Fig. 5: TRF1 is required for preventing telomere fusion at the bouquet stage

a Telomere fusions occurred after Trf1 depletion. Meiotic chromosome spreads from the Trf1^Flox/Flox and Stra8-Trf1^−/− testis were immunostained with antibody against SYCP3 (red) and Tel-FISH (green). In the pachytene-like Stra8-Trf1^−/− spermatocytes, FISH signal can be found in the middle of the SYCP3 threads, instead of at the ends. Schematic illustrations represent the structures. b and c Telomeric FISH signal intensities and areas per telomere were increased in Stra8-Trf1^−/− spermatocytes. Telomeric FISH signal intensities and areas were measured, and the mean Tel-FISH signal intensities and areas were shown. Intensity: Trf1^Flox/Flox (gray bar, 116.22 ± 5.42) Stra8-Trf1^−/− (white bar, 142.77 ± 5.35). Area: Trf1^Flox/Flox (gray bar, 121.98 ± 7.58 px²), Stra8-Trf1^−/− (white bar, 158.28 ± 8.87 px²). Data are presented as mean ± SEM. ***P < 0.001. d Telomere length was increased in the Trf1-deficient spermatocytes. Quantitative PCR was performed to measure telomere length and the relative telomere to reference single copy gene (T/R) ratio was calculated to represent the average telomere length in the Trf1^Flox/Flox and Stra8-Trf1^−/− spermatocytes at pachytene or pachytene-like stage. Trf1^Flox/Flox (gray bar, 0.78 ± 0.12), Stra8-Trf1^−/− (white bar, 1.23 ± 0.09). Data are presented as mean ± SEM. ***P < 0.001. e and f Speedy A and Cdk2 are not recruited to the telomeres in the Trf1-deficient spermatocytes. Trf1^Flox/Flox and Stra8-Trf1^−/− chromosome spreads of spermatocytes were immunolabelled with antibodies against SYCP3 (red) and Speedy A (green) or Cdk2 (green). g Selected shelterin and LINC complexes protein levels were decreased after Trf1 depletion. Immunoblotting for shelterin and LINC proteins in Trf1^Flox/Flox and Stra8-Trf1^−/− spermatocytes. TIN2, TRF2, POT1, Speedy A, and Cdk2 were all decreased to some extent in the Stra8-Trf1^−/− spermatocytes. GAPDH was used as loading control.