Low translational activity of meioc mutant spermatogonia

(A) Schema of the development of spermatogonial cysts surrounded by Sertoli cells and progression of spermatogenesis in zebrafish. (B) Histology (HE) and immunostaining against Plzf and spermatocyte markers (Sycp1, 3) in the wild-type and the moto-/- testes. Arrowheads: sperm. SC: spermatocytes. Scale bar: 10 µm. (C, D) OP-Puro fluorescence analysis (C) and quantification of the signal intensities (D) in wild-type and meiocmo/mospermatogenic cells. Dotted lines; 1-2 cell spermatogonia. Scale bars: 10 µm. (E-F) Effect of cycloheximide (CHX, 0.2 µM) on differentiation of SSCs in culture. Dotted lines; germ cell clumps. Sycp3; Immunostaining of Sycp3. Arrowheads; examples of a cell with a large nucleolus. The graph (F) presents the percentage of clumps of SSCs and differentiated cells (Differ) shown in Panel E. Scale bar; 50 mm. Data are represented as mean ± SD.

Defect on upregulation of rRNA transcription in meiocmo/mo spermatogonia

(A) In situ hybridization of 5S, 5.8S, 18S, and 28S rRNA and immunohistochemistry with anti-Rpl15 antibody in spermatogonia (gonia) and spermatocytes (cyte) in wild-type and meiocmo/mo. Yellow dotted lines indicate 1-2-cell spermatogonia. Percentages represents the frequency of low and high 1-2-cell spermatogonia. (B-D) Fluorescent in situ hybridization of 28S rRNA in wild-type. The graphs present quantification of signal intensities of 28S rRNA in nucleoli (C) and in cytoplasm (D) in spermatogenic cells. Arrowheads; nucleoli. cytes; spermatocytes. (E) qRT-PCR analysis of rRNAs and R2 between wild-type and meiocmo/mopurified sox17::egfp positive spermatogonia. Two-way arrows in the schema indicates position of primers on the rRNA and R2 element. (F) Northern blot analysis of pre-rRNA processing in wild-type and meiocmo/mo testes using probes for 5’ external transcribed spacer (ETS) and internal transcribed spacer 1 (ITS1). Right panel: schema of 45S pre-rRNA and pre-rRNA processing intermediates in zebrafish (Tao et al. 2017). Left panel: Northern blot analysis of pre-rRNA processing in wild-type and meiocmo/mo testes using 5’ETS and ITS1 probes. A probe for the 7SL RNA was used as a loading control. Graphs summarize relative signal intensity of 45S pre-rRNA and intermediates normalized to 7SL in three wild-type and meiocmo/mo testes. (G) Bisulfite-sequencing analysis of the tandem repeat region in the IGS region of the 45S-S rDNA locus in purified undifferentiated spermatogonia of wild-type and meiocmo/mo. Arrows; position of bisulfite primers in the tandem repeat elements (blue, magenta and white boxes), black dots; methylated CpG sites, white dots; unmethylated sites. *p < 0.05, **p < 0.01. Scale bars: 10 µm.

ythdc2 mutant spermatogonia have different defects from meiocmo/mo.

(A) Histology (HE) and immunostaining against Plzf and spermatocyte markers (Sycp1, 3) in the ythdc2-/-testes. (B) Representative image of meiocmo/moand ythdc2-/- testes sections stained with PAS (periodic acid Schiff) and hematoxylin. Dotted lines; 1-2-cell cyst spermatogonia (black) and 4≤-cell cysts (yellow). (C) The number of 1-2-, 4-, and 8-cell cyst spermatogonia per mm2 of sections in wild-type, meiocmo/mo and ythdc2-/- testes. (D) Ratio of the number of 4-8-cell cyst spermatogonia to 1-2-cell cysts in wild-type, meiocmo/mo and ythdc2-/- testes. (E) In situ hybridization of 5S, 5.8S, 18S, and 28S rRNA and immunohistochemistry with anti-Rpl15 antibody in the ythdc2-/- testes. Yellow dotted lines; 1-2-cell spermatogonia. *p < 0.05, **p < 0.01. Scale bars: 10 µm.

Meioc directly binds with Piwil1 and affects the localization of Piwil1.

(A) Immunostaining of Ddx4 and Piwil1, Piwil2, Tdrd1 and Tdrd6 in wild-type and meiocmo/mospermatogonia. The arrowhead; the Piwil1 signal in the nucleolus. (B) Immunostaining against Piwil1 and fibrillarin (left panels) and quantification of nucleolar Piwil1 (right panel) in wild-type and meiocmo/mo spermatogonia. Arrowheads; Fibrillarin positive nucleolus. (C) Immunostaining of Piwil1 and Ddx4 (left panels) and quantification of Piwil1 in germ granules (right panel) in wild-type and meiocmo/mospermatogonia. Arrowheads; Ddx4 positive germ granules. (D) Coimmunoprecipitation of Meioc and Piwil1 using testis lysate. Meioc signals were detected in Piwil1 immunoprecipitate and vice versa. Benzonase: addition of benzonase nuclease. **p < 0.01. Scale bars: 10 µm.

Reduction of Piwil1 compensated phenotypes of meiocmo/mo.

(A, B) In situ hybridization of 28S rRNA in wild-type and meiocmo/mo;piwil1+/- spermatogonia (1-2-cell cysts). Graphs (B) show the relative signal intensity in the cytoplasm normalized to the intensity of lobule myoid cells (left) and nucleoli normalized to the intensity of the nucleoplasm (right). (C, D) Differentiated spermatogonia in meiocmo/mo and meiocmo/mo;piwil1+/-testes. Yellow dotted lines; differentiated spermatogonia. Graphs (D) show the number of 16-cell and 32-cell cyst spermatogonia per mm2 of sections. ND: not detected. (E-G) meiocmo/mo and meiocmo/mo piwil1+/-testis sections stained with PAS and hematoxylin. Cysts of 1-2-cell spermatogonia (black) and 4≤-cell cysts (yellow) are indicated by dotted lines. Graphs show numbers of 1-, 2-, 4-, and 8-cell cysts per mm2 in sections of meiocmo/mo and meiocmo/mo;piwil1+/- testes (F), and ratio of the number of 4-8-cell cysts to 1-2-cell cysts in wild-type, meiocmo/moand meiocmo/mo;piwil1+/- (G). *p < 0.05, **p < 0.01. Scale bars: 10 µm.

Nucleolar Piwil1 interacted with Setdb1 and causes silenced epigenetic state of rDNA loci.

(A) Fold enrichment of pre-rRNA (5’ETS-18S rRNA) in Piwil1 immunoprecipitated RNA relative to the control IgG in wild-type and meiocmo/mo testes. (B) Immunostaining of Piwil1 (left panels) and the percentage of spermatogonia with detectable nucleolar Piwil1 (right panel) in the meiocmo/mo testes treated with a-amanitin (Am), actinomycin D (Ac) and BMH-21 (B). Arrows; Piwil1 detectable nucleoli, arrowheads; Piwil1 undetectable nucleoli, C; control without inhibitors, IC; initial control. (C) Immunostaining of Setdb1 and fibrillarin in wild-type and meiocmo/mospermatogonia. Arrowheads; nucleoli. (D) Co-IP of Piwil1 and Setdb1 using meiocmo/motestes lysate. Piwil1 was detected in Setdb1 IP. (E) Intensities of Setdb1 in nucleoli in wild-type and meiocmo/mo spermatogonia. (F, G) ChIP-qPCR analysis of H3K9me3 (F) and Piwil1 (G) levels in 45S-rDNA region in wild-type, piwil1+/-, and meiocmo/mo testes. Position of primers were indicated in Supplemental Figure S4. Mean ± s.d. are indicated. (H) Immunostaining of HP1α and Piwil1 in wild-type and meiocmo/mo spermatogonia. Arrowheads; nucleolus. (I) Co-IP of Piwil1 and HP1α using meiocmo/mo testis lysate. HP1α was detected in Piwil1 IP. (J) Intensities of HP1α in nucleoli in wild-type and meiocmo/mo spermatogonia. *p < 0.05, **p < 0.01. Scale bars: 10 µm.

Meioc was required for upregulation of 28S rRNA.

(A) Expression pattern of 28 rRNA in isolated sox17::egfp spermatogonia, based on the amount of Meioc granules and the localization. Right panels are intensities of 28 rRNA for each class of the purified sox17::egfp spermatogonia. 51≤ C and N; 51≤ cytoplasmic and nuclear Meioc granules, respectively. *p < 0.05, **p < 0.01, n: number of analyzed spermatogonia. Scale bars: 10 µm. (B) Expression patterns of Piwil1 in 5 classes of Meioc expressing spermatogonia in testis sections. The graph shows intensities Piwil1 in wild-type testes sections for each class of the spermatogonia. Arrows; Piwil1 detectable nucleoli, arrowheads; Piwil1 undetectable nucleoli. 51≤ C and N; 51≤ cytoplasmic and nuclear Meioc granules, respectively. **p < 0.01. Scale bars: 10 µm. (C) Graphical abstract. Meioc prevents the nucleolar localization of Piwil1 and its associated Setdb1 and HP1α to upregulate rRNA transcripts that are required for zebrafish SSCs to differentiate.