PSMA8 deficiency causes an accumulation of CDK1 and Cyclin B1 in spermatocytes.

(A) HEK293T cells were transfected with Flag-PSMA8 and GFP-CDK1. Protein complexes were immunoprecipitated with either an anti-Flag or anti-EGFP or IgGs (negative control) and were analyzed by immunoblotting with the indicated antibody. PSMA8 co-immunoprecipitates with CDK1 (as well as reciprocally). (B) Double labeling of endogenous CDK1 (green) and SYCP3 (red) in mouse spermatocytes at metaphase I. Chromatin was stained with DAPI (blue). During metaphase I, CDK1 labels in a slight and disperse way the chromosomes and in a more intensely fashion the centromeres of bivalents. This labeling pattern is enhanced in a normal Psma8-deficient metaphase I. Plot under the panel represents the quantification of the fluorescence intensity from Psma8+/+ and Psma8-/- metaphase I cells. (C) Double labeling of endogenous CDK1-Tyr15phosphorylated (green) and SYCP3 (red) in mouse spermatocytes at metaphase I showing similar expression levels in Psma8+/+ and Psma8-/-. Chromatin was stained with DAPI (blue). (D) Double labeling of endogenous cyclin B1 (green) and SYCP3 (red) in mouse spermatocytes at metaphase I showing higher expression levels in Psma8-/-. Plot under the panel represents the quantification of the fluorescence intensity from Psma8+/+ and Psma8-/- metaphase I cells. Welch´s t-test analysis: * p<0.01; ** p<0.001; *** p<0.0001. (E) CDK1 and CyclinB1 were measured by western blot analysis of protein extracts from whole testis of Psma8+/+ (WT) and Psma8-/- (KO) (n = 2 mice). Bar in panels, 10 μm. Welch´s t-test analysis: * p<0.05; ** p<0.001; *** p<0.0001.