Modulation of Prdm9-controlled meiotic chromosome asynapsis overrides hybrid sterility in mice

Hybrid sterility is one of the reproductive isolation mechanisms leading to speciation. Prdm9, the only known vertebrate hybrid-sterility gene, causes failure of meiotic chromosome synapsis and infertility in male hybrids that are the offspring of two mouse subspecies. Within species, Prdm9 determines the sites of programmed DNA double-strand breaks (DSBs) and meiotic recombination hotspots. To investigate the relation between Prdm9-controlled meiotic arrest and asynapsis, we inserted random stretches of consubspecific homology on several autosomal pairs in sterile hybrids, and analyzed their ability to form synaptonemal complexes and to rescue male fertility. Twenty-seven or more megabases of consubspecific (belonging to the same subspecies) homology fully restored synapsis in a given autosomal pair, and we predicted that two or more DSBs within symmetric hotspots per chromosome are necessary for successful meiosis. We hypothesize that impaired recombination between evolutionarily diverged chromosomes could function as one of the mechanisms of hybrid sterility occurring in various sexually reproducing species

X chromosome control of meiotic chromosome synapsis in mouse inter-subspecific hybrids.

Hybrid sterility (HS) belongs to reproductive isolation barriers that safeguard the integrity of species in statu nascendi. Although hybrid sterility occurs almost universally among animal and plant species, most of our current knowledge comes from the classical genetic studies on Drosophila interspecific crosses or introgressions. With the house mouse subspecies Mus m. musculus and Mus m. domesticus as a model, new research tools have become available for studies of the molecular mechanisms and genetic networks underlying HS. Here we used QTL analysis and intersubspecific chromosome substitution strains to identify a 4.7 Mb critical region on Chromosome X (Chr X) harboring the Hstx2 HS locus, which causes asymmetrical spermatogenic arrest in reciprocal intersubspecific F1 hybrids. Subsequently, we mapped autosomal loci on Chrs 3, 9 and 13 that can abolish this asymmetry. Combination of immunofluorescent visualization of the proteins of synaptonemal complexes with whole-chromosome DNA FISH on pachytene spreads revealed that heterosubspecific, unlike consubspecific, homologous chromosomes are predisposed to asynapsis in F1 hybrid male and female meiosis. The asynapsis is under the trans- control of Hstx2 and Hst1/Prdm9 hybrid sterility genes in pachynemas of male but not female hybrids. The finding concurred with the fertility of intersubpecific F1 hybrid females homozygous for the Hstx2(Mmm) allele and resolved the apparent conflict with the dominance theory of Haldane's rule. We propose that meiotic asynapsis in intersubspecific hybrids is a consequence of cis-acting mismatch between homologous chromosomes modulated by the trans-acting Hstx2 and Prdm9 hybrid male sterility genes

Data from: Hybrid sterility locus on Chromosome X controls meiotic recombination rate in mouse

Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies

Data from: Hybrid sterility locus on Chromosome X controls meiotic recombination rate in mouse

Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies

Expression profiling of a cluster of MiRNA genes within the <i>Hstx1/Hstx2</i> critical region.

<p>(A) Log fold-change of expression ratio of PWD versus B6 MiRNA genes in flow-sorted testicular cells. Significant overexpression of Mir465 cluster in PWD germ cells is shown in red. (B) Validation of Mir465 overexpression in PWD primary spermatocytes by qRT PCR. Data normalized to U6 non-coding RNA (C) Log fold-change of expression ratio of MiRNA genes in (PWD×B6)F1 versus (B6×PWD)F1 (abbreviated PB6F1 and B6PF1) 14.5 d old testes. Significant upregulation in red (P<0.05). (D) qRT PCR validation of differences in Mir465 expression. Data normalized to Mir152.</p

Asynapsis of individual chromosomes in pachytene oocytes of intersubspecific F1 hybrids and parental controls.

a<p>Each column represents the sum of two or three independent biological replicas. Asynapsis of each Chr was measured in a separate experiment in a separate set of cells. Incidence of asynapsis of a particular Chr is shown for cells with at least one asynapsis. Value in parenthesis is an estimate of overall frequency of asynapsis of a given Chr considering the overall frequency of cells with any asynapsis (Any Chr column). n - number of cells with asynapsis. N - total number of cells examined.</p>b<p>Abbreviations: DX.1sD17F1 – (B6.PWD-Chr X.1s×B6.PWD-Chr 17)F1, DX.1sPF1 – (B6.PWD-ChrX 1s×PWD)F1, PD17F1 – (PWD×B6.PWD-Chr 17)F1.</p

Super-resolution microscopy of synaptonemal complexes on spreads of (B6.PWD-Chr X.1s×PWD) pachytene spermatocytes.

<p>(A) Detail of a pachytene spermatocyte of a sterile male immunostained by SYCP3 (red) and SYCP1 (green) antibodies. Properly synapsed bivalents (arrows) show two parallel threads of transverse filaments decorated by SYCP1 antibody which form the central region embedded in SYCP3 lateral elements. Unsynapsed chromosomes lack transverse filaments but display some irregular SYCP1 spots (arrowheads). (B) Example of a nonhomologous pairing and/or translocations, and asynapsis in pachynema of (B6.Chr X.1s×PWD)F1 sterile male. Bar 2000 nM.</p

Meiotic asynapsis in female hybrids.

<p>Abbreviations of consomic strains and their hybrids: DX.1 – B6.PWD-Chr X.1; DX.1PF1 – (B6.PWD-Chr X.1×PWD)F1; DX.1sPF1 – (B6.PWD-Chr X.1s×PWD)F1; DX.1B6F1 – (B6.PWD-Chr X.1×B6)F1; DX.1sB6F1 – (B6.PWD-Chr X.1s×B6)F1; DX.1sD17F1 – (B6.PWD-Chr X.1s×B6.PWD-Chr 17)F1; D17B6F1 – (B6.PWD-Chr 17×B6)F1; B6PF1 – (B6×PWD)F1, PB6F1 – (PWD×B6)F1; PD17F1 – (PWD×B6.PWD-Chr 17)F1. (A) Chromosome synapsis in pachytene oocytes of B6 and (B6.PWD-Chr X.1s×PWD)F1 18.5–19.5 dpc female fetuses was analyzed by combination of SYCP1, SYCP3 and CREST (centromeric heterochromatin) immunostaining or by HORMAD2 and SYCP3 to detect unsynapsed chromosomes. Bar, 10 µm. (B) The frequency of oocytes showing one or more asynaptic chromosomes is similar (>40%) irrespective of <i>Hstx2</i> and <i>Prdm9/Hst1</i> genotype. (C) Although the (PWD×B6), (B6.PWD-Chr X.1s×PWD), (B6.PWD-Chr X.1×PWD) and (B6.PWD-Chr 17×PWD)F1 hybrid females do not differ in percentage of pachytene oocytes with asynapsis, the (B6.PWD-Chr 17×PWD)F1 females, conspecific for Chr 17^PWD, carry significantly less asynapsed chromosomes per cell. (D) The frequency of diplonemas in spread oocyte preparations was significantly lower (p<0.01, χ² test) in intersubspecific hybrids than in parental inbred strains..</p

The proposed sequence of events leading to male limited sterility of intersubspecific hybrids of house mouse.

<p>Susceptibility of heterosubspecific homologs to asynapsis is common to both sexes. <i>Prdm9/Hst1</i> and <i>Hstx2</i> hybrid sterility genes can modulate this sensitivity from 0% to >95% in spermatogenesis but not in oogenesis, depending on allelic combinations of epistatic DMIs. Multiple asynaptic autosomes provoke MSCI, contributing to hybrid male sterility. Approximately one half of unaffected oocytes ensure fertility of hybrid females. It remains to be established what is the cause of asynapsis of heterosubspecific homologs.</p

Single QTL scan for autosomal loci supporting <i>Hstx2</i> independent intrameiotic arrest of (<i>Mmd</i>×<i>Mmm</i>)F1 intersubspecific hybrids.

<p>(A) Testes weight QTLs (red) reached significance on Chrs 3 (marker UNC030163295, Chr3: 104,320,699) and 13 (JAX00357337 Chr 13:47,975,634) and QTLs for sperm count on Chrs 3, 9 (JAX00171568, Chr 9:56,491,601) and 13. Sperm count was evaluated as a binary trait (SC = 0, SC>0). (B) and (C) Additive effect of QTLs on testes weight and sperm count. For map positions and possible candidate genes see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004088#pgen.1004088.s009" target="_blank">Table S4</a>.</p