Shu complex SWS1-SWSAP1 promotes early steps in mouse meiotic recombination

Abstract

The DNA-damage repair pathway homologous recombination (HR) requires factors that promote the activity of strand-exchange protein RAD51 and its meiosis-specific homolog DMC1. Here we show that the Shu complex SWS1-SWSAP1, a candidate for one such HR regulator, is dispensable for mouse viability but essential for male and female fertility, promoting the assembly of RAD51 and DMC1 on early meiotic HR intermediates. Only a fraction of mutant meiocytes progress to form crossovers, which are crucial for chromosome segregation, demonstrating crossover homeostasis. Remarkably, loss of the DNA damage checkpoint kinase CHK2 rescues fertility in females without rescuing crossover numbers. Concomitant loss of the BRCA2 C terminus aggravates the meiotic defects in Swsap1 mutant spermatocytes, suggesting an overlapping role with the Shu complex during meiotic HR. These results demonstrate an essential role for SWS1-SWSAP1 in meiotic progression and emphasize the complex interplay of factors that ensure recombinase function.

Fig. 1

Defective meiotic progression in Sws1 and Swsap1 mutant males and females. a, b Sws1 (a) and Swsap1 (b) genomic structure, TALEN target sequences (red and blue), mutations analyzed, and the predicted truncated proteins, if translated (shown on the right). c Testis to body weight ratios are reduced in adult single and double mutants, but not in juveniles. Error bars, mean ± s.d. Adult mice: control, n = 45 (black circles); Sws1^−/− alleles combined [∆1(A), n = 20 (purple circles); ∆1(G), n = 4 (purple triangles); ∆1(T), n = 3 (purple inverted triangles)], Swsap1^−/− alleles combined [∆131, n = 10 (dark blue circles); (+1), n = 8 (dark blue triangles)], Sws1^−/− Swsap1^−/− (∆1(A) / (+1)), n = 5 (light blue diamonds). Juvenile mice at 7.5 dpp: control, n = 15; Sws1^−/−∆1(A), n = 3; Swsap1^−/−∆131, n = 4. ns not significant; ****P ≤ 0.0001; Student’s t-test, two-tailed. d Ovary to body weight ratios are reduced in adult single and double mutants. Error bars, mean ± s.d. Adult mice: control, n = 23; Sws1^−/− [∆1(A), n = 11; ∆1(G), n = 3; ∆1(T), n = 3], Swsap1^−/− [∆131, n = 4; (+1), n = 5], Sws1^−/− Swsap1^−/− (∆1(A) / (+1)), n = 4. ***P ≤ 0.001; ****P ≤ 0.0001; Student’s t-test, two-tailed. e Shu mutant spermatocytes arrest at pachynema. Sections were stained with periodic acid-Schiff (PAS) and counterstained with hematoxylin. Scale bars, 100 μm and 50 μm for left and right columns, respectively. Control, Sws1^−/−, Swsap1^−/−, n ≥ 3. f Ovaries from Shu mutant adults lack follicles. Sections were stained with PAS. Scale bar, 500 μm. Control, Sws1^−/−, Swsap1^−/−, n ≥ 3

Fig. 2

SWS1-SWSAP1 is required for meiotic homolog synapsis and RAD51 and DMC1 focus assembly. a Histone H1t staining indicates that few spermatocytes in Sws1 and Swsap1 mutants progress to mid-pachynema and beyond. Total number of mid-pachytene, late-pachytene, and diplotene spermatocytes divided by the total number of spermatocytes analyzed from adult testes. Mice: control, n = 6; Sws1^−/−, Swsap1^−/−, n = 3. ****, P ≤ 0.0001; Fisher’s exact test, two-tailed. b, c Sws1 and Swsap1 mutants show altered meiotic progression and abnormal chromosome synapsis. Percentage of spermatocytes in each of the indicated meiosis prophase I stages in b with representative images in c. Sws1^−/− and Swsap1^−/− cells at leptonema are indistinguishable from controls. At later stages abnormal cells with synaptic defects are observed; because synapsis is abnormal these stages are appended with the word “-like” (orange bars). At early zygonema, chromosomes begin to synapse in the majority of mutant cells, although the number of synaptic stretches are usually reduced; however, delayed synapsis onset is apparent in a subset of mutant cells, as indicated by the lack of SYCP1 stretches. At late zygonema, abnormal cells with fully formed chromosome axes (SYCP3) but little or no synapsis (SYCP1) predominate. Fully-synapsed chromosomes with thicker and shorter SYCP3 axes characterize early pachynema, indicative of chromatin condensation, however, abnormal cells with unsynapsed chromosomes and chromosomes with non-homologous synapsis and/or partial asynapsis are frequent in the mutants. Mid-pachytene (H1t-positive) abnormal cells contain unsynapsed chromosomes, broken bivalents, or parts of chromosomes involved in non-homologous synapsis. Scale bars, 10 µm. Boxes in c in early pachytene-like cells highlight non-homologous synapsis and arrows indicate unsynapsed chromosomes. d–g RAD51 and DMC1 focus counts are reduced in Sws1 and Swsap1 mutant spermatocytes. Representative chromosome spreads from adult mice at early zygonema (-like) are shown in d, f with focus counts for all stages in e, g. n = 3. Error bars, mean ± s.d. Scale bars, 10 µm. Each circle in e, g indicates the total number of foci from a single nucleus. Solid circles in e, g normal cells. Open circles in e, g cells with abnormal synapsis. Abnormal cells at early zygonema in the Sws1 and Swsap1 mutants are not indicated because they are a fraction of cells and are not distinguishable without SYCP1 costaining. **P ≤ 0.01; ****P ≤ 0.0001; Mann–Whitney test, one-tailed

Fig. 3

Sws1 and Swsap1 mutants accumulate resected DNA and have reduced HR foci later in meiosis. a, b MEIOB focus counts are increased in Sws1^−/− and Swsap1^−/− spermatocytes, but not as much as in Dmc1^−/− spermatocytes. Representative chromosome spreads at early zygonema (-like) from adult mice in a with focus counts at indicated stages in b. Mice: Control, n = 2; Sws1^−/−, Swsap1^−/−, n = 3, Dmc1^−/−, n = 4. c, d MSH4 focus counts are reduced in Sws1^−/− and Swsap1^−/− spermatocytes. Focus counts at indicated stages in c with representative images of chromosome spreads at late zygonema (-like) in d. White lines in the images demark boundaries of other cells in the field. Mice: control, n = 2; Sws1^−/−, Swsap1^−/−, n = 3. e, f MLH1 focus counts are reduced on average by ~20% in Sws1^−/− and Swsap1^−/− spermatocytes at mid-/late pachynema (-like). Representative chromosome spreads at mid-pachynema in e with focus counts in f. Mice: Control, n = 4; Swsap1^−/− (∆131, circles; (+1), triangles), Sws1^−/−, n = 3. g Although most Sws1^−/− and Swsap1^−/− bivalents have at least one MLH1 focus (left), most spermatocytes have at least one bivalent which lacks a focus. Left, percentage of bivalents in mid-/late pachynema (-like) with indicated number of MLH1 foci per bivalent. Right, number of bivalents without an MLH1 focus per spermatocyte; total percentage of spermatocytes containing bivalents without an MLH1 focus is indicated. Solid circles and triangles, spermatocytes with fully synapsed homologs. Open circles, abnormal spermatocytes containing homologs partially synapsed and/or one or two homolog pairs fully unsynapsed. h, i MLH1 focus counts are reduced on average by ~30% in Swsap1^−/− oocytes. Representative chromosome spreads at mid-pachynema (-like) from mice at embryonic day 18.5 in h with focus counts in i. Control, n = 2; Swsap1^−/−, n = 3. While a fraction of mutant oocytes show completely synapsed bivalents (solid circles in i), the majority have chromosomal synapsis defects (open circles), i.e., fully unsynapsed and/or partially synapsed bivalents. In controls, a small fraction of oocytes also have one or two chromosome ends that are not fully synapsed. Each circle in b, c, f, i indicates the total number of foci from a single nucleus. Solid circles, normal cells. Open circles/triangles, cells with abnormal synapsis. Scale bars in a, d, e, h, 10 µm. Error bars in b, c, f, g, i, mean ± s.d. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; Mann–Whitney test, one-tailed

Fig. 4

Chk2 mutation rescues fertility of Sws1 and Swsap1 mutant females. a Ovary to body weight ratios of Sws1^−/− Chk2^−/− and Swsap1^−/− Chk2^−/− adult mice are similar to controls, but testis to body weight ratios remain significantly reduced. Female mice: Control, n = 16; Chk2^−/−, n = 7; Sws1^−/−, n = 5; Sws1^−/− Chk2^−/−, n = 3; Swsap1^−/−, Swsap1^−/− Chk2^−/−, n = 3. Male mice: control, n = 15; Chk2^−/−, n = 14; Sws1^−/−, n = 4; Sws1^−/− Chk2^−/−, n = 2; Swsap1^−/−, n = 10; Swsap1^−/− Chk2^−/−, n = 5. ns not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; Student’s t-test; two-tailed. b–d Adult Swsap1^−/− Chk2^−/− ovaries have follicles at various stages of oocyte development, unlike Swsap1^−/− ovaries. Primordial follicles in Chk2^−/− and Swsap1^−/− Chk2^−/− are highlighted in b in insets and by arrows in c; primordial follicles in Chk2^−/−, Sws1^−/− Chk2^−/− and Swsap1^−/− Chk2^−/− are quantified in d. Sections were stained with hematoxylin and eosin (H&E) in b, and with hematoxylin and an antibody for DDX4/Vasa in c. Scale bar, 500 μm and 50 μm insets. Mice: control, n = 4; Chk2^−/−, n = 5; Sws1^{-/ -}, n = 4; Sws1^−/− Chk2^−/−, n = 3; Swsap1^{-/ -}, Swsap1^−/− Chk2^−/−, n = 3. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; Student’s t-test, two-tailed. e MLH1 foci are reduced in Swsap1^−/− Chk2^−/− oocytes from mice at embryonic day 18.5 similar to the level observed in Swsap1^−/− oocytes, and synaptic defects are also observed in the majority of Swsap1^−/− Chk2^−/− oocytes as also seen in Swsap1^−/− mice (open circles). Mice: Control, Swsap1^−/−, n = 1 (from Fig. 3i); Swsap1^−/− Chk2^−/−, Chk2^−/−, n = 1. ****P ≤ 0.0001; Mann–Whitney test, one-tailed. f Swsap1^−/− Chk2^−/− spermatocytes that progress to mid-pachynema and beyond are increased in number relative to that observed in Swsap1^−/− mice, as demonstrated by histone H1t staining. Total number of mid-pachytene, late-pachytene, and diplotene spermatocytes divided by the total number of spermatocytes analyzed from adult testes. Mice: Control, Swsap1^−/−, from Fig. 2a; Chk2^−/−, Swsap1^−/− Chk2^−/−, n = 2. *P ≤ 0.05; ****P ≤ 0.0001; Fisher’s exact test, two-tailed. g Spermatocytes in adult Swsap1^−/− Chk2^−/− mice mostly arrest at pachynema, but tubules occasionally contain round and elongated spermatids (blue rectangles and insets). Sections were stained with H&E. Scale bars, 100 μm (top panel), 50 μm (bottom panel), and 20 μm (inset). Error bars in a, d, e, mean ± s.d

Fig. 5

BRCA2 C terminus promotes Swsap1 spermatocyte progression. a Swsap1^−/−(+1) Brca2^∆27/∆27 spermatocytes do not progress past early pachynema in contrast to Swsap1^−/−(+1) in which a fraction progresses. Mice: Control, n = 8 (combined with those from Fig. 2a); Brca2^∆27/∆27, n = 3; Swsap1^−/−(+1) Brca2^∆27/∆27, n = 4; Swsap1^−/−(+1), n = 4 (combined with those from Supplementary Fig. 3a). ***P ≤ 0.001; ****P ≤ 0.0001; Fisher’s exact test, two-tailed. b, c Swsap1^−/−(+1) Brca2^∆27/∆27 spermatocytes show altered meiotic progression and aggravated synapsis defects relative to Swsap1^−/−. Percentage of spermatocytes in the indicated stages in b with representative images in c. Most Swsap1^−/−(+1) Brca2^∆27/∆27 early-zygotene (-like) cells have delayed synapsis, while is only seen in some Swsap1^−/−(+1) spermatocytes (light orange bars). By early pachynema, all double-mutant spermatocytes display incomplete homolog synapsis and/or nonhomologous synapsis. Boxes in early pachytene-like cells in c highlight non-homologous synapsis, and arrows indicate unsynapsed chromosomes. d–g RAD51 and DMC1 focus counts are further reduced in Swsap1^−/−(+1) Brca2^∆27/∆27 spermatocytes relative to Swsap1^−/−(+1). Representative chromosome spreads are shown in d, f with focus counts for the indicated stages in e, g. Control, n = 5 (RAD51, combined with 2 mice from Fig. 3e) and n = 5 (DMC1, combined with 2 mice from Fig. 2g); Swsap1^−/−(+1), n = 3 (RAD51, combined with that from Supplementary Fig. 3f) and n = 4 (DMC1, combined with that from Supplementary Fig. 3g); Brca2^∆27/∆27, n = 4 (RAD51) and n = 6 (DMC1); Swsap1^−/−(+1) Brca2^∆27/∆27, n = 3 (RAD51) and n = 4 (DMC1). h MEIOB focus counts at early zygonema (-like) are further increased in Swsap1^−/−(+1) Brca2^∆27/∆27 spermatocytes relative to Swsap1^−/−(+1), but still lower than that in Dmc1^−/−. Mice: control, n = 3 (combined with those from Fig. 3b); Swsap1^−/−(+1), n = 3 (combined with that from Supplementary Fig. 5c); Brca2^∆27/∆27, n = 2; Swsap1^−/−(+1) Brca2^∆27/∆27, n = 3; Dmc1^−/−, n = 4 (from Fig. 3b). Scale bars in c, d, f: 10 µm. Each symbol in e, g, h is the total number of foci from a single nucleus. Solid symbols in e, g, h: normal cells. Open symbols in e, g, h: abnormal cells. Error bars in e, g, h: mean ± s.d. *P ≤ 0.05; ****P ≤ 0.0001; Mann–Whitney test, one-tailed. i Functional interaction between the mouse Shu complex and the BRCA2 C terminus during meiotic HR. While SWS1-SWSAP1 is critical for stable RAD51 and DMC1 nucleoprotein filament assembly during meiosis, the BRCA2 C terminus can provide some activity in the absence of this complex