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First published online June 20, 2006
doi: 10.1242/10.1242/jcs.03022

Journal of Cell Science 119, 2819-2825 (2006)
Published by The Company of Biologists 2006
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Research Article

Interplay of PIWI/Argonaute protein MIWI and kinesin KIF17b in chromatoid bodies of male germ cells

Noora Kotaja1, Haifan Lin2, Martti Parvinen3 and Paolo Sassone-Corsi1,*,{ddagger}

1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, B.P. 10142, 67404 Illkirch-Strasbourg, France
2 Department of Cell Biology, Duke University Medical Center, PO Box 3709, Durham, NC 27710, USA
3 Department of Anatomy, University of Turku, FIN-20520, Turku, Finland

* Author for correspondence (e-mail: psc{at}uci.edu)

Accepted 20 April 2006


   Summary
Top
 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Chromatoid bodies are thought to act as male-germ-cell-specific platforms for the storing and processing of haploid transcripts. The molecular mechanisms governing the formation and function of these germ-cell-specific structures have remained elusive. In this study, we show that the kinesin motor protein KIF17b, which is involved in the nucleocytoplasmic transport of RNA and of a transcriptional coactivator, localizes in chromatoid bodies. The chromatoid body moves actively and non-randomly in the cytoplasm of round spermatids, making frequent contacts with the nuclear envelope. The localization of KIF17b thereby offers a potential mechanism for microtubule-dependent mobility of chromatoid bodies, as well as for the transport of the specific components in and out of the chromatoid body. Interestingly, we demonstrate that KIF17b physically interacts with a testis-specific member of the PIWI/Argonaute family, MIWI, a component of chromatoid bodies implicated in RNA metabolism. A functional interplay between KIF17b and MIWI might be needed for the loading of haploid RNAs in the chromatoid body. Importantly, chromatoid bodies from round spermatids of miwi-null mice are not fully compacted and remain as a diffuse chromatoid material, revealing the essential role played by MIWI in the formation of chromatoid bodies. These results shed new light on the function of chromatoid bodies in the post-transcriptional regulation of gene expression in haploid germ cells.

Key words: Spermatogenesis, Chromatoid body, MIWI, Kinesin, RNA processing


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The chromatoid body is a germ-cell-specific cytoplasmic structure that was first described more than one hundred years ago (Benda, 1891Go). Its presence has been reported in various mammals and other species [see Fawcett et al. (Fawcett et al., 1970Go) and references therein]. Using electron microscopy, the chromatoid body appears to comprise thin filaments consolidated into a compact mass or into dense strands of varying thickness that branch to form an irregular network. In mouse, the chromatoid body appears for the first time in the cytoplasm of meiotic pachytene spermatocytes as an electron-dense fibrous-granular structure in the interstices of mitochondria clusters (Fawcett et al., 1970Go). After meiotic divisions in round spermatids, it condenses to one single finely filamentous lobulated perinuclear granule, and it stays as a distinctive feature in the cytoplasm of post-meiotic spermatids until the nucleus starts to elongate. In elongating spermatids, it forms a ring around the base of the flagellum together with the annulus. It diminishes in size while migrating with the annulus to the caudal end of the developing middle piece of the flagellum, and finally disappears late in spermiogenesis (Fawcett et al., 1970Go).

On the basis of its structural features and protein composition, the chromatoid body was suggested to be a specialized form of germplasm or nuage, the structure that in many species determines the germ cell status of cells (Parvinen, 2005Go). The ATP-dependent RNA helicase of the DEAD-box protein family, VASA, is the best-characterized component of germplasm in Drosophila (Fujiwara et al., 1994Go; Toyooka et al., 2000Go). Homologues of the vasa gene have been isolated in various species, such as Caenorhabditis elegans, planarian, Xenopus, zebrafish, mouse and rat (Raz, 2000Go). The VASA protein in Drosophila binds many important target mRNAs involved in germ cell establishment, such as Oskar and Nanos, or Gurken in oogenesis, and controls the onset of translation (Styhler et al., 1998Go). Females carrying the vasa mutation give rise to sterile progenies because of the lack of pole cells (Styhler et al., 1998Go).

The mouse vasa homologue, Mvh, is expressed specifically in both female and male germ cells, first in primordial germ cells during embryogenesis, and later in germ cells during oogenesis and spermatogenesis (Fujiwara et al., 1994Go; Toyooka et al., 2000Go). In male germ cells, MVH is a cytoplasmic protein that is closely associated with chromatoid bodies in round spermatids (Toyooka et al., 2000Go). Interestingly, mutation of the Mvh gene in the mouse gives rise to a male-sterile phenotype, and spermatogenesis in Mvh–/– males is blocked at a zygotene stage (Tanaka et al., 2000Go). In contrast to Drosophila, Mvh–/– female mice are fertile (Tanaka et al., 2000Go). In addition to MVH, several proteins have been reported to localize in chromatoid bodies in the mouse, many of which are known to be involved in RNA metabolism. These proteins include ribonucleoproteins (snRNPs and hnRNPs) (Biggiogera et al., 1993Go; Moussa et al., 1994Go), actin (Walt and Armbruster, 1984Go), histone H4 (Werner and Werner, 1995Go), two cytochrome c isozymes (Hess et al., 1993Go), germ-cell-specific RNA-binding protein p48/52 (Oko et al., 1996Go), and gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) (Tsai-Morris et al., 2004Go).

DNA is not present in the chromatoid body (Biggiogera et al., 1990Go). However, the presence of RNA has been suggested during early spermiogenesis from step 1 to step 8 (Söderström and Parvinen, 1976Go; Walt and Armbruster, 1984Go; Figueroa and Burzio, 1998Go; Saunders et al., 1992Go). Treatment of spermatids with actinomycin D, an inhibitor of transcription, caused structural changes in the chromatoid body and disturbed the labeling of the chromatoid body with [3H]uridine, suggesting that mRNAs synthesized in the nucleus are stored in the chromatoid body (Söderström, 1977Go; Parvinen et al., 1978Go). The chromatoid body moves rapidly in the cytoplasm of spermatids in both parallel and perpendicular fashion related to the nuclear envelope (Parvinen and Parvinen, 1979Go; Ventelä et al., 2003Go). Rapidly moving chromatoid bodies have been suggested to collect gene products from the nucleus, and be involved in nucleocytoplasmic RNA transport (Parvinen, 2005Go). The chromatoid body was also reported to move through cytoplasmic bridges to neighboring cells, suggesting that this organelle could provide a mechanism to share haploid products between adjacent spermatids (Ventelä et al., 2003Go).


Figure 1
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  		Fig. 1. Localization of KIF17b and MIWI in the chromatoid body. (A,B) Squash preparations at stage VI (A), or drying down slides of male germ cells from stages II-VI (B) were immunostained with anti-KIF17b antibody (red). (C,D) Concentration of MIWI in chromatoid bodies as shown by immunostaining of squash preparation (C) or drying down slides (D) with anti-MIWI antibody (red). The parallel phase contrast image in panels B and D demonstrate the location of chromatoid bodies. Alexa Fluor 594 anti-rabbit IgG was used as a secondary antibody, and nuclei are stained blue with DAPI. Bar in A and C, 10 µm; bar in B and D, 5 µm.

 
Recently, we demonstrated that the RNA-binding protein MIWI is also concentrated in chromatoid bodies (Kotaja et al., 2006Go). MIWI belongs to the Argonaute family of highly basic proteins that contain two common domains, PAZ and PIWI (Deng and Lin, 2002Go). The family members are classified into two subfamilies based on sequence comparison: those that are more similar either to Arabidopsis Argonaute1 or to Drosophila Piwi (Carmell et al., 2002Go; Sasaki et al., 2003Go). Mammals contain four Argonaute1 subfamily members, Ago1 to Ago4, which have been shown to constitute core components of RNA-induced silencing complex (RISC) in microRNA and RNA interference (RNAi) pathways (Liu et al., 2004Go; Meister et al., 2004Go). All four members of the PIWI subfamily are expressed mainly in testis (Sasaki et al., 2003Go), and two family members, MIWI and MILI, have been shown to be crucial for normal spermatogenesis in mouse (Deng and Lin, 2002Go; Kuramochi-Miyagawa et al., 2004Go).

Here, we show that MIWI interacts with KIF17b. The testis-specific kinesin KIF17b has been shown to shuttle between nuclear and cytoplasmic compartments and to transport the coactivator of CREM in testis (ACT) from the nucleus to the cytoplasm, thus regulating CREM-dependent transcription (Macho et al., 2002Go; Kotaja et al., 2005Go). We have found that the localization of KIF17b in the cytoplasm of round spermatids is concentrated in chromatoid bodies. Thus, KIF17b provides a potential mechanism for the active movements of the chromatoid body, as well as for the transport of RNAs from the nucleus to the chromatoid bodies. The physical interaction of KIF17b and MIWI suggests a functional interpretation of the role played by the chromatoid body in the differentiation of male germ cells. Finally, the essential role of MIWI in the structuring of chromatoid bodies was demonstrated by the use of miwi-null mice.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Localization of kinesin KIF17b in chromatoid bodies
KIF17b shuttles between nuclear and cytoplasmic compartments in round spermatids (Macho et al., 2002Go; Kotaja et al., 2005Go). We performed immunofluorescence experiments on squash preparations to study the localization of KIF17b with great detail. Interestingly, we observed that, when in the cytoplasm, the signal was concentrated in one spot (Fig. 1A). We prepared drying down cell preparations containing cells from stages II-V of the seminiferous epithelial cycle to study if this granule corresponded to chromatoid bodies. In these preparations, the cytoplasm is partially lost and chromatoid bodies that usually stay in contact with the nuclear envelope are clearly visible as dense structures by phase contrast microscopy. The KIF17b signal was shown to overlap with the phase contrast image of each chromatoid body, thus confirming the localization of KIF17b in this structure (Fig. 1B). We have previously shown that MIWI localizes in chromatoid bodies (Kotaja et al., 2006Go) and therefore we used MIWI antibody as a positive control for chromatoid body staining (Fig. 1C,D).

KIF17b interacts with MIWI, a component of the chromatoid body
KIF17b and MIWI have both been shown to be involved in CREM-dependent regulation of gene expression (Macho et al., 2002Go; Deng and Lin, 2002Go). Since KIF17b and MIWI co-localized in the same subcellular structure in haploid male germ cells, we explored the possibility that these two proteins might physically interact. In co-immunoprecipitation experiments after co-expression of FLAG-MIWI and Myc-KIF17b in transfected cultured cells, we could clearly demonstrate that KIF17b co-precipitates with MIWI (Fig. 2).


Figure 2
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  		Fig. 2. KIF17b interacts with MIWI. Interaction of the full-length KIF17b with MIWI. COS-1 cells were transfected with expression plasmids encoding Myc-tagged KIF17b and FLAG-MIWI. Immunoprecipitation (IP) was performed from the cell lysates with anti-FLAG antibody ({alpha}-FLAG), and the samples were immunoblotted either by anti-Myc antibody ({alpha}-Myc) to detect co-immunoprecipitated KIF17b or by anti-FLAG antibody to detect MIWI. A non-specific band crossreacting with the anti-FLAG antibody is indicated by an asterisk.

 
Compaction of chromatoid bodies in step 1 spermatids
The co-localization and interaction of MIWI with KIF17b prompted us to study the role of this protein in chromatoid body function by analyzing spermatids from miwi-null mice. First, we used wild-type mice to characterize the formation of chromatoid bodies in stage I cells, a time when they compact to their final form (Parvinen, 2005Go). We have divided stage I round spermatids into three subgroups, step Ia, Ib and Ic spermatids, on the basis of the appearance of chromatoid bodies. Immediately after meiotic division, in very early step Ia spermatids, the chromatoid material is still dispersed in the cytoplasm (Fig. 3A). In the mid-stage I, chromatoid-body-like structures appear in the cytoplasm of step Ib spermatids. At this point, chromatoid bodies are not fully condensed but appear as a more diffuse material. At late stage I, in step Ic spermatids, the chromatoid body condenses to form a dense granule that becomes typical of later stages of round spermatid development. As MVH is the specific marker of chromatoid bodies in the mouse (Toyooka et al., 2000Go), we used anti-MVH antibodies combined with squash preparations of specific phases of stage I to localize chromatoid bodies during their condensation process (Fig. 3B). In secondary spermatocytes, before the second meiotic division at stage XII, MVH is already concentrated in cytoplasmic granules. During meiotic division, granular staining disappears and MVH disperses in the cytoplasm (not shown). Step Ia and Ib spermatids show concentrated granular MVH staining, but the signal is more diffused than at step Ic spermatids. From step Ic onwards, chromatoid bodies are fully compacted (Fig. 3B). Thus, MVH is present in the chromatoid material during the whole process of chromatoid body formation.


Figure 3
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  		Fig. 3. Formation of the chromatoid body at stage I. (A) Phase contrast microscopy of early chromatoid bodies. Squash preparations at specific stages (indicated in the lower right corner of each picture) were observed by phase contrast microscopy. Stage I was divided into three subgroups (Ia, Ib and Ic) on the basis of the progress of the chromatoid body compaction. In step Ia spermatids, chromatoid body material is still dispersed in the cytoplasm. In step Ib, chromatoid bodies start condensing and, in step Ic, they are finally condensed to the final form corresponding to the mature chromatoid bodies seen also at later stages. Arrows point to chromatoid bodies; asterisks show the location of the developing acrosome. (B) Localization of MVH in the forming chromatoid bodies. Immunostaining of squash preparations at specific stages was performed using polyclonal anti-MVH antibody ({alpha}-MVH) and Alexa Fluor 594 anti-rabbit IgG as a secondary antibody (red). Nuclei are stained blue with DAPI. Bars, 5 µm.

 

Disruption of the chromatoid body in miwi-null mice
In miwi-null mice, spermatogenesis is fully blocked at very early stages of round spermatid differentiation. Germ cell development seems to continue normally until the round spermatid stage, and then stops for most haploid cells at stage I (Deng and Lin, 2002Go). Some spermatids were reported to continue until steps 4-5 of spermiogenesis (Deng and Lin, 2002Go), and we have been able to identify some rare cases of acrosomes at step 6 (not shown). The presence of normal pre-meiotic and meiotic cell types enabled us to identify the stages of seminiferous epithelial cycle in a given piece of seminiferous tubule. The identification was assessed on the basis of the presence of type A4, type B and intermediate spermatogonia, as well as pre-leptotene, leptotene and zygotene spermatocytes, and the size of pachytene spermatocytes (Kotaja et al., 2004bGo). We analyzed specific stages in the seminiferous epithelium of miwi–/– mice, and noticed that chromatoid bodies had an abnormal structure (Fig. 4). In contrast to normal round spermatids, under phase contrast microscopy it was difficult to identify chromatoid bodies. Whenever present, chromatoid bodies looked more dispersed and diffused compared with wild-type mice. Fig. 4A presents several examples of round spermatids at various stages. Note that the acrosomal system is not normally developed.


Figure 4
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  		Fig. 4. The chromatoid body is disrupted in miwi–/– mice. (A) Phase contrast microscopy of miwi-null round spermatids. Squash preparations of specific stages were observed under the phase contrast optics and identified on the basis of the presence or absence of the other specific cell types in the preparations, as well as on the basis of the size of pachytene spermatocytes. Some diffuse chromatoid material is present in the cytoplasm of round spermatids at stages I, III, IV, V, VI, VII, but fully compacted chromatoid bodies were absent. The development of a normal acrosomal system is also compromised (asterisks). Bar, 5 µm. (B-E) Electron microscopy of the chromatoid body. In B and D, the wild-type fully condensed chromatoid body is shown; whereas, C and E show the chromatoid body in a miwi–/– mouse. Arrowheads point to the chromatoid bodies. Bar in B and C, 3 µm; bar in D and E, 1 µm. (F,G) The pattern of MVH staining in the chromatoid body is changed in miwi–/– mice. Squash preparation at stage II of the wild-type (F) or miwi–/– (G) mouse was immunostained with anti-MVH antibody ({alpha}-MVH) and Alexa Fluor 594 secondary antibody (red). Nuclei are stained blue with DAPI. Bar, 3 µm.

 

To examine in more detail the structure of chromatoid bodies in miwi–/– mice, we performed electron microscopy (Fig. 4B-E). In wild-type mice, chromatoid bodies are clearly visible as filamentous electron-dense non-membrane-bound areas (Fig. 4B,D). By contrast, we were unable to identify fully compacted chromatoid bodies in miwi-null mice, and observed instead thin threads and/or granules of non-compacted chromatoid material (Fig. 4C,E). Immunofluorescence of round spermatids using the anti-MVH antibody combined with confocal microscopy revealed that MVH still has a granular cytoplasmic staining, indicating that MIWI is not required for MVH localization. However, MVH staining appeared more diffuse. In chromatoid bodies of wild-type mice, the MVH signal is not homogenous, but is instead concentrated in specific areas that correspond to the lobes of chromatoid bodies (Fig. 4F). This type of localization pattern was disrupted in miwi-null mice (Fig. 4G).


   Discussion
Top
 Summary
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Highly specialized genetic and epigenetic pathways of gene regulation govern the complex differentiation program of male germ cells (Kimmins and Sassone-Corsi, 2005Go). Genes implicated in meiotic division and differentiation of haploid germ cells are highly expressed in spermatocytes and spermatids, respectively. During late steps of spermiogenesis, transcription ceases, mostly because of the compaction of the haploid genome following the transition from histones to protamines (Sassone-Corsi, 2002Go). Because a large number of specific gene products are still required for the last steps of sperm development, mRNA storing and processing play a crucial role (Sassone-Corsi, 1997Go; Braun, 1998Go; Steger, 2001Go). Interestingly, various mRNA-binding proteins that control the stability and translation of target mRNAs have been identified in male germ cells.

Increasing evidence supports the hypothesis that chromatoid bodies serve as platforms for specialized, male-germ-cell-specific RNA processing in the cytoplasm of round spermatids (Parvinen, 2005Go). The involvement of chromatoid bodies in RNA metabolism is supported by our recent results demonstrating that components of the microRNA (miRNA) pathway, such as miRNAs, Argonaute (Ago) proteins and the Dicer endonuclease, accumulate in these perinuclear granules (Kotaja et al., 2006Go). Our findings suggest the involvement of the chromatoid body in miRNA-dependent regulation of specific mRNAs, providing an attractive interpretation of the phenomenon of translational repression that occurs post-meiotically (Sassone-Corsi, 1997Go). The testis-specific PIWI/Argonaute family member MIWI also accumulates in the chromatoid body, suggesting that it might function as a germ-cell-specific component of the miRNA pathway (Kotaja et al., 2006Go).


Figure 5
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  		Fig. 5. The chromatoid body in post-meiotic male germ cells. Hypothetical model of how the chromatoid body might function. After transcription, haploid gene products are assembled in the ribonucleoprotein particles containing RNA-binding proteins. A kinesin KIF17b transports mRNAs through nuclear pore complexes into the cytoplasm. The chromatoid body in the cytoplasm of haploid spermatids moves actively, makes frequent contacts with the nuclear envelope and collects mRNAs, KIF17b and other material directly from nuclear pores. In the chromatoid body, KIF17b interacts with the testis-specific PIWI/Argonaute family member, MIWI. Chromatoid bodies also contain other RNA-binding and RNA-processing proteins, such as the ATP-dependent DEAD-box RNA helicase MVH (mouse VASA homolog), and components of the RNA decay pathway and the miRNA pathway such as miRNAs, Dicer and Argonaute proteins Ago2 and Ago3 (Kotaja et al., 2006Go). MIWI is proposed to function as a testis-specific component of the miRNA pathway. RNA-processing enzymes act on their target mRNAs, which might be either degraded by the RNA decay enzymes or translationally repressed and stored by order of miRNAs. The presence of several separate processing pathways suggests that the chromatoid body functions as a sorting center that determines the destiny of mRNAs. KIF17b could also be involved in the regulation of the active movements of the chromatoid body. Txn, transcription.

 
One distinct feature of chromatoid bodies is their rapid and non-random movements, which include frequent contacts with the nuclear envelope and transient movements into the indentations of the nuclear envelope (Parvinen, 2005Go). On the basis of these characteristics, chromatoid bodies are thought to be involved in the transport of RNAs or other nuclear components in the cytoplasm of haploid male germ cells. Electron microscopy has revealed material continuities between nucleus and chromatoid body, proposing that chromatoid bodies collect their contents from the nucleus (Söderström and Parvinen, 1976Go; Parvinen and Parvinen, 1979Go). This hypothesis is supported by the observation that nuclear pores tend to be more concentrated in the area adjacent to the chromatoid body (Fawcett et al., 1970Go).

The movements of chromatoid bodies are disturbed by microtubule-depolymerizing drugs, demonstrating the involvement of the intracellular microtubular network in their mobility (Ventelä et al., 2003Go). Interestingly, we have shown that a microtubule-binding kinesin protein, KIF17b, accumulates in chromatoid bodies (Fig. 1), which is suggestive of possible mechanisms accounting for the active movements. In addition, the finding that KIF17b interacts with MIWI (Fig. 2) is revealing of the intimate interplay that this kinesin has with the chromatoid body. Indeed, ablation of MIWI results in aberrant formation of chromatoid bodies (Fig. 4).

Kinesins are motor proteins that bind microtubules and carry various types of cargos, including vesicles, proteins or RNAs along the microtubules in an ATP-dependent-manner (Woehlke and Schliwa, 2000Go). KIF17b shuttles between nuclear and cytoplasmic compartments in haploid round spermatids, and regulates CREM-dependent transcription by determining the subcellular localization of the coactivator protein ACT (Fimia et al., 1999Go; Macho et al., 2002Go; Kotaja et al., 2004aGo). This function was demonstrated to be microtubule independent and not requiring the motor domain of KIF17b (Kotaja et al., 2005Go). In addition to the transport of ACT, evidence indicates that KIF17b might be involved in the transport of RNA: KIF17b binds ribonucleoparticles containing specific mRNAs, such as CREM target mRNAs, and mediates the transport of these particles from the nucleus into the cytoplasm (Chennathukuzhi et al., 2003Go). We favor a scenario in which KIF17b is involved in the transport of RNAs from the nucleus to the chromatoid bodies of male germ cells.

Importantly, both MIWI and KIF17b associate with CREM target mRNAs in ribonucleoprotein complexes, suggesting that they are functionally connected (Deng and Lin, 2002Go; Chennathukuzhi et al., 2003Go). Interestingly, miwi-null and crem-null mice share very similar spermatogenic phenotypes, with arrest in early post-meiotic cells, which reflects the misregulation of important genes required for the differentiation of haploid cells (Deng and Lin, 2002Go; Nantel et al., 1996Go). Here, we show that KIF17b and MIWI not only co-localize in chromatoid bodies, but also physically interact, thus providing an intriguing functional link between this motor protein and a RNA-binding PIWI/Argonaute family member. Localization of KIF17b and MIWI overlaps both temporally and spatially in post-meiotic round spermatids: MIWI is a cytoplasmic protein that is concentrated in chromatoid bodies, whereas KIF17b shuttles between the two compartments, transiently localizing in chromatoid bodies. It is plausible that MIWI could serve as an anchoring point for KIF17b, whose function would be to collect RNAs from the nucleus and transport them to the cytoplasm. Interestingly, MIWI is not required for KIF17b localization in chromatoid bodies (data not shown). Thus, the interaction between KIF17b and MIWI is compatible with a mechanism of material exchange between these two proteins (Fig. 5).

The accumulation of MIWI in chromatoid bodies and its interaction with KIF17b (Fig. 2) and MVH, the well-characterized chromatoid body component (Kuramochi-Miyagawa et al., 2004Go), indicated that MIWI is likely to play a role in the chromatoid body function. MIWI is essential for spermatogenesis, as clearly demonstrated by the complete block of germ cell differentiation at early stages of spermiogenesis in miwi-null mouse (Deng and Lin, 2002Go). We have shown that chromatoid bodies of miwi-null mice are either absent, or display an aberrant, uncompacted organization. Electron microscopy revealed the complete absence of a highly organized structure of the chromatoid body of miwi-null mice. It has been suggested that the chromatoid body originates from thin fibrous cytoplasmic structures that consolidate into a dense particle in late step I spermatids (Fawcett et al., 1970Go). By electron microscopy, we have demonstrated that the condensation of chromatoid material in miwi–/– mice is aberrant, indicating that MIWI is required for the formation of chromatoid bodies (Fig. 4). MVH still accumulates in perinuclear dots in miwi-null mice, but the lobulated and compartmentalized staining observed in normal spermatids becomes diffuse in miwi–/– mice, corresponding to the lack of higher order structure as shown by the electron microscopy.

The role of the chromatoid body in the development of male germ cells is gradually being revealed, although many attractive aspects remain elusive. The characterization of the kinesin KIF17b as a chromatoid body component, as well as its functional interaction with MIWI, a protein crucial for chromatoid body formation, provide new exciting clues for future studies. Our findings pave the way to novel routes of investigation of the role played by this intriguing organelle in male germ cell differentiation.


   Materials and Methods
Top
 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Plasmid construction
The plasmid pcdna-FLAG-MIWI has been described elsewhere (Kuramochi-Miyagawa et al., 2004Go). The plasmid pCS2-MTK-MIWI was cloned by transferring MIWI cDNA from pcdna-FLAG-MIWI to pCS2-MTK vector with EcoRI and XbaI. pCS2-MTK-KIF17b and KIF17b deletions have been described before (Kotaja et al., 2005Go).

Squash preparations and phase contrast microscopy
Testes of an adult C57B1/6 mouse or miwi–/– mouse (Deng and Lin, 2002Go) were dissected and decapsulated in phosphate-buffered saline (PBS). After identification of the waves of the seminiferous epithelium by transillumination technique, stage-specific short tubule segments were cut (Kotaja et al., 2004bGo). For squash preparations, the tubule segments were transferred with a pipette on microscope slides in 15 µl of PBS. A coverslip was placed carefully onto the tubule segment, and the excess fluid was removed by blotting, which allowed the cells to float out from the tubule. The exact stage was identified under phase contrast microcopy.

Immunofluorescence
The squash preparation slides were snap-frozen in liquid nitrogen, the coverslip was removed, and the cells were fixed with 97% ethanol. After fixation, the slides were air dried at room temperature. To prepare the stage-specific drying-down slides, segments of seminiferous tubules were isolated as described above, and transferred in 20 µl of 100 mM sucrose solution in a small petri dish. Cells were released from the tubules by squeezing carefully with fine forceps, and the cells were suspended by pipetting up and down. The cell suspension was spread on a slide dipped in the fixing solution [1% paraformaldehyde (PFA), 0.15% Triton X-100, pH 9.2], and the slides were dried in a highly humidified box overnight. For immunofluorescence, the squash preparations were postfixed with 4% PFA. The squash preparation or drying down slides were permeabilized with 0.2% Triton X-100 for 5 minutes. Non-specific sites were blocked by incubating slides in 5% BSA for 60 minutes. The primary antibody incubation was carried out at 4°C in 1% BSA solution with anti-MVH polyclonal antibody (1:200), anti-KIF17 polyclonal antibody (1:200, K3638; Sigma), anti-MIWI-C polyclonal antibody (1:150) (Kuramochi-Miyagawa et al., 2004Go). Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes) and Alexa Fluor 488 goat anti-mouse IgG were used as secondary antibodies. Nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI), and the preparations were mounted in Vectashield (Vector Laboratories).

Electron microscopy
Tubule segments (2 mm in length) from stages I-IV of the cycle were isolated by transillumination (Kotaja et al., 2004bGo), fixed in 5% glutaraldehyde in PBS at 20°C and prepared according to standard procedures.

Immunoprecipitations
COS-1 cells grown on 10 cm cell culture plates were transfected with the indicated plasmids using the FuGENE transfection reagent (Roche). At 40 hours after transfection, cells were collected and lysed in a buffer containing 50 mM Tris-HCl pH 8.0, 170 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 mM dithiothreitol (DTT) and 1:1000 protease inhibitor cocktail. The lysate was cleared by centrifuging at full speed for 10 minutes, and the supernatant was precleared with protein-G-Sepharose. Immunoprecipitation was performed with anti-FLAG M2 (Sigma) monoclonal antibody or anti-Myc 9E10 monoclonal antibody, and protein-G-Sepharose (Amersham Biosciences). After binding, beads were washed with lysis buffer and proteins were released in 2x Laemmli sample buffer. Samples were run on SDS polyacrylamide gels, and immunoblotting was performed by monoclonal anti-Myc 9E10 (1:1000) or anti-FLAG M2 (1:1000) antibodies, or polyclonal anti-KIF17b (1:1000) antibody.


   Acknowledgments
 
We thank W. Filipowicz, S. Kimmins, C. Ziegler-Birling, N. Fischer, A. Gansmuller and all members of the Sassone-Corsi laboratory for help, stimulating discussions and reagents. N.K. was supported by the European Molecular Biology Organization. Our studies are supported by grants from Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire Régional, Fondation de la Recherche Médicale, Université Louis Pasteur et La Ligue contre le Cancer (Equipe Labelisée), and NIH (HD42012 to H.L.).


   Footnotes
 
{ddagger} Present address: Department of Pharmacology, Gillespie Neuroscience, University of California, Irvine, CA 92697-4625, USA Back


   References
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 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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