Analysis of the sex-specific variability of blood parameters in C3H inbred mice by using data from a long-term, high-throughput project.

Mice are important models for biomedical research by providing the possibility of standardizing genetic background and environmental conditions, which both affect phenotypic variability. Use of both sexes in experiments is strongly recommended because of possible differences in the outcome. However, sex-specific phenotypic variability is discussed with regard to putative consequences on the group size which is necessary for achieving valid and reproducible results. Here, we retrospectively analyzed the sex-specific variability of 25 blood parameters of C3H inbred mice in two different mouse facilities withinthe long-term, high-throughput Munich ENU mouse mutagenesis project. Using the 95 % data range, data of4,780-20,706 mice per parameter were analyzed and resulted in ratios of the coefficient of variation (= female CV / (female CV + male CV)) from 0.44 to 0.58 for the 25 parameters, with an overall mean of 0.51 in both facilities. Together with data analyses of three additional, smaller studies with 72-247 animals per parameter examined and various genetic backgrounds (inbred strains, F1 hybrids) included, hints for reproducible sex-specific variability were observed for particular parameters. Thus, the overall analysis comprising all 25 clinical chemical and hematological parameters of the standardized, long-term analysis of a high number of group housed, young adult, twelve-week-old C3H inbred mice showed no evidence for substantial sex-specific variability. The results may provide a basis for the examination of sex-specific variability in particular blood parameters.

A paternal methyl donor-rich diet altered cognitive and neural functions in offspring mice.

Dietary intake of methyl donors, such as folic acid and methionine, shows considerable intra-individual variation in human populations. While it is recognized that maternal departures from the optimum of dietary methyl donor intake can increase the risk for mental health issues and neurological disorders in offspring, it has not been explored whether paternal dietary methyl donor intake influences behavioral and cognitive functions in the next generation. Here, we report that elevated paternal dietary methyl donor intake in a mouse model, transiently applied prior to mating, resulted in offspring animals (methyl donor-rich diet (MD) F1 mice) with deficits in hippocampus-dependent learning and memory, impaired hippocampal synaptic plasticity and reduced hippocampal theta oscillations. Gene expression analyses revealed altered expression of the methionine adenosyltransferase Mat2a and BK channel subunit Kcnmb2, which was associated with changes in Kcnmb2 promoter methylation in MD F1 mice. Hippocampal overexpression of Kcnmb2 in MD F1 mice ameliorated altered spatial learning and memory, supporting a role of this BK channel subunit in the MD F1 behavioral phenotype. Behavioral and gene expression changes did not extend into the F2 offspring generation. Together, our data indicate that paternal dietary factors influence cognitive and neural functions in the offspring generation.

Genetic characterization of a mouse line with primary aldosteronism.

In an attempt to define novel genetic loci involved in the pathophysiology of primary aldosteronism, a mutagenesis screen after treatment with the alkylating agent N-ethyl-N-nitrosourea was established for the parameter aldosterone. One of the generated mouse lines with hyperaldosteronism was phenotypically and genetically characterized. This mouse line had high aldosterone levels but normal creatinine and urea values. The steroidogenic enzyme expression levels in the adrenal gland did not differ significantly among phenotypically affected and unaffected mice. Upon exome sequencing, point mutations were identified in seven candidate genes (Sspo, Dguok, Hoxaas2, Clstn3, Atm, Tipin and Mapk6). Subsequently, animals were stratified into wild-type and mutated groups according to their genotype for each of these candidate genes. A correlation of their genotypes with the respective aldosterone, aldosterone-to-renin ratio (ARR), urea and creatinine values as well as steroidogenic enzyme expression levels was performed. Aldosterone values were significantly higher in animals carrying mutations in four different genes (Sspo, Dguok, Hoxaas2 and Clstn3) and associated statistically significant adrenal Cyp11b2 overexpression as well as increased ARR was present only in mice with Sspo mutation. In contrast, mutations of the remaining candidate genes (Atm, Tipin and Mapk6) were associated with lower aldosterone values and lower Hsd3b6 expression levels. In summary, these data demonstrate association between the genes Sspo, Dguok, Hoxaas2 and Clstn3 and hyperaldosteronism. Final proofs for the causative nature of the mutations have to come from knock-out and knock-in experiments.

High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice.

OBJECTIVE: Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. METHODS: We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. RESULTS: Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. CONCLUSIONS: We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and during changes in hepatic insulin action in liver alter membrane properties - in particular those of mitochondria which are highly abundant in hepatocytes. In turn, a progressive decrease in the abundance of mitochondrial membrane proteins throughout HF-exposure likely impacts on mitochondrial energy metabolism, substrate exchange across mitochondrial membranes, contributes to oxidative stress, mitochondrial damage, and the development of insulin resistance in liver.

Peri-conceptional obesogenic exposure induces sex-specific programming of disease susceptibilities in adult mouse offspring.

Vulnerability of the fetus upon maternal obesity can potentially occur during all developmental phases. We aimed at elaborating longer-term health outcomes of fetal overnutrition during the earliest stages of development. We utilized Naval Medical Research Institute (NMRI) mice to induce pre-conceptional and gestational obesity and followed offspring outcomes in the absence of any postnatal obesogenic influences. Male adult offspring developed overweight, insulin resistance, hyperleptinemia, hyperuricemia and hepatic steatosis; all these features were not observed in females. Instead, they showed impaired fasting glucose and a reduced fat mass and adipocyte size. Influences of the interaction of maternal diet∗sex concerned offspring genes involved in fatty liver disease, lipid droplet size regulation and fat mass expansion. These data suggest that a peri-conceptional obesogenic exposure is sufficient to shape offspring gene expression patterns and health outcomes in a sex- and organ-specific manner, indicating varying developmental vulnerabilities between sexes towards metabolic disease in response to maternal overnutrition.

Enhanced oxidative stress and endocrine pancreas alterations are linked to a novel glucokinase missense mutation in ENU-derived Munich Gck(D217V) mutants.

In the large-scale Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project murine models recapitulating human diseases were generated. In one strain, a novel missense mutation (D217V) in the glucokinase (Gck) gene was identified, resulting in decreased glucokinase activity. Heterozygous mutants display mild hyperglycaemia, disturbed glucose tolerance, and decreased glucose-induced insulin secretion. In contrast, homozygous mutants exhibit severe but not survival affecting hyperglycaemia, mild growth retardation, diminished oxidative capacity, and increased abundance of CHOP protein in the islets. Furthermore, the total islet and ß-cell volumes and the total volume of isolated ß-cells are significantly decreased in adult homozygous mutants, whereas in neonatal mice, ß-cell mass is not yet significantly decreased and islet neogenesis is unaltered. Therefore, reduced total islet and ß-cell volumes of adult homozygous mutants might predominantly emerge from disturbed postnatal islet neogenesis. Thus, we identified a novel Gck mutation in mice, with relevance in humans, leading to glycaemic disease.

Phenotypic and pathomorphological characteristics of a novel mutant mouse model for maturity-onset diabetes of the young type 2 (MODY 2).

Several mutant mouse models for human diseases such as diabetes mellitus have been generated in the large-scale Munich ENU (N-ethyl-N-nitrosourea) mouse mutagenesis project. The aim of this study was to identify the causal mutation of one of these strains and to characterize the resulting diabetic phenotype. Mutants exhibit a T to G transversion mutation at nt 629 in the glucokinase (Gck) gene, leading to an amino acid exchange from methionine to arginine at position 210. Adult Munich Gck(M210R) mutant mice demonstrated a significant reduction of hepatic glucokinase enzyme activity but equal glucokinase mRNA and protein abundances. While homozygous mutant mice exhibited growth retardation and died soon after birth in consequence of severe hyperglycemia, heterozygous mutant mice displayed only slightly elevated blood glucose levels, present from birth, with development of disturbed glucose tolerance and glucose-induced insulin secretion. Additionally, insulin sensitivity and fasting serum insulin levels were slightly reduced in male mutant mice from an age of 90 days onward. While beta-cell mass was unaltered in neonate heterozygous and homozygous mutant mice, the total islet and beta-cell volumes and the total volume of isolated beta-cells were significantly decreased in 210-day-old male, but not female heterozygous mutant mice despite undetectable apoptosis. These findings indicate that reduced total islet and beta-cell volumes of male mutants might emerge from disturbed postnatal islet neogenesis. Considering the lack of knowledge about the pathomorphology of maturity-onset diabetes of the young type 2 (MODY 2), this glucokinase mutant model of reduced total islet and total beta-cell volume provides the opportunity to elucidate the impact of a defective glucokinase on development and maintenance of beta-cell mass and its relevance in MODY 2 patients.

The German Mouse Clinic: a platform for systemic phenotype analysis of mouse models.

The German Mouse Clinic (GMC) is a large scale phenotyping center where mouse mutant lines are analyzed in a standardized and comprehensive way. The result is an almost complete picture of the phenotype of a mouse mutant line--a systemic view. At the GMC, expert scientists from various fields of mouse research work in close cooperation with clinicians side by side at one location. The phenotype screens comprise the following areas: allergy, behavior, clinical chemistry, cardiovascular analyses, dysmorphology, bone and cartilage, energy metabolism, eye and vision, host-pathogen interactions, immunology, lung function, molecular phenotyping, neurology, nociception, steroid metabolism, and pathology. The German Mouse Clinic is an open access platform that offers a collaboration-based phenotyping to the scientific community (www.mouseclinic.de). More than 80 mutant lines have been analyzed in a primary screen for 320 parameters, and for 95% of the mutant lines we have found new or additional phenotypes that were not associated with the mouse line before. Our data contributed to the association of mutant mouse lines to the corresponding human disease. In addition, the systemic phenotype analysis accounts for pleiotropic gene functions and refines previous phenotypic characterizations. This is an important basis for the analysis of underlying disease mechanisms. We are currently setting up a platform that will include environmental challenge tests to decipher genome-environmental interactions in the areas nutrition, exercise, air, stress and infection with different standardized experiments. This will help us to identify genetic predispositions as susceptibility factors for environmental influences.

Genome-wide, large-scale production of mutant mice by ENU mutagenesis.

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).

Identification of immunological relevant phenotypes in ENU mutagenized mice.

The immunology screen focuses on the identification of novel gene products involved in the mammalian immune response and on the establishment of mouse models for immunological disorders. For this purpose, high throughput and semi-automated techniques were developed and optimized for low cost per sample and reproducibility. All assays are designed to be nonconsumptive and are based on peripheral blood or direct PCR amplification.

Large-scale N-ethyl-N-nitrosourea mutagenesis of mice--from phenotypes to genes.

The most important tool for obtaining insight into the function of genes is the use of mutant model organisms. Homologous recombination in embryonic stem cells allows the systematic production of mouse mutants for any gene that has been cloned. Gene trap strategies have been designed to interrupt even unknown genes which are tagged by the inserted vector and can be characterised structurally and functionally. Complementary to such 'gene-driven' approaches, 'phenotype-driven' approaches are necessary to identify new genes or gene products through a search for mutants with specific defects, uncovering the function of genetic pathways in physiological and pathological processes. Mutagenesis using the alkylating agent N-ethyl-N-nitrosourea (ENU) is a powerful approach for the production of such mouse mutants. Since ENU induces mainly point mutations in premeiotic spermatogonia, this strategy allows the production of multiple alleles of a particular gene, which is pivotal for a fine tuned analysis of its function.

Identification of leucocyte surface antigens in paraffin-embedded bovine tissues using a modified formalin dichromate fixation.

A modified fixative of formalin dichromate was combined with a cold embedding procedure for the preservation of bovine leucocyte surface antigens. Fourteen monoclonal antibodies recognizing seven bovine leucocyte surface antigens (BoCD1w2, BoCD4, BoCD8, BoWC1, BoWC3, BoWC4 and BoIgM) were applied as primary antisera in a sensitive avidin-biotin-peroxidase complex detection method. The staining results were compared with those obtained in cryostat and routinely formalin-fixed sections of corresponding tissue samples. Using the modified formalin dichromate fixative and the cold embedding procedure, all the leucocyte surface antigens tested were detectable immunohistologically in paraffin sections with a generally more distinct staining than in traditionally processed tissues. Morphological structures were better preserved than in cryostat sections but, to some extent, were poorer when compared with routinely formalinfixed tissues. However, this method suggests that there are only mild masking effects and provides an alternative to the use of unfixed material, particularly for morphological-immunohistochemical investigations.