Supporting Information

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Fig. 6. Impaired spermatogenesis in Tdrd1^tm1/tm1 mutants. (A) Wild-type (Left) and Tdrd1^tm1/tm1 (Right) testes at 6 months of age. (B) A H&E section of a wild-type testis. (Inset) Higher-magnification view. (C and D) H&E sections of a Tdrd1^tm1/tm1 testis. Some seminiferous tubules contain few germ cells (arrowheads), whereas in others, more differentiating germ cells are observed (arrows). (E and F) H&E sections of wild-type (E) and Tdrd1^tm1/tm1 (F) ovaries. [Scale bars = (A) 1 mm; (B and C) 200 mm; (D) 50 mm; (E and F) 100 mm.]

Fig. 7. Spermatogenic defects during prepubertal development of Tdrd1^tm1/tm1 testes. (A-C) H&E-stained sections of wild-type (Upper) and Tdrd1^tm1/tm1 (Lower) testes at 7 (A), 14 (B), and 20 (C) dpp. The lack of spermatogenic cells becomes evident in seminiferous tubules at 20 dpp (C, arrowheads). (D and E) TUNEL staining (brown) of sections of wild-type (Upper) and Tdrd1^tm1/tm1 (Lower) testes at 14 (D) and 20 (E) dpp. Apoptosis is increased among Tdrd1^tm1/tm1 spermatocytes at 14 dpp (D, arrowheads), and among both spermatocytes and round spermatids at 20 dpp (E, arrowheads). Nuclei were counterstained with hematoxylin (blue). (F) Sections of wild-type (Upper) and Tdrd1^tm1/tm1 (Lower) adult testes stained with anti-activated caspase-3 antibody (green) and Hoechst 33258 (blue). [Scale bars = (A-C) 50 mm; (D and E) 100 mm; (F) 20 mm.]

Fig. 8. Differential localization of TDRD1 and MVH. Sections of a wild-type testis were immunostained for TDRD1 and MVH. (A) Spermatogonia and leptotene-zygotene spermatocytes are indicated by arrowheads and arrows. The dotted line demarcates the seminiferous tubule. (B and C) Spermatocytes at mid- (B) and late- (C) pachytene stages. Granular accumulation of TDRD1 precedes that of MVH (from B to C, arrowheads). (D) In round spermatids, TDRD1 and MVH show precise colocalization that corresponds to chromatoid bodies (arrowheads). Nuclei were counterstained with Hoechst 33258 (blue). [Scale bars = 10 mm.]

Table 1. Intermitochondrial cement in pachytene spermatocytes

Data were collected from 11, 12, and 6 seminiferous tubules of two adult testes for each genotype.

*Mean values, with minimum and maximum in parentheses, are shown.

†Staining intensities of intermitochondrial cement were digitized from electron microscope images (8-bit gray scale of 0, black and 255, white). Mean values ± standard deviations are presented.

‡This cell was scored as containing intermitochondrial cement. The structure could instead be a staining artifact, other cellular components, or a chromatoid body adjoining mitochondria, which were sometimes difficult to distinguish from intermitochondrial cement. Six among 69 Tdrd1^tm1/tm1 spermatocytes contained chromatoid bodies.

NA, not applicable.

Table 2. Chromatoid bodies in round spermatids

Data were collected from five and eight seminiferous tubules of two adult testes for each genotype. Round spermatids were not observed in the sections of 14 seminiferous tubules of two Mvh^1098/1098 testes examined in this study.

*Mean values, with minimum and maximum in parentheses, are shown.

†Staining intensities of chromatoid bodies were digitized from electron microscope images (8-bit gray scale of 0, black and 255, white). Mean values ± standard deviations are presented.

Table 3. Intermitochondrial cement in fetal prospermatogonia

Data were collected from two fetal testes at 17.5 days postcoitum for each genotype.

*Mean values, with minimum and maximum in parentheses, are shown.

†Staining intensities of intermitochondrial cement were digitized from electron microscope images (8-bit gray scale of 0, black and 255, white). Mean values ± standard deviations are presented.

‡These cells were scored as containing intermitochondrial cement. The structure could instead be staining artifacts or other cellular components, such as ruptured mitochondria, which were occasionally difficult to distinguish from nuage material.

Table 4. Intermitochondrial cement in postnatal spermatogonia

Data were collected from 10, 7, and 9 seminiferous tubules of two adult testes for each genotype.

*Mean values, with minimum and maximum in parentheses, are shown.

†Staining intensities of intermitochondrial cement were digitized from electron microscope images (8-bit gray scale of 0, black and 255, white). Mean values ± standard deviations are presented.

NA, not applicable.

Table 5. Intermitochondrial cement in postnatal oocytes

Data were collected from primordial to preantral follicles of two adult ovaries for each genotype.

*Mean values, with minimum and maximum in parentheses, are shown.

†Staining intensities of intermitochondrial cement were digitized from electron microscope images (8-bit gray scale of 0, black and 255, white). Mean values ± standard deviations are presented.

NA, not applicable.

Supporting Text

The Tudor domain containing 1 (Tdrd1) cDNA that was previously reported was 4.9 kb and contained a full ORF with an in-frame stop codon in the 5'-UTR (1), but the 5' ends had not been determined. As a basis for designing a targeting vector, we carried out 5'-RACE on Tdrd1 mRNA and obtained four transcript variants. Each transcript variant had a different transcription initiation site (Fig. 2A). The promoter regions of these transcripts lacked TATA-box sequences like several other genes expressed in spermatogenic cells (2). The Tdrd1 gene consisted of 27 exons that spanned »50 kb on chromosome 19. Transcript variants 1-3 encoded the common ORF of 1,172 aa with the first coding ATG in exon 3. Transcript variant 4, which was less abundant in the 5'-RACE analysis, contained a potential ORF of 928 aa, with the first ATG in exon 8. However, the translation efficiency of transcript variant 4 is probably very low, because (i) three small ORFs precede the ATG in exon 8, and (ii) Western blotting of adult testes with anti-TDRD1 C-terminal antibody showed a single signal at »120 kDa, which corresponded to the putative molecular mass of the 1,172 aa from transcript variants 1-3 (Fig. 2D).

We examined the first occurrence of spermatogenic defects during prepubertal development of Tdrd1^tm1/tm1 testes. At 7 days postpartum (dpp), when spermatogonia are proliferating but before the first appearance of spermatocytes (3), no histological differences were discernible between wild-type and Tdrd1^tm1/tm1 testes (Fig. 7A). At 14 dpp, when spermatocytes derived from the first wave of spermatogenesis reach the pachytene-diplotene stages, some Tdrd1^tm1/tm1 seminiferous tubules appeared to be less full compared to wild type, but the difference was still not clear (Fig. 7B). Then, the defects among spermatocytes and spermatids became evident at 20 dpp, when haploid round spermatids first appear in a subset of seminiferous tubules (Fig. 7C).

Mouse vasa homologue/DEAD box polypeptide 4 (MVH/DDX4) is another component of mouse nuage that functions in postnatal spermatogenesis (5, 6). To reveal a possible relationship between Tdrd1 and Mvh, we compared the localization pattern of these two proteins. In spermatogonia, TDRD1 showed a fine granular appearance that corresponds to intermitochondrial cement, whereas MVH was diffusely observed in the cytoplasm (Fig. 8A, arrowhead). In spermatocytes at the leptotene-zygotene to early pachytene stages (Fig. 8A, arrow), TDRD1 became undetectable, whereas MVH was continually present in the cytoplasm. In spermatocytes later than the mid-late pachytene stages, TDRD1 was again detected and formed distinct granules: at these stages, MVH also showed granular distribution that merged with TDRD1, but such localization of MVH was more gradual and occurred later than TDRD1 (from Fig. 8 B to C). Then in round spermatids, TDRD1 and MVH precisely colocalized to larger discrete aggregates (Fig. 8D) that correspond to chromatoid bodies (1, 6). In oocytes, TDRD1 clearly exhibited a fine granular appearance, whereas MVH was diffuse in the cytoplasm (see Fig. 4 E and G). No specific colocalization of these two proteins was observed during oocyte development. In all, TDRD1 more consistently localized to nuage than MVH during germ-cell differentiation, whereas MVH translocated from the cytoplasm to nuage in spermatocytes, suggesting that nuage changes its molecular properties during spermatogenesis in mice.

The cDNA sequence of Tdrd1, which contained an ORF of 1,172 aa with an in-frame stop codon in the 5'-UTR, was previously reported (GenBank/EMBL/DDBJ accession no. AB067571) (1). Poly(A)⁺ mRNAs isolated from the adult testes of Jcl:ICR mice (Clea, Tokyo, Japan) using oligo dT-bound magnetic beads (Dynabeads mRNA direct kit, DYNAL, Norway) were ligated to an RNA oligomer in a 5'-cap structure-dependent manner (Gene Racer, Invitrogen, Carlsbad, CA) and reverse-transcribed by using Tdrd1 specific reverse transcription primers in exons 8 and 9 (5'-CACCCGCTGT-3' and 5'-GGGTGCTTGA-3'). The resulting Tdrd1 cDNA pool was used as a template for nested PCR using the 5' oligonucleotide forward primer and Tdrd1 specific reverse primers in exons 5 and 6 (5'-GCGGTCCTAACGAGTTGAAGAGTGCT-3' and 5'-TGGCAGGCCGTGGAGCAGTAGTA-3') using LA taq (TakaraBio, Tokyo, Japan) and PfuTurbo (Stratagene, La Jolla, CA). Amplified products of three different lengths were cloned into pBlueScript SK (Stratagene), and sequencing of each product revealed four transcript variants of Tdrd1, where two were almost identical in length. Consensus sequences from at least 12 clones for each transcript variant were submitted to GenBank/EMBL/DDBJ under accession no. AB183526-183529.

A Tdrd1 cDNA fragment encoding the C-terminal region of amino acids 1040-1172 was appended with BamHI and SalI sites and cloned into pQE-30 (Qiagen, Cologne, Germany). The 6´ histidine-tagged TDRD1 fragment produced in Escherichia coli M15[pREP4] was purified by using Ni-NTA agarose beads under denaturing conditions. Rabbits were immunized with the denatured protein, and polyclonal antibodies were affinity-purified from the antisera by using the same antigen coupled to cellufine beads (Chisso, Japan). The differences between the previously reported anti-TDRD1 antibody (1) and the present one were the form of the antigen protein and the immunized species; rats were immunized with native-form antigen to generate the previous antibody, whereas in this study, rabbits were immunized with the denatured form of the same region of TDRD1.

A 6-kb PstI-HindIII fragment downstream of Tdrd1 exon 3 was obtained from a Lambda FIX II library of 129/SvJ mouse genomic DNA (Stratagene). A 1.5-kb fragment upstream of the first coding ATG was PCR-amplified by using the primers 5'-CCATCGATATTAGGTGTAATTTAACATTCA-3' and 5'-GGGTCGACGCATATTTAGAACCATTTCAG-3'. These two genomic fragments were ligated to an AscI fragment of pKO SelectNeo (Lexicon Genetics, The Woodlands, TX) that contains the neomycin resistance gene driven by the Pgk promoter with a downstream poly(A) signal. The diphtheria toxin A fragment gene driven by the MC1 promoter from pND3 was appended to the 3' end of the 6-kb genomic fragment. The Tdrd1 targeting vector was electroporated to 5 ´ 10⁶ R1 ES cells (7). After drug selection with G418 (Sigma-Aldrich, St. Louis, MO), 360 colonies were screened for homologous recombination by PCR using the primers 5'-TGTGGTTGGTACTGTGGCTTTACG-3' and 5'-CTACCGGTGGATGTGGAATGTGTG-3', and 11 positive clones were obtained. Chimeric mice were produced from two recombinant ES clones by injection into C57BL/6 blastocysts, and male chimeras of one line were mated with C57BL/6 females to obtain heterozygous mice. The genotyping of Mvh gene-targeted mice was carried out as described (5). All experiments on the mice were performed according to institutional guidelines.

Total RNAs from adult testes were isolated by a modified AGPC method (TRIzol, Invitrogen, Carlsbad, CA), treated with DNase I (Promega, Madison, WI), and reverse-transcribed by using SuperScript III (Invitrogen) with random hexamers. For the detection of truncated mRNAs from the Tdrd1^tm1 allele, RT-PCR was carried out by using the testis cDNA and forward primers in exon 1 (GenBank accession no. AB183526, 36-55 bp), exon 2 (AB183527, 5-24 bp), and exon 3 (AB183528, 11-32 bp), and reverse primers in exon 5 (AB067571, 485-464 bp) and exon 26 (AB067571, 3496-3475 bp). Amplified products were gel-extracted after electrophoresis and directly sequenced. Other PCR primers used in this study were as follows: glyceraldehyde-3-phosphate dehydrogenase, Gapdh (M32599, 983-1006 and 1166-1143 bp) (8); synaptonemal complex protein 3, Sycp3 (Y08485, 788-812 and 1112-1088 bp) (9); synaptonemal complex protein 1, Sycp1 (Z38118, 130-154 and 382-358 bp) (10); preproacrosin, Acr (NM_013455, 75-98 and 282-259 bp) (11, 12); outer dense fiber of sperm tails 1, Odf1 (NM_008757, 176-199 and 410-387 bp) (13); transition protein 2, Tnp2 (NM_013694, 116-139 and 357-334 bp) (14); and protamine 1, Prm1 (K02926, 135-158 and 330-307 bp) (15). Amplified products were gel-electrophoresed and detected with ethidium bromide.

Fifteen micrograms of tail genomic DNAs were digested with SpeI, electrophoresed in 0.9% agarose gel, and transferred to a nylon membrane (Hybond-N+, Amersham Pharmacia, Uppsala, Sweden). The blot was hybridized with a [³²P]dCTP-labeled genomic fragment of Tdrd1, which was PCR-amplified with the primers 5'-GTGGTCGACATGAGACAGATCTAA-3' and 5'-CCGTCTTCTTTGCAACTCTCTTAC-3'. Signals were detected with x-ray film (Kodak, Rochester, NY). For Western blotting, adult testis lysates containing 50 mg of total protein were subjected to 5-20% gradient SDS/PAGE and transferred to nitrocellulose membranes [Protran BA, Schleicher & Schuell (Whatman), Germany]. The blots were probed with anti-TDRD1 and anti-b-actin (AC-15, Sigma) antibodies followed by alkaline phosphatase-conjugated secondary antibodies, and signals were detected with CDP-Star with NitroBlock II (Perkin-Elmer, Wellesley, MA) and x-ray film.

For histology, 7-mm paraffin sections of tissues fixed in Bouin's solution were stained with H&E. For immunostaining, 10-mm cryosections of testes and ovaries fixed in 2% paraformaldehyde (PFA) in PBS were immunostained with anti-TDRD1, MVH (6), GENA (16), SYCP3/SCP3 (17), and Golgi-58K (58K-9, Sigma) antibodies. The secondary antibodies used were Alexa 488-conjugated anti-rat, -rabbit, and -mouse IgG (Invitrogen) and rhodamine-conjugated anti-rabbit immunoglobulins (BioSource, Carlsbad, CA) antibodies. Acrosomes were stained with rhodamine-conjugated peanut agglutinin (PNA) (Vector Laboratories, Burlingame, CA). Nuclei were counterstained with 1 mg/ml Hoechst 33258 dye (Sigma).

The terminal deoxynucleotidyl TUNEL method was carried out as described (18). Briefly, cryosections of testes fixed in 2% PFA in PBS were treated with proteinase K, and the 3' ends of the genomic DNA were labeled with DIG-dUTP by TdT (Roche, Basel, Switzerland). DIG was detected with horseradish peroxidase-conjugated anti-DIG antibody and diaminobenzidine (Roche). Nuclei were counterstained with hematoxylin. The activation of caspase-3 was detected by immunostaining cryosections with anti-active caspase-3 (C92-605, BD Biosciences, San Jose, CA) and Alexa 488-conjugated anti-rabbit IgG (Invitrogen) antibodies.

Testes and ovaries were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), postfixed with 1% OsO₄ and 0.1 M sucrose in 0.1 M phosphate buffer, and then dehydrated with graded concentrations of ethanol and embedded in epoxy resin (19). Semithin 1-mm sections were stained with 0.1% toluidine blue for light microscopy. Seventy- to 90-nm sections were placed on 150-mesh copper grids, stained with uranyl acetate followed by lead citrate, and examined by using an electron microscope (Hitachi H7000, Tokyo, Japan). For immunoelectron microscopy, testes and ovaries were fixed in 2% PFA/0.02% glutaraldehyde in 0.1 M phosphate buffer, and embedded in epoxy resin. Seventy- to 90-nm sections were incubated with anti-TDRD1 antibodies, followed by 15-nm gold-labeled secondary antibodies. After postfixation with 2% glutaraldehyde in PBS, sections were stained with uranyl acetate and lead citrate. The quantification of nuage structures was carried out by digitizing the areas and staining intensities of intermitochondrial cement and chromatoid bodies in electron microscope images using ImageJ software (by W. S. Rasband, National Institutes of Health, Bethesda, MD).

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