Abstract
Homology recognition and DNA-strand invasion ensure faithful homolog pairing and segregation during the first meiotic division1. RAD51 and DMC1 recombinases catalyze these steps2, with BRCA2 promoting their assembly into nuclear foci3. The recently identified human SWS1-SWSAP1 complex, related to the Shu complex in yeast, promotes RAD51 focus formation in cell lines4,5. We show here that mouse SWS1-SWSAP1 is critical for meiotic homologous recombination (HR) by promoting the assembly of RAD51 and DMC1 on early recombination intermediates. Absence of the complex perturbs meiotic progression in males and females and both sexes are sterile, although a fraction of meiocytes form crossovers. Remarkably, loss of the DNA damage checkpoint kinase CHK2 rescues fertility specifically in females without rescuing crossover numbers. Unlike the Shu complex, the BRCA2 C terminus (known to be required for RAD51 stabilization6,7) is dispensible for RAD51 and DMC1 focus formation. However, concomitant loss of the BRCA2 C terminus aggravates the meiotic defects in Shu mutant spermatocytes. These results point to a complex interplay of factors that ensure recombinase function and hence meiotic progression in the mouse.
Regulating the assembly, stabilization, and disassembly of nucleoprotein filaments of RAD51 and its meiosis-specific paralog DMC1 is critical for productive meiotic HR2. Transgenic mouse studies have shown that BRCA2, a multi-domain protein with several interaction sites for both recombinases7-9 is necessary for RAD51 and DMC1 focus formation during meiosis, such that BRCA2 loss causes meiotic arrest and sterility3. Other proteins are also expected to play a role in this process, including the canonical RAD51 paralogs, but understanding their meiotic role has been hampered by the embryonic lethality of mutants10. More recently, “Shu” complexes have been identified in several organisms; these interact with RAD51 and RAD51 paralogs and modulate RAD5111. Shu complexes are comprised of a defining member with a conserved Zn-coordinating motif and one or more members with RAD51-like structural motifs4,5,11-13. In budding yeast, mutations in any of the four subunits of the Shu complex suppress the slow growth and hydroxyurea sensitivity (“Shu”) of sgs1 and top3 mutants14, but also promote meiotic HR by stabilizing Rad51 nucleoprotein filaments and promoting inter-homolog bias13, 15, 16. Structurally diverse Shu complexes found in Caenorhabditis elegans and Schizosaccharomyces pombe also play critical roles in meiotic HR4,17, but the role of the mammalian complex in this process is unknown.
To investigate the role of the mouse Shu complex, we disrupted Sws1 (formally Zswim7) or Swsap1 in fertilized eggs using TALE nuclease pairs directed to exon 1 downstream of the translation start site (Fig. 1a,b). From the several mutations obtained (Supplementary Table 1a,b), three frame-shift alleles for Sws1 and two for Swsap1 were selected for further analysis (Fig. 1a,b). Because results are similar for all alleles, most experiments in the main text focus on one mutant for each gene, Sws1Δ1(A)/Δ1(A) and Swsap1Δ131/Δ131, hereafter Sws1-/- and Swsap1-/-, unless indicated otherwise in the figure legends. Surprisingly, unlike other RAD51 paralog knockout mice10, Sws1-/- and Swsap1-/- homozygous animals are viable, as are Sws1-/- Swsap1-/- double mutants (Supplementary Table 2a,b). RT-PCR analysis using testis cDNA derived from Sws1-/- and Swsap1-/- mice confirmed the respective frame-shift alleles (Supplementary Fig. 1a). Mutant mice show no obvious gross morphological defects and have normal body weights (Supplementary Fig. 1b, 2a). However, neither male nor female Sws1-/- and Swsap1-/- mutants are fertile (Supplementary Table 3a). Testis weights from adult single and double mutants are 3- to 4-fold smaller than in control animals, and ovary weights are reduced 3- to 8-fold (Fig. 1c,d; Supplementary Fig. 1b and 2a). Notably, testis weights from mutant juveniles obtained before meiotic arrest (7.5 days postpartum, dpp) has occurred are similar to controls (Fig. 1c).
In testis sections from adult Shu-mutant mice, seminiferous tubules have substantially reduced cellularity and are devoid of post-meiotic germ cells (Fig. 1e and Supplementary Fig. 1c). Spermatocytes appear to arrest during mid-pachynema, possibly at stage IV of the seminiferous epithelial cycle18. TdT-mediated dUTP nick end-labeling (TUNEL) demonstrates widespread apoptosis (Supplementary Fig. 2b). Apoptosis in mutant juvenile testes at 7.5 dpp is rarely observed, as in controls, suggesting that pre-meiotic cells are not affected (Fig. 1c and Supplementary Fig. 2c). Adult ovary sections from mutants lack follicles at any developmental stage (Fig. 1f and Supplementary Fig. 2d). At 3 dpp, ovaries stained for c-Kit, a marker for diplotene and dictyate stage oocytes in primordial and primary follicles, have significantly reduced oocyte numbers in Sws1-/- and Swsap1-/- mice, with some oocytes appearing to be apoptotic (Supplementary Fig. 2e). Together, our data demonstrate that SWS1 and SWSAP1 are essential for meiotic progression in both male and female mice.
Testes and ovaries from Shu-mutant mice resemble those of HR- and synapsis-defective mutants, such as Dmc1-/- and Sycp1-/- (Ref.19-22). To test if the Shu complex is required for HR and/or synapsis, we analyzed the synaptonemal complex (SC), a tripartite proteinaceous structure that forms between the homolog axes as they pair, by immunostaining surface-spread spermatocytes for the SC central region (SYCP1) and axial/lateral elements (SYCP3)1. Spermatocytes were also stained for the testis-specific histone H1 variant (H1t), which specifically labels cells at mid-pachynema and beyond23. H1t-positive spermatocytes are significantly reduced in mutant testes, indicating an early-pachytene arrest that is bypassed in only a fraction of cells (Fig. 2a,b and Supplementary Fig. 3a,b). The ability of some cells to progress contrasts with Dmc1-/-, in which H1t-positive cells are absent24. Unlike later stages, early meiotic prophase cells at leptonema and zygonema are increased in Shu single- and doublemutant mice. Synaptic abnormalities begin to be observed at early zygonema, such that chromosomes are seen with long axes but no synapsis, which becomes more pronounced by late zygonema, where chromosomes with fully formed axes are found with little or no synapsis (“early- and late zygonema-like”, respectively; Fig. 2b,c and Supplementary Fig. 3b,d). In contrast to wild-type pachytene cells in which all of the homologs are typically synapsed, the majority of mutant cells are abnormal at this stage, displaying unsynapsed or partially synapsed chromosomes and frequent synapsis between non-homologous chromosomes (“pachynemalike”; Fig. 2b,c and Supplementary Fig. 3b,d,e). While severe, the synaptic defects are not as profound as reported for Dmc1-/- mutants19,20. Synaptic defects are also observed in the sex chromosomes, with less than half of mutant spermatocytes at early pachynema having synapsed XY pairs (Supplementary Fig. 3c). Mutant cells with autosomal synapsis defects are more likely to also have unsynapsed XY pairs, whereas those with full autosomal synapsis typically have synapsed XY pairs. The few cells that reach mid-pachynema tend to have fewer synaptic abnormalities (Fig. 2b and Supplementary Fig. 3b,d).
Synapsis defects in Shu-mutant spermatocytes could reflect meiotic HR defects, as the human Shu complex promotes HR in cultured cells5. Indeed, Sws1-/- and Swsap1-/- spermatocytes display an ~2-fold reduction in RAD51 and DMC1 focus numbers at leptonema (Fig. 2d-g and Supplementary Fig. 3f,g). RAD51 and DMC1 focus numbers increase substantially by early zygonema in control cells, but remain low in mutant cells. At later stages, focus numbers progressively decrease in all genotypes, including the mutants. Shu double-mutant spermatocytes have similarly reduced RAD51 and DMC1 focus numbers at all stages (Supplementary Fig. 3f,g).
Sterility can occur in mutants where a similar reduction in RAD51 and DMC1 foci is attributable to fewer DSBs25. To rule out effects of Shu complex loss on DSB formation and/or their resection, we quantified chromatin-bound γH2AX26, a marker for DSBs, and foci of MEIOB, a meiosis-specific, single-stranded DNA (ssDNA)-binding protein27,28. At leptonema and early zygonema, γH2AX levels are indistinguishable from controls (Supplementary Fig. 4a,b), suggesting that DSB formation is unaffected. Further, there are more MEIOB foci at these stages in Shu mutant spermatocytes (2.4-fold at leptonema and 1.2-fold at early zygonema; Fig. 3a,b and Supplementary Fig. 4c), indicating an increase in the number of end-resected intermediates that are unable to stably assemble RAD51 and DMC1. Notably, the increase in MEIOB foci is not as great as in Dmc1-/- spermatocytes (3.3- and 1.8-fold, respectively). We interpret these findings to indicate that DSBs are formed in normal numbers and are resected, to be initially bound by MEIOB, in Sws1-/- and Swsap1-/- mutants, but that the mouse Shu complex fosters the stable assembly of RAD51 and DMC1 nucleoprotein filaments during meiotic HR, which in turn promotes homolog synapsis.
Given the early meiotic prophase I defects in Shu-mutant spermatocytes, we expected that HR is impaired later as well. Consistent with defects in DSB repair, mutant spermatocytes at early pachynema display γH2AX on autosomes, which is even more evident in early pachytenelike cells with synapsis defects (Supplementary Fig. 4d). γH2AX mostly disappears from autosomes in control cells and remains concentrated in the unsynapsed XY chromatin forming the sex body26. In contrast, autosomal γH2AX in Shu-mutant cells is accompanied by defects in sex body formation/maturation, especially in those cells with a high degree of autosome asynapsis (Supplementary Fig. 4d,e). Interestingly, the rare mid-pachytene Shu-mutant spermatocytes display less autosomal γH2AX than those at early pachynema and occasionally mature sex bodies (Supplementary Fig. 4f), suggesting that some mutant cells do not trigger the pachytene checkpoint due to greater proficiency in DSB repair. However, γH2AX remnants are still observed in these Sws1-/- and Swsap1-/- “escapers”, in agreement with evidence suggesting that the pachytene checkpoint tolerates some unrepaired DSBs29.
Because a small fraction of Shu-mutant spermatocytes are apparently repair-proficient and progress to mid-pachynema, we asked whether mutant cells could form later HR intermediates. MSH4 stabilizes DNA-strand exchange intermediates, some of which will become crossovers, whereas MLH1 specifically marks crossovers1. Mutant spermatocytes have 2- to 3fold fewer MSH4 foci from early zygonema to early pachynema, proportional to the earlier reduction in the RAD51 and DMC1 foci (Fig. 3c,d). Remarkably, however, MLH1 foci are reduced on average only ~20% in mid-pachytene cells, and the majority of bivalents have at least one MLH1 focus even though most cells have one or more chromosome pairs lacking a focus (Fig. 3e-g). Considering the number of MLH1 foci, it is striking that none of the mutant cells at early pachynema have MSH4 focus numbers within one standard deviation of the mean in control cells (Fig. 4c). These results suggest that crossover homeostasis30,31 operates in the Shu mutants.
As Shu-mutant females are sterile, we also tested for evidence of HR defects in oocytes from embryonic day 18.5, when most have entered pachynema (Supplementary Fig. 4g). MLH1 foci are present in Swsap1-/- oocytes at mid-pachynema, but fewer as in spermatocytes (Fig. 3h,i). Furthermore, most mutant oocytes have ≥1 chromosome pair that is not synapsed and/or lacks an MLH1 focus (Supplementary Fig. 4h). Thus, the Shu complex is essential for crossover formation in both male and female meiosis.
Although some mutant oocytes form normal numbers of MLH1 foci, oocytes are mostly eliminated within a few days after birth (Supplementary Fig. 2e), resembling other DNA repair-defective mutants like Dmc1-/- (Ref.22). Oocyte loss in Dmc1-/- mice at 3 weeks can be partially rescued by eliminating the DNA damage checkpoint kinase CHK2, although these mice still lack primordial follicles and most oocytes are depleted in adult Dmc1-/- Chk2-/- females (2-month-old)32. By contrast, Swsap1-/- Chk2-/- adults show a complete rescue of ovary size (Fig. 4a,b and Supplementary Fig. 5a). Double mutant ovaries contain primordial follicles, unlike ovaries from Swsap1-/- mice, although the rescue is incomplete (Fig. 4c,d). Loss of CHK2, however, does not alleviate the synapsis defects (open circles; Fig. 4e) or improve MLH1 focus numbers (Fig. 4e and Supplementary Fig. 5b). Nonetheless, Swsap1-/- Chk2-/- mutant females produce viable, fertile offspring, although with about half the litter size of controls (Supplementary Table 3b).
This rescue is remarkable, given the absence of MLH1 foci on one or more chromosomes (Supplementary Fig. 5b). CHK2 loss also suppresses infertility in females with a hypomorphic Trip13 mutation32, although in this mutant MLH1 focus numbers in oocytes are inferred to be nearly normal (as reported for spermatocytes33,34). It would be interesting to determine whether viable pups from Swsap1-/- Chk2-/- dams arise from oocytes with normal MLH1 focus counts or from fortuitous segregation of non-recombinant chromosomes35.
Unlike females, Swsap1-/- Chk2-/- males exhibit only minimal rescue, with only marginally larger testes. Some tubules exhibit greater cellularity, H1t-positive spermatocytes are increased in number, and round and elongated spermatids are occasionally observed (Fig. 4a,f,g and Supplementary Fig. 5c). Nonetheless, tubules still mostly lack the full complement of germ cells (Fig. 4g) and mice remain infertile (Supplementary Table 3b). The minimal rescue by CHK2 loss in males could reflect a DSB-independent arrest tied to sex-body defects29.
Because some chromosomes in Shu-mutant meiocytes synapse and some cells progress to have MLH1 foci, we reasoned that another mediator protein(s) promotes some level of DSB repair in the absence of the Shu complex. One obvious candidate is BRCA2. Loss of BRCA2 abolishes assembly of both RAD51 and DMC1 into foci, such that spermatocytes do not progress past early pachynema3. Although expression of BRCA2 lacking the C-terminal domain in Brca2Δ27/Δ27 mice36 causes an HR defect in somatic cells37, there is little discernible effect on RAD51 and DMC1 foci in early meiotic cells and cells progress to later prophase I stages (Fig. 5a-g). This truncated BRCA2 protein lacks C-terminal RAD51 and DMC1 interaction sites6, 7, 9, but contains several other RAD51 and DMC1 interaction sites which can promote their assembly into foci8,10 (Fig. 5d-g). Brca2Δ27/Δ27 mice are fertile, although testes are smaller (Supplementary Fig. 5d), likely due to late meiotic prophase defects (C.M.A. and M.J., unpublished results).
We asked whether the BRCA2 C terminus plays a role in the absence of the Shu complex to support RAD51 and DMC1 foci and thus inter-homolog repair. Indeed, loss of the BRCA2 C terminus aggravates the homolog synapsis defects in Swsap1-/- mice, and H1t-positive cells are absent from Swsap1-/- Brca2Δ27/Δ27 mice, indicating a fully penetrant meiotic arrest (Fig. 5a-c). Testis weights are also slightly reduced (Supplementary Fig. 5d). Unlike Swsap1-/- spermatocytes, most double mutant cells at early zygonema show delayed synapsis, and all cells at early pachynema display asynapsis and/or nonhomologous synapsis, with fewer fully synapsed chromosomes (Fig. 5b,c and Supplementary Fig. 5e). Importantly, Swsap1-/- Brca2Δ27/Δ27 spermatocytes show even fewer foci at leptonema and early zygonema for both RAD51 (2.4 and 1.6-fold reduction, respectively) and DMC1 (1.1 and 1.6-fold reduction, respectively) compared with Swsap1-/- (Fig. 5d-g). There is a concomitant further increase in MEIOB foci (1.2-fold; Fig. 5h), although interestingly, MEIOB foci are still fewer than in Dmc1-/- spermatocytes. We conclude that, although the BRCA2 C terminus is largely dispensible for meiotic HR in the presence of SWSAP1, it functions in the absence of the Shu complex to support stable assembly of RAD51 and DMC1 on resected DNA ends. However, it is clearly not sufficient to overcome the loss of the Shu complex at most DSBs.
Strand invasion by RAD51 and DMC1 is a central step in meiotic HR. Our studies reveal a key requirement for the mouse Shu complex in stable assembly of sufficient numbers of both RAD51 and DMC1 nucleoprotein filaments to promote homolog pairing during meiosis. As a result, resected DNA ends accumulate in the Sws1-/- and Swsap1-/- mutants, although not to the level seen when DMC1 itself is absent. As in mouse, Rad51 focus formation is severely reduced in budding yeast Shu mutants during meiosis; however, our observations contrast with those from yeast, where Dmc1 focus formation is relatively unaffected13. Other yeast mediator proteins likely have Dmc1-specialized roles, in particular the Mei5-Sae3 complex2. Because of the embryonic lethality associated with mutation of canonical RAD51 paralogs10, it is unclear if or how these proteins contribute to mammalian meiosis. One hint comes from mice expressing a hypomorphic Rad51c allele, as prophase spermatocytes from these mice show reduced RAD51 focus formation, although DMC1 was not examined38. Thus, as in mitotic cells, multiple protein complexes are likely needed to promote recombinase activity in meiotic cells, although how they functionally interact remains to be elucidated.
We envision the mouse Shu complex stabilizing both RAD51 and DMC1 nucleoprotein filaments (Fig. 5i), and possibly remodeling them, as reported for Rad51 by Shu complexes and canonical RAD51 paralogs in other organisms13, 39, 40. By contrast, the primary role of BRCA2 is nucleation of RAD51 and DMC1 nucleoprotein filaments, a role ascribed to the BRC repeats in the center of the protein8,10. However, the BRCA2 C terminus has also been implicated in RAD51 filament stabilization by selectively binding to the interface between two RAD51 protomers6,7. It will be interesting to determine if the BRCA2 C terminus also promotes DMC1 filament stabilization. Thus, while the Shu complex and the BRCA2 C terminus have overlapping biochemical roles during meiotic HR, the Shu complex is clearly more critical given the infertility of Shu mutant mice.
Online Methods
Mouse care
The care and use of mice were performed in accordance with the Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee guidelines.
Generation and genotyping of Shu mutant mice
We targeted Sws1 and Swsap1 with TALE nucleases directed to each gene’s first exon, close to the translational start sites. TALEN pairs (RNA) were injected into fertilized mouse eggs, derived from superovulated CBA/J x C57BL/6J F1 females mated with C57BL/6J males, which were then implanted into pseudo-pregnant females41,42. To initially genotype founder mice, at least 10 cloned PCR products from each of 22 Sws1 and 10 Swsap1 founders were sequenced. Founders were backcrossed to C57BL/6J to separate multiple alleles and then further backcrossed for 3-6 additional generations prior to generating experimental mice.
Genotyping for Sws1 was done by PCR-sequencing using the following PCR primers: Sws1-A: 5’-CCTGCAGGGCGCGTGAAGTTC, Sws1-B: 5’-ACCGGCTCGCACTCAGGGATC under the following conditions: 94 °C, 3 min; 35 cycles of 94 °C, 30 sec; 55 °C, 1 min and 65 °C, 30 sec; and a final extension of 72 °C, 5 min. The PCR product (259 bp) was sequenced using Sws1-A primer and sequencing reads were aligned against the wild-type controls to detect the 1-bp deletion.
Genotyping for Swsap1 was done using the following PCR primers Swsap1-C: 5’-TCTGTGAACTATAGCCAATGAGGC, and Swsap1-D: 5’-AACTGTCACTCAGGCGCGAACTAG under the following PCR conditions: 94 °C, 3 min; 35 cycles of 94 °C, 30 sec; 55 °C, 1 min and 65 °C, 30 sec; and a final extension of 72 °C, 5 min. The Swsap1(+1) allele was genotyped by PCR-sequencing using the Swsap1-C primer; the Swsap1Δ1A allele was genotyped by running PCR products on a 2.4% agarose gel. The wild-type product is 396 bp and the mutant is 265 bp.
Chk2 (Ref.43,44) and Brca2Δ27 (Ref.36,37) mice and genotyping were previously described.
RT-PCR
Twenty milligrams of mouse tissue was incubated with 1 ml Triazol and homogenized with a Dounce homogenizer. The extract was transferred to Eppendorf tubes and incubated for 5 min at room temperature. Extracts were centrifuged at 12,000 g for 10 min at 4 °C. Supernatants were transferred to another Eppendorf tube and RNA was extracted using chloroform followed by isopropanol precipitation. The RNA pellet was dissolved in H2O. To prepare the cDNA library, Superscript one-step RT-PCR kit was used (Invitrogen). To amplify cDNA for Sws1 and Swsap1, the following primers were used: Sws1-RT-A: 5’-AAGTTCGCAGCGCCCGGG, Sws1- RT-B: 5’-CTAGGCTTCTGTCTTTGAAGTCC, Swsap1-RT-A: 5’-ATGGCGGAGGCGCTGAGG, Swsap1-RT-B: 5’-TCAGGTCTTTGAATCTGCACCTG. The following conditions were used for PCR: 94 °C, 2 min; 30 cycles of 94 °C, 1 min; 65 °C, 1 min and 72 °C, 1 min; and a final extension of 72 °C, 10 min. PCR products were separated on 1.0% agarose gels, excised, and DNA was purified and sequenced. The Sws1-RT-A and Swsap1-RT-A primers were used for sequencing.
Histology
Ovaries and testes were dissected from animals at the stated ages and fixed in Bouin’s and stained with PAS, fixed in 4% PFA and stained with H&E, or fixed in 4% PFA and stained with hematoxylin and antibodies against DDX4/Vasa (Abcam, ab13840; 2.5 μg/ml) or c-Kit (Cell Signaling, 3074; 0.75 μg/ml) or were TUNEL-stained (Roche, 03333566001 and 11093070910). Staging of PAS- or H&E-stained testes sections was performed as described18. For follicle counts, ovaries were serially sectioned at 6 μm thickness, and follicles were counted in every fifth section, without further correction. The results were from one ovary from each animal.
Spermatocyte chromosome spreads and immunofluorescence microscopy
Testes were collected from 2-4 month-old mice and spermatocytes were prepared for surface spreading and processed using established methods for immunofluorescence45, using the following primary antibodies in dilution buffer (0.2% BSA, 0.2% fish gelatin, 0.05% Triton X-100, 1xPBS), with incubation overnight at 4 °C: mouse anti-SYCP3 (Santa Cruz Biotechnology, sc-74569; 1:200), rabbit anti-SYCP3 (Abcam, ab15093; 1:500), goat anti-SYCP3 (Santa Cruz Biotechnology, sc-20845; 1:200), rabbit anti-SYCP1 (Novus, NB-300-229; 1:200), mouse anti-γH2AX (Millipore, 05-636; 1:500), rabbit anti-RAD51 (Calbiochem, PC130; 1:200), rabbit anti-DMC1 (Santa Cruz Biotechnology, sc-22768; 1:200), rabbit anti-MEIOB (kindly provided by P.J. Wang, University of Pennsylvania; 1:200), rat anti-RPA2 (Cell Signaling Technology, 2208S; 1:100), rabbit anti-MSH4 (Abcam, ab58666; 1:100), mouse anti-MLH1 (BD Biosciences, 51-1327GR; 1:50) and guinea pig anti-H1t (kindly provided by M.A. Handel, Jackson Laboratory; 1:500). Slides were subsequently incubated with the following secondary antibodies at 1:200 to 1:500 dilution for 1h at 37 °C: 488 donkey anti-mouse (Life Technologies, A21202), 488 donkey anti-rabbit (Life Technologies, A21206), 488 goat anti-rat (Life Technologies, A11006), A568 goat anti-mouse (Molecular probes, A-11019), 568 goat antirabbit (Life Technologies, A11011), 594 donkey anti-mouse (Invitrogen, A21203), 594 goat anti-rabbit (Invitrogen, A11012), donkey 594 anti-goat (Invitrogen, A11058), 647 donkey antimouse (Life Technologies, A31571), 647 donkey anti-rabbit (Invitrogen, A31573), 647 goat anti-guinea pig (Life Technologies, A21450). Cover slips were mounted with ProLong Gold antifade reagent with or without DAPI (Invitrogen, P36935 and P36934, respectively). Immunolabeled chromosome spread nuclei were imaged on a Marianas Workstation (Intelligent Imaging Innovations; Zeiss Axio Observer inverted epifluorescent microscope with a complementary metal-oxide semiconductor camera) using 100× oil-immersion objective. Images were processed using Image J for foci analysis and Photoshop (Adobe) to make the figures.
Spermatocytes were staged by assessing the extent of SYCP3 staining and synapsis (based on SYCP1 staining for Fig. 2, 5 and Supplementary Fig. 3). Only foci colocalizing with the chromosome axis were counted. In controls, leptotene cells are characterized by the presence of small stretches of SYCP3 and no SYCP1 staining. At early zygonema, homolog synapsis initiates, marked by the presence of SYCP1, and longer SYCP3 stretches are visible as chromosome axes continue to elongate. By late zygonema, chromosome axis formation completes at the same time that SYCP1 appears between homologs (>50% of overall synapsis). At pachynema, homologs are fully synapsed (co-localization of SYCP3 and SYCP1), except in the non-pseudoautosomal region of the XY chromosomes. Due to chromatin condensation at this stage, pachytene chromosome axes are shorter and thicker. H1t staining is used whenever possible to distinguish mid-/late-pachytene from early-pachytene cells. Late pachytene cells are further characterized by thickening of chromosome ends and elongation/curling of XY chromosomes. In diplotene cells, chromosome desynapsis ensues. Sws1-/- and Swsap1-/- cells at leptonema are indistinguishable from controls. Abnormal cells in mutants are characterized as follows: Early zygotene-like cells have chromosomes with long axes (SYCP3) and no SYCP1 stretches. Late zygotene-like cells have chromosomes with fully formed axes (SYCP3) and little or no synapsis in addition to elongated chromosomes completing synapsis as in controls. Early pachytene-like cells have unsynapsed chromosomes and/or incompletely synapsed homologs as well as non-homologous synapsis, but also display fully synapsed autosomes with thicker and shorter SYCP3 axes indicative of chromatin condensation characteristic of this stage. Midpachytene-like cells, which are H1t-positive, also display synaptic abnormalities and may contain chromosome fragments. Cells displaying normal homolog synapsis but chromosome end-to-end fusions are considered normal cells in control and mutants.
Oocyte chromosome spreads
Prenatal ovaries were collected at embryonic day 18.5 and processed to obtain oocyte spreads as described46 with some modifications. Briefly, ovaries were placed in a 1.5 ml Eppendorf tube containing 0.7 ml isolation medium (TIM: 104 mM NaCl, 45 mM KCl, 1.2 mM MgSO4, 0.6 mM KH2PO4, 0.1% (w/v) glucose, 6 mM sodium lactate, 1 mM sodium pyruvate, pH 7.3, filter sterilized) and fragmented by pipetting up and down several times. After centrifuging for 3 min at 400 g and discarding the supernatant, 0.5 ml of 1 mg/ml collagenase (Sigma, C0130) in TIM was added to the ovarian fragments and tubes were incubated at 37 °C for 1 h with gentle shaking. Next, careful pipetting up and down was performed until large fragments of ovarian tissue were no longer visible. Following centrifugation for 3 min at 400 g, the supernatant was discarded, the pellet was carefully resuspended in 0.5 ml 0.05% trypsin (Sigma, T9935), and tubes were incubated at 37 °C for 5 min with gentle shaking. Then 0.5 ml DMEM containing 10% (v/v) FBS was added, carefully pippeted up and down, and centrifuged for 3 min at 400 g. The supernatant was discarded followed by the addition of 1 ml TIM to resuspend the cells by pipetting. Cells were again centrifuged for 3 min at 400 g, and after discarding the supernantant, cells were resuspended in 0.5 ml hypotonic solution (30 mM Tris-HCl pH 8.2, 50 mM sucrose, 17 mM Na-Citrate, 5 mM EDTA, 1× protease inhibitors), and incubated for 30 min to 1 h at room temperature. Positively charged, precleaned glass slides were placed in a humid chamber and a circle of ~1.0-1.5 cm diameter was marked in the center of the slide with a hydrophobic barrier pen; 40 μl of 1% (w/v) PFA containing 0.1% (v/v) Triton X-100 was placed into each circle and 10 μl cell suspension was slowly dropped on each slide within the PFA solution. Slides were slowly dried in a moist chamber that was closed for 2 h, then ajar for 30 min, and then open for 30 min. Slides were rinsed two times with milliQ H2O, and one time with 1:250 Photo-Flo 200 (Kodak, 1464510) solution. Slides were air-dried and stored at -80 °C. Staining of slides was then performed as for the spermatocyte chromosome spreads described above.
Statistical analysis
Statistical analyses were performed using a Chi square test for animal breeding, a nonparametric two-tailed Mann-Whitney test for pup analysis, a two-tailed Student’s t-test for testis, ovary, and body weight comparisons, a two-tailed Fisher’s test for H1t cell analysis, a nonparametric one-tailed Mann-Whitney test for foci number comparisons, as a normal distribution could not be assumed, and an ANOVA test (Kruskal Wallis) for primordial follicle counts, due to the lack of follicles in the mutant. Error bars, mean±s.d.; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
Author’s contributions
C.M.A., R.P., P.J.R., S.K., and M.J. designed experiments. C.M.A., R.P., and P.J.R. performed experiments. I.R., S.K., and M.J. supervised the research. C.M.A., R.P., S.K., and M.J. wrote the paper with input from I.R.
Competing financial interests
The authors declare no competing financial interests.
Acknowledgements
We thank Mary Ann Handel and P. Jeremy Wang for antibodies, Kara Bernstein (University of Pittsburgh) and members of the Jasin and Keeney labs for discussions and critical reading of the manuscript, and Katia Manova and members of the MSKCC Molecular Cytology core facility for technical help. This work was supported by MSK Cancer Center Support Grant/Core Grant (NIH P30CA008748), NIH F32GM110978 (R.P.), BFU2016-80370-P (I.R.), R35 GM118092 (S.K.), R35 GM118175 (M.J.), and R01CA185660 (M.J.).