C57BL/6J Research Articles

Slowing of fast-twitch muscle in the dystrophic mouse

Isometric twitch tension was measured in fast-twitch and slow-twitch muscles of normal and dystrophic ( C57Bl 6J dy 2J dy 2J ) mice in vivo. In dystrophic mice more than 6 months old the fast-twitch extensor digitorum longus (EDL) showed a prolongation of the time to peak tension as well as the time to relax to one-half peak tension ( 1 2 R T ) compared with age-matched controls. In younger dystrophic mice (4 to 6 weeks) the time to peak tension was prolonged but 1 2 R T not significantly so. This apparent “slowing” of dystrophic fast-twitch muscle was accompanied by a reduction in both cooling potentiation and post-tetanic potentiation toward values typical of slow-twitch muscle. Slow-twitch soleus muscle (SOL) of old mice was almost unaffected by the dystrophic process with regared to its contractile characteristics. However, there appeared to be a slight, but significant “speeding” of young dystrophic SOL compared with age-matched control muscles. This was apparent in reduced times to peak tension and half-relaxation as well as an enhanced cooling potentiation. We suggest that the altered contractile characteristics result from a change in some intrinsic property of the muscle fibers rather than from extrinsic factors such as the additional perimysial connective tissue seen in these muscles.

Serum creatine phosphokinase variations in dystrophic mice

Prior studies reported increased serum or plasma creatine phosphokinase (CPK) activity in dystrophic mice from strains C57BL 6J and 129 B 6F 1, but technique may have been a factor. In strain 129 REJ there are conflicting data with respect to CPK activities in the dystrophic mice and also with respect to any age effect on CPK activity. We compared serum CPK activity in all three dystrophic mouse strains and in a nondystrophic strain using a technique designed to eliminate or minimize muscle trauma. No significant difference in CPK activity with increasing age was noted in 129 B 6F 1 dystrophic or normal mice or in mice from the nondystrophic strain. Strains C57BL 6J and 129 B 6F 1 showed no difference in CPK activity between dystrophic and normal mice of the same strain. In strain 129 REJ, dystrophic mice had a significantly lower CPK activity than normal mice from the same strain. Significant interstrain differences in CPK activities for both normal and dystrophic mice were also observed among the three dystrophic strains as well as compared with the nondystrophic strain. Serum CPK activities are not elevated in mouse muscular dystrophy and, therefore, this disorder differs significantly from human dystrophy. Because of interstrain differences in CPK activities, studies using CPK activity as a dependent variable should use strain-specific controls.

Glutamine synthetase activity in the developing secondary palate and induction by dexamethasone

Glutamine synthetase (EC 6.3.1.2) (GS) and glutamyltransferase (EC 2.3.2.1) (GT) specific activity were examined in developing A/Jax and C57BL/6J (C57) mouse fetal secondary palates. In addition, the induction of palatal GS was also examined after maternal injection of dexamethasone. Palatal GT activity was uniformly higher in A/J than C57 palates with both strains showing highest activity late on day 13 of gestation and a drop in activity by early day 14. In contrast, A/J palatal GS activity peaked transiently late on day 13, dropped by early day 14 and remained lower throughout the remaining period of palatal development. Palatal GS activity in C57 mouse fetuses, although failing to show a discrete transient peak of activity, remained at a constant elevated level from early day 13 to late day 14 and did not decrease until day 15 of gestation. These elevated levels of palatal GS and GT activity correspond to the gestational period of maximal palatal glycoconjugate biosynthesis. Thus, palatal GS activity may play an important regulatory role in the synthesis of these macromolecules. A/J and C57BL/6J mice exhibit different susceptibilities to glucocorticoid-induced cleft palate. However, maternal administration of a non-teratogenic dose of dexamethasone on either late day 12 or late day 13 resulted in a dramatic stimulation of both A/J and C57 fetal palatal GS but not GT activity when assayed 18 h later. A/J palatal tissue responded to dexamethasone with greater induction of palatal GS activity than enzyme activity in C57 palates. Palatal GS, sensitive to glucocorticoid stimulation, may thus be an important link in expressing hormonal control of normal palatal differentiation.

Relationship between brain cortex and testis maturation rate in two inbred strains of mice

Two inbred strains of mice, SEC/1ReJ (SEC) and C57BL/6J (C57) showed significant differences in the maturation rate of two different anatomical districts: the brain cortex and the seminiferous epithelium. The cortical maturation rate, recorded by means of an electrocorticogram (ECoG), indicated that SEC mice are slower in the development of an adult wave pattern than C57 mice, which are more mature at birth. Furthermore, the C57 strain appears to have a faster maturation rate of the seminiferous epithelium. In fact, spermatogenesis in these mice shows the presence of spermatids in the seminiferous epithelium at 25 days, and mature spermatozoa in the epididymis at 40 days. Instead, the SEC strain, because of its slower development, shows a considerable delay of spermiogenesis and the epididymis is still empty at 40 days. These data, together with those on the ECoG differences of these strains, seem to indicate a relationship between brain and testis maturation.

Cell lineage analysis of cerebellar Purkinje cells in mouse chimeras

Murine chimeras provide an experimental system in which cell lineage analysis of the mammalian central nervous system (CNS) can be accomplished. Utilizing a cell marker system that permits the identification of cells of each genotype in various cell populations present in histologic sections of the CNS at different developmental periods, fate maps of the mammalian CNS can be constructed. Thus, the presence or persistence of clones of cells can be readily visualized in simply organized CNS regions, like the cerebellar cortex. The electrophoretic variants of the glycolytic enzyme, glucosephosphate isomerase (GPI, E.C. 5.3.1.9; GPI-1A, GPI-1B), are the genotype-specific cell markers most commonly used by experimental mammalian embryologists in studies of cell lineage utilizing mammalian chimeras. We have adapted this cell marker system to permit the visualization and unequivocal identification of cells containing the GPI-1B variant throughout the CNS of adult BALB cBy J a3 C57BL 6J chimeric mice. Utilizing allozyme-specific anti-GPI-1B antisera in immunocytochemical (PAP) staining techniques, we can score small as well as large cell populations, neurons as well as glia. We have reconstructed and statistically analyzed the location and distribution of chimerism present in the Purkinje cell population of four of these chimeric mice. We found the Purkinje cells in each of these animals existed as small (3–8) cell patches of like genotype that were not randomly arranged. This suggests that clones of cells may persist as contiguous groups of cells throughout mammalian cerebellar development.

Prostaglandin synthesis by primary cultures of mouse embryo palate mesenchyme cells

Primary cultures of mouse embryo palate mesenchyme cells could be stimulated to release compounds which behaved as authentic prostaglandins identified by thin layer chromatography and radioimmunoassay. Melittin was more effective in stimulating the release of incorporated [ 14C] arachidonic acid from C57BL 6J strain cells than from A J strain cells, suggesting a subtle strain difference in metabolic control over arachidonic acid metabolism.

Decreased in vivo acetaldehyde oxidation and hepatic aldehyde dehydrogenase inhibition in C57BL and DBA mice treated with carbon tetrachloride

Male C57BL 6J (C57) and DBA 2J (DBA) mice were dosed with 500 μl/kg ig CCl 4, 24 hr before a 3.25 g/kg ip dose of ethanol. The relative bioavailability of CCl 4 was similar in both mouse strains. CCl 4 pretreatment produced genotypically related decreases in blood ethanol elimination rates with DBA mice showing the greater decrease. Blood acetaldehyde concentrations were significantly elevated in the CCl 4-pretreated animals of both strains. DBA CCl 4-pretreated mice exhibited significantly higher blood acetaldehyde concentrations than C57 mice 1, 2, 3, and 4 hr after ethanol administration. CCl 4 pretreatment resulted in acetaldehyde elevations of approximately threefold in C57 mice four- to fivefold in DBA mice. Four hours after the ethanol dose, animals were sacrificed for enzymatic analyses of hepatic aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activity. The specific activities and activity per gram of cytosolic and mitochondrial ALDH were decreased in both mouse strains. ADH activity was not significantly altered by CCl 4 pretreatment. Liver necrosis, as measured by serum alanine aminotransferase activity, was similar in both strains (approx fivefold increase). These data indicate that CCl 4 increased blood acetaldehyde concentrations by inhibiting hepatic cytosolic and mitochondrial ALDH and that the degree of blood acetaldehyde elevation is different in the two inbred mouse strains C57 and DBA.

Effects of morphine and chlordiazepoxide on avoidance behaviour in two inbred strains of mice

Morphine and chlordiazepoxide were tested in the inbred strains of mice DBA/2J (DBA) and C57BL/6J (C57), subjected to three daily 50-tria1 avoidance sessions in the shuttle-box. The DBA strain reached higher avoidance levels in comparison to the C57 strain after the administration of saline. Morphine facilitated avoidance responding in the C57 strain but not in DBA mice. An improvement in avoidance behaviour was observed following the administration of chlordiazepoxide in both strains of mice. Some favourable effects were obtained in DBA mice by combining morphine with chlordiazepoxide, whereas no interaction between these drugs was found in the C57 strain.

Cortisone-induced cleft palate in the brachymorphic mouse.

Previous studies have shown that the autosomal recessive gene brachymorphic (bm/bm), which is maintained on a C57BL/6J (C57) background, reduces limb growth and sulfation of cartilage proteoglycans. Hydrocortisone administered on gestational days 11-14 resulted in 20% CP in the C57 mouse, but 95% CP in the bm/bm mouse. The bm/bm mouse had a median effective dose for CP of 45 mg/kg, compared to 325 mg/kg for C57 and 40 mg/kg for A/J. Morphometric analysis indicated that the time of palatal elevation was delayed in the bm/bm relative to the C57 mouse both with and without hydrocortisone treatment. The amount of cytoplasmic glucocorticoid receptor protein present in the bm/bm palate on day 14 was the same as the amount found in the C57 palate, and was not elevated as it is in the A/J palate. The levels of cyclic AMP in the bm/bm palate on day 14 were 30-70% higher than that found in the C57 palate with or without hydrocortisone. These results suggest that both bm/bm and A/J exhibit a delay in palatal shelf rotation and elevated levels of cyclic AMP, which appear to be predisposing factors for cortisone-induced cleft palate. These strains differ in that elevated levels of steroid receptors are present in A/J palate, whereas lower levels are found in the C57 and bm/bm mice.

Effects of morphine and chlordiazepoxide on locomotor activity in two inbred strains of mice

Summary The effects of morphine, given alone and in combination with chlordiazepoxide, have been investigated in the inbred strains of mice DBA/2J (DBA) and C57BL/6J (C57). Morphine increased the spontaneous locomotor activity only in the C57 strain, whereas no effect was found when it was administered to the DBA strain. Chlordiazepoxide, at dose levels which did not influence the locomotion, significantly increased the locomotor activity of DBA mice when given in combination with morphine. Conversely, chlordiazepoxide, at each dose tested in the present study, did not enhance the stimulation of locomotor activity elicited by morphine in the C57 strain.

The inhalation toxicology of benzene: Incidence of hematopoietic neoplasms and hematotoxicity in [formula omitted] and [formula omitted] mice

AKR J mice and C57BL 6J mice were given lifetime exposures to 100 and 300 ppm benzene, respectively. Peripheral blood cell counts were obtained biweekly throughout the exposures. Anemia and lymphocytopenia were produced in benzene-exposed AKR mice. Twenty percent of the exposed AKR mice developed bone marrow hypoplasia, compared to 2% for the controls. The benzene exposures did not alter the incidence or induction time of the viral-induced lymphomas commonly seen in AKR mice. In C57BL mice, exposure to benzene produced anemia, lymphocytopenia, and neutrophilia accompanied by a left shift. Thirteen (33%) of the exposed C57BL mice developed bone marrow hyperplasia and in four of these mice, hyperplasia was essentially limited to granulopoietic elements. None of the control C57BL mice developed bone marrow hyperplasia. In benzene-exposed C57BL mice there was a significant increase in the incidence of hematopoietic neoplasms including six cases (15%) of thymic lymphoma. Although two control mice (5%) died with lymphoma neither of these tumors involved the thymus. Thymic lymphoma is rare in C57BL mice but can be produced by ionizing radiation and chemical carcinogens.

Dependence of the porphyrogenic effect of 2,3,7,8-tetrachlorodibenzo( p)dioxin upon inheritance of aryl hydrocarbon hydroxylase responsiveness

Experimental porphyria caused by chlorinated aromatic hydrocarbons is associated with decreased uroporphyrinogen decarboxylase activity and induction of the microsomal mixed function oxygenase systems, especially cytochrome P 1-450 ( P-448). 2,3,7,8-Tetrachlorodibenzo( p)dioxin (TCDD) produces porphyria in mice and is a specific inducer of cytochrome P 1-450 and its associated aryl hydrocarbon hydroxylase (AHH) activity. To test the hypothesis that susceptibility to porphyria depends on responsiveness to induction of P 1-450, the susceptibility to TCDD-induced porphyria was studied in mice which were genetically responsive ( C57BL 6J ) or nonresponsive ( DBA 2J ) to induction of AHH. To compensate for nonspecific strain differences, mice were obtained by backcrossing the progeny from a C57BL 6J × DBA 2J mating with DBA 2J animals to yield the strain D2(B6D2)F 1. These animals were phenotyped for AHH responsiveness by determining the effect of β-naphthoflavone on zoxazolamine paralysis time. It was found that susceptibility to porphyria was inherited together with AHH responsiveness. Increase in urine porphyrin was similar in mice homozygous and heterozygous for AHH responsiveness but depression of uroporphyrinogen decarboxylase activity was intermediate in the heterozygotic group.

Assay for mouse tissue enzymes: Levels of activity and statistical variation for 29 enzymes of liver or brain

Automated methods for optimal rapid quantitation of the levels of 29 enzymes in the liver and brain of C57BL 6J mice using a miniature fast analyzer (MFA) are described. Enzyme assays were developed by varying reagent concentrations and pH until we obtained the highest activities with the lowest coefficient of variation for the 29 selected enzymes while maintaining linearity with protein concentration and time. Our techniques should be readily adaptable to the commercial prototypes of the MFA and other automated assay systems, for other strains or species, and a variety of applications such as the study of dietary effects or other types of perturbations or experimental manipulations on certain enzyme pathways. Using the described methods, we define the mean normal activity of 29 enzymes as well as the associated statistical parameters used to define these ranges of activity in C57BL 6J populations. These values are in turn useful in the identification of abnormal activities during routine analysis. The methods described are presently being incorporated into a mutagenesis test system to detect chemically induced mutations expressed as altered enzyme activity.

A defect of purine nucleotide cycle in the skeletal muscle of hereditary dystrophic mice

The purine nucleotide cycle in the hind leg skeletal muscle of hereditary dystrophic mice ( C57BL 6J - dy dy ) was investigated. The amount of adenine nucleotide produced from adenylosuccinate by the muscle extract in the dystrophic group was less than 3 % of that in the control group, while adenine nucleotide plus adenylosuccinate converted from IMP in the dystrophic group was about 70 % of that of the control group. Moreover, the activity of AMP deaminase of the dystrophic group was about 50 % of that of the control group. These results indicate that the purine nucleotide cycle is defective in the dystrophic muscle. This abnormality was suggested to be caused by the considerably low activity of adenylosuccinase.

Postweaning development of diabetes in [formula omitted] mice

In association with moderate diabetes, fully mature obese mice ( C57BL 6J ob ob ) display several disturbances of metabolism, behavior, and growth. As yet, no precise developmental relationships have been established between the major abnormalities of these mice, although obesity, hypometabolism, and reduced activity have been reported as early as 7 days of age and appear to precede the development of diabetes. In the present studies, examination of glucose regulation revealed that preweanling obese mice, which are already characterized by many metabolic and growth deficits, had significantly lower fasting glucose levels when compared with lean controls, and displayed subnormal glycemia in response to glucose or control stimulation. In contrast, 28-day-old mutant mice had mildly elevated fasting glucose levels and maintained severe hyperglycemia after either glucose or distilled water administration. In response to exogenous insulin (1 U/kg), obese mice at each age displayed significant reductions of serum glucose, but appeared to be mildly insulin-insensitive when their glucose responses were compared with those of lean mice. The present results suggest that several factors are likely to be responsible for the rapid postweaning development of diabetes in ob ob mice, including insulin resistance, dietary changes, and, possibly, an increased capacity for diabetogenic hormone production; however, the appearance of diabetes substantially later than several other metabolic disturbances indicates that abnormal glycoregulation is a relatively indirect consequence of the ob ob mutation.

Cobalt as a modifier of microsomal monooxygenases in mice

The influence of cobaltous chloride treatment on various functions of microsomal cytochromes was studied in C57BL 6J Han mice. Two daily doses of 40 mg/kg reduce the cytochrome P-450 content and the ethylmorphine N-demethylase activity to about 60%, based on microsomal protein. On the basis of microsomal cytochrome content, however, this activity and that of ethoxyresorufin dealkylation is not changed. In contrast, the deethylation of 7-ethoxycoumarin is even increased about fourfold by cobalt pretreatment. The inhibitor metyrapone decreases the 7-ethoxycoumarin-deethylation more strongly in cobalt-pretreated animals than in controls. α-Naphthoflavone has the opposite effect. The use of these “diagnostic” inhibitors suggests a certain prevalence of cytochrome P-450-like material in the microsomes. When ethoxyresorufin is the substrate, metyrapone inhibits the reaction apparently in two steps as manifested by two different inflection points of the inhibition curve that are apart from each other by four orders of magnitude in metyrapone concentration. Spectral measurements show a more pronounced binding spectrum of metyrapone in the cobalt-treated animals when the ligand is added in very low concentrations. However, no determination of ligand affinity was attempted. The described effects cannot be demonstrated after in vitro addition of cobaltous chloride to microsomes. The results suggest that cobalt administration to mice produces distinct and substrate-dependent alterations of the microsomal mixed function oxidase activity.

Inhibition of growth in vitro by glucocorticoids in mouse embryonic facial mesenchyme cells.

The growth of primary embryonic facial mesenchyme cells established from cleft palate sensitive A/J and resistant C57BL/6J (C57) mice is inhibited by glucocorticoid treatment. A reduction in cell number in both A/J and C57 culture is accompanied by a significant decrease in [3H] thymidine incorporation into both acid soluble and insoluble material. No significant changes in total cellular protein or [14C] leucine incorporation were observed in either cell type. A greater reduction in [3H] thymidine incorporation occurs in cells undergoing exponential growth following steroid exposure than in cells approaching stationary growth. In both A/J and C57 cultures the reduction in cell number exhibits a dose-dependent response to dexamethasone; is specific for glucocorticoids; and is dependent upon the concentration of serum in which the cells are maintained. A/J cells show a greater sensitivity to the inhibitory effect of dexamethasone on cell number and thymidine incorporation than comparably treated C57 cells. Specific, high affinity, saturable cytoplasmic receptors for [3H] dexamethasone are present in the maxillary cytosols from which the primary cultures were established. These receptors exhibit binding specificity for glucocorticoids, and have properties which are similar to glucocorticoid receptors identified in other systems. In both cell types, a correlation exists between the degree of growth inhibition or reduction of [3H] thymidine incorporation and the level of glucocorticoid receptors. These results provide evidence for a receptor-mediated set of responses to glucocorticoids in these cells.

Increased synthesis of l-glycerol 3-phosphate dehydrogenase during in vitro differentiation of reaggregating cerebellar cells

Reaggregating cell cultures of neonatal mouse cerebellar cells express many of the differentiated properties of normal developing cerebellum, including the transition for the embryonic and adult isozymes of l-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8). In order to determine the mechanism leading to increased levels of adult isozyme, aggregates in culture from 2 to 17 days were labeled with radioactive leucine and the relative rate of enzyme synthesis was measured after purification of the enzyme by affinity chromatography on Blue Sepharose 6B. During the course of in vitro differentiation, the relative rate of synthesis increased 100-fold, such that it represented 0.5% of the total protein synthesized in the cytoplasmic fraction of the cell. In vivo, BALB cBy mice have twice the level of enzyme activity in the cerebellum as do C57BL 6J mice. Reaggregating cell cultures of cerebellar cells from these strains of mice also express a difference in the activity level, but only when the cerebellar cells are taken from mice 4 days of age or less. When the relative rates of synthesis of l-glycerol 3-phosphate dehydrogenase were measured in cultures expressing the strain-dependent difference in activity, these rates were found to be approximately twofold greater in cultures of BALB cBy cells. In contrast, estimates of the relative rate of enzyme degradation by the double-isotope labeling technique indicate that neither specific enzyme degradation nor degradation of total protein is different in aggregates from the two strains of mice. The results suggest that the genetic mechanisms controlling the levels of l-glycerol 3-phosphate dehydrogenase in the cerebellum during development are intrinsic to the cells and, with the exception of serum factors, are independent of systemic influences.

Studies of the genetic and immunologic components of the maternal age effect

To assess the genetic component of the maternal age effect, the frequencies of aneuploid embryos derived from several inbred mouse strains were examined as a function of maternal age. Some of the strains were chosen to evaluate possible etiologic agents: CBA for premature reproductive aging and increased univalent frequency, and NZB J and A J for predisposition toward autoimmunity. The C3H HeJ and C57BL 6J strains also were examined. Although there was considerable variation between strains, an increased frequency of aneuploid fetuses was observed in all strains as a function of maternal aging. The CBA strain revealed the greatest increase in frequency of aneuploid fetuses at a relatively early maternal age. The effect of autoimmunity was assessed by analyzing the frequency of aneuploid embryos derived from thymectomized A J females. No significant difference in aneuploidy was found between thymectomized and control nonthymectomized animals.

Glutathione S-transferase activities in rat and mouse sperm and human semen

Glutathione S -transferase activity was found in sperm of the rat and DBA 2J and C57 BL 6J mice. In rat sperm activities with benzo(a)pyrene 4,5-oxide, styrene 7,8-oxide, and 1-chloro-2,4-dinitrobenzene were 0.88, 1.07, and 26.1 nmoles/min/mg protein, respectively. Δ 5-3-Ketosteroid isomerase activity of rat sperm was 4.9 nmoles/min/mg protein. These specific glutathione S -transferase and Δ 5-3-ketosteroid isomerase activities in sperm represent 0.4–4.1% of rat liver cytosol values. Human semen also contained significant glutathione S -transferase activity. It is postulated that these enzymes could function in the metabolism and detoxification of certain electrophilic xenobiotics, if present in sperm.