![]() b Percentage of seminiferous tubules containing spermatocytes positive for phosphorylated Histone H3 (pHH3). Some metaphase cells are enlarged on the right and indicated with arrows. ![]() a Hematoxylin & eosin (H&E) staining of testes derived from wild-type (WT) and Psma8 −/− males. PSMA8 deletion causes delayed M-phase entry and M-phase arrest. f Western blotting showing the levels of RAD51 and ubiquitination in testes electroporated with GFP, GFP-PSMA7, or GFP-PSMA8 Immunostaining of GFP was shown on the right to indicate the GFP-expressing cells, which were bordered with dashed circles. e Immunostaining of RAD51 in sections of testes electroporated with plasmids encoding green fluorescent protein (GFP), GFP-PSMA8, and GFP-PSMA7, respectively. c– d Immunostaining of RAD51 ( c) and RPA1 ( d) in testes sections derived from wild-type and Psma8 −/− males at the age of PD25. EZ early zygonema, Dia diakinesis spermatocytes. The regions bordered with a dashed box are enlarged on the right two panels. The different stages of seminiferous tubules are indicated by roman numerals. SYCP3 (red) and DAPI (blue) staining showing the stages of spermatogenesis. a, b Immunostaining of RAD51 ( a) and RPA1 ( b) in PD25 testes sections. The dashed line shows the weight of knockout testes at the age of PD90įailure in RAD51 and RPA1 degradation in PSMA8-deleted spermatocytes. 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. e Weights of testes derived from WT and Psma8 −/− males at the indicated ages. “+” represents the wild-type (WT) allele. d A representative image showing the morphology of testes derived from Psma8 +/− and Psma8 −/− males at the age of PD90. b, c Western blotting ( b) and immunofluorescent staining ( c) showing successful deletion of PSMA8 in spermatocytes at PD42. The primer sequences are provided in Supplementary Table 1. The locations of sgRNA and primers (P1–P5) are indicated. The null allele of Psma8 harbored a 5-bp deletion within the selected single-guide RNA (sgRNA) and introduced a premature stop codon (**). a Schematic diagram showing the gene structure of Psma8 and the CRISPR/Cas9 strategy used to generate the knockout allele. ![]() “−” represents the knockout allele and therefore “−/−” means knockout. d Immunofluorescent staining of PSMA8 (green) and SYCP3 (red) in sections from WT, Spo11 −/−, and Dmc1 −/− testes at PD21. Sertoli Sertoli cells, ES elongated spermatid, PreL pre-leptonema, LP late pachynema, M M phase. The representative cell types are enlarged on the right and indicated by dashed circles. c Co-staining of PSMA8 (green) and SYCP3 (red) in sections of WT testes. RS round spermatids, EP early pachynema, MP mid-pachynema, SG spermatogonia, Z zygonema, D diplonema. Different stages of seminiferous tubules are shown. The regions within the squares are enlarged on the right, and different types of cells were separated by dashed lines. ![]() b Immunofluorescent staining of PSMA7 in sections of PD42 wild-type (WT) testes. The molecular weights (kDa) are indicated on the right. The arrowhead indicates the specific band. The anti-α sub antibody detects all α subunits of the 20S core proteasomes. a Western blotting results showing the expression of PSMA7 (α4) and PSMA8 (α4s) during spermatogenesis. PSMA8 is expressed in spermatocytes from the pachytene stage. Thus, meiosis I progression in spermatogenesis, particularly entry into and exit from M-phase, requires the proteasomal activity of PSMA8-associated proteasomes. However, PSMA8 is neither expressed nor required for female meiotic progression. Moreover, PSMA8-null spermatocytes exhibit delayed M-phase entry and are finally arrested at this stage, resulting in male infertility. ![]() Meiotic proteins that are normally degraded at late prophase I, such as RAD51 and RPA1, remain stable in PSMA8-deleted spermatocytes. Deletion of PSMA8 decreases the abundance of proteasome in testes. PSMA8 is expressed in spermatocytes from the pachytene stage, and assembles a type of testis-specific core proteasome. Here, we show that PSMA8-associated proteasomes are essential for the degradation of meiotic proteins and the progression of meiosis I during spermatogenesis. However, the functions of proteasomal activity in meiosis I and II remain elusive. Spermatogenesis is tightly regulated by ubiquitination and proteasomal degradation, especially during spermiogenesis, in which histones are replaced by protamine. ![]()
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