Mcmdc2 is preferentially expressed in the gonads and required for fertility in both sexes

To address if MCMDC2 could play a role in meiotic recombination we asked if Mcmdc2 transcripts are present in testis. Thus, we used RT-PCR to assess expression levels of Mcmdc2 in 17 somatic tissues and testes of mice (Fig 1a). The RT-PCR analysis indicated that Mcmdc2 transcripts were indeed enriched in testis as compared to somatic tissues (Fig 1a). Furthermore, analysis of public databases (http://www.germonline.org/Homo_sapiens/geneview?gene=ENSG00000178460) [49] showed that human Mcmdc2 was preferentially expressed in the testis, particularly in spermatocytes. Thus, its expression suggested a role for Mcmdc2 in meiosis. Given that the Drosophila homologs of MCMDC2 are required for crossover formation [50–52], we speculated that mammalian MCMDC2 may also function in meiotic recombination. To test this hypothesis, we attempted to generate antibodies against distinct fragments of mouse MCMDC2 both in rabbit and guinea pig, however none of our antibodies reliably detected MCMDC2, which precluded localization studies of MCMDC2. To directly test the biological functions of MCMDC2 we generated mice where Mcmdc2 was disrupted after the 4^th exon (Fig 1b–1d). The targeting strategy was designed to terminate the 681-amino acid-long MCMDC2 protein after the 95th amino acid (Fig 1b). This was due to the combined effects of the removal of the 5-7^th exons (encodes 96–237 amino acids of MCMDC2) causing a frameshift, and the insertion of a strong ectopic splice acceptor site and a transcriptional terminator into the 4^th intron. RT- PCR analysis confirmed strongly reduced expression of Mcmdc2 exons beyond the 4^th exon (including exon 8–11, which are not deleted from the genome) in testes of the Mcmdc2^-/- mice (Fig 1d). Even transcripts of exon 3–4, which are upstream of the deletion, were detected at a lower level in Mcmdc2^-/- testes than in wild-type testes. MCMDC2 protein fragments that may be produced from these residual transcripts are unlikely to be functional. This is because deletion of the 5-7^th exons would allow only a short 95 amino acid N-terminal fragment to be produced from exons 1–4. Even if alternative splicing generated rare transcripts where sequences from the 4^th exon were linked to sequences downstream of the deleted 5-7^th exons, protein products of these transcripts would lack most parts of MCMDC2, including the entire conserved MCM-like region (SMART: SM00350, amino acids 177–623 of MCMDC2), because these transcripts would be subject to a frameshift mutation.

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Fig 1. Preferential expression of Mcmdc2 in the gonads, and Mcmdc2 targeting in mice.

(a) Expression of Mcmdc2 and a “house-keeping” gene (S9) in testis and a somatic tissue mix measured by RT-PCR. cDNAs were prepared from four RNA mixtures: (1) Equal amounts of RNAs from 17 somatic tissues (see Materials and Methods for the tissue list) were mixed and 1μg of the resulting mixture was used for RT (17 somatic tissues). (2) Mixture “1” supplemented with testis RNA at a concentration equal to that of the individual somatic RNAs (17 somatic tissues + 1x testis). (3) Mixture “1” supplemented with testis RNA at a concentration equal to five times that of the individual somatic RNAs (17 somatic tissues + 5 x testis) (4) Mixture “3” with no RT (17 somatic tissues + 5xtestis noRT). Mcmdc2-specific PCR-products were amplified preferentially from templates that contained testis cDNA. (b) Mcmdc2 targeting strategy. Schematics of the targeting construct, the wild-type (WT) and the modified Mcmdc2 genomic locus. Black boxes represent exons (not to scale). Recombination at the homology arms (HA) of the targeting construct modifies intron 4 by introducing: 1) an additional exon (SA-IRES-LacZ) that contains a strong splice acceptor site (SA) and poly-adenylation site (left grey box), 2) a transcriptional unit that contains the strong housekeeping human ß-Actin promotor (hBactP) driving the neomycin (Neo) resistance gene as a selection marker. This modification of intron 4 also disrupts the Mcmdc2 open reading frame after the 95th codon (Mcmdc2^insertion allele). Recombination catalyzed by FLPe at FRT sites removes the SA-IRES-LacZ exon and the hBactP-Neo gene, and restores the MCMDC2 ORF (Mcmdc2^restored). Mcmdc2^restored is a functional allele that can be disrupted by Cre-mediated recombination between loxP sites (Mcmdc2^deletion). Excision of exon 5–7 causes a frameshift after the 80th codon. Cre-mediated recombination between loxP sites of a Mcmdc2^insertion allele results in Mcmdc2^{insertion-deletion} allele. The positions of PCR-genotyping primers are indicated. Red bars mark the 3`and the internal Southern blot probes; the predicted length of restriction fragments is indicated. (c) Southern blot of DNA from wild-type (+/+) and targeted Mcmdc2^+/insertion (+/i) embryonic stem cell clones (C6 and F7) that were used to derive two independent mouse lines. DNA was digested with Eco31I and hybridized with an internal probe for LacZ (left panel), or DNA was digested with BclI and hybridized with a 3’ probe (right panel). The blots indicate a single integration of the targeting cassette in the Mcmdc2 locus. (d) RT-PCR was used to detect Mcmdc2 and "house-keeping" Rps9 (S9) transcripts in testes of wild-type and Mcmdc2^-/- (insertion-deletion) mice. Oligo-pairs specific to Mcmdc2 exon 3 and 4, 5 and 6, 6 and 7, 8 and 9, or 10 and 11 were used.

https://doi.org/10.1371/journal.pgen.1006393.g001

Mcmdc2^-/- mice were viable and did not show any obvious somatic defects. Although the previously published Mcm8^-/- and Mcm9^-/- mice were viable, Mcm8^-/- and Mcm9^-/- mouse embryonic fibroblasts (MEFs) displayed slow growth and sensitivity to the DNA replication inhibitor aphidicolin [45]. These phenotypes were attributed to the functions of MCM8 and MCM9 in mitotic homologous recombination. To test if MCMDC2 had a defect in mitotic cell cycle due to a possible function in mitotic recombination we established MEFs from Mcmdc2^-/- and Mcmdc2^+/+ litter-mate embryos. MEFs of Mcmdc2^-/- and wild-type mice did not differ significantly in their growth rate or in their sensitivity to aphidicolin (Fig 2a and 2b). This suggests that unlike MCM8 and MCM9, MCMDC2 does not play an important role during mitotic growth.

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Fig 2. Mcmdc2^-/- mice are deficient in germ cells from late meiotic prophase onwards in both sexes.

(a, b) Growth curves of five (a) or three (b) independent lines of Mcmdc2^+/+ (+/+) and Mcmdc2^-/- mouse embryonic fibroblasts. Cells were grown either without aphidicolin treatment (a) or with aphidicolin treatment for the first 24 hours (b), where 1μM aphidicolin was added at day 0. (a, b) Cell numbers were determined at the indicated time points in three technical replicates of each fibroblast line. Means and standard deviations of the medians of technical triplicates are shown. Growth curves of Mcmdc2^+/+ and Mcmdc2^-/- mouse embryonic fibroblasts are not significantly different (a: p = 0.8201, b: p = 0.9932, two-way ANOVA test). (c) Images of Mcmdc2^+/+ (+/+) and Mcmdc2^-/- (-/-) testes (upper panel) and ovaries (lower panel). Scale bars; 500μm. (d) Cryosections of testes from adult Mcmdc2^+/+ and Mcmdc2^-/- mice. DNA was detected by DAPI, histone H1T (marker of spermatocytes after mid-pachytene) and nuclear cleaved PARP1 (marker of apoptotic cells) were detected by immunostaining. Outlines of testis tubules are marked by dashed lines. The upper panels of d show stage V-VI and VII-VIII wild-type testis tubules, which contain several layers of germ cells at distinct spermatogenic stages: Sertoli cells (Se), spermatogonia B (SgB, stage V-VI), preleptotene (pl, stage VII-VIII), mid-pachytene (pa, stage V-VI), late-pachytene (pa, stage VII-VIII) spermatocytes, post-meiotic spermatids (sd) and spermatozoa (sp). Lower panels of d show that Mcmdc2^-/- meiocytes underwent apoptosis at a stage corresponding to wild-type mid-pachytene in stage IV tubules. Consequently, spermatocytes were not found in the inner layers of testis tubules beyond stage IV, and post-meiotic spermatids and spermatozoa were also missing from Mcmdc2^-/- testes. To illustrate this, stage IV, V-VI and VII-VIII tubules of Mcmdc2^-/- mice are shown. Apoptotic (ap) and non-apoptotic early-mid pachytene (pa) spermatocytes are shown in the stage IV tubule, which was identified by the presence of mitotic intermediate spermatogonia (m) and intermediate spermatogonia (Int). Stage V-VI and VII-VIII tubules contain somatic Sertoli cells (Se) and spermatogonia B (SgB) or preleptotene (pl) spermatocytes, respectively, but more advanced spermatogenic cells are missing. Due to elimination at mid-pachytene, histone H1T positive cells are missing from Mcmdc2^-/- testis tubules. (e) NOBOX (oocyte marker) was detected by immunofluorescence on cryosections of ovaries from 6-week-old mice. DNA was stained by DAPI. Oocytes in primordial (pd) and secondary (s) follicles are shown in the section of a wild-type ovary. In contrast, oocytes are not detected in the shown Mcmdc2^-/- ovary section. (d, e) Scale bars; 50μm.

https://doi.org/10.1371/journal.pgen.1006393.g002

Importantly, while we observed no obvious somatic defects, both sexes of Mcmdc2^-/- mice were infertile (no pups after 113 breeding weeks, n = 3 males and n = 3 females) because both oogenesis and spermatogenesis were blocked (Fig 2c–2e). Ovaries of 6 weeks old Mcmdc2^-/- females were atrophic, barely discernible, and completely devoid of oocytes (n = 3 mice, Fig 2c and 2e). This was due to an apparent loss of oocytes perinatally or soon after birth, as oocytes were still present in ovaries of fetal and newborn Mcmdc2^-/- mice. Spermatogenesis takes place within testis tubules, which can be found at 12 distinct stages of the seminiferous epithelial cycle (stages I-XII). Each stage is identified by the distinct combinations of spermatogenic cells found within [53]. We observed no late prophase spermatocytes and postmeiotic cells in Mcmdc2^-/- males. This was due to apoptosis of spermatocytes in epithelial cycle stage IV testis tubules (Fig 2d); in the wild type, stage IV tubules contain spermatocytes at the mid pachytene stage. Consistent with a complete elimination of spermatocytes at stage IV no histone H1T (a late prophase marker [54]) positive cells were found in Mcmdc2^-/- testis tubules (n>200 tubules, Fig 2d). In contrast, histone H1T was increasingly expressed in wild-type spermatocytes beyond stage IV as expected.

Mcmdc2 is required for efficient synaptonemal complex formation

Elimination of meiocytes at the observed stages indicated a possible defect in meiotic recombination in Mcmdc2^-/- mice. Specifically, persistent asynapsis and failure in DNA break repair is known to elicit elimination of spermatocytes and oocytes in stage IV testis tubules and perinatal ovaries, respectively [55–58]. Therefore we examined synaptonemal complex formation and markers of meiotic recombination in Mcmdc2^-/- meiocytes. While chromosome axes readily formed (as judged by SYCP3 staining), synaptonemal complex formation was severely defective in Mcmdc2^-/- meiocytes (as shown by disrupted SYCP1 localization along chromosome axes) (Fig 3). In the most advanced stages full axes formed along the core of each chromosome in Mcmdc2^-/- spermatocytes. Fully assembled chromosome axes can be observed from late zygotene to diplotene in wild-type spermatocytes. Given that Mcmdc2 ^-/- spermatocytes were eliminated at a stage equivalent to wild-type mid-pachytene we postulate that Mcmdc2^-/- spermatocytes with fully formed axes reached a prophase stage equivalent to wild-type late zygotene to mid pachytene, hence we refer to this stage in Mcmdc2 ^-/- spermatocytes as late zygotene-pachytene.

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Fig 3. Synaptonemal complex formation is defective in Mcmdc2^-/- mice.

(a, c) SYCP3 (axis marker) and SYCP1 (synaptonemal complex marker) were detected by immunofluorescence on nuclear surface spreads of Mcmdc2^+/+ pachytene and Mcmdc2^-/- zygotene-pachytene spermatocytes (a) and oocytes (c). Two distinct categories of Mcmdc2^-/- zygotene-pachytene meiocytes were found. They either had no synapsis with no or weak punctate SYCP1 along unsynapsed axes (middle rows, a and c), or stretches of SYCP1 formed between some chromosomes that have managed to synapse (bottom panels, a and c). The spermatocyte shown in the bottom panels of a illustrates the maximum extent of synaptonemal complex formation observed in Mcmdc2^-/- spermatocytes. Although some chromosomes evidently managed to fully synapse (asterisks in a and c) most synaptonemal complexes are incomplete and often form between apparently non-homologous axes (a inset, arrowheads mark synaptonemal complex stretches between apparently non-homologous chromosomes that have different lengths). Note that X and Y sex chromosomes are synapsed only in their short PAR regions in spermatocytes (a, top). Scale bars; 10μm. (b, d) Quantification of synaptonemal complex formation in Mcmdc2^+/+ and Mcmdc2^-/- spermatocytes (b) or oocytes (d). Spermatocytes were collected from testes of adult mice, and oocytes were collected from the ovaries of 16 or 18 days post coitum (dpc) fetuses. Synaptonemal complex development was assessed by detecting SYCP3 (axis marker) and SYCP1 (synaptonemal complex marker) on nuclear spreads of meiocytes from littermate pairs of wild-type and Mcmdc2^-/- mice. Three categories of synaptonemal complex formation were distinguished in cells with fully formed continuous axes: 1) No synapsis (characterized by either no SYCP1 stain or punctate weak SYCP1 stain along unsynapsed axes), 2) incomplete synapses (stretches of axes have formed but there is at least one chromosome without fully completed synaptonemal complex) or 3) full synapsis (all chromosomes are fully synapsed except the male sex chromosomes). Counted numbers of cells are indicated (n).

https://doi.org/10.1371/journal.pgen.1006393.g003

In the wild type, all late zygotene spermatocytes had partially synapsed chromosomes and by early pachytene all autosomes fully synapsed (n = 101 cells, of which 20% were late zygotene and 80% were early pachytene, Fig 3b). In contrast, in Mcmdc2 ^-/- spermatocytes, synapsis of all chromosomes was never observed (n>500 spermatocytes) as chromosome axes were unaligned and remained mostly unsynapsed in late zygotene-pachytene. The transverse filament component SYCP1, which marks synapsed axes [59], was detectable in a punctate pattern at very low levels along unsynapsed chromosome axes, or was detected only at a few foci at intersections of chromosome axes in 36% of late zygotene-pachytene Mcmdc2 ^-/- spermatocyte nuclear spreads (n = 412 cells, Fig 3b). In the rest of the spermatocytes, stretches of SYCP1 were detected along sections of juxtaposed axes, but the numbers of these synaptonemal complexes were generally low (a median number of 4 SYCP1 stretches in each spermatocyte, n = 131 cells). Chromosomes with different axis lengths were engaged in synapsis with each other, and chromosomes formed synapsis with more than one partner indicating that synaptonemal complexes frequently formed between non-homologous chromosomes (53% of synaptonemal complex stretches were unequivocally identified as non-homologous, n = 232 synaptonemal complex stretches in 62 spermatocytes). Nevertheless, in a significant fraction of cells (14 out of 116 cells) we observed apparently fully synapsed chromosomes (median 1 fully synapsed chromosome, range 1–4). We observed a similar chromosome alignment and synaptonemal complex formation defect in Mcmdc2^-/- oocytes that were collected from 16 or 18dpc fetuses. At these stages most wild-type oocytes were in early (16dpc) or late (18dpc) pachytene stages, and all chromosomes in oocytes with fully formed axes were either fully synapsed (16dpc: 89%, n = 500, 18dpc: 90%, n = 325 oocytes) or partially synapsed (16dpc: 11%, 18dpc: 10%) (Fig 3d). In contrast, a large fraction of Mcmdc2 ^-/- oocytes with fully formed axes (16dpc: 55%, n = 136, 18dpc: 41%, n = 174 oocytes) lacked synapsis completely or formed only punctate/very short stretches of synaptonemal complexes at intersections of axes (Fig 3d). Stretches of synaptonemal complexes formed in 45% (16dpc) or 59% (18dpc) of oocytes. Among those oocytes with SYCP1 stretches, the median number of stretches was 4 at 16dpc (n = 61) and 5 at 18dpc (n = 103). We also observed apparently fully synapsed chromosomes in 10 out of 61 (16dpc) or 73 out of 103 (18dpc) oocytes where SYCP1 stretches were observed. In cells which had fully synapsed chromosomes, a median number of 1 fully synapsed chromosome was observed at 16dpc (n = 10 oocytes) and 2 at 18dpc (n = 73). The highest number of fully synapsed chromosomes we observed was 8. We detected low levels of punctate SYCP1 signals along unsynapsed axes in Mcmdc2^-/- meiocytes in both sexes (male: Fig 3a, female: Fig 3c), although this SYCP1 staining pattern was more obvious in oocytes. This suggested that synaptonemal complex transverse filament assembly was initiated, although synaptonemal complex formation mostly failed in the absence of effective homologue alignment in Mcmdc2 ^-/- meiocytes. A similar weak association of SYCP1 with unsynapsed chromosome axes has been described in DNA strand invasion-defective Dmc1^-/- and Hop2^-/- meiocytes [60]. Thus, SYCP1 accumulation along unsynapsed chromosome axes may be a general phenomenon that can occur when synaptonemal complex formation is initiated but cannot be completed along unpaired chromosome axes. Taken together, these observations showed that MCMDC2 is critical for homologue alignment and synaptonemal complex formation in both sexes.

MCMDC2 is required for the repair of programmed meiotic DNA breaks

The observed defects in homologue alignment and synaptonemal complex formation suggested that early stages of recombination may be defective in the absence of MCMDC2. Single-stranded DNA ends that are produced after DSB formation are bound by recombinases RAD51 and DMC1, which form foci along chromosome axes. These foci have been defined as early recombination nodules by electron-microscopy and are thought to represent recombinosome complexes [61–66]. DMC1 and RAD51 promote strand-invasion of DNA ends into homologues [9–11]. This leads to synaptonemal complex formation, and as DNA repair progresses, the early recombinosomes/recombination nodules lose DMC1 and RAD51 and progress to become transitional recombinosomes/nodules [61,62,64,65]. Hence, quantification of DMC1 and RAD51 foci is informative about the number of unrepaired DNA breaks involved in early stages of recombination in meiocytes. We found that foci of both RAD51 and DMC1 accumulate with similar kinetics in wild-type and Mcmdc2^-/- spermatocytes in leptotene and early zygotene stages of prophase (Fig 4c and 4d). Numbers of RAD51 and DMC1 foci dropped as wild-type spermatocytes progressed to early-mid pachytene. In contrast, high numbers of RAD51 and DMC1 foci persisted in late zygotene-pachytene Mcmdc2^-/- spermatocytes. The high RAD51 and DMC1 foci numbers in Mcmdc2^-/- spermatocytes required SPO11 (Fig 4a and 4b). This observation is consistent with the idea that a delay in the repair of SPO11-generated programmed DSB breaks causes accumulation of RAD51 and DMC1 foci in Mcmdc2^-/- spermatocytes. To further test if meiotic DSB repair is delayed in the absence of MCMDC2 we detected phospho-serine 139 histone H2AX (ɣH2AX), which accumulates on chromatin in response to unrepaired DNA breaks and asynapsis in meiotic cells. ɣH2AX decorated chromatin in leptotene and zygotene stages but largely disappeared from autosomal chromatin due to progression of DSB repair and synapsis in wild-type cells. It remained associated only with the chromatin of the largely unsynapsed sex chromosomes, which form the sex body at the early pachytene stage (Fig 4e). In stark contrast to wild-type spermatocytes, late zygotene-pachytene Mcmdc2^-/- spermatocytes failed to form sex bodies and ɣH2AX persisted on autosomal chromatin (n>200 cells). Persistent widespread ɣH2AX accumulation on chromatin was dependent on SPO11, as Mcmdc2^-/- Spo11^-/- spermatocytes formed only more localized ɣH2AX-rich chromatin domains, so called pseudo-sex bodies, which are characteristic of mutants defective in programmed DSB formation (Fig 4e) [55,67].

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Fig 4. RAD51 and DMC1 foci persist in Mcmdc2^-/- spermatocytes.

(a, b, e) Immunostaining showing SYCP3 together with RAD51 (a), DMC1 (b) or γH2AX (e) on nuclear surface spreads of pachytene Mcmdc2^+/+, late zygotene-pachytene Mcmdc2^-/-, Spo11^-/-, and Spo11^-/- Mcmdc2^-/—spermatocytes. RAD51 and DMC1 foci are present at comparatively high density along the axes of unsynapsed sex chromosomes (a, b, asterisk), and are largely absent from synapsed autosomes of Mcmdc2^+/+ spermatocytes. Both RAD51 and DMC1 foci are present in high numbers along the unpaired axes of Mcmdc2^-/- spermatocytes. Absence of RAD51 and DMC1 foci is shown in Spo11^-/- and Spo11^-/- Mcmdc2^-/—spermatocytes. (e) γH2AX preferentially accumulates on the partially synapsed sex chromosomes of the Mcmdc2^+/+ spermatocyte. γH2AX associates with chromatin throughout the nucleus in the Mcmdc2^-/- spermatocytes. γH2AX is largely restricted to the sex chromatin in wild-type pachytene spermatocytes, and to pseudo-sex bodies in Spo11^-/- and Spo11^-/- Mcmdc2^-/—spermatocytes. Scale bars; 10μm. (c, d) Numbers of RAD51 (c) or DMC1 (d) foci are shown in leptotene (lepto), early zygotene (e zygo) in Mcmdc2^+/+ and Mcmdc2^-/-, late zygotene (l zygo) and early-mid pachytene (e-m pa) in Mcmdc2^+/+ and zygotene-pachytene (zyg-pa) in Mcmdc2^-/- spermatocytes. Median numbers of foci are marked, and n corresponds to the number of analyzed spermatocytes in three pooled experiments. DMC1 and RAD51 foci numbers are significantly higher in zygotene-pachytene Mcmdc2^-/- spermatocytes than in late-zygotene or early-mid-pachytene Mcmdc2^+/+ spermatocytes (Mann Whitney test).

https://doi.org/10.1371/journal.pgen.1006393.g004

We found a similar delay in meiotic recombination in Mcmdc2^-/- oocytes (Fig 5). We observed low numbers of DMC1 foci (median 29.5 foci, n = 66 cells) or RAD51 foci (median 21 foci, n = 62 cells) in late pachytene wild-type oocytes at 18dpc (Fig 5b and 5d). Correspondingly, the chromatin of pachytene oocytes was depleted of ɣH2AX (n>100 oocytes, Fig 5e). In contrast, both foci of RAD51 (median 162.5 foci, n = 66 cells) and DMC1 (median 173 foci, n = 64 cells) persisted along unsynapsed axes and ɣH2AX accumulated to high levels throughout the genome (n>100 oocytes, Fig 5e) in oocytes from Mcmdc2^-/- females.

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Fig 5. RAD51 and DMC1 foci persist in Mcmdc2^-/- oocytes.

(a, c, e) Immunostaining of SYCP3 along with RAD51 (a), DMC1 (c) or γH2AX (e) on nuclear surface spreads of pachytene Mcmdc2^+/+, or zygotene-pachytene Mcmdc2^-/- oocytes. Oocytes were collected from the ovaries of littermate fetuses at 18dpc, which is a time point when most wild-type oocytes are in the late pachytene stage. RAD51 and DMC1 foci are largely absent from synapsed chromosomes in Mcmdc2^+/+ oocytes. Both RAD51 and DMC1 foci are present in high numbers along the unpaired axes of Mcmdc2^-/- oocytes. (e) γH2AX is largely absent from the synapsed chromosomes of the Mcmdc2^+/+ oocyte. γH2AX associates with chromatin throughout the nucleus in the Mcmdc2^-/- oocyte. Scale bars; 10μm. (b, d) Numbers of RAD51 (b) or DMC1 (d) foci are shown in Mcmdc2^+/+ and Mcmdc2^-/- oocytes at 18dpc. Median numbers of foci are marked, and n corresponds to the number of analyzed oocytes in two pooled experiments. DMC1 and RAD51 foci numbers are significantly higher in Mcmdc2^-/- than in Mcmdc2^+/+ oocytes (Mann Whitney test).

https://doi.org/10.1371/journal.pgen.1006393.g005

The combination of these observations suggests that MCMDC2 is not required for DSB formation or the loading of recombinases on single-stranded DNA ends. Yet, RAD51/DMC1 appears to be ineffective in promoting homologue alignment and DNA break repair is severely impaired in the absence of MCMDC2.

Formation of MSH4- and MLH1-marked recombination intermediates requires MCMDC2

In wild-type meiosis, successful homologue alignment is accompanied by the appearance of axis-associated MSH4 foci that are thought to represent MSH4/5 (MutSγ) complexes within transitional recombinosomes/recombination nodules [38,61,64]. MutSγ is necessary for robust homologue pairing and alignment, most likely because it stabilizes strand invasion intermediates [23,36–39]. Hence, MSH4 foci are inferred to mark stabilized post-strand invasion recombination intermediates that are needed for efficient homologue alignment. Therefore, we tested if MSH4 forms axis-associated foci in Mcmdc2^-/- meiocytes (Fig 6a–6d). MSH4 foci numbers were significantly lower in Mcmdc2^-/- spermatocytes (median 11.5 foci, in late zygotene-pachytene cells, n = 48) and oocytes (median two foci in 16dpc late zygotene-pachytene oocytes, n = 48) than in wild-type meiocytes (median 80 foci in early-mid pachytene spermatocytes, n = 49, median 139 foci in 16dpc late zygotene and pachytene oocytes, n = 64).

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Fig 6. MutSγ and MutLγ foci formation are defective in Mcmdc2^-/- meiocytes.

(a, b, e, f) Immunostaining of SYCP3 together with MSH4 (a, b) or MLH1 (e, f) on nuclear surface spreads of pachytene Mcmdc2^+/+ or zygotene-pachytene Mcmdc2^-/- meiocytes. (a, b) MSH4 foci are readily detected along synapsed axes of pachytene spermatocytes and oocytes (16dpc). MSH4 foci numbers are much lower in Mcmdc2^-/- meiocytes. (e, f) Typically, a single MLH1 focus is detected along each synapsed axis pair of Mcmdc2^+/+ pachytene spermatocytes and oocytes (from ovaries of newborn mice). MLH1 foci are not present along the unsynapsed axes of Mcmdc2^-/- meiocytes. Scale bars; 10μm. (c, d) Numbers of MSH4 foci in Mcmdc2^+/+ and Mcmdc2^-/- spermatocytes and oocytes. (c) Spermatocytes were examined at leptotene (lepto), early zygotene (e zygo) in Mcmdc2^+/+ and Mcmdc2^-/-, late zygotene (l zygo) and early-mid pachytene (e-m pa) in Mcmdc2^+/+ and zygotene-pachytene (zyg-pa) in Mcmdc2^-/- mice. MSH4 foci numbers are significantly lower in Mcmdc2^-/- than in Mcmdc2^+/+ spermatocytes from early-zygotene stage onwards (Mann Whitney test). (d) Oocytes with fully formed axes (late zygotene and early pachytene) were examined from fetal ovaries at the 16dpc developmental time point. MSH4 foci numbers are significantly lower in Mcmdc2^-/- than in Mcmdc2^+/+ oocytes (Mann Whitney test). (c, d) Median numbers of foci are marked, and n corresponds to the number of analyzed meiocytes in two pooled experiments.

https://doi.org/10.1371/journal.pgen.1006393.g006

MSH4 foci numbers were marginally higher in late zygotene-pachytene than leptotene Mcmdc2^-/- spermatocytes (p = 0.0138, Mann Whitney test). This may indicate that MSH4-marked intermediates still form with low efficiency in the absence of MCMDC2. However, we observed an increasing punctate anti-MSH4 signal throughout the nuclei of wild-type meiocytes upon progression to pachytene (see Fig 6a upper panel). This pan-nuclear signal is unlikely to represent MutSγ complexes bound to recombination intermediates. Thus, a “background” anti-MSH4 signal in Mcmdc2^-/- spermatocytes may provide an explanation for the low MSH4 foci counts, which show a small increase upon progression from leptotene to late zygotene-pachytene. Regardless, the strongly reduced MSH4 foci numbers of Mcmdc2^-/- meiocytes suggest severe impairment in MutSγ function and/or in a recombination step that precedes recruitment of MutSγ to recombination intermediates.

Most MSH4-marked intermediates are repaired as non-crossovers in late pachytene, but a minority of them (at least one per homologue pair and on average 23 per cell) is thought to develop into MLH1-marked late recombinosomes (defined as late recombination nodules by electron-microscopy) [61,62,64,65,68], which are sites of future crossovers [5,69,70]. Consistent with an impairment in MutSγ-containing recombinosome formation, and consistent with the elimination of spermatocytes in mid-pachytene, we found no MLH1 foci in Mcmdc2^-/- spermatocytes (n = 35 spermatocytes, Fig 6e). We also detected a similar defect in MLH1 foci formation in Mcmdc2^-/- oocytes that had full axis (n = 23 oocytes at 18dpc, and n = 47 oocytes at 20.5dpc/newborn, Fig 6f). Thus, the recombination defect in Mcmdc2^-/- meiocytes ultimately prevents the formation of MLH1 foci, which likely represent precursors of a large majority of meiotic crossovers.

Inhibition of non-homologous synapsis formation is dependent on SPO11 in the absence of MCMDC2

While analyzing synaptonemal complexes in Mcmdc2^-/- meiocytes, we noted that synaptonemal complex formation was more severely affected in Mcmdc2^-/- meiocytes than in Spo11^-/- meiocytes (Fig 7). Spo11^-/- meiocytes lack programmed DNA breaks, and thus fail in homologue alignment and homologous synaptonemal complex formation. Nevertheless, synaptonemal complexes extensively formed between non-homologous chromosomes often creating a meshwork of interconnected chromosomes in Spo11^-/- meiocytes of the most advanced stages (Fig 7a middle panel). Our observations suggest that MCMDC2 is needed for meiotic DSB repair and progression beyond the early stages of recombination. Thus, early recombination intermediates may inhibit non-homologous synaptonemal complex formation in Mcmdc2^-/- meiocytes. Alternatively, it is possible that MCMDC2 has a DSB-independent function that is needed for non-homologues synaptonemal complex formation when homologue alignment is defective. To distinguish between these possibilities we tested the epistatic relationship between Mcmdc2 and Spo11. We reasoned that if non-homologous synapsis formation was similarly limited in Mcmdc2^-/- and Spo11^-/- Mcmdc2^-/- meiocytes then this would indicate a DSB-independent role for MCMDC2 in non-homologous synaptonemal complex formation. Conversely, extensive non-homologous synaptonemal complex formation in Spo11^-/- Mcmdc2^-/- meiocytes would indicate a role for SPO11 and SPO11-dependent recombination intermediates in the inhibition of non-homologous synaptonemal complex formation in Mcmdc2^-/- meiocytes. We found that Mcmdc2^-/- did not significantly reduce non-homologous synaptonemal complex formation in a Spo11^-/- background, and accordingly, we observed significantly more non-homologous synaptonemal complex stretches in Spo11^-/- (p = 0.0001, Mann Whitney test) and Spo11^-/- Mcmdc2^-/- (p = 0.0001, Mann Whitney test) spermatocytes than in Mcmdc2^-/- spermatocytes (Fig 7b). Thus, MCMDC2 is not required for non-homologous synapsis that forms in the absence of SPO11 and programmed DSBs. We conclude that Spo11 is epistatic to Mcmdc2 in “erroneous” non-homologous synaptonemal complex formation, and that SPO11-dependent recombination intermediates, which fail to promote homologous synaptonemal complex formation in the absence of MCMDC2, most likely interfere with the formation of extensive non-homologous synapsis in Mcmdc2^-/- spermatocytes.

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Fig 7. MCMDC2 is not required for extensive non-homologous synaptonemal complex formation in the Spo11^-/- background.

(a) SYCP3 (axis marker) and SYCP1 (synaptonemal complex marker) were detected by immunofluorescence on nuclear surface spreads of zygotene-pachytene Mcmdc2^-/-, Spo11^-/- or Spo11^-/- Mcmdc2^-/—spermatocytes. Whereas comparatively few synaptonemal complex stretches are detected in the Mcmdc2^-/-spermatocyte, extensive non-homologous synaptonemal complex formation is seen in the Spo11^-/- or Spo11^-/- Mcmdc2^-/—spermatocytes. Scale bars; 10μm (b) Quantification of SYCP1 stretch numbers in zygotene-pachytene spermatocytes with fully condensed chromosome axes of the indicated genotypes. The numbers of synaptonemal complex stretches is significantly higher in Spo11^-/- or Spo11^-/- Mcmdc2^-/—spermatocytes than in Mcmdc2^-/- (Mann Whitney test). The numbers of synaptonemal complex stretches are not significantly different in Spo11^-/- or Spo11^-/- Mcmdc2^-/—spermatocytes (p = 0.8639, Mann Whitney test). Median numbers of foci are marked, and n corresponds to the number of analyzed spermatocytes in two (Spo11^-/- or Spo11^-/- Mcmdc2^-/-) or three (Mcmdc2^-/-) pooled experiments.

https://doi.org/10.1371/journal.pgen.1006393.g007

MCMDC2 is needed for early steps in recombination mediated DNA DSB repair

Our work revealed that the MCM domain-containing protein MCMDC2 is essential for meiotic recombination, and hence gametogenesis. In contrast to MCM8 and MCM9, which have been implicated in homologous recombination during stalled replication fork restart and interstrand crosslink repair in mitotically growing cells [45–48], we found no evidence of an important role for MCMDC2 in mitotic cells (Fig 2a and 2b). We found that Mcmdc2^-/- meiocytes were defective in recombination-mediated repair of programmed meiotic DSBs, alignment of homologues, and synaptonemal complex formation.

DSB repair and synaptonemal complex formation are mutually dependent on each other in mammalian meiosis [24–28,37–39,60,71,72]. Thus, the severe synapsis formation defect observed could either be a cause or a consequence of the failed DNA DSB break repair in Mcmdc2^-/- meiocytes. We favor the hypothesis that the primary role of MCMDC2 is in DSB repair and not in synaptonemal complex formation. In support of this hypothesis, while synaptonemal complex formation is not required for correct alignment of homologue axes [24–28], MCMDC2 and initial steps of recombination that involve the formation of stable inter-homologue strand invasion intermediates are required [37–39,60,71,72]. This implicates MCMDC2 in early recombination steps that are needed for homologue pairing.

Additional support is provided by the observation that DNA DSB repair seems to progress further in synaptonemal complex-defective mutants than in Mcmdc2^-/- meiocytes. RAD51/DMC1-marked early recombinosomes seem to develop into MSH4-marked transitional recombinosomes in mutant spermatocytes that lack structural components of the synaptonemal complex [24–26]. MSH4 is thought to stabilize inter-homologue recombination intermediates [11,23,35,36], which is likely important for the extensive homologue pairing that takes place in synaptonemal complex deficient meiocytes. In contrast, MSH4 foci counts remained low in Mcmdc2^-/- meiocytes, indicating an earlier impairment in recombination that provides a likely reason for the observed failure in homologue alignment. Thus, defective synaptonemal complex formation cannot account for the observed defect in recombination in Mcmdc2^-/- meiocytes. These observations strongly indicate that defective synaptonemal complex formation is the consequence of failed recombination in Mcmdc2^-/- meiocytes, and not vice versa. Consistent with this conclusion, we found that MCMDC2 was not required for synaptonemal complex formation in the DSB formation defective Spo11^-/- spermatocytes, which form extensive non-homologous synapsis. This observation suggests that MCMDC2 is not involved directly in synaptonemal complex formation, although we cannot formally exclude the possibility that MCMDC2 plays a direct role specifically in homologous synaptonemal complex formation in a DSB formation-proficient background. Synaptonemal complex formation was much more limited in Mcmdc2^-/- than in the DSB formation defective Spo11^-/- and Spo11^-/- Mcmdc2^-/- meiocytes. Interestingly, synaptonemal complex formation is also reduced in strand-invasion defective Dmc1^-/- and Hop2^-/- meiocytes as compared to the DSB formation defective Spo11-deficient meiocytes [60]. Thus, accumulation of SPO11-dependent recombination intermediates may interfere with “erroneous” non-homologous synaptonemal complex formation in mutants where homologue pairing is defective due to an early block in recombination. Recombination intermediates might also inhibit non-homologous synaptonemal complex formation in unperturbed meiosis, which could help to ensure that synaptonemal complexes form between homologous substrates. Unrepaired DSBs may inhibit non-homologous synaptonemal complex formation directly. Alternatively, unrepaired DSBs may have an indirect effect by altering cell cycle-progression. Although, spermatocytes are eliminated in stage IV testis tubules in both DSB repair defective (e.g. Dmc1^-/-, or Mcmdc2^-/-) and the DSB formation defective Spo11^-/- spermatocytes, it has been proposed that Spo11^-/- spermatocytes progress further in meiotic prophase [55,73]. This is because the mid-late pachytene marker histone H1T was observed in Spo11^-/- spermatocytes but not in DSB repair defective spermatocytes, which indicates that unrepaired DSBs likely delay progression through meiotic prophase [55,73].

What is the function of MCMDC2 in recombination?

The accumulation of RAD51 and DMC1 foci in Mcmdc2^-/- meiocytes indicates that DNA ends were resected, and single-stranded DNA overhangs recruited strand-invasion promoting recombinases at break sites. Yet, these inferred RAD51 and DMC1 coated single-stranded overhangs were unable to efficiently promote homologue pairing. This might indicate that RAD51 and DMC1 recombinases cannot promote strand invasions effectively in the absence of MCMDC2. Alternatively, strand invasions and D-loop formation may still occur, but these recombination intermediates are not stabilized sufficiently to ensure alignment of homologues and the formation of extensive synaptonemal complexes. The observation that MSH4 foci numbers are low in Mcmdc2^-/- meiocytes is consistent with both of these scenarios. In the former scenario, recombination intermediates that could recruit the MutSγ complex would not form in Mcmdc2^-/- meiocytes, hence MSH4 foci could not form either. In the latter scenario, accumulation of MutSγ complex at strand invasion intermediates/D-loops would be defective in Mcmdc2^-/- meiocytes. Hence, these recombination intermediates would be unstable and might be dissolved by helicases, e.g. the BLM helicase, which has been proposed to antagonize MutSγ in its function of stabilizing recombination intermediates [31,35]. It follows, that MCMDC2 would play an important role in MutSγ function in the latter scenario.

MCM proteins are AAA+ ATPases that form hexameric rings on duplex DNA and promote the melting of double-stranded DNA in an ATP dependent-manner (reviewed in [40]). MCM2-7 are primarily involved in the initiation of DNA replication [74], but MCM8 and MCM9 are particularly important for homologous recombination in mitotic cells in vertebrates [45–48]. MCM8 and MCM9 interact, and are thought to also form hexameric helicase complexes [45,46,48]. MCM8 and MCM9 promote DNA repair by facilitating resection of DNA ends at DSB sites [47], promoting an as yet undefined post-strand-invasion steps of recombination [45,46], and melting DNA at sites of mismatches during mismatch repair in somatic cells [75]. Despite being crucial for recombination and mismatch repair in mitotically dividing cells, MCM9 does not have an essential role in meiotic recombination [45,76]. Thus, MCM8 functions independent of MCM9 in meiosis. The Drosophila orthologues of MCM8 and MCMDC2 form a protein complex that is presumed to stabilize strand invasion intermediates of recombination specifically in meiosis [43]. Furthermore, the meiotic phenotypes of Mcm8^-/- [45] and Mcmdc2^-/- mice appear very similar, although no data was reported on MutSγ behavior in Mcm8^-/-. Thus, it is tempting to speculate that MCM8 and MCMDC2 collaborate in mouse meiosis, and that MCMDC2 replaces MCM9 in MCM8-containing helicase complexes in meiosis. Curiously, the sequences of Walker A and B motifs, which are domains that are necessary for the ATPase activity of MCMs, are apparently not conserved in either Drosophila or mammalian MCMDC2 proteins [43]. This suggests that MCMDC2 is unlikely to function as an ATPase, but it may function as a modulator in putative meiotic helicase complexes that contain other MCMs (e.g. MCM8) with an active ATPase domain. Interestingly, the Drosophila MCM8 orthologue, REC, was proposed to facilitate repair DNA synthesis during meiotic recombination, because meiotic gene conversion tracks were significantly shortened in rec mutants [41]. Putative mammalian MCM8/MCMDC2-containing helicase complexes may have similar functions. One possibility could be that MCMDC2 promotes unwinding of the invaded DNA at sites of strand invasions. This could facilitate the formation of extended strand invasions, and/or may be needed for efficient DNA repair-synthesis starting from the 3´end of the invading strands. These hypothesized functions would be expected to stabilize strand invasions. Unwinding invaded homologous sequences to promote the formation of extended and stable D-loops might be particularly important for inter-homologue recombination during meiosis. The reason is that mismatches that can occur between homologues would likely interfere with extension of D-loops thereby antagonizing the stabilization of inter-homologue strand-invasion intermediates.

MCMDC2 and MutSγ

The observation that MCMDC2 is required for the accumulation of MSH4 at recombination intermediates during meiosis raises the interesting possibility that a putative MCMDC2-containing helicase complex and the MutSγ complex may physically interact and collaborate in stabilizing D-loops. At sites of DNA mismatches, MCM9 forms a complex with MSH2 and MSH6, which are homologs of MutSγ components MSH4 and MSH5, and the complex of these proteins is thought to be crucial for correct mismatch repair in mitotically active cells [75]. Thus, it is possible that functional and/or physical interaction between MSH proteins and “DNA repair-promoting” MCMs is a conserved principle in distinct DNA repair pathways. Relevant to this point is the observation that the MutSγ complex is missing from Schisophora, a taxon that includes Drosophila [43]. It has been proposed that a complex of REC (Drosophila melanogaster MCM8) and MEI-217/218 (two Drosophila melanogaster orthologues of MCMDC2) proteins substitutes for the functions of the missing MutSγ complex in antagonizing BLM helicase, stabilizing strand invasion intermediates, and promoting crossover formation in meiosis in Drosophila [43]. The REC/MEI-217/218 complex may have been capable of replacing MutSγ in Schisophora because MCMDC2-containing complexes and MutSγ might have interacted and had shared functions in stabilizing DNA strand invasion intermediates in ancestral taxa where both of these complexes existed. Thus, loss of MutSγ in Schisophora may have required only a modification to an already pre-existing (and possibly conserved) function in MCMDC2-containing complexes. This speculative scenario would be certainly consistent with a putative conserved functional interplay of MutSγ and MCMDC2 in stabilizing recombination intermediates during mammalian meiosis. Thus, an important aim of future studies of MCMDC2 functions will be to address if MCMDC2 forms helicase complexes with other MCMs and if these complexes interact physically and functionally with MutSγ to stabilize D-loops.

RNA-isolation and RT-PCR

To test Mcmdc2 expression in testes of wild-type and Mcmdc2^-/- mice, RNA was isolated and RT-PCR was performed as described earlier [20,77]. The RNA of the somatic tissue mix in (Fig 1a) originated from 17 distinct tissues: liver, brain, thymus, heart, lung, spleen, kidney, mammary gland, pancreas, placenta, salivary gland, skeletal muscle, skin, small intestine, spinal cord, tongue and uterus. The sequence of transcript-specific primers for RT-PCR were:

Mcmdc2 1R (Fig 1a) 5’-CGTTCCCTGTTGCAGTCTCT

Mcmdc2 1F (Fig 1a) 5’-CCCCACACAGCAAAAGTTCC

s9for (Fig 1a) 5’-GGCCAAATCTATTCACCATGC

s9rev (Fig 1a) 5’-TAATCCTCTTCCTCATCATCAC

Mcmdc2 Exon3 fw (Fig 1d, exon 3/4) 5’-ATTCAAAGCAGAGTTATGCTG

Mcmdc2 Exon4 rv (Fig 1d, exon 3/4) 5’-TTGAGTTTCAGTCTGTAACTGT

Mcmdc2 Exon5 fw (Fig 1d, exon 5/6) 5’-ATCAATATTGTGCTGAAGTTAAC

Mcmdc2 Exon6 rv (Fig 1d, exon 5/6) 5’-ACCAAGTACTCTAAATTTTCTGT

Mcmdc2 Exon6 fw (Fig 1d, exon 6/7) 5’-GATTTCAGTATGTGAGAGTCC

Mcmdc2 Exon7 rv (Fig 1d, exon 6/7) 5’-CTCTTAGGAAAATACCAAGTGA

Mcmdc2 Exon8 fw (Fig 1d, exon 8/9) 5’-ATGAACTAGTGAATAAGATGAAAA

Mcmdc2 Exon9 rv (Fig 1d, exon 8/9) 5’-CTGTCTACAAGCAGAGTGTC

Mcmdc2 Exon10 fw (Fig 1d, exon 10/11) 5’-ACTTTTGAATTTTAGCATGAATCT

Mcmdc2 Exon11 rv (Fig 1d, exon 10/11) 5’-CATCTGACCAATCAGAGTACT

Generation of knockouts and genotyping

Mcmdc2 was targeted in JM8A3.N1.C2 embryonic stem (ES) cells by the EUCOMM-IKMC project (project: 93238, ES line:HEPD0781_2_C06 and project: 118859, ES line: HEPD0800_2_F07). Targeting was based on a so called ‘knockout first’ multipurpose allele strategy [78] (Fig 1). Chimeras were generated by laser assisted C57BL/6 morula injections with ES cell clones heterozygote for the Mcmdc2 ^insertion allele (Fig 1c). Progeny of the chimeric animals were crossed to the outbred wild-type CD-1 mouse line, and to pCAGGs-FLPo [79] and PGK-Cre [80] transgenic mice to generate Mcmdc2 ^restored, Mcmdc2 ^deletion, and Mcmdc2 ^{insertion–deletion} alleles from the Mcmdc2 ^insertion allele (Fig 1b). Mice were maintained on the outbred ICR (CD-1) background. Mice were genotyped by PCR using tail-tip genomic DNAs. Genotyping primers:

LacZfor 5’-TGGCTTTCGCTACCTGGAGAGAC

LacZrev 5’-AATCACCGCCGTAAGCCGACCAC

CreFw 5’-GCCTGCATTACCGGTCGATGCAACGA

CreRv 5’-GTGGCAGATGGCGCGGCAACACCATT

FlpOFw 5’-GCTATCGAATTCCACCATGGCTCCTAAGAAGAA

FlpORv 5’-CAATGCGATGAATTCTCAGATCCGCCTGTTGATGTA

o566 5’-GCAAGAAAACTATCCCGACC

o1046 5’-CACAGTGAGGCCCAATATAAA

o1047 5’-TCCACAGGAAAAGGCAAACG

o1049 5’-GGTGCTAGCCCCTTCCTTTT

o1050 5’-TCACTTGGTATTTTCCTAAGAG

o1147 5’-TGAAAGTTGATATGAAACTGTATA

o1163 5’-AAGGTTGTAGAATTACAGCAGC

PCR product sizes: with LacZfor/LacZrev primers, Mcmdc2^insertion and Mcmdc2^{insertion–deletion} allele 208 bp, other alleles-no specific product; with o1046/o1047 primers, wild-type allele 244 bp, Mcmdc2^insertion allele 225bp, Mcmdc2^restored allele 225 bp, Mcmdc2^deletion allele no product, Mcmdc2^{insertion–deletion} allele no product; with o566/o1047/o1050 primers, wild-type allele 561bp, Mcmdc2^insertion allele 542bp, Mcmdc2^restored allele 542bp, Mcmdc2^deletion allele no product, Mcmdc2^{insertion–deletion} allele 751bp; with o1049/o1147 primers, wild-type allele 310bp, Mcmdc2^insertion allele effectively not amplifiable product (7370 bp), Mcmdc2^restored allele 467bp, Mcmdc2^deletion allele no product, Mcmdc2^{insertion–deletion} allele no product; with o1049/o1163/o1147 primers, wild-type allele 310 bp (and 3421bp), Mcmdc2^insertion allele effectively no amplifiable products (7370bp and 10462bp), Mcmdc2^restored allele 467bp (and 3559bp), Mcmdc2^deletion allele 690 bp, Mcmdc2^{insertion–deletion} allele effectively not amplifiable product (5683bp). FlpOFw/FlpORv were used to detect FlpO recombinase transgene (1500 bp), CreFw/CreRv were used to detect Cre recombinase transgene (750 bp).

Animal experiments

Mice carrying Spo11-null alleles were described earlier [6,7]. Histology in testis, analysis of RAD51/DMC1 foci or the synaptonemal complex were carried out in mice lines derived from both independent clones. The phenotypes of all the listed alleles were examined. No obvious differences were detected in testis histology, RAD51/DMC1 foci accumulation and synaptonemal complex formation between mice derived from the different ES clones and between the Mcmdc2 ^{insertion/insertion}, Mcmdc2 ^{deletion/deletion}, and Mcmdc2 ^{insertion–deletion/insertion–deletion} strains. We chose the HEPD0800_2_F07 derived Mcmdc2 ^{insertion–deletion} allele for complete phenotypic analysis, hence this line was used in all the reported experiments. Given that Mcmdc2 ^{insertion–deletion/insertion–deletion} mice lack three exons, which causes a frameshift we refer to this genotype as Mcmdc2 ^-/-. Mcmdc2 ^{restored/ restored} mice were fertile and their spermatocytes were indistinguishable from wild-type spermatocytes reconfirming the specificity of the observed phenotypes in the Mcmdc2 ^{insertion/insertion}, Mcmdc2 ^{deletion/deletion}, and Mcmdc2 ^{insertion–deletion/insertion–deletion} strains. Whenever possible, experimental animals were compared with littermate controls or with age-matched non-littermate controls from the same colony. All animals were used and maintained in accordance with the German Animal Welfare legislation (“Tierschutzgesetz”), the Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes and its German implementation (Tierschutz-Versuchstierverordnung–TierSchVersV). All procedures pertaining to animal experiments were approved by the Governmental IACUC ("Landesdirektion Sachsen”) and overseen by the animal ethics committee of the Technische Universität Dresden. The license numbers concerned by the present experiments are DD24-5131/287/1 and 24–9168.24-1/2006-13 (tissue collection without prior in vivo experimentation).

Growth curve measurements

Wild-type and Mcmdc2^-/- mouse embryonic fibroblasts were derived from 12.5–14.5dpc embryos using standard procedures [81]. We plated 10,000 mouse embryonic fibroblasts in triplicates in 1ml DMEM (GIBCO) in 24-well plates. Live cells were counted using Miltenyi Biotec MACSQuant on day 3, 6, 9, 13 and 15 of cultures without aphidicolin. For aphidicolin treatment, 10000 cells were plated and incubated with media containing 1μM aphidicolin for the first 24 hour of the culture. Live cells were counted on day 5, 9, 13, 17 after plating. The media was changed every 3rd day for both types of cultures.

Antibodies

In addition to antibodies that were previously described [20,21] we used two commercial antibodies: mouse anti-MLH1 (IF 1:50, BD Biosciences, order number 551092) and rabbit anti-MLH1 (IF 1:50, Calbiochem, order number D00122409). We also used a chicken anti-SYCP3 antibody that was raised against a His-tagged version of a 99 amino acid-long (from 13E to 111E amino acids) peptide of SYCP3, which we overexpressed in Escherichia coli and purified using metal ion affinity chromatography. IgYs from the yolk of eggs of immunized chicken were extracted using a published protocol [82]. Anti-SYCP3 IgYs were affinity purified on immunizing-antigen coupled NHS-Activated Sepharose 4 Fast Flow beads (Cat#17-0906-01, Amersham, GE Healthcare) according to standard methods [83].

Immunofluorescence microscopy

Preparation and immunostaining of testis-ovary cryosections and nuclear surface spreads of meiocytes were carried out as described before [20,21,84,85]. Recombination foci and synaptonemal complex stretches were counted manually on matched exposure images with the use of the count tool of Photoshop CS5. We counted anti-RAD51, -DMC1, -MSH4 or -SYCP1 signals that were associated with SYCP3-marked chromosome axes, to avoid counting signals that do not represent genuine recombination foci (RAD51, DMC1 and MSH4) or synaptonemal complexes (SYCP1).

Statistics

Statistical analysis was carried out with GraphPad Prism 5. For the comparison of independent samples, the two-tailed non-parametric Mann_Whitney (two-sample Wilcoxon rank-sum) test was used.