Genetic analysis of X-linked hybrid sterility in the house mouse

Hybrid sterility is a common postzygotic reproductive isolation mechanism that appears in the early stages of speciation of various organisms. Mus musculus musculus and Mus musculus domesticus represent two recently separated mouse subspecies particularly suitable for genetic studies of hybrid sterility. Here we show that the introgression of Chr X of M. m. musculus origin (PWD/Ph inbred strain, henceforth PWD) into the genetic background of the C57BL/6J (henceforth B6) inbred strain (predominantly of M. m. domesticus origin) causes male sterility. The X-linked hybrid sterility is associated with reduced testes weight, lower sperm count, and morphological abnormalities of sperm heads. The analysis of recombinant Chr Xs in sterile and fertile males as well as quantitative trait locus (QTL) analysis of several fertility parameters revealed an oligogenic nature of the X-linked hybrid sterility. The Hstx1 locus responsible for male sterility was mapped near DXMit119 in the central part of Chr X. To ensure full sterility, the PWD allele of Hstx1 has to be supported with the PWD allelic form of loci in at least one proximal and/or one distal region of Chr X. Mapping and cloning of Hstx1 and other genes responsible for sterility of B6–X^PWDY^B6 males could help to elucidate the special role of Chr X in hybrid sterility and consequently in speciation.     

Polymorphism in hybrid male sterility in wild-derived Mus musculus musculus strains on proximal chromosome 17

The hybrid sterility-1 (Hst1) locus at Chr 17 causes male sterility in crosses between the house mouse subspecies Mus musculus domesticus (Mmd) and M. m. musculus (Mmm). This locus has been defined by its polymorphic variants in two laboratory strains (Mmd genome) when mated to PWD/Ph mice (Mmm genome): C57BL/10 (carrying the sterile allele) and C3H (fertile allele). The occurrence of sterile and/or fertile (wild Mmm × C57BL)F₁ males is evidence that polymorphism for this trait also exists in natural populations of Mmm; however, the nature of this polymorphism remains unclear. Therefore, we derived two wild-origin Mmm strains, STUS and STUF, that produce sterile and fertile males, respectively, in crosses with C57BL mice. To determine the genetic basis underlying male fertility, the (STUS × STUF)F₁ females were mated to C57BL/10 J males. About one-third of resulting hybrid males (33.8%) had a significantly smaller epididymis and testes than parental animals and lacked spermatozoa due to meiotic arrest. A further one-fifth of males (20.3%) also had anomalous reproductive traits but produced some spermatozoa. The remaining fertile males (45.9%) displayed no deviation from values found in parental individuals. QTL analysis of the progeny revealed strong associations of male fitness components with the proximal end of Chr 17, and a significant effect of the central section of Chr X on testes mass. The data suggest that genetic incompatibilities associated with male sterility have evolved independently at the proximal end of Chr 17 and are polymorphic within both Mmd and Mmm genomes.     

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.     

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.     

Prdm9 Incompatibility Controls Oligospermia and Delayed Fertility but No Selfish Transmission in Mouse Intersubspecific Hybrids

PR-domain 9 (Prdm9) is the first hybrid sterility gene identified in mammals. The incompatibility between Prdm9 from Mus musculus domesticus (Mmd; the B6 strain) and the Hstx2 region of chromosome (Chr) X from M. m. musculus (Mmm; the PWD strain) participates in the complete meiotic arrest of mouse intersubspecific (PWD×B6)F1 hybrid males. Other studies suggest that also semisterile intersubspecific hybrids are relevant for mouse speciation, but the genes responsible remain unknown. To investigate the causes of this semisterility, we analyzed the role of Prdm9 and Chr X in hybrids resulting from the crosses of PWK, another Mmm-derived inbred strain. We demonstrate that Prdm9 and Chr X control the partial meiotic arrest and reduced sperm count in (PWK×B6)F1 males. Asynapsis of heterosubspecific chromosomes and semisterility were partially suppressed by removal of the B6 allele of Prdm9. Polymorphisms between PWK and PWD on Chr X but not in the Prdm9 region were responsible for the modification of the outcome of Prdm9 - Chr X F1 hybrid incompatibility. Furthermore, (PWK×B6)F1 hybrid males displayed delayed fertility dependent on the Prdm9 incompatibility. While the Drosophila hybrid sterility gene Overdrive causes both delayed fertility and increased transmission of its own chromosome to the offspring, the segregation of Chr X and the Prdm9 region from the mouse (PWK×B6)F1 males was normal. Our results indicate extended functional consequences of Prdm9 - Chr X intersubspecific incompatibility on the fertility of hybrids and should influence the design of fertility analyses in hybrid zones and of laboratory crosses between Mmm and Mmd strains.     

Comparison of interspecific to intersubspecific backcrosses demonstrates species and sex differences in recombination frequency on mouse Chromosome 16

One hundred fourteen progeny from an interspecific backcross between laboratory mice and M. spretus were typed for six markers spanning most of mouse Chromosome (Chr) 16. Additional maps of 9–10 markers of this chromosome were derived from analysis of over 500 progeny from four backcrosses between inbred laboratory strains and members of the Mus musculus group, M.m. musculus and M.m. molossinus (subspecies). The results of these analyses confirmed the gene order: (CEN)-Prm-1/Prm-2-Igl-1-Smst-Mtv-6-Gap43-Pit-1(dw)-D21S16h-App-Sod-1-Ets-2-Mx. Maps produced from these five crosses were of similar lengths, but recombination in several regions was affected by sex of the F₁ parent or by the combination of strains used in the cross. As reported previously, recombination frequencies were elevated significantly at the distal end of the chromosome in a cross using F₁ males. The male map showed significant compression in the interval Smst to Gap43. Both male and female intersubspecific maps were expanded near the proximal and distal ends of the chromosome relative to the interspecific cross. The spretus cross was compressed in the proximal interval, Prm-1-Igl-1-Smst, and was slightly expanded in the Smst-Gap43 interval, relative to intersubspecific crosses using F₁ females. Female intersubspecific maps were expanded about 50% near the distal end of the chromosome when compared to the interspecific cross. The expansion or compression of maps using different strain or sex combinations has implications for the efficient production of high resolution recombinational maps of the mouse genome.     

Genetic dissection of a key reproductive barrier between nascent species of house mice

Reproductive isolation between species is often caused by deleterious interactions among loci in hybrids. Finding the genes involved in these incompatibilities provides insight into the mechanisms of speciation. With recently diverged subspecies, house mice provide a powerful system for understanding the genetics of reproductive isolation early in the speciation process. Although previous studies have yielded important clues about the genetics of hybrid male sterility in house mice, they have been restricted to F1 sterility or incompatibilities involving the X chromosome. To provide a more complete characterization of this key reproductive barrier, we conducted an F2 intercross between wild-derived inbred strains from two subspecies of house mice, Mus musculus musculus and Mus musculus domesticus. We identified a suite of autosomal and X-linked QTL that underlie measures of hybrid male sterility, including testis weight, sperm density, and sperm morphology. In many cases, the autosomal loci were unique to a specific sterility trait and exhibited an effect only when homozygous, underscoring the importance of examining reproductive barriers beyond the F1 generation. We also found novel two-locus incompatibilities between the M. m. musculus X chromosome and M. m. domesticus autosomal alleles. Our results reveal a complex genetic architecture for hybrid male sterility and suggest a prominent role for reproductive barriers in advanced generations in maintaining subspecies integrity in house mice.     

Genetic analysis of protein variations in Mus musculus using two-dimensional electrophoresis

We have investigated the variation of proteins from crude homogenates of mouse kidneys in several strains of Mus musculus by means of two-dimensional electrophoresis. In this study, we have used the strains C57BL/6J, DBA/2J, CD-1, M. m. castaneus, and M. m. molossinus, as well as offspring from crosses among these strains. Out of the 100 loci screened, we have found nine loci showing interstrain differences. We have been able to identify three proteins as Id-1, Car-2, and Sep-1. The remaining variants are probably new loci in the mouse. Most of the variants (seven) can be mapped to a chromosome. We have found also that differences in the protein pattern as seen on two-dimensional gels are small among subspecies of Mus musculus.     

Genetic linkage analysis of X-ray hypersensitivity in the LEC mutant rat

The LEC rat has been reported to exhibit X-ray hypersensitivity and deficiency in DNA double-strand break (DSB) repair. The present study was performed to map the locus responsible for this phenotype, the xhs (X-ray hypersensitivity), as the first step in identifying the responsible gene. Analysis of the progeny of (BN × LEC)F₁× LEC backcrosses indicated that the X-ray hypersensitive phenotype was controlled by multiple genetic loci in contrast to the results reported previously. Quantitative trait loci (QTL) linkage analysis revealed two responsible loci located on Chromosomes (Chr) 4 and 1. QTL on Chr 4 exhibited very strong linkage to the X-ray hypersensitive phenotype, while QTL on Chr 1 showed weak linkage. The Rad52 locus, mutation of which results in hypersensitivity to ionizing radiation and impairment of DNA DSB repair in yeast, was reported to be located on the synteneic regions of mouse Chr 6 and human Chr 12. However, mapping of the rat Rad52 locus indicated that it was located 23 cM distal to the QTL on Chr 4. Furthermore, none of the radio-sensitivity-related loci mapped previously in the rat chromosome were identical to the QTL on Chrs 4 and 1 in the LEC rat. Thus, it seems that X-ray hypersensitivity in the LEC rat is caused by mutation(s) in as-yet-undefined genes. Received: 14 February 2000 / Accepted: 17 May 2000     

Maternal–foetal genomic conflict and speciation: no evidence for hybrid placental dysplasia in crosses between two house mouse subspecies

Interspecific hybridization between closely related mammalian species, including various species of the genus Mus, is commonly associated with abnormal growth of the placenta and hybrid foetuses, a phenomenon known as hybrid placental dysplasia (HPD). The role of HPD in speciation is anticipated but still poorly understood. Here, we studied placental and foetal growth in F₁ crosses between four inbred mouse strains derived from two house mouse subspecies, Mus musculus musculus and Mus musculus domesticus. These subspecies are in the early stage of speciation and still hybridize in nature. In accordance with the maternal–foetal genomic conflict hypothesis, we found different parental influences on placental and foetal development, with placental weight most affected by the father's body weight and foetal weight by the mother's body weight. After removing the effects of parents’ body weight, we did not find any significant differences in foetal or placental weights between intra‐subspecific and inter‐subspecific F₁ crosses. Nevertheless, we found that the variability in placental weight in inter‐subspecific crosses is linked to the X chromosome, similarly as for HPD in interspecific mouse crosses. Our results suggest that maternal–foetal genomic conflict occurs in the house mouse system, but has not yet diverged sufficiently to cause abnormalities in placental and foetal growth in inter‐subspecific crosses. HPD is thus unlikely to contribute to speciation in the house mouse system. However, we cannot rule out that it might have contributed to other speciation events in the genus Mus, where differences in the levels of polyandry exist between the species.     

GENETICS OF STERILITY IN HYBRIDS BETWEEN TWO SUBSPECIES OF DROSOPHILA

Hybrids between D. pseudoobscura bogotana and D. pseudoobscura pseudoobscura are fertile except for males produced in one of the two reciprocal crosses. As there is no premating isolation between these subspecies, nonreciprocal male sterility represents the first step in speciation. Genetic analysis reveals two causes of hybrid F₁ sterility: a maternal effect and incompatibilities between chromosomes within males. The maternal effect appears to play the greatest role in hybrid sterility. The X chromosome has the largest effect on fertility of any chromosome, a ubiquitous result in analyses of hybrid sterility and inviability in Drosophila. This effect is entirely attributable to a region comprising less than 30% of the X chromosome. These results are compared to those from a similar study of D. pseudoobscura-D. persimilis hybrids, an older and more reproductively isolated species pair in the same lineage. Such comparisons may allow one to identify the genetic changes characterizing the early versus late stages of speciation.     

The uncharacterized gene 1700093K21Rik and flanking regions are correlated with reproductive isolation in the house mouse,Mus musculus

Reproductive barriers exist between the house mouse subspecies, Mus musculus musculus and M. m. domesticus, members of the Mus musculus species complex, primarily as a result of hybrid male infertility, and a hybrid zone exists where their ranges intersect in Europe. Using single nucleotide polymorphisms (SNPs) diagnostic for the two taxa, the extent of introgression across the genome was previously compared in these hybrid populations. Sixty-nine of 1316 autosomal SNPs exhibited reduced introgression in two hybrid zone transects suggesting maladaptive interactions among certain loci. One of these markers is within a region on chromosome 11 that, in other studies, has been associated with hybrid male sterility of these subspecies. We assessed sequence variation in a 20 Mb region on chromosome 11 flanking this marker, and observed its inclusion within a roughly 150 kb stretch of DNA showing elevated sequence differentiation between the two subspecies. Four genes are associated with this genomic subregion, with two entirely encompassed. One of the two genes, the uncharacterized 1700093K21Rik gene, displays distinguishing features consistent with a potential role in reproductive isolation between these subspecies. Along with its expression specifically within spermatogenic cells, we present various sequence analyses that demonstrate a high rate of molecular evolution of this gene, as well as identify a subspecies amino acid variant resulting in a structural difference. Taken together, the data suggest a role for this gene in reproductive isolation.     

Identification of quantitative trait loci for locomotor activation and anxiety using closely related inbred strains

We carried out a quantitative trait loci (QTL) mapping experiment in two phenotypically similar inbred mouse strains, C57BL/6J and C58/J, using the open‐field assay, a well‐established model of anxiety‐related behavior in rodents. This intercross was initially carried out as a control cross for an ethylnitrosurea mutagenesis mapping study. Surprisingly, although open‐field behavior is similar in the two strains, we identified significant QTL in their F₂ progeny. Marker regression identified a locus on Chr 8 having associations with multiple open‐field measures and a significant interaction between loci on Chr 13 and 17. Together, the Chr 8 locus and the interaction effect form the core set of QTL controlling these behaviors with additional loci on Chr 1 and 6 present in a subset of the behaviors.     

Genetic analysis of genome-scale recombination rate evolution in house mice

The rate of meiotic recombination varies markedly between species and among individuals. Classical genetic experiments demonstrated a heritable component to population variation in recombination rate, and specific sequence variants that contribute to recombination rate differences between individuals have recently been identified. Despite these advances, the genetic basis of species divergence in recombination rate remains unexplored. Using a cytological assay that allows direct in situ imaging of recombination events in spermatocytes, we report a large (∼30%) difference in global recombination rate between males of two closely related house mouse subspecies (Mus musculus musculus and M. m. castaneus). To characterize the genetic basis of this recombination rate divergence, we generated an F2 panel of inter-subspecific hybrid males (n = 276) from an intercross between wild-derived inbred strains CAST/EiJ (M. m. castaneus) and PWD/PhJ (M. m. musculus). We uncover considerable heritable variation for recombination rate among males from this mapping population. Much of the F2 variance for recombination rate and a substantial portion of the difference in recombination rate between the parental strains is explained by eight moderate- to large-effect quantitative trait loci, including two transgressive loci on the X chromosome. In contrast to the rapid evolution observed in males, female CAST/EiJ and PWD/PhJ animals show minimal divergence in recombination rate (∼5%). The existence of loci on the X chromosome suggests a genetic mechanism to explain this male-biased evolution. Our results provide an initial map of the genetic changes underlying subspecies differences in genome-scale recombination rate and underscore the power of the house mouse system for understanding the evolution of this trait.     

Quantitative trait loci affecting growth in high growth (hg) mice

A genome-wide scan was performed in order to identify Quantitative Trait Loci (QTL) associated with growth in a population segregating high growth (hg), a partially recessive mutation that enhances growth rate and body size in the mouse. A sample of 262 hg/hg mice was selected from a C57BL/6J-hg/hg× CAST/EiJ F₂ cross and typed with 79 SSLP markers distributed across the genome. Eight significant loci were identified through interval mapping. Loci on Chromosomes (Chrs) 2 and 8 affected the growth rate of F₂ mice. Loci on Chr 2 and 11 affected growth rate and carcass lean mass (protein and ash). A locus on Chr 9 modified femur length and another one in Chr 17 affected both carcass lean mass and femur length, but none of these had significant effects on growth rate. Loci on Chrs 5 and 9 modified carcass fat content. Additive effects were positive for C57BL/6J alleles, except for the two loci affecting carcass fatness. Typing of selected markers in 274 +/+ F₂ mice revealed significant interactions between hg and other growth QTL, which were detected as changes in gene action (additive or dominant) and in allele substitution effects. Knowledge about interactions between loci, especially when major genes are involved, will help in the identification of positional candidate genes and in the understanding of the complex genetic regulation of growth rate and body size in mammals. Received: 29 June 2000 / Accepted: 22 November 2000     

Quantitative trait locus analysis for hemostasis and thrombosis

Susceptibility to thrombosis varies in human populations as well as many in inbred mouse strains. The objective of this study was to characterize the genetic control of thrombotic risk on three chromosomes. Previously, utilizing a tail-bleeding/rebleeding assay as a surrogate of hemostasis and thrombosis function, three mouse chromosome substitution strains (CSS) (B6-Chr5^A/J, Chr11^A/J _, Chr17^A/J) were identified (Hmtb1, Hmtb2, Hmtb3). The tail-bleeding/rebleeding assay is widely used and distinguishes mice with genetic defects in blood clot formation or dissolution. In the present study, quantitative trait locus (QTL) analysis revealed a significant locus for rebleeding (clot stability) time (time between cessation of initial bleeding and start of the second bleeding) on chromosome 5, suggestive loci for bleeding time (time between start of bleeding and cessation of bleeding) also on chromosomes 5, and two suggestive loci for clot stability on chromosome 17 and one on chromosome 11. The three CSS and the parent A/J had elevated clot stability time. There was no interaction of genes on chromosome 11 with genes on chromosome 5 or chromosome 17. On chromosome 17, twenty-three candidate genes were identified in synteny with previously identified loci for thrombotic risk on human chromosome 18. Thus, we have identified new QTLs and candidate genes not previously known to influence thrombotic risk. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Prdm9 Intersubspecific Interactions in Hybrid Male Sterility of House Mouse

The classical definition posits hybrid sterility as a phenomenon when two parental taxa each of which is fertile produce a hybrid that is sterile. The first hybrid sterility gene in vertebrates, Prdm9, coding for a histone methyltransferase, was identified in crosses between two laboratory mouse strains derived from Mus mus musculus and M. m. domesticus subspecies. The unique function of PRDM9 protein in the initiation of meiotic recombination led to the discovery of the basic molecular mechanism of hybrid sterility in laboratory crosses. However, the role of this protein as a component of reproductive barrier outside the laboratory model remained unclear. Here, we show that the Prdm9 allelic incompatibilities represent the primary cause of reduced fertility in intersubspecific hybrids between M. m. musculus and M. m. domesticus including 16 musculus and domesticus wild-derived strains. Disruption of fertility phenotypes correlated with the rate of failure of synapsis between homologous chromosomes in meiosis I and with early meiotic arrest. All phenotypes were restored to normal when the domesticus Prdm9^dom2 allele was substituted with the Prdm9^dom2H humanized variant. To conclude, our data show for the first time the male infertility of wild-derived musculus and domesticus subspecies F1 hybrids controlled by Prdm9 as the major hybrid sterility gene. The impairment of fertility surrogates, testes weight and sperm count, correlated with increasing difficulties of meiotic synapsis of homologous chromosomes and with meiotic arrest, which we suppose reflect the increasing asymmetry of PRDM9-dependent DNA double-strand breaks.     

Chromosome X modulates incidence of testicular germ cell tumors in <Emphasis Type="Italic">Ter</Emphasis> mice

Germ cell tumor development in humans has been proposed to be part of testicular dysgenesis syndrome (TDS), which manifests as undescended testes, sterility, hypospadias, and, in extreme cases, as germ cell tumors. Males of the Ter mouse strain show interesting parallels to TDS because they either lack germ cells and are sterile or develop testicular germ cell tumors. We found that these defects in Ter mice are due to mutational inactivation of the Dead-end (Dnd1) gene. Here we report that chromosome X modulates germ cell tumor development in Ter mice. We tested whether the X or the Y chromosome influences tumor incidence. We used chromosome substitution strains to generate two new mouse strains: 129-Ter/Ter that carry either a C57BL/6J (B6)-derived chromosome (Chr) X or Y. We found that Ter/Ter males with B6-Chr X, but not B6-Chr Y, showed a significant shift in propensity from testicular tumor development to sterile testes phenotype. Thus, our studies provide unambiguous evidence that genetic factors from Chr X modulate the incidence of germ cell tumors in mice with inactivated Dnd1. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.