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The Drosophila parkin homologue is required for normal mitochondrial dynamics during spermiogenesis
Developmental Biology 303 (2007) 108 – 120 www.elsevier.com/locate/ydbio
The Drosophila parkin homologue is required for normal mitochondrial dynamics during spermiogenesis Maria Giovanna Riparbelli, Giuliano Callaini ⁎ University of Siena, Department of Evolutionary Biology, Via A. Moro 4, I-53100 Siena, Italy Received for publication 12 July 2006; revised 20 October 2006; accepted 26 October 2006 Available online 10 November 2006
Abstract Drosophila parkin, the ortholog of the human parkin gene, responsible for a familiar form of autosomal recessive juvenile parkinsonism, has been shown previously to be involved in Drosophila male fertility. Loss-of-function mutations in the parkin gene cause failure of spermatid individualization by affecting the proper progression of the actin-based investment cones that assemble in the nuclear region, but fail to translocate in synchrony down the cyst. In parkin mutants, the investment cones are scattered along the post-elongated spermatid bundles and fail to act properly in the process of sperm individualization. Using phase-contrast and electron microscopy analysis, we demonstrate that the parkin spermatids assemble a seemingly normal onion-stage nebenkern, but when the axoneme elongates only one mitochondrial derivative unfurls from the nebenkern. This unique mitochondrial derivative undergoes abnormal shaping and condensation during spermatid elongation. Our results indicate that parkin gene function is necessary for mitochondrial morphogenesis during earlier and later phases of spermiogenesis. The failure of cyst individualization may be due to the sensitivity of investment cone movement to the perturbation of mitochondrial morphology during spermatid elongation. © 2006 Elsevier Inc. All rights reserved. Keywords: Drosophila; Parkin; Spermatogenesis; Individualization complexes; Mitochondria
Introduction Spermiogenesis in Drosophila melanogaster is a complex process that involves a series of elaborate transformations of the spermatid components. These dramatic transformations from an immotile cell to a motile one include chromatin condensation, cell and axoneme elongation, mitochondrial rearrangement, and sperm individualization (Fuller, 1993). Successful spermiogenesis requires the proper execution of these developmentally regulated processes. Recent investigations have focused attention on the individualization process at the end of spermiogenesis. This process is mediated by 64 distinct conical complexes, or individualization complexes (ICs) (Tokuyasu et al., 1972), mainly composed of actin. The actin cones, or investment cones, move along the axoneme from the nucleus to the tail end, sequestering the excess cytoplasm, remodeling the minor mitochondrial derivative, and completing the uneven spermatid ⁎ Corresponding author. Fax: +39 577 334476. E-mail address: [email protected] (G. Callaini). 0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.10.038
plasma membrane to enclose each sperm axoneme. Some cytoskeletal proteins, such as unconventional myosin V and VI (Rogat and Miller, 2002; Mermall et al., 2005; Noguchi et al., 2006), dynein (Li et al., 2004; Ghosh-Roy et al., 2005), and actin (Noguchi and Miller, 2003), seem to be directly involved in the individualization complex function. Although the cell remodeling mechanisms that transform fully elongated spermatids into individual sperm are understood to some extent, much less is known about the morphogenetic processes of the earlier phase of spermatid elongation, including mitochondrial transformation and tail growth. Our knowledge of this early phase of sperm formation is mainly based on morphological studies in which cell remodeling was investigated by transmission electron microscopy (Tokuyasu et al., 1972; Tates, 1971). However, these studies did not investigate in detail the mechanisms driving cell elongation and sperm tail growth. Recent observations suggest that axoneme elongation in Drosophila does not require the ubiquitous process of intraflagellar transport (Han et al., 2003; Sarpal et al., 2003), but relies on unknown mechanisms, perhaps in part driven by the
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cytoskeletal reorganization mediated by cytoplasmic dynein– dynactin complex (Ghosh-Roy et al., 2004). Genetic analysis indicates that genes involved in vesicular transport and fusion may play a major role in sperm cell growth (Bergeret et al., 2001; Farkas et al., 2003). Although mitochondrial behavior during spermiogenesis is fairly well understood, the molecular pathway involved in this complex process of transformation remains obscure. It is, for example, still unclear which signals direct the aggregation of the mitochondria in two equivalent and distinct components, how these structures wrap to form the onion-stage nebenkern, and how the onion resolves into two derivatives that undergo different fates during later spermiogenesis. The study of mutations that alter the usual pattern of mitochondrial transformation could help decipher the role of these organelles during sperm maturation and provide further insight into mitochondrial biogenesis. Mutational analysis has identified several phenotypes in which sperm do not undergo proper maturation and show slight defects of mitochondrial organization within the tail (Fabrizio et al., 1998; Castrillon et al., 1993; Wakimoto et al., 2004). These studies, however, were not specifically designed to elucidate the relationship between sperm maturation and mitochondrial transformation and did not investigate mitochondrial morphogenesis in detail. Up to now, only two genes, fuzzy onion (fzo) (Hales and Fuller, 1997) and rhomboid-7 (McQuibban et al., 2006), have been characterized and correlated with the process of mitochondrial dynamics and morphogenesis in Drosophila spermatogenesis. Mutations in these genes, that encode proteins required for the developmentally regulated fusion of mitochondria, result in onion-stage nebenkern defects and male sterility. Loss-of-function mutations in the parkin gene, the Drosophila ortholog of the human parkin gene, responsible for a familiar form of Parkinson disease known as autosomal recessive juvenile parkinsonism (AR-JP), result in sperm with abnormally sized mitochondrial derivatives (Greene et al., 2003). This indicates a potential function of this gene in mitochondrial morphogenesis during sperm maturation in the fly, especially when combined with the finding that mitochondrial pathology is a prominent feature in flight muscles of Drosophila parkin null mutants (Greene et al., 2003; Pesah et al., 2004). In addition, the Drosophila homologue of a human AR-JP-linked gene, PTEN-induced putative kinase 1 (PINK1), acts in a common pathway with parkin in maintaining mitochondrial integrity; the ortholog of PINK1 is required for proper mitochondrial structure and function in Drosophila (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). We confirm by careful cytological analysis a previous hypothesis (Greene et al., 2003) that mutations in parkin do not affect mitochondrial dynamics during spermatogenesis, but rather lead to the failure of proper remodeling of these organelles during earlier post-meiotic stages. We demonstrate that only one mitochondrial derivative assembles from the onion-stage nebenkern in parkin sperm. This unique mitochondrion behaves irregularly during spermatid elongation, leading to defective progression of the individualization complexes and thus to the failure of sperm maturation.
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Mitochondrial dysfunctions have been suggested for a key role in Parkinson disease in humans (for a review, see Beal, 2003; Shen and Cookson, 2004). However, definitive evidence is lacking. The careful analysis of a system, such as spermiogenesis in Drosophila, in which strictly regulated mitochondrial dynamics is required for proper cell development and differentiation could be useful to investigate potential functions and cellular targets of the parkin gene. Materials and methods Drosophila strains and crosses All fly stocks were maintained and crossed on standard culture medium at 24°C. Since the stock containing park[25] also carries a linked homozygous lethal, we crossed this strain with flies carrying the Df(3L)Pc-MK deletion to obtain the viable null utilized for cytological studies. Oregon-R flies and the revertant park[rvA] line were used as controls to obtain wild-type testes. The Df (3L)Pc-MK deletion was obtained from the Bloomington Drosophila Stock Center (Bloomington, IN). park[25] and park[rvA] lines were a generous gift of Leo Pallanck (University of Washington, Seattle, WA, USA).
Reagents A mouse monoclonal anti-β-tubulin (Boehringer, Mannheim UK) was used at a 1:200 dilution; a rabbit anti-Pav-KLP polyclonal Rb3301 (Adams et al., 1998) at 1:100. Goat anti-mouse or anti-rabbit secondary antibodies coupled to fluorescein or rhodamine (Cappel, West Chester, PA) were used at 1:600 dilution. DNA was visualized with Hoechst 33258 (Sigma, St. Louis, MO). The actin cytoskeleton was visualized with rhodamine-phalloidin (Molecular Probes, Eugene, OR) prepared by dissolving 10 μl of vacuum-dried methanol stock solution in 100 μl of PBS. Bovine serum albumin (BSA) was obtained from Sigma.
Examination of spermiogenesis by phase-contrast microscopy Cytological preparations were made with testes from 3- to 4-day-old pupae and from 4-day-old adult males. Testes were dissected in phosphate-buffered saline (PBS) and transferred to a drop of PBS on a glass slide. The testes were open and squashed under a cover slip. The morphology of early spermatids was analyzed using an Axio Imager Z1 (Carl Zeiss, Jena, Germany) microscope equipped with 100× phase-contrast objective. Images were obtained with an AxioCam HR cooled charge-coupled camera (Carl Zeiss).
Immunofluorescence Immunostaining of testes from pupae, newly eclosed, and older flies was performed by either the methanol/acetone fixation method or by the ethanol/ formaldehyde fixation method (Glover and Gonzalez, 1993; Cenci et al., 1994; Hime et al., 1996). After fixation, the material was washed in PBS and incubated 1 h in PBS containing 0.1% bovine serum albumin (PBS-BSA). For localization of microtubules and Pav-KLP, testes were incubated overnight at 4°C with the PAV-KLP antisera and then with anti-β-tubulin antibody for 4–5 h at room temperature. After washing in PBS-BSA, the slides were incubated 1 h at room temperature with the appropriate secondary antibodies. For actin and tubulin visualization, the slides were incubated with anti-βtubulin antibody for 1 h at room temperature. After washing in PBS, the slides were incubated 1 h in the appropriate secondary antibody to which rhodaminelabeled phalloidin was added. Nuclei were then visualized with incubation for 3–4 min in Hoechst. Testes were mounted in small drops of 90% glycerol containing 2.5% n-propyl-gallate. The fluorescent images were taken by using an Axio Imager Z1 (Carl Zeiss) microscope equipped with an HBO 50-W mercury lamp for epifluorescence and with an AxioCam HR cooled charge-coupled camera (Carl Zeiss). Gray-scale
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digital images were collected separately and then pseudocolored and merged using Adobe Photoshop 7.0 software (Adobe Systems).
Transmission electron microscopy Testes dissected in PBS from 3- to 4-day-old pupae, and from young (1 to 2day-old) and old (15 to 20-day-old) adult males were fixed for 2 h at 4°C in 2.5% glutaraldehyde in PBS. After rinsing 20 min in PBS, the material was post-fixed in 1% osmium tetroxide in PBS. After extensive washing in distilled water, the samples were dehydrated in a graded series of alcohols, embedded in an Epon– Araldite mixture, and polymerized at 60°C for 48 h. Ultrathin sections obtained with an LKB Nova ultramicrotome were collected on copper grids and stained with uranyl acetate and lead citrate. Sections were observed with a Philips CM10 electron microscope.
Spermatid length measurements Since mutations in parkin result in sperm individualization defects and empty seminal vesicles, to obtain more reliable data, we measured fully elongated spermatid cysts from both mutant and control testes. Testes from 4- to 5-day-old adult males were dissected in PBS, fixed as described above, and stained for F-actin to visualize ICs. Fully elongated spermatid cysts can be unambiguously recognized on the basis of the presence of actin-rich cones that make their appearance after the cellular elongation has been completed. Seventy fully elongated spermatid cysts were scored for mutant and control males each. The measures were performed under a fluorescence microscope connected to a CCD camera, as previously described, using the AxioVision 4.0 software (Carl Zeiss).
Results Investment cones move asynchronously through parkin spermatid bundles In Drosophila spermatogenesis, cysts of 16 primary spermatocytes undergo two meiotic divisions to produce 64 haploid round spermatids interconnected by cytoplasmic bridges. Mutations in the steps of this process lead to the production of abnormal spermatids that arrest in development or undergo abnormal differentiation, leading to male sterility. It was previously demonstrated that, in parkin males, spermatogenesis is normal until post-meiotic spermatid differentiation (Greene et al., 2003). The primary spermatocytes enter meiosis to produce syncytial cysts of 64 round spermatids, as in wild-type. However, while wild-type seminal vesicles accumulate motile sperm, in mutant testes, the seminal vesicles are devoid of coiled mature sperm. To pinpoint the specific stage of spermiogenesis that requires parkin function, we compared wild-type and parkin spermatids at various stages of elongation. Post-meiotic spermatids undergo extensive syncytial cellular differentiation including chromatin condensation, mitochondrial remodeling, axoneme assembly, spermatid elongation, individualization, and coiling. The 64 interconnected spermatids develop in a syncytium that extends through the length of the testis. Elongating spermatids are orientated with their heads (apical region of the cyst) toward the basal end of the testis and their tail ends (distal region of the cyst) toward the apical region of the testis. Spermatid elongation in parkin mutants follows the usual pattern observed in wild-type testis: the nuclei align within the cyst opposite the tip of the tails. At the distal region of the
spermatid cyst, the ring canals form a cluster that becomes more compact with the elongation process. The proper execution of cytokinesis in germ cells can be unambiguously detected by the presence of ring canals. Since ring canals are formed by stabilization of the contractile rings following cytokinesis, they can be readily observed with antibodies against the kinesin-like protein encoded by pavarotti (Pav-KLP) that is associated with the central region of the spindle at anaphase/telophase and is required for the correct organization of the spindle during cytokinesis (Adams et al., 1998). This protein associates with the ring canals which form at the sites of incomplete cytokinesis in the germline cysts of both testes (Carmena et al., 1998) and ovaries (Minestrini et al., 2002). Staining for Pav-KLP shows that, as spermatids enter the elongation process, the ring canals are driven toward the distal region of the spermatid bundle in both wild-type (Fig. 1A) and mutant (Fig. 1B) testes. To determine whether the elongation process proceeds normally in mutants, we measured fully elongated cysts at the beginning of individualization in both park and control testes. The overall length of the fully elongated spermatid cysts varies from 1.65 to 2.03 mm in parkin and 1.85 to 2.13 mm in control flies. We cannot, however, exclude the presence within mutant fully elongated cysts of some shorter spermatids. After elongation, the syncytial spermatids become mature sperm cells. This process is driven by an individualization complex that translocates along the length of the spermatid bundles from the nuclei to the tail ends, eliminating excess cytoplasm and surrounding each spermatid in its own plasma membrane. Thus, the failure of spermatid maturation in parkin mutants might result from sperm individualization defects as demonstrated in other mutants (Fabrizio et al., 1998). Since the major component of the individualization complex is a cone-shaped actin-rich structure, we compared wild-type and mutant spermatid bundles at the end of elongation phase, using markers for F-actin, microtubules, and DNA, to more closely examine the possibility that the parkin phenotype might be correlated to individualization defects. In normal development, the elongated spermatid nuclei align in parallel at the apical region of each cyst. F-actin staining is absent or gives only a feeble signal in this region when spermatid nuclei have partially condensed their chromatin. As the chromatin condenses further and the spermatid nuclei become needle-shaped, Rh-phalloidin labeling reveals distinct thin cone-shaped structures associated with the aligned nuclear bundles, representing the actin-rich investment cones (ICs) that have just formed but have not yet begun the individualization process (Fig. 1C). In testes from park mutants, not all the spermatid nuclei are aligned in register; some of them are mispositioned along the cyst length. Actin staining was associated with both aligned and out-of-place nuclei (Fig. 1D). In wild-type testes, the actin-rich ICs traverse the cyst in a compact mass from the spermatid heads to the tips of the tails. This was clearly visualized by Rh-phalloidin staining showing distinct parallel bundles of ICs at various distances from the nuclei of different spermatid bundles (Figs. 2A, B). As they move, the ICs resolve the syncytial spermatids into individual sperm cells, and excess cytoplasm, membranes, and the ICs
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Fig. 1. Fluorescence preparations of wild-type (A) and parkin (B) elongating spermatids stained for tubulin (green), Pav-KLP (red) and DNA (blue). Ring canal remnants (arrows) are found at the growing edge of the spermatid bundles in both wild-type (A) and parkin (B) spermatids; note that Pav-KLP also accumulates at the poles (smaller arrowheads) and midzone (larger arrowhead) of late anaphase spindles. Nuclear region of pre-individualization wild-type (C) and parkin (D) spermatids stained for tubulin (green), F-actin (red) and DNA (blue). Nuclei are aligned in wild-type (C) and investment cones (ICs) are associated with the posterior nuclear heads (arrows); by contrast, some nuclei (arrows) and ICs (arrowheads) are scattered in parkin (D). Scale bar: 20 μm.
themselves are collected in large “waste bags” at the tip of the spermatid tail (Fig. 2C). In mutant testes, the ICs were located in positions distant from the nuclear region, indicating that they have translated along the spermatid bundles. However, they were not aligned as in wild-type, but were scattered along the sperm tail, suggesting that they did not move in synchrony (Fig. 2D). The individual ICs are cone-shaped as in wild-type. Normally, their apex points toward the apical region of the cyst, where the nuclei are aligned, and their basis is toward the tips of the tails (Fig. 2E). Among the fully elongated spermatid cysts examined (n = 189), 19% of them (n = 36) display 9–10% of misoriented ICs. Fig. 2F shows a detail of a spermatid bundle in which scattered ICs had their pointed ends oriented in opposite directions. “Waste bags” in parkin testes were not distinct as in wild-type, suggesting that the process of individualization is not properly executed in these mutants. This was confirmed by the finding of irregularly coiled distal ends of the spermatid bundles (Fig. 2G).
the end of meiosis. In early spermatids, the aggregated mitochondria form a spherical compact structure, the onionstage nebenkern, recognizable in phase-contrast microscopy as a dark round structure, closely apposed to the white round spermatid nucleus. No significant differences in nebenkern morphology were observed in young wild-type (Fig. 3C) or mutant (Fig. 3D) spermatids. During earlier stages of elongation, the round wild-type spermatids still show a spherical nucleus, but the nebenkern starts to elongate and a distinct paired structure is readily observed (Fig. 3E). park mutant spermatids at the same stage show an elongated nebenkern that does not resolve into two parts (Fig. 3F). Thus, in parkin spermiogenesis, there is an obvious departure from normal development during the first steps of mitochondrial morphogenesis.
parkin spermatids have only one mitochondrial derivative
Examination of parkin testes using electron microscopy confirmed defects in the remodeling of the mitochondria during early stages of spermatid differentiation. Cross-sections of round post-meiotic wild-type spermatids at the onion stage reveal that the mitochondria cluster in a spherical structure slightly larger than the nucleus (Fig. 4A). As expected from the phase-contrast observations, this structure is
To determine whether the park phenotype was associated with defects of mitochondrial behavior, we did a cytological analysis of wild-type and mutant testes using squash preparations. Mitochondria aggregate in a loose cluster around the nuclei in both wild-type (Fig. 3A) and mutant (Fig. 3B) spermatids at
Early steps of mitochondrial morphogenesis are aberrant in parkin testes
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Fig. 2. IC organization in wild-type (A, B, C) and parkin (D, E, F, G) testes stained with Rh-phalloidin. Wild-type (A) pre-individualization spermatid bundles show orderly arranged cluster of ICs (arrows) that appear conical in shape (B). (C) Detail of “waste bags” showing the presence of ICs with bent pointed ends (arrows). The travel of ICs is not synchronous in parkin testes (D), and they are scattered along the length of the spermatids (arrowheads). Although the apex of the conical ICs is mainly directed toward the head region (arrowheads, E), in some cases, the pointed ends of the ICs are opposed (arrowheads, F). The terminal ends of the individualizing spermatids (arrow, G) are often coiled and lack distinct “waste bags”. Scale bar: 40 μm in panels A, D; 15 μm in panels B, E, F; 12 μm in panels C, G.
also visible in mutant spermatocytes (Fig. 4B). However, whereas mitochondria form concentric ring-like structures in wild-type nebenkern, their arrangement is more irregular in mutant spermatocytes. The parkin nebenkern is, indeed, formed by a large portion of rather ring-like concentric mitochondria, and a small portion of loose peripheral mitochondria, that do not seem to integrate into the tightly packed main cluster (Fig. 4B). The concentric mitochondria, moreover, appear more electrondense in mutant than in wild-type spermatids. The morphogenesis of the nebenkern changes during axoneme elongation. Two mitochondrial derivatives of about the same size are associated with the axoneme in wild-type testes when the spermatid starts to elongate (Fig. 4C). Mitochondria appear in cross-section as large circular structures
surrounding a cytoplasmic round area and containing numerous cristae. Cross-sections through parkin spermatocytes at the same developmental stage reveal only one large mitochondrion associated with the growing flagellum (Fig. 4D). To investigate further mitochondrial morphogenesis, we examined subsequent stages of spermatid elongation and differentiation. The level of the sections in both wild-type and mutant spermatids was compared by examining axoneme decoration. As the axoneme elongates further in wild-type testes, the mitochondrial derivatives shrink and transform into tubular structures of about the same size with a few inner membranes (Fig. 5A). In the axoneme, the B-subfiber of each peripheral doublet shows a short external arm, indicating that accessory tubules have started to assemble from outgrowth of
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Fig. 3. Comparison of earlier stages of mitochondrial morphogenesis in wild-type (left) and parkin (right) testes as viewed by phase-contrast microscopy. Large postmeiotic spermatocytes in both wild-type (A) and mutant (B) testes have many small mitochondria (arrowheads) clustered near the uniformly sized nuclei (arrows). Wild-type (C) and parkin (D) onion-stage spermatids; the nuclei (arrows) and their adjacent nebenkerns (arrowheads) are present in a 1:1 ratio. Wild-type spermatids (E) show pairs of mitochondrial derivatives (arrowheads) associated with round nuclei (arrows) during early elongation stages, whereas parkin mutant spermatids (F) exhibit only one oblong mitochondrial derivative (arrowheads) associated with each nucleus (arrows). The preparation has been flattened slightly, and a few of cytoplasmic bridges between the cells are broken, revealing the cytoplasmic continuity between spermatids. Thus, some of the spermatids appear syncytial due to opening of cytoplasmic bridges between cells during sample preparations. Scale bar: 20 μm.
external microtubule doublets (Fig. 5A, inset). Sections through elongating post-meiotic spermatids of parkin males, in which the B-tubule of the axoneme also shows a thin external arm (Fig. 5B, inset), display mitochondria arranged in irregular rings, either outside of or enveloping the flagellum (Fig. 5B). Cross-sections through mutant spermatid bundles that appear slightly elongated, as judged by the hooked peripheral arms growing by the B-subfiber (Fig. 5C, inset), show both ring-like and spherical mitochondria (Fig. 5C). This suggests that parkin
mitochondria undergo compaction as the spermatid elongates. There was, however, usually only one mitochondrial derivative, and only occasionally did we observe additional mitochondria in the same spermatid. Small light-staining vesicles, containing microtubules, were usually found (Fig. 5C). Two mitochondrial derivatives, clearly different in size and shape, are seen in wild-type pre-individualized spermatogenic cysts. The minor mitochondrial derivative is round and smaller, whereas the major one is larger, ovoid in cross-section and
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Fig. 4. Electron microscopy (EM) analysis of post-meiotic spermatids from wild-type (A, C) and parkin (B, D) testes. The onion-stage nebenkern appears to be formed of concentric cisternae in wild-type (arrow, A) spermatids, whereas it is somewhat irregular in mutant (arrow, B) spermatids; N, nucleus. As the wild-type spermatids (C) start to elongate, a pair of mitochondrial derivatives (arrows) lie adjacent to the axoneme (arrowhead). By contrast, only one large mitochondrion is observed in mutant spermatids (arrow, D). Scale bar: 2.5 μm in panels A, B; 1 μm in panels C, D.
appears slightly darker (Fig. 6A). Dark-staining material accumulates in the major derivative next to the axoneme. Highly organized sheaths of cytoplasmic microtubules surround the mitochondria (Fig. 6B). The axoneme at this point has nine distinct accessory tubules embedded in a dense material (Fig. 6B, inset). Spermatids are still connected by cytoplasmic bridges and the axoneme–mitochondrial complexes are in an orderly arrangement within the cyst. At a comparable developmental stage, the pre-individualization parkin spermatids maintain some cytoplasmic ground substance and are connected by intercellular bridges. The spermatids usually contain a single irregularly shaped and sized mitochondrion surrounded by disorganized sheaths of microtubules (Fig. 6C). These microtubules are scattered in the spermatid cytoplasm and occasionally localized near the axoneme. The organization of the axonemal microtubules is normal (Fig. 6D, inset), whereas the orientation of the mitochondrial derivatives varies within the same cyst. Some
spermatids show abnormally sized circular mitochondria, reminiscent of the mitochondrial whorls observed in crosssections of the wild-type individualization complexes (Tokuyasu et al., 1972). Mitochondrial condensation is highly defective: some mitochondria are filled with dense material in the region adjacent to the axoneme, whereas others do not show appreciable signs of condensation or the condensation is barely detectable. Although the dark material that filled the mitochondria is mostly restricted to the region close to the axoneme, circular mitochondria also display distinct condensation in regions far from the axoneme (Figs. 6C, D). Closely paired axonemes are also found in these cysts (Fig. 6D), whereas wild-type cysts contain evenly spaced axonemes. Detailed examination reveals that the dynein arms often appear oriented both clockwise and anti-clockwise in adjacent parkin axoneme pairs. The dynein arms appear clockwise when cross-sections of the axoneme are seen from head to tail and anti-clockwise when the same sections are seen in the
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7A). Dense material envelops the accessory tubules that are no longer distinguishable. In the same stage in mutant spermatids, one mitochondrion alone, abnormally shaped and of varying size, is usually found (Fig. 7B). Mitochondrial condensation is also defective. We observed mitochondria filled with large dark masses and pale mitochondria with evident cristae remnants in the same cyst (Fig. 7B). The minor mitochondrial derivatives shrink again after the passage of the individualization complex in wild-type. Cross-sections of wild-type individualized spermatid bundles display well-ordered sperm cells with tightly packed condensed major mitochondrial derivatives, each surrounded by its own plasma membrane (Figs. 7C, E). In contrast, sections of mutant spermatids that appear quite mature on the basis of axonemal decoration reveal that only a few spermatids are apparently cellularized (Figs. 7D, F). Most of the spermatids are immersed in a large amount of cytoplasmic ground substance, and their mitochondrial derivatives appear irregularly condensed and abnormally sized. These data suggest that investment cone movement was defective in parkin mutants and that further fusion events could occur within mitochondria. Discussion
Fig. 5. Cross-sections of wild-type (A) and mutant (B, C) elongating spermatids. Mitochondrial derivatives are transformed into pairs of tubular structures in wild-type (arrows, A), whereas they are more convoluted in same-stage parkin spermatids (arrows, B); insets in panels A and B represent magnifications of axonemes showing thin external arms (arrowheads) outgrowing from external microtubule doublets. Slightly elongated parkin spermatids (C) show ring-like (arrows) and spherical (larger arrowheads) mitochondria, together with lightstaining vesicles containing microtubules (smaller arrowheads); note the hooked peripheral arms growing from the B-subfiber (arrowheads, inset). Scale bar: 1 μm in panels A, B, C; 0.3 μm in insets.
opposite direction. This observation could, therefore, indicate that close axoneme pairs are sections of the same flagellum that had folded back on itself. Toward the distal region of post-elongating wild-type spermatid bundles, the size of the mitochondrial derivatives decreases. The major mitochondrial derivative is mostly filled with dark material, whereas the minor one is less dense (Fig.
Syncytial spermatids must be individualized at the end of differentiation in Drosophila to become mature sperm. Cytoplasmic bridges are broken and the cell membranes are sealed, the excess cytoplasmic material within the flagella is stripped away, and mitochondrial derivatives change dramatically in size and shape (Fuller, 1993). Mature sperm can be thus identified on the basis of their full cellularization, the absence of microtubules within the reduced cytoplasm, the small size of the minor mitochondrial derivative, and the large paracrystalline structure within the major mitochondrial derivative. Spermiogenesis appears to proceed normally to the elongated spermatid stage in parkin testes, but most of the spermatids have cytoplasmic bridges, abundant cytoplasm and abnormal mitochondrial derivatives. Only a few spermatids show the usual characteristics of mature sperm, although their mitochondria appear irregularly sized and shaped. These findings are consistent with defects in the process of individualization in Drosophila spermiogenesis, as previously suggested (Greene et al., 2003). This process is achieved by ICs that assemble at the aligned nuclear heads of the spermatid bundle and proceed down the spermatid tails as a linear cluster, resolving the syncytial spermatids of a cyst into 64 cells with individual membranes (Tokuyasu et al., 1972). Since spermatogenesis appears to proceed normally in the mutant, the absence of parkin alone is unable to block the spermatogonial cell divisions and meiosis and, thus, spindle assembly. However, in the absence of parkin, though the actin cones are still formed around the nuclei, their synchronous movement and alignment along the spermatid bundle are disrupted and they are scattered along the entire length of the tail. Several mutations have been characterized that disrupt the proper progression of the ICs (Fabrizio et al., 1998). In most of these cases, the ICs are normally assembled at the spermatid heads, but then break down or become scattered after migration
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Fig. 6. Wild-type (A, B) and mutant (C, D) cysts. Wild-type pre-individualization spermatids (A, B) contain major (arrows) and minor (larger arrowheads) mitochondrial derivatives adjacent to axonemes showing nine distinct accessory tubules embedded in a dense material (B, inset); note the regular bands of microtubules surrounding the mitochondria (smaller arrowheads). Representative examples of pre-individualization spermatids in mutants (C, D) showing abnormally sized mitochondria; the intra-mitochondrial dense material is sometimes lacking (larger arrowheads) or out-of-place (smaller arrows). Scattered microtubules are observed close to the mitochondria (smaller arrowheads). The organization of the axonemal tubules is normal (D, inset). When present in the same spermatid, close axoneme pairs (larger arrows, D) show dynein arms and accessory structures oriented in opposite directions. Scale bar: 2 μm in panels A, C; 0.9 μm in panels B, D; 0.3 μm in insets.
starts. Since the movement of the ICs is actin-based and a number of actin-binding proteins are located in or near these structures (Hicks et al., 1999; Rogat and Miller, 2002; Noguchi and Miller, 2003; Noguchi et al., 2006; Mermall et al., 2005), the defects observed in many individualization mutants could be due to mutations in genes coding for actin-associated proteins or motor proteins. Our results suggest that, although parkin function is not required for IC formation, parkin may play an indirect role in IC movement. Genetic control of sperm maturation is not a linear cascade of developmental stages. Thus, early defects in spermatid differentiation often do not block late stages of spermiogenesis (Lifschytz, 1987; Fuller, 1993). Therefore, a subtle defect in cyst organization introduced before individualization in parkin mutant males may interrupt the continuous travel of the ICs along the cyst, resulting in the loss of synchrony of ICs as reported in other individualization mutants (Fabrizio et al., 1998). Thus, the failure of post-meiotic spermatids to be individualized could be an indirect consequence of the defective execution of the differentiation processes that might result in a defective scaffolding for IC movement. Spermatid bundle elongation and individualization represent two aspects of the male germ cell development in Drosophila
that are accompanied by extensive mitochondrial remodeling. In early post-meiotic spermatids, dispersed mitochondria aggregate beside each haploid nucleus and fuse to form the spherical nebenkern (Tates, 1971; Tokuyasu, 1975). During further development, nebenkern mitochondria unfurl, forming minor and major mitochondrial derivatives that elongate and undergo final shaping during the individualization phase. Thus, the accurate dynamics of the pair of elongating tubular mitochondrial derivatives, adjacent to the growing axoneme, might be essential for the movement of ICs along the spermatid cytoplasm. It seems possible that the large size of mitochondrial derivatives could cause steric hindrance that prevents passage of ICs. Phase-contrast microscopy revealed that parkin males assemble a normal-looking onion-stage nebenkern, but fail to produce a normal pair of mitochondrial derivatives. Electron microscopic analysis confirmed that only one mitochondrial derivative is present during sperm elongation in mutant testes. Most of these mitochondria fail to undergo final shaping and appear to be highly convoluted and large, instead of being tubular and small. These unexpected shape changes might represent an obstacle for the proper passage of the ICs through the sperm tail.
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Fig. 7. Fully elongated wild-type (A) and mutant (B) spermatids: note the regular disposition of the major (arrows) and minor (arrowheads) mitochondrial derivatives and the homogeneous distribution within the major mitochondria of the dense material in wild-type (A); by contrast, the condensation of the mitochondria is variable in mutant spermatids (B) and there are fully condensed mitochondria (arrows) alongside mitochondria in which the cristae are still visible (arrowheads). (C, E) Wild-type cyst following individualization: note the closely packed sperm each surrounded by its own plasma membrane. (D, F) Example of one of the most mature cysts found in parkin testis: note the large amount of cytoplasmic ground substance in which many axonemes and large abnormally sized mitochondria are immersed; only a few sperm (arrows) are surrounded by a plasma membrane. Scale bar: 0.9 μm in panels A, B, D, F; 2 μm in panels C, E.
The lack of a mitochondrial derivative implies that some problems occur during earlier steps of mitochondrial morphogenesis in parkin mutants at the end of meiosis II. The formation of the mitochondrial derivatives, a central feature of the spermatid differentiation in the fly, involves the coordinated aggregation and fusion of smaller mitochondria, dispersed during cell division, into greatly enlarged structures near the spermatid nuclei. Flies mutant for fuzzy onion (Fzo) (Hales and Fuller, 1997) and rhomboid-7 (McQuibban et al., 2006) have defects in mitochondrial fusion at the end of meiosis and display
abnormal onion-stage nebenkern that resolve into fragmented mitochondrial derivatives. Thus, these gene products play a central role during mitochondrial morphogenesis in the fly to realize the giant nebenkern. However, it is unclear how the fusogenic properties of Fzo and rhomboid-7 can be correlated with the observation that there are two distinct mitochondrial derivatives at the start of spermatid elongation. It is still unclear if these derivatives originate in Drosophila by the fusion of two separate populations of mitochondria that form two intertwined structures within the onion-stage nebenkern, or alternatively, if
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the dispersed mitochondria aggregate into a unique nebenkern that bisects equally at the beginning of spermatid elongation. Although definitive and convincing evidence is lacking, it is remarkable that ultrastructural serial section studies made in D. melanogaster (Tates, 1971) and other insects (Pratt, 1968) indicate that the onion-stage nebenkern contains two topologically distinct compartments. This suggests that the dispersed mitochondria aggregate in two distinct clusters at one side of the cell, close to the basal body to form a bipartite onion-stage nebenkern. Then, the pair of derivatives unfurl from each other and elongate beside the growing axoneme. Since only one tubular structure resolves from the onion-stage nebenkern in parkin testes, perhaps an earlier defect occurs at the time of the clustering of the scattered mitochondria into two distinct groups. Numerous observations in many cells types indicate that mitochondria are highly dynamic structures that continuously change size and dimensions (Mozdy and Shaw, 2003). Since membrane fusions and fissions are emerging as regulators of mitochondrial biology (Chen and Chan, 2004), the disturbance of the balance among these events might lead to the disruption of mitochondrial architecture and function. Thus, the parkin product could be part of a mechanism that coordinates these events to realize the bipartite onion-stage nebenkern. Our studies indicate that the parkin mutation does not interfere with mitochondrial dynamics during spermatogenesis; it affects post-meiotic differentiation of the male germ cell, when there is an essential mitochondrial fusion process. Many gene products needed for spermatid differentiation are typically translated at the beginning of spermiogenesis (Schafer et al., 1995). Although parkin transcripts are present in embryos and egg chambers, mutations do not have striking effects on viability, female fertility or embryogenesis. Perhaps normal mitochondrial morphogenesis requires a threshold level of parkin activity, or the parkin product might be functionally redundant with other proteins or pathways involved in mitochondrial dynamics in cells outside of the testis. On the other hand, since the mutation in parkin is a null allele, perhaps the effect is not due to threshold levels of protein activity, but rather is only apparent in a sensitive system that puts great demands on its function. Spermiogenesis, in which all the mitochondria in the spermatid coalesce adjacent to the nucleus and undergo a process of massive membrane fusion, may be such a sensitive system, and the wild-type function of parkin could be mainly required for assembly and/or maintenance of the complex architecture of the mitochondrial derivative, which forms from mitochondria dispersed throughout the male germ cell after the meiotic divisions. A role for the parkin protein in mitochondrial morphogenesis or function during the process of sperm maturation is suggested by the observation of unusual electron-dense onion-stage nebenkern and by the finding of mitochondria of variable size and shape that might represent abnormal fusion events. Moreover, since paracrystalline bodies are abnormal in number, size, and condensation, mitochondrial metabolism could be in part affected by lesions in parkin. It has been recently shown that removal of the Drosophila PTEN-
induced kinase 1 (PINK1) homologue function results in male sterility and defects in mitochondrial morphology (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). Since genetic evidence has established that pink1 and parkin act in a common pathway in maintaining mitochondrial integrity, with pink1 functioning upstream of parkin, it is noteworthy that the individualization defects found in parkin spermatid cysts share a marked phenotypic similarity with pink1 loss-of-function mutants. Parkin was reported to be associated in mouse brain and cultured cells with different subcellular compartments, such as cytoplasmic vesicles, Golgi complexes, and endoplasmic reticulum, as well as the outer mitochondrial membrane (Kubo et al., 2001; Darios et al., 2003; Mouatt-Prigent et al., 2004). Parkin can also binds to α/β tubulin in rat brain and human embryonic kidney cells (Ren et al., 2003) and it is found at the centrosome of human cultured cells in response to inhibition of proteasomes (Zhao et al., 2003). Because the frequency of fusion is limited by cytoskeletal motors and transport events that bring mitochondria into close proximity (Bereiter-Hahn and Voth, 1994), excessive fusion could be possible if mitochondrial motility is increased or mitochondrial-cytoskeleton attachments are disturbed. parkin could, therefore, also operate in a non-mitochondrial compartment and could be involved in mechanisms, such as transport or positioning, essential to proper mitochondrial distribution during the early steps of spermatid differentiation. However, a role for parkin in actin cytoskeleton dynamics has not been demonstrated, even if the association of the parkin gene product with actin filaments in kidney and neuronal cells has been reported (Huynh et al., 2000). It is also possible that the defects seen were in the stability of the bipartite onion structure rather than in the proper establishment of its architecture. In the absence of functional parkin, the equilibrium between the two distinct clusters of mitochondria may be altered, destabilizing the initial structure and ultimately leading to the improper fusion in a unique structure of the two closely wrapped mitochondrial slices. Since mitochondrial morphogenesis is a necessary process during differentiation and establishment of final structures within the mature spermatozoon of the fly, it is understandable that mutations in parkin lead to male sterility. However, mice lacking parkin have been reported to be fertile (Goldberg et al., 2003; Itier et al., 2003). This suggests that mitochondrial transformation and morphogenesis during sperm maturation in mice and flies may require different routes or they are differently sensitive to the proper function of parkin gene product. It does not seem surprising because mouse spermiogenesis has no individualization process, the sperm are shorter and their elongation relies on IFT. Drosophila is rather unusual in how spermiogenesis is carried out, likely making this process uniquely sensitive to parkin dysfunction. Thus, Drosophila may be the only model in which parkin can be productively studied in spermiogenesis. It could be argued that, while this may not translate directly to mammalian spermiogenesis, this model still is likely to shed light on parkin function in general. parkin was detected in various organisms including flies, frogs, birds, and mice, indicating that this gene may play a general role
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that has been conserved in different species (Horowitz et al., 2001). Acknowledgments We are very indebted to Leo Pallanck (University of Washington) for kindly providing the park[25] and park[rvA] strains used in this work. The authors are grateful to Luigi Federico Falso for assistance with transmission electron microscope. This work was supported by grants from PRIN and PAR (University of Siena) to GC. We would also like to thank Fondazione MPS for financial support. References Adams, R.R., Tavares, A.A.M., Salzberg, A., Bellen, H.J., Glover, D.M., 1998. Pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev. 12, 1483–1494. Beal, M.F., 2003. Mitochondria, oxidative damage, and inflammation in Parkinson's disease. Ann. N. Y. Acad. Sci. 991, 120–131. Bereiter-Hahn, J., Voth, M., 1994. Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria. Microsc. Res. Tech. 27, 198–219. Bergeret, E., Pignot-Paintrand, I., Guichard, A., Raymond, K., Fauvarque, M.O., Cazemajor, M., Griffin-Shea, R., 2001. Rotund RacGAP functions with Ras during spermatogenesis and retinal differentiation in Drosophila melanogaster. Mol. Cell. Biol. 21, 6280–6291. Carmena, M., Riparbelli, M.G., Minestrini, G., Tavares, A.M., Adams, R., Callaini, G., Glover, D.M., 1998. Drosophila polo kinase is required for cytokinesis. J. Cell Biol. 143, 659–671. Castrillon, D.H., Gonczy, P., Alexander, S., Rawson, R., Eberhart, C.G., Viswanathan, S., DiNardo, S., Wasserman, S.A., 1993. Toward a molecular genetic analysis of spermatogenesis in Drosophila melanogaster: characterization of male-sterile mutants generated by single P element mutagenesis. Genetics 135, 489–505. Cenci, G., Bonaccorsi, S., Pisano, C., Verni, F., Gatti, M., 1994. Chromatin and microtubule organization during premeiotic, meiotic and early postmeiotic stages of Drosophila melanogaster spermatogenesis. J. Cell Sci. 107, 3521–3534. Chen, H., Chan, D.C., 2004. Mitochondrial dynamics in mammals. Curr. Top. Dev. Biol. 59, 119–144. Clark, I.E., Dodson, M.W., Jiang, C., Cao, J.H., Huh, J.R., Seol, J.H., Yoo, S.J., Hay, B.A., Guo, M., 2006. Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441, 1162–1166. Darios, F., Corti, O., Lücking, C.B., Hampe, C., Muriel, M.-P., Abbas, N., Gu, W.-J., Hirsch, E.C., Rooney, T., Ruberg, M., Brice, A., 2003. Parkin prevents mitochondrial swelling and cytochrome c release in mitochondriadependent cell death. Hum. Mol. Genet. 12, 517–526. Fabrizio, J.J., Hime, G., Lemmon, S.K., Bazinet, C., 1998. Genetic dissection of sperm individualization in Drosophila melanogaster. Development 125, 1833–1843. Farkas, R.M., Giansanti, M.G., Gatti, M., Fuller, M.T., 2003. The Drosophila Cog5 homologue is required for cytokinesis, cell elongation, and assembly of specialized Golgi architecture during spermatogenesis. Mol. Biol. Cell 14, 190–200. Fuller, M.T., 1993. Spermatogenesis. In: Bates, M., Arias, A.M. (Eds.), The Development of Drosophila melanogaster, vol. I. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 71–147. Ghosh-Roy, A., Kulkarni, M., Kumar, V., Shirolikar, S., Ray, K., 2004. Cytoplasmic dynein–dynactin complex is required for spermatid growth but not axoneme assembly in Drosophila. Mol. Biol. Cell 15, 2470–2483. Ghosh-Roy, A., Desai, B.S., Ray, K., 2005. Dynein light chain 1 regulates dynamin-mediated F-actin assembly during sperm individualization in Drosophila. Mol. Biol. Cell 16, 3107–3116. Glover, D.M., Gonzalez, C., 1993. Techniques for studying mitosis in Droso-
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The Drosophila parkin homologue is required for normal mitochondrial dynamics during spermiogenesis Maria Giovanna Riparbelli, Giuliano Callaini ⁎ University of Siena, Department of Evolutionary Biology, Via A. Moro 4, I-53100 Siena, Italy Received for publication 12 July 2006; revised 20 October 2006; accepted 26 October 2006 Available online 10 November 2006
Abstract Drosophila parkin, the ortholog of the human parkin gene, responsible for a familiar form of autosomal recessive juvenile parkinsonism, has been shown previously to be involved in Drosophila male fertility. Loss-of-function mutations in the parkin gene cause failure of spermatid individualization by affecting the proper progression of the actin-based investment cones that assemble in the nuclear region, but fail to translocate in synchrony down the cyst. In parkin mutants, the investment cones are scattered along the post-elongated spermatid bundles and fail to act properly in the process of sperm individualization. Using phase-contrast and electron microscopy analysis, we demonstrate that the parkin spermatids assemble a seemingly normal onion-stage nebenkern, but when the axoneme elongates only one mitochondrial derivative unfurls from the nebenkern. This unique mitochondrial derivative undergoes abnormal shaping and condensation during spermatid elongation. Our results indicate that parkin gene function is necessary for mitochondrial morphogenesis during earlier and later phases of spermiogenesis. The failure of cyst individualization may be due to the sensitivity of investment cone movement to the perturbation of mitochondrial morphology during spermatid elongation. © 2006 Elsevier Inc. All rights reserved. Keywords: Drosophila; Parkin; Spermatogenesis; Individualization complexes; Mitochondria
Introduction Spermiogenesis in Drosophila melanogaster is a complex process that involves a series of elaborate transformations of the spermatid components. These dramatic transformations from an immotile cell to a motile one include chromatin condensation, cell and axoneme elongation, mitochondrial rearrangement, and sperm individualization (Fuller, 1993). Successful spermiogenesis requires the proper execution of these developmentally regulated processes. Recent investigations have focused attention on the individualization process at the end of spermiogenesis. This process is mediated by 64 distinct conical complexes, or individualization complexes (ICs) (Tokuyasu et al., 1972), mainly composed of actin. The actin cones, or investment cones, move along the axoneme from the nucleus to the tail end, sequestering the excess cytoplasm, remodeling the minor mitochondrial derivative, and completing the uneven spermatid ⁎ Corresponding author. Fax: +39 577 334476. E-mail address: [email protected] (G. Callaini). 0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.10.038
plasma membrane to enclose each sperm axoneme. Some cytoskeletal proteins, such as unconventional myosin V and VI (Rogat and Miller, 2002; Mermall et al., 2005; Noguchi et al., 2006), dynein (Li et al., 2004; Ghosh-Roy et al., 2005), and actin (Noguchi and Miller, 2003), seem to be directly involved in the individualization complex function. Although the cell remodeling mechanisms that transform fully elongated spermatids into individual sperm are understood to some extent, much less is known about the morphogenetic processes of the earlier phase of spermatid elongation, including mitochondrial transformation and tail growth. Our knowledge of this early phase of sperm formation is mainly based on morphological studies in which cell remodeling was investigated by transmission electron microscopy (Tokuyasu et al., 1972; Tates, 1971). However, these studies did not investigate in detail the mechanisms driving cell elongation and sperm tail growth. Recent observations suggest that axoneme elongation in Drosophila does not require the ubiquitous process of intraflagellar transport (Han et al., 2003; Sarpal et al., 2003), but relies on unknown mechanisms, perhaps in part driven by the
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cytoskeletal reorganization mediated by cytoplasmic dynein– dynactin complex (Ghosh-Roy et al., 2004). Genetic analysis indicates that genes involved in vesicular transport and fusion may play a major role in sperm cell growth (Bergeret et al., 2001; Farkas et al., 2003). Although mitochondrial behavior during spermiogenesis is fairly well understood, the molecular pathway involved in this complex process of transformation remains obscure. It is, for example, still unclear which signals direct the aggregation of the mitochondria in two equivalent and distinct components, how these structures wrap to form the onion-stage nebenkern, and how the onion resolves into two derivatives that undergo different fates during later spermiogenesis. The study of mutations that alter the usual pattern of mitochondrial transformation could help decipher the role of these organelles during sperm maturation and provide further insight into mitochondrial biogenesis. Mutational analysis has identified several phenotypes in which sperm do not undergo proper maturation and show slight defects of mitochondrial organization within the tail (Fabrizio et al., 1998; Castrillon et al., 1993; Wakimoto et al., 2004). These studies, however, were not specifically designed to elucidate the relationship between sperm maturation and mitochondrial transformation and did not investigate mitochondrial morphogenesis in detail. Up to now, only two genes, fuzzy onion (fzo) (Hales and Fuller, 1997) and rhomboid-7 (McQuibban et al., 2006), have been characterized and correlated with the process of mitochondrial dynamics and morphogenesis in Drosophila spermatogenesis. Mutations in these genes, that encode proteins required for the developmentally regulated fusion of mitochondria, result in onion-stage nebenkern defects and male sterility. Loss-of-function mutations in the parkin gene, the Drosophila ortholog of the human parkin gene, responsible for a familiar form of Parkinson disease known as autosomal recessive juvenile parkinsonism (AR-JP), result in sperm with abnormally sized mitochondrial derivatives (Greene et al., 2003). This indicates a potential function of this gene in mitochondrial morphogenesis during sperm maturation in the fly, especially when combined with the finding that mitochondrial pathology is a prominent feature in flight muscles of Drosophila parkin null mutants (Greene et al., 2003; Pesah et al., 2004). In addition, the Drosophila homologue of a human AR-JP-linked gene, PTEN-induced putative kinase 1 (PINK1), acts in a common pathway with parkin in maintaining mitochondrial integrity; the ortholog of PINK1 is required for proper mitochondrial structure and function in Drosophila (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). We confirm by careful cytological analysis a previous hypothesis (Greene et al., 2003) that mutations in parkin do not affect mitochondrial dynamics during spermatogenesis, but rather lead to the failure of proper remodeling of these organelles during earlier post-meiotic stages. We demonstrate that only one mitochondrial derivative assembles from the onion-stage nebenkern in parkin sperm. This unique mitochondrion behaves irregularly during spermatid elongation, leading to defective progression of the individualization complexes and thus to the failure of sperm maturation.
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Mitochondrial dysfunctions have been suggested for a key role in Parkinson disease in humans (for a review, see Beal, 2003; Shen and Cookson, 2004). However, definitive evidence is lacking. The careful analysis of a system, such as spermiogenesis in Drosophila, in which strictly regulated mitochondrial dynamics is required for proper cell development and differentiation could be useful to investigate potential functions and cellular targets of the parkin gene. Materials and methods Drosophila strains and crosses All fly stocks were maintained and crossed on standard culture medium at 24°C. Since the stock containing park[25] also carries a linked homozygous lethal, we crossed this strain with flies carrying the Df(3L)Pc-MK deletion to obtain the viable null utilized for cytological studies. Oregon-R flies and the revertant park[rvA] line were used as controls to obtain wild-type testes. The Df (3L)Pc-MK deletion was obtained from the Bloomington Drosophila Stock Center (Bloomington, IN). park[25] and park[rvA] lines were a generous gift of Leo Pallanck (University of Washington, Seattle, WA, USA).
Reagents A mouse monoclonal anti-β-tubulin (Boehringer, Mannheim UK) was used at a 1:200 dilution; a rabbit anti-Pav-KLP polyclonal Rb3301 (Adams et al., 1998) at 1:100. Goat anti-mouse or anti-rabbit secondary antibodies coupled to fluorescein or rhodamine (Cappel, West Chester, PA) were used at 1:600 dilution. DNA was visualized with Hoechst 33258 (Sigma, St. Louis, MO). The actin cytoskeleton was visualized with rhodamine-phalloidin (Molecular Probes, Eugene, OR) prepared by dissolving 10 μl of vacuum-dried methanol stock solution in 100 μl of PBS. Bovine serum albumin (BSA) was obtained from Sigma.
Examination of spermiogenesis by phase-contrast microscopy Cytological preparations were made with testes from 3- to 4-day-old pupae and from 4-day-old adult males. Testes were dissected in phosphate-buffered saline (PBS) and transferred to a drop of PBS on a glass slide. The testes were open and squashed under a cover slip. The morphology of early spermatids was analyzed using an Axio Imager Z1 (Carl Zeiss, Jena, Germany) microscope equipped with 100× phase-contrast objective. Images were obtained with an AxioCam HR cooled charge-coupled camera (Carl Zeiss).
Immunofluorescence Immunostaining of testes from pupae, newly eclosed, and older flies was performed by either the methanol/acetone fixation method or by the ethanol/ formaldehyde fixation method (Glover and Gonzalez, 1993; Cenci et al., 1994; Hime et al., 1996). After fixation, the material was washed in PBS and incubated 1 h in PBS containing 0.1% bovine serum albumin (PBS-BSA). For localization of microtubules and Pav-KLP, testes were incubated overnight at 4°C with the PAV-KLP antisera and then with anti-β-tubulin antibody for 4–5 h at room temperature. After washing in PBS-BSA, the slides were incubated 1 h at room temperature with the appropriate secondary antibodies. For actin and tubulin visualization, the slides were incubated with anti-βtubulin antibody for 1 h at room temperature. After washing in PBS, the slides were incubated 1 h in the appropriate secondary antibody to which rhodaminelabeled phalloidin was added. Nuclei were then visualized with incubation for 3–4 min in Hoechst. Testes were mounted in small drops of 90% glycerol containing 2.5% n-propyl-gallate. The fluorescent images were taken by using an Axio Imager Z1 (Carl Zeiss) microscope equipped with an HBO 50-W mercury lamp for epifluorescence and with an AxioCam HR cooled charge-coupled camera (Carl Zeiss). Gray-scale
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digital images were collected separately and then pseudocolored and merged using Adobe Photoshop 7.0 software (Adobe Systems).
Transmission electron microscopy Testes dissected in PBS from 3- to 4-day-old pupae, and from young (1 to 2day-old) and old (15 to 20-day-old) adult males were fixed for 2 h at 4°C in 2.5% glutaraldehyde in PBS. After rinsing 20 min in PBS, the material was post-fixed in 1% osmium tetroxide in PBS. After extensive washing in distilled water, the samples were dehydrated in a graded series of alcohols, embedded in an Epon– Araldite mixture, and polymerized at 60°C for 48 h. Ultrathin sections obtained with an LKB Nova ultramicrotome were collected on copper grids and stained with uranyl acetate and lead citrate. Sections were observed with a Philips CM10 electron microscope.
Spermatid length measurements Since mutations in parkin result in sperm individualization defects and empty seminal vesicles, to obtain more reliable data, we measured fully elongated spermatid cysts from both mutant and control testes. Testes from 4- to 5-day-old adult males were dissected in PBS, fixed as described above, and stained for F-actin to visualize ICs. Fully elongated spermatid cysts can be unambiguously recognized on the basis of the presence of actin-rich cones that make their appearance after the cellular elongation has been completed. Seventy fully elongated spermatid cysts were scored for mutant and control males each. The measures were performed under a fluorescence microscope connected to a CCD camera, as previously described, using the AxioVision 4.0 software (Carl Zeiss).
Results Investment cones move asynchronously through parkin spermatid bundles In Drosophila spermatogenesis, cysts of 16 primary spermatocytes undergo two meiotic divisions to produce 64 haploid round spermatids interconnected by cytoplasmic bridges. Mutations in the steps of this process lead to the production of abnormal spermatids that arrest in development or undergo abnormal differentiation, leading to male sterility. It was previously demonstrated that, in parkin males, spermatogenesis is normal until post-meiotic spermatid differentiation (Greene et al., 2003). The primary spermatocytes enter meiosis to produce syncytial cysts of 64 round spermatids, as in wild-type. However, while wild-type seminal vesicles accumulate motile sperm, in mutant testes, the seminal vesicles are devoid of coiled mature sperm. To pinpoint the specific stage of spermiogenesis that requires parkin function, we compared wild-type and parkin spermatids at various stages of elongation. Post-meiotic spermatids undergo extensive syncytial cellular differentiation including chromatin condensation, mitochondrial remodeling, axoneme assembly, spermatid elongation, individualization, and coiling. The 64 interconnected spermatids develop in a syncytium that extends through the length of the testis. Elongating spermatids are orientated with their heads (apical region of the cyst) toward the basal end of the testis and their tail ends (distal region of the cyst) toward the apical region of the testis. Spermatid elongation in parkin mutants follows the usual pattern observed in wild-type testis: the nuclei align within the cyst opposite the tip of the tails. At the distal region of the
spermatid cyst, the ring canals form a cluster that becomes more compact with the elongation process. The proper execution of cytokinesis in germ cells can be unambiguously detected by the presence of ring canals. Since ring canals are formed by stabilization of the contractile rings following cytokinesis, they can be readily observed with antibodies against the kinesin-like protein encoded by pavarotti (Pav-KLP) that is associated with the central region of the spindle at anaphase/telophase and is required for the correct organization of the spindle during cytokinesis (Adams et al., 1998). This protein associates with the ring canals which form at the sites of incomplete cytokinesis in the germline cysts of both testes (Carmena et al., 1998) and ovaries (Minestrini et al., 2002). Staining for Pav-KLP shows that, as spermatids enter the elongation process, the ring canals are driven toward the distal region of the spermatid bundle in both wild-type (Fig. 1A) and mutant (Fig. 1B) testes. To determine whether the elongation process proceeds normally in mutants, we measured fully elongated cysts at the beginning of individualization in both park and control testes. The overall length of the fully elongated spermatid cysts varies from 1.65 to 2.03 mm in parkin and 1.85 to 2.13 mm in control flies. We cannot, however, exclude the presence within mutant fully elongated cysts of some shorter spermatids. After elongation, the syncytial spermatids become mature sperm cells. This process is driven by an individualization complex that translocates along the length of the spermatid bundles from the nuclei to the tail ends, eliminating excess cytoplasm and surrounding each spermatid in its own plasma membrane. Thus, the failure of spermatid maturation in parkin mutants might result from sperm individualization defects as demonstrated in other mutants (Fabrizio et al., 1998). Since the major component of the individualization complex is a cone-shaped actin-rich structure, we compared wild-type and mutant spermatid bundles at the end of elongation phase, using markers for F-actin, microtubules, and DNA, to more closely examine the possibility that the parkin phenotype might be correlated to individualization defects. In normal development, the elongated spermatid nuclei align in parallel at the apical region of each cyst. F-actin staining is absent or gives only a feeble signal in this region when spermatid nuclei have partially condensed their chromatin. As the chromatin condenses further and the spermatid nuclei become needle-shaped, Rh-phalloidin labeling reveals distinct thin cone-shaped structures associated with the aligned nuclear bundles, representing the actin-rich investment cones (ICs) that have just formed but have not yet begun the individualization process (Fig. 1C). In testes from park mutants, not all the spermatid nuclei are aligned in register; some of them are mispositioned along the cyst length. Actin staining was associated with both aligned and out-of-place nuclei (Fig. 1D). In wild-type testes, the actin-rich ICs traverse the cyst in a compact mass from the spermatid heads to the tips of the tails. This was clearly visualized by Rh-phalloidin staining showing distinct parallel bundles of ICs at various distances from the nuclei of different spermatid bundles (Figs. 2A, B). As they move, the ICs resolve the syncytial spermatids into individual sperm cells, and excess cytoplasm, membranes, and the ICs
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Fig. 1. Fluorescence preparations of wild-type (A) and parkin (B) elongating spermatids stained for tubulin (green), Pav-KLP (red) and DNA (blue). Ring canal remnants (arrows) are found at the growing edge of the spermatid bundles in both wild-type (A) and parkin (B) spermatids; note that Pav-KLP also accumulates at the poles (smaller arrowheads) and midzone (larger arrowhead) of late anaphase spindles. Nuclear region of pre-individualization wild-type (C) and parkin (D) spermatids stained for tubulin (green), F-actin (red) and DNA (blue). Nuclei are aligned in wild-type (C) and investment cones (ICs) are associated with the posterior nuclear heads (arrows); by contrast, some nuclei (arrows) and ICs (arrowheads) are scattered in parkin (D). Scale bar: 20 μm.
themselves are collected in large “waste bags” at the tip of the spermatid tail (Fig. 2C). In mutant testes, the ICs were located in positions distant from the nuclear region, indicating that they have translated along the spermatid bundles. However, they were not aligned as in wild-type, but were scattered along the sperm tail, suggesting that they did not move in synchrony (Fig. 2D). The individual ICs are cone-shaped as in wild-type. Normally, their apex points toward the apical region of the cyst, where the nuclei are aligned, and their basis is toward the tips of the tails (Fig. 2E). Among the fully elongated spermatid cysts examined (n = 189), 19% of them (n = 36) display 9–10% of misoriented ICs. Fig. 2F shows a detail of a spermatid bundle in which scattered ICs had their pointed ends oriented in opposite directions. “Waste bags” in parkin testes were not distinct as in wild-type, suggesting that the process of individualization is not properly executed in these mutants. This was confirmed by the finding of irregularly coiled distal ends of the spermatid bundles (Fig. 2G).
the end of meiosis. In early spermatids, the aggregated mitochondria form a spherical compact structure, the onionstage nebenkern, recognizable in phase-contrast microscopy as a dark round structure, closely apposed to the white round spermatid nucleus. No significant differences in nebenkern morphology were observed in young wild-type (Fig. 3C) or mutant (Fig. 3D) spermatids. During earlier stages of elongation, the round wild-type spermatids still show a spherical nucleus, but the nebenkern starts to elongate and a distinct paired structure is readily observed (Fig. 3E). park mutant spermatids at the same stage show an elongated nebenkern that does not resolve into two parts (Fig. 3F). Thus, in parkin spermiogenesis, there is an obvious departure from normal development during the first steps of mitochondrial morphogenesis.
parkin spermatids have only one mitochondrial derivative
Examination of parkin testes using electron microscopy confirmed defects in the remodeling of the mitochondria during early stages of spermatid differentiation. Cross-sections of round post-meiotic wild-type spermatids at the onion stage reveal that the mitochondria cluster in a spherical structure slightly larger than the nucleus (Fig. 4A). As expected from the phase-contrast observations, this structure is
To determine whether the park phenotype was associated with defects of mitochondrial behavior, we did a cytological analysis of wild-type and mutant testes using squash preparations. Mitochondria aggregate in a loose cluster around the nuclei in both wild-type (Fig. 3A) and mutant (Fig. 3B) spermatids at
Early steps of mitochondrial morphogenesis are aberrant in parkin testes
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Fig. 2. IC organization in wild-type (A, B, C) and parkin (D, E, F, G) testes stained with Rh-phalloidin. Wild-type (A) pre-individualization spermatid bundles show orderly arranged cluster of ICs (arrows) that appear conical in shape (B). (C) Detail of “waste bags” showing the presence of ICs with bent pointed ends (arrows). The travel of ICs is not synchronous in parkin testes (D), and they are scattered along the length of the spermatids (arrowheads). Although the apex of the conical ICs is mainly directed toward the head region (arrowheads, E), in some cases, the pointed ends of the ICs are opposed (arrowheads, F). The terminal ends of the individualizing spermatids (arrow, G) are often coiled and lack distinct “waste bags”. Scale bar: 40 μm in panels A, D; 15 μm in panels B, E, F; 12 μm in panels C, G.
also visible in mutant spermatocytes (Fig. 4B). However, whereas mitochondria form concentric ring-like structures in wild-type nebenkern, their arrangement is more irregular in mutant spermatocytes. The parkin nebenkern is, indeed, formed by a large portion of rather ring-like concentric mitochondria, and a small portion of loose peripheral mitochondria, that do not seem to integrate into the tightly packed main cluster (Fig. 4B). The concentric mitochondria, moreover, appear more electrondense in mutant than in wild-type spermatids. The morphogenesis of the nebenkern changes during axoneme elongation. Two mitochondrial derivatives of about the same size are associated with the axoneme in wild-type testes when the spermatid starts to elongate (Fig. 4C). Mitochondria appear in cross-section as large circular structures
surrounding a cytoplasmic round area and containing numerous cristae. Cross-sections through parkin spermatocytes at the same developmental stage reveal only one large mitochondrion associated with the growing flagellum (Fig. 4D). To investigate further mitochondrial morphogenesis, we examined subsequent stages of spermatid elongation and differentiation. The level of the sections in both wild-type and mutant spermatids was compared by examining axoneme decoration. As the axoneme elongates further in wild-type testes, the mitochondrial derivatives shrink and transform into tubular structures of about the same size with a few inner membranes (Fig. 5A). In the axoneme, the B-subfiber of each peripheral doublet shows a short external arm, indicating that accessory tubules have started to assemble from outgrowth of
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Fig. 3. Comparison of earlier stages of mitochondrial morphogenesis in wild-type (left) and parkin (right) testes as viewed by phase-contrast microscopy. Large postmeiotic spermatocytes in both wild-type (A) and mutant (B) testes have many small mitochondria (arrowheads) clustered near the uniformly sized nuclei (arrows). Wild-type (C) and parkin (D) onion-stage spermatids; the nuclei (arrows) and their adjacent nebenkerns (arrowheads) are present in a 1:1 ratio. Wild-type spermatids (E) show pairs of mitochondrial derivatives (arrowheads) associated with round nuclei (arrows) during early elongation stages, whereas parkin mutant spermatids (F) exhibit only one oblong mitochondrial derivative (arrowheads) associated with each nucleus (arrows). The preparation has been flattened slightly, and a few of cytoplasmic bridges between the cells are broken, revealing the cytoplasmic continuity between spermatids. Thus, some of the spermatids appear syncytial due to opening of cytoplasmic bridges between cells during sample preparations. Scale bar: 20 μm.
external microtubule doublets (Fig. 5A, inset). Sections through elongating post-meiotic spermatids of parkin males, in which the B-tubule of the axoneme also shows a thin external arm (Fig. 5B, inset), display mitochondria arranged in irregular rings, either outside of or enveloping the flagellum (Fig. 5B). Cross-sections through mutant spermatid bundles that appear slightly elongated, as judged by the hooked peripheral arms growing by the B-subfiber (Fig. 5C, inset), show both ring-like and spherical mitochondria (Fig. 5C). This suggests that parkin
mitochondria undergo compaction as the spermatid elongates. There was, however, usually only one mitochondrial derivative, and only occasionally did we observe additional mitochondria in the same spermatid. Small light-staining vesicles, containing microtubules, were usually found (Fig. 5C). Two mitochondrial derivatives, clearly different in size and shape, are seen in wild-type pre-individualized spermatogenic cysts. The minor mitochondrial derivative is round and smaller, whereas the major one is larger, ovoid in cross-section and
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Fig. 4. Electron microscopy (EM) analysis of post-meiotic spermatids from wild-type (A, C) and parkin (B, D) testes. The onion-stage nebenkern appears to be formed of concentric cisternae in wild-type (arrow, A) spermatids, whereas it is somewhat irregular in mutant (arrow, B) spermatids; N, nucleus. As the wild-type spermatids (C) start to elongate, a pair of mitochondrial derivatives (arrows) lie adjacent to the axoneme (arrowhead). By contrast, only one large mitochondrion is observed in mutant spermatids (arrow, D). Scale bar: 2.5 μm in panels A, B; 1 μm in panels C, D.
appears slightly darker (Fig. 6A). Dark-staining material accumulates in the major derivative next to the axoneme. Highly organized sheaths of cytoplasmic microtubules surround the mitochondria (Fig. 6B). The axoneme at this point has nine distinct accessory tubules embedded in a dense material (Fig. 6B, inset). Spermatids are still connected by cytoplasmic bridges and the axoneme–mitochondrial complexes are in an orderly arrangement within the cyst. At a comparable developmental stage, the pre-individualization parkin spermatids maintain some cytoplasmic ground substance and are connected by intercellular bridges. The spermatids usually contain a single irregularly shaped and sized mitochondrion surrounded by disorganized sheaths of microtubules (Fig. 6C). These microtubules are scattered in the spermatid cytoplasm and occasionally localized near the axoneme. The organization of the axonemal microtubules is normal (Fig. 6D, inset), whereas the orientation of the mitochondrial derivatives varies within the same cyst. Some
spermatids show abnormally sized circular mitochondria, reminiscent of the mitochondrial whorls observed in crosssections of the wild-type individualization complexes (Tokuyasu et al., 1972). Mitochondrial condensation is highly defective: some mitochondria are filled with dense material in the region adjacent to the axoneme, whereas others do not show appreciable signs of condensation or the condensation is barely detectable. Although the dark material that filled the mitochondria is mostly restricted to the region close to the axoneme, circular mitochondria also display distinct condensation in regions far from the axoneme (Figs. 6C, D). Closely paired axonemes are also found in these cysts (Fig. 6D), whereas wild-type cysts contain evenly spaced axonemes. Detailed examination reveals that the dynein arms often appear oriented both clockwise and anti-clockwise in adjacent parkin axoneme pairs. The dynein arms appear clockwise when cross-sections of the axoneme are seen from head to tail and anti-clockwise when the same sections are seen in the
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7A). Dense material envelops the accessory tubules that are no longer distinguishable. In the same stage in mutant spermatids, one mitochondrion alone, abnormally shaped and of varying size, is usually found (Fig. 7B). Mitochondrial condensation is also defective. We observed mitochondria filled with large dark masses and pale mitochondria with evident cristae remnants in the same cyst (Fig. 7B). The minor mitochondrial derivatives shrink again after the passage of the individualization complex in wild-type. Cross-sections of wild-type individualized spermatid bundles display well-ordered sperm cells with tightly packed condensed major mitochondrial derivatives, each surrounded by its own plasma membrane (Figs. 7C, E). In contrast, sections of mutant spermatids that appear quite mature on the basis of axonemal decoration reveal that only a few spermatids are apparently cellularized (Figs. 7D, F). Most of the spermatids are immersed in a large amount of cytoplasmic ground substance, and their mitochondrial derivatives appear irregularly condensed and abnormally sized. These data suggest that investment cone movement was defective in parkin mutants and that further fusion events could occur within mitochondria. Discussion
Fig. 5. Cross-sections of wild-type (A) and mutant (B, C) elongating spermatids. Mitochondrial derivatives are transformed into pairs of tubular structures in wild-type (arrows, A), whereas they are more convoluted in same-stage parkin spermatids (arrows, B); insets in panels A and B represent magnifications of axonemes showing thin external arms (arrowheads) outgrowing from external microtubule doublets. Slightly elongated parkin spermatids (C) show ring-like (arrows) and spherical (larger arrowheads) mitochondria, together with lightstaining vesicles containing microtubules (smaller arrowheads); note the hooked peripheral arms growing from the B-subfiber (arrowheads, inset). Scale bar: 1 μm in panels A, B, C; 0.3 μm in insets.
opposite direction. This observation could, therefore, indicate that close axoneme pairs are sections of the same flagellum that had folded back on itself. Toward the distal region of post-elongating wild-type spermatid bundles, the size of the mitochondrial derivatives decreases. The major mitochondrial derivative is mostly filled with dark material, whereas the minor one is less dense (Fig.
Syncytial spermatids must be individualized at the end of differentiation in Drosophila to become mature sperm. Cytoplasmic bridges are broken and the cell membranes are sealed, the excess cytoplasmic material within the flagella is stripped away, and mitochondrial derivatives change dramatically in size and shape (Fuller, 1993). Mature sperm can be thus identified on the basis of their full cellularization, the absence of microtubules within the reduced cytoplasm, the small size of the minor mitochondrial derivative, and the large paracrystalline structure within the major mitochondrial derivative. Spermiogenesis appears to proceed normally to the elongated spermatid stage in parkin testes, but most of the spermatids have cytoplasmic bridges, abundant cytoplasm and abnormal mitochondrial derivatives. Only a few spermatids show the usual characteristics of mature sperm, although their mitochondria appear irregularly sized and shaped. These findings are consistent with defects in the process of individualization in Drosophila spermiogenesis, as previously suggested (Greene et al., 2003). This process is achieved by ICs that assemble at the aligned nuclear heads of the spermatid bundle and proceed down the spermatid tails as a linear cluster, resolving the syncytial spermatids of a cyst into 64 cells with individual membranes (Tokuyasu et al., 1972). Since spermatogenesis appears to proceed normally in the mutant, the absence of parkin alone is unable to block the spermatogonial cell divisions and meiosis and, thus, spindle assembly. However, in the absence of parkin, though the actin cones are still formed around the nuclei, their synchronous movement and alignment along the spermatid bundle are disrupted and they are scattered along the entire length of the tail. Several mutations have been characterized that disrupt the proper progression of the ICs (Fabrizio et al., 1998). In most of these cases, the ICs are normally assembled at the spermatid heads, but then break down or become scattered after migration
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Fig. 6. Wild-type (A, B) and mutant (C, D) cysts. Wild-type pre-individualization spermatids (A, B) contain major (arrows) and minor (larger arrowheads) mitochondrial derivatives adjacent to axonemes showing nine distinct accessory tubules embedded in a dense material (B, inset); note the regular bands of microtubules surrounding the mitochondria (smaller arrowheads). Representative examples of pre-individualization spermatids in mutants (C, D) showing abnormally sized mitochondria; the intra-mitochondrial dense material is sometimes lacking (larger arrowheads) or out-of-place (smaller arrows). Scattered microtubules are observed close to the mitochondria (smaller arrowheads). The organization of the axonemal tubules is normal (D, inset). When present in the same spermatid, close axoneme pairs (larger arrows, D) show dynein arms and accessory structures oriented in opposite directions. Scale bar: 2 μm in panels A, C; 0.9 μm in panels B, D; 0.3 μm in insets.
starts. Since the movement of the ICs is actin-based and a number of actin-binding proteins are located in or near these structures (Hicks et al., 1999; Rogat and Miller, 2002; Noguchi and Miller, 2003; Noguchi et al., 2006; Mermall et al., 2005), the defects observed in many individualization mutants could be due to mutations in genes coding for actin-associated proteins or motor proteins. Our results suggest that, although parkin function is not required for IC formation, parkin may play an indirect role in IC movement. Genetic control of sperm maturation is not a linear cascade of developmental stages. Thus, early defects in spermatid differentiation often do not block late stages of spermiogenesis (Lifschytz, 1987; Fuller, 1993). Therefore, a subtle defect in cyst organization introduced before individualization in parkin mutant males may interrupt the continuous travel of the ICs along the cyst, resulting in the loss of synchrony of ICs as reported in other individualization mutants (Fabrizio et al., 1998). Thus, the failure of post-meiotic spermatids to be individualized could be an indirect consequence of the defective execution of the differentiation processes that might result in a defective scaffolding for IC movement. Spermatid bundle elongation and individualization represent two aspects of the male germ cell development in Drosophila
that are accompanied by extensive mitochondrial remodeling. In early post-meiotic spermatids, dispersed mitochondria aggregate beside each haploid nucleus and fuse to form the spherical nebenkern (Tates, 1971; Tokuyasu, 1975). During further development, nebenkern mitochondria unfurl, forming minor and major mitochondrial derivatives that elongate and undergo final shaping during the individualization phase. Thus, the accurate dynamics of the pair of elongating tubular mitochondrial derivatives, adjacent to the growing axoneme, might be essential for the movement of ICs along the spermatid cytoplasm. It seems possible that the large size of mitochondrial derivatives could cause steric hindrance that prevents passage of ICs. Phase-contrast microscopy revealed that parkin males assemble a normal-looking onion-stage nebenkern, but fail to produce a normal pair of mitochondrial derivatives. Electron microscopic analysis confirmed that only one mitochondrial derivative is present during sperm elongation in mutant testes. Most of these mitochondria fail to undergo final shaping and appear to be highly convoluted and large, instead of being tubular and small. These unexpected shape changes might represent an obstacle for the proper passage of the ICs through the sperm tail.
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Fig. 7. Fully elongated wild-type (A) and mutant (B) spermatids: note the regular disposition of the major (arrows) and minor (arrowheads) mitochondrial derivatives and the homogeneous distribution within the major mitochondria of the dense material in wild-type (A); by contrast, the condensation of the mitochondria is variable in mutant spermatids (B) and there are fully condensed mitochondria (arrows) alongside mitochondria in which the cristae are still visible (arrowheads). (C, E) Wild-type cyst following individualization: note the closely packed sperm each surrounded by its own plasma membrane. (D, F) Example of one of the most mature cysts found in parkin testis: note the large amount of cytoplasmic ground substance in which many axonemes and large abnormally sized mitochondria are immersed; only a few sperm (arrows) are surrounded by a plasma membrane. Scale bar: 0.9 μm in panels A, B, D, F; 2 μm in panels C, E.
The lack of a mitochondrial derivative implies that some problems occur during earlier steps of mitochondrial morphogenesis in parkin mutants at the end of meiosis II. The formation of the mitochondrial derivatives, a central feature of the spermatid differentiation in the fly, involves the coordinated aggregation and fusion of smaller mitochondria, dispersed during cell division, into greatly enlarged structures near the spermatid nuclei. Flies mutant for fuzzy onion (Fzo) (Hales and Fuller, 1997) and rhomboid-7 (McQuibban et al., 2006) have defects in mitochondrial fusion at the end of meiosis and display
abnormal onion-stage nebenkern that resolve into fragmented mitochondrial derivatives. Thus, these gene products play a central role during mitochondrial morphogenesis in the fly to realize the giant nebenkern. However, it is unclear how the fusogenic properties of Fzo and rhomboid-7 can be correlated with the observation that there are two distinct mitochondrial derivatives at the start of spermatid elongation. It is still unclear if these derivatives originate in Drosophila by the fusion of two separate populations of mitochondria that form two intertwined structures within the onion-stage nebenkern, or alternatively, if
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the dispersed mitochondria aggregate into a unique nebenkern that bisects equally at the beginning of spermatid elongation. Although definitive and convincing evidence is lacking, it is remarkable that ultrastructural serial section studies made in D. melanogaster (Tates, 1971) and other insects (Pratt, 1968) indicate that the onion-stage nebenkern contains two topologically distinct compartments. This suggests that the dispersed mitochondria aggregate in two distinct clusters at one side of the cell, close to the basal body to form a bipartite onion-stage nebenkern. Then, the pair of derivatives unfurl from each other and elongate beside the growing axoneme. Since only one tubular structure resolves from the onion-stage nebenkern in parkin testes, perhaps an earlier defect occurs at the time of the clustering of the scattered mitochondria into two distinct groups. Numerous observations in many cells types indicate that mitochondria are highly dynamic structures that continuously change size and dimensions (Mozdy and Shaw, 2003). Since membrane fusions and fissions are emerging as regulators of mitochondrial biology (Chen and Chan, 2004), the disturbance of the balance among these events might lead to the disruption of mitochondrial architecture and function. Thus, the parkin product could be part of a mechanism that coordinates these events to realize the bipartite onion-stage nebenkern. Our studies indicate that the parkin mutation does not interfere with mitochondrial dynamics during spermatogenesis; it affects post-meiotic differentiation of the male germ cell, when there is an essential mitochondrial fusion process. Many gene products needed for spermatid differentiation are typically translated at the beginning of spermiogenesis (Schafer et al., 1995). Although parkin transcripts are present in embryos and egg chambers, mutations do not have striking effects on viability, female fertility or embryogenesis. Perhaps normal mitochondrial morphogenesis requires a threshold level of parkin activity, or the parkin product might be functionally redundant with other proteins or pathways involved in mitochondrial dynamics in cells outside of the testis. On the other hand, since the mutation in parkin is a null allele, perhaps the effect is not due to threshold levels of protein activity, but rather is only apparent in a sensitive system that puts great demands on its function. Spermiogenesis, in which all the mitochondria in the spermatid coalesce adjacent to the nucleus and undergo a process of massive membrane fusion, may be such a sensitive system, and the wild-type function of parkin could be mainly required for assembly and/or maintenance of the complex architecture of the mitochondrial derivative, which forms from mitochondria dispersed throughout the male germ cell after the meiotic divisions. A role for the parkin protein in mitochondrial morphogenesis or function during the process of sperm maturation is suggested by the observation of unusual electron-dense onion-stage nebenkern and by the finding of mitochondria of variable size and shape that might represent abnormal fusion events. Moreover, since paracrystalline bodies are abnormal in number, size, and condensation, mitochondrial metabolism could be in part affected by lesions in parkin. It has been recently shown that removal of the Drosophila PTEN-
induced kinase 1 (PINK1) homologue function results in male sterility and defects in mitochondrial morphology (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). Since genetic evidence has established that pink1 and parkin act in a common pathway in maintaining mitochondrial integrity, with pink1 functioning upstream of parkin, it is noteworthy that the individualization defects found in parkin spermatid cysts share a marked phenotypic similarity with pink1 loss-of-function mutants. Parkin was reported to be associated in mouse brain and cultured cells with different subcellular compartments, such as cytoplasmic vesicles, Golgi complexes, and endoplasmic reticulum, as well as the outer mitochondrial membrane (Kubo et al., 2001; Darios et al., 2003; Mouatt-Prigent et al., 2004). Parkin can also binds to α/β tubulin in rat brain and human embryonic kidney cells (Ren et al., 2003) and it is found at the centrosome of human cultured cells in response to inhibition of proteasomes (Zhao et al., 2003). Because the frequency of fusion is limited by cytoskeletal motors and transport events that bring mitochondria into close proximity (Bereiter-Hahn and Voth, 1994), excessive fusion could be possible if mitochondrial motility is increased or mitochondrial-cytoskeleton attachments are disturbed. parkin could, therefore, also operate in a non-mitochondrial compartment and could be involved in mechanisms, such as transport or positioning, essential to proper mitochondrial distribution during the early steps of spermatid differentiation. However, a role for parkin in actin cytoskeleton dynamics has not been demonstrated, even if the association of the parkin gene product with actin filaments in kidney and neuronal cells has been reported (Huynh et al., 2000). It is also possible that the defects seen were in the stability of the bipartite onion structure rather than in the proper establishment of its architecture. In the absence of functional parkin, the equilibrium between the two distinct clusters of mitochondria may be altered, destabilizing the initial structure and ultimately leading to the improper fusion in a unique structure of the two closely wrapped mitochondrial slices. Since mitochondrial morphogenesis is a necessary process during differentiation and establishment of final structures within the mature spermatozoon of the fly, it is understandable that mutations in parkin lead to male sterility. However, mice lacking parkin have been reported to be fertile (Goldberg et al., 2003; Itier et al., 2003). This suggests that mitochondrial transformation and morphogenesis during sperm maturation in mice and flies may require different routes or they are differently sensitive to the proper function of parkin gene product. It does not seem surprising because mouse spermiogenesis has no individualization process, the sperm are shorter and their elongation relies on IFT. Drosophila is rather unusual in how spermiogenesis is carried out, likely making this process uniquely sensitive to parkin dysfunction. Thus, Drosophila may be the only model in which parkin can be productively studied in spermiogenesis. It could be argued that, while this may not translate directly to mammalian spermiogenesis, this model still is likely to shed light on parkin function in general. parkin was detected in various organisms including flies, frogs, birds, and mice, indicating that this gene may play a general role
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