Galectins in the mouse ovary: concomitant expression of galectin-3 and progesterone degradation enzyme (20alpha-HSD) in the corpus luteum.

Galectin, an animal lectin that recognizes beta-galactosides of glycoconjugates, is involved in multiple biological functions such as cell growth, differentiation, apoptosis, and signal transduction. The present study using in situ hybridization revealed the predominant expression of galectin-1 and galectin-3 in the mouse ovary. Galectin-1 mRNA was diffusely expressed in the ovarian stroma, including the interstitial glands and theca interna, and intensely expressed in the corpus luteum (CL) at particular stages of regression. Transcripts of galectin-3 were restricted to CL and always coincident to the expression of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a progesterone degradation enzyme. In the non-pregnant ovary, signals for both galectin-1 and -3 were intense in the old, regressing CL formed at previous estrous cycles. In the newly formed CL, the signal intensity of galectin-1 first increased at the starting point of regression followed by increasing galectin-3/20alpha-HSD expressions. Under gestation with active progesterone production, signals for both galectin-1 and -3 in CL completely disappeared. At the perinatal stage, intense expressions of galectin-3/20alpha-HSD recovered in the remaining CL of gestation with the temporal expression of galectin-1 and continued until weaning. These findings suggest that galectin-1 and -3 may mediate progesterone production and metabolism in luteal cells via different mechanisms.

Immunohistochemical and in situ hybridization analysis of galectin-3, a beta-galactoside binding lectin, in the urinary system of adult mice.

Galectin is an animal lectin that has high affinity to beta-galactoside of glycoconjugates. In the present study, cellular expression of galectin subtypes in the urinary system of adult mice was examined by in situ hybridization and immunohistochemistry. The major subtype expressed in the murine urinary system was galectin-3, which was expressed continuously from the kidney to the distal end of the urethra. The renal cortex expressed galectin-3 more intensely than the medulla. Renal galectin-3 immunoreactivity was strongest in the cortical collecting ducts, where principal cells were the sole cellular source. All cell layers of the transitional epithelium from the renal pelvis to the urethra strongly expressed galectin-3 at the mRNA and protein levels. An electron microscopic study demonstrated diffuse cytoplasmic localization of galectin-3 in principal cells of the collecting ducts and in the bladder epithelial cells. Urethral galectin-3 expression at the pars spongiosa decreased in intensity near the external urethral orifice, where the predominant subtype of galectin was substituted by galectin-7. The muscular layer of the ureter and urinary bladder contained significant signals for galectin-1. Taken together, the observations indicate that the adult urinary system shows intense and selective expression of galectin-3 in epithelia of the uretic bud- and cloaca-derivatives.

Histochemical demonstration of a Na(+)-coupled transporter for short-chain fatty acids (slc5a8) in the intestine and kidney of the mouse.

Short-chain fatty acids in the intestinal lumen affect colonic cell proliferation as well as function as an energy source for intestinal epithelial cells. A novel transporter of monocarboxylates, Slc5a8, is expressed abundantly in the colon, where it may participate in the Na(+)-coupled absorption of short-chain fatty acids produced by bacterial fermentation of dietary fiber. The present study examined the cellular localization of Slc5a8 in the murine gastrointestinal tract and kidney by in situ hybridization and immunohistochemistry. The hybridization signals were recognized in the terminal ileum and whole length of the large intestine, and were especially intense in the distal colon and rectum. The immunoreactivity of Slc5a8 was restricted to the striated border (the brush border) of enterocytes, and was not present in goblet cells, Paneth cells, or lamina propria cells. In the kidney, proximal tubules of both the cortex and the outer stripe of the outer medulla intensely expressed Slc5a8 mRNA, while the distal portions, including the loop of Henle, lacked the signals. The renal Slc5a8 immunoreactivity was localized only in the brush border of proximal tubules, not along the basolateral membrane. Thyroid follicular cells were immunoreactive for Slc5a8, with predominant labeling on the apical membrane. No other organs, including the esophagus, stomach, liver, pancreas, and salivary glands contained any notable signals of Slc5a8. These findings on the cellular and subcellular localization of Slc5a8 under normal conditions are helpful for understanding the physiological and pathological roles of Slc5a8.

Differential cellular expression of galectin family mRNAs in the epithelial cells of the mouse digestive tract.

Galectin is an animal lectin that recognizes beta-galactosides of glycoconjugates and is abundant in the gut. This study revealed the cellular expression of galectin subtypes throughout the mouse digestive tract by in situ hybridization. Signals for five subtypes (galectin-2, -3, -4/6, and -7) were detected exclusively in the epithelia. In the glandular stomach, galectin-2 and -4/6 were predominantly expressed from gastric pits to neck of gastric glands, where mucous cells were the main cellular sources. The small intestine exhibited intense, maturation-associated expressions of galectin-2, -3, and -4/6 mRNAs. Galectin-2 was intensely expressed from crypts to the base of villi, whereas transcripts of galectin-3 gathered at villous tips. Signals for galectin-4/6 were most intense at the lower half of villi. Galectin-2 was also expressed in goblet cells of the small intestine but not in those of the large intestine. In the large intestine, galectin-4/6 predominated, and the upper half of crypts simultaneously contained transcripts of galectin-3. Stratified epithelium from the lip to forestomach and anus intensely expressed galectin-7 with weak expressions of galectin-3. Because galectins in the digestive tract may be multi-functional, information on their cell/stage-specific expression contributes to a better understanding of the functions and pathological involvements of galectins.

Single point mutation in tick-borne encephalitis virus prM protein induces a reduction of virus particle secretion.

Flaviviruses are assembled to bud into the lumen of the endoplasmic reticulum (ER) and are secreted through the vesicle transport pathway. Virus envelope proteins play important roles in this process. In this study, the effect of mutations in the envelope proteins of tick-borne encephalitis (TBE) virus on secretion of virus-like particles (VLPs), using a recombinant plasmid expression system was analysed. It was found that a single point mutation at position 63 in prM induces a reduction in secretion of VLPs. The mutation in prM did not affect the folding of the envelope proteins, and chaperone-like activity of prM was maintained. As observed by immunofluorescence microscopy, viral envelope proteins with the mutation in prM were scarce in the Golgi complex, and accumulated in the ER. Electron microscopic analysis of cells expressing the mutated prM revealed that many tubular structures were present in the lumen. The insertion of the prM mutation at aa 63 into the viral genome reduced the production of infectious virus particles. This data suggest that prM plays a crucial role in the virus budding process.

Noc2 is essential in normal regulation of exocytosis in endocrine and exocrine cells.

Rab3 is a subfamily of the small GTP-binding protein Rab family and plays an important role in exocytosis. Several potential effectors of Rab3, including rabphilin3 and Rims (Rim1 and Rim2), have been isolated and characterized. Noc2 was identified originally in endocrine pancreas as a molecule homologous to rabphilin3, but its role in exocytosis is unclear. To clarify the physiological function of Noc2 directly, we have generated Noc2 knockout (Noc2(-/-)) mice. Glucose intolerance with impaired insulin secretion was induced in vivo by acute stress in Noc2(-/-) mice, but not in wild-type (Noc2(+/+)) mice. Ca(2+)-triggered insulin secretion from pancreatic isles of Noc2(-/-) mice was markedly impaired, but was completely restored by treatment with pertussis toxin, which inhibits inhibitory G protein Gi/o signaling. In addition, the inhibitory effect of clonidine, an alpha(2)-adrenoreceptor agonist, on insulin secretion was significantly greater in Noc2(-/-) islets than in Noc2(+/+) islets. Impaired Ca(2+)-triggered insulin secretion was rescued by adenovirus gene transfer of wild-type Noc2 but not by that of mutant Noc2, which does not bind to Rab3. Accordingly, Noc2 positively regulates insulin secretion from endocrine pancreas by inhibiting Gi/o signaling, and the interaction of Noc2 and Rab3 is required for the effect. Interestingly, we also found a marked accumulation of secretory granules in various exocrine cells of Noc2(-/-) mice, especially in exocrine pancreas with no amylase response to stimuli. Thus, Noc2, a critical effector of Rab3, is essential in normal regulation of exocytosis in both endocrine and exocrine cells.

Cellular expression of murine Ym1 and Ym2, chitinase family proteins, as revealed by in situ hybridization and immunohistochemistry.

Ym is one of the chitinase family proteins, which are widely distributed in mammalian bodies and can bind glycosaminoglycans such as heparin/heparan sulfate. Ym1 is a macrophage protein produced in parasitic infections, while its isoform, Ym2, is upregulated in lung under allergic conditions. In the present study, we revealed the distinct cellular expression of Ym1 and Ym2 in normal mice by in situ hybridization and immunohistochemistry. Ym1 was principally expressed in the lung, spleen, and bone marrow, while Ym2 was found in the stomach. Ym1-expressing cells in the lung were alveolar macrophages, and the immunoreactivity for Ym1 was localized in rough endoplasmic reticulum. In the spleen, Ym1-expressing cells gathered in the red pulp and were electron microscopically identified as immature neutrophils. In the bone marrow, immature neutrophils were intensely immunoreactive, but lost this immunoreactivity with maturation. Moreover, needle-shaped crystals in the cytoplasm of macrophages, which formed erythroblastic islands, also showed intense Ym1 immunoreactivity. Ym2 expression was restricted to the stratified squamous epithelium in the junctional region between forestomach and glandular stomach. The function of Ym1 and Ym2 is still unclear; however, the distinct cellular localization under normal conditions suggests their important roles in hematopoiesis, tissue remodeling, or immune responses as an endogenous lectin.

Immunohistochemical demonstration of acidic mammalian chitinase in the mouse salivary gland and gastric mucosa.

Acidic mammalian chitinase (AMCase) is the sole chitinolytic enzyme that has been identified thus far in the gastrointestinal tract of mammals. AMCase mRNA expression has been demonstrated in the salivary gland and stomach of mice and in the stomach of humans, while a bovine homologue of AMCase is produced in the liver and secreted into the blood. The present study using antibody raised against bovine AMCase demonstrates the cellular distribution of AMCase in salivary and gastric secretions at the protein level. Immunostaining using mouse tissues detected intense immunoreactivity for AMCase in serous-type secretory cells of the parotid gland and von Ebner's gland. Gastric chief cells, localized at the bottom of gastric glands, were also immunoreactive for AMCase. Electron-microscopically, the immunoreactivity was localized in granules in the apical cytoplasm of these secretory cells, and not in other structures. Western blot analysis confirmed the existence of AMCase in the parotid gland and stomach, and in their secretions in mice. However, no immunoreactive band was clearly detectable in immunoblots of the human parotid saliva and gastric juice. At least in the mouse, AMCase is secreted into the saliva and gastric juice, and may function as a digestive enzyme or play a defensive role against chitinous pathogens.

Three-dimensional ultrastructure of synoviocytes in the knee joint of rabbits and morphological changes in osteoarthritis model.

The synovial intima is composed of two types of synoviocytes: absorptive macrophages and secretory, fibroblast-like F cells. Many studies have tried to observe synoviocytes by scanning electron microscopy (SEM) but failed to reveal the entire shape of synoviocytes because they are deeply embedded in the interstitial matrix. The present study, primarily employing SEM observation of NaOH macerated samples, reveals the distribution and three-dimensional ultrastructure of the synoviocytes in the normal knee joint of rabbits, and the morphological changes of synoviocytes in an osteoarthritis model of this animal. F cells were broadly distributed throughout the synovial intima, while macrophages showed a restricted distribution on fatty tissues around the patella. F cells were classified into a flat type, which covered the surface of synovial membrane like an epithelium, and a dendritic type, which extended long processes to form a characteristic meshwork on the surface. The flat type predominated in regions adhering to the femur, while the dendritic type predominated in ambilateral parts of both the patella and tendon of the musculus quadriceps femoris, and on the peripatellar fatty tissue. Intermediate forms of flat and dendritic types appeared in middle regions between the patella and periphery of the joint capsule. In the synovial membrane of the osteoarthritis model, both types of synoviocytes increased in number and changed their morphology, indicating their elevated activities in absorption and secretion. It is suggested that the ultrastructural changes in synoviocytes reflect pathological conditions of the synovial membrane, and synoviocytes play important roles in the pathogenesis of osteoarthritis.

Cellular architecture of the synovium in the tendon sheath of horses: an immunohistochemical and scanning electron microscopic study.

The intimal lining cells of the synovium in joints have been studied morphologically and histochemically and shown to consist of macrophagic cells (type A) and fibroblast-like cells (type B). It is believed that the structure of the synovium in the tendon sheath is similar to that in the joint, but there have been only a few morphological studies of the tendon sheath. The present study revealed the cellular architecture of synovium in the tendon sheath of horses by histochemistry and scanning electron microscopy (SEM). Like the joint, the inner surface of the tendon sheath was covered with a cell-rich intimal layer. Acid phosphatase-positive A cells accumulated in the mesotendon but few in other regions. B cells were selectively immunolabeled with protein gene product (PGP) 9.5 antiserum and distributed in the entire length of the synovial intima in the tendon sheath. The synovial intima consisted of a surface layer rich in the processes of B cells and a deep layer containing cell bodies of B cells. Using SEM, B cells could be classified into two types according to the morphology of their processes. B cells of dendritic type were located mainly in the joint-side of the tendon sheath and extended branched processes to form a meshwork on the intimal surface. B cells of flat type were located in the skin-side of the tendon sheath and in the mesotendon. Their membranous processes extended in a horizontal direction and covered the intimal surface, resembling epithelium. It appears likely that the morphology and distribution of synovial intimal cells are influenced by various factors, such as the nature of the underlying tissues and the magnitude of mechanical stress.