Distribution patterns of uterine glands and embryo spacing in the mouse.

To clarify the mechanism of implantation, relationship between positioning of the mouse embryo in the uterus and distribution of uterine glands along the long axis of the uterine horn was examined by three-dimensional remodelling of the uterine endometrium. There were two unique regions in the endometrium. Uterine glands were distributed widely from mesometrial to anti-mesometrial side in one region. It was localized from lateral to anti-mesometrial side in another. These different regions were alternately aligned throughout the uterine horn. The number and position of embryos was consistent with that of the latter region. This study suggests that the type of distribution of uterine glands is closely related to the positioning of the embryo in mice.

Immunohistochemical evaluation of the muscularis mucosae in the ruminant forestomach.

The muscularis mucosae and condensed fibrous layer of the ruminant forestomach were studied by immunohistochemistry using specific antibodies against alpha-smooth muscle actin (alphaSMA) and gamma-smooth muscle actin (gammaSMA). The specimens were collected from the rumen, reticulum and omasum of cattle, water buffalo, sheep, goat, Barbary sheep, Japanese serow, sika deer and mouse deer. The muscularis mucosae showed immunoreactivity for both alphaSMA and gammaSMA. On the other hand, the condensed fibrous layer appearing between the propria mucosa and tela submucosa was immunoreactive only for alphaSMA except for that in the goat and Barbary sheep reticulum which is intermingled with gammaSMA immunoreactivity. The distribution of muscularis mucosae and/or condensed fibrous layer varied among the compartments of forestomach and ruminant species. In the rumen, only the condensed fibrous layer was detected. On the other hand, the omasum contained only the muscularis mucosae. In the reticulum, both were detected. The amount of the condensed fibrous layer in the reticulum varied among different species in the following order of abundance: goat > Barbary sheep > sika deer> sheep > water buffalo > cattle and Japanese serow. Smooth muscle cells of external muscle layer were immunoreactive for alphaSMA and gammaSMA whereas those of blood vessels and pericytes were immunoreactive only for alphaSMA. The present findings on the actin immunoreactivity and distribution profile of muscularis mucosae and the condensed fibrous layer provide additional knowledge to further understand the histophysiological specialization of the different compartments of the ruminant forestomach.

Expression of mRNA of chemokine receptor CXCR4 in feline mammary adenocarcinoma.

The expression of mRNA of the chemokine receptor CXCR4 in 65 surgically resected mammary adenocarcinomas from cats was investigated by in situ hybridisation. No expression of the receptor's mRNA was detectable in the mammary tissue of healthy cats, but it was expressed in areas adjacent to necrosis, surrounding blood vessels and cells infiltrating the lymphatics of 47 (72.3 per cent) of the 65 samples. There was a significant relationship between lymphatic infiltration by neoplastic cells and the expression of the receptor's mRNA (P < 0.005), but there was no significant relationship between its expression and the one-year survival of the cats.

Immunohistochemical study of the ontogeny of prochymosin--and pepsinogen-producing cells in the abomasum of sheep.

The appearance and development of prochymosin- and pepsinogen-producing cells were investigated in the ovine abomasum from fetus to adult using immunohistochemistry. Prochymosin immunoreactivity appeared first in the proper gastric glands of the 100-day-old fetus. The intensity and distribution of prochymosin-immunoreactive cells increased gradually with the progress of gestation, and their most intense immunoreactivities and widest distribution were observed in 3-day-old lambs. They were subsequently reduced throughout postnatal growth. A few prochymosin-immunoreactive cells were scattered in the glands of adult sheep. Pepsinogen immunoreactivity appeared at first in a small number of cells in the base of some proper gastric glands of 120-day-old fetuses. After 130 days, pepsinogen-immunoreactive cells increased their staining intensities and distribution. They reached a peak in area at 21 days, which is comparable to adult sheep. In the pyloric glands, prochymosin- and pepsinogen-immunoreactive cells appeared from 100 and 130 days, respectively. Numbers were reduced in comparison to gastric glands and their occurrence was capricious. The results demonstrated that the ontogeny of prochymosin- and pepsinogen-immunoreactive cells in the abomasum of sheep is more similar to that in cattle than to that in the goat. The present data will contribute to the overall understanding of the development of ruminant gastric proteases.

Cycle of the seminiferous epithelium in the Java fruit bat (Pteropus vampyrus) and the Japanese lesser horseshoe bat (Rhinolophus cornutus).

The cycle of the seminiferous epithelium in the Java fruit bat, Pteropus vampyrus, and the Japanese lesser horseshoe bat, Rhinolophus cornutus, was investigated by light microscopy and the characteristics of spermiogenesis were compared between these two species. In the Java fruit bat, the cycle of the seminiferous epithelium was divided into 11 stages and developing spermatids were subdivided into 13 steps. While in the Japanese lesser horseshoe bat, the cycle of the seminiferous epithelium was divided into 10 stages and developing spermatids were subdivided into 13 steps. Excepting slight morphological differences, the characteristics of acrosomal formation in both species were almost similar with each other. In the Java fruit bat after stage VII, the acrosome gradually elongated, flattened and finally became scoop-like in shape. In the Japanese lesser horseshoe bat after stage VIII, the acrosome elongated, flattened and then slightly shortened. Before spermiation, the acrosome became long spatula-like in shape. The elongation and flattening of spermatids in these two species were similar to those in insectivores. The finding may reflect the fact that the order Chiroptera is phylogenetically close to the order Insectivora.

A study of reproductive performance in pregnant, IL-2 receptor beta-chain overexpressed transgenic mice.

Relationships between female reproductive performance and uterine natural killer (uNK) cells were investigated in pregnant IL-2 receptor beta-chain overexpressed transgenic (Tg2Rbeta) mice. At 8 days of pregnancy, all fetuses were alive, suggesting that implantation normally occurred in these mice. However, 47% of fetuses were dead at 10 days of pregnancy and at 12 days all fetuses were resorbing, indicating that fetal loss progressed with the advance of pregnancy. The placenta of Tg2Rbeta mice gradually decreased in weight with the advance of pregnancy. At 10 days the placental labyrinth, decidua basalis, and metrial gland in Tg2Rbeta mice were poorly developed, and more uNK cells were found in Tg2Rbeta mice than in the control mice. We propose that Tg2RPbeta mice are the first and interesting model that uNK cells can cause abortion, to clarify the involvement of uNK cell function in female reproductive performance.

Immunolocalization of gastrin-releasing peptide in the bovine uterus and placenta.

Gastrin-releasing peptide (GRP), a mammalian homologue of amphibian bombesin, has been suggested to be a novel regulatory peptide in the reproductive tract during pregnancy. In this study, the localization of GRP in the bovine uterus and placenta was demonstrated by immunohistochemistry. Uterine and placental samples were collected from nonpregnant and pregnant specimens, respectively. Tissue sampling was done from the caruncle and intercaruncle of the uterus, and from the placentome (caruncle and cotyledon) and intercotyledon of the placenta. In all the tissues examined, GRP was detected although its immunoreactivity was observed at various degrees. In the uterus, moderate immunoreactivity for GRP was observed in the uterine gland epithelial cells. In the placenta, strong immunoreactivity for GRP was demonstrated in the uterine gland epithelial cells; moderate in superficial epithelial cells; and weak in the trophoblasts, trophoblastic giant cells and cryptal epithelial hybrid cells. In both nonpregnant and pregnant animals, GRP was immunolocalized in the uterine gland secretions and was found predominantly in the supranuclear region of the uterine gland epithelial cells. These findings may suggest that GRP is secreted into the uterine lumen and regulates the intrauterine environment of both the nonpregnant and pregnant bovine by exocrine, autocrine and/or paracrine manner.

Immunohistochemical localization of inhibin subunits in the testis of the bull.

The differential localization of the inhibin beta subunits betaA and betaB in the testis of adult bull was studied using specific monoclonal and polyclonal primary antibodies. Inhibin betaA- and betaB-subunits were localized only in the Sertoli cells. The inhibin betaA-subunit was observed in the cytoplasm while the betaB-subunit was localized in the nucleus. No specific findings depending on spermatogenic stages were observed among the seminiferous tubules. Moreover, the inhibin alpha-subunit was not detected in the testis of the bulls. In addition, no inhibin subunits were detected in the Leydig cells and spermatogenic cells. These findings indicate the presence of betaA- and betaB-subunits in the bull, which may suggest a possibility that activin is produced and/or stored in the Sertoli cells and regulates spermatogenesis in an autocrine/paracrine manner. Moreover, the inhibin betaB-subunit may be produced in the nucleus but the functional meaning of this is not yet clear.

A lectin-histochemical study on the seminiferous epithelium of the northern smooth-tailed tree shrew (Dendrogale murina) and the Java tree shrew (Tupaia javanica).

Lectin-binding patterns in the testes of the northern smooth-tailed tree shrew, Dendrogale murina and Java tree shrew, Tupaia javanica were studied by light microscopy and compared the data with those of the common tree shrew. Four lectins (PNA, SBA, BPA and GS-II) were used in this study. Peanut (Arachis hypogaea) agglutinin (PNA), soybean (Glycine max) agglutinin (SBA) and Bauhinia purpurea agglutinin (BPA) showed a strong reaction in the acrosomal region from Golgi to acrosome-phase spermatids in three species of tree shrews. These lectins also showed a granular positive reaction in the cytoplasm from acrosome to maturation-phase spermatids in three species, except that BPA revealed no granular reaction (though it was positive) in the spermatid cytoplasm of the northern smooth-tailed tree shrew and that PNA revealed no reaction in the spermatid cytoplasm of the common tree shrew. While, Griffonia simplicifolia-II agglutinin (GS-II) showed a positive reaction in the acrosomal region of Golgi-phase spermatids in three species of tree shrews. Although GS-II was positive in the spermatocyte cytoplasm of three species, it showed granular in the northern smooth-tailed tree shrew and common tree shrew but not granular in the Java tree shrew. Thus, the lectin-binding patterns in testes were similar among three species belonging to the Order Scandentia. However, slight differences were also detected even among these phylogenetically-close species.

Distribution of capsaicin-sensitive substance P- and calcitonin gene-related peptide-immunoreactive nerves in bovine respiratory tract.

The distribution of nerve fibers immunoreactive for substance P (SP) and calcitonin gene-related peptide (CGRP) was examined by means of immunohistochemical methods in the respiratory tract from nose to lung of normal and capsaicin-treated cattle. SP- and CGRP-immunoreactive (IR) nerve fibers with varicosities were detected in all portions. They were more numerous in calves than in cows. They were abundant in the nasal and laryngeal mucosae and tracheal bronchus, and few in number in the lung. SP- and CGRP-IR nerve fibers were mainly seen in the epithelium, in connective tissue beneath the epithelium and around blood vessels, and in the glands throughout the respiratory tract. In contrast, SP- and CGRP-IR nerve fibers were sparse in the smooth muscle layer. Capsaicin treatment of neonates caused a remarkable reduction in the number of SP- and CGRP-IR nerve fibers in the respiratory tract of calves. Double immunofluorescence experiments showed the colocalization of SP and CGRP in most of the nerve fibers. The present findings suggest that SP- and CGRP-IR nerve fibers are involved in the regulation of the bovine respiratory tract, and that capsaicin-sensitive SP- and CGRP-IR nerve fibers are sensory neurons of the bovine respiratory tract.

Differential localization of immunoreactive alpha- and beta-subunits of S-100 protein in feline testis.

This study investigates the differential localization of the alpha-subunit (S100-alpha) and the beta-subunit (S100-beta) of the S-100 protein in the feline testis, using immunohistochemistry with polyclonal antibodies to bovine S-100 protein (S-100) and monoclonal antibodies to bovine S100-alpha and S100-beta. Appreciable differences were observed in the cellular localization of the immunoreactivity of each subunit. S-100 was observed in the Sertoli cells, the epithelial cells of the transitional segment of the seminiferous tubules, Leydig cells and the peritubular cells of the seminiferous tubules, but was not observed in the epithelial cells of straight tubules and the rete testis or in the endothelial cells of blood and lymph vessels. S100-alpha immunoreactivity was localized in Sertoli cells, peritubular cells and the epithelial cells of the terminal segment of the tubules, whereas S100-beta immunoreactivity was localized in Leydig cells. The differential localization of the alpha- and beta-subunits of the S-100 protein in the feline testis suggests that this protein is multifunctional and be useful as an investigative tool in studying feline testis function.

Immunohistochemical localization of neuropeptides in bovine pancreas.

The occurrence and density of distribution of nerves and endocrine cells that are immunoreactive for neuropeptides in the bovine pancreas were studied by immunohistochemistry. The six neuropeptides localized were galanin (GAL), substance P (SP), methionine-enkephalin (MENK), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP). The exocrine pancreas was shown to have an appreciable number of GAL- and SP-immunoreactive nerve fibres but few fibres showing immunoreactivity for VIP and CGRP. Numerous MENK-, GAL-, SP-, and NPY-immunoreactive nerve fibres were seen in the endocrine portion of the pancreas. Nerve cell bodies in the intrapancreatic ganglia showed immunoreactivity for all of the neuropeptides except CGRP. Endocrine cells showing immunoreactivity for GAL and SP were observed in the large islets and islets of Langerhans, respectively. The present results indicate a characteristic distribution of neuropeptides in the bovine pancreas, which may regulate both exocrine and endocrine secretions of pancreas.

Immunohistochemical study on the distribution of endocrine cells in the gastrointestinal tract of the babirusa, Babyrousa babyrussa (Suidae).

The distribution and relative frequency of endocrine cells in the gastrointestinal tract of the babirusa were studied immunohistochemically using the avidin-biotin-peroxidase complex method. Thirteen types of gut endocrine cells were detected; they were immunoreactive for chromogranin, serotonin, somatostatin, gastrin, bovine pancreatic polypeptide (BPP), glucagon, secretin, cholecystokinin (CCK), methionine-enkephalin-Arg6-Gly7-Leu8 (MENK8), motilin, gastric inhibitory polypeptide (GIP) and peptide tyrosine tyrosine (PYY). Cells that were immunoreactive for chromogranin, serotonin, somatostatin and glucagon were found in all portions of the gastrointestinal tract. MENK8-immunoreactive cells were observed in the stomach and small intestine. Gastrin-immunoreactive cells were detected in the pyloric region and duodenum. PYY-immunoreactive cells were found in the small and large intestine. Cells immunoreactive for motilin, CCK, GIP, and secretin were observed in the proximal small intestine and those immunoreactive for neurotensin were found only in the ileum. Although the distribution pattern of endocrine cells in the gastrointestinal tract of babirusa was similar to those reported for pig, restricted distribution of several endocrine cells, gastrin, BPP, MENK8, motilin, CCK, GIP, secretin and neurotensin and wider distribution of glucagon and PYY were observed in the babirusa. The unexpected presence of MENK8 in all glandular regions of the stomach and PYY in the small intestine was also noted. The distribution of gut endocrine cells might be related to the regulatory characteristics of the babirusa digestive tract.

Galanin-like immunoreactive endocrine cells in bovine pancreas.

Pancreata of fetal, neonatal and adult cattle were studied immunohistochemically for galanin. The results revealed galanin-like immunoreactivity both in the endocrine cells and in the neural elements. The galanin-like immunoreactive endocrine cells (Gal-LIEC) were confined to the large islets, and were not observed in the islets of Langerhans and exocrine pancreas. They were first detected at the third prenatal month. Their developmental profile showed an increase from fetal to early neonatal stage with a subsequent decrease towards adulthood. The considerable number of Gal-LIEC from late prepartum to early postpartum stage may imply functional significance of galanin during the perinatal development of cattle. Coexistence of galanin and insulin was also observed which may suggest autocrine interaction between the 2 hormones.

Morphological study on pigmented cells in the horse testis.

One of the most attractive characteristics of a horse testis is the change of the weight during development. As the testicular weight changes and the number of Leydig cells decreases, pigments appear in interstitial tissues. In the present study, the characteristics of the pigments found in the interstitial tissues were examined histochemically and ultrastructurally. Specific stainings indicated that the pigmented granules showed almost all of the histological and histochemical characteristics of ceroid or ceroid-like pigment. The cells showed positive reaction for acid phosphatase while the pigmented cells contained a lot of lysosomes ultrastructurally. These results suggest that macrophages might phagocytize Leydig cells, and store their digested materials as ceroid-like pigment.

Immunohistochemical survey of the gut endocrine cells in the common tree shrew (Tupaia belangeri).

Regional distribution and relative frequency of endocrine cells in the gastrointestinal tract of the common tree shrew (Tupaia belangeri) were studied immunohistochemically. Ten types of immunoreactive endocrine cells were localized in the gastric mucosa, i.e., chromogranin-, serotonin-, gastrin-, somatostatin-, bovine pancreatic polypeptide (BPP)-, enteroglucagon-, pancreatic glucagon-, peptide tyrosine tyrosine (PYY)-, motilin-, and substance P (SP)-immunoreactive (IR) cells. In the intestine, 13 types of immunoreactive cells were observed, i.e., chromogranin-, serotonin-, somatostatin-, gastrin-, BPP-, enteroglucagon-, PYY-, secretin-, cholecystokinin (CCK)-, gastric inhibitory peptide (GIP)-, motilin-, neurotensin-, and SP-IR cells. The regional distribution and relative frequency of the cell types varied along the gastrointestinal tract. Basically, the types, distribution, and relative frequency of the gut endocrine cells were similar to those reported in other mammalian species. However, some characteristic findings were noted in the present study: (1) the considerably large number of gastrin-IR cells in the pyloric region; (2) numerous serotonin-IR cells in the stomach; (3) appreciable number of BPP-IR cells in the transitional region of the stomach; and (4) wide distribution of PYY- and motilin-IR cells in the gut.

An immunohistochemical survey of catecholamine-synthesizing enzyme-immunoreactive nerves and endocrine cells in the bovine pancreas.

The distribution of catecholamine-synthesizing enzyme-immunoreactive nerves and endocrine cells in the pancreas of the calf and cow was studied immunohistochemically using antisera against tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). TH- and DBH-immunoreactive nerve fibres were abundant both within and around the islet of Langerhans. A few TH- and DBH-immunoreactive nerve fibres were seen around the large islets characteristic of calf pancreas, but the majority of cells in the large islets, and some in islets of Langerhans, showed TH immunoreactivity. In the exocrine pancreas, both TH- and DBH-immunoreactive nerve fibres were distributed randomly among the acini, with the DBH-immunoreactive fibres being more numerous. Abundant TH- and DBH-immunoreactive nerve fibres were seen in close association with blood vessels and in the connective tissue around the interlobular duct. Immunoreactivity for both enzymes was also observed in the nerve cell bodies and fibres of the intrapancreatic ganglia. The findings suggest an important role for catecholamines in the regulation of bovine pancreatic function.

An osteometrical study of the cranium and mandible of the lesser mouse deer (Chevrotain), Tragulus javanicus.

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.

Expression of inhibin alpha-subunit in horse testis.

Inhibin is believed to play roles in the pituitary secretion of FSH and in the paracrine regulation of testicular function. Although it has been generally accepted that inhibin is produced in Sertoli cells, there was a recent evidence for the localization of inhibin in Leydig cells of primates, rat and sheep. However, there is no report on the expression of inhibin in the adult horse testis. Therefore, using immunohistochemistry, western blotting and in situ hybridization techniques, the present study examined inhibin alpha-subunit (Ih-alpha) expression in the adult horse testis. For the detection of Ih-alpha protein, we used anti-porcine Ih-alpha antibody in immunohistochemistry and western blotting. Furthermore, digoxigenin-labeled complementary RNA probes were prepared to detect intracellular messenger RNA (mRNA) of Ih-alpha. Immunostainings for Ih-alpha were found not only in Leydig cells but also in Sertoli cells. The intensity in Leydig cells was stronger than in Sertoli cells. Immunoreactivities for Ih-alpha were found at approximately 46 kDa, 56 kDa and 90 kDa in the homogenates from testicular interstitial tissues. The bands at 56 kDa and 90 kDa agree with previous report, but not at 46 kDa. Signals for mRNA of Ih-alpha by in situ hybridization were detected in Leydig cells and in the basal region of seminiferous epithelium including Sertoli cells. These results suggest that Ih-alpha is expressed in Leydig cells and Sertoli cells of horse testis, and the expression level should be higher in Leydig cells than Sertoli cells.

Inhibin secretion in the stallion.

To examine the physiological role of inhibin in the stallion, a heterologous radioimmunoassay (RIA) based on a bovine RIA was validated and used to measure immunoreactive (ir)-inhibin concentrations in plasma and testicular homogenates. The bioactivity of equine testicular inhibin was also examined using an assay for suppression of FSH secretion from rat anterior pituitary cells. In addition, to identify the cell responsible for secreting testicular inhibin, the localisation of inhibin in the testis was investigated by an immunohistochemical method using a polyclonal antibody against (Tyr30)-porcine inhibin alpha(1-30) NH2. In the RIA, parallel dose response curves were obtained for the bovine inhibin standard and serial dilutions of stallion plasma and equine testicular homogenates. Parallel FSH inhibition curves were also observed for the bovine inhibin standard and serial dilutions of equine testicular homogenates in the bioassay. The inhibition of FSH secretion from rat pituitary cells by equine testicular homogenates was neutralised by an antiserum against bovine inhibin in vitro. Plasma concentrations of ir-inhibin, testosterone and oestradiol-17beta in stallions decreased abruptly after bilateral gonadectomy and FSH and LH concentrations in the plasma subsequently increased. Therefore, circulating inhibin in the stallion appeared to be largely of testicular origin. The histochemical results showed for the first time that strong immunopositive staining for inhibin occurred in the Leydig cells of the testes. Sertoli cells were also stained by the inhibin antibody but the reaction was weaker than that in Leydig cells. These results indicate clearly that both Leydig and Sertoli cells are potential sources of testicular inhibin in the stallion. A clear increase in plasma ir-inhibin concentrations was observed during the natural breeding season. Similar seasonal changes in the plasma concentrations of testicular steroid hormones and pituitary gonadotrophins occurred throughout the year. In conclusion, the testes appear to be the main source of inhibin, and testicular inhibin is secreted by Leydig and Sertoli cells in stallions. The positive correlations between plasma ir-inhibin and testicular activity during both the breeding and nonbreeding seasons indicate that plasma ir-inhibin is a useful indicator of reproductive activity in the stallion.