Transmission Ratio Distortion,Sterility, and Control of the t-Complex Function in Sperm

Mouse t-complex located on chromosome 17 contains genes affecting only 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.     

New insights into the t-complex and control of sperm function

The mouse t-complex, located on chromosome 17, contains genes known to influence male, but not female, fertility. Although some t-complex genes are recessive lethals, t-chromosomes are maintained in the population by transmission ratio distortion. When male mice heterozygous for the t-chromosome mate with wild-type females, most offspring will possess the t-chromosome, indicating a link between t-complex genes and sperm function. Several proteins coded for by t-complex genes have been localised in the sperm flagellum, suggesting roles relating to motility. Another t-complex protein appears able to regulate the adenylyl cyclase/cAMP signal transduction pathway, known to play an important role in capacitation. Defective motility and/or failure to capacitate (“switch on”) would result in poorly fertile or infertile spermatozoa. Given the existence of human homologues for many genes in the t-complex and the prevalence of “male factor” infertility, information obtained about the t-complex not only will provide insight into basic biological mechanisms but may be of future clinical relevance as well. BioEssays 21:304–312, 1999. © 1999 John Wiley & Sons, Inc.     

Mapping of the Sod-2 locus into the t complex on mouse chromosome 17

A human DNA probe specific for the superoxide dismutase gene was used to identify the corresponding mouse gene. Under the chosen hybridizing conditions, the probe detected DNA fragments most likely carrying the mouse Sod-2 gene. Mapping studies revealed that the Sod-2 gene resides in the proximal inversion of the t complex on mouse chromosome 17. All complete t haplotypes tested showed restriction fragment length polymorphism which is distinct from that found in all wild-type chromosomes tested. The Sod-2 locus maps in the same region as some of the loci that influence segregation of t chromosomes in male gametes. The possibility that the Sod-2 locus is related to some of the t-complex distorter or responder loci is discussed. The data indicate that the human homolog of the mouse t complex has split into two regions, the distal region remaining on the p arm of human chromosome 6, while the proximal region has been transposed to the telomeric region of this chromosome's q arm.     

Tctex3, related to Drosophila Polycomblike, is expressed in male germ cells and mapped to the mouse t-complex

Tctex3 showing restricted expression in male germ cells has been isolated during the process of chromosome walking in the mouse t-complex region. The total sequence of Tctex3 cDNA predicts a protein of 580 amino acids with two C4HC3 type PHD fingers. The region containing this conserved motif is shared among members of the Polycomblike proteins that include the mouse M96 and Drosophila Polycomblike. A partial cDNA for a human homolog of Tctex3, HUTEX3, has also been isolated. Mouse Tctex3 gene was mapped adjacent to Tsc2 gene on mouse Chromosome (Chr) 17, and HUTEX3 was located closely to HSET gene in the HLA class II region of chromosome 6. Received: 10 April 1998 / Accepted: 22 June 1998     

Assignment of the Human Homologue of the mTRiC-P5 Gene (TRICS) to Band 1q23 by Fluorescence in Situ Hybridization

The TCP1 ring complex (TRiC) is a molecular chaperone involved in actin and tubulin folding. Little is known about the components of this complex. The first component identified was TCP1, a protein coded by a gene in the t -complex locus on mouse chromosome 17. This locus is involved in several embryonic defects, male sterility, and the transmission ratio distortion. In humans, the t-complex genes map to chromosome 6. Other components of TRiC are thought to be TCP1-related proteins. Recently, a mouse cDNA coding for one of these proteins has been cloned and named mTRiC-P5. Here we report the cloning of a partial human cDNA clone, homologous to mTRiC-P5, and its chromosome localization by fluorescence in situ hybridization. The human TRiC-P5 gene (TRIC5) maps to human chromosome 1q23, a region known to be a preferential chromosomal breakpoint involved in leukemia. Therefore, even if TCP1 and TRiC-P5 are related proteins and are found in the same protein complex, they are not coded by syntenic genes in humans.     

Recombination within mouse <Emphasis Type="Italic">t</Emphasis> haplotypes has replaced significant segments of <Emphasis Type="Italic">t</Emphasis>-specific DNA

Previous studies on the fourth inversion of the t complex, In17(4), suggest that loci near the center of this inversion have been subjected to segmental recombination during the past 1–2 million years. We have used a combination of PCR-based restriction site (PBR) analysis and DNA sequencing to perform a high-resolution analysis of a 2-million base pair (Mbp) segment in the middle of In17(4). We examined 21 restriction sites that are polymorphic between t haplotypes and their wild-type homologs, over nine distinct loci. In addition, we examined several other polymorphic sites through DNA sequence analysis of two of these nine loci. We analyzed several haplotypes in this way, including the “complete” t haplotypes t ^w2 , t ⁰ , t ^w32 , t ^w71 , and t ^w75 . We show that only t ^w32 is a true “complete” t haplotype; the remaining four t haplotypes have segments of wild-type DNA ranging from less than 100 bp to 2 Mbp. The sizes of these wild-type DNA segments are consistent with their being generated by gene-conversion events. The 2-Mbp segment is located in a region that may contain the t-complex distorter gene Tcd2. One of the nine loci examined in this study is Fgd2, a gene that has been proposed to encode Tcd2. Sequencing and PBR data show that at least a portion of the Fgd2 gene has been converted to the wild-type within t ^w71 and t ^w75 mice.     

The formation of endo-complexes between calixarenes and amines—a reinvestigation

The formation of an endo-complex between p-allylcalix[4]arene and t-butylamine was described by Gutsche in 1985. However, for a comparable system, it has been shown using NOE methods that the amine does not reside inside the calix. Instead, an exo-calix complex is formed. A reevaluation suggests that the previous conclusion is an artifact due to improper NMR-data processing. DFT (RB3LYP/6–31G(d)) calculations confirm the higher stability of the exo-complex over its endo-counterpart. Dedicated to Professor Dr. Paul von Ragué Schleyer on the occasion of his 75th birthday.     

Molecular organization of theD-Qa region oft-haplotypes suggests that recombination is an important mechanism for generating genetic diversity of the major histocompatibility complex

We have determined the molecular maps of theH-2D andQa regions of thet-complex haplotypest ^l2 andt ^w5 by chromosomal walking. Analysis with class I probes and other probes unique to theH-2D:Qa subregion indicates that the class I gene organization oft ¹² is:D1-D2-Q1-Q2-Q3-Qx-Q4-Q5-Q10, while that oft ^w5 is:D1-D2-Q1-Q2-Q4-Q5-Q10. Thus, the absence of theQ6-Q9 genes suggested previously int-haplotypes was confirmed. A comparison of the molecular maps of thet ¹² andt ^w5 chromosomes revealed an extremely mosaic pattern of diversity: The regions betweenD1 andD2, and betweenQ4 andQ10, are very similar in both chromosomes. However, theirQ1 toQ3 regions are strikingly different. Further comparisons of wild-type chromosomes and additionalt-haplotypes by molecular mapping and genomic Southern blot hybridization with probes to theQ1-Q3 region showed a high level of polymorphism among both wild-type chromosomes and amongt-haplotypes. The characteristics of the polymorphisms suggest that recombination may play an important role in generating this genetic diversity. Furthermore, recombination between wild-type andt-haplotype chromosomes may be involved.     

Genetic analysis of the organic cation transporter genes Orct2/Slc22a2 and Orct3/Slc22a3 reduces the critical region for the t haplotype mutant t w73 to 200 kb

Here we report an analysis of two candidate genes for the t ^w73 implantation mutation. The t ^w73 gene maps to a 20-cM region of mouse Chromosome (Chr) 17 known as the t-complex, which exists in a wild-type and t haplotype form in present-day mice. The t haplotype variants contain several mutant alleles affecting male fertility and embryonic viability and offer the opportunity to identify genes critical for these processes. t ^w73 homozygous embryos are defective in trophoblast production and fail to implant adequately, with death occurring at approximately 7.5 days post coitum (pc). Two recently described organic cation transporter genes, Slc22a2 (Orct2) and Slc22a3 (Orct3), fulfill criteria predicted for t ^w73 candidate genes, since both map to the previously defined 500-kb t ^w73 minimal region and both are also expressed in 7.5 days pc post-implantation embryos. The genomic locus of the Orct2 gene appears similar in wild-type and t ^w73 chromosomes. In contrast, the genomic locus of Orct3 is amplified and displays an altered expression profile in all t haplotype variant chromosomes tested. In addition, Orct3 shows a t ^w73 specific polymorphism. To test whether either Orct2 or Orct3 is involved in the t ^w73 phenotype, we have performed a genetic rescue experiment using YAC transgenes overexpressing Orct2, and genetic complementation with an allele in which the Orct3 gene was inactivated by homologous recombination. The results eliminate both Orct2 and Orct3 as candidates and further reduce the critical region containing the t ^w73 mutant from 500 kb to 200 kb. Received: 8 February 2001 / Accepted: 1 May 2001     

Cell surface galactosyltransferase as a recognition molecule during development

Recent results from our laboratory suggest that a variety of cellular interactions during development are mediated, in part, by the binding of a cell surface enzyme, galactosyltransferase (GalTase), to its specific lactosaminoglycan (LAG) substrate on adjacent cell surfaces and in the extracellular matrix. Our present interest in surface GalTase developed from earlier biochemical studies of a series of morphogenetic mutations in the mouse which map to the T/t-complex. These studies identified a specific defect in the regulation of surface GalTase activity on morphogenetically abnormal cells, while eight other enzymes showed normal activity. This led us to consider the unique function of surface GalTase in those cell interactions that are influenced by mutations of the T/t-complex. By using a multidisciplinary approach, which included genetic, biochemical and immunological probes, we have found that GalTase functions as a surface receptor during fertilization, early embryonic cell adhesions, and embryonic cell migration on basal lamina matrices. Recently, we have examined the expression of surface GalTase during spermatogenesis, as well as the fate of sperm GalTase following the acrosome reaction. This paper summarizes the results of these studies, as well as others, which suggest that GalTase functions as a surface receptor during those cell interactions regulated by the T/t-complex alleles.     

Sterility of Males Determined by Functional Features of the Mouse Spermatozoa Bearing t-Complex

The mechanisms underlying normal spermatogenesis and its pathology expressed as male sterility determined by t-complex located on chromosome 17 in mice are considered in this review. t-Complex is a very convenient model with diverse markers of expression of the genes involved in development of the functional features of the spermatozoa bearing t-complex. These features include defects of mobility, capacitation, and acrosome reactions, which determine full or partial male sterility. It has been proposed that the defects of capacitation are also inherent in humans and affect male fertility. This homology is confirmed by the presence of the male gene Tcp11 in humans and demonstration of the fact that the protein TCP11 plays a leading role in modulation of the capacitation of murine spermatozoa. Hence it follows that the defects of human genes leading to incomplete binding of the fertilization promoting peptide could play a certain role in a decreased male fertility. All this is essential not only for deeper understanding of the biology of spermatozoa, but also for development of new therapeutic methods of finding and treating the pathology of spermatozoa.     

Meiotic drive in mice carrying t-complex in their genome

The deviation of alleles and chromosomes from Mendelian inheritance is characteristic of the meiotic drive. This review describes the mechanism in question using the best-studied example of transmitted ratio distortion in the heterozygous male mice carrying t-haplotypes. The t-complex is best model for studying the meiotic drive under laboratory conditions. Putative mechanisms of meiotic drive that influence the frequency of t-haplotypes in natural populations are considered, of which prezygotic selection is the most important. The role of meiotic drive in male hybrid sterility is emphasized. The factors and models that determine the phenomenon of meiotic drive are discussed in detail.

     

The receptor function of galactosyltransferase during cellular interactions

Summary The molecular mechanisms that underly cellular interactions during development are still poorly understood. There is reason to believe that complex glycoconjugates participate in cellular interactions by binding to specific cell surface receptors. One class of carbohydrate binding proteins that could serve as receptors during cellular interactions are the glycosyltransferases. Glycosyltransferases have been detected on a variety of cell surfaces, and evidence suggests that they may participate during cellular interactions by binding their specific carbohydrate substrates on adjacent cells or in extracellular matrix (see Refs. 1–4 for review).This review will focus on the receptor function of galactosyltransferase, in particular, during fertilization, embryonic cell adhesion and migration, limb bud morphogenesis, immune recognition and growth control. In many of these systems, the galactosyltransferase substrate has been characterized as a novel, large molecular weight glycoconjugate composed of repeating N-acetyllactosamine residues. The function of surface galactosyl-transferase during cellular interactions has been examined with genetic and biochemical probes, including the T/t-complex morphogenetic mutants, enzyme inhibitors, enzyme modifiers, and competitive substrates. Collectively, these studies suggest that in the mouse, surface galactosyltransferase is under the genetic control of the T/t-complex, and participates in multiple cellular interactions during development by binding to its specific lactosaminoglycan substrate.     

TCP-11, the product of a mouse t-complex gene,plays a role in stimulation of capacitation and inhibition of the spontaneous acrosome reaction

Tcp-11 is a candidate for a distorter gene within the t-complex on mouse chromosome 17; although t-complex genes appear to affect sperm function, relatively little is known about mechanisms whereby these genes might play a specific physiological role. We present evidence that the protein TCP-11 is found on the surface of mature epididymal spermatozoa. Although detected on both the acrosomal cap region of the head and the flagellum of acrosome-intact cells, it is absent from the heads of acrosome-reacted cells. When epididymal spermatozoa were incubated in the presence of anti-TCP-11 IgG Fab fragments for a total of 120 min and assessed using chlortetracycline fluorescence, we observed a stimulation of capacitation and an inhibition of spontaneous acrosome loss, suggestive of enhanced fertility compared with untreated suspensions. In vitro fertilization experiments confirmed that Fab-treated suspensions became fertile more quickly and then maintained high fertility. Because these responses were remarkably similar to those obtained using the TRH-related peptide FPP (fertilization promoting peptide; pGlu-Glu-ProNH₂) and adenosine, we investigated responses to Fab fragments, FPP, and adenosine. Results indicated that the Fab fragments appear to work at the same extracellular site as FPP, one that is distinct from the adenosine site of action. Further evidence for this conclusion was obtained using pGlu-Gln-ProNH₂, an FPP-related tripeptide known to competitively inhibit responses to FPP; as with FPP, pGlu-Gln-ProNH₂ inhibited the stimulatory effect of Fab fragments in a concentration-dependent manner. From these results we suggest that TCP-11 may be the receptor for FPP and that the adenylate clyclase/cyclic AMP pathway may be the signal transduction pathway activated by interactions between extracellular effector molecules (e.g., Fab fragments or FPP acting as an agonist) and TCP-11. A mechanism such as this that promotes capacitation but inhibits spontaneous acrosome loss in vivo would play a very important role by helping to maximize the fertilizing potential of the few spermatozoa that reach the site of fertilization. The fact that there is a human homolog of Tcp-11 suggests that this gene could play an important role in regulation of human, as well as mouse, sperm function. Mol. Reprod. Dev. 48:375–382, 1997. © 1997 Wiley-Liss, Inc.     

Molecular mapping of gene Gm-6(t) which confers resistance against four biotypes of Asian rice gall midge in China

The Chinese rice cultivar Duokang #1 carries a single dominant gene Gm-6(t) that confers resistance to the four biotypes of Asian rice gall midge (Orseolia oryzae Wood-Mason) known in China. Bulked segregant analysis was performed on progeny of a cross between Duokang #1 and the gall midge-susceptible cultivar Feng Yin Zhan using the RAPD method. The RAPD marker OPM06₍₁₄₀₀₎ amplified a locus linked to Gm-6(t). The locus was subsequently mapped to rice chromosome 4 in a region flanked by cloned RFLP markers RG214 and RG163. Fine mapping of Gm-6(t) revealed that markers RG214 and RG476 flanked the gene at distances of 1.0 and 2.3 cM, respectively. Another gall midge resistance gene, Gm-2, mapped previously to chromosome 4, is located about 16 cM from Gm-6(t), to judge by data from a segregating population derived from a cross between Duokang #1 and the Indian cultivar Phalguna that carries Gm-2. We developed a PCR-based marker-assisted selection kit for transfer of the Gm-6(t) gene into Ming Hui 63 and IR50404, two parental lines commonly used in hybrid rice production in China. The kit contains PCR primer pairs based on the terminal sequences of the RG214 and RG476 clones. Polymorphism between Duokang #1 and the hybrid parental lines was found at these markers after digestion of the PCR products with specific restriction endonucleases. The kit will accelerate introduction of gall midge resistance into hybrid rice in China. Received: 18 May 2000 / Accepted: 9 March 2001     

Embryonal effects of <Emphasis Type="Italic">t</Emphasis>-haplotypes in mice

The effects of t-haplotypes on embryonic morphology in house mouse Mus musculus were described. Lethal mutations, t-haplotypes, in homozygotes induce abnormal embryogenesis and zygotic death at different developmental stages, which depends on the time of their action in ontogeny. Death commonly occurs in the first semester of pregnancy from the morula to the mature embryo stage (day 9–10), and the embryogenetic abnormalities and their timing were Specific for each t-haplotype. Such mutations were analyzed to identify the gene products (proteins) affecting the nervous system development. The t-complex proved to contain tandem repeats coding for regulatory factors modulating the expression of specific structural genes in mouse neurons.     

Sequence of the t complex Tcp-10a t gene and examination of the Tcp-10 t gene family

Transmission ratio distortion (TRD) is a property of complete t haplotypes which results in the preferential transmission of the t haplotype chromosome from heterozygous t/+ males to the majority of the offspring. A candidate gene for one of the primary genetic elements in TRD, the t complex responder locus has recently been suggested to be Tcp-10b ^t. There are multiple, functional Tcp-10 ^t genes, but genetic data suggest the presence of the Tcp-10a ^t gene alone is compatible with normal transmission ratios. Here we present the complete sequence and genomic structure of the Tcp-10a ^t gene which is compared with sequence data from a number of cDNAs and genomic subclones representing all active Tcp-10 ^t family genes. A detailed table of all sequence variants discovered in the course of our investigation is presented, and we have clarified the extent of 5 untranslated alternative splicing patterns exhibited by this gene family. A 60 base pair (bp) in-frame deletion from the 5 end of exon 3 of the Tcp-10a ^t gene is also presented and compared with the equivalent region of Tcp-10b ^t and Tcp-10c ^t. A search of the University of Edinburgh database has revealed a significant homology between the Tcp-10b ^t open reading frame and several cytosolic filament proteins. Interestingly, the region of homology is involved in the deletion from the Tcp-10a ^t gene.     

Molecular mapping of a resistance-specific PCR-based marker linked to a gall midge resistance gene (Gm4t) in rice

 A PCR-based marker (E20₅₇₀) linked to the gene Gm4t, which confers resistance to a dipteran pest gall midge (Orseolia oryzae), has been mapped using the restriction fragment length polymorphism (RFLP) technique in rice. Gm4t is a dominant resistance gene. We initially failed to detect useful polymorphism for this marker in a F₃ mapping population derived from a cross between two indica parents, ‘Abhaya’בShyamala’, with as many as 35 restriction enzymes. ‘Abhaya’ carries the resistance gene Gm4t and ‘Shyamala’ is susceptible to gall midge. Subsequently, E20₅₇₀ was mapped using another mapping population represented by a F₂ progeny from a cross between ‘Nipponbare’, a japonica variety, and ‘Kasalath’, an indica variety, in which the gene Gm4t was not known to be present. Gm4t mapped onto chromosome 8 between markers R1813 and S1633B. Our method, thus, presents an alternative way of mapping genes which otherwise would be difficult to map because of a lack of polymorphism between closely related parents differing in desired agronomic traits. Received: 1 April 1997 / Accepted: 13 May 1997     

The effects of increasing serum calcitriol on energy and fat metabolism and gene expression

Objective: Evidence from a number of investigations indicates that calcium intake could be inversely related to body weight through alterations in the 1,25‐OH₂‐D₃ metabolism. The objective of this study was to test whether energy and substrate metabolism and adipose tissue enzyme mRNA expression can be altered by changes in serum 1,25‐OH₂‐D₃ through oral cholecalciferol supplementation in non‐obese human subjects. Research Methods and Procedures: An intervention study was used with a treatment period of 7 days. During this intervention, energy expenditure (EE) and substrate metabolism were measured using indirect calorimetry at t = 0, 1, 3, and 7 days, and blood samples were obtained at t = ?1, 0, 1, 2, 3, 5 and 7 days. Fat biopsies were obtained at t = 0 and 7 days for determination of expression of genes involved in lipolytic and lipogenic pathways. Subjects from the general community were studied in an ambulatory setting at a university hospital. Ten healthy young men (age, 28 ± 3 years; BMI, 25.5 ± 0.5 kg/m²) were recruited by local announcement, and all completed the study. All subjects received 2000 IU cholecalciferol/d for 7 days, and they were instructed to consume a low‐cholecalciferol, low‐calcium diet. EE, fat oxidation, and adipose tissue enzyme mRNA were the main outcome measures. Results: Despite a significant increase in serum 1,25‐OH₂‐D₃ concentration at t = 5 and 7 days, no significant differences in substrate and energy metabolism nor mRNA concentrations of different lipid metabolism‐related proteins were observed. Discussion: Seven‐day supplementation with 2000 IU cholecalciferol/d together with a decrease in dietary calcium intake does not affect EE or substrate metabolism nor gene expression of proteins related to fat metabolism, despite a significant increase in serum 1,25‐OH₂‐D₃ concentration.