To reveal the developmental process of the chondrocranial base of the musk shrew, Suncus murinus (Insectivora), light microscopic observation of serial sections and macroscopic observation with whole-mount differential staining of bone and cartilage were performed on day-17 to -22 embryos. The following unusual findings were obtained: (1) The sphenoethmoidal commissure formed a part of the posterior edge of the tectum nasi, implying that the posterior portion of the paries nasi in the mammalian nasal capsule shares a common origin with the neurocranium. (2) The processus alaris arose independently from the hypophysial cartilage and also contributed to formation of the carotid foramen. (3) The hypophysial cartilage consisted of one medial and a pair of lateral cartilage nodules. The homology of the pair of cartilages with polar cartilages in lower vertebrates is discussed.

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SMXA recombinant inbred (RI) strains of mice were produced by systematic inbreeding from the F₂ generation of a cross between two progenitor inbred strains, A/J and SM/J, which differ considerably with respect to many characteristics, and consists of 28 inbred strains. In this study, we investigated the applicability of DNA fingerprinting with M13 phage DNA to the identification of these closely related strains. DNA fingerprints of the SMXA RI strains and their progenitors, SM/J and A/J, showed strain-specific patterns, with the same banding patterns within each strain. Linkage analysis by using strain distribution patterns of minisatellite loci with 108 genetic markers containing microsatellites, biochemical and immunological marker genes allowed 23 minisatellite loci to be assigned to 11 chromosomes. The results suggested that DNA fingerprinting with M13 phage DNA is applicable not only for strain identification but also for genetic monitoring of RI strains on almost all chromosomes.

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Two virus strains were isolated from the lungs of athymic rats and mice used as sentinel animals in 2 colonies of laboratory rats in Japan in which antibodies to the pneumonia virus of mice (PVM) had been detected. The new isolates were identified as PVM by the following characteristics: RNA virus, susceptibility to ether treatment, long filamentous viral structure in the cytoplasm of infected cells, and hemagglutinating activity in various erythrocytes, including those of mice and rats. In addition, cross neutralization with the prototype of PVM (No.15 strain) was observed. This is the first report of the isolation of PVM from laboratory animals in Japan.

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Tetraploid mouse embryos usually cease to develop early after implantation, though they can develop to blastocysts. To characterize the failure of development in detail, tetraploid mouse embryos at the preimplantation period were examined as to both their morphology and number of cells. The tetraploid embryos were produced by 12 hr treatment with cytochalasin B (CB) at the 2-cell stage of backcross of (C57BL/6 × C3H/He) F₁ × C3H/He. The tetraploid embryos in the preimplantation period exhibited compaction at 72 hr after hCG injection and blastocyst formation at 96 hr, as well as diploid embryos, but the number of cells composing the embryos was significantly smaller than that in the diploid embryos. At the term 60-96 hr after hCG injection, mean cell cycles were 14.03 hr in the tetraploid embryos, but 12.02 hr in the diploid. When tetraploid embryos were transferred into the oviducts of pseudopregnant recipients immediately after CB treatment, the number of cells in tetraploid blastocysts was increased compared with the embryos cultured in vitro, though the number did not reach that of diploid embryos. These results suggested that compaction and blastocyst formation in preimplantation development of tetraploid embryos depended on the time after hCG injection, irrespective of the number of cells or the length of the cell cycle. The lengthening of the cell cycle in tetraploid embryos may be one of the causes of failure in postimplantation development.

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Adult Wistar-Imamichi rats were induced to superovulate by an i.p. injection of PMSG followed by an i.p. injection of hCG 48 hr later. The doses of PMSG and hCG administered were 0, 75, 100, 150, 300 iu/kg body weight. In this study the doses were based on iu/kg body weight rather than iu/rat to facilitate accurate induction. The following results were obtained. In the adult rats (10 to 15 weeks old) PMSG induced an increase in the number of morphologically normal and fertilized oocytes, as well as in the total number of superovulated oocytes, dose-dependently upto 150 iu/kg. The increase was significant compared with control rats, but PMSG treatment had only half this effect in young rats (6 to 9 weeks old), compared with the adult rats. These results suggest that sensitivity to PMSG changes with age. The proportion of fertilized oocytes among the morphologically normal oocytes was not significantly different in young and adult rats, and both groups had high percentages. In view of the results of this study, the authors recommend that, to collect a large number of fertilized oocytes required for generation of transgenic rats, the induction of superovulation be performed in adult rats aged 10-15 weeks rather than young rats aged 6-9 weeks and giving PMSG at a dose of 150 iu/kg and hCG at a dose of 75 iu/kg.

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In order to develop genetic markers in rats, arbitrarily primed polymerase chain reaction (AP-PCR) was performed with single primers originally designed for microsatellite loci, instead of primers with short-sized and arbitrary nucleotide sequences. Each primer generated reliable and reproducible segments under optimal conditions. The fourteen amplification products have been successfully mapped to rat chromosomes by either linkage analysis using backcross progeny or chromosomal assignment with somatic cell hybrids. All of the loci were located on different chromosomes from those of the microsatellite loci, suggesting that sequence-specifically designed single primers can produce anonymous segments of genomic DNA showing polymorphisms. These markers should contribute to finding linkages for traits of interest in rats.

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The recombinant inbred (RI) mouse group is useful for genetic analysis of a phenotype which is regulated by multiple loci. The female adrenal cortex was compared histologically to detect the strain differences between A/J and SM/J for the purpose of genetic analysis of adrenocortical morphogenesis using the SMXA RI group constructed between A/J and SM/J. Clear morphological differences were found in the zona reticularis and X zone in addition to the significantly small body and adrenal weights in SM/J. The z. reticularis was markedly thicker in SM/J than in A/J and the X zone was constituted of vacuolated cells in A/J and non-vacuolated cells in SM/J. The genetic control of these morphological traits was analyzed using SMXA RI strains. The results suggest that the thickness of the z. reticularis may be controlled by single locus and that vacuolation of the X zone by 3 different loci between A/J and SM/J.

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Male golden hamsters were exposed to coal fly ash (FA) at the concentration of 0 (control), 1, 2, or 20 mg/m³ for 3 or 6 months (20 hr/day, 7 days/week). They were sacrificed at 0, 1, 3 and 6 months after exposure to undergo morphological investigation of the lung clearance of particles ingested by alveolar macrophages (AMs) following excessive deposition of FA particles. Particle-laden AMs in a cluster decreased in inverse proportion to the dust burden after the cessation of exposure. AMs with well-spread lamellipodia frequently appeared on the degenerated AMs in a cluster. In addition, neutrophils also agglomerated mainly in the alveoli proximal to the terminal bronchioles and ingested a small amount of particles. These results suggest that AMs and neutrophils might recruit into alveoli enclosing AMs in necrosis and reingest released particles. Macrophages in bronchioles generally ingested fewer particles than those clustered in alveoli. This finding suggests that AMs ingesting a small amount of released particles could move on the mucociliary escalator of the terminal bronchioles. On the other hand, AMs ingesting a large amount of particles would agglomerate in alveoli due to the loss of their migrating ability. The latter agglomeration of particle-laden AMs might be responsible for the retardation of lung clearance in proportion to the lung burden of particles.

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The induction of ovulation by hormone treatment, preparation of fertilized eggs by in vitro fertilization and recovery of offspring by embryo transfer were studied in five strains of mutant mice: C57BL/6-dy/dy progressive muscular dystrophy model, C57BL/6- ob/ob obesity model, and BALB/c-rl/rl , BALB/c- shi/shi and C57BL/6-mld/mld motor ataxia models. The homozygotes of these mutant mice are all affected with the disease about 2 weeks after birth, followed by reproductive disturbances. Ovulation could be induced by injection with PMSG-hCG in the females. Sperm was obtained from the cauda epididymis of males and used for in vitro fertilization. The success rate of the in vitro fertilization was as low as 71.6% in C57BL/6-dy/dy mice, but was over 85% in the other strains. When 2-cell embryos obtained by in vitro fertilization were transferred to the oviducts of pseudopregnant recipients, offspring were obtained from 39.2-57.7% of the transferred embryos. These offspring developed the expected diseases about 2 weeks after birth, and it was confirmed that the disease characters were reliably reproduced. These results demonstrate that the experimental system of in vitro fertilization and embryo transfer enables production of the offspring homozygous for a mutant gene and use of them for experiments before the onset of the disease which has been impossible.

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We attempted to convert the genetic background of transgenic (Tg) mice in a short period of time by applying in vitro fertilization techniques. Tg mice were obtained by injecting human interleukin-2 (hIL-2) gene into the fertilized eggs of the C57BL/6N strain. These Tg mice were back-crossed 8 times to the inbred C3H/HeN strain using the hIL-2 gene as the genetic selection marker. In order to shorten the length of the back-crossing time, in vitro fertilization was performed with eggs collected from immature Tg females and spermatozoa from mature C3H/HeN males, and successfully fertilized eggs (2-cell stage) were transferred to pseudopregnant recipients to obtain the offspring of next generation. When no offspring were obtained through the procedures using immature Tg females, in vitro fertilization was performed with mature Tg males and mature C3H/HeN females to continue successive back-crosses. With this method, it was possible to perform 8 successive back-crosses in 18 months.

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We examined the age-associated changes in tumor necrosis factor α (TNFα) receptor on peripheral blood monocytes and bone marrow macrophages obtained from slc:Wistar rats and slc:ICR mice. The TNFα receptor on monocytes and macrophages in both rats and mice showed high and low affinity sites. The Kd values of those sites gradually increased with age in both types of cells from both species. Binding capacities (receptor number) were low in 52-week-old animals. These results suggest that the TNFα receptor on monocytes and macrophages of these species might decrease with age in both quality and quantity.

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Restriction endonuclease analysis of amplified nucleocapsid protein genes from mouse hepatitis virus (MHV) was used to differentiate 12 strains isolated from mouse liver or transplantable tumors from five facilities, and the restriction patterns of the isolates were compared with those of five well-defined MHV strains, A59, JHM, 2, S and Nu-67. The patterns of 10 isolates from three facilities were the same as that of Nu-67. The remaining two isolates revealed different patterns from the five reference strains. This study showed that reverse transcription and the polymerase chain reaction assay based restriction analysis are feasible for the detection and genotyping of MHV, and the Nu-67 related strain was the most prevalent type found in the clinical samples.

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Myocardial infarction was induced by ligation of the coronary artery in fifteen 32-week-old male spontaneously hypertensive rats (SHR) and fifteen 32-week-old male normotensive Wistar-Kyoto rats (WKY). Age-matched male SHR (n=6) and WKY (n=7) without the operation were used as non-infarcted control rats. Four weeks after the operation, cartilaginous metaplasia and osseous metaplasia were observed histopathologically in all SHR and 13 of the 15 WKY with myocardial infarction, but not in the control rats. The mean rates of infarction to the heart and the mean areas of metaplasia per rat showing metaplasia were greater in SHR than in WKY. The metaplastic areas were well correlated with the infarct percentage in SHR. It is therefore considered that extensive myocardial infarction is associated with the pathogenesis of cartilaginous and osseous metaplasia.

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The distribution of deoxyribonuclease II (DNase II) in tissues and body fluids was examined in 12-week-old C3H/He mice. Activity was observed in most tissues and body fluids except erythrocytes and serum, but their levels were quite different among tissues. Activity was high in the spleen, salivary gland, and preputial gland, moderate in the liver, kidney, thymus, lung, heart, pancreas, seminal vesicle, coagulating gland and prostate and low in the brain, testis and muscles. Sex difference, males having a significantly higher DNase II activity level than females, was observed in salivary gland, kidney and urine.

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