available pAbp alleles are viable, and their spermatogenesis can be analysed by fluorescence microscopy (see Materials and methods section). Among homozygotes, viable flies can be found only in the case of pAbpEP310 allele. Most spermatogenesis abnormalities were found in the case of pAbpk10109/pAbpEY11561 heterozygotes. Those include absence of meiotic cytokinesis (5% cases, 25 among 500 meiotic cells), resulting in formation of polyploid spermatids (Figures 1E–1G) and defects in spermatid elongation (in about 100% of the cases, 1000 spermatids) (Figure 1H). Nuclei fail to elongate, keep a round shape and are dispersed along the cyst instead of grouping at the head of the cyst. Normally, mitochondrial DNA can be visualized by DAPI staining (Figures 1A–1C). In the case of pAbpk10109/pAbpEY11561 Nebenkern DAPI staining appears to be non-uniform, probably reflecting the effect of mutation on mitochondrial DNA organization at least in 60% of the cases (about 1000 cells inspected) (compare Figures 1A and 1G). The MitoTracker staining reveals no essential differences between the mutant and control showing that mitochondrial membranes keep their integrity, but in a mutant, the onion-stage Nebenkern looked larger than in a control, and its morphology differs from a perfect round (Figures 1B and 1F). Electron microscopic observations of Nebenkern structure in elongating onion and elongating spermatids of pAbpk10109/pAbpEY11561 males reveal abnormal mitochondrial membranes at onion-stage spermatids in all the cases studied (Figure 2B). Figure 2(D) also documents the abnormal shape of mitochondrial derivatives and its defective attachment to axonemes in the elongating spermatids. We detected the presence of a chimaeric protein in the cytoplasm of spermatocytes (Figure 3). In meiosis and onion stage spermatids, the level of the protein greatly decreased. Despite low viability, homozygotes for P{w+mC = PTT−GA}pAbp insertion can be found. We have not found any spermatogenesis abnormality in those homozygotes. The intracellular localization of the chimaeric protein in homo- and heterozygotes is the same. Thus, the chimaeric pAbp protein is fully functional in spermatogenesis, and its localization reflects the true pAbp distribution within the cell. 3.2. hyd phenotype In the case of three hyd alleles, viable flies were seen in the hydhs1/hydC017, hyd15/hydC017, hydC017 cases. Spermatogenic cells in hydC017 homozygotes could not develop up to spermatocytes. Qualitatively, the abnormalities in the case of hydhs1/hydC017 are the same with hyd15/hydC017, but are expressed stronger than in the former case [100% of cysts (among 100 inspected) contain at least one type of defect]. Figure 4(A) shows nuclei scattered through the elongated cysts and have a round shape instead of a needle one. In rare cases, the group of needle-shaped nuclei can be found in the correct place in the cyst; however, some of the nuclei are evidently incorrectly condensed (Figure 4B). About 90% of nuclei and micronuclei do not reach the correct cyst pole (totally about 100 cysts inspected). The formation of two spindles in one cell in meiosis we found in the case of hyd15/hydC017 shows the defect of meiotic cytokinesis (Figure 5A). We also note that the meiotic products in the mutants may have unequal amounts of chromatin or mitochondrial material. The example given in Figure 5(B) shows that onion-stage spermatid nuclei and Nebenkerns have unequal size. Centrosome behaviour is also affected. Figure 6 demonstrates the meiotic cell bearing four centrosomes, four asters per one nucleus. The formation of such a cell must be the consequence of incorrect centrosome disjunction. Really, in wild-type, the formation of meiotic asters takes place just before meiosis (Figure 7A). Some cells in the mutant at this stage completely lack an opportunity to organize meiotic cytoskeleton and particularly spindle asters (Figure 7B). Figure 7(C) depicts two cells, the upper one lacking a nuclei representing a product of meiosis-1 connected with the chromatin bearing a cell with two centrosomes and two asters separated just before the second meiotic division. Those examples show that meiotic chromosomes were not correctly attached to the spindle (this takes place in 50% of the meiotic figures: 100 among 200 meiotic cells in analysis). Spermatid nuclei vary in size; this is evident from the observation of different spermatogenesis stages, including the late ones. Elongated spermatid cyst in Figure 7(D) gives an example. Some of the nuclei contain only a small amount of chromatin and definitively constitute a single chromosome. We studied the process of spermatid differentiation in hyd15/hydC017 males by electron microscopy. Cross-sections depicted in Figures 8(A) and 8(B) document a cyst, containing less than 64 spermatids. Since the axoneme grows from a single centrosome attached to spermatid nuclei (Lindsley and Tokuyasu, 1980), the cysts shown in Figure 8 may be the consequence of abnormal centrosome distribution in meiosis. It can also be seen that one axoneme can be attached to a few mitochondrial bodies (Figure 8B), and one mitochondrial body can be attached to two axonemes (Figure 8A). All this shows that, in addition to the focus of action in meiosis, hyd is also functioning in the process of spermatid differentiation. 3.3. hyd and pAbp Genetic interaction Since pAbp and Hyd proteins have a common PABC domain, we checked the genetic interaction of their mutation in the diheterozygous condition. All possible diheterozygous combinations of pAbp and hyd alleles were fertile. Among those combinations, only pAbpk10109/hydhs1 and pAbpk10109/ hyd15 variants demonstrate some degree of spermatogenesis defects. In the case of pAbpk10109/hydhs1, 30% of head bundles in the sperm contain both round- and needle-shaped nuclei and look like those shown in Figure 4(B) (100 among 300 cysts). Nuclei scattering was mentioned but at a low frequency. In the case of pAbpk10109/ hyd15, the morphology of nuclei in the sperm head bundle was indistinguishable from the control, but the nuclei were frequently scattered instead. Also, in those two diheterozygotic combinations, some preponderance of cytokinesis abnormalities (in comparison with other diheterozygotic combinations) were found. 4. Discussion Human pAbp protein was identified on the basis of homology with Drosophila Hyd protein (Callaghan et al., 1998). D. melanogaster has a homologous protein pAbp (P21187), constituting the CG5119 ORF (open reading frame), whose molecular function was defined upon protein domains (four RRM and one PABC domain) binding to the 3′ poly(A) tail of mRNA and have very important roles in the pathways of gene expression. The protein provides a scaffold on which other proteins can bind and mediate processes such as export, translation and turnover of the transcripts. Moreover, pAbp acts as antagonists to the binding of factors that allow mRNA degradation, regulating mRNA longevity. Our analysis of pAbp function in Drosophila spermatogenesis reveals defects in cytokinesis and spermatid elongation. Male meiotic spindle defect in the pAbp (duo) mutant was described earlier (Fasulo et al., 1999). However, we have not found disconnection of asters and central mini-spindle, described in this thesis, despite trying to find this effect. The authors also described cytokinesis defects in this case, and this agrees with our finding. Insignificant discrepancy between those data may be a consequence of the different allelic combinations used. Moreover, in the RNAi (RNA interference) screening for cytokinesis regulators in Drosophila tissue culture, the pAbp was identified as one of the essential cytokinesis genes (Eggert et al., 2004). Thus, all the data agree well and show that protein has cytokinesis function both in meiosis and mitosis. The localization of pAbp in spermatogenesis almost perfectly coincides with that found in the case of the mRNA localization protein Imp (Nerusheva et al., 2009). The only difference found was that the Imp concentrates at the caudal end of fully elongated cysts, while pAbp did not. This structure, we named ‘pottle’ is currently thought of as the major protein synthetic activity site during spermatid elongation (Lindsley and Tokuyasu, 1980; Fuller, 1993). Thus, in general, pAbp intracellular localization follows the expected from its protein domain structure. If so, we must attribute the effect of pAbp mutations in meiotic cytokinesis and in the spermatid elongation to the disruption of its function in mRNA translation. The range of spermatogenesis abnormalities in the case of hyd mutants was significantly wider: chromosome condensation and attachment to the spindle, centrosome behaviour and cytokinesis in meiosis are greatly affected. Despite of the efforts done, we were not able to draw a conclusion as to what process is the primary target of hyd mutation in meiosis; it looks like all the processes were disrupted simultaneously, and the general picture of the abnormalities qualitatively differ from those observed in the cases where the structural meiotic protein was destroyed. The process of spermatid elongation was also greatly affected, nuclei are scattered along the cyst and have abnormal shapes; Nebenkern–axoneme angular relation and attachment is distorted; axonemes, themselves, may lose their correct structure. All this shows the presence of an additional hyd target in spermatid differentiation. Our study of hyd and pAbp genetic interaction revealed only the phenotype of defective nuclei shape at the final stage of spermatid differentiation. This suggests that, despite of the presence of common PABC domain, the proteins may have the only common target in spermatid differentiation, while all other spermatogenesis targets may differ. Looking at the spectrum of phenotypes of pAbp and hyd mutants, it is tempting to suppose that their effects may both be due to mRNA translation and the PABC domain. It would be more adequate to relate Hyd effects with its ubiquitin–ligase activity. This point agrees well with the already established role of the ubiquitin-dependent proteolytic system in spermatogenesis of mammals (Baarends et al., 2000). In Drosophila, the phenotype of scattered nuclei produced by the mutation in a gene cbx, coding for ubiquitin–ligase was also described (Fabrizio et al., 1998). Author contribution Julia Pertceva and Natalia Dorogova performed the cytological study of hyd and pAbp mutants and studied the phenotype of pAbp–hyd genetic interaction. Elena Bolobolova carried out the electron microscopy. Olga Nerusheva performed a pilot study of pAbp–GFP protein distribution in spermatogenesis and pAbp–hyd genetic interaction. Svetlana Fedorova performed the Feulgen–Giemsa study of hyd and pAbp–hyd mutants, supporting the data presented in the manuscript. Leonid Omelyanchuk helped in the design, co-ordination, writing, submission and correction of the manuscript. Acknowledgements We are grateful to A. Debec for the PTT insertion in the pAbp gene. Funding This study was supported by the REFERENCES Baarends, WM, van der Laan, R and Grootegoed, JA (2000) Specific aspects of the ubiquitin system in spermatogenesis. J Endocrinol Invest 23, 597-604 Callaghan, MJ, Russell, AJ, Woollatt, E, Sutherland, GR, Sutherland, RL and Watts, CK (1998) Identification of a human HECT family protein with homology to the Drosophila tumor suppressor gene hyperplastic discs. 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ISSN Print: 1065-6995
ISSN Electronic: 1095-8355 Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB) |
Abstract
