Zebrafish smc1b is conserved and expressed in germ cells. (A) Cartoon representing the meiotic cohesin complex. (B) Phylogenetic tree of zebrafish (z), mouse (m), and human (h) Smc protein family. Numbers to the right of protein names represent bootstrap confidence values. (C) RT-PCR of smc1b and control gene rpl13a using cDNA generated from brains, eyes, gills, hearts, kidneys, livers, ovaries, skin, testes, and viscera of wild-type adult zebrafish. (D,E)In situ hybridization (ISH) on testis (D) and ovary (E) sections of wild-type zebrafish. (D’,E’) ISH using sense probes were run as negative controls. Scale bars are 20 μm for testes and 100 μm for ovaries.

Smc1b mutant zebrafish display defects in spermatogenesis. (A)Smc1b mutations used in this study: smc1b^Q198X located in exon 4 and smc1b^Q261X in exon 5. Both mutations cause premature termination codons. (B–E) Hematoxylin-eosin (H&E) staining on adult testes of wild type (B), smc1b^{Q261X(–/–)}(C), smc1b^{Q198X(–/–)}(D), and smc1b^Q198X/Q261X(E). Dashed lines denote examples of: spermatogonia (yellow), spermatocytes (red), and spermatozoa (orange). (F,G) Caspase-3 (green) and DAPI (blue) staining on adult testes from wild type (F) and smc1b mutant (G). Caspase-3 positive cells are indicated by arrows. (H) Quantification of caspase-3 positive cells per testis area in the mutant (N = 3 testes) and wild type (N = 3 testes). Student’s t-test shows no significant (p = 0.3524) difference between wild-type and mutant caspase-3 positive cells numbers. Scale bars are 50 μm.

Absence of oogenesis in smc1b^Q198X mutants. Hematoxylin-eosin stain of wild-type and smc1b mutant gonads during sex-differentiation. (A,B) Undifferentiated gonads in wild-type (A) and mutant (B) fish at 4 weeks old. (C–H) Although oogenesis (C,F) and spermatogenesis (D,G) took place in wild-type gonads at 5- and 6-weeks old, only spermatogenesis progressed in the mutants (E,H). Gonads are outlined by red dashed line, scale is 50 μm. N = total number of individuals with the gonad histology pictured per total number of fish analyzed for each age group.

Spermatocytes in smc1b^Q198X mutants failed to progress beyond the leptotene stage. Labeling of Sycp3 and telomeres in adult wild-type and mutant testes sections: Sycp3 (green), telomere in situ hybridization (orange), DAPI nuclear stain (blue). (A–H) Leptotene stage spermatocytes are present in both wild-type and mutant spermatocytes, examples circled in white. Bouquet stage cells (circled red) are present in wild type but not in the mutant, however, partial telomere clustering is apparent in some mutant cells [e.g., panel (F), inset]. Insets are zoomed images of the areas boxed in purple. Scale bar is 20 μm.

Failed synapsis and anomalous pairing in smc1b^Q198X mutant spermatocytes. (A–F) Labeling of Sycp3 and Sycp1 proteins and telomeres (telo) on meiotic chromosome spreads from adult testes. (A–D) In wild-type spermatocytes, leptotene stage cells had Sycp3 protein localized near chromosome ends and exhibited either no Sycp1 signal or Sycp1 puncta. The synaptonemal complex proteins continued to assemble between homologous pairs through pachytene stages as homolog pairing and synapsis occurred. (D′) Sycp3 exhibited enrichment at or near telomeres. Images show enlarged views of the red boxed areas in D. (E,F)Smc1b mutant spermatocytes leptotene stage cells expressed Sycp3 in with a pattern similar to that of wild-type leptotene stage cells and Sycp1 was either absent or present in puncta. However, Sycp3 only labeled chromosomes near the ends and Sycp1 did not extend along chromosomes. A small percentage of mutant cells exhibited some telomere clustering one side of the nucleus (F), which we called bouquet-like and classified as leptotene stage as there was either no Sycp1 or only Sycp1 puncta visible. (G) Quantification of spermatocytes showed that all mutant cells are in leptotene stage compared to about 40% in wild type. Later stages were not observed in the mutants (N = 52 wild-type and 130 mutant cells). (H) Quantification of Sycp3 foci in wild-type bouquet (N = 13) and mutant “bouquet-like” (N = 23) nuclei shows abnormal pairing in smc1b mutant spermatocytes. Mutants have significantly fewer Sycp3 puncta than wild types (T-test, p = 0.000007). Horizontal bars in (H) show the averages. Scale bar is 10 μm.

The cohesin complex does not associate with meiotic chromosomes in zebrafish spermatocytes. (A–E) Immunolabeling of Smc3, and Sycp1 in wild-type (A–D) and smc1b mutant (E,F) chromosome spreads. (A–D) In wild-type cells, Smc3 showed a localization pattern similar to Sycp1. (D′) Smc3 was not enriched at telomeres. Images show enlarged views of the red-boxed areas in panel (D). (E,F) In mutant cells, Smc3 was expressed in leptotene stage but failed to load onto chromosomes. Scale is 10 μm.

DNA double strand break marker γ–H2Ax was expressed in smc1b^Q198X mutant testes. Immunolabeling of γ-H2Ax (green) and Telo-FISH (orange) on wild-type and mutant testis sections. (A–D) In wild-type early bouquet stage spermatocytes, γ-H2Ax appeared to localize more broadly in nuclei on testis sections (red dashed lines). Bouquet stage spermatocytes with tightly clustered telomeres (white dashed lines) exhibited γ-H2Ax localization near telomeres. (E–H) Early bouquet-stage spermatocytes in smc1b mutants display broad γ-H2Ax expression similar to wild type, however, tight clustering of telomeres and γ-H2Ax to one side of the nucleus was not observed. Scale bar is 20 μm.

DNA double strand break marker γ-H2Ax was expressed in smc1b^Q198X mutant testes. (A–F) Labeling of γ-H2Ax (green), Sycp1 (magenta), and Telo-FISH (orange) on wild-type (A–D) and mutant (E,F) chromosome spreads from testes. γ-H2Ax protein is expressed in both wild-type and mutant nuclei. (G) Quantification of γ-H2Ax fluorescence intensity in wild-type (N = 10) and mutant (N = 18) leptotene stage cells shows no statistically significant difference (P = 0.0945). The horizontal bars show the medians. Scale bar is 10 μm.

DNA double strand break and recombination marker Rad51 was expressed in smc1b^Q198X mutant spermatocytes. Immunolabeling of Rad51 (green) and Sycp1 (magenta) in wild-type and mutant spreads, combined with telomere (telo) in situ hybridization (orange). (A,B) Rad51 protein was detected in wild-type beginning in leptotene stage and was initially located near the chromosome ends in leptotene and bouquet stages. (C,D) In more advanced wild-type spermatocytes, Rad51 localization become more distinct and resolved to one or two spots per chromosome. (E,F) Mutant spermatocytes had detectable levels of Rad51. (G) Quantification of Rad51 signal in wild-type (N = 10) and mutant (N = 15) leptotene stage cells demonstrates there is no significant difference (P = 0.0614). The horizontal bars show the medians. Scale bar is 10 μm.

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