Meiosis I progression in spermatogenesis requires a type of testis-specific 20S core proteasome

Abstract

Spermatogenesis is tightly regulated by ubiquitination and proteasomal degradation, especially during spermiogenesis, in which histones are replaced by protamine. However, the functions of proteasomal activity in meiosis I and II remain elusive. Here, we show that PSMA8-associated proteasomes are essential for the degradation of meiotic proteins and the progression of meiosis I during spermatogenesis. PSMA8 is expressed in spermatocytes from the pachytene stage, and assembles a type of testis-specific core proteasome. Deletion of PSMA8 decreases the abundance of proteasome in testes. Meiotic proteins that are normally degraded at late prophase I, such as RAD51 and RPA1, remain stable in PSMA8-deleted spermatocytes. Moreover, PSMA8-null spermatocytes exhibit delayed M-phase entry and are finally arrested at this stage, resulting in male infertility. However, PSMA8 is neither expressed nor required for female meiotic progression. Thus, meiosis I progression in spermatogenesis, particularly entry into and exit from M-phase, requires the proteasomal activity of PSMA8-associated proteasomes.

Fig. 1

PSMA8 is expressed in spermatocytes from the pachytene stage. a Western blotting results showing the expression of PSMA7 (α4) and PSMA8 (α4s) during spermatogenesis. The anti-α sub antibody detects all α subunits of the 20S core proteasomes. MVH is a germ cell marker. The arrowhead indicates the specific band. The molecular weights (kDa) are indicated on the right. b Immunofluorescent staining of PSMA7 in sections of PD42 wild-type (WT) testes. The regions within the squares are enlarged on the right, and different types of cells were separated by dashed lines. Different stages of seminiferous tubules are shown. RS round spermatids, EP early pachynema, MP mid-pachynema, SG spermatogonia, Z zygonema, D diplonema. Three testes were analyzed. Scale bars, 25 μm. c Co-staining of PSMA8 (green) and SYCP3 (red) in sections of WT testes. The representative cell types are enlarged on the right and indicated by dashed circles. Sertoli Sertoli cells, ES elongated spermatid, PreL pre-leptonema, LP late pachynema, M M phase. Scale bar, 50 μm. d Immunofluorescent staining of PSMA8 (green) and SYCP3 (red) in sections from WT, Spo11^−/−, and Dmc1^−/− testes at PD21. “−” represents the knockout allele and therefore “−/−” means knockout. Scale bars, 25 μm

Fig. 2

PSMA8 is required for male fertility. a Schematic diagram showing the gene structure of Psma8 and the CRISPR/Cas9 strategy used to generate the knockout allele. The null allele of Psma8 harbored a 5-bp deletion within the selected single-guide RNA (sgRNA) and introduced a premature stop codon (**). The locations of sgRNA and primers (P1–P5) are indicated. The primer sequences are provided in Supplementary Table 1. b, c Western blotting (b) and immunofluorescent staining (c) showing successful deletion of PSMA8 in spermatocytes at PD42. Scale bar, 100 μm. d A representative image showing the morphology of testes derived from Psma8^+/− and Psma8^−/− males at the age of PD90. “+” represents the wild-type (WT) allele. e Weights of testes derived from WT and Psma8^−/− males at the indicated ages. n = 6 testes for both WT and Psma8^−/− at PD21, and Psma8^−/− at PD25, PD30 and PD90; n = 8 testes for WT at PD30 and Psma8^−/− at PD16; n = 10 testes for WT at PD90 and Psma8^−/− at PD42; n = 12 testes for WT at PD16, PD25, and PD42. Error bars indicate S.E.M. **P < 0.01 and ***P < 0.001 by two-tailed Student’s t tests. n.s. not significant. The dashed line shows the weight of knockout testes at the age of PD90

Fig. 3

PSMA8 is required for proteasome assembly and protein degradation. a Western blotting results showing the elevated ubiquitination level in Psma8^−/− testes at the age of PD21. b PSMA8 deletion destabilized the proteasomes, as shown by α sub immunofluorescent staining in sections of PD25 testes. P pachynema. Scale bars, 25 μm. c Western blotting results showing the instability of proteasomes and failure of their ability to degrade prophase I proteins (RAD51 and RPA1) in Psma8^−/− testes at the age of PD42. The band intensity from three independent western blottings are quantified and the relative values are shown underneath each band. d Immunofluorescent staining of PSMA7 in testes sections derived from WT and Psma8^−/− males at PD42. Scale bars, 25 μm

Fig. 4

Failure in RAD51 and RPA1 degradation in PSMA8-deleted spermatocytes. a, b Immunostaining of RAD51 (a) and RPA1 (b) in PD25 testes sections. SYCP3 (red) and DAPI (blue) staining showing the stages of spermatogenesis. The different stages of seminiferous tubules are indicated by roman numerals. The regions bordered with a dashed box are enlarged on the right two panels. Scale bars, 25 μm. EZ early zygonema, Dia diakinesis spermatocytes. c–d Immunostaining of RAD51 (c) and RPA1 (d) in testes sections derived from wild-type and Psma8^−/− males at the age of PD25. Scale bars, 25 μm. e Immunostaining of RAD51 in sections of testes electroporated with plasmids encoding green fluorescent protein (GFP), GFP-PSMA8, and GFP-PSMA7, respectively. Immunostaining of GFP was shown on the right to indicate the GFP-expressing cells, which were bordered with dashed circles. Scale bars, 50 μm. f Western blotting showing the levels of RAD51 and ubiquitination in testes electroporated with GFP, GFP-PSMA7, or GFP-PSMA8

Fig. 5

Meiotic recombination is less affected by PSMA8 deletion. a, b Staining of γH2AX (a) and SYCP1 (b) in nuclear surface spreads derived from wild-type (WT) and Psma8^−/− males at PD42. Scale bars, 10 μm. c, d Immunostaining of RAD51 (d) in the nuclear surface spreads derived from WT and Psma8^−/− males at PD42 and the quantification of RAD51 foci (c) in WT and Psma8^−/− spermatocytes at pachytene stage. Scale bar, 10 μm. n = 39 for WT spermatocytes and n = 20 for Psma8^−/− spermatocytes. Median focus numbers are marked. Error bars indicate S.E.M. e, f Immunostaining of MLH1 (f) in nuclear surface spreads derived from WT and Psma8^−/− males at PD42. The quantification of MLH1 foci in WT and Psma8^−/− spermatocytes at the pachytene stage is shown in e. Scale bar, 10 μm. n = 37 for WT spermatocytes and n = 34 for Psma8^−/− spermatocytes. Median focus numbers are marked. Error bars indicate S.E.M.

Fig. 6

PSMA8 deletion causes delayed M-phase entry and M-phase arrest. a Hematoxylin & eosin (H&E) staining of testes derived from wild-type (WT) and Psma8^−/− males. Some metaphase cells are enlarged on the right and indicated with arrows. Scale bar, 50 μm. b Percentage of seminiferous tubules containing spermatocytes positive for phosphorylated Histone H3 (pHH3). n = 3 sections for both WT and Psma8^−/− testes at PD16 and PD21, and Psma8^−/− testes at PD42; n = 4 sections for both WT and Psma8^−/− testes at PD25 and PD30, and WT testes at PD42. Error bars indicate S.E.M. **P < 0.01 and ***P < 0.001 by two-tailed Student’s t tests. n.s. not significant. c Staining of pHH3 on testes sections derived from WT and Psma8^−/− males at PD21 and PD42. Arrows indicate metaphase cells. Scale bar, 100 μm. d Staining of phosphorylated H2AX (γH2AX) in testes sections derived from WT and Psma8^−/− males at PD42. The stages of seminiferous tubules are indicated. Scale bar, 50 μm. e Overexpression of PSMA8 partially rescued spermatogenesis defects. Green arrows indicate round spermatids. Scale bar, 50 μm

Fig. 7

PSMA8 is not required for female fertility. a, b Immunostaining of RAD51 (a) and RPA1 (b) in ovaries at the indicated ages. Scale bars, 25 μm. Oocytes at different stages are indicated with white arrows. Dic dictyate. c, d Immunostaining of RAD51 (c) and RPA1 (d) in wild-type (WT) and Psma8^−/− ovary sections at PD3. Scale bars, 25 μm. e Immunohistochemistry (IHC) staining of PSMA8 in ovary and testes sections derived from WT and Psma8^−/− mice. Black arrows indicate oocytes. Black arrows indicate individual oocytes. Scale bar, 50 μm