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Considering the usefulness of the immunopotentiator from Pantoea agglomerans 1 (IP-PA1), which is a purified lipopolysaccharide (LPS) derived from symbiotic gram-negative bacteria of food crops, in controlling immunosuppression in poultry husbandry, in this study, we examined its immune-recovery effects in dexamethasone-treated stressed chicken models. Three-week-old chickens daily administered 10 microg/kg of dexamethasone for 35 days to induce stress showed more whole body weight loss; relative thymic, bursal, and splenic weight losses; and decrease in the number of peripheral blood lymphocytes, as compared with the control chickens on day 35; the IP-PA1-pretreated, dexamethasone-treated chickens showed reduced weight losses. Five- to eight-week-old chickens administered 5 mg/kg of dexamethasone showed excessive apoptosis of thymic and bursal lymphocytes 24 hr after a single dexamethasone treatment; apoptosis was inhibited in the IP-PA1-pretreated, dexamethasone-treated chickens. Chickens daily administered 10 microg/kg of dexamethasone for 35 days and injected with commercial Salmonella Enteritidis (SE) vaccine or sheep red blood cells (SRBC) on days 7 and 21 showed about 8- or 2-fold lower antibody production in response to SE or SRBC, respectively, as compared with the control chickens on day 35; the antibody production in response to SE or SRBC was increased in the IP-PA1-pretreated, dexamethasone-treated chickens. These results indicate that IP-PA1 exerts inhibitory effects on dexamethasone-induced immunosuppression and that it may be useful in controlling immunosuppression in poultry husbandry.

This study was performed to isolate a velogenic Newcastle disease virus (NDV) strain currently found in Indonesia for establishing a domestic reference virus for future pathological and molecular epidemiological studies. A chicken suspected to have contracted Newcastle disease (ND) in a local outbreak in Bali was selected for NDV isolation. Atrophy of lymphoid tissues such as the bursa of Fabricius, thymus, and spleen; intestinal haemorrhage; and oedema of the brain were observed in the chicken. Histopathological examination revealed severe non-suppurative meningoencephalomyelitis characterised by neuronal necrosis, multifocal to diffuse gliosis, and perivascular cuffing of mononuclear cells, hemorrhagic necrosis of the trachea, intestines and bursa of Fabricius, and various degree of lymphoid depletion and necrosis of the lymphoid tissues. After ND was confirmed immunohistochemically, the NDV was propagated by inoculating tissue homogenate of the diseased chicken in embryonated eggs. Phylogenetic analysis based on the F gene nucleotide sequence revealed that this isolate belonged to genotype VII. The deduced amino acid sequence of the isolated NDV F protein at the cleavage site was (112)RRQKRF(117), which is typically found in virulent NDV isolates. Pathogenicity indexes such as the mean death time (MDT) and intracerebral pathogenicity index (ICPI) were 54 hr and 1.77, respectively. Pathological findings, phylogenetic analysis, amino acid sequence of the F protein cleavage site, and pathogenicity index test results revealed the NDV isolate, designated as NDV/Bali-1/07, to be a novel Indonesian velogenic NDV strain belonging to group VII.

Interferon (IFN)-gamma is essential but not sufficient to control leishmaniasis. It is known that IFN-gamma is one of the major macrophage-activating cytokines, and the activated macrophages are a principal source of interleukin (IL)-12, which induces autocrine macrophage activation. In this study, the combined effect of IFN-gamma and IL-12 on the susceptibility of macrophages to Leishmania major infection was evaluated. Macrophages pretreated with IFN-gamma and/or IL-12 were infected with the parasites. Four hr post-infection (p.i.), the levels of infection and parasite load in the macrophages treated with the combination of IFN-gamma and IL-12 (IFN-gamma/IL-12) were significantly lower than those in the nontreated cells. However, the macrophages treated with either IFN-gamma or IL-12 did not show resistance to L. major infection. In addition, 72 hr p.i., the IFN-gamma/IL-12-treated and IFN-gamma-treated macrophages showed significantly lower levels of infection and parasite load than the nontreated cells, and higher levels of resistance was observed in the IFN-gamma/IL-12-treated macrophages than in the IFN-gamma-treated macrophages. Although IFN-gamma/IL-12 treatment of macrophages prior to the infection led to the induction of resistance, as described above, this resistance was not induced when these cytokines and the parasites were added simultaneously to the macrophage culture. These results suggest that IFN-gamma/IL-12 treatment prior to the infection restricts the early phase of the infection.

Recently we reported that a chimeric molecule containing mouse transferrin receptor and immunoglobulin G1 (IgG1) Fc, mTR-Fc, induced higher immune responses and can be used as a vaccine adjuvant. In this study, the immunological property of the molecule was investigated. Although, the mTR-Fc did not activate complement classical pathway, it was recognized by activated macrophage as like intact IgG Fc, which is recognized by macrophage via Fcgamma receptor. In addition, we found that splenocyte simultaneously exposed to lipopolysaccaride (LPS) and mTR-Fc produced higher amount of interleukin-10, comparing to that exposed to only LPS. These results suggest that the mTR-Fc molecules conserved the IgG Fc property to biasing immune responses via modulation of cytokine production by antigen presenting cell.

We osteometrically examined the skulls of the black giant squirrel (Ratufa bicolor) from three mainland populations (M. Malayan Peninsula, V. South Vietnam, and B. Burma, India and North Thailand) and from two island populations (T. Tioman, and S. Sumatra Islands). The skull in the Malayan peninsula population was significantly smaller than that of the two other mainland populations. It is consistent with Bergmann's rule as shown in the gray-bellied squirrel. The two island populations did not show obvious differences in comparison with the Malayan population in many measurements. In the proportion analysis eliminating the size factor, the differences among populations were not easily confirmed and we concluded that the osteological characters peculiar to each population could not be shown in this species. The first and second principal component scores of M, S, and T populations were intermingled, whereas the V and B populations of V and B were not separated in the chart. We pointed out that the morphological differences were demonstrated between northern and southern groups of the Isthmus of Kra in the mainland populations, and that the two island populations did not show the island-isolation effect in comparison with the M population. The adaptational variation related to feeding and locomotion could not be confirmed among populations of the black giant squirrel as shown in the proportion analysis.

The geographical variations of the skulls were osteometrically examined in the gray-bellied squirrel (Callosciurus caniceps) from the populations of Korat, Ranong, southernmost Thailand, and Terutau Island. The skull size was larger in northern population than in the southern population in the continental mainland. The zoogeographical influences of the Isthmus of Kra remained unclear, since the plots from Korat population were intermingled with those from southernmost Thailand population in the principal component charts. Although Korat population has been thought to belong to north group, we suggest that Ranong and southernmost Thailand populations may contain individuals from both north and south groups separated by the ancient Kra barrier. Terutau Island population was similar to southernmost Thailand population in skull size, although Terutau population has been isolated in the island and separated from the south group of the Isthmus of Kra. In the proportional analysis the interorbital space was narrower and the binocular sense has been well-developed in Terutau population. It suggests that this population has been highly adapted to arboreal behavior. In contrast, the skull with larger interorbital space was more adaptive for terrestrial life in Korat population. The canonical discriminant analysis could clearly separate the four populations in the scattergrams of discriminant scores.

To study the role of estrogen in the testes, testosterone and testicular steroidogenic enzyme mRNA levels were investigated in male Sprague-Dawley rats 24 hr after intramuscular administration of a single dose of estradiol-3-benzoate (EB). EB administration resulted in a greater decrease in intra-testicular and serum testosterone in 10-week-old rats than in 3- or 5-week-old rats. A dose of 2 microg EB/kg had the lowest observed effect. The level of serum luteinizing hormone (LH) was unchanged at any dose. Semiquantitative RT-PCR analysis revealed that, of the four major testicular steroidogenic enzymes, mRNA levels of cytochrome P450 side-chain cleavage and 17beta-hydroxysteroid dehydrogenase type-III were significantly reduced, and mRNA levels of cytochrome P450 17alpha-hydroxylase/ C17-20 lyase (P450c17) were reduced severely and significantly, by EB administration. However, the level of 3beta-hydroxysteroid dehydrogenase type-I mRNA was not changed. In addition, the P450c17 mRNA level in EB-treated rats was much lower than that in the testes of hypophysectomized rats, with the level in the latter being equal to that in control rats. LH is secreted into blood periodically, the effects of estrogen on the LH secretion pattern of the pituitary gland, for example, in frequency and amplitude of LH pulse, were difficult to detect with the methods of the present study. The results indicated, at least, that EB administration down-regulates P450c17 gene expression predominantly, resulting in the inhibition of testosterone production. From the differences in the steroidogenic enzyme expressions between hypophysectomized and EB-treated rats, it was suggested that EB acts on the testis directly or indirectly though not via alteration of LH secretion and induces reduction of P450c17 mRNA level.

The proximal straight tubules of the female mouse kidney exhibit heavy periodic acid Schiff (PAS) staining in their brush borders and numerous cytoplasmic granules. In the present study, the female DBA/2Cr mouse kidney was examined, using various fixatives (formalin, PFA, PLP, Zamboni's, Bouin, or Carnoy solution) and various staining methods (HE, PAS, alcian blue, periodic acid methenamine-silver (PAM), toluidine blue, azan, or Congo red). Under azan and PAM, the staining pattern of the brush border was similar to that of PAS, and few effects of the fixative were observed. Cytoplasmic granules were clearly detected with PAM as well as PAS. However, these granules were not detected with Carnoy solution. Furthermore, distribution of granules differed between PAS and PAM.

Although mice kidney morphology shows various sexual dimorphisms, the effect of the estrous cycle has not previously been discussed. In this study, we investigated the effects of the estrous cycle on kidney morphology, including renin-positive areas, of female DBA/2 mice. No effects were confirmed in most of the histometrical parameters, however, the percentage of the renal corpuscles in which cuboidal epithelium covered under 50% of the parietal layer was significantly higher during estrus compared to that during anestrus.

The muscles of mastication of the polar bear (Ursus maritimus) and those of the brown bear (U. arctos) were examined by anatomical approach. In addition, the examination of the skull was carried out in the polar bear, brown bear and giant panda (Ailuropoda melanoleuca). In the polar bear, the rostro-ventral part of the superficial layer of the M. masseter possessed the abundant fleshy portion folded in the rostral and lateral directions like an accordion. Moreover, the rostro-medial area of the superficial layer became hollow in the nuchal direction when the mouth was closed. The M. temporalis of the polar bear covered up the anterior border of the coronoid process of the mandible and occupied the almost entire area of the cranial surface. The M. pterygoideus medialis of the polar bear was inserted on the ventral border of the mandible and on the ventral part of the temporal bone more widely than that of the brown bear. As results of our measurements of the mandible, an effect of the leverage in the polar bear was the smallest in three species. In the polar bear, the skull was flat, and the space between zygomatic arch and ventral border of the mandible, occupied by the M. masseter was the narrowest. It is suggested that the muscles of mastication of the polar bear is adapted to the flat skull feature for supplementing the functions.

The hump attachment structure was morphologically examined in the two-humped camel (Camelus bactrianus). The cranial hump is fixed by the trapezius and rhomboid muscles in the thoracic region. The strong collagen sheet in the basement of the hump is attached to the segmented bellies of the trapezius muscle, and the thoracic rhomboid muscle and the nuchal-supraspinous ligament support the attachment function of the trapezius muscle. The basement sheet possesses the line structure of collagen fibers, which are fitted to the segmented bundles of the trapezius muscle, and we observed that the muscle cells of the trapezius muscle are intermingled with the collagen fibers around the attachment line structure. In contrast, the caudal hump is directly attached to the subcutaneous tissue in the superficial region of the lumbar longissimus and lumbar iliocostal muscles. These findings demonstrated that the caudal hump of the two-humped camel is consistent with the hump of the one-humped camel in the attachment structure.

The skull size and shape were osteometrically examined in the four species of the tree shrews (Tupaia tana, T. javanica, T. minor and T. dorsalis). We suggest that the skull characters were affected by the species specific behavior and terrestrial, arbo-terrestrial and arboreal life, among the genus Tupaia. The neurocranium was laterally narrower in the braincase area, and the splanchnocranium was longer only on dorsal side in T. tana, and these characters were opposite to T. minor. The principal component analysis confirmed the obviously separated clusters among T. tana, T. javanica and T. minor, affected by the adaptation for each behavior. T. dorsalis was considered as terrestrial species from the results of proportion analysis and the principal component analysis.

The Isthmus of Kra in Southern Thailand consists of a lowland of about 70 km in length. It has been suggested that the Isthmus may sink beneath the sea surface according to the change of level of the sea, and may function as a zoogeographical barrier in land mammals in this region. So, the geographical variation was osteometrically examined in skull of the common tree shrew (Tupaia glis) from the both sides of Isthmus of Kra. The osteometrical examination demonstrated that the skull is larger in southern population than in northern one. In the charts of the principal component analysis, however, the morphological separation between two populations can not be obviously seen in female. While, the results of the discriminant analysis indicated the morphological separation between the two populations. These findings suggest that the zoogeographical barrier of Isthmus of Kra may have influence on the osteometrical variation in the common tree shrew, when the Isthmus was covered with sea.

Following stimulation by the transforming growth factor-beta (TGF-beta) family in the cytoplasm, the Smad family is phosphorylated and translocated to the nucleus and activates several gene transcriptions. In this study, the mouse Smad3 cDNA including the open reading frame (ORF) was cloned from the mouse brain using a RACE (rapid amplification of cDNA ends) technique, and its expression pattern was analyzed in mouse tissue using northern blot. The predicted amino acid (aa) sequences of mouse Smad3 showed a high homology with human Smad3 (99.3%) and mouse Smad2 (85.4%). It revealed that this protein may be highly conserved in different species of mammals. Northern blot analyses revealed that Smad3 was highly expressed in the brain and ovary, and that the size of major transcript was about 5.7 kb. In situ hybridization analyses revealed the high expression of Smad3 was detected in the pyramidal cells of the hippocampus, the granule cells of the dentate gyrus, the granular cells of the cerebral cortex and the granulosa cells of the ovary. Smad3 may be essential transducer of signals from TGF-beta and activin in these cells.

The distribution of sugar residues in gonocytes of the differentiating mouse testis was examined by light microscopy using 22 different kinds of lectins. Characteristic binding patterns of sWGA, VVA, and LEA in gonocytes were observed during prespermatogenesis. sWGA preferentially bound to the cytoplasm and plasma membrane of gonocytes on 16.5 days post coitus (dpc). Its reaction decreased thereafter and almost disappeared on 1.5 days post partum (dpp), but reaction reappeared on 4.5 dpp and continued until 6.5 dpp. The VVA reaction was recognized in a few gonocytes on 0.5 dpp, and remained strong until 6.5 dpp. LEA reacted strongly in the plasma membrane and cytoplasm of gonocytes from 0.5 dpp to 6.5 dpp. The present study indicates that sWGA, VVA, and LEA are useful markers for gonocytes, and the appearance or disappearance of sWGA and VVA may be related to the differentiation of gonocytes during prespermatogenesis.

The MRI examination was carried out in a formalin-fixed specimen of the Ganges River dolphin (Platanista gangetica), one of the evolutionary primitive species of cetaceans. We could morphologically elucidate the tracheobronchial ramification in the intact whole body. We demonstrated from the MRI sections that the characteristic tracheal bronchus branches from the trachea at the cranial portion. These findings suggest the phylogenetic relationships between cetaceans and artiodactyls. The left bronchus is obviously larger in diameter than the right one. We suggest that the right bronchus has smaller capacity of gas exchange than the left one, because the dolphin possesses the tracheal bronchus in the right lung. The MRI method will be important in the non-invasive study of the anatomy in endangered animal carcass as Ganges River dolphin.

Morphological features of the testicular artery and vein in the spermatic cord of the musk shrew (Suncus murinus) were evaluated by light microscopy, transmission electron microscopy, corrosion cast technique combined with scanning electron microscopy and immunohistochemistry. The vascular architecture in the spermatic cord of the musk shrew was simple. The testicular artery in the musk shrew was straight and accompanied by 1 to 3 branches of testicular vein. The testicular vein was also straight and anastomosed with each other in some points along its length, but it did not form a delicate pampiniform plexus. In the middle and distal portions of the spermatic cord, the tunica adventitia of the artery and vein was joined together to form a single connective tissue septum. Clusters of cells were found in this connective tissue septum in the middle portion of the cord. These cells were located close to the arterial wall and nerve endings, but they did not appear inside of neurium. They showed several typical characteristics similar to Leydig cells, and they were positive for 3beta hydroxysteroid dehydrogenase (HSD) antibody. Ultrastructural and immunohistochemical studies also indicated that the cells in cluster found in the vascular wall of the musk shrew spermatic cord may be equivalent to Leydig cells in testes. These extratesticular Leydig cells had characteristics of the active steroid-producing cell and seemed to be another source of testosterone.

Since about 1630, the Imperial Palace has been biologically isolated from other habitats by the development and urbanization of Tokyo. We morphologically examined the skulls of the lesser Japanese mole (Mogera imaizumii) from the Imperial Palace and compared them with those from Kanto District, while the sequences of the cytochrome b and 12S rRNA genes were also analyzed to clarify the genetic status of this isolated population. The skulls from the Imperial Palace were much larger than those from Kanto District in the length items. We suggest that the Imperial Palace skulls morphologically may compose a cluster as a large body-sized type in Kanto District within the dots of Mogera imaizumii in charts of principal component analysis. The mitochondrial DNA sequences of the Imperial Palace population were highly homologous to those of other Tokyo population at the level of 98.5% in cytochrome b and 98.7% in 12S rRNA genes.

We measured adult mandibles of Ryukyu wild pigs from Amami-Oshima, Kakeroma, Okinawa, Ishigaki and Iriomote Islands. The size cline was not statistically recognized among the populations of Nansei Islands. The Ishigaki population was significantly larger than Iriomote one in mandible length. Some measurement ratios indicated that the ramus is enlarged laterally and that the body of mandible is dorso-ventrally developed in Ishigaki and Iriomote populations. The Amami-Oshima mandibles were relatively smaller than those of Okinawa and Iriomote populations in length item of body of mandible. These results will contribute to the zoo-archaeology on the historical change of size and shape in the island populations, and to the evolutionary biology on the morphological adaptation of this animal.

Mitochondrial capsule selenoprotein (MCS) has been known as a structural protein of the mitochondrial sheath in spermatozoa. In the present study, to determine the expression pattern of MCS mRNA in the hamster testis, northern blot and in situ hybridization analyses using digoxigenin-labeled riboprobes for the hamster MCS were performed in the testes of 10-week-old golden hamsters. According to the northern blot analysis, hamster MCS was detected as a single transcript of about 1 kb in the testis. During spermatogenesis, the hamster MCS mRNA first appeared in step 6 spermatids, gradually increased in round spermatids during spermiogenesis, reached a peak in step 8 spermatids, and persisted a high level until step 13 spermatids. After step 14, the signal began to show a progressive decline in the spermatids and was weakly detected in the tails of step 17 spermatids. However, the signal was not observed in spermatogonia, spermatocytes, Sertoli cells, peritubular myoid cells, and interstitial cells. These findings indicate that hamster MCS is mainly related to the spermiogenesis during spermatogenesis.

The lesser mouse deer (Tragulus javanicus) is one of the most primitive ruminants. Skulls of lesser mouse deer were measured to evaluate their growth. The age was estimated from the eruption and attrition of molars on the mandible. A total of 95 specimens was divided into five age groups, and the growth pattern in each sex was established for some measurements. The relative growth coefficients were calculated for the head and body length and several parts of the cranium and mandible against the profile length. Sexual dimorphism could not be found on the skull in any age group except for measurements related to the upper canine. However, females showed a higher value in head and body length in the oldest group. This feature was consistent with an interpretation of mouse deer society as monogamous. Growth was more rapid in length than in width. Various statistical analyses showed that the visceral cranium related to masticatory facility was much better developed than the neurocranium. However, observations on the shape of the mandible ramus and of the premolars implied that the masticatory function was not suitable for taking roughage. This speculation agreed with previous reports on the feeding habits and digestive organs of the mouse deer.

The monoclonal antibody (MAb), named TSd-1, specific to spermatogenic cells of the common tree shrew (Tupaia glis) was established and characterized using immunohistochemistry and immunoblotting. MAb TSd-1 reacted with elongating and matured spermatids in a stage-dependent manner. TSd-1 recognized a 94 kilodalton (kDa) peptide in the plasma membrane and cytosol. Additionally, an extremely weak 107 kDa band was detected only in the cytosol. The reactions were not detected in round spermatids. In elongating Stage VI spermatids, the plasma membrane and the granular structure within the cytoplasm were intensely positive, and most intense after the appearance of new round spermatids in the lower layer (Stage I). The reactions were observed neither in the other organs of the common tree shrew nor in the testes of other animals, indicating that TSd-1 antigen is specific to the spermatogenic cells of the common tree shrew, and may act on elongating or matured spermatids.

This study was conducted to evaluate protective efficiency of three different protocols for vaccination in canine heartworm infection. To evaluate the three protocols of immunization, dogs were separately immunized with living larvae; 1) immunization with gamma-attenuated infective larvae, 2) with 50 micrograms/kg ivermectin-abbreviation, and 3) with chemical abbreviation plus Freund's complete adjuvant (FCA). Each group was composed of two dogs. All dogs used for this study were subcutaneously challenged with 100 intact third-stage larvae (L3) various days after the last immunization, and the worms in the pulmonary arteries and the right ventricle of the heart were recovered 17 to 25 weeks post-infection. The numbers and the sexes of the worms were determined. A mean of 38 worms was burdened in the group immunized with irradiated L3, 36 worms in the chemically-abbreviated group, but 15.5 worms in the group with chemical abbreviation plus FCA. The percentages of the protection in the former two groups were nearly 50%, but 72.3% in the group with ivermectin plus FCA. The adjuvant enhanced the protective immunity against L3 challenge. Obvious eosinophilia was observed in both immunized and control dogs except for two dogs. There was no correlation between the suppression of eosinophilia and the protective immunity in the present study.

The distribution of desmin and fibronectin in the chick embryo gonad in and after sexual differentiation was investigated immunohistochemically. In the undifferentiated gonad (stage 27), fibronectin was detected in the dorsal mesentery, basement membrane and part of the parenchyma, while desmin was not recognized. After sexual differentiation (stages 31 to 34), desmin-positive cells gradually increased in number with the progress of development. They were obviously seen in developing interstitial regions between testicular cords. However, fibronectin was hardly observed at the same stages. At stage 37, desmin-positive cells were present only in a part of the Leydig cells and myoid cells, while fibronectin was discontinuous in the basement membrane surrounding testicular cords. In addition, the expressions of desmin and fibronectin were simultaneously and adjacently recognized. These results suggest that myoid cells may produce fibronectin protein and that fibronectin mainly contributes to the formation of testicular cord basement membrane in chick embryos. Thus, the cell-to-cell and cell-to-matrix interactions may be important in testicular morphogenesis of chick embryos.

To clarify the features of the seminiferous epithelium of the Japanese rat snake, Elaphe climacophora, the identification of spermatogonium and the examination of features of cell to cell junctions were performed in the present study. As for the identification, 5-bromo-2-deoxyuridine (BrdU) incorporation was examined immunohistochemically to mark spermatogonia. The seminiferous epithelium was observed throughout a year at the electron microscopic level. BrdU immunoreactivity was detected not only in the cells of the first layer of the seminiferous epithelium but also in the second and/or third layers. The cells immunoreactive in the first layer did not seem to attach to the basement membrane and were recognized throughout a year. To investigate cell to cell junctions, we performed actin filament detection by phalloidin staining. Distribution of actin filaments was different from that in mammalian species. At the ultrastructural level, Sertoli-Sertoli cell junctions were observed. Sertoli cells formed junctional complexes. Tight junctions were clearly found, but lacked the backing by actin filaments. These results indicate that the blood-testis barrier of the Japanese rat snakes was structurally different from that of mammalian species. In conclusion, the seminiferous epithelium of the Japanese rat snake is intermediate in morphology between amphibians and mammals.

The classification of the cycle of the seminiferous epithelium was carried out in the common tree shrew (Tupaia glis). The tree shrew captured in Thailand were fixed with Bouin's fixative, embedded in paraffin wax, and stained with PAS-hematoxylin. The cycle was classified into twelve stages on the basis of the acrosomal changes of spermatids. Relative frequencies of stages form I to XII were 11.9, 7.2, 8.9, 22.5, 12.9, 9.7, 8.0, 5.9, 4.0, 3.2, 2.9, and 3.6%, respectively. Different stages did no appear in a cross-sectioned tubule as did in primates. The head of matured spermatid was discoidal in shape and different from that of primates and rodents. Spermatogenesis of the common tree shrew is different from that of primates and rodents according to its morphological features.

Apart from apoptosis, a type of parenchymal cell death by the cell protruding into the capillary lumen was observed in the adrenal gland of normal Sprague-Dawley rats. Ultrathin sections were prepared in the conventional manner and were examined by electron microscopy. The protruded cells (p-cells) had the electron lucent cytoplasm and the p-cells with ruptured cell membranes were observed in the capillaries. The egress of p-cells was either through the endothelial gaps, or following the rupture of capillary endothelia. The cytoplasmic matrices of the dying p-cells were seen to scatter in the capillary lumen where the nuclei, mitochondria and granules remained morphologically intact. The p-cells were seen in the capillaries of medulla, but restricted to those of zona reticularis in the cortex.

Effects of estrogen and progesterone on the first abdomino-inguinal mammary gland and associated blood vessels in ovariectomized adult mice were investigated morphometrically and by light and electron microscopy. Although there were no significant differences in the area of the mammary gland fat pad among ovariectomized groups, development of mammary parenchyma reached maximal degree in ovariectomized mice treated with estradiol (E) + progesterone (P). Epithelial cells of the mammary buds and ducts contained a small number of mitochondria and ribosomes, under-developed Golgi apparatus and rough-surfaced endoplasmic reticulum, and lipid droplets. Each organelle was more abundant in E+P treated mice than in other experimental groups. In adipose tissues of the mammary stroma in intact mice, fat cells were of a multilocular type in peripheral regions around the main vessel in the rear of the inguinal lymph node and of a unilocular type in other regions. In E and E+P treated mice, however, most of fat cells were unilocular. These findings suggest that mammary fat tissues may be formed in advance of mammary parenchyma. Blood capillaries around buds and ducts of the mammary gland were most densely distributed in E+P treated mice. Although there were no significant differences in the density of pinocytotic vesicles of endothelial cells of blood capillaries among the overiectomized groups, marginal folds and microvillous processes reached maximal length in E+P treated mice. The diameters of blood vessels supplying the mammary gland, i.e., V. circumflexa ilium profunda and V. epigastrica caudalis superticialis, reached maximal size in ovariectomized mice treated with E+P. These observations prove that administration of E+P induces development of the mammary gland and associated blood vessels in ovariectomized mice.

We established a mouse antiserum by immunizing BALB/c mice with sexually undifferentiated 6-day chick embryonic gonads. The antiserum had a higher affinity to the plasma membrane and cytoplasm of primordial germ cells (PGCs) than to the somatic cells in 6-day chick gonads. PGCs at this stage appear to have a higher specificity with more antigenic capacity than gonadal somatic cells; thus, the antiserum should be useful as a cell marker of chick PGCs. Subsequently, an immunohistochemical examination of PGCs settling in the genital ridges of chick embryos was carried out by the use of the antiserum. The reaction was clearly detected in the plasma membrane and cytoplasm of 5-day PGCs. After sexual differentiation, the reaction changed in distribution in females. At 8 days, it was limited to the perinuclear region of PGCs. In males, although the reaction persisted for longer period than in females, only a weak reaction was detectable at 9 days. These results suggest that the occurrence of periodic molecular changes may be different between male and female PGCs.

The effects of starvation on fat cells and blood capillaries of the first abdomino-inguinal mammary gland in mice were investigated by light and transmission electron microscopy. The body weight of starved mice abruptly decreased to approximately 70% of that of controls at 3 days of starvation and, thereafter, gradually decreased. In adipose tissues of mammary stroma, multilocular fat cells increased in number and clustered during starvation to a glandular appearance at 6 days. Collagen fibers increased in amount around mammary ducts and buds. By electron microscopy, multilocular fat cells possessed numerous mitochondria, small lipid droplets, and plasmalemmal vesicles, while endothelial cells of the blood capillaries showed numerous pinocytotic vesicles plus short marginal folds and microvillous processes. These observations prove that the number of pinocytotic vesicles in blood capillary endothelium is closely related with the increased amount of lipid of fat cells in the mammary gland during starvation.

Cardiac myocytes in the left ventricle and atrium of the greater bandicoot rat (Bandicota indica) were examined by transmission electron microscopy. The fine structure showed typical features of mammalian cardiac myocytes. In atrial myocytes, however, the areas occupied by mitochondria were much smaller than that filled with myofibrils. The decrease in mitochondria and the abundance of myofibrils are thought to be an ultrastructural adaptation to the large body size of this species. Many conducting myocytes were observed in both atrium and ventricle. The atrial conducting myocytes were ultrastructurally different from the Purkinje fibers of the ventricle. We suggest that the abundance and the ultrastructural variation of conducting myocytes are related to the physiological aspects, such as body size, of this animal.

We measured crania and mandibles of Ryuku wild pigs from Iriomote Island. Sex and age were determined by observation of lower teeth. From the present data, the growth pattern was established for some items. For several parts of the cranium and mandible, the relative growth coefficient was compared. The results obtained here are summarized as follows: (1) Although sexual dimorphism was already shown in the younger group, a significant difference in profile length and length from the angle was not seen in the adult group. (2) As the animal grew, the proportion of length to width became larger in the skull. (3) The visceral cranium grew more rapidly in length than the cerebral cranium. On the other hand, growth of the cerebral cranium contributed to width in cranial development. (4) The body of the mandible was shown to grow more rapidly in length than the ramus of the mandible. (5) The growth pattern of some items was related to that of their associated muscles. These basic data are expected to be compared with data from other population of the same subspecies and Japanese wild pig. The comparisons will contribute to the establishment of origin and phylogeny of this animal.

Seasonal changes in spermatogenesis and the ultrastructure of developing spermatids in the Japanese rat snake, Elaphe climacophora, were observed by light and transmission electron microscopy. Spermatogenesis in this species began in August, continued through October and ceased in November. No spermatozoa were found during other periods. Japanese rat snakes mate from May to July, and spermatogenesis occurs after the mating season. Therefore, according to Girons' classification (1982), the reproduction of this species was classified as a postnuptial type. Although the morphological changes in developing spermatids during spermiogenesis in these snakes fundamentally resembled those in mammals, some unique features were detected. The most prominent characteristic of the developing spermatids was lipid-like structures. These structures first appeared in early round spermatids, gradually increased in size and number, and, finally, aligned around the nucleus of mature spermatids. Although spermatozoa with lipid-like structures were released from the epithelium, stored spermatozoa in vas deferenses had none of these structures. Lipid-like structures that apparently separated from spermatozoa were scattered in the vas deferens. Prominent elongation of mitochondria was also remarkable in elongated spermatids.

The permeability of mammary gland capillaries to ferritin was investigated by transmission electron microscopy. In virgin mice, the concentration of tracers in perivascular spaces increased with the advance of time after injection. The ferritin never passed through the intercellular junction of the endothelial cells. In the early to middle stages of lactation, numerous ferritin particles were observed in the basal cytoplasm of alveolar epithelial cells after only 1 min of circulation. These findings indicate that a large amount of materials quickly pass through the capillary walls during these periods for milk production. The number of vesicles labelled with ferritin per micron2 of endothelial cytoplasm decreased remarkably in the late stage of lactation. This result suggests that the permeability of vesicles declines along with the degeneration of alveolar cells.

The ultrastructure of the Shiba goat Sertoli cell was observed using light and transmission electron microscopy. In the basal portion of the Sertoli cell, the nucleus was located near the basement membrane and was irregular in outline. A number of filaments with intermediate width were observed around the nucleus. A multivesicular nuclear body, composed of numerous vesicles of different sizes and ribosome-like structures, was detected within the nucleus. The majority of cell organelles was situated in this basal portion of the cytoplasm. Oval mitochondria were randomly distributed. A well-developed laminated smooth endoplasmic reticulum (sER) was frequently found within the cytoplasm of Sertoli cell. Lysosomes and large lipid droplets were also recognized. While, the outline of the basal lamina was almost straight. No nodular processes were observed in the lamina. The Golgi complex appeared only in the supranuclear region. In the middle and apical portions of the Sertoli cell, elongated mitochondria and microtubules were oriented parallel to the long axis of the cell. The laminated sER was also detected in these portions. The apical head of slightly-elongated spermatids was occasionally surrounded by the laminated sER. The head of maturing spermatids without laminated sER revealed a bleb-like shape. The microfilament bundles at the ectoplasmic specialization site were regularly arranged. Judging from the present observations, we have suggested that only the multivesicular nuclear body and the laminated sER may be the peculiar structures which are commonly found in the Sertoli cell of ruminants.

The morphology of the testis of the mutant As (aspermia) strain rat with severely interrupted spermatogenesis was investigated in the present study. The most advanced development in spermatogenesis was observed at the step 8 at which spermatids had basally-oriented acrosomes. Multinucleate giant cells derived from round spermatids were frequently encountered within the seminiferous tubules. The functionally normal status of the blood-testis barrier between adjacent Sertoli cells was confirmed by morphological and lanthanum tracer studies. By light microscopy, a peculiar structure was found within the pachytene spermatocyte, consisting of numerous particles of approximately 25 nm in diameter. By histochemical and electron microscopic studies, this structure was identified as an aggregate of ribosomes. Neither immature spermatogenic cells up to the pachytene phase, nor Sertoli cells nor interstitial regions showed a recognizable abnormality. It seems that the testicular disruption in the As mutant rat is due to the abnormality in the protein synthetic pathway in pachytene spermatocytes.

Age-related morphological changes in the testis of the BDF1 mouse were studied by light and transmission electron microscopy. No apparent changes were detected until 12 months of age. After 18 months of age, vacuoles firstly appeared in the seminiferous epithelium. These vacuoles were gradually increased in number and showed a tendency to cluster with each other in accordance with age. While, germ cells were decreased in number. The sloughing of germ cells caused a thin seminiferous epithelium. In the tubule with a thin epithelium, spermatogenesis was severely interrupted. After 30 months of age, extremely thin seminiferous epithelia were observed. In these epithelia, most of spermatids and spermatocytes disappeared, and most of Sertoli cells lost their polarity to be flattened. On the other hand, in the interstitial region, PAS-positive cells (mononuclear phagocytes) tended to increase in number after 24 months of age. PAS-positive extracellular matrix newly appeared at 27 months of age. In the cytoplasm of Leydig cells, a whorl of sER was frequently found. Degeneration of testes proceeded with age. The regressive tubules occupied only 2.2% at 18 months of age, but extended to 63.0% at 33 months.

The cardiac musculature of cranial and caudal venae cavae and pulmonary vein was examined to clarify its distribution pattern in the fowl using both light and electron microscopies. The musculature was distributed from the heart to the root of subclavian vein in the cranial vena cava, to the cranial margin of the liver in the caudal vena cava, and to the left and right distal pulmonary veins in the pulmonary vein, respectively. Judging from the morphology and distribution pattern in the venous wall, the cardiac musculature in the fowl is thought to share the same phylogenic origin with that in mammals. The ultrastructure of cardiac myocytes including transitional cells in the cranial vena cava and the pulmonary vein resembled that of atrial myocytes. While, the typical specialized myocytes such as Purkinje fiber were found in the caudal vena cava of the fowl.

Microvascular changes in the mammary gland in mice during pregnancy and lactation were investigated by scanning electron microscopy (SEM) with a corrosion cast method, transmission electron microscopy (TEM) and morphometry. By SEM, duct-associated capillary plexuses were sparsely distributed to branch into adipocytes during virgin period. With advance in pregnancy, both branches from the capillary plexuses and branches from the vessels surrounding adipocytes extended further to form capillary networks. The basket-like architecture was completed by day 18 of pregnancy. These findings may indicate that angiogenesis occurs frequently during this period. During lactation, the basket-like architecture still remained and the capillaries surrounding alveoli meandered. After weaning, the regression of microvasculature followed the degeneration of alveoli. By TEM and morphometry, the density of pinocytotic vesicles (PVs) (number of PVs per microns 2 of endothelium cytoplasm) increased twofold from day 18 of pregnancy to day 5 of lactation, furthermore increased threefold from days 10 to 20 of lactation, and subsequently decreased after weaning. Marginal folds and microvillous processes gradually increased in length with advance in pregnancy, reached the maximum from days 5 to 15 of lactation, and thereafter decreased. In addition, the capillaries with thinner walls were in close contact with alveoli during the late stage of pregnancy and during lactation. Furthermore, the alveolar epithelial cells had well-developed basal infoldings during lactation. These findings suggest that the capillaries play an important role in transporting materials necessary for milk production.

Changes in the intracellular and cell surface localization of Lewis x (Le(x)) determinants in germ cells during fetal development in mice were examined by light and electron microscopy. Light microscopically, in undifferentiated gonads on day 12 post coitum (p.c.), the anti-Le(x) monoclonal antibody (MAb) was specifically bound to the plasma membranes and to the cytoplasmic granule-like structures of germ cells. In the testes on day 13 p.c., most of the germ cells were enclosed within the testicular cords and showed an MAb-positive reaction which was restricted mainly to the cytoplasmic granule-like structures. The reaction on the plasma membranes almost disappeared. On the other hand, the ectopic germ cells still showed a positive reaction on their plasma membranes. In the ovaries on day 13 p.c., the germ cells also exhibited positive reactions both on the plasma membranes and on the granule-like structures. Immunoelectron microscopic observations agreed well with these light microscopic observations in such a way that both the plasma membranes and the "small dense bodies" (SDB) were positive in undifferentiated gonads on day 12 p.c. In the germ cells organized into the testicular cords, the reaction to anti-Le(x) MAb became restricted to the SDB. These results may indicate that such intracellular changes in Le(x) determinants during germ cell differentiation are associated with the enclosure of germ cells within the testicular cords.

To clarify the functions of the equine guttural pouch, the distribution of various immunoglobulin isotypes and subisotypes in the guttural pouch mucosa were examined in healthy horses. IgGa was present in the mucosa of guttural pouch, mucosal lymph nodules and submucosal lymph nodules. IgM was scattered in the mucosal lymph nodules and in the germinal centers of the submucosal lymph nodules. IgGc was recognized only in the submucosal lymph nodules. These immunoglobulin isotypes and subisotypes were found in lymphocytes and plasma cells. On the other hand, IgA was detected in glandular epithelial cells and the surface layer of the mucosal epithelium, as well as in free cells. This finding suggests that IgA is secreted through the glandular epithelium. Based on the above findings, we conclude that the guttural pouch has phylactic ability.

The localization of alpha, beta A and beta B subunits of inhibin/activin polypeptides was studied in the ovary and testis of sexually mature, immature, and embryonic rats. Specific staining with these three subunits was also evident in the oocytes from embryonic to mature female rats. This result suggests that inhibin- and activin-like substances may be produced in the oocytes and these substances may play a role in the oocyte growth and differentiation. Judging from the intensity of immunoreaction in mature female rats, the three subunits should be produced more abundantly in luteal cells than in the granulosa cells. Immunoreactive alpha, beta A and beta B subunits were observed in the cummulus oophorous in the morning (11:00), but not in the evening (23:00) on proestrus. The results are in well agreement with the previous report that inhibin alpha and beta A subunit mRNA signals decline on proestrus evening. It is supposed that the cyclic change may be related with physiological phenomena prior to the ovulation, such as primary gonadotropin surges, loss of cummulus-oocyte gap junctions, or germinal vesicle breakdown. In both germ cells and Sertoli cells of the testis, alpha, beta A and beta B subunits were more abundant in the embryonic rat than in the mature rat. Although clear reactions with beta A and beta B subunits were detected in Leydig cells, alpha subunit was not detectable in the cells throughout the developmental stages examined.

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.