Fig. 3: Zfp541 knockout spermatocytes fail to complete meiotic prophase.

a Chromosome spreads of Zfp541± and Zfp541 KO spermatocytes at postnatal day 21 (P21) were immunostained as indicated. Enlarged images are shown to highlight asynapsed chromosomes in pachytene-like cell. Lep: leptotene, Zyg: zygotene, Pac: pachytene, Dip: diplotene. Quantification of meiotic prophase stage spermatocytes per total SYCP3+ spermatocytes in Zfp541± and Zfp541 KO mice is shown on the right. n: the number of cells examined. Scale bars: 25 μm. b XY chromosome pairing in Zfp541± (n = 52) and Zfp541 KO (n = 50) pachytene spermatocytes was examined by immune-FISH using Y painting probe (green) and X point probe (red). X and Y chromosomes were in close proximity in most of the Zfp541 KO pachytene spermatocytes that we examined (100% = 52/52 in the control, 98% = 49/50 in Zfp541 KO, Chi-square test: p = 0.1474). Scale bar: 5 μm. c Seminiferous tubule sections (3-weeks and 8-weeks old) were stained for SYCP3, H1t, and DAPI. Pa: pachytene spermatocyte, rS: round spermatid. Shown on the right is the quantification of the seminiferous tubules that have H1t+/SYCP3+ cells per the seminiferous tubules that have SYCP3+ spermatocyte cells in Zfp541± and Zfp541 KO mice (mean with SD). n: the number of animals examined for each genotype. Statistical significance is shown (two-tailed t-test). N.S.: Statistically not significant. Scale bar: 25 μm. d Chromosome spreads of Zfp541± and Zfp541 KO spermatocytes were stained for MLH1, SYCP3, and SYCP1. The number of MLH1 foci is shown in the scatter plot with median (right). Statistical significance is shown (two-tailed Mann–Whitney U-test). n: number of spermatocytes examined. N.S.: Statistically not significant. Scale bar: 5 μm. e Seminiferous tubule sections (8-weeks old) were stained for SYCP3, H3S10p, and DAPI. M I: Metaphase I spermatocyte. Shown on the right is the quantification of the seminiferous tubules that have H3S10p+/SYCP3+ Metaphase I cells per the seminiferous tubules that have SYCP3+ spermatocyte cells in Zfp541± and Zfp541 KO testes (mean with SD). n: the number of animals examined for each genotype. Statistical significance is shown by *p = 0.0068 (two-tailed t-test). Scale bar: 25 μm. f Seminiferous tubule sections from 3-weeks and 8-weeks old mice were subjected to TUNEL assay with immunostaining for SYCP3. L: leptotene, Pa: pachytene. Shown on the right is the quantification of the seminiferous tubules that have TUNEL+ cells per total tubules in Zfp541± (8w; n = 3) and Zfp541 KO (8w; n = 3) testes (mean with SD). Statistical significance is shown by *p = 0.0165 (two-tailed t-test). Scale bar: 25 μm.