The PSMA8 subunit of the spermatoproteasome is essential for proper meiotic exit and mouse fertility

Abstract

The ubiquitin proteasome system regulates meiotic recombination in yeast through its association with the synaptonemal complex, a 'zipper'-like structure that holds homologous chromosome pairs in synapsis during meiotic prophase I. In mammals, the proteasome activator subunit PA200 targets acetylated histones for degradation during somatic DNA double strand break repair and during histone replacement during spermiogenesis. We investigated the role of the testis-specific proteasomal subunit α4s (PSMA8) during spermatogenesis, and found that PSMA8 was localized to and dependent on the central region of the synaptonemal complex. Accordingly, synapsis-deficient mice show delocalization of PSMA8. Moreover, though Psma8-deficient mice are proficient in meiotic homologous recombination, there are alterations in the proteostasis of several key meiotic players that, in addition to the known substrate acetylated histones, have been shown by a proteomic approach to interact with PSMA8, such as SYCP3, SYCP1, CDK1 and TRIP13. These alterations lead to an accumulation of spermatocytes in metaphase I and II which either enter massively into apoptosis or give rise to a low number of aberrant round spermatids that apoptose before histone replacement takes place.

Fig 1. Expression analysis and localization of PSMA8 in the mouse.

(A) Western blot analysis of protein extracts from mouse testis (from P8 to adult) and cell lines (TM3, TM4 and GC1) with a specific antibody against the C-terminal (α4S) and whole recombinant PSMA8 protein (PSMA8-R2). β-Actin was used as loading control. The corresponding bands to PSMA8 and PSMA7 are indicated in the right of the panel. Note that from P16 to adult the intensity of both PSMA8 and PSMA7 bands impedes its independent observation. (B) Double immunolabeling of spermatocyte spread preparations with PSMA8 (green) and SYCP3 (red) by Stimulated emission depletion (STED) microscopy, showing that PSMA8 localizes to the central region of the SC. PAR (pseudo-autosomal region) of the XY bivalent is indicated with an arrow. (C) Immuno-localization of PSMA8 in mouse testis after in vivo electroporation of a plasmid encoding a protein fusion of PSMA8 with GFP (GFP-PSMA8). PSMA8 was detected with anti-GFP antibody (green) and endogenous SYCP3 was detected using mouse anti-SYCP3 (red). (D) Triple labeling of PSMA8 (green), SYCP3 (blue) and SYCP1 (red) in Rec8^-/- and Six6os1^-/-. PSMA8 is detected in the pseudosynapsed AEs of the meiotic Rec8 cohesin mutant but is absent from the unsynapsed AEs in Six6os1^-/- spermatocytes. Bar in panels, 5 μm (B, upper panel), 1 μm (B, lower panel) and 10 μm (C, D).

Fig 2. PSMA8 deficiency leads to azoospermia.

(A-B) Genetic ablation of Psma8 leads to a reduction of the testis size (A) (n = 6, WT and KO; Welch´s t-test analysis: p<0.0001), and (B) the accumulation of metaphase I (black asterisks), apoptotic meiotic division (red asterisks), round spermatids entering apoptosis (arrowheads), and apoptotic round spermatids (blue asterisks) in PAS stained testis sections. The spermatogenic arrest leads to empty epididymides and azoospermia. Bar in upper panels 100 μm, lower panels 200 μm and in right panels, 5 μm. (St) Seminiferous tubules and (Ep) Epididymides. (C) Immunofluorescence analysis of p-ser10-H3 (green) of paraffin sections of Psma8^+/+ and Psma8^-/- tubules. Nuclei were counterstained with DAPI. Bar represents 10 μm. The diagram represents the quantification of the fraction of tubules showing the indicated number of metaphase I/II. Number of tubules counted for each genotype is expressed in S2B Table. (D) Low magnification view of a representative squash preparation of seminiferous tubules showing the accumulation of metaphases I and metaphases II in knock-out Psma8 in comparison with a representative wild-type view. The identity of metaphases I /metaphases II (asterisks) was confirmed by the immunolabeling of SYCP3 (red) in squash preparations. Chromosomes were counterstained with DAPI (blue). The diagram represents the percentage of spermatocytes at metaphase I and II (normal and apoptotic) in relation with the total number of spermatocytes from Psma8^+/+ and Psma8^-/- tubules (right). Quantification and number of cells analyzed are described in S2C Table. Welch´s t-test analysis: * p<0.01; ** p<0.001; *** p<0.0001.

Fig 3. Apoptosis, FACs and aberrant metaphase II and spermatid cells in Psma8-deficient mice.

(A) Double immunolabeling of Caspase3 (green) and (B) TUNEL (green) with SYCP3 (red). Non-apoptotic metaphase I cells from Psma8^+/+ show absence of green staining whereas apoptotic metaphases I from Psma8^-/- show intense Caspase-3 and TUNEL labeling. Chromatin was counterstained with DAPI. (C) Acrosome positive labeling of round spermatids by PNA staining (green). (D) FACs analysis of cells from whole seminiferous tubules from wild type and Psma8^-/- showing in both genotypes (N = 2) the presence of 4C, 2C and 1C compartment as a result of the early spermatid arrest. Source data describing the gating strategy is shown in S5B Fig. (E) Double immunolabeling of metaphase I cells with tubulin (green) and ACA (red) showing normal (Psma8^+/+) and abnormal spindles (Psma8^-/-). (F) Double immunolabeling of SYCP3 (green) with ACA (red) in wild-type and Psma8^-/- spermatocytes at metaphase II which shows aberrant accumulation of SYCP3 at the centromeres. (G) Double immunolabeling of PA200 (green) and SYCP3 (red) in chromosome spreads. PA200 is detected at the chromosome axes of the autosomal and XY bivalents during pachytene in wild type spermatocytes in contrast to the absence of labeling in Psma8^-/- spermatocytes. Bar in panels (C, E) 5 μm and 10 μm (A, B, F and G).

Fig 4. Histone acetylation, nuclei ubiquitylation and proteasome activity in PSMA8-deficient mice.

(A-D) Plots represent the quantification of the fluorescence intensity from Psma8^+/+ and Psma8^-/- spermatocytes at early pachytene (EP), mid pachytene (MP), late pachytene (LP), early diplotene (ED), late diplotene (LD), diakinesis (DK), metaphase I (MI) and round spermatid (RS) corresponding to the immunolabeling of (A) H2AK5ac, (B) H3ac, (C) H4ac, and (D) H4K16ac. Representative figures for each immunofluorescence are presented in S9–S12 Figs. (E) Proteasome activity of Psma8-deficient testis. 100 μg of protein from whole testis extracts of Psma8^+/+and Psma8^−/− mice were inoculated into 96-well plate and the proteasome peptidases activities were measured. The enzymatic activities relative to WT are shown. (F) Plots represent the quantification of the fluorescence intensity from Psma8^+/+ and Psma8^-/- spread (upper) and squashed (lower) spermatocytes. Welch´s t-test analysis: * p<0.01; ** p<0.001; *** p<0.0001.

Fig 5. SYCP1 interacts with PSMA8 and is accumulated in Psma8-deficient metaphase I cells.

(A) HEK293T cells were transfected with Flag-PSMA8 and GFP-SYCP1. Protein complexes were immunoprecipitated overnight 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 SYCP1. (B) Double immunolabeling of squashed tubules with SYCP1 (green) and SYCP3 (red) in wild-type and Psma8^-/- spermatocytes at metaphase I. Chromatin was stained with DAPI (blue). Bar in panel, 10 μm.

Fig 6. 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.

Fig 7. TRIP13 and MAD2 levels are increased in Psma8-deficient spermatocytes.

(A) HEK293T cells were transfected with a plasmid encoding GFP-TRIP13 and Flag-PSMA8. Protein complexes were immunoprecipitated with either an anti-Flag or anti-EGFP or IgGs (negative control), and immunoblotted with the indicated antibody. (B) Double immunolabeling of TRIP13 (green) and SYCP3 (red). TRIP13 labels the telomeres at pachytene and the intensity of the labeling decreases through desynapsis at diplotene and diakinesis. This labeling is enhanced during prophase I in the Psma8 mutants but its main pattern is not altered. At metaphase I, a faint labeling of sister kinetochores is observed in the Psma8^-/- spermatocytes that is absent in the wild type. Plot over the panel represents the quantification of the fluorescence intensity from Psma8^+/+ and Psma8^-/- spermatocytes at pachytene and late diplotene. (C) MAD2 (green) labels with enhanced intensity the centromeres of the chromosomes from Psma8^-/- metaphase I cells in comparison with the WT controls. Plot right to the panel represents the quantification of the fluorescence intensity from Psma8^+/+ and Psma8^-/- spermatocytes at metaphase I spermatocytes. Bar in panels, 10 μm. Welch´s t-test analysis: * p<0.01; ** p<0.001; *** p<0.0001.

Fig 8. PSMA8 interacts with proteins of the SC.

(A) PSMA8 co-immunoprecipitates with SIX6OS1 and SYCE3. HEK293T cells were transfected with plasmids encoding Flag-PSMA8 and GFP-SIX6OS1 or GFP-SYCE3. Protein complexes were immunoprecipitated overnight with either an anti-Flag or anti-EGFP or IgGs (negative control), and were analyzed by immunoblotting with the indicated antibody. (B) Double immunofluorescence of transfected HEK293T cells with plasmids encoding Flag-PSMA8 and Flag-PSMA7 alone or together with plasmid encoding SYCP3-HA and immuno-detected with antibodies against Flag (green) or HA (red). Transfected PSMA8 alone is delocalized and occupies the whole cell whereas when co-transfected with SYCP3-HA is recruited to form polycomplexes. PSMA7 do not form polycomplexes when co-transfected with SYCP3-HA. (C-D) SYCP3 is accumulated in vivo in Psma8^-/- spermatocytes. (C) Double immunolabeling of squashed tubules with SYCP3 (red) and SYCP1 (green) in wild-type and Psma8^-/- spermatocytes at prophase I showing large SYCP3 aggregates surrounding the nuclei (arrows). (D) Double immunolabeling of squashed tubules with SYCP3 (green) and ACA (red) in wild-type and Psma8^-/- spermatocytes at metaphase I and II. Psma8^-/- metaphases I show labeling of SYCP3 in aggregates (arrows, absent in the WT) in addition to its typical labeling at the centromeres. Metaphases II from Psma8^-/- show labeling for SYCP3 at the centromeres between the sister kinetochores and as aggregates in the cytosol (arrows) whereas wild type metaphases II show barely visible SYCP3 labeling. (E) SYCP3 was 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.