To increase the efficiency of Syrian hamster reproduction, individual males were caged with 2 or 4 estrous females. The fertility of the males caged with 2 or 4 females was lower than that of those caged with a single estrous female. To clarify the cause of this, we observed the copulating behavior of males for 30 minutes and examined their fertility. The median frequency of mounting, intromission and ejaculation was 27, 36 and 3, respectively, for the males caged with a single female, 37, 27 and 4, respectively, for the males caged with 2 females and 13, 2 and 0, respectively, for the males caged with 4 females. Thus, these parameters were markedly reduced in the 1: 4 mating group as compared with the 1: 1 and 1: 2 mating groups. In the 1: 2 mating group, spermatozoa were detected in the vaginas of 12 of the 14 females, but only 6 of these 12 females delivered litters. In the 1: 4 mating group, complete sequences of copulating behaviors were rarely seen, and spermatozoa were detected in the vaginas of only 6 of the 28 females, and only one of these 6 females delivered. Thus, the efficiency of offspring production was not increased by raising the number of estrous females exposed to a male, probably due to a decrease in the frequency of male copulation in the presence of 2 females and due to inhibition of the male' s copulating behavior in the presence of 4 females.

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Mouse renal juxtaglomerular cells (JGC) were vitally stained with neutral red, and then fixed with glutaraldehyde to study by electron microscopy. Ultrastructural observation showed a large number of characteristic low electron dense juxtaglomerular granules (JGG) or low dense granules (LDG) in the cytoplasm of JGC. Many LDG contained a flocculent material with a dense core, which was as dense as the contents of JGG. Two kinds of LDG were observed. One of LDG was surrounded by single membrane and partial membrane, and another was surrounded by double membranes. Some LDG did not contain electron dense flocculent material. LDG were surrounded by rough endoplasmic reticulum (r-ER) . Alteration of Golgi apparatus and r-ER was observed in JGC. Golgi complex did not associate with JGG and LDG.

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Optimal conditions for detection of calcitonin mRNA were examined by in situ hybridization (ISH) method using a non-radioactive probe in the rat thyroid gland. An oligonucleotide complementary to rat calcitonin mRNA was synthesized with a DNA synthesizer, labelled at 3'-end by using digoxigenin-11-deoxyuridine triphosphate (dUTP) and terminal transferase (Boehringer Mannheim), and used as a probe. Hybrid chains formed by probe and calcitonin mRNA were visualized by anti-digoxigenin-alkaline phosphatase conjugate, nitroblue tetrazolium (NBT) and X-phosphate (Boehringer Mannheim) . To determine the optimal conditions for ISH, relations between tissue fixation and proteinase K treatment, and between hybridization temperature and time were mainly surveyed in the present study. As for the relation between tissue fixation and proteinase K treatment, good results were obtained in sections fixed by immersion in 10% formalin at 4°C for 2 hr, and digested with 1 μg/ml proteinase K at 37°C for 20 min. Under this condition, the most intense signals were obtained after hybridization at 37°C overnight. Alternative application of ISH and immunostaining to each of the adjacent sections revealed a small number of cells which were immunonegative but displayed hybridization signals.

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Fine structure of the vomeronasal organ (VNO) was examined in the chinchilla (Chinchilla laniger) from a viewpoint of comparative anatomy. The VNO of chinchilla was a pair of tubular structure, about 6mm in length, and situated bilaterally along the base of the nasal septum. The VNO was encircled rostrally by the vomeronasal cartilage, but caudally by a bony capsule. The VNO communicated with the nasal cavity via a small pore at its rostral end, while it ended blindly at its caudal end. Its lumen was crescent to elliptical in a transverse plane, and lined medially with the vomeronasal sensory epithelium (VSE), but laterally with the vomeronasal respiratory epithelium (VRE) . Jacobson's glands were tubulo-alveolar in type and distributed from the dorsolateral to the ventrolateral region of the VNO and opened with the duct to the lumen in the transitional region from the VSE to the VRE. Their secretions were periodic acid-Schiff (PAS) -positive but alcian blue (AB) -negative. The VSE consisted of sensory, supporting and basal cells. Supporting cells were characteristic of a large number of huge dense bodies in the perinuclear cytoplasm. The other ultrastructural features in sensory, supporting and basal cells of the VSE were similar to those in the previous reports. The VRE consisted of ciliated, non-ciliated and basal cells. Acinar cells of Jacobson's gland possessed two types of secretory granules. Secretory granules of one type were homogeneous and electronlucent, about 1, 700nm in diameter, while granules of the other type were about 2, 200nm in diameter, and various in electron density. The present findings suggest that the VNO is functionally active in the chinchilla.

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Anti-human CD4 monoclonal antibodies have been successfully used to label Tlymphocytes in Cynomolgus monkeys by two different methods. A magnetisable bead separation was used prior to immunogold labelling of lymphocytes in one of the methods. In addition, an assay for soluble CD4 has been applied to the sera of these monkeys.

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The perikarya of sensory neurons have been reported frequently from elecrophysiological studies of the plexus submucosus of many mammalian digestive tracts, but the fine structural characteristics of those cells or ganglia have rarely been demonstrated. Therefore, we conducted an electron microscopic study in search of possible sensory perikarya in the plexus of the mouse small intestine. Many ganglion cells scattered over the plexus submucosus possessed a few areas of fine granules frequently adjacent to dense bodies in their perikaryonal cytoplasm, as revealed first by Wilsonet al. [23], who were unable to explain the significance, of this finding. On the other hand, one type of ganglion with a special three-dimensional structure was observed in the same plexus. This ganglion was composed of one central perikaryon, one innermost cellular semiperfect cover of the Schwann cell, a middle two-layered cellular coat, an outer neuropil and an outermost external lamina. The surface of the central perikaryon, which differed from those of the other ganglion cells, was almost entirely covered with one Schwann cell, most probably for isolation from various kinds of external electrical and/or chemical inputs. Dumbbellshaped vesicles containing endings that were presumed to be sensory, were frequently localized in the outer neuropil of this ganglion. It is likely that this perikaryon belongs to the sensory ganglion cells and so acts as an important factor in the peristaltic reflex arc.

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In order to analyze renal abnormalities in mice with polycystic kidney disease (PKD), we produced a series of monoclonal antibodies reactive with the murine kidney by hybridizing P3U1 myeloma cells with spleen cells from DBA/2 mice immunized with the kidney of adult-type PKD mice, DBA/2FG-pcy. One clone, D28, reacted specifically with the basement membrane of the proximal tubules of DBA/2 mice and DBA/2FG-pcymice. It did not react with other parts of the murine kidney nor with other tissues such as the skin, ovary, fallopian tube, testis, lung and small intestine. While other components such as collagen IV, laminin and the core protein of proteoglycan could be found, the D28 epitope could not be found in the basement membrane of renal cysts formed in adult-type (DBA/ 2FG-pcy) and infant-type (C57BL/6J-cpk) PKD mice. The D28 epitope did not, however, disappear from the basement membrane of proximal tubules in other types of renal abnormalities. These results suggest that the formation of renal cysts in the proximal tubules is associated with an alteration to the proximal tubule-specific structure of the basement membrane. The D28 monoclonal antibody should prove a useful tool with which to analyze basement membrane-associated abnormalities in genetic PKD.

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Identification of substrains or sublines of inbred mice and assessment of genetic relationships among them were performed on the basis of DNA fingerprinting using M13 phage DNA as a probe. We used eight C57BL/6 sublines (J//Jcl, J//Jms, J//Sic, J//Nrs, Yok, Jah, N//Crj, N//Jcl) and eleven C3H/He sublines (J, J//Jcl, J//Yok, J//Nrs, Jms, N, N//Jcl, N//Crj, Sic, Jah, Nrs) . Two kinds of restriction endonucleases (HinfI andPstI) were used. It was found that : 1) DNA fingerprint within each subline showed identical patterns. 2) Most sublines of C57BL/6 and C3H/He could be identified using DNA fingerprinting withHinfI except between N//Crj and Sic, and among J, J//Nrs and J//Yok in C3H/He. DNA fingerprints withPstI endonucleases showed low polymorphic banding patterns. 3) A dendrogram constructed from DNA fingerprint patterns reflected generally the genealogy of the sublines used. 4) DNA fingerprinting, therefore, seemed to be suitable for the genetic monitoring and assessment of genetic relationships among sublines of inbred mice having close relationships.

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There is a paucity of model animals for naturally occurring nephrosis. The nephrotic mouse strain (ICGN) found from ICR mouse colony at National Institute of Health could be one of the most suitable model for nephrosis. We maintained the strain of mice which was originated from the hybrid between the nephrotic ICGN mice and ICR mice. Nephrosis is diagnosed with the presence of albumin band on SDS-PAGE of the urine. The detection of urinary albumin using SDS-PAGE could be valuable for early diagnosis of nephrosis in the mice. The total urinary protein concentration was determined on the course of nephrosis. The nephrotic mice showed slightly higher protein concentration between 2 and 6 days old as compared to control mice. Until 16 day old, it was maintained relatively low level. Thereafter, the total urinary protein increased gradually. However the diagnosis of nephrosis with total urinary protein alone may be limited due to the major urinary protein which can be detected even in normal rodents.

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The total blood volume of SPF female rats of Wistar-Imamichi strain, a strain that shows a 4-day estrous cycle at very high rates and is widely used in the study of reproductive biology in Japan, was estimated from blood radioactivity after the intravenous administration of¹²⁵I-rat serum albumin. The radioactivity of blood showed a rapid decrease until 10 min after injection and a slow decay thereafter. Radioactivity at time zero was estimated by extrapolation from this slow decay curve, and the blood volume was calculated. From 30 rats weighing 150-300g we obtained the following equation between blood volume (BLV) and body weight (BW), and a high correlation coefficient (r) : BLV=8.4007+0.0430×BW, r²=0.92. The blood volume per body weight decreased linearly with increasing body weight, and we obtained the following equation: BLD/100g BW=12.25-0.0174×BW at r²=0.92.

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An attempt was made to use a diabetic dog as a model recipient of pancreas transplantation. The diabetic state was induced by total pancreatectomy, and was shown to be stable by oral and intravenous glucose tolerance tests. In dogs that underwent total pancreatectomy under isoflurane anesthesia (GOT), blastogenesis of peripheral blood lymphocytes had a tendency to be reduced in comparison with non-treated dogs until 2 weeks after surgery. However, from 3 weeks after surgery, their responses became comparable to those of non-treated dogs. These results suggest that stable diabetes mellitus is induced, in dogs, by total pancreatectomy, and that this animal is a useful model of a pancreas transplantation recipient.

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Levels of cytokines released from endotoxin-mediated monocytes were investigated in mice of DBA/2, BALB/c, C57BL/6, C3H/He, ddY and ICR strains and in ICR mice of various ages (4-52 weeks old) . Interleukin-1α (IL-1α), interleukin-1β (IL-1β) and tumor necrosis factorα (TNFα) levels from monocytes were low in DBA/2 and BALB/c mice, and high in ddY and ICR strains. In addition, interleukin-6 (IL-6) levels were low in DBA/2, and high in ddY and ICR strains. Therefore, monocytes obtained from ddY and ICR strain mice released large amounts of cytokines as compared to those from the DBA/2 strain. The levels of IL-1α, IL-1β and IL-6 released from monocytes of ICR mice were constant at 418 weeks old, and low at 26 and 52 weeks old. In contrast, the TNFα level was low at 18 weeks and high at 26 and 52 weeks. These results indicate that ageassociated changes in cytokine release from monocytes correlate well with the cytokine levels in bone marrow.

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Administration of anti-inflammatory non-steroidal drugs to pregnant animals causes constriction of the fetalductus arteriosus (DA) . To clarify the mechanism of the indomethacin-induced constriction of the rat fetal DA, extracellular matrix (ECM) components were observed by immunohistochemical techniques with antibodies 2B1, 6B6, 3B3, 9A2 and others to various kinds of proteoglycans (PGs) . In the 10mg/kg group, the large PG revealed with antibody 2B1 was increased in the inner and outer media at constriction, and chondroitin 6-sulfate PG was strongly immunostained with antibody 3B3 as granular substances in the outer media, although no difference in stainability of chondroitin 4-sulfate PG revealed with the antibody 9A2 was observed between experimental and control groups. This suggests that the cells composing the fetal DA showed a tendency of proliferation and resultant production and aggregation of large PG with chondroitin 6-sulfate chains during constriction induced by indomethacin-treatment.

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The production of Interleukin-6 (IL-6), which is known as a B cell differentiation factor, by peritoneal macrophages from mice with a chronic lactic dehydrogenase virus (LDV) infection was compared with that from uninfected mice. The same amounts of IL-6 were detected in the culture supernatant of macrophages from LDV-infected mice as those from uninfected mice. Furthermore IL-6 production of macrophages from LDV-infected and uninfected mice was not affected by the addition of indomethacin. These results suggested that many immunological alterations seen in LDV-infected mice may not be due to, at least in part, altered IL-6 production ability of macrophages and the IL-6 production may not be affected by cyclooxygenase-derived products.

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The relationship between the hair cycle and the age or daily body weight gain (daily gain) in rabbits was investigated. A survey of the ages of rabbits used for the dermal toxicological studies in our laboratories over the last 4 years was done, followed by an experiment to confirm the results of the survey. Although there seemed to be no relationship between the hair cycle and the daily gain, the results showed that the hair cycle was closely related to the age of the animal, and the animals in the telogen stage may be obtained efficiently at about 17 wks of age.

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The influences of sex hormones on the dilatation of the urinary tubules and acidophil bodies were histologically investigated in NON (Non-Obese Non-diabetic) mice. Although the dilatation of the proximal tubules and acidophil bodies in NON mice were observed only in female but not in male, a slight dilatation and a few bodies were also observed in castrated male NON mice. Moreover, in ovariectomized female NON mice the dilatation and bodies were less compared with intact female NON mice. Estradiol administration induced prominent dilatation and numerous acidophil bodies, while the administration of testosterone showed a complete preventive effect. Therefore, it is suggested that the dilatation of the tubules and the acidophil bodies can be profoundly influenced by sex hormones.

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Changes in measurements of lumbar vertebrae (L3-L5) during a six-month interval in African green monkeys were demonstrated using dual-energy X-ray absorptiometry (DEXA, DPX-α, Lunar, USA) . Area, mean width and bone mineral content (BMC) of L3-L5 were measured, and bone mineral density (BMD) was calculated using 62 laboratory-bred and 23 wild animals. Laboratory-bred animals were divided into 4 groups according to their gender and age, and in addition, two groups of wild animals were constructed according to gender. Although no remarkable change in BMD of laboratorybred animals aged less than two years was observed at a six-month interval, significant decreases in BMD were detected in laboratory-bred animals aged over 6 years as well as in wild animals. Thus, DEXA seems to be useful for investigating the kinetics of BMD in aged animals during a relatively short period such as six-months.

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Testis weight and seminal vesicle weight (relative to total body weight) inrdwrats and N rats were compared with advancing age. The results obtained were as follows 1) The increase in testis weight became remarkable at 15-16 weeks of age. The weight remained constant up to 23-30 weeks of age and decreased thereafter with age. 2) Seminal vesicle weight increased rapidly inrdwrat at 15-16 weeks of age, reflecting elevated androgen secretion and sexual maturation. 3) From the viewpoint of histology, one of causes of the increase in testis weight may be edema. The increase in testis weight coincided with the increase in androgen secretion at 15-16 weeks of age as suggested by the relative increase in seminal vesicle weight.

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Sexual dysfunction in diabetic men is thoroughly recognized, while not yet done in diabetic women. Recently, Streptozotocin (STZ) -induced male rats showed a significantly depressed copulatory behavior, compared with normal animals. We investigated whether STZ-induced diabetic female rats would produce observable deficits in sexual behavior. Results in the present study are the first to show that STZ-induced diabetic female rats have a depressed sexual behavior.

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In this study, an alkaline treatment was applied to the observation of implantation site in rabbits. Embryos in pregnant rabbits were stabbed to death on days 7, 8, 9, 10 and 18 of gestation. These animals were sacrificed on day 29. The uteri were removed, immersed in 2% sodium hydroxide and fixed with 10% buffered formalin. After the alkaline treatment, all of the implantation sites were clearly recognizable as white tissue since the other parts became fairly transparent. Even after the formalin fixation, all the implantation sites were similarly detectable . The mean area of implantation sites visibly increased after day 10. From all the results, the simple and accurate procedure including alkaline treatment was concluded to be useful for estimating implantation and pregnancy conditions in rabbits.

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