Fig 1.
Super-resolution localization of RPA, DMC1 and MEIOB on meiotic chromosomes.
Spermatocytes from adult testes were subjected to immunofluorescent analysis with various antibodies. (A-F) Localization of RPA1 in leptotene through diplotene spermatocytes. Early, mid, and late pachytene stages were distinguished based on the XY morphology, SYCP3 staining intensity and the accumulation of SYCP3 at the SC ends as previously described [41]. (G) Double immunolocalization analysis of RPA2 and DMC1 in early pachytene spermatocytes. (H) Co-localization of RPA2 and MEIOB in early pachytene spermatocytes. Enlarged view of boxed individual chromosomes (A-H) is shown in the bottom panels (A′-H′) respectively. Scale bars, 10 μm.
Fig 2.
Tamoxifen-inducible Cre-mediated Rpa1 deletion strategy during male meiosis.
(A) Schematic diagram of various Rpa1 alleles: Rpa1tm1a, Rpa1fl (floxed allele), and Rpa1 knockout. Cre-mediated deletion of exon 8 (encoding residues 182–226) is expected to cause a frameshift in the resulting mutant transcript. FLP, FLP1 recombinase; Cre, Cre recombinase. Frt and loxP sites are shown. (B) Tamoxifen treatment regimen. (C) Absence of RPA1, RPA2, and RPA3 foci in Rpa1cKO zygotene-like spermatocytes. RPA1, RPA2, and RPA3 form many foci on synaptonemal complexes in control leptotene spermatocytes but none in zygotene-like spermatocytes from Rpa1cKO testes at 4 days post-tamoxifen treatment. Asterisks indicate the non-specific immunofluorescence signals (RPA2) on sperm heads. (D) Western blot analysis of various ssDNA-binding proteins (RPA1, RPA2, RPA3, MEIOB, SPATA22, DMC1, RAD51) in testes from 8-week control and Rpa1cKO mice at 2, 4, 6, and 8 days post-tamoxifen treatment (dpt). ACTB serves as a loading control.
Fig 3.
RPA1 is essential for meiosis in males.
(A) Histological analysis of testes from 8-week-old control and tamoxifen-treated Rpa1fl/fl Ddx4-CreERT2 mice. Cross-sections of Stage IX and XII seminiferous tubules are shown. Dash lines indicate missing germ cells. Arrowheads designate the apparently apoptotic spermatocytes (day 12, Stage XII). Lep, leptotene; Zyg, zygotene; Pa, pachytene; M, metaphase spermatocytes; ES, elongating spermatids. Scale bar, 50 μm. (B) Surface spread analysis of spermatocytes from testes at 4 days post-TMX treatment (dpt). (C) Composition of spermatocytes. Three males per timepoint were analysed. ~200 spermatocytes from each mouse were counted and categorized into different spermatocyte types. The percentage (average ± SD) was plotted. (D) Time-dependent progressive loss of spermatocytes in tamoxifen-treated Rpa1fl/fl Ddx4-CreERT2 males. Lines indicate the presence of spermatocytes. Two cell cycle checkpoints (pachytene checkpoint at stage IV and spindle assembly checkpoint at stage XII) are shown. Note that zygotene-like is separated from zygotene for illustration purpose and is not a separate stage of meiosis.
Fig 4.
RPA1 is required for pre-meiotic S-phase DNA replication in preleptotene spermatocytes.
Pulse labelling of DNA replicating cells was performed with intraperitoneal injection of BrdU. (A) Immunofluorescence analysis of BrdU incorporation in stage VII-VIII seminiferous tubules. The acrosome morphology shown by SP10 immunofluorescence was used for seminiferous tubule staging. All positive tubule: all preleptotene spermatocytes are BrdU-positive; All negative tubule: all preleptotene spermatocytes are BrdU-negative; Partial positive tubule: a fraction of preleptotene spermatocytes are BrdU-positive; Pre-lep loss tubule: absence of preleptotene spermatocytes in stage VII-VIII seminiferous tubules. (B) Percentage of two types of stage VII-VIII tubules in control mice. (C) Percentage of four types of stage VII-VIII tubules from Rpa1cKO at 0 day post-tamoxifen treatment. (B, C) Three pairs of control and Rpa1cKO males (0 day post tamoxifen treatment) were analyzed. At least thirty five stage VII-VIII tubules were counted for each mouse.
Fig 5.
RPA1 is required for focal localization of RAD51/DMC1 on meiotic chromosomes but not MEIOB/SPATA22 at the zygotene stage.
(A) Immunolocalization of ssDNA-binding proteins in zygotene (control) and zygotene-like (Rpa1fl/fl Ddx4-CreERT2, 4 dpt) spermatocytes. Synaptonemal complexes were immunostained with anti-SYCP2 antibody. Note that γH2AX signal is much stronger in mutant zygotene-like spermatocytes than control zygonema. (B) Dot plots of foci of RAD51, DMC1, MEIOB, and SPATA22 in zygotene (control) and zygotene-like (Rpa1cKO) spermatocytes. Solid lines show the average ± SD. n, number of cells counted from two experiments; ***, p < 0.001 (Student’s t test); ns, non-significant. (C) A role for RPA in pre-synaptic filament formation. Upper panel, control; bottom panel, absence of RPA.
Fig 6.
Reduction in the number of TEX11 and MSH4 foci in early/mid pachytene spermatocytes from Rpa1cKO mice at 6 days post-tamoxifen treatment.
Spermatocytes from untreated adult males were used as controls. The number of TEX11 foci (A) or MSH4 foci (B) in mid-pachytene spermatocytes is lower than in early pachytene spermatocytes. Early and mid-pachytene spermatocytes were categorized by the morphology of XY chromosomal axis and the intensity of the synaptonemal complex staining as previously described [41]. Early pachytene stage is characterized by an extended XY configuration and low intensity of the synaptonemal complex staining, whereas the mid-pachytene spermatocytes exhibit a U-shaped XY axis and higher synaptonemal complex staining. n, number of spermatocytes counted from four experiments (A) or three experiments (B); **, p< 0.01; ***, p<0.001.
Fig 7.
RPA1 regulates the formation of meiotic crossovers.
(A) Reduction in MLH1 foci in Rpa1cKO pachytene spermatocytes. Asterisks mark the chromosomes without MLH1 foci. (B) Counts of MLH1 foci in pachytene spermatocytes. n, number of cells counted from four experiments. (C) Presence of univalent chromosomes (indicated by arrows) in Rpa1cKO metaphase I spermatocytes. (D) Quantification of chiasmata from control and Rpa1cKO metaphase I cells. n, number of cells counted from three experiments. (E) TUNEL analysis of stage XII seminiferous tubules from adult control and Rpa1cKO males. Note the TUNEL-positive (green) metaphase I spermatocytes in the Rpa1cKO tubule. Scale bar, 25 μm. (F) Quantification of apoptotic cells of stage XII tubules from adult control and Rpa1cKO males. n, number of stage XII tubules from two experiments. ns, no significance; ***, p ≤ 0.001 (Student’s t-Test); dpt, days post tamoxifen injection. (G) Dual functions of RPA in meiotic recombination. The upper diagram illustrates the distribution of recombination proteins and timing of key recombination events during male meiosis. The lower diagram depicts the defects in the absence of RPA: depletion of RAD51/DMC1, reduction in MLH1 foci, and two rounds of apoptosis (the first round at stage IV and the second round at stage XII). Abbreviations: Lep, leptotene spermatocytes; Zyg, zygotene; Pac, pachytene; Dip, diplotene; Met I, metaphase I; Ana I, anaphase I.