Male sterility of the mouse t-complex is due to homozygosity of the distorter genes

Evidence is presented that the male sterility produced by the mouse t-complex is due to interaction of at least three sterility factors. These factors are carried in the same partial haplotypes as the three distorter genes, Tcd-1, Tcd-2, and Tcd-3 and are suggested to be identical with them. When heterozygous, the distorter/sterility genes act on the wild-type form of the responder gene, rendering sperm carrying it nonfunctional, thus leading to high transmission of the t form of the responder. When homozygous, the harmful effects of the distorter genes are stronger and affect both forms of the responder, leading to sterility. If homozygous sterility is an inescapable part of ratio distortion, then the t-lethals confer a selective advantage in removing sterile males from the population. Thus, the relationship between the various properties of the t-complex can now be understood.     

Narrowing the critical regions for mouse t complex transmission ratio distortion factors by use of deletions

Previously a deletion in mouse chromosome 17, T(22H), was shown to behave like a t allele of the t complex distorter gene Tcd1, and this was attributed to deletion of this locus. Seven further deletions are studied here, with the aim of narrowing the critical region in which Tcd1 must lie. One deletion, T(30H), together with three others, T(31H), T(33H), and T(36H), which extended more proximally, caused male sterility when heterozygous with a complete t haplotype and also enhanced transmission ratio of the partial t haplotype t(6), and this was attributed to deletion of the Tcd1 locus. The deletions T(29H), T(32H), and T(34H) that extended less proximally than T(30H) permitted male fertility when opposite a complete t haplotype. These results enabled narrowing of the critical interval for Tcd1 to between the markers D17Mit164 and D17Leh48. In addition, T(29H) and T(32H) enhanced the transmission ratio of t(6), but significantly less so than T(30H). T(34H) had no effect on transmission ratio. These results could be explained by a new distorter located between the breakpoints of T(29H) and T(34H) (between T and D17Leh66E). It is suggested that the original distorter Tcd1 in fact consists of two loci: Tcd1a, lying between D17Mit164 and D17Leh48, and Tcd1b, lying between T and D17Leh66E.     

The nucleoside diphosphate kinase gene Nme3 acts as quantitative trait locus promoting non-Mendelian inheritance

The t-haplotype, a variant form of the t-complex region on mouse chromosome 17, acts as selfish genetic element and is transmitted at high frequencies (> 95%) from heterozygous (t/+) males to their offspring. This phenotype is termed transmission ratio distortion (TRD) and is caused by the interaction of the t-complex responder (Tcr) with several quantitative trait loci (QTL), the t-complex distorters (Tcd1 to Tcd4), all located within the t-haplotype region. Current data suggest that the distorters collectively impair motility of all sperm derived from t/+ males; t-sperm is rescued by the responder, whereas (+)-sperm remains partially dysfunctional. Recently we have identified two distorters as regulators of RHO small G proteins. Here we show that the nucleoside diphosphate kinase gene Nme3 acts as a QTL on TRD. Reduction of the Nme3 dosage by gene targeting of the wild-type allele enhanced the transmission rate of the t-haplotype and phenocopied distorter function. Genetic and biochemical analysis showed that the t-allele of Nme3 harbors a mutation (P89S) that compromises enzymatic activity of the protein and genetically acts as a hypomorph. Transgenic overexpression of the Nme3 t-allele reduced t-haplotype transmission, proving it to be a distorter. We propose that the NME3 protein interacts with RHO signaling cascades to impair sperm motility through hyperactivation of SMOK, the wild-type form of the responder. This deleterious effect of the distorters is counter-balanced by the responder, SMOK(Tcr), a dominant-negative protein kinase exclusively expressed in t-sperm, thus permitting selfish behaviour and preferential transmission of the t-haplotype. In addition, the previously reported association of NME family members with RHO signaling in somatic cell motility and metastasis, in conjunction with our data involving RHO signaling in sperm motility, suggests a functional conservation between mechanisms for motility control in somatic cells and spermatozoa.     

Transmission ratio distortion in mouse t-haplotypes is due to multiple distorter genes acting on a responder locus

Transmission ratios of male mice heterozygous for various combinations of partial t-haplotypes provide evidence in support of a model for the genetic basis of ratio distortion, involving two or more distorter genes acting on a responder locus. The t form of the responder locus, Tcr, in the medial part of the haplotype, must be present and heterozygous for distortion to occur. When the responder alone is present, as in t^low haplotypes, the chromosome carrying it is transmitted in a low ratio (<50%). The t forms of the distorter loci act additively, in cis or trans, to raise the transmission of whichever chromosome carries Tcr. Identified distorter loci are Tcd-1, in the proximal part of the haplotype, Tcd-2, distal to Tcr, and probably Tcd-3, lying between Tcr and Tcd-2. In the absence of Tcr the distorters are transmitted normally. The system is compared with the SD system of Drosophila.     

Gene dosage effects on transmission ratio distortion and fertility in mice that carry t haplotypes

Complete t haplotypes can be transmitted at distorted ratios from heterozygous +/t male mice as a consequence of t-specific alleles at a series of t complex distorter loci (Tcd-1t through Tcd-4t) and a t complex responder locus. Partial t haplotypes that lack the Tcd-2t allele cannot be transmitted at the very high ratios characteristic of complete t haplotypes. The breeding studies reported here tested the possibility that the absence of Tcd-2t could be compensated for by the presence of double doses of other Tcdt alleles. The results indicate that a double dose of Tcd-4t alone will not work, but that a double dose of both Tcd-1t and Tcd-4t can promote a very high transmission ratio in the absence of Tcd-2t. These results suggest that the extent to which transmission ratios are distorted is dependent upon the absolute level of expression of the individual Tcd genes. Further studies of genotypic effects on transmission ratio distortion, as well as fertility, lead to the suggestion of a fifth t complex distorter (Tcd-5) locus within t haplotypes.     

Elevated galactosyltransferase activity on t-bearing sperm segregates with T/t-complex distorter loci-2

Sperm bearing complete t-haplotypes are preferentially transmitted during fertilization from heterozygous +/t males, often in excess of 95% relative to their (+)-bearing meiotic partner. Sperm from t-bearing males have an approximate two- to fourfold increase in beta 1,4-galactosyltransferase (GalTase) activity, a cell surface protein that mediates sperm binding to the egg zona pellucida. The elevated GalTase activity strictly correlates with the preferential transmission of t-sperm from +/t males, since eight other enzymes show normal levels of activity on t-sperm. Furthermore, sperm bearing proximal partial t-haplotypes, which are no longer favoured during fertilization, have normal levels of GalTase activity. Nevertheless, it has been unclear whether the elevated sperm GalTase activity on t-sperm is due to specific loci in the distal segment of the T/t-complex, or rather, is an indirect consequence of the abnormal sperm function characteristic of +/t and tx/ty males. In this study, it is shown that the elevated sperm GalTase activity is due specifically to factors that reside within the distal segment of the T/t complex, which also contains Tcd-2, the strongest of the distorter loci. Since the structural locus for GalTase is located on mouse chromosome 4, these results also show that T/t-complex alleles on chromosome 17 are regulatory in nature and affect the expression of sperm surface components critical for normal fertilization. Models are presented to explain how elevated GalTase activity could contribute to sperm transmission distortion.     

Genetic and Molecular Analysis of the Proximal Region of the Mouse T-Complex Using New Molecular Probes and Partial T-Haplotypes

The t-complex is located on the proximal third of chromosome 17 in the house mouse. Naturally occurring variant forms of the t-complex, known as complete t-haplotypes, are found in wild mouse populations. The t-haplotypes contain at least four nonoverlapping inversions that suppress recombination with the wild-type chromosome, and lock into strong linkage disequilibrium loci affecting normal transmission of the chromosome, male gametogenesis and embryonic development. Partial t-haplotypes derived through rare recombination between t-haplotypes and wild-type homologs have been critical in the analysis of these properties. Utilizing two new DNA probes. Au3 and Au9, and several previously described probes, we have analyzed the genetic structure of several partial t-haplotypes that have arisen in our laboratory, as well as several wild-type chromosomes deleted for loci in this region. With this approach we have been able to further our understanding of the structural and dynamic characteristics of the proximal region of the t-complex. Specifically, we have localized the D17Tul locus as most proximal known in t-haplotypes, achieved a better structural analysis of the partial t-haplotype t6, and defined the structure and lethal gene content of partial t-haplotypes derived from the lethal tw73 haplotype.     

Deletion analysis of male sterility effects of t-haplotypes in the mouse

We present data on the effects of three chromosome 17 deletions on transmission ratio distortion (TRD) and sterility of several t-haplotypes. All three deletions have similar effects on male TRD: that is, Tdel/tcomplete genotypes all transmit their t-haplotype in very high proportion. However, each deletion has different effects on sterility of heterozygous males, with TOr/t being fertile, Thp/t less fertile, and TOrl/t still less fertile. These data suggest that wild-type genes on chromosomes homologous to t-haplotypes can be important regulators of both TRD and fertility in males, and that the wild-type genes concerned with TRD and fertility are at least to some extent different. The data also provide a rough map of the positions of these genes.     

Transmission ratio disruption, sterility, and control of t-complex function in sperm

Mouse t-complex located on chromosome 17 contains genes affecting solely male fertility. Some genes of this complex are recessive lethals; nonetheless, the high frequency of the t-complex carriers in a population is maintained due to a mechanism referred to as transmission ratio distortion (TRD), i.e., after crosses with wild-type females, males heterozygous for the t-complex transmit the t-bearing chromosome to nearly all their offspring, which suggests that the t-complex genes control sperm function. Analysis of this phenomenon shows that the resultant TRD is determined by the ratio between the distorter genes (Tcd) and a responder gene (Tcr) located within the t-complex region. Many authors believe that two to six distorter genes currently known have an additive effect. A genetic model of the non-Mendelian inheritance in the progeny of heterozygous male mice specifically explains sterility of animals carrying the t-complex with complementary lethal genes. The model suggests that some distorter gene products interacting with the responder gene have a selective effect on motility of both mutant and wild-type sperm. Insufficient sperm motility and/or their unsuccessful capacitation result in poor if any fertilization. Information on the t-complex genes is necessary for understanding the biological mechanisms of male sterility and may be used in medical practice.     

The Influence of Genetic Background and the Homologous Chromosome 17 on t-Haplotype Transmission Ratio Distortion in Mice

Transmission ratio distortion is a characteristic of complete t-haplotypes, such that heterozygous males preferentially transmit the t-haplotype bearing chromosome 17 to the majority of their progeny. At least two genes contained within the t-haplotype have been identified as being required for such high transmission ratios. In this study we examine the effects of the genetic background and the chromosome homologous to the t-haplotype on transmission ratio distortion. We use two different congenic lines: BTBRTF/Nev.Ttf/t12, in which the t12 haplotype has a transmission ratio of 52%, and C3H/DiSn.Ttf/t12, in which the t12 haplotype has a transmission ratio of 99%. By intercrossing these two strains to produce reciprocal F1 and F2 generations, we have isolated the effects of the homologous chromosome 17 from the effects of the genetic background. We demonstrate that both the homologous chromosome and the genetic background have profound effects on t-haplotype transmission ratio distortion. Furthermore, it is evident that the t-haplotype transmission ratio behaves as a quantitative character rather than an intrinsic property of t-haplotypes.     

Search for differences among t haplotypes in distorter and responder genes

Transmission ratio distortion due to the mouse t complex is though to be due to harmful effects of trans-acting distorter genes acting on a responder, with the t complex form of the responder being relatively resistant to this harmful action of the distorters. Previous work had indicated that naturally occurring t haplotypes differed in their responders or in distorters lying near the responder, with the result that animals doubly heterozygous for two responder-carrying haplotypes transmitted these haplotypes unequally. In the present work t haplotypes could be divided into three types on the basis of their transmission when doubly heterozygous with the responder-carrying partial haplotype tlowH. The majority, t0, t6, tw1, tw2 and tw73, were transmitted equally with tlowH, a second group, including tw5 and two haplotypes derived from it, were transmitted less frequently than tlowH, and the single member of a third group, tw32, was transmitted in excess of tlowH. This last result suggests that the underlying differences are in the responder itself, rather than in the distorters. Search for differences among t haplotypes in distorters produced some equivocal results possibly resulting from effects of genetic background. In particular, results of others suggesting presence of a fourth distorter, Tcd-4, were not confirmed.     

Sperm motility-activating complex formed by t-complex distorters

Transmission ratio distortion is a dramatic example of non-Mendelian transmission. In mice, t-haplotype males produce dysfunctional +-sperm and normal t-sperm, leading to transmission in favor of t-sperm. Genetic studies have indicated that the t-complex responder locus, Tcr, rescues t-sperm but not +-sperm from defective products of t-complex distorter loci, Tcds. Light chain 1 (LC1) and LC3 from sea urchin sperm outer arm dynein have sequence similarities to Tctex2 and Tctex1, respectively, both of which are wild-type products of Tcds. We show here that LC1 and LC3 are able to make a 1:1 complex. Since Tcr is a member of the Smok (sperm motility kinase) family and LC1 is phosphorylated at the activation of sperm motility in a cAMP-dependent manner, this complex in a dynein motor molecule might be a direct target of Smok/Tcr kinase in a signal cascade that regulates sperm motility. Thus, we designate it as Smoac (sperm motility activating complex).     

Physical mapping of male fertility and meiotic drive quantitative trait loci in the mouse t complex using chromosome deficiencies

The t complex spans 20 cM of the proximal region of mouse chromosome 17. A variant form, the t haplotype (t), exists at significant frequencies in wild mouse populations and is characterized by the presence of inversions that suppress recombination with wild-type (+) chromosomes. Transmission ratio distortion and sterility are associated with t and affect males only. It is hypothesized that these phenomena are caused by trans-acting distorter/sterility factors that interact with a responder locus (Tcr(t)) and that the distorter and sterility factors are the same because homozygosity of the distorters causes male sterility. One factor, Tcd1, was previously shown to be amorphic using a chromosome deletion. To overcome limitations imposed by recombination suppression, we used a series of deletions within the t complex in trans to t chromosomes to characterize the Tcd1 region. We find that the distorter activity of Tcd1 is distinct from a linked sterility factor, originally called tcs1. YACs mapped with respect to deletion breakpoints localize tcs1 to a 1.1-Mb interval flanked by D17Aus9 and Tctex1. We present evidence for the existence of multiple proximal t complex regions that exhibit distorter activity. These studies demonstrate the utility of chromosome deletions for complex trait analysis.     

Genetic Basis of Mating Preferences in Wild House Mice

This paper reviews work conducted over the last several yearson the effect of genetic differences within the t-complex ofwild house mice on female mating preference. Wild mice are polymorphicfor a mutation within the t complex on chromosome 17. About25% of wild mice are heterozygous (+/t) for a t-haplotype andthe remainder are +/+. These t-haplotypes have a number of deleteriouseffects when homozygous and hence t/t individuals are rarelyfound in wild populations. We have examined preferences of +/+and +/t females for males of both genotypes. We have found that+/t, but not +/+ females have strong preferences for +/+ males.These preferences can be modified by a variety of factors includingestrous condition of the female (the preferences are strongeramong estrous than diestrous females) and the dominance statusof the male (when forced to choose, females give priority tomale dominance status over t complex genotype in choosing males).The restiction of preference to +/t females indicates that geneson t haplotypes modulate these preferences. Because t haplotypesinclude the major histocompatibility complex (MHC) of the mousewe designed a study to ascertain whether the preferences of+/t females were associated with the MHC. Results of the studyindicate that the preferences are independent of the MHC. Furtherwork testing females carrying a partial t-haplotype (t^w18) indicatesthat the genes for mating preference are localized in the regionof the t complex distal to the MHC. A large number of t haplotypesare found in wild mouse populations. Females that are themselves+/t when forced to choose between 2 +/t males (one carryinga haplotype that is the same as their own and one carrying ahaplotype that is different) prefer males carrying t-haplotypesthat differ from their own. Finally, we conclude that matingpreference may only be a weak force regulating the frequencyof t-mutations in wild mouse populations. The impact of matingpreference on population genetics of genes within this regionis muted because of the great importance of male dominance rankin determining mating patterns within interacting social groups.     

Functional analysis of mutations on murine chromosome 17 using tertiary trisomy

Analysis of the functional nature of mutations can be based on their manifestation in organisms with a deletion or a duplication of a particular chromosome segment. With the use of reciprocal translocation T(16;17)43H it is feasible to produce mice with tertiary trisomy for proximal region of chromosome 17. The mutations on chromosome 17 we tested included brachyury (T), hairpin tail (Thp), kinky (Fuki), quaking (qk), tufted (tf), as well as tct (t-complex tail interaction) and tcl (t complex lethal), that are specific for t haplotypes. The set of dominant and recessive mutations was assigned to two groups, one obligatory manifesting itself in the phenotype independently of the number of normal alleles in di- and trisomics, and the other facultative, phenotypically manifesting itself, depending upon the dosage of mutant alleles. A model was derived from analysis of the interaction of the T and Thp mutations with t haplotypes which is to explain the morphogenetic effects of the mutations observed in mice of different genotypes. The tir gene is postulated to reside on chromosome 17 within its framework. It is suggested that the gene dosage ratio at the tir and tct loci determines tail length.     

Female Preferences Based on Male Quality in House Mice: Interaction between Male Dominance Rank and t-Complex Genotype

Considerable evidence indicates that female house mice (Mus domesticus) prefer dominant over subordinate males as mates. In addition, male genotype at the t-complex seems to be an important characteristic used by females in mate choice. Specifically, female mice that carry a t-haplotype at the t-complex prefer +/+ over +/t males as mates. The purpose of the present study was to examine the relative contributions of male dominance rank and male t-complex genotype to female mating preference when both factors were systematically varied. We tested females of three genotypes (+/+, +/t, and t/t) in a preference apparatus using pairs of stimulus males varying in relative dominance status and t-complex genotype. In general, when given the choice, females preferred dominant over subordinate males regardless of the male's t-complex genotype. The preference for dominant males was manifested when both stimulus males were of the same t-complex genotype but differed in dominance rank. In addition, when forced to choose between a dominant +/+ and subordinate +/t or between a dominant +/t and subordinate +/+, females continued to prefer the dominant male. Preference for dominant males was independent of female genotype. Only when both males were dominant but differed in t-complex genotype (i.e. one male was +/+ and the other +/t) or when males were unranked (i.e. had not been used in aggressive encounters to determine dominance rank) did females carrying t-haplotypes manifest preferences for +/+ males. Quite unexpectedly, when both males were subordinate but differed in t-complex genotype, preferences of all females shifted in the direction of the +/t male. It is not clear from present data whether the propensity of females to give greater weight to male dominance rank than to t-complex genotype in choosing mates results in greater fitness. However, if these trends are found in natural populations, it would indicate that the role of mating preference in regulating the frequency of t-haplotypes in wild populations is less straightforward than had been previously thought.     

New Molecular Markers for the Distal End of the T-Complex and Their Relationships to Mutations Affecting Mouse Development

Many mutations affecting mouse development have been mapped to the t-complex of mouse chromosome 17. We have obtained 17 cosmid clones as molecular markers for this region by screening a hamster-mouse chromosome 17 and 18 cell hybrid cosmid library with mouse-specific repetitive elements and mapping positive clones via t-haplotype vs. C3H restriction fragment length polymorphism (RFLP) analysis. Twelve of the clones mapping distal to Leh66B in t-haplotypes are described here. Using standard RFLP analysis or simple sequence length polymorphism between t-haplotypes, exceptional partial t-haplotypes and nested sets of inter-t-haplotype recombinants, five cosmids have been mapped in or around In(17)3 and seven in the most distal inversion In17(4). More precise mapping of four of the cosmids from In(17)4 shows that they will be useful in the molecular identification of some of the recessive lethals mapped to the t-complex: two cosmids map between H-2K and Crya-1, setting a distal limit in t-haplotypes for the position of the tw5 lethal, one is inseparable from the tw12 lethal, and one maps distal to tf near the t0(t6) lethal and cld.     

Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes

Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.     

LC2, the Chlamydomonas Homologue of the t Complex-encoded Protein Tctex2, Is Essential for Outer Dynein Arm Assembly

Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility.