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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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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