A: Expression of sycp2 mRNA in sycp2^+/+, sycp2^its/its and sycp2^-/- testes. The mRNA levels relative to actb1 are shown (n = 5). The vertical bars indicate the SD. ** and *** indicate p<0.01 and p<0.001, respectively (Student’s t-test). B: HE-stained sections of sycp2^+/+, sycp2^-/-, sycp2^its/its, and sycp2^its/- testes. All samples were prepared from the same siblings. Sg: spermatogonia, Sc: spermatocytes, St: spermatids, Sz: spermatozoa. Spermatids and spermatozoa were observed only on the sycp2^+/+ section. Scale bar, 10 μm.

A: cDNA sequences of the exon 8–9 junction of sycp2. The sequences of one sycp2^+/+ (WT) and three sycp2^its/its (its-1 to its-3) cDNA clones are shown. Two of the three sycp2^its/its cDNA clones had a 5-bp insertion (in yellow) containing a premature stop codon (in red letters) at the exon 8–9 junction. B: Genomic sequences of the intron 8-exon 9 junction of sycp2. Sequences from sycp2^+/+ (WT) and sycp2^its/its (its) testes are shown. The sycp2^its allele harbors a T-to-A substitution (in red) upstream of exon 9. The its and wild-type (canonical) splicing sites are indicated with vertical arrows. C: Splicing at sycp2 exon 8-exon 9 was examined by RT-PCR. The target sites of the PCR primers are shown above. Template cDNA was prepared from sycp2^+/+ (WT) and sycp2^its/its (its) testes from three individual fish. The PCR products were examined on a 20% acrylamide gel. D: Mini-gene splicing assay in the wild-type genetic background. RT-PCR was performed with caudal fin cDNA from transgenic fish with either wild-type (WT E8-9) or its-type (its E8-9) sycp2 mini-genes (S3 Fig). Wild-type fish without mini-genes were used as controls (-). See S3 Fig for more clones and the results of control PCR with a primer pair specific to EGFP.

Immunostaining of SC components on wild-type (A), sycp2^-/- and sycp2^its/its (B) spermatocyte chromosomal spreads. Individual images with anti-Sycp3, anti-Sycp2, or anti-Sycp1 antibodies and a merged image are shown for each nucleus. Wild-type nuclei are staged according to the staining patterns of Sycp3, Sycp2 and Sycp1 (A-i to A-v) [26]. The white line on A-ii indicates a border with another nucleus on the top right. Nuclei from two individual sycp2^its/its males (M1 and M2) are shown (B-ii to B-v). Wild-type-like nuclei were observed only among M1 spermatocytes. All spreads were prepared and stained at the same time, and all images were processed in the same manner. Scale bars, 5 μm.

A: Costaining of telomeres and Sycp1 on sycp2^+/+ (i-ii), sycp2^its/its (iii), and sycp2^-/- (iv) spermatocyte chromosomal spreads. The regions outlined with white are shown at a higher magnification at the bottom. The nuclei of sycp2^+/+ spermatocytes are at zygonema (i; telomere detected at one end of each Sycp1 filament) and pachynema (ii; telomeres detected at both ends of Sycp1 filaments). Scale bars, 5 μm. B: Colocalization of Sycp1 fragments with telomere foci stained by the telomere-targeting polyamide at their ends in sycp2^+/+, sycp2^its/its and sycp2^-/- spermatocytes. The percentages of Sycp1 filaments that did not colocalize or that colocalized at one or both ends with telomeres are shown in gray, light green and green, respectively. Each pool corresponds to the nuclei counted in S6 Fig. Error bars indicating the SD are shown only for the plus direction. A small fraction of Sycp1 ends did not colocalize with telomere foci, but two telomere foci were observed in close proximity (Fig 4A, arrowhead). These sites were possible locations at which synapsis or desynapsis was in progress. C: Numbers of telomere foci in sycp2^+/+, sycp2^its/its and sycp2^-/- spermatocytes. Telomere foci stained by the telomere-targeting polyamide were counted in nuclei with Sycp1 filaments of each genotype. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; data points are plotted as open circles. Chromosomal spreads of one (wild-type) or two (sycp2^its/its and sycp2^-/-) individual fish were used for counting. sycp2^+/+, n = 34; sycp2^its/its, n = 41; and sycp2^-/-, n = 45. *** indicates p<0.0001 (Student’s t-test).

A: Fluorescent in situ hybridization with a BAC probe in sycp2^+/+, sycp2^its/its and sycp2^-/- spermatocytes. The regions outlined with white in A-i, A-iii and A-iv are shown at a higher magnification at the bottom. White arrowheads indicate BAC-probe-stained foci. A-i, A-iii and A-iv show nuclei with paired BAC foci with (A-i and A-iii) and without (A-iv) colocalization on an Sycp1 fragment. A-ii, A-v, A-vi and A-vii show nuclei with multiple unpaired BAC foci. Scale bars, 5 μm. B: Quantification of pairing of BAC foci in sycp2^+/+ (n = 34), sycp2^its/its (n = 67) and sycp2^-/- (n = 61) spermatocytes. The percentage of nuclei stained for one to four BAC foci is shown for each genotype. When BAC signals in a nucleus were observed as one focus or two foci with an intervening distance of <3 μm, they were considered to be paired, a localization on Sycp1 filaments was also evaluated. Proportions corresponding to one BAC focus with localization (paired on Sycp1) and without localization (paired not on Sycp1) on Sycp1 filaments are shown in dark and light green, respectively. Chromosomal spreads of one (wild-type) or two (sycp2^its/its and sycp2^-/-) individual fish were used for counting.

A: Immunostaining of Dmc1/Rad51, Sycp1 and Sycp3 on wild-type (i to iv), spo11^-/- (v) and sycp2^-/- (vi) spermatocyte chromosomal spreads. The wild-type nuclei are at leptonema (i), early zygonema (ii), late zygonema (iii) and pachynema (iv) according to the Sycp1 and Sycp3 staining patterns. A spo11^-/- nucleus at an early zygotene-like stage, according to Sycp3 staining patterns, is shown (v). An early zygotene-like sycp2^-/- nucleus stained with short Sycp1 fragments is shown (vi). Scale bars, 5 μm. B: Quantification of the Dmc1/Rad51-stained area. The sum of the area stained for the Dmc1/Rad51 foci in each nucleus was measured in wild-type nuclei at leptonema (L, n = 14), early zygonema (EZ, n = 21), late zygonema (LZ, n = 17), and pachynema (P, n = 26), in leptotene- or early zygotene-like spo11^-/- (n = 40), and in early zygotene-like sycp2^-/- (n = 39) spermatocytes. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; data points are plotted as open circles. Chromosomal spreads of two individual fish were used for each genotype. *** indicates p<0.0001 (Student’s t-test). N.S. indicates not significant.

A: Staining of sycp2^+/+ and sycp2^-/- spermatocyte chromosomal spreads with anti-human RPA, anti-Sycp1 and anti-Sycp3 antibodies. Note that the human RPA protein (NP_002936.1) shares 63.5% identity and 80.9% similarity with the zebrafish protein (NP_956105.2). In sycp2^+/+ spermatocytes, RPA appears at leptonema in faint foci, which become brighter at zygonema and disappear at pachynema. In sycp2^-/- spermatocytes, RPA foci were rarely detected; only a few foci were observed in some cells. B: Immunostaining of sycp2^+/+ (i, ii) and sycp2^-/- (iii, iv) spermatocyte chromosomal spreads with anti-γH2AX, anti-Sycp3 and Sycp1 antibodies. Individual images with each antibody and a merged image are shown. The wild-type (sycp2^+/+) nuclei are at early to mid-zygonema. Scale bars, 5 μm.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

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