Developmental Biology 301 (2007) 38–52 www.elsevier.com/locate/ydbio

Tudor-related TDRD1/MTR-1, TDRD6 and TDRD7/TRAP: Domain composition, intracellular localization, and function in male germ cells in mice

Mihoko Hosokawa a, Masanobu Shoji a, Kouichi Kitamura a, Takashi Tanaka a, ⁎ Toshiaki Noce b, Shinichiro Chuma a, , Norio Nakatsuji a

a Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan b Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya Machida-shi, Tokyo 194-8511, Japan Received for publication 24 March 2006; revised 27 October 2006; accepted 31 October 2006 Available online 6 November 2006

Abstract

The germ-line cells of many animals possess a characteristic cytoplasmic structure termed nuage or germinal granules. In mice, nuage that is prominent in postnatal male germ cells is also called intermitochondrial cement or chromatoid bodies. TDRD1/MTR-1, which contains Tudor domain repeats, is a specific component of the mouse nuage, analogously to Drosophila Tudor, a constituent of polar granules/nuage in oocytes and embryos. We show that TDRD6 and TDRD7/TRAP, which also contain multiple Tudor domains, specifically localize to nuage and form a ribonucleoprotein complex together with TDRD1/MTR-1. The characteristic co-localization of TDRD1, 6 and 7 was disrupted in a mutant of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), which encodes another evolutionarily conserved component of nuage. In vivo over-expression experiments of the TDRD proteins and truncated forms during male germ cell differentiation showed that a single Tudor domain is a structural unit that localizes or accumulates to nuage, but the expression of the truncated, putative dominant negative forms is detrimental to meiotic spermatocytes. These results indicate that the Tudor-related proteins, which contain multiple repeats of the Tudor domain, constitute an evolutionarily conserved class of nuage components in the germ-line, and their localization or accumulation to nuage is likely conferred by a Tudor domain structure and downstream of Mvh, while the characteristic repeated architecture of the domain is functionally essential for the differentiation of germ cells. © 2006 Elsevier Inc. All rights reserved.

Keywords: Germ cell; Spermatogenesis; Tdrd; Tudor; Mvh; Vasa; Intermitochondrial cement; Chromatoid body; Nuage; Electroporation

Introduction nuage in oocytes and early embryos, and these granules are asymmetrically partitioned to germ cell precursors (Mahowald, In a wide variety of animals, germ cells exhibit particular 1962; Lehmann and Ephrussi, 1994; Saffman and Lasko, 1999). cytoplasmic structures called nuage or germinal granules (Eddy, Similarly, P granules in C. elegans (Strome and Wood, 1982) 1975). The structures are characterized by an amorphous shape, and germinal granules in Xenopus (Czolowska, 1969) are the absence of surrounding membranes, the abundance of RNAs segregated to prospective germ cells during early development, and proteins and a close association with mitochondria or and these structures are thought to participate in the partitioning nuclei. In Drosophila, several products of posterior-group and/or accumulation of germ cell determinants. such as oskar, vasa and tudor, which function in pole cell In mice, prospective germ cells are induced among and abdominal formation, localize to polar granules, a form of pluripotent epiblast cells at around gastrulation stages (Lawson and Hage, 1994; Lawson et al., 1999; Tam and Zhou, 1996; ⁎ Corresponding author. Fax: +81 75 751 3890. McLaren, 2003), and the presence of nuage during this E-mail address: [email protected] (S. Chuma). determination process remains unclear. On the other hand,

0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.10.046 M. Hosokawa et al. / Developmental Biology 301 (2007) 38–52 39 mammalian nuage becomes clearly discernible at the later suggesting that the Tudor related proteins retain an evolutiona- stages of differentiation of the germ-line, such as in rily conserved mechanism that regulates their intracellular spermatogonia and developing oocytes, and in mice, nuage localization. To investigate the possible correlation between the becomes most prominent in postnatal meiotic spermatocytes repeated architecture of the Tudor domain and their localization and haploid spermatids (Fawcett et al., 1970; Eddy, 1974; and function, we carried out in vivo over-expression experi- Russell and Frank, 1978; Parvinen, 2005). Nuage in ments of the TDRD proteins and truncated forms in male germ spermatogonia, spermatocytes and oocytes is seen among cells. The results showed that a single Tudor domain can clusters of mitochondria and is called “intermitochondrial localize or accumulate to nuage, while the Tudor domain repeats cement/material/bar,” whereas in spermatocytes and sperma- are essential for meiotic spermatocyte differentiation. Our tids, larger solitary aggregates of nuage, termed “chromatoid results demonstrate that Tudor-related proteins constitute a bodies,” are prominent in the cytoplasm. Mammalian nuage novel class of nuage components, with their characteristic does not appear to be asymmetrically partitioned in these cells, Tudor domains being important for their localization and thus its developmental function may differ from nuage in early function in the germ-line. embryos of other species. Meanwhile, close morphological similarities among nuages of divergent species, including Materials and methods mice, at different developmental stages suggest that they share common properties that are essential and conserved in the Mice germ-line. Vasa , a DEAD-box RNA helicase, is a component of Jcl: ICR mice were obtained from CLEA Japan and maintained in a controlled environment with 12:12 light: dark cycles. Mvh -targeted mice Drosophila polar granules, and its homologues are widely were genotyped as previously described (Tanaka et al., 2000). All experiments conserved components of nuage (Raz, 2000). In mice, the on mice were carried out in accordance with the institutional guidelines and mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4) regulations. is expressed in differentiating germ cells rather than during germ cell specification, and the MVH protein localizes to Cloning of Tdrd6 cDNA chromatoid bodies (Fujiwara et al., 1994; Toyooka et al., 2000). Interestingly, the targeted disruption of Mvh leads to male- The mouse genomic sequence Genbank/EMBL/DDBJ AZ647796 was found to contain an open reading frame (ORF) for two Tudor domains. An specific sterility due to postnatal defects in early spermatocytes approximately 260 bp fragment of this partial ORF was PCR-amplified from (Tanaka et al., 2000), although the gene is expressed in both mouse testis cDNA with the primers 5′-TTTATCGATTATGGCAACATGTCT- male and female germ cells. 3′ and 5′-ACCTGCTAATCATATCTTCAGCTA-3′. A lambda gt11 library of Tudor is another component of polar granules in Drosophila mouse testis cDNA (a kind gift from Dr. M. Nozaki) was probed with this cDNA ′ and is genetically downstream of vasa in respect to its fragment, and four overlapping clones were obtained. 5 -cap structure dependent rapid amplification of cDNA ends (5′-RACE) was carried out (Gene Racer, intracellular localization. The tudor gene maternally functions Invitrogen, USA) using adult testis mRNA (Dynabeads mRNA direct kit, Dynal, in pole cell and abdominal formation, as well as participating in Norway). Amplified products of four different lengths were cloned into the localization of mitochondrial RNAs to polar granules. The pBlueScript SK (Stratagene, USA), and at least 12 clones were sequenced for protein contains 11 Tudor domains, but the biochemical and each transcript variant. The consensus sequence of transcript variant 1 ′ physiological importance of the domain repeats remains assembled from the lambda library clones and 5 -RACE products was submitted to Genbank/EMBL/DDBJ under the accession number AB097085. unknown (Boswell and Mahowald, 1985; Golumbeski et al., 1991; Bardsley et al., 1993; Ponting, 1997; Amikura et al., Northern blot and RT-PCR analyses 2001; Thomson and Lasko, 2004). We previously reported that the Tudor domain containing 1/mouse tudor repeat 1 (Tdrd1/ 20 μg of total RNA isolated from tissues of Jcl: ICR mice using a modified Mtr-1; hereafter referred to as Tdrd1 according to Mouse AGPC method (Trizol, Invitrogen, USA) was electrophoresed in a 0.9% Genome Informatics) is expressed in differentiating germ cells, formaldehyde gel, transferred to a nylon membrane (Hybond-N+, Amersham, USA) and probed with a 1.3 kb 3′-fragment of Tdrd6 cDNA labeled with [32P] and encodes four Tudor domains and a zinc-finger MYND dCTP. Signals were detected with X-ray film (Kodak, USA). For RT-PCR, 1 μg domain (Wang et al., 2001; Chuma et al., 2003). The TDRD1 of total RNA was treated with DNase I (Promega, USA) and reverse-transcribed protein localizes to both intermitochondrial cement in male and using random 9 mer and SuperScript II (Invitrogen). PCR primers were 5′- female germ cells and to chromatoid bodies in the male (Chuma TTTATCGATTATGGCAACATGTCT-3′ and 5′-ACCTGCTAATCATATCTT- ′ ′ ′ et al., 2003; Chuma et al., 2006). The germ-line expression, CAGCTA-3 for Tdrd6 and 5 -GTCGTACCACGGGCATTGTGATGG-3 and 5′-GCAATGCCTGGGTACATGGTGG-3′ for β-actin. Amplified products domain composition and intracellular localization to nuage are were gel-electrophoresed and stained with ethidium bromide. analogous features shared by Drosophila Tudor and mouse TDRD1. Production of anti-TDRD6 and 7 antibodies, and Western blot In this study, we report the characterization of TDRD6, a analysis putative orthologue of Drosophila Tudor, and TDRD7/TRAP (Hirose et al., 2000) which also contains Tudor domain repeats. A Tdrd6 cDNA fragment encoding amino acids 1911–2134 with a 6xHis TDRD6 and 7 specifically localize to nuage and form a complex tag was cloned into pGEX-6P-1 (Amersham). The fusion protein produced in E. coli BL21 was purified using Glutathione Sepharose 4B and PreScission together with TDRD1. The co-localization of the TDRD protease (Amersham). Rabbits were immunized with the TDRD6 C-terminal proteins was disrupted in a mutant of Mvh, and this is analogous fragment, and specific antibodies were affinity-purified from the antisera using to the relationship between Drosophila vasa and Tudor, the same antigen coupled to cellufine beads (Chisso, Japan). A Tdrd7 cDNA 40 M. Hosokawa et al. / Developmental Biology 301 (2007) 38–52 fragment encoding amino acids 1–246 was cloned into pQE-32 (Qiagen, Plasmids and in vivo electroporation of the testis Germany). The 6xHis-tagged TDRD7 N-terminal fragment produced in E. coli M15[pREP4] was purified using Ni-NTA agarose under denaturing conditions. pCAG-TDRD1-TDx4, -ΔTDRD1-TDx2 and -ΔTDRD1-TDx1, which Rabbits were immunized with the denatured protein, and specific antibodies contain four, two, and one Tudor domain(s), respectively, and tagged with a were affinity-purified from the antisera using the same antigen. For Western 6xHis, were described previously (Chuma et al., 2003). pCAG-TDRD6-TDx7, blotting of TDRD6, lysates of adult testes and NIH/3T3 cells transfected with -ΔTDRD6-TDx2 and -ΔTDRD6-TDx1 contained the full-length or truncated pCAG-TDRD6 or control pCXN2 (Niwa et al., 1991) were subjected to a 5– Tdrd6 cDNAs, encoding amino acids 1–2134, 1–415 and 1–160 tagged with 20% gradient SDS-PAGE and transferred to a nitrocellulose membrane (Protran FLAG, respectively, in an XhoI site downstream of the CAG enhancer/promoter BA, Schleicher and Schuell, Germany). The blots were probed with anti- of pCXN2. Point mutations Tyr1019 to Asn (TAT to AAT) and Glu1023 to Lys TDRD6 antibodies followed by alkaline phosphatase-conjugated secondary (GAA to AAA) were introduced into the Tudor domain of pCAG-ΔTDRD1- antibodies. Signals were detected with CDP-Star with NitroBlock II TDx1 by a PCR-based over-lap extension method (Sambrook and Russell, (PerkinElmer, USA) and X-ray film. 2001) using Pfu DNA polymerase (Stratagene). These plasmids were mixed with the transfection marker pCAG-enhanced cyan fluorescent protein (ECFP) Immunofluorescence and immunoelectron microscopy and injected into seminiferous tubules of testes of juvenile mice undergoing the first wave of spermatogenesis. The testes were subjected to in vivo 10 μm cryosections of testes fixed in 2% paraformaldehyde (PFA) in 1× electroporation as described (Shoji et al., 2005). Spermatogenic cells were phosphate buffered saline (PBS) were immunostained with anti-TDRD1 identified by their morphology and position in seminiferous tubules and by (Chuma et al., 2003), TDRD6, TDRD7, SYCP3/SCP3 (Chuma and immunostaining for germ cell marker proteins. Nakatsuji, 2001), MVH (Toyooka et al., 2000), Sm (Y12) (Lab vision, USA), FLAG (Sigma) and 6xHis (Bethyl laboratories, USA). The secondary antibodies used were FITC-conjugated anti-rat immunoglobulin (Ig), Results Rhodamine B-anti-rabbit Ig (BioSource, USA), Alexa Fluor 488 anti-rabbit IgG, and Alexa Fluor 568 and 555 anti-mouse IgG (Molecular Probes, USA). For double-immunostaining using two rabbit antibodies, Zenon Rabbit IgG Tdrd6 encodes seven Tudor domains and is abundant in the Labeling Kits for Alexa Fluor 555 and 568 (Molecular Probes) were used. testis Nuclei were stained with 1 μg/ml Hoechst 33258 dye (Sigma). For μ immunoelectron microscopy of TDRD6, 8 m cryosections of testes fixed Tblastn search was carried out for cDNAs and ESTs encoding in 4% PFA, 0.05% glutaraldehyde in 0.1 M phosphate buffer (pH. 7.4) were incubated with anti-TDRD6 antibodies or preimmune serum as a control, multiple Tudor domains, and the human cDNA fragment followed by incubation with 1.4 nm gold-conjugated secondary antibodies Genbank/EMBL/DDBJ AF039442 was found to contain an (Nanoprobes, USA). The signals were intensified using a silver enhancement incomplete ORF for two Tudor domains. By blastn search, the kit, HQ silver and sections were post-fixed in 1% OsO4 in 0.1 M phosphate mouse genomic sequence AZ647796 was found to contain the – buffer, dehydrated, embedded in epoxy resin and cut into 70 90 nm sections mouse homologue of this human cDNA. A corresponding mouse for electron microscopy. For TDRD7, testes were fixed in 4% PFA, 0.02% glutaraldehyde in 0.1 M phosphate buffer, and embedded in epoxy resin. 70– cDNA fragment, amplified by RT-PCR using primers designed 90 nm sections were incubated with anti-TDRD7 antibodies, followed by based on the genomic sequence, was then used to probe a mouse 15 nm gold-labeled secondary antibodies. After postfixation with 2% testis cDNA library, and four overlapping clones were obtained. glutaraldehyde in PBS, sections were stained with uranyl acetate followed The 5′ ends of the cDNA were determined by 5′-RACE, and the by lead citrate. Samples were examined using an electron microscope H7000 assembled sequences revealed four transcript variants of 4.8– (Hitachi, Japan). 7.1 kb (Fig. 1A). The longest transcript variant 1 was most Immunoprecipitation abundant in the RACE analysis. Correspondingly, Northern blotting showed a predominant band of about 7 kb in the testis, Single cell suspension of adult testes was prepared by collagenase and indicating that transcript variant 1 is the major transcript (Fig. 1B). trypsin treatment (Shoji et al., 2005). For Western blotting, the cells were The transcripts were not detected by RT-PCR in fetal male and – suspended in hypotonic buffer (50 mM KCl, 10 mM Tris Cl (pH 7.4), 5 mM female gonads or in adult ovaries (Fig. 1C). MgCl ), homogenized using a Dounce homogenizer, added with one volume 2 Transcript variant 1 contained an ORF encoding 2134 amino of 280 mM KCl, 60 mM NaCl, 10 mM Tris–Cl, 5 mM MgCl2, then centrifuged at 160×g. The supernatant was supplemented with 0.1% NP-40, a acids. Dot matrix plotting and motif searches using the protease inhibitor cocktail (Sigma) and SUPERaseIn (Ambion, USA). PROSITE and Pfam databases identified seven copies of the Immunoprecipitation was carried out using anti-TDRD1, 6 and 7 antibodies Tudor domain (Figs. 1D, E), and the gene was designated tudor and a normal rabbit immunoglobulin G bound to Protein G coupled magnetic domain containing 6 (Tdrd6) according to the Mouse Genome beads (Dynal). The precipitates were eluted in standard SDS-PAGE buffer with 6 M urea, and Western blots were probed with anti-TDRD1, 6 and 7 and Informatics. The Tdrd6 gene consists of a long first exon anti-MVH (Abcam, UK) antibodies, followed by alkaline phosphatase- followed by four short exons (Fig. 1A) spanning about 50 kb on conjugated secondary antibodies. Signals were detected with CDP-Star with 17. The promoter region of the Tdrd6 gene lacked NitroBlock II and X-ray film. For protein and RNA detection, single cell TATA box sequences, similarly to several other genes expressed suspension of testes was cross-linked with 1% formaldehyde in 1× PBS, in spermatogenic cells (Kleene, 2005). Tdrd6 is a putative quenched with 0.25 M glycine, then washed with 1× PBS three times. The cells were re-suspended in standard RIPA buffer, sonicated and immunopre- orthologue of Drosophila tudor as was predicted by a unique cipitation was carried out as above. Proteins were eluted in standard SDS- best reciprocal hit by BLAST, Ensemble v.32. PAGE buffer with 6 M urea, subjected to 5–20% gradient SDS-PAGE and stained with SyproRuby dye (Invitrogen, USA). RNAs were extracted from TDRD6 is a component of intermitochondrial cement and the immunoprecipitates using a modified AGPC method (ISOGEN, Nippon chromatoid bodies in male germ cells Gene, Japan), treated with DNase I (Ambion) and end-labeled with [γ-32P] ATP using T4 polynucleotide kinase (New England BioLabs, USA). Denaturing electrophoresis was carried out on 7 M urea 6% PAGE gel and We raised polyclonal antibodies against a C-terminal the signals were detected by X-ray film. fragment of TDRD6. Western blotting of adult testes using M. Hosokawa et al. / Developmental Biology 301 (2007) 38–52 41

Fig. 1. Tdrd6 encodes seven Tudor domains and is abundant in the testis. (A) Exon structure and transcript variants of Tdrd6. The first ATGs and stop codons are indicated by vertical arrows and arrowheads. Numbered boxes represent regions encoding Tudor domains. The horizontal arrow indicates the position of Tdrd6- specific primer used in 5′-RACE. The right panel shows the 5′-RACE products. TAP, tobacco acid pyrophosphatase. (B) Upper panel, Northern blot of total RNA isolated from adult tissues probed with Tdrd6 cDNA. Lower panel, 18S rRNA stained with ethidium bromide. (C) RT-PCR of Tdrd6 and beta-actin from total RNA of adult and fetal 13.5 days post coitum (dpc) testes and ovaries. (D) Domain composition of mouse TDRD1, 6 and 7 and Drosophila Tudor. Black and shaded boxes represent Tudor domains and a MYND domain. (E) Alignment of selected Tudor domain sequences by ClustalW. Identical and similar residues are in reverse and shaded fonts, respectively. The residue numbers are on the right. Accession numbers: mouse TDRD6, AB097085; Drosophila Tudor, X62420; human SMN, Q16637.

the antibody showed a band of approximately 250 kDa, the size at intermitochondrial cement in spermatocytes (Fig. 2D, arrows) of the putative molecular mass of TDRD6 (Fig. 2A). By and chromatoid bodies in both spermatocytes and round immunofluorescence staining of adult testis sections, TDRD6 spermatids (Figs. 2D, E, arrowheads). Thus, TDRD6 is a signals were observed as fine cytoplasmic granules or larger novel specific component of nuage in male germ cells, and this solitary aggregates in germ cells (Figs. 2B, C). Double-staining localization agrees with the granular distribution of TDRD6 for TDRD6 and SYCP3, a component of meiotic synaptonemal signals observed by immunofluorescence staining (Figs. 2B, C). complex (Lammers et al., 1994), showed that the cells with fine TDRD6 granules were pachytene spermatocytes (Fig. 2C, TDRD1, 6 and 7 co-localize to nuage and form an arrows). Larger aggregates of TDRD6 were observed in haploid ribonucleoprotein complex in male germ cells round spermatids (Fig. 2C, arrowheads). No significant signal was detected in adult ovaries and in male and female fetal TDRD7/TRAP is another protein that contains Tudor domain gonads (data not shown). repeats and is abundant in the testis (Hirose et al., 2000). To To further localize TDRD6, immunoelectron microscopy examine the intracellular localization of TDRD7, we produced was carried out. TDRD6 signals were predominantly observed anti-TDRD7 antibodies and carried out immunofluorescence 42 M. Hosokawa et al. / Developmental Biology 301 (2007) 38–52

Fig. 2. TDRD6 localizes to intermitochondrial cement and chromatoid bodies. (A) Western blot of lysates of adult testes, NIH/3T3 cells transfected with pCAG- TDRD6 or control pCXN2 were probed with anti-TDRD6 antibodies. (B, C) Sections of an adult testis immunostained for TDRD6 (B, green) and for TDRD6 and SYCP3/SCP3 (C, green and red) counterstained with Hoechst dye (blue). Dotted lines demarcate seminiferous tubules, and arrows and arrowheads mark spermatocytes and round spermatids. (D–F) Immunoelectron microscopy of adult testis sections stained with anti-TDRD6 antibodies (D, E) or with preimmune serum (F). TDRD6 localized to intermitochondrial cement in spermatocytes (D, arrows) and chromatoid bodies in spermatocytes and round spermatids (D, E, arrowheads). No significant signals were observed in the control (F, the arrowhead indicates a chromatoid body). Scale bars: (B), 25 μm; (C), 10 μm; (D–F), 1 μm. staining of testis sections. TDRD7 was diffusely observed in These results showed that TDRD7, together with TDRD1 spermatogonia (Figs. 3A, C, open arrowheads), but the signals (Chuma et al., 2003) and 6, constitute a novel class of nuage diminished in leptotene-zygotene spermatocytes. TDRD7 components in male germ cells in mice. became detectable again in early-pachytene spermatocytes, The immunostaining results of TDRD1, 6 and 7 appeared then in mid-late pachytene spermatocytes, the signals exhibited mostly similar (Figs. 4A–C). However, the precise localization fine granular appearance (Figs. 3A, B, arrows). Then in patterns of the TDRD proteins differed from each other. In spermatids, TDRD7 formed larger solitary aggregates similarly spermatogonia, TDRD1 exhibited a fine granular appearance to TDRD1 and 6 (Figs. 3A, B, arrowheads). The intracellular that corresponds to intermitochondrial cement (Chuma et al., localization of TDRD7 was further examined by immunoelec- 2006), while TDRD7 was diffused (Fig. 4E, asterisks) and tron microscopy. TDRD7 signals were enriched in chromatoid TDRD6 was undetectable. The three TDRD proteins were not bodies in spermatocytes and spermatids (Figs. 3D–F) and to a detected in leptotene-zygotene spermatocytes (Figs. 2B, and lesser extent, to intermitochondrial cement in spermatocytes. 3A, and data not shown). In pachytene spermatocytes, TDRD1 The signals were also observed in particulate cytoplasmic again showed granular distribution, which corresponds to both structures whose characteristics were unclear (data not shown). intermitochondrial cement and chromatoid bodies, and this

Fig. 3. TDRD7/TRAP is a component of chromatoid bodies. (A–C) Sections of adult (A, B) and postnatal day 6 (C) testes immunostained for TDRD7 (green) counterstained with Hoechst dye (blue). Spermatocytes and spermatids are indicated by arrows and arrowheads in panels A, B, and spermatogonia by open arrowheads in panels A, C. Inset in panel C shows a higher magnification view. On postnatal day 6 (C), spermatogonia are present both at the base and in the lumen of seminiferous tubules. Dotted lines demarcate seminiferous tubules. (D–F) Immunoelectron microscopy of adult testis sections stained with anti-TDRD7 antibodies (D, E) or with nonimmune rabbit immunoglobulins (F). TDRD7 localizes to chromatoid bodies in spermatocytes (D) and round spermatids (E) as indicated by arrowheads. No significant signals are seen in the control (F). Scale bars: (A, C), 25 μm; (B), 10 μm; (D–F), 0.5 μm. M. Hosokawa et al. / Developmental Biology 301 (2007) 38–52 43

Fig. 4. Nuage localization and a complex formation of TDRD1, 6 and 7 in male germ cells. (A–C) Sections of an adult testis immunostained for TDRD1 (A), 6 (B) and 7 (C) (green) and counterstained with Hoechst dye (blue). Spermatocytes and spermatids are indicated by arrows and arrowheads, respectively. (D, E) Double immunostaining of adult testis sections for TDRD1, 6 (D) and for TDRD6, 7 (E) counterstained with Hoechst dye (blue). (D) In spermatocytes, TDRD1 shows granular distribution at earlier stages than TDRD6 (arrow), while in spermatids, TDRD6 remains aggregated longer than TDRD1 (arrowheads). (E) TDRD6 and 7 exhibit granular appearances at about the same stage in spermatocytes (arrows), while in spermatids, TDRD7 remains detectable later than TDRD6 (arrowheads). Spermatogonia that show diffused signals of TDRD7 are also indicated by asterisks. In panels A–E, dotted lines demarcate seminiferous tubules. Scale bars: 10 μm. (F) Sequential localization of TDRD1, 6 and 7 to nuage. The arrows depict the differentiation stages of spermatocytes and spermatids when nuage localization is discernible. (G) Immunoprecipitation of TDRD1, 6 and 7 from adult testis. (Left panel) SDS-PAGE of immunoprecipitates with anti-TDRD1, 6 and 7 antibodies and normal rabbit immunoglobulin G as a control from adult testis lysate cross-linked with formaldehyde. Immunoglobulin heavy and light chains are indicated with arrowheads. Proteins were detected with SyproRuby dye. (Middle) Denaturing PAGE of RNAs extracted from the immunoprecipitates and end-labeled with 32P. (Right) Western blots of the immunoprecipitates obtained under a non-cross linked condition. The blots were probed with anti-TDRD1, 6 and 7 (upper panel) or with anti-MVH (lower panel) antibodies. The input lanes represent 1 (left), 0.1 (middle) and 3 (right) % of the input, respectively. localization of TDRD1 was observed at earlier stages than earlier than TDRD6 and 7 (Fig. 4D, arrowheads), and TDRD6 TDRD6 and 7 (for TDRD1 and 6, Fig. 4D, arrows). TDRD6 disappeared prior to TDRD7 (Fig. 4E, arrowheads). Taken and 7 subsequently co-localized with TDRD1 (for TDRD6 and together, the TDRD proteins are nuage components in common, 7, Fig. 4E, arrows), but considering the immunoelectron but they also show differential localization or the accessibility microscopy observations (Figs. 2D, and 3D), TDRD6 becomes of their epitopes change, as summarized in Fig. 4F. This localized to nuage earlier than TDRD7. In haploid round suggests that nuage undergoes compositional changes along spermatids, TDRD1, 6 and 7 strongly co-localized to with germ cell differentiation and the TDRD proteins may take chromatoid bodies. However, TDRD1 became undetectable part in this molecular rearrangement of nuage. 44 M. Hosokawa et al. / Developmental Biology 301 (2007) 38–52

We then examined whether the TDRD proteins are physically accumulation, while TDRD6 and 7 formed discrete cytoplasmic associated in vivo. Adult testis lysate was subjected to granules. However, the distribution of TDRD6 and 7 granules was immunoprecipitation with anti-TDRD1, 6 and 7 antibodies distinct and independent. The TDRD proteins in Mvh1098/1098 and normal rabbit immunoglobulin G as a control. The immuno- spermatocytes did not co-localize with Sm proteins of small precipitates contained several proteins and also, RNAs, as nuclear ribonucleoproteins (snRNPs) (data not shown), which shown in Fig. 4G. Western blotting demonstrated that the TDRD also are conserved components of nuage (Biggiogera et al., 1990; proteins reciprocally co-precipitated each other together with Moussa et al., 1994; Chuma et al., 2003). This indicates that each MVH/DDX4, another characteristic component of nuage. Thus, TDRD complex was compositionally distinct from or incomplete the TDRD proteins are in a large ribonucleoprotein complex, compared to wild-type nuage. The results showed that TDRD1, 6 consistently with their characteristic localization to nuage. and7requireMvh for their proper localization and assembly, and Future identification of the protein and RNA species in the that each TDRD protein likely functions in distinct complexes at TDRD complex would help address the molecular architecture different intracellular loci before their co-localization to nuage. of nuage structure. A single Tudor domain can localize or accumulate to nuage, Co-localization of TDRD proteins is disrupted in Mvh mutant but Tudor domain repeats are essential for germ cell differentiation Mvh/Ddx4 is a mouse homologue of vasa and encodes a well conserved component of nuage (Toyooka et al., 2000). In mice, It was speculated that Tudor domain repeats are closely Mvh1098/1098 gene-targeted mutants are male-sterile and arrest associated with intracellular localization and possible func- spermatogenesis during meiotic prophase of spermatocytes tion. To address this issue, we carried out in vivo over- (Tanaka et al., 2000). In Mvh1098/1098 spermatocytes, intermito- expression experiments of the TDRD proteins and their chondrial cement is not discernible and the localization of TDRD1 truncated forms in male germ cells (Fig. 6A). Testes of is disrupted (Chuma et al., 2006). Here, we compared the premature mice on different postnatal days were used for in intracellular localization of TDRD1, 6 and 7 in Mvh1098/1098 vivo electroporation of the constructs to achieve preferential spermatocytes. While all the TDRD proteins were detected, they transfection into mitotic spermatogonia, meiotic spermato- failed to co-localize in Mvh1098/1098 spermatocytes (Fig. 5). cytes and haploid round spermatids (Fig. 6B) (Shoji et al., TDRD1 was diffusely distributed or exhibited peri-nuclear 2005).

Fig. 5. Co-localization of TDRD1, 6 and 7 is disrupted in Mvh mutant. Sections of Mvh+/1098 (A, B) and Mvh1098/1098 (C, D) testes were immunostained for TDRD1, 6 and 7 as indicated. (A, B) In control Mvh+/1098 spermatocytes, the granules of TDRD1, 6 and 7 show precise co-localization that corresponds to nuage (arrowheads). (C, D) In Mvh1098/1098 spermatocytes, TDRD1, 6 and 7 exhibit separate, distinct distributions. TDRD1 is diffusely distributed or shows peri-nuclear accumulation, while TDRD6 and 7 form cytoplasmic granules, but they do not merge each other. Nuclei were counterstained with Hoechst dye (blue). Scale bar: 10 μm. M. Hosokawa et al. / Developmental Biology 301 (2007) 38–52 45

Fig. 6. In vivo over-expression experiments of TDRD1, 6 and their truncated forms during male germ cell differentiation. (A) Constructs used for in vivo electroporation of the testis. Full-length and truncated TDRD1 and 6 were tagged with 6xHis or FLAG. Shaded boxes represent Tudor domains. Arrowheads indicate the positions of amino acid substitutions introduced into ΔTDRD1-TDx1 (see Fig. 10). (B) Schematic of the experimental time course. Testes of juvenile mice on different postnatal days were used for preferential transfection into spermatogonia (d5–d8), spermatocytes (d15–d17 and d16–d18) and round spermatids (d18–d23). d5–d8 represents transfection on postnatal day 5 and sample recovery on day 8. (C–F) In vivo over-expression in spermatogonia. Sections of testes transfected for postnatal days 5–8 with the constructs indicated on the left were immunostained for 6xHis or FLAG and MVH. TDRD1-TDx4 (C) and TDRD6-TDx7 (D) were mostly diffused in the cytoplasm. ΔTDRD1-TDx1 (E) and ΔTDRD6-TDx1 (F) were detected both in the cytoplasm and nuclei. TDRD1-TDx4 and ΔTDRD1-TDx1 also localized to speckle-like structures in the nucleus. For all constructs examined, the distribution of MVH was not altered. Nuclei were counterstained with Hoechst dye (blue). Dotted lines demarcate seminiferous tubules. Scale bar: 10 μm.

In spermatogonia, full-length TDRD1-TDx4 (TDx4 denotes granular localization to nuage in spermatocytes and spermatids. four Tudor domains) was primarily diffused in the cytoplasm. ΔTDRD1-TDx1 and ΔTDRD6-TDx1, that contained a single The fine granular pattern, as seen for endogenous TDRD1 Tudor domain, were detected both in the cytoplasm and nuclei (Chuma et al., 2006), was not discernible, possibly due to the (Figs. 6E, F). The nuclear localization is presumably due to large increase in the diffused signal (Fig. 6C). Full-length passive diffusion through the nuclear pore because of their TDRD6-TDx7 was also diffusely observed in the cytoplasm reduced molecular masses. ΔTDRD1-TDx1 was also localized (Fig. 6D). The expression of these two full-length constructs did to speckle-like structures in the nucleus similarly to full-length not trigger aggregate formation of MVH (Figs. 6C, D) and Sm (Figs. 6C, E) and endogenous TDRD1. The nuclear bodies of proteins of snRNPs (data not shown), both of which show TDRD1 co-localize with those of Sm proteins of snRNPs that 46 M. Hosokawa et al. / Developmental Biology 301 (2007) 38–52 are called Cajal bodies (Chuma et al., 2003). ΔTDRD6-TDx1, Discussion on the other hand, occasionally showed mitochondrial localization (data not shown). This difference between We showed that TDRD1, 6 and 7 are abundant in the testis ΔTDRD1-TDx1 and ΔTDRD6-TDx1 suggests that these and constitute a novel class of nuage components in male germ Tudor domains associate with different binding partners cells. The molecular property and function of mammalian nuage located at different intracellular loci, or that the fragments remain largely unclarified. The chromatoid body in spermatids contain distinct localization signals to each intracellular has been relatively well described among the mammalian destination, although we did not find canonical signal peptide nuage, because of its prominence by light and electron sequences in these TDRD fragments. microscopy. This structure is implicated in RNA regulation In spermatocytes, full-length TDRD1-TDx4 and TDRD6- based on the abundance of RNA and RNA binding proteins TDx7 showed discrete cytoplasmic granules that co-localized such as transition protein 2 mRNA (Saunders et al., 1992) and with endogenous nuage components, TDRD1, 6, MVH (Figs. p48/p52 protein (Oko et al., 1996). Recently, Kotaja et al. 7A–D, arrowheads) and Sm proteins of snRNPs (data not (2006) reported that the chromatoid body in spermatids contains shown). This indicated that the two full-length constructs were Dicer and microRNAs, proposing that the structure is involved properly targeted to nuage. In contrast, ΔTDRD1-TDx1 and in a microRNA pathway. Intracellular and intercellular ΔTDRD6-TDx1 did not show particular localization, and the movements of the structure also suggest that it functions in distribution of endogenous nuage components was disrupted RNA/protein trafficking and gene dosage compensation (Figs. 7E–H). The spermatocytes expressing these truncated (Parvinen, 2005). In contrast, nuage in germ cells at other constructs were morphologically impaired, and mostly solitary stages of development, including spermatocytes, is less studied, among surrounding non-transfected spermatocytes. Deleterious and only a few components have so far been reported. TDRD1, effects of ΔTDRD1-TDx1 and ΔTDRD6-TDx1 on spermato- 6 and 7 are common components of nuage in both spermato- cytes were also observed as disorganization of meiotic synap- cytes and spermatids, and these proteins sharing Tudor domain tonemal complexes (Figs. 8A–F). Similar results were also seen repeats may represent characteristics of nuage in these cells. for ΔTDRD1-TDx2 and ΔTDRD6-TDx2 (data not shown). Meanwhile, TDRD1 also localizes to nuage in oocytes, while Quantification of the disorganization of synaptonemal TDRD6 and 7 were only detectable in the male. The TDRD complexes in spermatocytes by the expression of each proteins may confer more diversity of components and construct is shown in Fig. 8G. The data clearly demon- functions onto the male nuage than female. strated that the truncated TDRD1 and 6, which presumably In this report, we addressed the possible correlation of functioned as dominant negative forms, were detrimental to Tudor domain repeats with their intracellular location and meiotic spermatocytes. function. The result obtained by in vivo over-expression In round spermatids, TDRD1-TDx4 and TDRD6-TDx7 were experiments showed that the truncated, but not wild-type localized to chromatoid bodies, as demonstrated by co- TDRD1 and 6 proteins, were deleterious to meiotic sperma- localization with MVH and Sm proteins of snRNPs (Figs. tocytes. The truncated proteins that contained only one or two 9A–D, arrowheads). ΔTDRD1-TDx2 and ΔTDRD1-TDx1 also Tudor domains presumably functioned as dominant negative localized to chromatoid bodies (Figs. 9E–H, arrowheads), while forms that interfered with full-length proteins, providing these truncated forms exhibited diffused signals both in the evidence that Tudor domain repeats are actually essential for cytoplasm and nuclei as was also seen in spermatogonia. The germ cell differentiation. It is probable that the phenotype over-expression of both full-length and truncated proteins observed in spermatocytes resulted from functional inhibition showed no discernible differences in deleterious effects on of the TDRD proteins localized to nuage, although it remains spermatids. possible that smaller diffused fractions of the TDRD proteins The single Tudor domain of the Survival of Motor Neuron are responsible for the phenotype. In spermatids, on the other (SMN) protein interacts with its binding partners via aromatic hand, both the full-length and truncated TDRD proteins and charged residues at the binding surface (Selenko et al., showed clear localization to chromatoid bodies. Thus, Tudor 2001; Cote and Richard, 2005). These amino acids are domain repeats are not a prerequisite for nuage localization or conserved among Tudor domain sequences, and we intro- accumulation. It is reported that multiple domains or motifs of duced point mutations at residues, Y1019N and E1023K, in several proteins make up one contiguous unit of a substrate ΔTDRD1-TDx1 (Fig. 6A). The expression levels of the two binding site, such as the Armadillo repeats of importin alpha mutated ΔTDRD1-TDx1 were confirmed to be comparable to that associate with nuclear localization signals (Conti et al., that of wild-type ΔTDRD1-TDx1 when examined in NIH/ 1998). The data on ΔTDRD1-TDx1 in spermatids suggest that 3T3 cells (data not shown). In round spermatids, the mutated this is not the case for Tudor domains in TDRD1 and that a ΔTDRD1-TDx1-Y1019N and -E1023K were both diffusely single Tudor domain can be a unit for nuage localization or distributed and did not exhibit specific localization, despite accumulation. that endogenous chromatoid bodies were clearly observed The single Tudor domain in the SMN protein binds to Sm (Figs. 10A–F, and quantification in Fig. 10G). This result proteins of snRNPs (Buhler et al., 1999; Selenko et al., 2001) suggests that the single Tudor domain functions as a and two neighboring Tudor folds of 53BP1, which comprise a structural unit that localizes or accumulates to chromatoid single globular domain, associate with histone H3 (Charier et bodies/nuage. al., 2004; Huyen et al., 2004). These Tudor domains have been .Hskw ta./DvlpetlBooy31(07 38 (2007) 301 Biology Developmental / al. et Hosokawa M. – 52

Fig. 7. In vivo over-expressions of TDRD1, 6 and truncated forms in spermatocytes. Sections of testes transfected with the constructs indicated for postnatal days 15–17 or 16–18 were immunostained for 6xHis or FLAG and for TDRD1, 6 or MVH. Transfected and adjoining non-transfected spermatocytes are demarcated by dotted and solid lines, respectively. (A–D) TDRD1-TDx4 (A, B) and TDRD6-TDx7 (C, D) formed granules that co-localized with endogenous nuage components as indicted (arrowheads). (E–H) Spermatocytes expressing ΔTDRD1-TDx1 (E, F) and ΔTDRD6-TDx1 (G, H) showed aberrant morphologies, and endogenous nuage components were irregularly distributed or the signals were undetectable. Nuclei were counterstained with Hoechst dye (blue). Scale bar: 10 μm. 47 48 .Hskw ta./DvlpetlBooy31(07 38 (2007) 301 Biology Developmental / al. et Hosokawa M. – 52

Fig. 8. Truncated TDRD1 and 6 are detrimental to meiotic spermatocytes. (A–F) Sections of testes, transfected as described in Fig. 7, were immunostained for 6xHis or FLAG and for meiotic synaptonemal complex protein SYCP3. Spermatocytes expressing TDRD1-TDx4 (A) and TDRD6-TDx7 (D) were clustered and showed normal fibrous structures of synaptonemal complexes. In contrast, spermatocytes expressing ΔTDRD1- TDx1 (B, C) and ΔTDRD6-TDx1 (E, F) were mostly solitary, and synaptonemal complexes were disorganized, or SYCP3 signals were undetectable. (G) The percentages of spermatocytes showing different patterns of synaptonemal complexes stained by SYCP3. Spermatocytes transfected with each construct were immunostained for 6xHis or FLAG and for SYCP3, and classified as follows: normal, fibrous structures of SYCP3 were observed as in non-transfected spermatocytes; disorganized, fibrous patterns of SYCP3 were fragmented or disrupted; and signal(−), SYCP3 signals were invisible despite adjoining non-transfected spermatocytes showed normal SYCP3 staining. Data were collected from at least 50 spermatocytes from two independent experiments. Scale bar: 10 μm. .Hskw ta./DvlpetlBooy31(07 38 (2007) 301 Biology Developmental / al. et Hosokawa M. – 52

Fig. 9. In vivo over-expressions of TDRD1, 6 and truncated forms in spermatids. Sections of testes, transfected with the constructs indicated for postnatal days 18–23, were immunostained for 6xHis or FLAG and for endogenous nuage components MVH or Sm proteins of snRNPs. Full-length TDRD1-TDx4 (A, B) and TDRD6-TDx7 (C, D), and also truncated ΔTDRD1-TDx2 (E, F) and ΔTDRD1-TDx1 (G, H) all clearly localized to chromatoid bodies (arrowheads). Dotted lines demarcate transfected spermatids. Scale bar: 10 μm. 49 50 .Hskw ta./DvlpetlBooy31(07 38 (2007) 301 Biology Developmental / al. et Hosokawa M. – 52

Fig. 10. A Tudor domain is a structural unit that localizes or accumulates to chromatoid bodies. (A–F) Spermatids transfected with wild-type ΔTDRD1-TDx1 (A, B), mutated ΔTDRD1-TDx1-Y1019N (C, D) and -E1023K (E, F) were immunostained for 6xHis and for endogenous nuage components MVH (A, C, E) or TDRD6 (B, D, F). The point mutations in ΔTDRD1-TDx1 (C–F) disrupt the localization to chromatoid bodies (arrowheads). Dotted and solid lines demarcate transfected and adjoining non-transfected spermatids. (G) The percentages of spermatids in which transfected constructs localize to chromatoid bodies. Spermatids expressing 6xHis signals were classified as follows: TDRD6 cb (+)/6xHis cb (+), aggregates of endogenous TDRD6, that corresponds to chromatoid bodies (cb), are visible and 6xHis signals of transfected constructs merge with the TDRD6 aggregates; TDRD6 cb (+)/6xHis cb (−), TDRD6 aggregates are seen, but 6xHis signals are diffused and do not co-localize with the TDRD6 aggregates; TDRD6 cb (−)/6xHis cb (−), both TDRD6 and 6xHis signals are diffused and no specific localization is observed. Data were collected from at least 100 spermatids from two independent experiments. Scale bar: 10 μm. M. Hosokawa et al. / Developmental Biology 301 (2007) 38–52 51 shown to interact with the dimethylated arginines of their Technically, this study involves an attempt to apply in vivo binding partners, and that aromatic clusters and negative transfection of the testis to characterize gene functions in male charges at the binding pocket surfaces are essential for these germ cells. To achieve preferential transfection into spermato- interactions (Selenko et al., 2001; Charier et al., 2004; Huyen et genic cells at the intended stages of differentiation, testes of al., 2004; Cote and Richard, 2005). Point mutations at the juvenile mice during the first wave of spermatogenesis were corresponding residues, Y1019N and E1023K, in ΔTDRD1- subjected to in vivo electroporation, as we recently reported TDx1 abolished its localization to chromatoid bodies. This (Shoji et al., 2005). When many constructs are to be examined, strongly suggests that the Tudor domain structure and its this approach can be an alternative to producing transgenic mice. interaction with dimethylated arginines of putative binding However, a disadvantage of this technique is its limited partners are essential for nuage localization or accumulation of transfection efficiencies. Thus, the method is more suitable for the TDRD proteins. analyzing genes that function cell-autonomously, such as those A possible function of Tudor domain repeats is to assemble encoding organelle components, rather than genes that function molecules that associate with each Tudor domain into via cell–cell interactions, like those encoding growth factors etc. macromolecular complexes. This notion agrees with the result In summary, this study showed that the Tudor-related proteins observed in spermatocytes that the localization of endogenous constitute a novel class of nuage components in the germ-line nuage components was disrupted following over-expression of and that the characteristic domain architecture is closely truncated TDRD proteins carrying one or two Tudor domains. associated with their intracellular localization and function. The scaffold function of Tudor domain repeats is also in The analogy between vasa-Tudor in Drosophila and Mvh-the agreement with the reduced number and size of polar granules TDRDs in mice suggests that the molecular pathway has been in hypomorphic and null mutants of Drosophila tudor (Boswell retained and plays an important role in germ-line cells. Nuage is and Mahowald, 1985; Thomson and Lasko, 2004). However, a site of assembly of these evolutionarily conserved molecules, over-expression of full-length TDRD1 and 6 did not trigger the and the Tudor-related proteins would serve as valuable probes to formation of nuage-like aggregates in spermatogonia. Nuage study this intriguing structure, whose molecular and develop- components that associate with these TDRD proteins may not mental characteristics remain largely unknown. be expressed or present in the cytoplasm of spermatogonia. For instance, Sm proteins of snRNPs are confined to the nucleus in Acknowledgments spermatogonia, and during meiosis of spermatocytes, these proteins are released into the cytoplasm and localize to nuage We thank Y. Nishimune and M. Nozaki for the mouse testis forming a complex with TDRD1 (Moussa et al., 1994; Chuma cDNA lambda library, T. Hirose and M. Matsuoka for anti- et al., 2003). human and mouse TDRD7/TRAP antibodies used for our Recently, RNF17, originally identified as a RING finger preliminary experiments, M. Fujioka for electron microscopy, protein, was shown to contain four Tudor domains. RNF17 is and M. Moribe and M. Tanaka for experimental assistance. This abundant in the testis, and a targeted disruption of Rnf17 results study was supported by Grants-in-Aid from the Ministry of in spermiogenic defect (Pan et al., 2005). Interestingly, RNF17 Education, Culture, Sports, Science, and Technology, Japan. M. is enriched in cytoplasmic granules similar to nuage, but the H. was supported by a Grant-in-Aid for the 21st Century COE RNF17-granules are distinct from intermitochondrial cement program, Japan. and chromatoid bodies where TDRD1, 6 and 7 accumulate. Provided that a Tudor domain has the activity to localize or References accumulate to nuage, a RING finger domain may bring about the different distribution of RNF17 from that of the TDRD Amikura, R., Hanyu, K., Kashikawa, M., Kobayashi, S., 2001. Tudor protein is proteins. essential for the localization of mitochondrial RNAs in polar granules of The characteristic co-localization of TDRD1, 6 and 7 was Drosophila embryos. Mech. Dev. 107, 97–104. abrogated in Mvh1098/1098 spermatocytes. Since Tudor localiza- Bardsley, A., McDonald, K., Boswell, R.E., 1993. Distribution of tudor protein tion is also regulated by vasa in Drosophila oocytes and in the Drosophila embryo suggests separation of functions based on site of localization. Development 119, 207–219. embryos, the molecular pathway, vasa/Mvh-Tudor/TDRD, is Biggiogera, M., Fakan, S., Leser, G., Martin, T.E., Gordon, J., 1990. likely evolutionarily conserved, suggesting its importance in the Immunoelectron microscopical visualization of ribonucleoproteins in the germ-line of diverse species and at different stages of chromatoid body of mouse spermatids. Mol. Reprod. Dev. 26, 150–158. development. The independent, separate distribution of each Boswell, R.E., Mahowald, A.P., 1985. Tudor, a gene required for assembly of – TDRD protein in Mvh1098/1098 spermatocytes indicates that the the germ plasm in Drosophila melanogaster. Cell 43, 97 104. Buhler, D., Raker, V., Luhrmann, R., Fischer, U., 1999. Essential role for the TDRD proteins possess distinct molecular properties that tudor domain of SMN in spliceosomal U snRNP assembly: implications for function at different intracellular loci before their trafficking spinal muscular atrophy. Hum. Mol. Genet. 8, 2351–2357. or accumulation to nuage. The sequential localization of the Charier, G., Couprie, J., Alpha-Bazin, B., Meyer, V., Quemeneur, E., Guerois, TDRD proteins to nuage, as shown in Fig. 4, may then bring R., Callebaut, I., Gilquin, B., Zinn-Justin, S., 2004. The Tudor tandem of about compositional changes of the structure during germ cell 53BP1: a new structural motif involved in DNA and RG-rich peptide binding. Structure (Cambridge) 12, 1551–1562. differentiation. Future investigation to identify molecules that Chuma, S., Nakatsuji, N., 2001. Autonomous transition into meiosis of mouse associate similarly and differently with each TDRD protein fetal germ cells in vitro and its inhibition by gp130-mediated signaling. Dev. would help study nuage and its assembly at the molecular level. Biol. 229, 468–479. 52 M. Hosokawa et al. / Developmental Biology 301 (2007) 38–52

Chuma, S., Hiyoshi, M., Yamamoto, A., Hosokawa, M., Takamune, K., Lehmann, R., Ephrussi, A., 1994. Germ plasm formation and germ cell Nakatsuji, N., 2003. Mouse Tudor Repeat-1 (MTR-1) is a novel component determination in Drosophila. Ciba Found. Symp. 182, 282–296 (discussion of chromatoid bodies/nuages in male germ cells and forms a complex with 296–300). snRNPs. Mech. Dev. 120, 979–990. Mahowald, A.P., 1962. Fine structure of pole cells and polar granules in Dro- Chuma, S., Hosokawa, M., Kitamura, K., Kasai, S., Fujioka, M., Hiyoshi, M., sophila melanogaster. J. Exp. Zool. 151, 201–216. Takamune, K., Noce, T., Nakatsuji, N., 2006. Tdrd1/Mtr-1, a tudor-related McLaren, A., 2003. Primordial germ cells in the mouse. Dev. Biol. 262, 1–15. gene, is essential for male germ-cell differentiation and nuage/germinal Moussa, F., Oko, R., Hermo, L., 1994. The immunolocalization of small nuclear granuleformationinmice.Proc.Natl.Acad.Sci.U.S.A.103, ribonucleoprotein particles in testicular cells during the cycle of the 15894–15899. seminiferous epithelium of the adult rat. Cell Tissue Res. 278, 363–378. Conti, E., Uy, M., Leighton, L., Blobel, G., Kuriyan, J., 1998. Crystallographic Niwa, H., Yamamura, K., Miyazaki, J., 1991. Efficient selection for high- analysis of the recognition of a nuclear localization signal by the nuclear expression transfectants with a novel eukaryotic vector. Gene 108, 193–199. import factor karyopherin alpha. Cell 94, 193–204. Oko, R., Korley, R., Murray, M.T., Hecht, N.B., Hermo, L., 1996. Germ cell- Cote, J., Richard, S., 2005. Tudor domains bind symmetrical dimethylated specific DNA and RNA binding proteins p48/52 are expressed at specific arginines. J. Biol. Chem. 280, 28476–28483. stages of male germ cell development and are present in the chromatoid Czolowska, R., 1969. Observations on the origin of the ‘germinal cytoplasm’ in body. Mol. Reprod. Dev. 44, 1–13. Xenopus laevis. J. Embryol. Exp. Morphol. 22, 229–251. Pan, J., Goodheart, M., Chuma, S., Nakatsuji, N., Page, D.C., Wang, P.J., 2005. Eddy, E.M., 1974. Fine structural observations on the form and distribution of RNF17, a component of the mammalian germ cell nuage, is essential for nuage in germ cells of the rat. Anat. Rec. 178, 731–757. spermiogenesis. Development 132, 4029–4039. Eddy, E.M., 1975. Germ plasm and the differentiation of the germ cell line. Int. Parvinen, M., 2005. The chromatoid body in spermatogenesis. Int. J. Androl. 28, Rev. Cytol. 43, 229–280. 189–201. Fawcett, D.W., Eddy, E.M., Phillips, D.M., 1970. Observations on the fine Ponting, C.P., 1997. Tudor domains in proteins that interact with RNA. Trends structure and relationships of the chromatoid body in mammalian Biochem. Sci. 22, 51–52. spermatogenesis. Biol. Reprod. 2, 129–153. Raz, E., 2000. The function and regulation of vasa-like genes in germ-cell Fujiwara, Y., Komiya, T., Kawabata, H., Sato, M., Fujimoto, H., Furusawa, M., development. Genome Biol. 1, reviews 1017.1–1017.6. Noce, T., 1994. Isolation of a DEAD-family protein gene that encodes a Russell, L., Frank, B., 1978. Ultrastructural characterization of nuage in murine homolog of Drosophila vasa and its specific expression in germ cell spermatocytes of the rat testis. Anat. Rec. 190, 79–97. lineage. Proc. Natl. Acad. Sci. U. S. A. 91, 12258–12262. Saffman, E.E., Lasko, P., 1999. Germline development in vertebrates and Golumbeski, G.S., Bardsley, A., Tax, F., Boswell, R.E., 1991. tudor, a posterior- invertebrates. Cell. Mol. Life Sci. 55, 1141–1163. group gene of Drosophila melanogaster, encodes a novel protein and an Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual. mRNA localized during mid-oogenesis. Genes Dev. 5, 2060–2070. Cold Spring Harbor Laboratory Press, New York. Hirose, T., Kawabuchi, M., Tamaru, T., Okumura, N., Nagai, K., Okada, M., Saunders, P.T., Millar, M.R., Maguire, S.M., Sharpe, R.M., 1992. Stage-specific 2000. Identification of tudor repeat associator with PCTAIRE 2 (Trap). A expression of rat transition protein 2 mRNA and possible localization to the novel protein that interacts with the N-terminal domain of PCTAIRE 2 in rat chromatoid body of step 7 spermatids by in situ hybridization using a brain. Eur. J. Biochem. 267, 2113–2121. nonradioactive riboprobe. Mol. Reprod. Dev. 33, 385–391. Huyen, Y., Zgheib, O., Ditullio, R.A., Gorgoulis, V.G., Zacharatos, P., Petty, Selenko, P., Sprangers, R., Stier, G., Buhler, D., Fischer, U., Sattler, M., 2001. T.J., Sheston, E.A., Mellert, H.S., Stavridi, E.S., Halazonetis, T.D., 2004. SMN tudor domain structure and its interaction with the Sm proteins. Nat. Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand Struct. Biol. 8, 27–31. breaks. Nature 432, 406–411. Shoji, M., Chuma, S., Yoshida, K., Morita, T., Nakatsuji, N., 2005. RNA Kleene, K.C., 2005. Sexual selection, genetic conflict, selfish genes, and the interference during spermatogenesis in mice. Dev. Biol. 282, 524–534. atypical patterns of in spermatogenic cells. Dev. Biol. 277, Strome, S., Wood, W.B., 1982. Immunofluorescence visualization of germ-line- 16–26. specific cytoplasmic granules in embryos, larvae, and adults of Caenor- Kotaja, N., Bhattacharyya, S.N., Jaskiewicz, L., Kimmins, S., Parvinen, M., habditis elegans. Proc. Natl. Acad. Sci. U. S. A. 79, 1558–1562. Filipowicz, W., Sassone-Corsi, P., 2006. The chromatoid body of male germ Tam, P.P., Zhou, S.X., 1996. The allocation of epiblast cells to ectodermal and cells: similarity with processing bodies and presence of Dicer and germ-line lineages is influenced by the position of the cells in the microRNA pathway components. Proc. Natl. Acad. Sci. U. S. A. 103, gastrulating mouse embryo. Dev. Biol. 178, 124–132. 2647–2652. Tanaka, S.S., Toyooka, Y., Akasu, R., Katoh-Fukui, Y., Nakahara, Y., Suzuki, Lammers, J.H., Offenberg, H.H., van Aalderen, M., Vink, A.C., Dietrich, A.J., R., Yokoyama, M., Noce, T., 2000. The mouse homolog of Drosophila Vasa Heyting, C., 1994. The gene encoding a major component of the lateral is required for the development of male germ cells. Genes Dev. 14, 841–853. elements of synaptonemal complexes of the rat is related to X-linked Thomson, T., Lasko, P., 2004. Drosophila tudor is essential for polar granule lymphocyte-regulated genes. Mol. Cell. Biol. 14, 1137–1146. assembly and pole cell specification, but not for posterior patterning. Lawson, K.A., Hage, W.J., 1994. Clonal analysis of the origin of primordial Genesis 40, 164–170. germ cells in the mouse. Ciba Found. Symp. 182, 68–84 (discussion 84–91). Toyooka, Y., Tsunekawa, N., Takahashi, Y., Matsui, Y., Satoh, M., Noce, T., Lawson, K.A., Dunn, N.R., Roelen, B.A., Zeinstra, L.M., Davis, A.M., 2000. Expression and intracellular localization of mouse Vasa-homologue Wright, C.V., Korving, J.P., Hogan, B.L., 1999. Bmp4 is required for the protein during germ cell development. Mech. Dev. 93, 139–149. generation of primordial germ cells in the mouse embryo. Genes Dev. 13, Wang, P.J., McCarrey, J.R., Yang, F., Page, D.C., 2001. An abundance of 424–436. X-linked genes expressed in spermatogonia. Nat. Genet. 27, 422–426.