Skip to main content
NCBI home page
Journal of Heredity logoLink to Journal of Heredity
. 2018 Nov 19;110(2):183–193. doi: 10.1093/jhered/esy060

Hybrid Sterility with Meiotic Metaphase Arrest in Intersubspecific Mouse Crosses

Risako Nishino 1,2, Sabrina Petri 3, Mary Ann Handel 3, Tetsuo Kunieda 4,✉, Yasuhiro Fujiwara 1,3,5,✉
PMCID: PMC6399516  PMID: 30452700

Abstract

Although organisms belonging to different species and subspecies sometimes produce fertile offspring, a hallmark of the speciation process is reproductive isolation, characterized by hybrid sterility (HS) due to failure in gametogenesis. In mammals, HS is usually exhibited by males, the heterogametic sex. The phenotypic manifestations of HS are complex. The most frequently observed are abnormalities in both autosomal and sex chromosome interactions that are linked to meiotic prophase arrest or postmeiotic spermiogenesis aberrations and lead to defective or absent gametes. The aim of this study was to determine the HS phenotypes in intersubspecific F1 mice produced by matings between Mus musculus molossinus-derived strains and diverse Mus musculus domesticus-inbred laboratory mouse strains. Most of these crosses produced fertile F1 offspring. However, when female BALB/cJ (domesticus) mice were mated to male JF1/MsJ (molossinus) mice, the (BALBdomxJF1mol)F1 males were sterile, whereas the (JF1molxBALBdom)F1 males produced by the reciprocal crossings were fertile; thus the sterility phenotype was asymmetric. The sterile (BALBdomxJF1mol) F1 males exhibited a high rate of meiotic metaphase arrest with misaligned chromosomes, probably related to a high frequency of XY dissociation. Intriguingly, in the sterile (BALBdomxJF1mol)F1 males we observed aberrant allele-specific expression of several meiotic genes, that play critical roles in important meiotic events including chromosome pairing. Together, these observations of an asymmetrical HS phenotype in intersubspecific F1 males, probably owing to meiotic defects in the meiotic behavior of the XY chromosomes pair and possibly also transcriptional misregulation of meiotic genes, provide new models and directions for understanding speciation mechanisms in mammals.

Keywords: hybrid sterility, meiosis, metaphase, Spo11

Crosses between 2 separate populations that originated from the same taxon can often produce progeny. However, if they have evolutionarily diverged and become genetically distinct through the process of speciation, the hybrid progeny are infertile, because of failure to produce normal gametes. This phenomenon of reproductive isolation, known as hybrid sterility (HS), plays an important role in speciation (Orr et al. 2004). In general, the heterogametic sex is more likely to reflect this genetic isolation mechanism. In species with XY sex chromosomes (e.g., flies and mammals), the male is more severely affected than is the female, whereas females tend to show fertility defects in species with ZW sex chromosomes (e.g., butterflies and birds). This phenomenon is known as Haldane’s rule (Haldane 1922). A widely accepted genetic explanation for HS is the Dobzhansky–Muller (DM) model, which proposes that incompatibilities between genes derived from genetically divergent populations exert deleterious effects in their hybrids (Muller 1942; Dobzhansky 1951; Wu and Ting 2004). In eukaryotes, only a few genes have been found to be involved in HS (Ting et al. 1998; Sun et al. 2004; Masly et al. 2006; Bayes and Malik 2009; Mihola et al. 2009; Phadnis and Orr 2009; Sawamura et al. 2010).

The first report of HS in the house mouse (Mus musculus) was published in 1974, for a cross between the Mus musculus musculus (hereafter musculus) subspecies and the C57BL/10 strain derived from the Mus musculus domesticus (hereafter domesticus) subspecies (Forejt and Ivanyi 1974). Although several hybrid sterility (Hst) loci have been mapped in the mouse genome (Forejt 1996; Dzur-Gejdosova et al. 2012), only one has been identified as a protein-coding gene: Prdm9 on mouse chromosomes 17, encoding the PR-domain-containing 9 protein, with histone methyltransferase activity (Mihola et al. 2009). Prdm9 in mouse and PRDM9 in primates, including humans, display a high level of polymorphism in the DNA-binding zinc-finger-containing domain (Mihola et al. 2009; Berg et al. 2010, 2011; Kong et al. 2010; Groeneveld et al. 2012; Buard et al. 2014; Kono et al. 2014). These polymorphic variations are the source of incompatibilities causing spermatogenic failure in intersubspecific F1 hybrids between musculus and domesticus (Mihola et al. 2009). Interestingly, many cases of male-specific HS are asymmetric, meaning that different phenotypes in the reciprocal hybrids are distinguished by an effect of the inherited maternal chromosome X and specific X linked loci (Storchova et al. 2004; Good et al. 2008a, 2008b; White et al. 2011). For example, recent studies have revealed that the Hstx2 locus on the mouse X chromosome controls asymmetrical abnormal spermatogenesis in reciprocal intersubspecific F1 hybrids (Bhattacharyya et al. 2014; Balcova et al. 2016).

As part of recent forward genetic studies for linkage mapping, we generated F1/F2 hybrid mice from matings between domesticus-derived laboratory strains and the JF1/MsJ (JF1mol) strain, which was derived from the Mus musculus molossinus (hereafter molossinus) subspecies (Asano et al. 2009; Khalaj et al. 2014; Fujiwara et al. 2015). We observed that the F1 and F2 hybrid males thus frequently produced spermatogenic abnormalities. The molossinus subspecies is a Japanese wild mouse that is genetically close to the musculus subspecies (Yonekawa et al. 1988; Sakai et al. 2005), and it is postulated that it emerged from a hybrid population of musculus and the Mus musculus castaneus Southeast Asian subspecies (Koide et al. 2011). Approximately 95% of the JF1mol genome is derived from the musculus subspecies (Yang et al. 2011). Male-specific HS cases with meiosis-related spermatogenesis defects have been reported in intersubspecific F1 hybrids between molossinus- and domesticus-derived strains. A characteristic of these hybrids is a high rate of precocious physical separation (hereafter referred to as dissociation) of the XY chromosomes pair during meiotic divisions (Matsuda et al. 1983), although the XY chromosome pair should remain attached during metaphase I and until the onset of anaphase I. Additionally, Oka et al. (2010) reported that the HS-associated meiotic phenotypes were often observed at 3 distinct stages (premeiotic, mid-pachytene, and metaphase). However, the extent to which the HS phenotype is present in F1 hybrids of a wider range of inbred domesticus strains and molossinus mice is not currently known. In addition, although dissociation of the XY chromosomes, which can result in meiotic arrest during metaphase, is common (Imai et al. 1981; Matsuda et al. 1982), the frequency of meiosis arrest at specific substages in F1 males of intersubspecific hybrids between molossinus and domesticus strains remains to be determined. Arrest during meiotic metaphase is of particular genetic interest, because it can impair the successful chromosome segregation required to ensure faithful transfer of genetic information to the next generation, often leading to chromosome abnormalities and sterility (Nagaoka et al. 2012; Watanabe 2012). The sex chromosomes seem particularly vulnerable to metaphase abnormalities, perhaps because pairing and meiotic recombination are limited to the small distal region of homology termed the pseudoautosomal region (PAR; Kauppi et al. 2011).

The goal of the present study was to determine the HS phenotypes of intersubspecific F1 mice between JF1mol and a wider array of domesticus strains than previously studied, with a focus on the behavior of chromosomes and the stages of meiotic arrest phenotypes. We found that JF1mol males produced sterile male F1 offspring, with meiotic arrest at metaphase, when crossed to BALB/cJ (domesticus) (hereafter BALBdom) females, but not when crossed to other domesticus strains tested. This HS phenotype is asymmetric, depending on the maternal BALBdom chromosome X. The sterile hybrids exhibited unique defects in meiosis, including arrest at metaphase as a result of misaligned chromosomes. Unexpectedly, we found abnormal allele-specific expression of meiotic genes and polymorphisms in a region of the BALBdom X that is adjacent to the XY-pairing region. These findings suggest that allelic incompatibilities drive these meiotic defects.

Materials and Methods

Animals

Mus musculus domesticus-inbred strains C57BL/6J (B6dom), C3HeB/FeJ (C3Hdom), DBA/2J (DBAdom), BALB/cJ(BALBdom), and wild-derived WSB/EiJ (WSBdom) were purchased from the Jackson Laboratory (JAX, Bar Harbor, ME). The M. m. molossinus wild-derived strains and JF1mol mice had been imported to JAX or Okayama University (Okayama, Japan) from the National Institute of Genetics (NIG, Mishima, Japan) and maintained over 30 generations. The designation of the hybrid mice indicates the female parent first, followed by the male parent, for example, (BALBdomxJF1mol)F1 is the offspring of a female BALBdom mouse mated to a male JF1mol mouse). The experimental crosses are listed in Supplementary Table S1.

Mice were maintained according to protocols approved by the committees for the regulation of animal protection and animal husbandry at Okayama University and JAX. The protocols for animal care and use were approved by the JAX Animal Care and Use Committee. Mice were euthanized at 10–12 weeks of age unless indicated.

For fertility testing, F1 mice were mated twice at 8–12 weeks of age with B6dom mice for 3 weeks. After the mating period, the F1 males or females were removed from the B6dom mating pair, and these female (both F1 and B6dom) mice were maintained for another 3 weeks for parturition. The number of offspring obtained from each mating was measured as the litter size.

Histological Analysis and TdT-Mediated dUTP Nick End Labeling (TUNEL) Assay

Testes and epididymides were fixed in Bouin’s solution overnight at 4 °C. Tissues were paraffin-embedded, and 5 μm-thick sections were stained with hematoxylin and eosin (HE) or periodic acid-Schiff (PAS). The stages of the seminiferous epithelium cycle were identified using established morphological criteria (Russell et al. 1990). The TUNEL assay was performed using the in situ Cell Death Detection Kit (11684817910, Roche, Basel, Switzerland) according to the manufacturer’s instruction.

Air-Dried Chromosome Preparations

To assess spread chromatin of metaphase spermatocytes, air-dried chromosome preparations from spermatocytes were produced as previously described (Fujiwara et al. 2013). Briefly, a cluster of seminiferous tubules obtained from testes was disentangled in 2.2% sodium citrate and treated with 1% sodium citrate for 30 min at room temperature. The hypotonic solution was replaced with fixative (methanol:acetic acid = 3:1) and kept at 4 °C for 10 min. The fixed tubules were then transferred to 50% acetic acid until the germ cells were dispersed. Cells were collected by centrifugal separation at 300 × g for 10 min and resuspended in the same fixative. A few drops of cell suspension were dropped on a glass slide and dried overnight. The spread chromatin was visualized via staining with 5% Giemsa solution for 5 min.

Surface-Spread Chromatin Preparation and Immunostaining

Surface-spread chromatin was prepared from spermatocytes obtained from 12- to 16-week-old mice. Briefly, seminiferous tubules were removed from the tunica albuginea of the testes and minced in 1 mL of ice-cold PBS containing a protease inhibitor cocktail (Roche), using a blade. The cell suspension was transferred into PBS containing a protease inhibitor and washed twice. The cells were collected by centrifugation at 500 × g for 10 min, resuspended in 2% PFA in H2O containing 0.03% SDS, and then mixed with an equal volume of 0.04% Photo-Flo in H2O (Kodak, Rochester, NY). The cell suspension was applied to the wells of a 12-well Shandon slide and incubated in a humid chamber at room temperature for 1 h. After fixation, the slides were briefly air-dried and passed through 2% PFA with 0.03% SDS for 3 min, followed by 2% PFA without SDS for 3 min. The slides were then washed 3 times with 0.04% Photo-Flo. The slides were then used immediately for immunolabeling.

The slides were blocked with 5% blocking reagent (170–6404, Bio-Rad Laboratories, Hercules, CA) in PBS and incubated with primary antibodies at 4 °C overnight, and then secondary antibodies at 37 °C for 1 h in the same blocking buffer. All antibodies are listed in Supplementary Table S2. The slides were counterstained with DAPI and observed using an SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Germ Cell Isolation

Germ cells were isolated as previously described, with some modifications (Cobb et al. 1997; La Salle et al. 2009; Ball et al. 2016). Briefly, seminiferous tubules were transferred into 20 mL DMEM (Gibco, Life Technologies, Carlsbad, CA) containing 0.5 mg of Liberase TL Research Grade (05401020001, Roche) and incubated for 20 min at 32 °C. To remove interstitial cells, the tubules were washed 3 times with the same media. The tubules were then fragmented by pipetting several times and further digested with 0.5 mg of Liberase and 10 μg of DNase in 20 mL DMEM for 13 min at 32 °C in a shaking water bath. The germ cells were isolated by filtering through a Nitex mesh (53–70 μm pore size). The crude germ cells were washed 3 times by centrifugation for 7 min at 1700 rpm using 10 mL of media containing 10 μg of DNase. The cells were resuspended in 350 μL of RLT buffer. The purity of germ cells in the samples was generally 85–90%, as determined by a phase-contrast microscopy.

RNA Extraction and cDNA Sequencing

Total RNA of the isolated germ cells was obtained from 3 individual animals (12 weeks old) using the RNeasy Mini Kit (Qiagen, Valencia, CA). A 1 μg RNA aliquot of each sample was reverse-transcribed using the QuantiTect Reverse Transcription Kit (Qiagen) and random primers, according to the manufacturer’s instructions. A 50 ng aliquot of cDNA was used to amplify the meiotic genes, using the set of primers listed in Supplementary Table S3. Primers were designed using the NCBI Primer-BLAST program (www.ncbi.nlm.nih.gov/tools/primer-blast/). For allele-specific expression analysis (Wang et al. 2007; Zwemer et al. 2012; Eckersley-Maslin et al. 2014), fragments of cDNA containing single-nucleotide polymorphism (SNPs) were amplified using primer sets (Supplementary Table S3). After purification of the PCR amplicons using HighPrep PCR (MAGBIO, Gaithersburg, MD), the purified amplicons (approximately 5–10 ng) were treated with BigDye Terminator v3.1 (Applied Biosystems, Waltham, MA), 5× buffer (SBUF-100, MCLAB, South San Francisco, CA) and BDX64 BigDye Enhancing buffer (1:128 dilution, MCLAB). The treated amplicons were washed using HighPrep DTR (MAGBIO) to remove BigDye terminator and run on a 3730xl DNA analyzer (Applied Biosystems). Sequencing data from the triplicate biological samples were analyzed and visualized using SnapGene Viewer 4.2.1 software (www.snapgene.com).

Statistical Analyses

The statistical significance for assays with multiple samples was assessed using 1-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test in GraphPad Prism version 7.0d for Mac OS X (GraphPad Software, La Jolla, CA).

Bioinformatics Analysis

The SNPs of the X chromosome for each strain were obtained from the Mouse Phylogeny Viewer database (http://msub.csbio.unc.edu; Yang et al. 2011; Wang et al. 2012). In this study, we focused only on SNPs that differed between the parental strains of the F1 mice. A histogram of X-chromosome SNPs was created using RStudio version 0.98.1103 (http://www.rstudio.com/), with the ggplot2 package (Wickham 2009). The genomic region of the PAR was determined according to previously published studies (Perry et al. 2001; White et al. 2012). Genomic locations were based on NCBI37/mm9.

Results

Fertility Parameters of Hybrid F1 Males from Intersubspecific Crosses

Fertility parameters were assessed in F1 males derived from reciprocal crosses between JF1mol mice and mice of various domesticus-derived strains (Figure 1A, Supplementary Table S1). The F1 males derived from all but one crosses were fertile and capable of producing litters of normal sizes (5.8–8.33 pups/litter). However, all F1 males produced by matings between BALBdom females and JF1mol males (BALBdomxJF1mol)F1 were infertile, whereas (BALBdomxJF1mol)F1 females and both (JF1molxBALBdom)F1 males and females were fertile (Figure 1A, Supplementary Table S1). In spite of their fertility, the average testes-to-body-weight ratios of (B6domxJF1mol)F1, (C3HdomxJF1mol)F1, and (DBAdomxJF1mol)F1 males were significantly lower than those of both F1 males derived from the reciprocal crosses and the respective parental strains (Figure 1B, Supplementary Table S1). However, the average weight of the testes of the infertile (BALBdomxJF1mol)F1 males was even lower (46.50 ± 2.00 mg, N = 3 mice), approximately half of that of the parental strains (BALB: 112.86 ± 1.76 mg, N = 3 mice; JF1: 82.63 ± 5.84 mg, N = 4 mice; Figure 1B, Supplementary Table S1). This is consistent with germ-cell depletion and the loss of mature spermatozoa (see below).

Figure 1.

Figure 1.

Open in a new tab

Fertility and testis weights of intersubspecific F1 mice. (A). Litter size of F1 mice and their parental strains. Data are presented as the mean litter size with SD, and the actual litter size of each mating is represented as a yellow circle. The litter size of domesticus strains was obtained from a previous report (Nagasawa et al. 1973). n, number of matings. (B) Comparison of testis weights. Data are represented as the mean testis weight of the testes (mg) divided by body weight (g) with SD. The actual measurement is represented as a yellow circle. n, number of mice examined; SD, standard deviation; n.d., not detected, n.s., not significant; F, fertile; IF, infertile; brackets and asterisks, P values for indicated comparison (ordinary 1-way ANOVA followed by Tukey’s multiple comparison test), *P = 0.01–0.05, **P = 0.001–0.01, ***P < 0.001. See online version for full colors.

Arrest and Apoptosis of Meiotic Metaphase I Spermatocytes in Infertile (BALBdomxJF1mol)F1 Males

To reveal the cause of the reduced testis weight in F1 males, we analyzed histological sections of testes obtained from F1 males and their parental strains. More degenerating cells were observed in the tubules of F1 testes, especially those with the Xdom chromosome [(B6domxJF1mol)F1, (C3HdomxJF1mol)F1, (DBAdomxJF1mol)F1, and (BALBdomxJF1mol)F1] than in their parental strains (Figure 2A–D, Supplementary Figure S1). Notably, in the testes of infertile (BALBdomxJF1mol)F1 males, very few round or elongating spermatids were detected (Figure 2D). During normal spermatogenesis, metaphase spermatocytes are found in tubules at histological stage XII of the seminiferous epithelium. A TUNEL assay revealed that the degenerating apoptotic cells were predominantly spermatocytes with typical metaphase morphology in the stage XII tubules of the infertile F1 males (Figure 2A–D, Supplementary Figure S1). Moreover, at a later stage of development (i.e., when postmeiotic round spermatids are abundant in fertile males), residual degenerating (TUNEL-positive) cells with metaphase morphology were observed. The apoptotic cells were frequently observed in the tubules of the infertile males (Figure 2E). In addition, there were significantly fewer TUNEL-positive apoptotic metaphase spermatocytes in the (JF1molxBALBdom)F1 males, and even fewer in the parental strains (Figure 2A). Combined, these results indicate that asymmetric genetic incompatibility between domesticus and molossinus, probably related to inheritance of the Xdom chromosome, causes defects and cell death at the time of meiotic divisions. This may explain the sterility of these hybrids.

Figure 2.

Figure 2.

Open in a new tab

Meiotic metaphase arrest in (BALBdomxJF1mol)F1 males. (A–D) TUNEL assay of seminiferous tubules at stage XII (A–D) obtained from adult BALBdom (A), JF1mol (B), (JF1molxBALBdom)F1 (C), and (BALBdomxJF1mol)F1 (D) males. Apoptotic cells are stained in brown (black arrow), and nuclei are counterstained with hematoxylin. The tubules at stage XII were identified by the presence of leptotene/zygotene spermatocytes and elongating spermatids. (E) Boxplot of the ratio of apoptotic to total metaphase spermatocytes in the tubules at stages XII. The number of darkly stained apoptotic metaphase spermatocytes showing a metaphase plate was divided by the total number of metaphase spermatocytes. n, number of number of tubule examined (3 mice for each strain); n.s., not significant; brackets and asterisks, P values for indicated comparison (ordinary 1-way ANOVA followed by Tukey’s multiple comparison test), *P = 0.01–0.05, ***P < 0.001. See online version for full colors.

In normal metaphase spermatocytes, chromosomes were aligned in a characteristic way along the equatorial plane or metaphase plate (approximately 90% in parental strains and 75.5% in fertile (JF1molxBALBdom)F1 mice, Figure 3A,C). However, in (BALBdomxJF1mol)F1 testes, similarly aligned metaphase chromosomes were observed in only 28.1% of the spermatocytes (Figure 3B,C), which was significantly fewer than in parental strains and reciprocal fertile F1 males (P < 0.001; Figure 3C). Instead, most metaphase I spermatocytes obtained from the sterile hybrid males exhibited misalignment of one or more chromosomes (Figure 3B). Moreover, there was a high rate of dissociation of the XY chromosomes in both metaphase and pachytene spermatocytes of (BALBdomxJF1mol)F1 males (Figure 4). The abnormal dynamics of the XY chromosome pairing as well as misalignment of homologous chromosomes may contribute to the prolonged division-phase arrest and apoptosis in the sterile (BALBdomxJF1mol)F1 male mice.

Figure 3.

Figure 3.

Open in a new tab

Chromosomal misalignment in (BALBdomxJF1mol)F1 males and F1 males from wild-derived domesticus and wild-derived molossinus mice. (A–D) PAS-stained sections of testes obtained from F1 males revealed that metaphase spermatocytes in tubules at stage XII from fertile (JF1molxBALBdom)F1 males exhibit normal formation of the metaphase plate (inset, A) and the presence of elongating spermatids (black arrowheads, A). In contrast, elongating spermatids were rarely observed in the tubules of infertile (BALBdomxJF1mol)F1 males (black arrowheads, B), and their metaphase spermatocytes exhibited unaligned chromosomes (red arrows, inset, B). HE stained sections of testis from (WSBdomxJF1mol)F1 males (D) also exhibited chromosomal misalignment in metaphase spermatocytes (red arrow) and a few elongating spermatids (black arrowhead). The percentage of metaphase spermatocytes showing normal or misaligned chromosomes was compared using mice at 30 days postpartum (C). The gray column represents the mean percentage of metaphase spermatocytes with a normal metaphase plate, and the red column represents that of metaphase spermatocytes with misaligned chromosomes. Bars represent SD. n, number of metaphase spermatocytes [pooled from 3 mice; (WSBdomxJF1mol)F1 males exhibit no metaphase spermatocytes]; SD, standard deviation; brackets and asterisks, P values for indicated comparison (ordinary 2-way ANOVA followed by Tukey’s multiple comparison test), *P = 0.01–0.05, ***P < 0.001. See online version for full colors.

Figure 4.

Figure 4.

Open in a new tab

XY pairing defect in spermatocytes obtained from (BALBdomxJF1mol)F1 males. (A–E) The spread chromatin of pachytene spermatocytes was immunolabeled using an anti-SYCP3 antibody (red) to mark the lateral element of synaptonemal complex (SC) aligning each homologous and XY chromosomes; an anti-SYCP1 antibody (green) to mark the central element of the SC, which is expressed only where homologous or XY chromosomes are synapsed (paired); and an anti-phosphorylated histone H2AFX (P-H2AFX) antibody (white) to mark the XY body (A, B). In normal pachytene spermatocytes obtained from BALBdom males, the XY body contained the XY chromosomes paired at the PAR (A). In pachytene spermatocytes obtained from (BALBdomxJF1mol)F1 males, the XY chromosomes of pachytene spermatocytes were often dissociated, but were still within the XY body (B). Chromosome spreads of metaphase spermatocytes showed that the XY chromosomes in metaphase spermatocytes obtained from (BALBdomxJF1mol)F1 males were often dissociated (red arrowhead in D), whereas those obtained from BALBdom males were usually paired (black arrowhead in C). The X and Y chromosomes were identified as the pair of 2 chromosomes whose sizes were different but without signs of chiasmata. Quantification of the percentage of cells showing the paired XY chromosomes in the pachytene and metaphase substages (E). The number of cells counted is given below the graph. See online version for full colors.

The HS Phenotype Depends on domesticus Genome Content

We hypothesized that the HS phenotype of metaphase arrest and defective chromosome alignment is related to genomic incompatibilities between the molossinus and domesticus subspecies, especially BALBdom. Interestingly, a recent phylogenetic study of SNPs and variable-intensity oligonucleotides (VINOs; Yang et al. 2011) showed that 97.47% of the BALBdom genome contained SNPs and VINOs of domesticus, whereas the other domesticus strains used in the present study had fewer domesticus variants (92.84–95.04%; Supplementary Figure S2A). Therefore, the HS phenotype we have assessed may depend on the genomic content of domesticus, especially since the other F1 mice we analyzed exhibited less-severe phenotypes (Figures 1 and 2, Supplementary Figure S1). We, therefore, determined the HS defects in F1 males obtained from a mating of JF1mol males to wild-derived WSBdom females, in which 100% of the autosomes are derived from domesticus (Supplementary Figure S2A). Histological analysis revealed that the (WSBdomxJF1mol)F1 males exhibited a similar HS phenotype to that observed in (BALBdomxJF1mol)F1 males, with a greatly reduced number of spermatids and a higher frequency of chromosomal misalignments in metaphase spermatocytes (Figure 3B–D). Additionally, attempted to identify the region of the domesticus X chromosome that contributed the HS phenotypes by identifying SNP-dense region(s) between C3Hdom (compatible with fertility in F1 males produced by mating with JF1mol males) and either BALBdom and/or WSBdom (showing a spermatogenic arrest phenotype in F1 males produced by mating with JF1mol males). We found that the SNPs different between C3Hdom and either BALBdom and/or WSBdom were highly enriched within a ~27.5 Mb region near the PAR (Supplementary Figure S2B), indicating that this region in BALBdom and WSBdom mice is derived from the same origin, which is distinct from that of C3Hdom. Based on these analyses, we concluded that genomic incompatibilities between domesticus and molossinus, contribute to chromosomal misalignment and metaphase arrest and that a region near the PAR of the BALBdom X chromosome may play a role in the HS phenotype.

Abnormal Allele-Specific Meiotic Gene Expression in Sterile F1 Hybrids

F1 hybrid mice often exhibit random monoallelic gene expression in neuron cells or embryos (Wang et al. 2007; Zwemer et al. 2012; Eckersley-Maslin et al. 2014). The mammalian spermatogenesis process is under the control of strictly regulated gene expression (Handel and Schimenti 2010), so the suppression of a single allele may disrupt the spermatogenic process. To determine possible involvement in HS phenotypes, we examined allele-specific expression by analyzing sequencing electropherograms. We sequenced the amplified cDNA segments harboring the polymorphic regions of several meiotic genes that contain polymorphisms between the domesticus and molossinus strains and are required for successful meiosis and fertility. As expected, Rec8 and Cntd1 exhibited biallelic expression (i.e., peaks of both alleles were observed at the polymorphic sites Rec81245 and Cntd1768, respectively; Figure 5). In contrast, the X-linked Tex11 gene, as a marker of monoallelic expression, showed only a single peak at the polymorphic site, Tex11971, because heterogametic F1 males inherit the X chromosome only from their maternal parent (Figure 5). The Spo11 gene contains several polymorphic sites and, surprisingly, 2 of these (Spo11834 and Spo11954), exhibited only the JF1-specific peaks in both sterile (BALBdomxJF1mol)F1 males and reciprocal fertile (JF1molxBALBdom)F1 males (Figure 5). Thus, the expression of the Spo11BALB allele was suppressed in both hybrids, indicating monoallelic expression. We investigated whether this allele-specific expression was also the case for several other important meiotic genes, such as Mlh3, which is required for successful crossing over, and thus for the recombination between homologous chromosomes (Lipkin et al. 2002). The Mlh3 gene exhibited BALB allele-biased expression in both reciprocal F1 males at its polymorphic site, Mlh33526 (Figure 5), indicating suppression of JF1 alleles in both reciprocal hybrids. Here, we found evidence of allele-specific expression in both of these genes, but the direction of the bias was not consistent. Although the mechanisms of allelic bias in expression and its contribution to the HS phenotypes remains unclear, these observations suggest that genomic incompatibilities between domesticus and molossinus directly or indirectly disrupt the allelic expression of at least some meiotic genes.

Figure 5.

Figure 5.

Open in a new tab

Random monoallelic gene expression in the testes of F1 males. Allele-specific expression of meiotic genes in testes obtained from F1 males. Rec8 and Cntd1 are biallelic-expressing genes, while Tex11 (X-linked gene) is a monoallelic expressing gene. The black arrows represent the sites of polymorphisms. The analyses were performed in triplicates, but only one result from each is presented. See online version for full colors.

Discussion

Here, we have determined the prevalence of HS phenotypes in offspring derived from crosses between molossinus JF1 and a variety of common inbred laboratory strains of domesticus. Although most hybrid progeny exhibit some fertility, the (BALBdomxJF1mol)F1 males produced by crossing BALB (domesticus) females to JF1 (molossinus) males were sterile. This phenotype was asymmetrical, that is, males produced by the reciprocal cross were fertile. The spermatocytes of sterile (BALBdomxJF1mol)F1 males exhibited the most definitive HS phenotype, involving metaphase arrest and misaligned XY chromosomes. This is the first study to report these adverse meiotic effects of the BALB domesticus genome and X chromosome in combination with the molossinus genome. Notably, there was an allele-biased expression of meiotic genes, including Spo11 and Mlh3, in both reciprocal hybrids.

Dissociation of the XY Chromosomes and Aberrant Expression of Spo11

A phenotype that was consistently observed in this study was dissociation of the XY chromosomes in spermatocytes obtained from sterile (BALBdomxJF1mol)F1 males. Previously, XY dissociation in spermatocytes was found in F1 males produced by crosses of inbred domesticus females with wild-derived molossinus males (Matsuda et al. 1983, 1991, 1992). On the X and Y chromosomes, recombination and synapsis occur only within the PAR. The formation of double-strand breaks (DSBs) in the PAR is mediated by SPO11A, a shorter isoform of SPO11, acting after the late zygotene substage (Kauppi et al. 2011). The absence of Spo11α is associated with XY pairing defects in pachytene and metaphase spermatocytes (Kauppi et al. 2011). This phenotype is similar to that observed in sterile (BALBdomxJF1mol)F1 males. Intriguingly, we found that the BALB allele of the Spo11 gene was apparently suppressed in both reciprocal F1 hybrids, which exhibited monoallelic expression of Spo11JF1/MsJ (Figure 5). With respect to the HS phenotype, a possible explanation for this may be that aberrant expression of the Spo11 gene is a consequence of disrupted trans-regulation of gene expression (Yvert et al. 2003). The cause of this disruption remains unknown, since we also found monoallelic expression of Mlh3, which is harbored on other chromosome. Nevertheless, monoallelic expression results in ablation of gene expression in cis, affecting the splicing process and reducing the expression of Spo11α. Reduced allelic dosage is often compensated either at the transcript level, regardless of monoallelic expression (Eckersley-Maslin et al. 2014), or at the protein level (Homma et al. 2006; Wheway et al. 2013). However, it is possible that the suppression of a single allele may lead to a reduction in global gene expression, affecting the functions of dosage-sensitive genes, such as Spo11 (Cole et al. 2012; Kauppi et al. 2013), because a reduced Spo11 allele dosage cannot be compensated at either the transcript (Bellani et al. 2010) or the protein level (Cole et al. 2012). Aberrant expression of Spo11 in F1 males mimics that observed in heterozygous Spo11-null mutants. Thus, we speculate that downregulation of Spo11 expression in (BALBdomxJF1mol)F1 males may ultimately contribute to XY pairing defects, impairing the formation of DSBs at the PAR.

Defective XY pairing in these sterile hybrids leads to chromosomal misalignment during the meiotic metaphase, accounting for spermatogenic arrest and infertility. This monoallelic expression was also observed for other meiotic gene, although not always in the same direction (Figure 5). Random monoallelic expression has been reported in the neural cells of F1 mice during early embryonic development (Wang et al. 2007; Eckersley-Maslin et al. 2014) and is implicated in genetic disorders (Chess 2013). Based on the DM model, this biased-gene expression of some genes may be incompatible with X-linked loci or genes. Thus, it is possible that Spo11JF1/MsJ is not compatible with the XBALB/cJ chromosome of (BALBdomxJF1mol)F1 males resulting in XY chromosome asynapsis and sterility due to a shortage of DSBs in the PAR, while reciprocal (JF1molxBALBdom)F1 males with Spo11JF1/MsJ and the chromosome X JF1/MsJ, which are compatible, hence they are fertile. The mechanisms underlying the regulation of allele-specific gene expression are thought to be associated with certain histone modifications (Keverne 2009; Eckersley-Maslin et al. 2014) or variants of cis-regulatory elements such as promoters and enhancers (Mack and Nachman 2017). However, the mechanism underlying the aberrant gene expression in (BALBdomxJF1mol)F1 males remains to be determined.

Genomic Incompatibility between domesticus and molossinus Strains Contributes to HS in Intersubspecific F1 Mice

A previous study found dissociation of the XY chromosomes in infertile intersubspecific F1 males from a mating of female BALBdom mice to male Japanese wild mice (molossinus) that was associated with a locus within the PAR termed Sxa (Matsuda et al. 1983). The majority of the X chromosome in the molossinus strains is derived from musculus, while all or the majority of that in the domesticus laboratory strains is derived from domesticus (Yang et al. 2011; Supplementary Figure S2A). Thus, genomic incompatibilities within the X chromosomes may explain the HS phenotype in F1 males. We speculate that the extent of differences in the genomic origin of the X chromosome and the autosomes might affect the severity of any observed HS phenotypes. Our SNP assay identified a region on the X chromosome showing a relatively high density of polymorphic SNPs among some of the tested domesticus laboratory strains (Supplementary Figure S2B). It is tempting to speculate that this region contains a gene (or genes) contributing mechanistically to HS. Phylogenic clustering based on SNPs revealed that BALBdom is distinct from the other tested domesticus laboratory strains (Frazer et al. 2007; Supplementary Figure S2C), due to a higher genomic contribution from domesticus than in other domesticus laboratory stains (Frazer et al. 2007; Yang et al. 2011; Supplementary Figure S2A). Thus, this greater contribution of the domesticus genome, the identified specific region of the Xdom chromosome, or the combination of these may reduce compatibility with the molossinus genome in molossinus–domesticus hybrids, leading to loss of fertility.

The molossinus genome is genetically similar to that of musculus (Yonekawa et al. 1988; Sakai et al. 2005). This finding is thus particularly intriguing, in light of the fact that a musculus-derived X chromosome in a domesticus genetic background causes the HS phenotype in (PWDmusxB6dom)F1 mice (Storchova et al. 2004). In other words, the direction of the effect on the X-chromosome is opposite to that observed in the (BALBdomxJF1mol)F1 HS model. Further genetic analyses, such as genetic mapping to identify chromosome loci and candidate genes, will be important for discovering the mechanisms by which the X chromosome and/or the autosomal region(s) are involved in HS mechanisms between closely related mouse subspecies. Moreover, although most HS mechanisms are thought to be related to the X chromosome (Storchova et al. 2004; Oka et al. 2007; Good et al. 2008a, 2008b; Bhattacharyya et al. 2014; Turner et al. 2014), the roles of the Y chromosome (Campbell et al. 2012), or other autosomes (Turner et al. 2014) cannot be discounted.

In summary, metaphase arrest phenotypes are a hallmark of the asymmetric HS in F1 males produced from a cross between BALB (domesticus) females and JF1 (molossinus) males. In this HS model, disruption of an unknown mechanism regulating the transcription of important meiotic genes affected control of the precise segregation of the XY chromosomes, leading to spermatogenic arrest in these inter-subspecific F1 mice. In the (PWDmusxB6dom)F1 model, Prdm9 plays a key role in controlling homologous pairing and meiotic recombination (Bhattacharyya et al. 2013). The (BALBdomxJF1mol)F1 HS model can also be viewed as a study model for reproductive isolation, since the mechanism behind this HS model may be common among hybrid mammals, in the laboratory and the wild, because a similar phenotype has been reported for both interspecific and intersubspecific mouse hybrids (Matsuda et al. 1991, 1992; Hale et al. 1993). Our constantly accruing knowledge of the different HS mechanisms will ultimately lead to a greater understanding of the early steps in the speciation processes, and will thus contribute to practical endeavors such as the domestication of wild animals.

Funding

This work was supported in part by a program grant from the National Institute of Health (NIH; GM099640), and in part by a fellowship from the Japan Society for the Promotion of Science (JSPS), the Strategic Young Researcher Overseas Visits Program for Accelerating Brain Research.

Data Availability

Details of experimental crosses, antibodies, and primer sequences used in this study are available in the Supplementary Material.

Supplementary Material

Supplementary Figure
Click here for additional data file. (11.5MB, pdf)
Supplementary Table
Click here for additional data file. (17.5KB, docx)

Acknowledgments

We are indebted to the members of the Kunieda, Handel and Okada laboratories, particularly Dr Tanmoy Bhattacharyya, for the discussion of this work in progress. We thank Drs Tanmoy Bhattacharyya, Yuki Okada, Laura Reinholdt, Beverly Richards-Smith, and Hiroki Shibuya for their review on this article.

References

  1. Asano Y, Akiyama K, Tsuji T, Takahashi S, Noguchi J, Kunieda T. 2009. Characterization and linkage mapping of an ENU-induced mutant mouse with defective spermatogenesis. Exp Anim. 58:525–532. [DOI] [PubMed] [Google Scholar]
  2. Balcova M, Faltusova B, Gergelits V, Bhattacharyya T, Mihola O, Trachtulec Z, Knopf C, Fotopulosova V, Chvatalova I, Gregorova S, et al. 2016. Hybrid sterility locus on chromosome X controls meiotic recombination rate in mouse. PLoS Genet. 12:e1005906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ball RL, Fujiwara Y, Sun F, Hu J, Hibbs MA, Handel MA, Carter GW. 2016. Regulatory complexity revealed by integrated cytological and RNA-seq analyses of meiotic substages in mouse spermatocytes. BMC Genomics. 17:628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bayes JJ, Malik HS. 2009. Altered heterochromatin binding by a hybrid sterility protein in Drosophila sibling species. Science. 326:1538–1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bellani MA, Boateng KA, Mcleod D, Camerini-Otero RD. 2010. The expression profile of the major mouse SPO11 isoforms indicates that SPO11beta introduces double strand breaks and suggests that SPO11alpha has an additional role in prophase in both spermatocytes and oocytes. Mol Cell Biol. 30:4391–4403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berg IL, Neumann R, Lam KW, Sarbajna S, Odenthal-Hesse L, May CA, Jeffreys AJ. 2010. PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans. Nat Genet. 42:859–863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Berg IL, Neumann R, Sarbajna S, Odenthal-Hesse L, Butler NJ, Jeffreys AJ. 2011. Variants of the protein PRDM9 differentially regulate a set of human meiotic recombination hotspots highly active in African populations. Proc Natl Acad Sci USA. 108:12378–12383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bhattacharyya T, Gregorova S, Mihola O, Anger M, Sebestova J, Denny P, Simecek P, Forejt J. 2013. Mechanistic basis of infertility of mouse intersubspecific hybrids. Proc Natl Acad Sci USA. 110:E468–E477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bhattacharyya T, Reifova R, Gregorova S, Simecek P, Gergelits V, Mistrik M, Martincova I, Pialek J, Forejt J. 2014. X chromosome control of meiotic chromosome synapsis in mouse inter-subspecific hybrids. PLoS Genet. 10:e1004088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Buard J, Rivals E, Dunoyer De Segonzac D, Garres C, Caminade P, De Massy B, Boursot P. 2014. Diversity of Prdm9 zinc finger array in wild mice unravels new facets of the evolutionary turnover of this coding minisatellite. PLoS One. 9:e85021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Campbell P, Good JM, Dean MD, Tucker PK, Nachman MW. 2012. The contribution of the Y chromosome to hybrid male sterility in house mice. Genetics. 191:1271–1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chess A. 2013. Random and non-random monoallelic expression. Neuropsychopharmacology. 38:55–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cobb J, Reddy RK, Park C, Handel MA. 1997. Analysis of expression and function of topoisomerase I and II during meiosis in male mice. Mol Reprod Dev. 46:489–498. [DOI] [PubMed] [Google Scholar]
  14. Cole F, Kauppi L, Lange J, Roig I, Wang R, Keeney S, Jasin M. 2012. Homeostatic control of recombination is implemented progressively in mouse meiosis. Nat Cell Biol. 14:424–430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dobzhansky T. 1951. Experiments on sexual isolation in Drosophila: X. Reproductive isolation between Drosophila pseudoobscura and Drosophila persimilis under natural and under laboratory conditions. Proc Natl Acad Sci USA. 37:792–796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dzur-Gejdosova M, Simecek P, Gregorova S, Bhattacharyya T, Forejt J. 2012. Dissecting the genetic architecture of F1 hybrid sterility in house mice. Evolution. 66:3321–3335. [DOI] [PubMed] [Google Scholar]
  17. Eckersley-Maslin MA, Thybert D, Bergmann JH, Marioni JC, Flicek P, Spector DL. 2014. Random monoallelic gene expression increases upon embryonic stem cell differentiation. Dev Cell. 28:351–365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Forejt J. 1996. Hybrid sterility in the mouse. Trends Genet. 12:412–417. [DOI] [PubMed] [Google Scholar]
  19. Forejt J, Ivanyi P. 1974. Genetic studies on male sterility of hybrids between laboratory and wild mice (Mus musculus L.). Genet Res. 24:189–206. [DOI] [PubMed] [Google Scholar]
  20. Frazer KA, Eskin E, Kang HM, Bogue MA, Hinds DA, Beilharz EJ, Gupta RV, Montgomery J, Morenzoni MM, Nilsen GB, et al. 2007. A sequence-based variation map of 8.27 million SNPs in inbred mouse strains. Nature. 448:1050–1053. [DOI] [PubMed] [Google Scholar]
  21. Fujiwara Y, Matsumoto H, Akiyama K, Srivastava A, Chikushi M, Ann Handel M, Kunieda T. 2015. An ENU-induced mutation in the mouse Rnf212 gene is associated with male meiotic failure and infertility. Reproduction. 149:67–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fujiwara Y, Ogonuki N, Inoue K, Ogura A, Handel MA, Noguchi J, Kunieda T. 2013. t-SNARE Syntaxin2 (STX2) is implicated in intracellular transport of sulfoglycolipids during meiotic prophase in mouse spermatogenesis. Biol Reprod. 88:141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Good JM, Dean MD, Nachman MW. 2008a. A complex genetic basis to X-linked hybrid male sterility between two species of house mice. Genetics. 179:2213–2228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Good JM, Handel MA, Nachman MW. 2008b. Asymmetry and polymorphism of hybrid male sterility during the early stages of speciation in house mice. Evolution. 62:50–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Groeneveld LF, Atencia R, Garriga RM, Vigilant L. 2012. High diversity at PRDM9 in chimpanzees and bonobos. PLoS One. 7:e39064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Haldane JBS. 1922. Sex ratio and unisexual sterility in hybrid animals. J Genet. 12:101–109. [Google Scholar]
  27. Hale DW, Washburn LL, Eicher EM. 1993. Meiotic abnormalities in hybrid mice of the C57BL/6J x Mus spretus cross suggest a cytogenetic basis for Haldane’s rule of hybrid sterility. Cytogenet Cell Genet. 63:221–234. [DOI] [PubMed] [Google Scholar]
  28. Handel MA, Schimenti JC. 2010. Genetics of mammalian meiosis: regulation, dynamics and impact on fertility. Nat Rev Genet. 11:124–136. [DOI] [PubMed] [Google Scholar]
  29. Homma S, Iwasaki M, Shelton GD, Engvall E, Reed JC, Takayama S. 2006. BAG3 deficiency results in fulminant myopathy and early lethality. Am J Pathol. 169:761–773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Imai HT, Matsuda Y, Shiroishi T, Moriwaki K. 1981. High frequency of X-Y chromosome dissociation in primary spermatocytes of F1 hybrids between Japanese wild mice (Mus musculus molossinus) and inbred laboratory mice. Cytogenet Cell Genet. 29:166–175. [DOI] [PubMed] [Google Scholar]
  31. Kauppi L, Barchi M, Baudat F, Romanienko PJ, Keeney S, Jasin M. 2011. Distinct properties of the XY pseudoautosomal region crucial for male meiosis. Science. 331:916–920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kauppi L, Barchi M, Lange J, Baudat F, Jasin M, Keeney S. 2013. Numerical constraints and feedback control of double-strand breaks in mouse meiosis. Genes Dev. 27:873–886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Keverne B. 2009. Monoallelic gene expression and mammalian evolution. Bioessays. 31:1318–1326. [DOI] [PubMed] [Google Scholar]
  34. Khalaj M, Abbasi A, Yamanishi H, Akiyama K, Wakitani S, Kikuchi S, Hirose M, Yuzuriha M, Magari M, Degheidy HA, et al. 2014. A missense mutation in Rev7 disrupts formation of Polzeta, impairing mouse development and repair of genotoxic agent-induced DNA lesions. J Biol Chem. 289:3811–3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Koide T, Ikeda K, Ogasawara M, Shiroishi T, Moriwaki K, Takahashi A. 2011. A new twist on behavioral genetics by incorporating wild-derived mouse strains. Exp Anim. 60:347–354. [DOI] [PubMed] [Google Scholar]
  36. Kong A, Thorleifsson G, Gudbjartsson DF, Masson G, Sigurdsson A, Jonasdottir A, Walters GB, Jonasdottir A, Gylfason A, Kristinsson KT, et al. 2010. Fine-scale recombination rate differences between sexes, populations and individuals. Nature. 467:1099–1103. [DOI] [PubMed] [Google Scholar]
  37. Kono H, Tamura M, Osada N, Suzuki H, Abe K, Moriwaki K, Ohta K, Shiroishi T. 2014. Prdm9 polymorphism unveils mouse evolutionary tracks. DNA Res. 21:315–326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. La Salle S, Sun F, Handel MA. 2009. Isolation and short-term culture of mouse spermatocytes for analysis of meiosis. Methods Mol Biol. 558:279–297. [DOI] [PubMed] [Google Scholar]
  39. Lipkin SM, Moens PB, Wang V, Lenzi M, Shanmugarajah D, Gilgeous A, Thomas J, Cheng J, Touchman JW, Green ED, et al. 2002. Meiotic arrest and aneuploidy in MLH3-deficient mice. Nat Genet. 31:385–390. [DOI] [PubMed] [Google Scholar]
  40. Mack KL, Nachman MW. 2017. Gene regulation and speciation. Trends Genet. 33:68–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Masly JP, Jones CD, Noor MA, Locke J, Orr HA. 2006. Gene transposition as a cause of hybrid sterility in Drosophila. Science. 313:1448–1450. [DOI] [PubMed] [Google Scholar]
  42. Matsuda Y, Hirobe T, Chapman VM. 1991. Genetic basis of X-Y chromosome dissociation and male sterility in interspecific hybrids. Proc Natl Acad Sci USA. 88:4850–4854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Matsuda Y, Imai HT, Moriwaki K, Kondo K. 1983. Modes of inheritance of X-Y dissociation in inter-subspecies hybrids between BALB/c mice and Mus musculus molossinus. Cytogenet Cell Genet. 35:209–215. [DOI] [PubMed] [Google Scholar]
  44. Matsuda Y, Imai HT, Moriwaki K, Kondo K, Bonhomme F. 1982. X-Y chromosome dissociation in wild derived Mus musculus subspecies, laboratory mice, and their F1 hybrids. Cytogenet Cell Genet. 34:241–252. [DOI] [PubMed] [Google Scholar]
  45. Matsuda Y, Moens PB, Chapman VM. 1992. Deficiency of X and Y chromosomal pairing at meiotic prophase in spermatocytes of sterile interspecific hybrids between laboratory mice (Mus domesticus) and Mus spretus. Chromosoma. 101:483–492. [DOI] [PubMed] [Google Scholar]
  46. Mihola O, Trachtulec Z, Vlcek C, Schimenti JC, Forejt J. 2009. A mouse speciation gene encodes a meiotic histone H3 methyltransferase. Science. 323:373–375. [DOI] [PubMed] [Google Scholar]
  47. Muller HJ. 1942. Isolating mechanisms, evolution and temperature. Biol Symp. 6:71–125. [Google Scholar]
  48. Nagaoka SI, Hassold TJ, Hunt PA. 2012. Human aneuploidy: mechanisms and new insights into an age-old problem. Nat Rev Genet. 13:493–504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Nagasawa H, Miyamoto M, Fujimoto M. 1973. Reproductivity in inbred strains of mice and project for their efficient production (author’s transl). Jikken Dobutsu (Exp Anim). 22:119–126. [DOI] [PubMed] [Google Scholar]
  50. Oka A, Aoto T, Totsuka Y, Takahashi R, Ueda M, Mita A, Sakurai-Yamatani N, Yamamoto H, Kuriki S, Takagi N, et al. 2007. Disruption of genetic interaction between two autosomal regions and the X chromosome causes reproductive isolation between mouse strains derived from different subspecies. Genetics. 175:185–197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Oka A, Mita A, Takada Y, Koseki H, Shiroishi T. 2010. Reproductive isolation in hybrid mice due to spermatogenesis defects at three meiotic stages. Genetics. 186:339–351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Orr HA, Masly JP, Presgraves DC. 2004. Speciation genes. Curr Opin Genet Dev. 14:675–679. [DOI] [PubMed] [Google Scholar]
  53. Perry J, Palmer S, Gabriel A, Ashworth A. 2001. A short pseudoautosomal region in laboratory mice. Genome Res. 11:1826–1832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Phadnis N, Orr HA. 2009. A single gene causes both male sterility and segregation distortion in Drosophila hybrids. Science. 323:376–379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Russell LD, Rttlin RA, Sinhahikim AP, Clegg ED. 1990. Histological and histopathological evaluation of the testis. St. Louis, MO: Cache River Press. [Google Scholar]
  56. Sakai T, Kikkawa Y, Miura I, Inoue T, Moriwaki K, Shiroishi T, Satta Y, Takahata N, Yonekawa H. 2005. Origins of mouse inbred strains deduced from whole-genome scanning by polymorphic microsatellite loci. Mamm Genome. 16:11–19. [DOI] [PubMed] [Google Scholar]
  57. Sawamura K, Maehara K, Mashino S, Kagesawa T, Kajiwara M, Matsuno K, Takahashi A, Takano-Shimizu T. 2010. Introgression of Drosophila simulans nuclear pore protein 160 in Drosophila melanogaster alone does not cause inviability but does cause female sterility. Genetics. 186:669–676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Storchova R, Gregorova SA, Buckiova D, Kyselova V, Divina P, Forejt J. 2004. Genetic analysis of X-linked hybrid sterility in the house mouse. Mamm Genome. 15:515–524. [DOI] [PubMed] [Google Scholar]
  59. Sun S, Ting CT, Wu CI. 2004. The normal function of a speciation gene, Odysseus, and its hybrid sterility effect. Science. 305:81–83. [DOI] [PubMed] [Google Scholar]
  60. Ting CT, Tsaur SC, Wu ML, Wu CI. 1998. A rapidly evolving homeobox at the site of a hybrid sterility gene. Science. 282:1501–1504. [DOI] [PubMed] [Google Scholar]
  61. Turner LM, White MA, Tautz D, Payseur BA. 2014. Genomic networks of hybrid sterility. PLoS Genet. 10:e1004162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wang J, Valo Z, Smith D, Singer-Sam J. 2007. Monoallelic expression of multiple genes in the CNS. PLoS One. 2:e1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wang JR, De Villena FP, Mcmillan L. 2012. Comparative analysis and visualization of multiple collinear genomes. BMC Bioinformatics. 13(Suppl 3):S13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Watanabe Y. 2012. Geometry and force behind kinetochore orientation: lessons from meiosis. Nat Rev Mol Cell Biol. 13:370–382. [DOI] [PubMed] [Google Scholar]
  65. Wheway G, Abdelhamed Z, Natarajan S, Toomes C, Inglehearn C, Johnson CA. 2013. Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel–Gruber syndrome. Dev Biol. 377:55–66. [DOI] [PubMed] [Google Scholar]
  66. White MA, Ikeda A, Payseur BA. 2012. A pronounced evolutionary shift of the pseudoautosomal region boundary in house mice. Mamm Genome. 23:454–466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. White MA, Steffy B, Wiltshire T, Payseur BA. 2011. Genetic dissection of a key reproductive barrier between nascent species of house mice. Genetics. 189:289–304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wickham H. 2009. ggplot2: Elegant graphics for data analysis. New York: Springer-Verlag. [Google Scholar]
  69. Wu CI, Ting CT. 2004. Genes and speciation. Nat Rev Genet. 5:114–122. [DOI] [PubMed] [Google Scholar]
  70. Yang H, Wang JR, Didion JP, Buus RJ, Bell TA, Welsh CE, Bonhomme F, Yu AH, Nachman MW, Pialek J, et al. 2011. Subspecific origin and haplotype diversity in the laboratory mouse. Nat Genet. 43:648–655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Yonekawa H, Moriwaki K, Gotoh O, Miyashita N, Matsushima Y, Shi LM, Cho WS, Zhen XL, Tagashira Y. 1988. Hybrid origin of Japanese mice “Mus musculus molossinus”: evidence from restriction analysis of mitochondrial DNA. Mol Biol Evol. 5:63–78. [DOI] [PubMed] [Google Scholar]
  72. Yvert G, Brem RB, Whittle J, Akey JM, Foss E, Smith EN, Mackelprang R, Kruglyak L. 2003. Trans-acting regulatory variation in Saccharomyces cerevisiae and the role of transcription factors. Nat Genet. 35:57–64. [DOI] [PubMed] [Google Scholar]
  73. Zwemer LM, Zak A, Thompson BR, Kirby A, Daly MJ, Chess A, Gimelbrant AA. 2012. Autosomal monoallelic expression in the mouse. Genome Biol. 13:R10. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure
Click here for additional data file. (11.5MB, pdf)
Supplementary Table
Click here for additional data file. (17.5KB, docx)

Data Availability Statement

Details of experimental crosses, antibodies, and primer sequences used in this study are available in the Supplementary Material.

Articles from Journal of Heredity are provided here courtesy of Oxford University Press

RESOURCES