A premature stop mutation of the sycp1 gene in the isa mutant line. (A) Genomic sequences of a part of exon 15 in the sycp1 gene. DNA sequences and peaks obtained from a wild-type and an isa mutant fish are shown with corresponding amino acids (above in 1-letter abbreviations). Positions of the isa mutation (base 1225 of the sycp1 coding sequence) are shown in red letters. (B) A schematic presentation of the Sycp1 protein sequence. The isa mutation site is indicated with an arrow. The antigen region (amino acids 1 to 408) of the Sycp1 antibody used in this study is shown below as a black bar. N: N-terminus, C: C-terminus. Numbers indicate corresponding amino acid positions. (C) Western blotting of the Sycp1 protein. Testis protein extracts of wild-type (sycp1^+/+) and isa homozygote mutant (sycp1^isa/isa) adult fish were blotted with anti-Sycp1 antibody and anti-Gapdh antibody as an internal control. The left side of the broken line is a part labeled with a molecular ladder on the same membrane. The predicted molecular sizes of full-length Sycp1 and Gapdh proteins are 116 kDa and 35.8 kDa, respectively. Non-specific bands were marked with asterisks (*). (D) HE-stained sections of sycp1^+/+, sycp1^isa/del5, sycp1^isa/+ and sycp1^del5/+ testes. All samples were prepared from siblings at 4 mpf (months post fertilization). Representative results of 3 individual fish are shown for each genotype. Magnified images are shown for sycp1^+/+ and sycp1^isa/del5 sections. Sc: spermatocytes, St: spermatids, Sz: spermatozoa. In the sycp1^isa/del5 section, lumens with no spermatozoa (asterisks) and spermatocytes with irregular nuclei (inside broken lines) were observed.

Sexual phenotypes of sycp1 mutant zebrafish. (A) Sexual phenotypes of offspring from sycp1 ^+/isa incrosses. Proportions of offspring with testes (males) and ovaries (females) are shown for each genotype. The results of two incross matings were pooled and shown. (B) HE-stained sections of juvenile sycp1^+/+ and sycp1^isa/isa gonads. Representative images of sycp1^+/+ (i and ii) and sycp1^isa/isa (iii and iv) gonads with primary oocytes (i to iii) or spermatocysts (iv) are shown. Their body size (mm) and age (dpf: days post fertilization) are indicated below. Scale bars: 20 μm. (C) Phenotypes of juvenile sycp1^+/+ and sycp1^isa/isa gonads. Phenotypes of gonads from 24 to 45 dpf of sycp1^+/+ and sycp1^isa/isa fish were examined by HE staining and categorized into gonads with oocytes (red dots), gonads with spermatocysts (blue dots) and N.D. (not determined: open circle) and were plotted by their body size. Pooled results of siblings from two crosses are shown. Representative images are shown in Figure 2B. Histological images of all analyzed samples are shown in Supplementary Figure 3.

Formation of chromosomal axes in sycp1 mutant spermatocytes. Immunostaining of Sycp3, Sycp2 and Sycp1 components in wild-type (sycp1^+/+, i to iii) and sycp1^isa/isa (iv to vii) spermatocyte chromosome spreads. Wild-type nuclei at leptotene (L, i), mid-zygotene (MZ, ii) and pachytene (P, iii) were staged according to the staining patterns of Sycp3, Sycp2 and Sycp1 (Blokhina et al., 2019). sycp1^isa/isa nuclei at leptotene (L, iv), mid-zygotene-like (MZ-like, v) and late zygotene/pachytene-like (LZ/P-like, vi) stages were staged by axis staining patterns. Regions indicated in white rectangles are shown at the bottom at a higher magnification.

Transient pairing of homologs at chromosome ends in sycp1 mutant spermatocytes. (A) costaining of telomeres (TPA) and Sycp3 in sycp1^+/+ (i and ii) and sycp1^isa/isa (iii and iv) spermatocytes. The arrowhead indicates complete axis formation between two telomeres, and the arrow indicates paired telomere foci in LZ/P-like sycp1^isa/isa spermatocytes. Numbers in the top panels indicate counts of TPA foci. Regions indicated in white rectangles are shown at a higher magnification at the bottom. (B) quantification of telomere polyamide (TPA) foci in sycp1^+/+ and sycp1^isa/isa spermatocytes. Black bars indicate means. Statistical significance was examined by a two-tailed Mann-Whitney test (****P < 0.0001; exact P value). (C) fish staining of a peritelomeric locus on chromosome 3 in sycp1^+/+ (i to iii) and sycp1^isa/isa (iv to vi) spermatocytes. (D) Proportion of nuclei with one or two FISH foci in sycp1^+/+ and sycp1^isa/isa spermatocytes. (E) quantification of distances between FISH foci of a peritelomeric locus on chromosome 3 in sycp1^+/+ and sycp1^isa/isa spermatocytes. Distances between FISH foci observed in Figure 4D. For nuclei with only one FISH focus, the radius of the FISH-stained area was considered the distance between two FISH foci. Black bars indicate means. Statistical significance was examined by a two-tailed Mann-Whitney test (****P < 0.0001, ns, not significant; exact P value).

Meiotic DSB formation in sycp1 mutant spermatocytes. (A) costaining of γH2AX and Sycp2 in sycp1^+/+ (i to iii) and sycp1^isa/isa (iv to vi) spermatocytes. (B) quantification of γH2AX signal intensity in sycp1^+/+ and sycp1^isa/isa spermatocytes. Black bars indicate means. Statistical significance was examined by a two-tailed Mann-Whitney test (****P < 0.0001, ns, not significant; exact P value).

DSB localization in sycp1 mutant spermatocytes. (A) costaining of Dmc1/Rad51, telomeres and Sycp3 in sycp1^+/+ (i to iii) and sycp1^isa/isa (iv to vi) spermatocytes. (B) quantification of Dmc1/Rad51 focus numbers in sycp1^+/+ and sycp1^isa/isa spermatocytes. Black bars indicate means. Statistical significance was examined by a two-tailed Mann-Whitney test (**P < 0.01, ***P < 0.001, ****P < 0.0001; exact P value). (C) costaining of RPA, Sycp1 and Sycp3 in sycp1^+/+ (i and ii) and sycp1^isa/isa (iii and iv) spermatocytes. Regions marked as a white rectangle in the middle panels are shown at a higher magnification at the bottom. The white line on (iii) indicates a border with another nucleus on the top right. (D) quantification of RPA signal intensity in sycp1^+/+ and sycp1^isa/isa spermatocytes. Black bars indicate means. Statistical significance was examined by two-tailed Mann-Whitney test (ns, not significant; exact P value). Since RPA is observed as short stretches rather than discrete foci, signal intensity rather than the number of foci in a nucleus was quantified here. Quantification of RPA focus numbers is shown in Supplementary Figure 5.

Hormad1 localization in sycp1 mutant spermatocytes. Costaining of Hormad1 and Sycp2 in sycp1^+/+(i to iv) and sycp1^isa/isa(v and vi) spermatocytes. Regions marked as a white rectangle are shown at a higher magnification at the bottom. Arrows indicate synapsed (sycp1^+/+) and paired (sycp1^isa/isa) regions of axes.

Iho1 localization in sycp1 and spo11 mutant spermatocytes. (A) costaining of Iho1 and Sycp2 in sycp1^+/+ (i to iv) and sycp1^isa/isa (v and vi) spermatocytes. Regions marked in white rectangles are shown at a higher magnification at the bottom. (B) Costaining of Iho1 and Sycp3 in spo11^+/+ (i and ii) and spo11^–/– (iii and iv) spermatocytes. Regions marked in white rectangles are shown at a higher magnification at the bottom.