Molecular cloning of the t complex responder genetic locus

Although mouse t haplotypes carry recessive mutations causing male sterility and embryonic lethality, they persist in wild mouse populations via male transmission ratio distortion (TRD). Genetic evidence suggests that at least five t-haplotype-encoded loci combine to cause TRD. One of these loci, called the t complex responder (Tcr), is absolutely required for any deviation from Mendelian segregation to occur. A candidate for the Tcr gene has previously been identified. Evidence that this gene represents Tcr is its localization to the appropriate genomic subregion and testis-specific expression pattern. Here, we report the molecular cloning of the region between recombinant chromosome breakpoints defining the Tcr locus. These results circumscribe Tcr to a 150- to 220-kb region of DNA, including the 22-kb candidate responder gene. This gene and two other homologs were created by large genomic duplications, each involving segments of DNA 10-fold larger than the individual genes.     

Targeted Mutagenesis of a Candidate T Complex Responder Gene in Mouse T Haplotypes Does Not Eliminate Transmission Ratio Distortion

Transmission ratio distortion (TRD) associated with mouse t haplotypes causes +/t males to transmit the t-bearing chromosome to nearly all their offspring. Of the several genes involved in this phenomenon, the t complex responder (Tcr(t)) locus is absolutely essential for TRD to occur. A candidate Tcr(t) gene called Tcp10b(t) was previously cloned from the genetically defined Tcr(t) region. Its location, restricted expression in testis, and a unique postmeiotic alternative splicing pattern supported the idea that Tcp10b(t) was Tcr(t). To test this hypothesis in a functional assay, ES cells were derived from a viable partial t haplotype, and the Tcp10b(t) gene was mutated by homologous recombination. Mutant mice were mated to appropriate partial t haplotypes to determine whether the targeted chromosome exhibited transmission ratios characteristic of the responder. The results demonstrated that the targeted chromosome retained full responder activity. Hence, Tcp10b(t) does not appear to be Tcr(t). These and other observations necessitate a reevaluation of genetic mapping data and the actual nature of the responder.     

Identification of a cryptic lethal mutation in the mouse t(w73) haplotype

t haplotypes are naturally occurring, variant forms of the t complex on mouse chromosome 17, characterized by the presence of four inversions with respect to wild-type. They harbour mutations causing male sterility, male transmission ratio distortion (TRD) and embryonic lethality. Mice carrying t haplotypes have been found throughout the world, and genetic studies of the lethal mutations have identified at least 16 complementation groups. The embryonic lethal phenotypes of many t haplotypes have been characterized in detail, and are thought to be the consequence of homozygosity for single gene mutations. However, the existence of additional mutations in genes that function at later stages of development would be obscured. Here we investigated the possibility of multiple mutations in t haplotypes by screening the t(w73) haplotype for the presence of novel mutations. Since genetic analysis of t haplotype mutations is hindered by recombination suppression due to the inversions, deletion complexes covering the proximal two-thirds of the t complex were used to uncover the presence of any new lethal alleles. This analysis revealed a novel mutation between D17Jcs41 and D17Mit100, causing mice carrying both t(w73) and selected deletions to die at birth, prior to feeding. The finding of a new, cryptic lethal mutation in t haplotypes is an indication that these recombinationally isolated chromosomes, which already contain at least one lethal mutation that prevents homozygosity, may serve as sinks for the accumulation of additional recessive mutations.     

Genes in the first and fourth inversions of the mouse t complex synergistically mediate sperm capacitation and interactions with the oocyte

The t haplotypes (t) are recent evolutionary derivatives of an alternate form of the mouse t complex region located at the proximal end of chromosome 17. This variant form of approximately 1% of the mouse genome is a source of mutations altering numerous sperm functions crucial for fertilization. Males that carry two t haplotypes (t/t) are invariably sterile. t haplotypes contain four inversions relative to the wild-type t complex (+), so that in matings involving a +/t heterozygote, t is usually transmitted as a single unit. However, rare recombinants have been recovered, which carry only part of the t genotype and express only some of the t-dependent phenotypes. Use of these partial t haplotypes in genetic crosses has resulted in the general location of the two major t male sterility factors, S1 and S2, within inversions 1 and 4, respectively. Since sterility can result from a plethora of sperm defects, we have made a detailed study of various functional parameters of sperm from mice carrying S1 or S2 heterozygously or homozygously or in combination. Both S1 and S2 contain mutations altering sperm functions, including motility, capacitation, binding to the zona pellucida, binding to the oocyte membrane, and penetration of the zona pellucida-free oocyte. Therefore it seems clear that each of these factors contains multiple genes contributing to sterility. Furthermore, our results indicate that genes within S1 interact with genes in S2 for all sperm functions examined. However, S1 and S2 genes affecting motility interact in a purely additive fashion, while S1 and S2 genes affecting most other sperm characteristics interact in a synergistic manner. Additionally, the patterns of synergism between S1 and S2 for abnormalities in capacitation, sperm-oolemma binding, and zona-free oocyte penetration are nearly identical. This suggests that these three defects are caused by mutation of the same gene within each sterility factor. These findings will not only be instrumental in matching the various t haplotype sperm defects to candidate genes for S1 and S2, but will facilitate a more comprehensive understanding of the cellular and genetic mechanisms underlying t haplotype male sterility.     

A candidate gene family for the mouse t complex responder (Tcr) locus responsible for haploid effects on sperm function

The mouse t complex responder (Tcr) locus plays a central haploid-specific role in the transmission ratio distortion phenotype expressed during germ cell differentiation in t-carrying males. The accumulated data map Tcr to a region of less than 500 kb. Over 400 kb of this region has been cloned and consists entirely of sequences associated with a clustered family of large cross-hybridizing elements of 30 kb to 70 kb in size. We have characterized a gene family within this region that is expressed uniquely in male germ cells with a complex pattern of RNA processing. Antibodies produced against a product of the putative open reading frame recognize a testes-specific polypeptide. Genetic data support the hypothesis that this polypeptide(s) functions to effect the Tcr phenotype.     

The mouse t complex distorter/sterility candidate, Dnahc8, expresses a gamma-type axonemal dynein heavy chain isoform confined to the principal piece of the sperm tail

Heterozygosity for a t haplotype (t) in male mice results in distorted transmission (TRD) of the t-bearing chromosome 17 homolog to their offspring. However, homozygosity for t causes male sterility, thus limiting the spread of t through the population at large. The Ca(2+)-dependent sperm tail curvature phenotypes, "fishhook", where abnormally high levels of sperm exhibit sharp bends in the midpiece, and "curlicue", where motile sperm exhibit a chronic negative curving of the entire tail, have been tightly linked to t-associated male TRD and sterility traits, respectively. Genetic studies have indicated that homozygosity for the t allele of Dnahc8, an axonemal gamma-type dynein heavy chain (gammaDHC) gene, is partially responsible for expression of "curlicue"; however, its involvement in "fishhook"/TRD, if any, is unknown. Here we report that the major isoform of DNAHC8 is copiously expressed, carries an extended N-terminus and full-length C-terminus, and is stable and equally abundant in both testis and sperm from +/+ and t/t animals. By in silico analysis we also demonstrate that at least three of the seventeen DNAHC8(t) mutations at highly conserved positions in wild-type DHCs may be capable of substantially altering normal DNAHC8 function. Interestingly, DNAHC8 is confined to the principal piece of the sperm tail. The combined results of this study suggest possible mechanisms of DNAHC8(t) dysfunction and involvement in "curlicue", and support the hypothesis that "curlicue" is a multigenic phenomenon. They also demonstrate that the accelerated "fishhook" phenotype of sperm from +/t males is not directly linked to DNAHC8(t) dysfunction.     

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.     

Sperm dysfunction in the Rb(6.16)- and Rb(6.15)-bearing mice revisited: involvement of Hyalp1 and Hyal5

Earlier we showed that Sperm adhesion molecule1 (Spam1), the best studied sperm hyaluronidase, is involved in the sperm dysfunction associated with Robertsonian translocations (Rb). The dysfunction results in reduced fertility in mice homozygous for the Rb(6.16) or the Rb(6.15) translocation and transmission ratio distortion (TRD) in heterozygous males. This conclusion was based on the finding that Spam1 in the Rbs harbors multiple point mutations and a genomic alteration at the locus [in the case of Rb(6.16)]; and is accompanied by reduced steady-state levels of the RNA and protein. Here we show that closely linked family members in the hyaluronidase gene cluster on mouse chromosome 6, Hyalp1 and Hyal5, also harbor point mutations in these Rbs, leading to nonconservative substitutions in both the encoded proteins. To test if Spam1 by itself is capable of producing TRD we analyzed the transmission of wild-type and null alleles of the gene in the progeny of carriers and show that there is no significant TRD. This lack of TRD in null carriers argues for only a contributory role of Spam1 in the TRD seen in the Rb-bearing mice, and supports the involvement of Hyalp1 and/or Hyal5 in the sperm dysfunction and the resulting TRD. It is proposed that the clustering of point mutations in all three genes results from the cumulative effect of spontaneous mutations that do not disperse in the population due to suppression of recombination that occurs at Rb junctions.     

Low Frequency of Mouse T Haplotypes in Wild Populations Is Not Explained by Modifiers of Meiotic Drive

t haplotypes are naturally occurring forms of mouse chromosome 17 that show non-Mendelian transmission from heterozygous +/t males. In laboratory studies, transmission ratios of >=0.90 or higher are typically observed. With transmission ratios of this level, theoretical analyses predict high frequencies of t haplotypes (~ 75%) in wild populations. In contrast, empirical frequencies of only 15-25% are typically found. This has led to the suggestion that modifiers of drive may play a role in reducing t frequencies. We have measured transmission ratio distortion (TRD) levels in wild +/t mice to examine this hypothesis. TRD was very high in both litters collected from wild-caught pregnant females, and in wild litters bred in the laboratory (mean = 0.9). Contrary to the results of other studies, we found no difference in TRD levels between semilethal and lethal t haplotypes nor between litters conceived from cycling or postpartum estrus. We found three litters with aberrantly low TRDs that were all multiply sired, although the role this might play in natural populations is unknown. These findings show a general absence of modifiers of drive in natural populations and suggest that other factors are responsible for the low observed frequencies of wild t haplotypes.     

Concerted evolution of the mouse Tcp-10 gene family: implications for the functional basis of t haplotype transmission ratio distortion.

The mouse Tcr locus is defined by its central role in the transmission ratio distortion phenotype characteristic of t haplotypes. A molecular candidate for Tcr has been identified in the form of a gene--Tcp-10b--expressed during spermatogenesis. Tcp-10b is one member of a multigene family present in two to four copies on different homologs of chromosome 17. The coding regions of the Tcp-10 genes present within two inbred strains were compared with those of the tw5 haplotype. The various gene family members are highly conserved relative to each other with a minimum nucleotide identity of 98.6% in all pairwise comparisons. Maximal parsimony analysis indicates that the Tcp-10 gene family has evolved in a concerted manner with the obliteration of nearly all individual gene-specific characteristics. As a consequence, the candidate for the full-length mutant Tcr gene product is distinguished by only a single, highly conservative, amino acid change. The data are consistent with the hypothesis that the effector of mutant Tcr activity is a second, alternatively spliced product that is expressed in a haploid- and allele-specific manner.     

Identification of the t Complex–encoded Cytoplasmic Dynein Light Chain Tctex1 in Inner Arm I1 Supports the Involvement of Flagellar Dyneins in Meiotic Drive

The cytoplasmic dynein light chain Tctex1 is a candidate for one of the distorter products involved in the non-Mendelian transmission of mouse t haplotypes. It has been unclear, however, how the t-specific mutations in this protein, which is found associated with cytoplasmic dynein in many tissues, could result in a male germ cell–specific phenotype. Here, we demonstrate that Tctex1 is not only a cytoplasmic dynein component, but is also present both in mouse sperm and Chlamydomonas flagella. Genetic and biochemical dissection of the Chlamydomonas flagellum reveal that Tctex1 is a previously undescribed component of inner dynein arm I1. Combined with the recent identification of another putative t complex distorter, Tctex2, within the outer dynein arm, these results support the hypothesis that transmission ratio distortion (meiotic drive) of mouse t haplotypes involves dysfunction of both flagellar inner and outer dynein arms but does not require the cytoplasmic isozyme.     

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).     

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.