2819Research Article

IntroductionThe chromatoid body is a germ-cell-specific cytoplasmicstructure that was first described more than one hundred yearsago (Benda, 1891). Its presence has been reported in variousmammals and other species [see Fawcett et al. (Fawcett et al.,1970) and references therein]. Using electron microscopy,the chromatoid body appears to comprise thin filamentsconsolidated into a compact mass or into dense strands ofvarying thickness that branch to form an irregular network. Inmouse, the chromatoid body appears for the first time in thecytoplasm of meiotic pachytene spermatocytes as an electron-dense fibrous-granular structure in the interstices ofmitochondria clusters (Fawcett et al., 1970). After meioticdivisions in round spermatids, it condenses to one single finelyfilamentous lobulated perinuclear granule, and it stays as adistinctive feature in the cytoplasm of post-meiotic spermatidsuntil the nucleus starts to elongate. In elongating spermatids,it forms a ring around the base of the flagellum together withthe annulus. It diminishes in size while migrating with theannulus to the caudal end of the developing middle piece ofthe flagellum, and finally disappears late in spermiogenesis(Fawcett et al., 1970).

On the basis of its structural features and proteincomposition, the chromatoid body was suggested to be aspecialized form of germplasm or nuage, the structure thatin many species determines the germ cell status of cells

(Parvinen, 2005). The ATP-dependent RNA helicase of theDEAD-box protein family, VASA, is the best-characterizedcomponent of germplasm in Drosophila (Fujiwara et al., 1994;Toyooka et al., 2000). Homologues of the vasa gene have beenisolated in various species, such as Caenorhabditis elegans,planarian, Xenopus, zebrafish, mouse and rat (Raz, 2000). TheVASA protein in Drosophila binds many important targetmRNAs involved in germ cell establishment, such as Oskar andNanos, or Gurken in oogenesis, and controls the onset oftranslation (Styhler et al., 1998). Females carrying the vasamutation give rise to sterile progenies because of the lack ofpole cells (Styhler et al., 1998).

The mouse vasa homologue, Mvh, is expressed specificallyin both female and male germ cells, first in primordial germcells during embryogenesis, and later in germ cells duringoogenesis and spermatogenesis (Fujiwara et al., 1994; Toyookaet al., 2000). In male germ cells, MVH is a cytoplasmic proteinthat is closely associated with chromatoid bodies in roundspermatids (Toyooka et al., 2000). Interestingly, mutation ofthe Mvh gene in the mouse gives rise to a male-sterilephenotype, and spermatogenesis in Mvh–/– males is blockedat a zygotene stage (Tanaka et al., 2000). In contrast toDrosophila, Mvh–/– female mice are fertile (Tanaka et al.,2000). In addition to MVH, several proteins have been reportedto localize in chromatoid bodies in the mouse, many of whichare known to be involved in RNA metabolism. These proteins

Chromatoid bodies are thought to act as male-germ-cell-specific platforms for the storing and processing of haploidtranscripts. The molecular mechanisms governing theformation and function of these germ-cell-specificstructures have remained elusive. In this study, we showthat the kinesin motor protein KIF17b, which is involvedin the nucleocytoplasmic transport of RNA and of atranscriptional coactivator, localizes in chromatoid bodies.The chromatoid body moves actively and non-randomly inthe cytoplasm of round spermatids, making frequentcontacts with the nuclear envelope. The localization ofKIF17b thereby offers a potential mechanism formicrotubule-dependent mobility of chromatoid bodies, aswell as for the transport of the specific components in andout of the chromatoid body. Interestingly, we demonstratethat KIF17b physically interacts with a testis-specific

member of the PIWI/Argonaute family, MIWI, acomponent of chromatoid bodies implicated in RNAmetabolism. A functional interplay between KIF17b andMIWI might be needed for the loading of haploid RNAs inthe chromatoid body. Importantly, chromatoid bodies fromround spermatids of miwi-null mice are not fullycompacted and remain as a diffuse chromatoid material,revealing the essential role played by MIWI in theformation of chromatoid bodies. These results shed newlight on the function of chromatoid bodies in the post-transcriptional regulation of gene expression in haploidgerm cells.

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

Summary

Interplay of PIWI/Argonaute protein MIWI and kinesinKIF17b in chromatoid bodies of male germ cellsNoora Kotaja1, Haifan Lin2, Martti Parvinen3 and Paolo Sassone-Corsi1,*,‡

1Institut de Génétique et de Biologie Moléculaire et Cellulaire, B.P. 10142, 67404 Illkirch-Strasbourg, France2Department of Cell Biology, Duke University Medical Center, PO Box 3709, Durham, NC 27710, USA3Department of Anatomy, University of Turku, FIN-20520, Turku, Finland*Author for correspondence (e-mail: psc@uci.edu)‡Present address: Department of Pharmacology, Gillespie Neuroscience, University of California, Irvine, CA 92697-4625, USA

Accepted 20 April 2006Journal of Cell Science 119, 2819-2825 Published by The Company of Biologists 2006doi:10.1242/jcs.03022

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include ribonucleoproteins (snRNPs and hnRNPs) (Biggiogeraet al., 1993; Moussa et al., 1994), actin (Walt and Armbruster,1984), histone H4 (Werner and Werner, 1995), two cytochromec isozymes (Hess et al., 1993), germ-cell-specific RNA-binding protein p48/52 (Oko et al., 1996), and gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) (Tsai-Morris et al., 2004).

DNA is not present in the chromatoid body (Biggiogera etal., 1990). However, the presence of RNA has been suggestedduring early spermiogenesis from step 1 to step 8 (Söderströmand Parvinen, 1976; Walt and Armbruster, 1984; Figueroa andBurzio, 1998; Saunders et al., 1992). Treatment of spermatidswith actinomycin D, an inhibitor of transcription, causedstructural changes in the chromatoid body and disturbed thelabeling of the chromatoid body with [3H]uridine, suggestingthat mRNAs synthesized in the nucleus are stored in thechromatoid body (Söderström, 1977; Parvinen et al., 1978).The chromatoid body moves rapidly in the cytoplasm ofspermatids in both parallel and perpendicular fashion relatedto the nuclear envelope (Parvinen and Parvinen, 1979; Venteläet al., 2003). Rapidly moving chromatoid bodies have beensuggested to collect gene products from the nucleus, and beinvolved in nucleocytoplasmic RNA transport(Parvinen, 2005). The chromatoid body was alsoreported to move through cytoplasmic bridges toneighboring cells, suggesting that this organellecould provide a mechanism to share haploidproducts between adjacent spermatids (Ventelä etal., 2003).

Recently, we demonstrated that the RNA-binding protein MIWI is also concentrated inchromatoid bodies (Kotaja et al., 2006). MIWIbelongs to the Argonaute family of highly basicproteins that contain two common domains, PAZand PIWI (Deng and Lin, 2002). The familymembers are classified into two subfamilies basedon sequence comparison: those that are moresimilar either to Arabidopsis Argonaute1 or toDrosophila Piwi (Carmell et al., 2002; Sasaki etal., 2003). Mammals contain four Argonaute1subfamily members, Ago1 to Ago4, which havebeen shown to constitute core components ofRNA-induced silencing complex (RISC) inmicroRNA and RNA interference (RNAi)pathways (Liu et al., 2004; Meister et al., 2004).All four members of the PIWI subfamily areexpressed mainly in testis (Sasaki et al., 2003), andtwo family members, MIWI and MILI, have beenshown to be crucial for normal spermatogenesis inmouse (Deng and Lin, 2002; Kuramochi-Miyagawa et al., 2004).

Here, we show that MIWI interacts withKIF17b. The testis-specific kinesin KIF17b hasbeen shown to shuttle between nuclear andcytoplasmic compartments and to transport thecoactivator of CREM in testis (ACT) from thenucleus to the cytoplasm, thus regulating CREM-dependent transcription (Macho et al., 2002;Kotaja et al., 2005). We have found that thelocalization of KIF17b in the cytoplasm of roundspermatids is concentrated in chromatoid bodies.

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Thus, KIF17b provides a potential mechanism for the activemovements of the chromatoid body, as well as for the transportof RNAs from the nucleus to the chromatoid bodies. Thephysical interaction of KIF17b and MIWI suggests a functionalinterpretation of the role played by the chromatoid body in thedifferentiation of male germ cells. Finally, the essential roleof MIWI in the structuring of chromatoid bodies wasdemonstrated by the use of miwi-null mice.

ResultsLocalization of kinesin KIF17b in chromatoid bodiesKIF17b shuttles between nuclear and cytoplasmiccompartments in round spermatids (Macho et al., 2002; Kotajaet al., 2005). We performed immunofluorescence experimentson squash preparations to study the localization of KIF17b withgreat detail. Interestingly, we observed that, when in thecytoplasm, the signal was concentrated in one spot (Fig. 1A).We prepared drying down cell preparations containing cellsfrom stages II-V of the seminiferous epithelial cycle to studyif this granule corresponded to chromatoid bodies. In thesepreparations, the cytoplasm is partially lost and chromatoidbodies that usually stay in contact with the nuclear envelope

Fig. 1. Localization of KIF17b and MIWI in the chromatoid body. (A,B) Squashpreparations at stage VI (A), or drying down slides of male germ cells fromstages II-VI (B) were immunostained with anti-KIF17b antibody (red).(C,D) Concentration of MIWI in chromatoid bodies as shown byimmunostaining of squash preparation (C) or drying down slides (D) with anti-MIWI antibody (red). The parallel phase contrast image in panels B and Ddemonstrate the location of chromatoid bodies. Alexa Fluor 594 anti-rabbit IgGwas used as a secondary antibody, and nuclei are stained blue with DAPI. Bar inA and C, 10 �m; bar in B and D, 5 �m.

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are clearly visible as dense structures by phase contrastmicroscopy. The KIF17b signal was shown to overlap with thephase contrast image of each chromatoid body, thus confirmingthe localization of KIF17b in this structure (Fig. 1B). We havepreviously shown that MIWI localizes in chromatoid bodies(Kotaja et al., 2006) and therefore we used MIWI antibody asa positive control for chromatoid body staining (Fig. 1C,D).

KIF17b interacts with MIWI, a component of thechromatoid bodyKIF17b and MIWI have both been shown to be involved inCREM-dependent regulation of gene expression (Macho et al.,2002; Deng and Lin, 2002). Since KIF17b and MIWI co-localized in the same subcellular structure in haploid malegerm cells, we explored the possibility that these twoproteins might physically interact. In co-immunoprecipitationexperiments after co-expression of FLAG-MIWI and Myc-KIF17b in transfected cultured cells, we could clearlydemonstrate that KIF17b co-precipitates with MIWI (Fig. 2).

Compaction of chromatoid bodies in step 1 spermatidsThe co-localization and interaction of MIWI with KIF17bprompted us to study the role of this protein in chromatoidbody function by analyzing spermatids from miwi-null mice.First, we used wild-type mice to characterize the formationof chromatoid bodies in stage I cells, a time when theycompact to their final form (Parvinen, 2005). We have dividedstage I round spermatids into three subgroups, step Ia, Ib andIc spermatids, on the basis of the appearance of chromatoidbodies. Immediately after meiotic division, in very early stepIa spermatids, the chromatoid material is still dispersed in thecytoplasm (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 butappear as a more diffuse material. At late stage I, in step Icspermatids, the chromatoid body condenses to form a densegranule that becomes typical of later stages of roundspermatid development. As MVH is the specific marker ofchromatoid bodies in the mouse (Toyooka et al., 2000),

we used anti-MVH antibodies combined with squashpreparations of specific phases of stage I to localizechromatoid bodies during their condensation process (Fig.3B). In secondary spermatocytes, before the second meioticdivision at stage XII, MVH is already concentrated incytoplasmic granules. During meiotic division, granularstaining disappears and MVH disperses in the cytoplasm (notshown). Step Ia and Ib spermatids show concentratedgranular MVH staining, but the signal is more diffused thanat step Ic spermatids. From step Ic onwards, chromatoidbodies are fully compacted (Fig. 3B). Thus, MVH is presentin the chromatoid material during the whole process ofchromatoid body formation.

Fig. 2. KIF17b interacts with MIWI. Interaction of the full-lengthKIF17b with MIWI. COS-1 cells were transfected with expressionplasmids encoding Myc-tagged KIF17b and FLAG-MIWI.Immunoprecipitation (IP) was performed from the cell lysates withanti-FLAG antibody (�-FLAG), and the samples wereimmunoblotted either by anti-Myc antibody (�-Myc) to detect co-immunoprecipitated KIF17b or by anti-FLAG antibody to detectMIWI. A non-specific band crossreacting with the anti-FLAGantibody is indicated by an asterisk.

Fig. 3. Formation of the chromatoid body at stage I. (A) Phasecontrast microscopy of early chromatoid bodies. Squash preparationsat specific stages (indicated in the lower right corner of each picture)were observed by phase contrast microscopy. Stage I was dividedinto three subgroups (Ia, Ib and Ic) on the basis of the progress of thechromatoid body compaction. In step Ia spermatids, chromatoidbody material is still dispersed in the cytoplasm. In step Ib,chromatoid bodies start condensing and, in step Ic, they are finallycondensed to the final form corresponding to the mature chromatoidbodies 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 wasperformed using polyclonal anti-MVH antibody (�-MVH) and AlexaFluor 594 anti-rabbit IgG as a secondary antibody (red). Nuclei arestained blue with DAPI. Bars, 5 �m.

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Disruption of the chromatoid body in miwi-null miceIn miwi-null mice, spermatogenesis is fully blocked at veryearly stages of round spermatid differentiation. Germ celldevelopment seems to continue normally until the roundspermatid stage, and then stops for most haploid cells at stageI (Deng and Lin, 2002). Some spermatids were reported tocontinue until steps 4-5 of spermiogenesis (Deng and Lin,2002), and we have been able to identify some rare cases ofacrosomes 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 ofseminiferous tubule. The identification was assessed on thebasis of the presence of type A4, type B and intermediatespermatogonia, as well as pre-leptotene, leptotene andzygotene spermatocytes, and the size of pachytenespermatocytes (Kotaja et al., 2004b). We analyzed specificstages in the seminiferous epithelium of miwi–/– mice, andnoticed that chromatoid bodies had an abnormal structure (Fig.4). In contrast to normal round spermatids, under phasecontrast microscopy it was difficult to identify chromatoidbodies. Whenever present, chromatoid bodies looked moredispersed and diffused compared with wild-type mice. Fig. 4Apresents several examples of round spermatids at variousstages. Note that the acrosomal system is not normallydeveloped.

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

DiscussionHighly specialized genetic and epigenetic pathways of generegulation govern the complex differentiation program of malegerm cells (Kimmins and Sassone-Corsi, 2005). Genesimplicated in meiotic division and differentiation of haploidgerm cells are highly expressed in spermatocytes andspermatids, respectively. During late steps of spermiogenesis,transcription ceases, mostly because of the compaction of thehaploid genome following the transition from histones toprotamines (Sassone-Corsi, 2002). Because a large number of

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Fig. 4. The chromatoid body is disrupted in miwi–/– mice. (A) Phasecontrast microscopy of miwi-null round spermatids. Squashpreparations of specific stages were observed under the phasecontrast optics and identified on the basis of the presence or absenceof the other specific cell types in the preparations, as well as on thebasis of the size of pachytene spermatocytes. Some diffusechromatoid material is present in the cytoplasm of round spermatidsat stages I, III, IV, V, VI, VII, but fully compacted chromatoid bodieswere absent. The development of a normal acrosomal system is alsocompromised (asterisks). Bar, 5 �m. (B-E) Electron microscopy ofthe chromatoid body. In B and D, the wild-type fully condensedchromatoid body is shown; whereas, C and E show the chromatoidbody in a miwi–/– mouse. Arrowheads point to the chromatoid bodies.Bar in B and C, 3 �m; bar in D and E, 1 �m. (F,G) The pattern ofMVH 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 (�-MVH) andAlexa Fluor 594 secondary antibody (red). Nuclei are stained bluewith DAPI. Bar, 3 �m.

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specific gene products are still required for the last steps ofsperm development, mRNA storing and processing play acrucial role (Sassone-Corsi, 1997; Braun, 1998; Steger, 2001).Interestingly, various mRNA-binding proteins that control thestability and translation of target mRNAs have been identifiedin male germ cells.

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

One distinct feature of chromatoid bodies is theirrapid and non-random movements, which includefrequent contacts with the nuclear envelope andtransient movements into the indentations of thenuclear envelope (Parvinen, 2005). On the basis ofthese characteristics, chromatoid bodies are thoughtto be involved in the transport of RNAs or othernuclear components in the cytoplasm of haploid malegerm cells. Electron microscopy has revealedmaterial continuities between nucleus andchromatoid body, proposing that chromatoid bodiescollect their contents from the nucleus (Söderströmand Parvinen, 1976; Parvinen and Parvinen, 1979).This hypothesis is supported by the observation thatnuclear pores tend to be more concentrated in the areaadjacent to the chromatoid body (Fawcett et al.,1970).

The movements of chromatoid bodies aredisturbed by microtubule-depolymerizing drugs,demonstrating the involvement of the intracellularmicrotubular network in their mobility (Ventelä etal., 2003). Interestingly, we have shown that amicrotubule-binding kinesin protein, KIF17b,accumulates in chromatoid bodies (Fig. 1), which issuggestive of possible mechanisms accounting forthe active movements. In addition, the finding thatKIF17b interacts with MIWI (Fig. 2) is revealing ofthe intimate interplay that this kinesin has with thechromatoid body. Indeed, ablation of MIWI results inaberrant formation of chromatoid bodies (Fig. 4).

Kinesins are motor proteins that bind microtubulesand carry various types of cargos, including vesicles,proteins or RNAs along the microtubules in an ATP-dependent-manner (Woehlke and Schliwa, 2000).KIF17b shuttles between nuclear and cytoplasmiccompartments in haploid round spermatids, andregulates CREM-dependent transcription bydetermining the subcellular localization of the

coactivator protein ACT (Fimia et al., 1999; Macho et al.,2002; Kotaja et al., 2004a). This function was demonstrated tobe microtubule independent and not requiring the motordomain of KIF17b (Kotaja et al., 2005). In addition to thetransport of ACT, evidence indicates that KIF17b might beinvolved in the transport of RNA: KIF17b bindsribonucleoparticles containing specific mRNAs, such asCREM target mRNAs, and mediates the transport of theseparticles from the nucleus into the cytoplasm (Chennathukuzhiet al., 2003). We favor a scenario in which KIF17b is involvedin the transport of RNAs from the nucleus to the chromatoidbodies of male germ cells.

Importantly, both MIWI and KIF17b associate with CREMtarget mRNAs in ribonucleoprotein complexes, suggesting thatthey are functionally connected (Deng and Lin, 2002;Chennathukuzhi et al., 2003). Interestingly, miwi-null andcrem-null mice share very similar spermatogenic phenotypes,with arrest in early post-meiotic cells, which reflects themisregulation of important genes required for thedifferentiation of haploid cells (Deng and Lin, 2002; Nantel etal., 1996). Here, we show that KIF17b and MIWI not only co-localize in chromatoid bodies, but also physically interact, thus

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Fig. 5. The chromatoid body in post-meiotic male germ cells. Hypotheticalmodel of how the chromatoid body might function. After transcription,haploid gene products are assembled in the ribonucleoprotein particlescontaining RNA-binding proteins. A kinesin KIF17b transports mRNAsthrough nuclear pore complexes into the cytoplasm. The chromatoid body inthe cytoplasm of haploid spermatids moves actively, makes frequent contactswith the nuclear envelope and collects mRNAs, KIF17b and other materialdirectly from nuclear pores. In the chromatoid body, KIF17b interacts with thetestis-specific PIWI/Argonaute family member, MIWI. Chromatoid bodiesalso contain other RNA-binding and RNA-processing proteins, such as theATP-dependent DEAD-box RNA helicase MVH (mouse VASA homolog),and components of the RNA decay pathway and the miRNA pathway such asmiRNAs, Dicer and Argonaute proteins Ago2 and Ago3 (Kotaja et al., 2006).MIWI is proposed to function as a testis-specific component of the miRNApathway. RNA-processing enzymes act on their target mRNAs, which mightbe either degraded by the RNA decay enzymes or translationally repressedand stored by order of miRNAs. The presence of several separate processingpathways suggests that the chromatoid body functions as a sorting center thatdetermines the destiny of mRNAs. KIF17b could also be involved in theregulation of the active movements of the chromatoid body. Txn, transcription.

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providing an intriguing functional link between this motorprotein and a RNA-binding PIWI/Argonaute family member.Localization of KIF17b and MIWI overlaps both temporallyand spatially in post-meiotic round spermatids: MIWI is acytoplasmic protein that is concentrated in chromatoid bodies,whereas KIF17b shuttles between the two compartments,transiently localizing in chromatoid bodies. It is plausible thatMIWI could serve as an anchoring point for KIF17b, whosefunction would be to collect RNAs from the nucleus andtransport them to the cytoplasm. Interestingly, MIWI is notrequired for KIF17b localization in chromatoid bodies (datanot shown). Thus, the interaction between KIF17b and MIWIis compatible with a mechanism of material exchange betweenthese two proteins (Fig. 5).

The accumulation of MIWI in chromatoid bodies and itsinteraction with KIF17b (Fig. 2) and MVH, the well-characterized chromatoid body component (Kuramochi-Miyagawa et al., 2004), indicated that MIWI is likely to playa role in the chromatoid body function. MIWI is essential forspermatogenesis, as clearly demonstrated by the completeblock of germ cell differentiation at early stages ofspermiogenesis in miwi-null mouse (Deng and Lin, 2002). Wehave shown that chromatoid bodies of miwi-null mice are eitherabsent, or display an aberrant, uncompacted organization.Electron microscopy revealed the complete absence of a highlyorganized structure of the chromatoid body of miwi-null mice.It has been suggested that the chromatoid body originates fromthin fibrous cytoplasmic structures that consolidate into a denseparticle in late step I spermatids (Fawcett et al., 1970). Byelectron microscopy, we have demonstrated that thecondensation of chromatoid material in miwi–/– mice isaberrant, indicating that MIWI is required for the formation ofchromatoid bodies (Fig. 4). MVH still accumulates inperinuclear dots in miwi-null mice, but the lobulated andcompartmentalized staining observed in normal spermatidsbecomes diffuse in miwi–/– mice, corresponding to the lack ofhigher order structure as shown by the electron microscopy.

The role of the chromatoid body in the development of malegerm cells is gradually being revealed, although manyattractive aspects remain elusive. The characterization of thekinesin KIF17b as a chromatoid body component, as well asits functional interaction with MIWI, a protein crucial forchromatoid body formation, provide new exciting clues forfuture studies. Our findings pave the way to novel routes ofinvestigation of the role played by this intriguing organelle inmale germ cell differentiation.

Materials and MethodsPlasmid constructionThe plasmid pcdna-FLAG-MIWI has been described elsewhere (Kuramochi-Miyagawa et al., 2004). The plasmid pCS2-MTK-MIWI was cloned by transferringMIWI cDNA from pcdna-FLAG-MIWI to pCS2-MTK vector with EcoRI and XbaI.pCS2-MTK-KIF17b and KIF17b deletions have been described before (Kotaja etal., 2005).

Squash preparations and phase contrast microscopyTestes of an adult C57B1/6 mouse or miwi–/– mouse (Deng and Lin, 2002) weredissected and decapsulated in phosphate-buffered saline (PBS). After identificationof the waves of the seminiferous epithelium by transillumination technique, stage-specific short tubule segments were cut (Kotaja et al., 2004b). For squashpreparations, the tubule segments were transferred with a pipette on microscopeslides in 15 �l 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 outfrom the tubule. The exact stage was identified under phase contrast microcopy.

ImmunofluorescenceThe squash preparation slides were snap-frozen in liquid nitrogen, the coverslip wasremoved, and the cells were fixed with 97% ethanol. After fixation, the slides wereair dried at room temperature. To prepare the stage-specific drying-down slides,segments of seminiferous tubules were isolated as described above, and transferredin 20 �l of 100 mM sucrose solution in a small petri dish. Cells were released fromthe tubules by squeezing carefully with fine forceps, and the cells were suspendedby pipetting up and down. The cell suspension was spread on a slide dipped in thefixing solution [1% paraformaldehyde (PFA), 0.15% Triton X-100, pH 9.2], and theslides were dried in a highly humidified box overnight. For immunofluorescence,the squash preparations were postfixed with 4% PFA. The squash preparation ordrying 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. Theprimary 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.,2004). Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes) and Alexa Fluor488 goat anti-mouse IgG were used as secondary antibodies. Nuclei were stainedusing 4�,6-diamidino-2-phenylindole (DAPI), and the preparations were mounted inVectashield (Vector Laboratories).

Electron microscopyTubule segments (2 mm in length) from stages I-IV of the cycle were isolated bytransillumination (Kotaja et al., 2004b), fixed in 5% glutaraldehyde in PBS at 20°Cand prepared according to standard procedures.

ImmunoprecipitationsCOS-1 cells grown on 10 cm cell culture plates were transfected with the indicatedplasmids using the FuGENE transfection reagent (Roche). At 40 hours aftertransfection, cells were collected and lysed in a buffer containing 50 mM Tris-HClpH 8.0, 170 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 mM dithiothreitol (DTT) and1:1000 protease inhibitor cocktail. The lysate was cleared by centrifuging at fullspeed for 10 minutes, and the supernatant was precleared with protein-G-Sepharose.Immunoprecipitation was performed with anti-FLAG M2 (Sigma) monoclonalantibody or anti-Myc 9E10 monoclonal antibody, and protein-G-Sepharose(Amersham Biosciences). After binding, beads were washed with lysis buffer andproteins were released in 2� Laemmli sample buffer. Samples were run on SDSpolyacrylamide gels, and immunoblotting was performed by monoclonal anti-Myc9E10 (1:1000) or anti-FLAG M2 (1:1000) antibodies, or polyclonal anti-KIF17b(1:1000) antibody.

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

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