Fig 2: Depletion of RPA1 in oocytes causes loss of the RPA complex and female infertility.(A) Generation of Rpa1 oocyte-specific conditional knockout (OcKO) mice. (B) Immunofluorescence of RPA1, RPA2, and RPA3 in control (Rpa1 fl/fl without Cre or Rpa1 fl/+ with Cre) and Rpa1-ZcKO GV oocytes. Note the non-specific background signals in the cytoplasm of both genotypes in RPA1 and RPA3 images (4- to 5-week-old, n=3 per genotype). (C) Representative images of GV oocytes collected from adult control and Rpa1-ZcKO mice, before and after the removal of cumulus cells. For convenient comparison, some scale bars were placed adjacent to the oocytes instead of in the corners. (D, E) Number of GV oocytes obtained from the controls and Rpa1-ZcKOs in juvenile (4- to 5-week-old, CTR=9, Rpa1-ZcKO=10) and adult mice (8-week-old, n=6 per genotype) after hormone PMSG treatment. (F) Mating test showed the sterility of both Rpa1-ZcKO females and Rpa1-DcKO females. CTR=6, Rpa1-ZcKO=5, Rpa1-DcKO=3. Data are presented as mean ± SD, *P<0.05, **P<0.01, ****P<0.0005. F, forward primer; R, reverse primer. tm, targeted mutation. fl, floxed allele. del, deletion allele. Scale bars: 50 μm.

Fig 3: Diagrams of the proposed functional roles of RNF20-mediated H2B ubiquitination and chloroquine during meiotic recombination. (A) In the initiation of programmed DSB repair, ATM and ATR recruit RNF20 to ubiquitinate H2B and promote chromatin relaxation. Simultaneously or subsequently, RNF20 or another unknown protein recruits the MRN (MRE11/RAD50/NBS1) complex to the programmed DSB sites on the de-condensed chromatin, followed by other DNA repair factors such as RPA1, BRCA1 RAD51 and DMC1 to finish the entire meiotic recombination process. (B) In the RNF20-deficient spermatocytes, H2B cannot be ubiquitinated. As such, meiotic chromatin cannot be relaxed, which affects the efficient recruitment of the MRN complex (MRE11/RAD50/NBS1) and other DNA repair factors (RPA1, BRCA1, RAD51 and DMC1) to the DSB sites. Thereafter, programmed DSBs cannot be repaired by meiotic recombination, resulting in spermatocyte cell death and ultimately male infertility in mice. (C) During meiotic recombination in Stra8-Rnf20−/− spermatocytes, the insertion of chloroquine into the chromatin may relax the chromatin, mimicking the function of H2B ubiquitination. Thus, the recruitment of repair and recombination factors is partially restored, which recovers the programmed DSB repair and meiotic homologous recombination in the Rnf20-deficient spermatocytes and partially rescues spermatogenesis in Stra8-Rnf20−/− mice.

Fig 4: The MEIOB D383A mutation causes meiotic arrest and infertility in mouse. (A) Conservation of the D383 residue in MEIOB across species. The conserved residues are highlighted in red. (B) Histological analysis of testes from 8-week-old wild-type and MeiobD383A/D383A mice. Pac-like, pachytene-like. Scale bar, 50 μm. (C) Western blot analysis of wild-type and MeiobD383A/D383A testes. ACTB serves as a loading control. (D–G) Immunofluorescence analysis of surface nuclear spreads of spermatocytes from adult wild-type and MeiobD383A/FS (founder tag# 74). Spread nuclei of spermatocytes were immunostained with anti-MEIOB (D), anti-SPATA22 (E), anti-RPA1 (F), and anti-RPA2 (G). DNA was stained with DAPI. Scale bars, 10 μm.

Fig 5: Meiotic crossover, synapsis, and strand invasion are impaired due to RNF20 depletion. (A) Rnf20Flox/Flox and Stra8-Rnf20−/− spermatocytes stained for SYCP3 (red) and MLH1 (green). Scale bars, 10 μm. (B) Quantification of the MLH1 foci number per cell in Rnf20Flox/Flox and Stra8-Rnf20−/− mice. Rnf20Flox/Flox, 20.55 ± 0.75; Stra8-Rnf20−/−, 3.95 ± 0.48. (C) Rnf20Flox/Flox and Stra8-Rnf20−/− spermatocytes stained for SYCP3 (red) and SYCP1 (green). Discontinuous SYCP1 staining was observed in Stra8-Rnf20−/− cells. Scale bars, 10 μm. (D) Number of SYCP1 stretches per Rnf20Flox/Flox or Stra8-Rnf20−/−spermatocyte. Continuous SYCP1 stretches: Rnf20Flox/Flox, 18.36 ± 0.41; Stra8-Rnf20−/−, 8.27 ± 0.48. Discontinuous SYCP1 stretches: Rnf20Flox/Flox, 2.41 ± 0.28; Stra8-Rnf20−/−, 12.05 ± 0.81. (E) Spermatocytes stained for SYCP3 (red) and RAD51 (green) from Rnf20Flox/Flox and Stra8-Rnf20−/− mice. (F) Quantification of the RAD51 foci per cell in Rnf20Flox/Flox and Stra8-Rnf20−/− mice. Leptotene stage: Rnf20Flox/Flox, 64.14 ± 5.70; Stra8-Rnf20−/−, 42.33 ± 3.50. Zygotene stage: Rnf20Flox/Flox, 165.40 ± 7.28; Stra8-Rnf20−/−, 86.75 ± 7.92. Pachytene stage: Rnf20Flox/Flox, 62.13 ± 6.67; Stra8-Rnf20−/−, 20.81 ± 1.84. (G) Spermatocytes stained for SYCP3 (red) and RPA1 (green) in Rnf20Flox/Flox and Stra8-Rnf20−/− mice. RPA1 was not sufficiently recruited to the chromosomes in the Stra8-Rnf20−/− spermatocytes. Scale bars, 10 μm. (H) Immunoblot results showed decreased RAD51 and RPA1 in Stra8-Rnf20−/− spermatocytes. The Rnf20Flox/Flox and Stra8-Rnf20−/− spermatocytes used for immunofluorescence and western blottings were from 8 weeks mice.