A quantitative trait locus that accounts for glucose intolerance maps to chromosome 8 in hereditary obese KK-A(y) mice.

KK and KK-A(y) mice serve as animal models of non-insulin-dependent diabetes mellitus with moderate obesity. Quantitative trait locus (QTL) analysis was performed to identify gene loci that account for fasting hyperglycemia, glucose intolerance and plasma insulin in 91 F(2) females of a C57BL/6JxKK-A(y) intercross. For glucose intolerance, a significant QTL was identified on chromosome 8, with a maximum lod score of 5.6. This locus had strong influence on the late phase of the intraperitoneal glucose tolerance test (IPGTT), suggesting that the locus may have role in glucose clearance rather than in mere hyperglycemia. In addition, three suggestive QTLs were identified on chromosomes 1 (fasting glucose), 3 (fasting insulin) and 4, (blood glucose at 120 min during IPGTT, and glucose intolerance).

Genetic analysis of inferior nurturing ability in RR mice.

Females of the inbred mouse RR strain have a limited ability to nurture their offspring, and frequently the young die during rearing. Quantitative trait locus analysis was carried out on the F2 progeny produced from a genetic cross between RR and KK, a strain of normal nurturing ability, to elucidate the putative genetic basis governing certain aspects of the inferior nurturing ability in the RR strain. One hundred and ninety-two F2 female mice were mated with C57BL/6J males. After the number of newborns was adjusted to six per dam, nurturing ability was evaluated on the basis of litter weight at days 7, 12 and 21 after parturition. The results showed that 147 of the 192 F2 dams were able to rear all six pups, although the litter weight varied considerably among litters. Significant evidence for linkage was identified on chromosome 5 near the microsatellite marker of D5Mit161 for litter weight at 12 days after parturition. The R allele (RR strain) at this locus was recessive to the K allele (KK strain), and reduced the litter weight. It is suggested that this locus is one of the heritable components that determine the inferior nurturing ability of RR mice.

Quantitative trait loci that modify the sootiness of yellow pigmentation in KK-A(y)/a mice.

Compared with C57BL/6J-A(y)/a, KK-A(y)/a mice have yellow fur that is markedly darker. Furthermore, there is a considerable variation in the tone of color with a continuous range in F(2) progeny produced from C57BL/6J females and KK-A(y)/a males. The aims of this study are to reveal the phenotypic differences between the two A(y) congenic strains and to elucidate the genetic factors responsible for the sooty yellow pigmentation in the KK background. On the basis of a chemical analysis, the sootiness in KK-A(y)/a was the result of increased eumelanin (PTCA) and decreased pheomelanin (AHP). A statistically significant QTL was identified on Chromosome (Chr) 15, responsible for the AHP content. No significant loci responsible for PTCA were identified. On the other hand, on the basis of an optical analysis for color difference and overall sootiness, significant evidence of linkage was identified on the proximal part of Chr 15, in the region similar to AHP QTL. The overall sootiness is thus controlled solely by the locus on Chr 15 in F(2) progeny; however, the KK allele at this locus significantly increased the AHP content.

Genetic analysis of neonatal death with growth retardation in F(1) male Dh/+ mice.

Nearly all F(1) male mice with Dh/+ genotype between DDD female and DH-Dh/+ male die within a few days after birth; however, this is not observed in the reciprocal cross. The F(1) Dh/+ males usually exhibit growth retardation prior to death. To identify the putative genetic locus or loci in DDD genome that cause the abnormalities in the presence of the Dh, a linkage analysis was carried out in backcross progeny of a cross of (DDD female x DH-+/+ male) F(1) female x DH-Dh/+ male. Appearance of growth retardation was examined from the day of birth, and both growth-retarded and normally weaned Dh/+ males were genotyped for microsatellite marker loci spanning autosomes and the X Chromosome (Chr). Significant evidence for linkage was identified on the distal edge of the X Chr, near the microsatellite marker of DXMit135. Furthermore, among mice from DDD female x reciprocal F(1)Dh/+ male produced between DH-Dh/+ and progenitor strains (C57BL/6J, C3H/HeJ and BALB/cA), only the progeny from female DDD x male (female DH-Dh/+ x male C3H/HeJ) F(1) Dh/+ male did not show any lethality and/or growth retardation. Thus, the lethality in F(1) Dh/+ males accompanied by growth retardation is caused by the interactions between the Dh gene, X Chr, and Y Chr. Based on the CAG repeat sequence length polymorphism among Mus musculus musculus Sry gene, C3H/HeJ was different from C57BL/6J, BALB/cA, and DH. These data suggest that there are at least two functional types of Y Chr in Mus musculus musculus.

Quantitative trait loci that regulate plasma lipid concentration in hereditary obese KK and KK-Ay mice.

To identify quantitative trait loci (QTLs) responsible for regulating plasma lipid concentration associated with obesity, linkage analysis was carried out on the 190 F2 progeny of a cross between C57BL/6J female and KK-Ay (Ay allele at the agouti locus congenic) male. In F2 a/a (agouti locus genotype) mice, two QTLs were identified on chromosome 1 and a QTL on chromosome 3 for total-cholesterol. A QTL for HDL-cholesterol was identified on chromosome 1 and a QTL for NEFA on chromosome 9. In F2 Ay/a mice, two QTLs for HDL-cholesterol were found on chromosome 1. Loci for other lipids with suggestive linkage were also identified. In both F2 mice, one QTL on chromosome 1 for total- and HDL-cholesterol was mapped near D1Mit150, in the vicinity of the apolipoprotein A-II (Apoa2) locus. Seven nucleotide substitutions out of 309 nucleotide apolipoprotein A-II cDNA sequences were identified between KK and C57BL/6J. The Ay allele may be an indication of the plasma lipid levels, but its influence was less apparent than in the case of weight control. The loci for lipids were not on identical chromosomes with those previously identified for obesity, suggesting that hyperlipidemia in KK does not coincidentally occur with obesity.

Genetics of obesity in KK mouse and effects of A(y) allele on quantitative regulation.

KK mouse is known as a polygenic model for noninsulin-dependent diabetes mellitus with moderate obesity. To identify the quantitative trait loci (QTLs) responsible for the body weight in KK, linkage analysis with 97 microsatellite markers was carried out into 192 F2 progeny, comprising 93 mice with a/a genotype at agouti locus and 99 mice with A(y)/a genotype, of a cross between C57BL/6J female and KK-A(y) (A(y) congenic) male, thereby the influence of A(y) allele on the quantitative regulation of body weight was also examined. In F2 a/a mice, we identified a QTL on Chromosome (Chr) 4, and two loci with suggestive linkage on Chrs 15 and 18. In F2 A(y)/a mice, a QTL was identified on Chr 6, and two loci with suggestive linkage were identified on Chrs 4 and 16. That the QTL on Chr 4 was held in common between F2 a/a and F2 A(y)/a progenies implies that this locus may be a primary component regulating body weight in KK and KK-A(y). These results suggest that the body weight in KK is controlled by multiple genes, and the different combination of loci is involved in the presence of A(y) allele. The QTL on Chr 6 seemed to determine the body weight by controlling fat deposition, because the linkage was identified on body weight and adiposity, and is suggested to be a component involved in the metabolic pathway in obesity caused by the A(y) allele.

Genetic analysis of non-insulin-dependent diabetes mellitus in KK and KK-Ay mice.

The KK mouse is considered suitable as a polygenic model for human non-insulin-dependent diabetes mellitus. To identify the quantitative trait loci (QTLs) responsible for hyperglycemia and impaired glucose tolerance in KK mice, linkage analysis using 97 microsatellite markers was carried out in a 192 F2 progeny, comprising 93 mice with the a/a genotype at the agouti locus (chromosome 2) and 99 mice with the Ay/a genotype, produced by a cross between a C57BL/6J female and a KK-Ay (Ay congenic) male. In F2 a/a progenies, we identified a QTL for fasting glucose levels on chromosome 6 (LOD score 6.0) and three loci with suggestive linkage on chromosomes 3, 5 and 14, but could not identify loci accounting for glucose tolerance and plasma insulin levels. In F2 Ay/a progenies, there were no loci with statistically significant linkage, but three suggestive loci were identified: a locus for fasting glucose on chromosome 9, and two loci for glucose tolerance on chromosomes 1 and 8. It would thus appear that. although the fasting glucose level is controlled by QTLs in KK mice, these QTLs may be masked by the strong hyperglycemic influence of the Ay allele. Suggestive loci accounting for glucose tolerance may interact with the Ay allele, since these loci were identified only in F2 Ay/a progeny. This is consistent with the finding that the impaired glucose tolerance in KK mice is moderate and becomes overt when associated with the Ay allele.

Microbial conversion of mevinolin.

About 3000 microorganisms (bacteria, Actinomyces, Zygomyces, Deuteromyces) were screened for their capacity to convert mevinolin. Absidia coerulea IDR 705 was found to produce two hydroxylated derivatives of mevinolin, 2 and 3. Compound 2 is a new transformation product while compound 3 was described as a chemical modification product of mevinolin. By combination of spectroscopic techniques, the structures of 2 and 3 were identified with beta,delta-dihydroxy-7-(1,2-dihydro-2-hydroxymethyl-6-methyl-naphthal en-1-yl)-heptanoic acid delta-lactone and beta,delta-dihydroxy-7-[1,2,3,5,6,7,8,8a-octahydro-3,5-dihydroxy-2, 6-dimethyl-8-(2-methyl-butyryloxy)-naphthalen-1-yl]-hepta noi c acid delta-lactone, respectively. The inhibitory effects of the two derivatives on the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase were similar to that of mevinolin.

Rectovesical fistula: a possible cause of postnatal lethality in F1 Dh/+ male mice between DDD female and DH (Dh/+) male.

To reveal a major cause of postnatal lethality in F1 Dh/+ male mice from DDD x DH (Dh/+) mating, the current study was performed. Dh/+ males with retarded growth appeared within a few days after birth. The unusual junction between the rectum and the urinary bladder (rectovesical fistula) was found in nine of 22 Dh/+ male mice, and the bladder contained unusual substances derived from the intestines. When the fistula was recognized, the urine was always gelatinous, and the anus was imperforate in eight cases, suggesting that an affected individual would have trouble in urination and evacuation. From these observations we conclude that the rectovesical fistula is a major cause of postnatal death in the F1 Dh/+ male.

Penetration by field vole spermatozoa of mouse and hamster zonae pellucidae without acrosome reaction.

Spermatozoa of the field vole (Microtus montebelli) that bound to the zona pellucida of field vole oocytes underwent the acrosome reaction before passing through it. In contrast, vole spermatozoa that bound to the zonae of mouse and hamster oocytes penetrated the zonae without any sign of the acrosome reaction. The presence or absence of proteinase/hyaluronidase inhibitors in the medium did not make any difference to zona penetration by acrosome-intact vole spermatozoa. These observations suggest that field vole spermatozoa can cross mouse and hamster zonae mechanically without assistance from zona-hydrolysing enzymes.

Effect of hypotaurine on in vitro fertilization and production of term offspring from in vitro-fertilized ova of the Japanese field vole, Microtus montebelli.

The present study was proposed to establish new in vitro fertilization (IVF)-embryo transfer techniques for wild mammals. We demonstrated that IVF of the Japanese field vole, Microtus montebelli, is improved by hypotaurine, and that IVF vole embryos can develop to normal offspring after transfer to recipient females. The rate of IVF was significantly increased when 0.1 or 1 mM of hypotaurine was added during sperm preincubation and fertilization (p < 0.05). Preincubation of spermatozoa with 1 mM hypotaurine prior to oocyte insemination did not have a significant effect on fertilization rate (p < 0.01). At 96 h after insemination, most cultured embryos developed to the 2-cell stage (68-83%), but development to blastocysts was very low (0-3%) for all treatments. Although 43 living offspring were produced after transfer of IVF pronuclear embryos to recipients, the delivery rate of recipients was very low (21%). The litter size of pseudopregnant recipients that produced a litter was 4-11, and the rate of in vivo development of transferred embryos in the pregnant recipients was 41%. These results suggest that hypotaurine affects IVF rather than sperm preincubation and that IVF embryos can develop to normal offspring.

Skeletal malformations caused by the Dh (Dominant hemimelia) gene in mice.

Morphological characterization of the skeletal malformations caused by the Dh gene was carried out. Skeletal preparations from newborn mice of Dh/Dh, Dh/+ and +/+ genotypes, and adult Dh/+ mice were examined. The Dh gene induced skeletal deformities at the axial regions from the thorax to the tail. Attachment of the ribs to the sternum was sometimes irregular, and normal segmentation of the sternum was also disrupted. Abnormal fusion of the ventral part of the rib was observed in some cases. Lumbar vertebrae were fewer in number and abnormal in shape. Although it was rare, a curled tail was also observed due to distortion of the caudal vertebrae.

High lethality of F1 (Dh/+) male mice from the cross between DDD female and DH (Dh/+) male.

The present study demonstrates the incidence of high lethality of F1 (Dh/+) male mice, the offspring of DDD females and inbred DH (Dh/+) males. Among the newborn pups, the numbers of F1 (Dh/+) males are comparable to those of mice having other genotypes (+/+ male, Dh/+ female and +/+ female), but many F1 (Dh/+) males died before weaning. On the other hand, among the F1 mice from the cross between DH (Dh/+) females and DDD males, there are no biased numbers of Dh genotype pups. These results suggest the existence of defects in the interaction between the male derived Dh gene and the genetic traits of the DDD female.

Incomplete development of the spleen and the deformity in the chimeras between asplenic mutant (Dominant hemimelia) and normal mice.

The semidominant gene Dh (Dominant hemimelia) induces skeletal and visceral abnormalities of various degrees and failure of the spleen in mice. The homozygous individual (Dh/Dh) seems to be lethal. The present experiment was designed to investigate the ability Dh cells to form a spleen and the genesis of the hind limb malformations by Dh/Dh and Dh/+ cells in chimeric mice. The Dh/Dh and Dh/+ embryos were produced in the F2 progeny of a cross between inbred strains of Dh/+ and DDD mice. They were aggregated with C3H/He or C57BL/6 embryos to make chimeras. Identification of Dh/Dh or Dh/+ embryos was carried out by Pep-3, and chimerism was analyzed by Gpi-1. Of 25 chimeras carrying the Dh gene, four mice formed a small spleen, two mice had a vestigial spleen, and the others no spleen. The tissues of the incompletely developed spleens were normal histologically and Dh cells were involved in the tissues of the spleen. In the chimeric mice, hindlimb malformation by the Dh gene was reduced in severity and the lethality of the homozygote (Dh/Dh) was rescued.

Production of term offspring by in vitro fertilization using old mouse spermatozoa.

We successfully produced offspring of old male BDF1 mice using in vitro fertilization. Although 7 old male mice (33 months of age) were infertile and revealed frequent degeneration in the seminiferous epithelia, 4 of them had spermatozoa in the caudae epididymides. The IVF rate of their sperm with eggs from young ICR mice was very high (82.0%). The production rate of offspring was also satisfactory (61.6%). The present study indicates that the IVF method is useful for producing offspring of aged male mice.

In vitro fertilization and embryo development of Japanese field voles (Microtus montebelli).

Optimal conditions for in vitro fertilization of Japanese field voles (Microtus montebelli) were analysed. The medium used was a modified Krebs-Ringer bicarbonate devised for in vitro fertilization in rats. Ovulated eggs and epididymal spermatozoa were co-incubated in vitro at 37 degrees C under 5% CO2 in air for 6 h, and the eggs were fixed with 2.5% (w/v) glutaraldehyde, stained with 0.25% (v/v) acetolacmoid and examined for evidence of fertilization at the pronuclear stage. Although the fertilization rate with spermatozoa preincubated at 1-2 x 10(8) cells ml-1 for 2 h was very low (1-13%), it was significantly increased (43-51%, P < 0.05) when spermatozoa were preincubated at a lower concentration (1-2 x 10(7) cells ml-1). Furthermore, the fertilization rate was significantly higher with 1 mmol hypotaurine l-1 (74.0%) than without hypotaurine (44.4%, P < 0.05). Fertilization rates of spermatozoa preincubated at 1-2 x 10(7) cells ml-1 for 0.5 or 2 h were similar (69.0% and 73.6%), but a longer preincubation (10 h) resulted in a significantly lower fertilization rate (56.8%, P < 0.01). Vole spermatozoa preincubated for 2 h penetrated the zona pellucida 2 h after insemination, and the sperm heads became decondensed 3 h after insemination. At 6 h after insemination, male and female pronuclei were found in most penetrated eggs. When the eggs were left in the fertilization medium without washing and cultured for 96 h after insemination, they developed to two-cell (82.6%), four-cell (60.9%), eight-cell (23.2%) and morula/blastocyst (8.7%) stages in modified Krebs-Ringer bicarbonate supplemented with 1 mmol hypotaurine l-1.