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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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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


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.

 

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.

 

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.

 

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.

 

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.

 

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