Proper synapsis of homologous chromosomes is an important meiotic checkpoint preventing germline transfer of harmful genic and chromosomal mutations to the next generations (Schimenti, 2005; Zickler and Kleckner, 2015; Rinaldi et al., 2017). Synapsis of homologous chromosomes is initiated at the leptotene stage of the first meiotic prophase by induction of developmentally programmed, SPO11-induced DNA double-strand breaks (DSBs) (Keeney et al., 1997; Keeney et al., 1999; Romanienko and Camerini-Otero, 2000). After 5’ to 3’ resection of each end of DSB, replication protein A (RPA) binds the 3’ overhang to save it from degradation, later being displaced by RAD51 and DMC1 recombinases (Inagaki et al., 2010) but see (Moens et al., 2007; Chan et al., 2018). The resulting nucleoprotein filament is engaged in homology search in the process leading to DSBs repair by homologous recombination and to synapsis of homologous chromosomes. The SPO11-induced DSBs are nonrandomly clustered into narrow 1–2 kilobase-pair intervals called recombination hotspots and their localization is predetermined by the PRDM9 binding to specific motifs inside these intervals and PRDM9-driven induced trimethylation of histone H3 at lysine 4 and lysine 36 on adjacent nucleosomes (Baudat et al., 2010; Myers et al., 2010; Parvanov et al., 2010; Eram et al., 2014; Lange et al., 2016; Powers et al., 2016, for recent reviews see Grey et al., 2018 and Paigen and Petkov, 2018).

The Prdm9 gene, besides determining position of the recombination hotspots, acts as the major hybrid sterility gene in certain hybrids between house mouse subspecies of Mus m. musculus (mouse strain PWD) and Mus m. domesticus (mouse strain C57BL/6, hereafter B6) (Mihola et al., 2009; Dzur-Gejdosova et al., 2012; Forejt et al., 2012; Bhattacharyya et al., 2013; Bhattacharyya et al., 2014). Disrupted synapsis of homologous chromosomes and dysregulation of meiotic sex chromosome inactivation are two major cellular phenotypes controlled by the Prdm9 gene in sterile (PWD x B6)F1 (hereafter PBF1) hybrids (Forejt and Iványi, 1974; Mihola et al., 2009; Bhattacharyya et al., 2014; Gregorova et al., 2018).

When the mouse Prdm9 gene was ‘humanized’ by substitution of the C2H2 zinc-finger (ZnF) DNA-binding domain for its human ortholog, the humanized PBF1-Prdm9^Hu/PWD meiocytes regained normal meiotic pairing and hybrid males became fertile. This unexpected finding provided direct evidence for the role of PRDM9 ZnF array in the control of hybrid sterility (Davies et al., 2016). The asynapsis and male sterility were proposed to be mainly a consequence of the evolutionary erosion of PRDM9 binding sites (Figure 1). Because the heterozygous allelic sites with lower PRDM9 binding affinity are used preferentially as a template for DSB repair in gene-conversion events, the sites with higher binding affinity mutate much faster than the rest of the genome. As a result, the majority of the PRDM9^PWD-determined hotspots in PBF1 sterile males are found on B6 homologs and vice versa. Such hotspot asymmetry can result in a delay or inability to repair DSBs using homologous chromosome as a template, thus preventing successful pairing and synapsis of homologs (Davies et al., 2016).

Figure 1

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We recently rescued synapsis of homologous chromosomes in meiosis of PBF1 sterile hybrids by elimination of PRDM9 hotspot asymmetry in random chromosomal intervals ($\ge$27 Mb), with paternal and maternal copies originating from the same PWD subspecies (Gregorova et al., 2018). When synapsis of the four chromosomes most strongly affected by asynapsis in the sterile hybrids was restored in this way, male fertility was regained (Gregorova et al., 2018). To further test the idea that the failure of proper meiotic synapsis in hybrid males is due to an insufficient number of timely repaired 'symmetric' DSBs and to evaluate the unlikely possibility that the rescue was caused by multiple recessive genetic factors of PWD origin, we increased the number of DSBs per cell by inflicting random exogenous DSBs to meiotic cells. We report that the exogenous DSBs generated by chemotherapeutic drug cisplatin (Basu and Krishnamurthy, 2010) enhanced meiotic synapsis of homologous chromosomes in sterile mouse inter-subspecies hybrids, thus bringing independent evidence on the mechanism of meiotic chromosome asynapsis (Gregorova et al., 2018) and supporting the 'asymmetry' hypothesis (Davies et al., 2016).

Cisplatin (cis-platinum diamminedichloride, hereafter cisPt) is known to create DNA inter-strand (ICL) and intra-strand cross-links. In replicating yeast and mammalian somatic cells removal of ICLs results in DNA DSBs, which can be repaired by the nonhomologous end joining or by homologous recombination, the latter being favored in the germline (Lawrence et al., 2016). Removal of the cisPt-DNA adducts without creating DSBs was reported in quiescent somatic cells (Frankenberg-Schwager et al., 2005). In the mouse, cisPt was reported to increase meiotic crossing-over (Hanneman et al., 1997). Moreover, significant improvement of meiotic chromosome synapsis was observed in SPO11-/- meiocytes treated with cisPt or X rays, indicating that exogenous DSBs can at least partially substitute the role of the SPO11-induced DSBs in pairing of homologous chromosomes (Romanienko and Camerini-Otero, 2000; Carofiglio et al., 2018).

To assess an effect of exogenous DSBs on meiotic pairing in sterile hybrids we treated the adult (4–8 weeks) PBF1 hybrid males with cisPt and with the 5-ethynyl-2’-deoxyuridine (EdU), a nucleoside analog of thymidine (Salic and Mitchison, 2008) to distinguish the spermatogenic cells replicating their DNA at the moment of cisPt injection (Figure 2A). The males received a single i.p. injection of cisPt at a dose of 1, 5 or 10 mg/kg body weight together with 50 mg/kg of EdU. Based on the published estimates of duration of the meiotic S-phase (20 hr), leptotene (24–48 hr), zygotene (24–32 hr) and pachytene (160 hr) stages of the first meiotic prophase (OAKBERG, 1956; Oud et al., 1979; Goetz et al., 1984) the males were sacrificed 40 hr after cisPt and EdU injection to quantify the DSBs at the first meiotic prophase, or after 8 days to monitor the chromosome synapsis at the pachytene stage.

Figure 2

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Figure 2—source data 1

Distribution of Edu + and EdU- spermatocytes at the first prophase 40 hr after EdU and cisPt injection.

https://doi.org/10.7554/eLife.42511.004

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CisPt induced DSBs in early meiotic prophase of sterile male hybrids

Forty hours after cisPt and EdU injection, 84.1 ± 3.3% (mean ±SE) of leptonemas and 49.3 ± 2.2% of zygonemas were EdU-positive, thus being at the S-phase at the time of injection or shortly after that. The EdU-negative leptonemas (15.9%) most likely started their S-phase 20 hr or more after EdU injection, after assumed depletion of free EdU (Figure 2B, Figure 2—source data 1), while EdU-negative zygonemas finished their DNA replication before CisPt and EdU injection. All pachynemas finished DNA replication before the treatment and were EdU-negative (Figure 2B). The occurrence of EdU-positive cells fitted better with the shorter reported estimates of the duration of leptotene and zygotene stages.

Since the RPA protein was reported to bind ssDNA soon after resection of DSBs in mitotic and meiotic cells (Ribeiro et al., 2016; Pacheco et al., 2018), we used the RPA foci as an early cytological marker of DSBs (Figure 3A). We found out that in spite of the large variation in the number of RPA foci in individual leptonemas and zygonemas (see also Kauppi et al., 2013), cells treated with 10 mg/kg of cisPt showed significant increase of RPA foci in leptotene and zygotene stages (Figure 3B, Figure 3—source data 1). The leptotene median number of 162 RPA foci per cell in control males increased to 229 foci in males treated with 10 mg/kg of cisPt (p=0.0313 Mann-Whitney U test). Control zygotene median of 194 RPA foci increased to 210.5 foci after treatment with 10 mg/kg of cisPt (p=0.0483). The numbers of RPA foci decline at the pachytene stage of meiotic prophase in fertile male mice (Li et al., 2007; Inagaki et al., 2010) but persist in high numbers in sterile untreated hybrids (median 119 RPA foci per pachynema). CisPt did not affect frequency of RPA foci in pachynemas 40 hr after injection (Figure 3B). To evaluate the impact of RPA foci on pachynemas and because beside asynapsis, the unrepaired DSBs are known to induce apoptosis, we compared the frequency of RPA foci in early, mid and late pachynemas of control (0 mg/kg of cisPt) sterile PBF1 males with fertile PWD and B6 parental controls (Figure 3, Figure 3—figure supplement 1). Unexpectedly, but in accord with Moens et al. (2007), the RPA foci persisted in early pachynemas of fertile controls, but significantly dropped in mid pachynemas (median 38 and 14 RPA foci in PWD and B6 compared to 98 foci in PBF1, p<0.0001) and virtually disappeared at the late pachytene stage.

Figure 3 with 1 supplement see all

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Figure 3—source data 1

RPA foci in leptonemas (L), zygonemas (Z) and pachynemas (P) of PBF1 hybrid males treated with cisPt.

https://doi.org/10.7554/eLife.42511.008

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Figure 3—source data 2

RPA foci in EdU-negative (E-) and EdU-positive (E+) zygonemas of PBF1 hybrid males treated with cisPt.

https://doi.org/10.7554/eLife.42511.009

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Surprisingly, when zygonemas were split to EdU-positive and negative, only EdU-positive cells showed a significant increase in the cisPt dosage-dependent RPA foci (Figure 3B, Figure 3—source data 2). The doses of 1 mg/kg, 5 mg/kg and 10 mg/kg of injected cisPt increased the median of RPA foci from 200.5 in controls to 239, 255 and 250, respectively (p=0.0043, 0.0026 and 0.0006, Mann-Whitney U test). As shown above, approximately half of the zygonemas were EdU-negative, apparently finishing S-phase before EdU and CisPt injection, while the EdU-positive zygonemas were in the mid or late S-phase at the time of the treatment. Since little is known about the timing of enzymatic removal of cisPt interstrand crosslinks (Johnsson et al., 1995), the formation of DSBs cannot be precisely specified in respect of the end of DNA replication. The cisPt-induced DSBs could arise anytime during the meiotic S-phase and/or at the beginning of leptotene stage.

Since RPA is an ssDNA-binding protein, it could mark other forms of DNA damage beside DSBs (Wang et al., 2005); therefore, we quantified the foci of DNA meiotic recombination 1 (DMC1), a meiosis-specific strand exchange protein, which is recruited to SPO11-induced DSBs (Figure 4A). The DMC1 response to cisPt was similar to that of RPA. The combined EdU-positive and -negative zygonemas showed an enhancing effect of cisPt on the frequency of DMC1 foci (Figure 4B, Figure 4—source data 1). The median number of DMC1 foci increased from 215 to 241, 233 and 251 after 1, 5 and 10 mg/kg of cisPt (p=0.0208, 0.0263 and 0.0048, Mann-Whitney U), respectively. CisPt treatment did not influence the high frequency of DMC1 foci in PBF1 spermatocytes at pachytene stage. Contrary to RPA, the DMC1 foci significantly dropped (Figure 4, Figure 4—figure supplement 1) in early pachynemas of fertile PWD and B6 controls (median 60 and 31 DMC1 foci in PWD and B6 compared to 111 foci in PBF1, p<0.0001) and virtually disappeared at the mid pachytene stage (median 10 and 0 of DMC1 foci in PWD and B6 compared to 85 foci in PBF1, p<0.0001).

Figure 4 with 1 supplement see all

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Figure 4—source data 1

DMC1 foci in zygonemas (Z) and pachynemas (P) of PBF1 hybrid males treated with cisPt.

https://doi.org/10.7554/eLife.42511.013

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Figure 4—source data 2

DMC1 foci in EdU-negative (E-) and EdU-positive (E+) zygonemas of PBF1 hybrid males treated with cisPt.

https://doi.org/10.7554/eLife.42511.014

Download elife-42511-fig4-data2-v2.xlsx

When split according to the EdU phenotype, the EdU-positive zygonemas showed a significant increase of DMC1 foci at all three concentrations (Figure 4C, Figure 4—source data 2), from 225.5 to 260.5, 269 and 259.5 foci, respectively (p=0.0025, 0.0192 and 0.0442, Mann-Whitney U test), while in EdU-negative cells the steady DMC1 increase became significant at 10 mg/kg dose (p=0.0246).

CisPt treatment enhances meiotic synapsis of homologous chromosomes in sterile hybrids

Provided that the paucity of symmetric DSBs hotspots is indeed the main cause of meiotic synapsis failure (Davies et al., 2016; Gregorova et al., 2018) and that the increased frequency of DMC1 foci reflects cisPt-induced DSBs, then in spite of cytotoxicity of cisPt to proliferating cells, the exogenous DSBs should improve synapsis of the homologous chromosomes in the PBF1 testis. To verify this assumption, we analyzed the asynapsis rate by immunostaining the lateral elements of synaptonemal complexes with antibody against synaptonemal complex protein 3 (SYCP3) and unsynapsed axial cores of homologous chromosomes with antibodies specific for HORMA domain containing two proteins (HORMAD 2) (Kogo et al., 2012; Wojtasz et al., 2012) (Figure 5A). First, we tested in a pilot experiment the optimal effect of cisPt treatment by comparing the frequency pachynemas with a complete set of synapsed autosomes (hereafter ‘fully synapsed pachynemas’) at 4, 5, 7 and 8 days after a single injection of 10 mg/kg of cisPt. The percentage of pachynemas with fully synapsed bivalents dramatically increased from 5.66% in the control male (0 mg/kg of cisPt) to 48.78% and 46.70% in the males on the 7th and 8th day after treatment (Figure 5B, Figure 5—source data 1). In the next experiment, we combined the injection of cisPt (5 mg/kg or 10 mg/kg) with EdU (50 mg/kg) to distinguish the spermatogenic cells replicating their DNA at the moment of cisPt injection. For each cisPt dose, three males were sacrificed on day 8. The results confirmed the positive effect of cisPt on meiotic synapsis seen in the pilot experiment. The control males displayed the mean frequency of 8.61% (5.80; 12.12) (95% CI) of fully synapsed pachynemas in contrast to the males treated with 5 mg/kg and 10 mg/kg of cisPt, which showed a threefold increase of fully synapsed pachynemas, 24.68% (19.41; 30.49) (p=1.6 × 10⁻⁹, Tukey’s post-hoc test) and 28.71% (22.86; 35.08) (p=7.3 × 10⁻¹³), respectively (Figure 5C, Figure 5—source data 2).

Figure 5

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Figure 5—source data 1

The effect of cisPt (10 mg/kg) on chromosome synapsis at pachytene in sterile PBF1 hybrid males 0 to 8 days after treatment.

Pilot experiment, one male per column.

https://doi.org/10.7554/eLife.42511.016

Download elife-42511-fig5-data1-v2.xlsx

Figure 5—source data 2

Evaluation of meiotic chromosome synapsis at pachytene stage in PBF1 males treated with cisPt.

Syn = apparently complete synapsis of all chromosomes. Asyn = one or more pairs of homologous chromosomes are asynapsed.

https://doi.org/10.7554/eLife.42511.017

Download elife-42511-fig5-data2-v2.xlsx

We assessed synapsis of homologous chromosomes at early, mid and late pachytene stages. However, for statistical evaluation the mid and late pachynemas were merged because of the scarcity of the latest stage. While the cisPt treatment caused a nonsignificant increase of fully synapsed early pachynemas, the enhancement of synapsis was dramatic in mid-late pachynemas (Figure 5D, Figure 5—source data 2). We assume that the efficiency of repair of cisPt-induced DSBs by standard homologous recombination is low in general and/or that a significant fraction of spermatocytes carrying them do not survive until early pachytene stage. Those spermatocytes with multiple asynapsed autosomes that survive to the early pachytene stage are mostly eliminated before reaching the mid-late pachytene stage as reported previously (Bhattacharyya et al., 2013). This multiple filtering effect thus enhances the apparent efficiency of cisPt monitored as a proportion of fully synapsed pachynemas at the mid-late stage.

When divided according the cell cycle stage at the time of cisPt injection, the synapsis was marginally significantly more frequent (p=0.0433, GLM model) in EdU-negative than in EdU-positive pachynemas (Figure 5E), on average 1.69 times (1.02; 2.84, 95 % CI; Figure 5—source data 2). The EdU-negative fully synapsed pachynemas could arise from a subset of cells with exogenous DSBs generated at leptotene/early zygotene at the time of cisPt and EdU injection. It is tempting to suggest that also the EdU-positive cells that were at the preleptotene S-phase at the time of injection removed the cisPt induced ICLs at early leptotene stage.

No sperm was found in the ductus epididymis of the males 30 days after injection. Histological sections showed atrophy of seminiferous tubules caused by the lethal effect of cisPt on proliferating spermatogonia and somatic cells of seminiferous tubules (not shown). Beside the increased frequency of fully synapsed pachynemas, a short-term effect was apparent from the increased relative incidence of late pachynemas. While in untreated control hybrids the late pachynemas represented 2.03% (4/107) of all pachynemas recorded from the meiotic spreads, the frequency increased to 12.12% (28/231) and 13.10% (30/229) after 5 mg/kg and 10 mg/kg cisPt treatment, respectively.

All data generated or analyzed during this study are included in the manuscript and source files.

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Thank you for submitting your article "Cisplatin-induced DNA double-strand breaks promote meiotic chromosome synapsis in PRDM9-controlled hybrid sterility" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Patricia Wittkopp as the Senior and Reviewing Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This "Research Advance" report by Forejt and colleagues provides orthogonal support for the hypothesis that male hybrid sterility with allelic incompatibility between Prdm9 and chromosomal haplotypes is caused by insufficient "asymmetric" DSBs, leading to failed homologous chromosome synapsis that triggers the meiotic checkpoint. This short manuscript is in support of a previous eLife paper that had remarkable genetic support for this model.

The basic idea is that both homologs need to receive about equal numbers of DSBs to promote interhomolog repair. The argument goes that if one homolog gets the majority of DSBs (because the PRDM9 allele's binding sites are underrepresented on one homolog), these will tend to be repaired by intersister recombination, which isn't productive in driving synapsis. It isn't clear to me why this would be, and the paper doesn't really address this potential mechanism. Nevertheless, the authors show that exogenous DSBs induced by cisplatin, presumably which occur randomly across all chromosome homologs and thus are "symmetric," substantially rescues synapsis and spermatocytes.

The manuscript itself is nicely done, and the results are convincing that cisplatin-induced DSBs rescue synapsis in a substantial fraction of hybrid spermatocytes. This would be an interesting addition to the meiosis literature, in that it supports the findings of older papers indicating that exogenous DSBs can, to some extent, substitute for SPO11 DSBs. Regarding its contribution to the hybrid sterility literature, these results strongly support the idea that a DSB deficiency underlies the synapsis defect. However, the manuscript does not provide direct evidence for the "asymmetric DSB" hypothesis; this would require a comparison of intersister vs. interhomolog repair of DSBs either genome-wide or in chromosomal intervals that are particularly susceptible to asynapsis in hybrids. This is an important shortfall of the paper with respect to linking to the "parent" paper, but it isn't clear how this can actually be addressed directly.

Essential revisions:

1) Add a cogent description (or cartoon figure) of the symmetric DSB model. This concept is confusing and not widely known in the meiosis field. From reading this in isolation, one might erroneously assume that symmetric DSBs are those that occur on both homologs at the same hotspot in the same spermatocyte, whereas asymmetric ones only occur on one homolog. Another interpretation is that symmetric sites are where PRDM9 binds the same locus of both homologs, but only one receives a DSB.

2) Propose an explanation for why asymmetric DSBs are primarily repaired via sister chromatid recombination…this implies that such DSBs along chromosome intervals somehow communicate with one another, i.e., that if all/most DSBs are on one homolog, then they are repaired via the sisters, but if the DSBs are distributed equally between homologs, then interhomolog repair is favored. Are there hints from other model organisms in which intersister events can be monitored directly?

3) Address the following concerns related to the data in Figure 4:i) the conclusion that cisplatin improves synapsis at "mid-late" pachynema requires accurate substaging of cells, which is not the case based on Figure 4A. The bottom cell is clearly at late pachynema, as shown by the bulging chromosome ends, behaviour of the XY bivalent, and DAPI-staining. But the top and middle cells are at early pachynema (compare the chromosome morphology and DAPI). The issue is magnified by the apparently very high levels of asynapsis in the untreated mice at mid-late pachynema (Figure 4E). As the authors and others have found, cells with asynapsis are eliminated at mid pachynema, so I'm not sure how these high levels of asynapsis can be possible at late prophase I. A systematic problem in stage-matching should be considered here.

ii) It isn't clear to me why the authors don't also find improved synapsis at early pachynema. Sure, the frequency of asynapsis will be higher at early than at mid-late pachynema because of the midpachytene checkpoint, but shouldn't a difference be observed between the cisplatin-treated and -untreated mice at early pachynema?

4) The effect of cisplatin on other aspects of the hybrid sterile phenotype are not presented. The authors state in data not shown that cisplatin causes atrophy due to toxic effect. But couldn't they assay in the short-term whether the number of cells reaching late pachynema after treatment is increased? And how do toxic effects of cisplatin on pachytene substages influence the interpretations in Figure 4?

5) EdU treatment.

i) To the non-initiated, the expectations and interpretations of the EdU+ and – cells will not be easy to understand. The authors could do a better job of explaining this.

ii) Why are DSB counts assayed at zygonema and pachynema but not leptonema? Isn't the latter stage the important one to focus on given the DSB hypothesis?

6) Clarify the following: Why is there so much asynapsis in the early pachytenes (assuming correct staging), even with CP treatment? Is it possible that the timing was such that this cohort was derived from cells that weren't in S phase? And so many asynapsed "Mid-late" pachytenes doesn't make sense in terms of checkpoint elimination…perhaps H1t staining was needed? These potential issues with stage-matching and the lack of an objective measure (beyond incidence of full synapsis) with which to ascertain that cisplatin rescues F1 hybrid cells are major concerns.

[…] The basic idea is that both homologs need to receive about equal numbers of DSBs to promote interhomolog repair.

We are sorry that we have not been clear enough when describing the asymmetric hotspot hypothesis. An unequal mean number of DSBs on homologs is not the explanation of our model of hybrid sterility. The proposed mechanism of asymmetric DSB hotspots (Davies et al., 2016) is based on the subspecies-specific erasure of PRDM9 binding sites, as demonstrated in the scheme newly added in Figure 1. The asymmetry in sterile F1 hybrids results in unrepairable or hardly repairable DSBs trying to use the homologous chromosome as a template. These unrepaired DSBs and the failure of pairing due to the deficiency of non-sister repaired DSBs results in elimination of meiotic cells.

The argument goes that if one homolog gets the majority of DSBs (because the PRDM9 allele's binding sites are underrepresented on one homolog), these will tend to be repaired by intersister recombination, which isn't productive in driving synapsis. It isn't clear to me why this would be, and the paper doesn't really address this potential mechanism. Nevertheless, the authors show that exogenous DSBs induced by cisplatin, presumably which occur randomly across all chromosome homologs and thus are "symmetric," substantially rescues synapsis and spermatocytes.

We believe that this premise is based on misunderstanding the asymmetric hotspot hypothesis. The Prdm9 allele-specific DSBs occuring only on one homolog (being heterozygous according to Grey, Baudat and de Massy, 2018) are present on both homologs, PWD on B6 chromosome and B6 on PWD chromosome (see Figure 2A in Davies et al., 2016). The asymmetry model is based on the subspecies-specific erosion of PRDM9 binding sites due to the meiotic drive (see the newly added Figure 1). If the majority of DSBs cannot be repaired by recombination (CO or NCO) from the homologous chromosomes and the number of sucessfully repaired DSBs falls below a certain threshold, then the 100% efficiency of homologous chromosome synapsis becomes compromised.

The manuscript itself is nicely done, and the results are convincing that cisplatin-induced DSBs rescue synapsis in a substantial fraction of hybrid spermatocytes. This would be an interesting addition to the meiosis literature, in that it supports the findings of older papers indicating that exogenous DSBs can, to some extent, substitute for SPO11 DSBs. Regarding its contribution to the hybrid sterility literature, these results strongly support the idea that a DSB deficiency underlies the synapsis defect. However, the manuscript does not provide direct evidence for the "asymmetric DSB" hypothesis; this would require a comparison of intersister vs. interhomolog repair of DSBs either genome-wide or in chromosomal intervals that are particularly susceptible to asynapsis in hybrids. This is an important shortfall of the paper with respect to linking to the "parent" paper, but it isn't clear how this can actually be addressed directly.

We think that the argument is based on misunderstanding the asymmetric hotspot concept. The question we asked was whether, as predicted by the asymmetry hypothesis, exogenous DSBs can improve synapsis of homologous chromosomes in sterile interspecies hybrids by increasing the number of presumably repairable DSBs. The apoptosis of primary spermatocytes can be provoked by persisting unrepaired DSBs, or to be a response to the failure of meiotic synapsis. We newly compared the rates of DMC1 and RPA foci at the pachytene stage in the fertile untreated parents, B6 and PWD, with sterile (PWD x B6)F1 hybrids (Figure 3—figure supplement 1 and Figure 4—figure supplement 1) to emphasize the large numbers of persisting DSBs in pachynemas of sterile F1 hybrids. The results are summarized in the subsection “CisPt induced DSBs in early meiotic prophase of sterile male hybrids”, second and fourth paragraphs. To our best knowledge, no method is available to provide direct evidence for intersister chromatid repair in mouse/mammalian meiosis.

Essential revisions:

1) Add a cogent description (or cartoon figure) of the symmetric DSB model. This concept is confusing and not widely known in the meiosis field. From reading this in isolation, one might erroneously assume that symmetric DSBs are those that occur on both homologs at the same hotspot in the same spermatocyte, whereas asymmetric ones only occur on one homolog. Another interpretation is that symmetric sites are where PRDM9 binds the same locus of both homologs, but only one receives a DSB.

We thank the reviewers for the comment. We agree that a cogent description of the symmetric DSB model was missing, which might have led to misunderstandings. We added a schematic drawing of a pair of homologous chromosomes with the asymmetric DSBs in the new Figure 1 and supplied the detailed legend.

2) Propose an explanation for why asymmetric DSBs are primarily repaired via sister chromatid recombination…this implies that such DSBs along chromosome intervals somehow communicate with one another, i.e., that if all/most DSBs are on one homolog, then they are repaired via the sisters, but if the DSBs are distributed equally between homologs, then interhomolog repair is favored. Are there hints from other model organisms in which intersister events can be monitored directly?

In the manuscript we do not propose that the asymmetric DSBs are primarily repaired via sister chromatid recombination. Rather, we think that the asymmetric DSBs primarily remain unrepaired, as mentioned above. Our only reference to sister chromatid recombination is: “DSBs in these hotspots are difficult to repair or they repair too late, perhaps using sister chromatids as a template (Faieta et al. 2016; Li et al. 2018).” However, we do present direct cytological evidence for the persisting DSBs at the pachytene stage and for asynapsis, both of them being able to arrest the first meiotic division on its own. Contrary to the clearance of DSBs on unpaired parts of X and Y chromosomes or on certain chromosomal translocations where DMC1/RAD51 foci disappear by late pachytene most likely by inter-sister-chromatid repair, the high numbers of RPA and DMC1 foci persist through the pachytene stage in (PWD x B6)F1 hybrid males. The cells are eliminated before the DSBs could have been repaired by inter-sister recombination (see newly added Figure 3—figure supplement 1 and Figure 4—figure supplement 1). It is probable that the sister chromatid recombination also contributes to the reduction of DSB numbers at the pachytene stage (compared with zygonemas), but we do not possess tools to experimentally verify this idea.

3) Address the following concerns related to the data in Figure 4:i) the conclusion that cisplatin improves synapsis at "mid-late" pachynema requires accurate substaging of cells, which is not the case based on Figure 4A.

The conclusion is based on the quantitative evaluation of 645 pachynemas classified as early, middle and late pachynemas (See Figure 5—source data 2. Figure 4 became Figure 5 after revision). Figure 5A illustrates the reliability of the method to detect asynapsis but legend does not specify the pachytene stage.

The bottom cell is clearly at late pachynema, as shown by the bulging chromosome ends, behaviour of the XY bivalent, and DAPI-staining. But the top and middle cells are at early pachynema (compare the chromosome morphology and DAPI).

We agree with the reviewer that the cell is a late pachynema and admit the pachytene stages were not specified in the legend to Figure 5A. To avoid misunderstanding, the pachytene substage of all three cells is now added to the legend.

The issue is magnified by the apparently very high levels of asynapsis in the untreated mice at mid-late pachynema (Figure 4E). As the authors and others have found, cells with asynapsis are eliminated at mid pachynema, so I'm not sure how these high levels of asynapsis can be possible at late prophase I. A systematic problem in stage-matching should be considered here.

Previously, we analysed the ratio of asynaptic cells in PBF1 hybrids (Bhattacharyya et al., 2013 Figure 2C). It shows that the cells with four or less univalents can reach mid pachytene and a few of them late pachytene. Pachynemas with > 4 univalents were detected at early pachytene but were missing later. In this manuscript, we deliberately classified pachynemas into two groups, fully synapsed and pachynemas with asynapsis, to quantify the overall effect of cisPt on meiotic synapsis.

ii) It isn't clear to me why the authors don't also find improved synapsis at early pachynema. Sure, the frequency of asynapsis will be higher at early than at mid-late pachynema because of the midpachytene checkpoint, but shouldn't a difference be observed between the cisplatin-treated and -untreated mice at early pachynema?

We thank the reviewer for the comment. Indeed, we did not expect such a difference between early and mid-late pachynemas and failed to comment on it in the text. The detailed explanation was added to the second paragraph of the subsection “CisPt treatment enhances meiotic synapsis of homologous chromosomes in sterile hybrids”. See also our response to point 6.

4) The effect of cisplatin on other aspects of the hybrid sterile phenotype are not presented. The authors state in data not shown that cisplatin causes atrophy due to toxic effect. But couldn't they assay in the short-term whether the number of cells reaching late pachynema after treatment is increased? And how do toxic effects of cisplatin on pachytene substages influence the interpretations in Figure 4?

We thank the reviewers for the suggestion to focus on the late pachynemas after treatment. The comparison indeed shows the increase of late pachynemas. The analysis was included as follows “Beside the increased frequency of fully synapsed pachynemas, a short-term effect was apparent from the increased relative incidence of late pachynemas. While in untreated control hybrids the late pachynemas represented 2.03% (4/107) of all pachynemas recorded from the meiotic spreads, the frequency increased to 12.12% (28/231) and 13.10% (30/229) after 5 mg/kg and 10 mg/kg cisPt treatment, respectively”.For the interpretation of the toxic effect of cisPt in Figure 5, see also our answer to Essential revisions No 6.

5) EdU treatment.

i) To the non-initiated, the expectations and interpretations of the EdU+ and – cells will not be easy to understand. The authors could do a better job of explaining this.

We thank the reviewer for comment. The rationale of EdU treatment was explained as follows: “we treated the adult (4-8 weeks) PBF1 hybrid males with cisPt and with 5-ethynyl-2’-deoxyuridine (EdU), a nucleoside analog of thymidine (Salic and Mitchison, 2008) to distinguish the spermatogenic cells replicating their DNA at the moment of cisPt injection.”

We also added an additional sentence of explanation to the first paragraph of the subsection “CisPt treatment enhances meiotic synapsis of homologous chromosomes in sterile hybrids”.

ii) Why are DSB counts assayed at zygonema and pachynema but not leptonema? Isn't the latter stage the important one to focus on given the DSB hypothesis?

We had two reasons for assaying zygonemas. The spread preparations contain very few spermatocytes at the leptotene stage, and DMC1 and RAD51 foci are more numerous at zygotene than in leptotene (see e.g. Cole et al., Nat Cell Biol. 2012 or Kauppi et al., Gen Dev 2013). The most probable reason is that the foci accumulate gradually during the leptotene stage and survive for the major part of the zygotene stage.

6) Clarify the following: Why is there so much asynapsis in the early pachytenes (assuming correct staging), even with CP treatment? Is it possible that the timing was such that this cohort was derived from cells that weren't in S phase?

We thank the reviewers for the comment. The missing explanation was added on as follows: “We assume that the efficiency of repair of cisPt-induced DSBs by meiotic homologous recombination is low and/or that a significant fraction of spermatocytes carrying them do not survive until early pachytene stage. […] This multiple filtering effect thus enhances the apparent efficiency of cisPt monitored as a proportion of fully synapsed pachynemas at mid-late stage.”

And so many asynapsed "Mid-late" pachytenes doesn't make sense in terms of checkpoint elimination…perhaps H1t staining was needed?

The question was answered above: The ratio of asynaptic cells in PBF1 hybrids is given in Bhattacharyya (Bhattacharyya et al., 2013 Figure 2C). It shows that only cells with four or less univalents can reach mid pachytene and a few of them late pachytene. Pachynemas with > 4 univalents were detected only in the early pachytene stage. In this manuscript, we deliberately classified pachynemas as fully synapsed and pachynemas with asynapsis to verify the overall effect of cisPt on meiotic synapsis.

These potential issues with stage-matching and the lack of an objective measure (beyond incidence of full synapsis) with which to ascertain that cisplatin rescues F1 hybrid cells are major concerns.

We hope the concerns about the stage-matching were dispelled. We checked the effect of exogenous DSBs at the level of synapsis, which was verified. The ratio of the three pachytene substages was added to show the significant increase of late pachynemas after cisPt treatment. See also the answer to point 4.

Author details

1. Liu Wang

   BIOCEV Division, Institute of Molecular Genetics, Czech Academy of Sciences, Vestec, Czech Republic

   Present address

   Department of Genetics and Genome Sciences, School of Medicine, Case Western Reserve University, Cleveland, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Barbora Valiskova

   Competing interests

   No competing interests declared

2. Barbora Valiskova

   1. BIOCEV Division, Institute of Molecular Genetics, Czech Academy of Sciences, Vestec, Czech Republic
   2. Faculty of Science, Charles University, Prague, Czech Republic

   Contribution

   Validation, Investigation, Visualization, Methodology, Writing—original draft

   Contributed equally with

   Liu Wang

   Competing interests

   No competing interests declared

3. Jiri Forejt

   BIOCEV Division, Institute of Molecular Genetics, Czech Academy of Sciences, Vestec, Czech Republic

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   jforejt@img.cas.cz

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2793-3623

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