Immunohistochemical localization of ornithine decarboxylase in the rat ovary

Summary The present report describes the immunocytochemical localization of ornithine decarboxylase in the prepubertal rat ovary after administration of human chorionic gonadotropin (HCG). Numerous ornithine decarboxylase immunoreactive cells appeared in the thecal layer as well as in the interstitial gland tissue after treatment with HCG. The granulosa cells, the ovum and the ovarian stroma were devoid of immunoreactive ornithine decarboxylase. In contrast to the ovary of HCG-treated rats, the ovary of prepubertal rats given the vehicle alone contained only a few weakly immunoreactive cells.     

Immunohistochemical localization of metallothionein in developing rat tissues

Metallothionein (MT) is a cysteine-rich, low molecular weight protein inducible by heavy metal ions and various endogenous factors. Using an indirect immunofluorescent technique, we studied the localization of MT in developing rat tissues (kidney, small intestine, and liver). In kidney of the neonate and fetus, MT was found in both the cytoplasm and the nucleus of renal tubular epithelia. Localization of MT changed with shift of zonation in the renal cortex during development. Metallothionein was found mainly in the inner zone of the cortex but not in tubules of the neogenic zone on Day 4. Until Day 18, tubular cells containing MT were observed in a part of the cortex adjacent to the medulla, followed by a significant decrease in immunostaining by Day 27. In small intestine of the neonate, MT was localized predominantly in Paneth and goblet cells which play secretory roles. The number of goblet cells with strong immunostaining for MT was maximal on Day 27. In liver of 20-day fetuses and of 4-day-old neonates, both the cytoplasm and the nucleus of hepatocytes exhibited strong immunofluorescence. The intensity of MT staining diminished with development, and by 18-27 days after birth no immunofluorescence was observed in the nucleus. We further studied a possible association of MT with development by localizing MT in livers obtained from partially hepatectomized and laparotomized rats. Hepatectomy led to the appearance of MT not only in the nucleus and cytoplasm of hepatocytes but also in sinusoids and bile canaliculi. After laparotomy, MT immunofluorescence was observed only in the cytoplasm. The present results suggest a possible involvement of MT in cell proliferation and differentiation, as well as in transport and secretion of this metal-binding protein.     

Immunohistochemical localization of follistatin in rat tissues.

We have used immunohistochemistry to localize follistatin/activin-binding protein in adult male and female rats. A polyclonal antibody directed against a follistatin peptide (residues 123-134) was used as a specific immunologic probe. Intense and specific follistatin immunoreactivity was evident in spermatogenic cells of seminiferous tubules in the testis. The predominant staining was in nuclei of spermatocytes and spermatids, but no immune reaction was observed in spermatogonia or spermatozoa. Moderate immunoreactivity was detected in Leydig cells. Sertoli cells were follistatin-negative. Significant immunoreactivity was evident in ovarian granulosa cells. The intensity of the staining changed with follicle development: no immunoreactivity was observed in granulosa cells of primordial to primary follicles, but the cells of secondary to Graafian follicles displayed moderate to strong staining and finally luteal cells of the corpus luteum became negative. The epithelial lining of the oviduct and the smooth muscle of the myometrium of the uterus were intensely immunoreactive. Immunoreactive follistatin staining was present in the pituitary: a group of round-shaped cells were specifically stained. Immunostainable follistatin was visible in the epithelial layers of renal tubules with moderate to strong staining reactivity. Hepatic cells in the liver demonstrated homogeneous immunoreactivity from moderate to strong. The cortex of the adrenal gland, white pulp of the spleen and the brain cortex were also stained weakly but distinctly with the antiserum. In conclusion, immunoreactive follistatin is widespread in rat tissues, suggesting that follistatin/activin-binding protein is a ubiquitous protein, regulating a wide variety of activin actions.     

Immunohistochemical localization of Zn-alpha 2-glycoprotein in normal human tissues

The Zn-alpha 2-glycoprotein (Zn-alpha 2-GP) is present at a high concentration in the seminal plasma and at significant levels in other human body fluids. Its precise localization, however, has remained unclear, as well as its physiological and pathological significance. The present study reports the immunohistochemical localization of this protein in normal adult human tissues. Localization of the reactive product to anti-human plasma Zn-alpha 2-GP antibody was demonstrated in the following cells: luminal and basal cells of the prostate gland, luminal epithelial cells of the acini and of some ducts of the mammary glands, luminal cells of the secretory portion of the eccrine and apocrine sweat glands, serous cells of the salivary, tracheal, and bronchial glands, acinar cells of the esophageal glands, exocrine acinar cells of the pancreas, hepatocytes of the liver, and epithelial cells of the proximal and distal tubules in the kidney. The present results suggest that Zn-alpha 2-GP exerts some unknown but fairly widespread exocrine function and may be produced in the various epithelial cells tested. Hepatocytes are also suggested to be a source of the protein in the blood plasma.     

Immunohistochemical localization of smooth muscle myosin in normal human tissues

Immunohistochemical localization of smooth muscle myosin, an immunologically distinct contractile protein, was achieved using rabbit anti-human uterine smooth muscle myosin antibodies. In immunodiffusion studies and in cryostat sections, these antibodies were highly specific and reacted with smooth muscle myosin but not with platelet, skeletal muscle, or cardiac muscle myosin. To evaluate comprehensively the structural profile of smooth muscle elements in normal human tissues, an indirect immunoperoxidase technique (peroxidase-antiperoxidase) was applied to a wide variety of specimens. Parallel studies comparing cryostat sections with fixed (10% formalin, B5, Bouin's, or Zenker's solution) paraffin-embedded tissues revealed optimal immunoreactivity, sensitivity, and specificity of staining for smooth muscle myosin using frozen tissues. Strong immunoreactivity was present in muscular tissues such as blood vessels and the muscularis of gastrointestinal and genitourinary tracts. Distinct delineation of smooth muscle elements, including individual smooth muscle cells, and their specific patterns of alignment and organization, were observed, e.g., cells comprising the muscularis mucosae and extending into the lamina propria of the gastrointestinal tract, and myoepithelial cells of skin, exocrine glands, and breast. This method provides excellent morphologic preservation and readily permits unambiguous identification of individual cells containing smooth muscle myosin.     

Immunogold localization of mitochondrial aspartate aminotransferase in mitochondria and on the cell surface in normal rat tissues

Mitochondrial aspartate aminotransferase (mAspAT) (E.C. 2.6.1.1), an important enzyme in amino acid metabolism, is identical to a fatty acid-binding protein (FABPpm) isolated from plasma membranes of several cell types. Employing a monospecific polyclonal antibody to rat mAspAT, we have used immunogold electron microscopy to study the subcellular distribution of mAspAT in various mammalian tissues. Immunogold labeling of rat tissue sections embedded in LR Gold resin showed strong labeling of mitochondria in all tissues examined (viz. liver, pancreas, pituitary, spleen, heart, kidney, submandibular gland). In addition, strong and specific labeling was also observed at a number of non-mitochondrial sites including various locations in kidney, such as on cell surface in distal tubules and cortical collecting ducts, in condensing vacuoles, along cell boundaries between adjoining cells, and in endothelial cells lining capillaries in the glomerulus. Surface labeling due to mAspAT was also seen in arteriolar endothelial cells and in lymphocytes. These findings support the previous identification of mAspAT as both a mitochondrial enzyme and a plasma membrane protein. It is suggested that in accordance with its established role in other cells and tissues, the surface-located mAspAT in kidney and endothelial cells is involved in the fatty acid transport process. The dual-localization of mAspAT, as well as a large number of other mitochondrial proteins (viz. Hsp60, Hsp10, Cytochrome c, TRAP-1 and P32 (gC1q-R)) in recent studies, within both mitochondria and at various specific extramitochondrial sites raises fundamental questions about the role of mitochondria in cell structure and function, and about the mechanisms that exist in normal cells for protein translocation from mitochondria to other compartments. These results have implications for the role of mitochondria in apoptosis and different diseases.     

Immunohistochemical localization of exocrine enzymes in normal rat pancreas.

On the basis of morphological and biochemical differences, the exocrine pancreatic tissue has been divided in peri- and teleinsular regions. In the present study the enzymatic profile of these regions has been investigated by the immunofluorescent technique using antibodies against nine pancreatic enzymes (alpha-amylase, lipase, chymotrypsinogen A, trypsinogen, elastase, carboxypeptidase A and B, DNase and RNase A). These antibodies were specific to their antigens without cross reaction. By immunofluorescence, most acinar cells of the normal rat pancreas were positive to the nine enzymes tested. However, an inhomogeneity in the staining pattern was found; specifically, the cells located in the periinsular region of many islets showed a brighter fluorescence than acinar cells in the teleinsular tissue. These data add a new parameter to describe the inhomogeneity of the exocrine pancreas.     

Complete amino acid sequence of rat kidney ornithine aminotransferase: identity with liver ornithine aminotransferase

The complete amino acid sequence of rat kidney ornithine aminotransferase [EC 2.6.1.13] is presented. The 404-residue sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with CNBr, Achromobacter protease I, arginylendopeptidase, or Staphylococcus aureus V8 protease. Mueckler and Pitot have reported the amino acid sequence of the rat liver enzyme (440 residues) as predicted from the nucleotide sequence of the cDNA [Mueckler, M.M. & Pitot, H.C. (1985) J. Biol. Chem. 260, 12993-12997]. The amino acid sequence of the rat kidney enzyme presented herein coincides with residue 36 (Gly) through 440 (Phe) of the predicted precursor protein, indicating that the liver and kidney enzymes are identical, and that the enzyme is processed at the amino-terminal region after translation.     

Immunohistochemical localization and quantitative analysis of cellular glutathione peroxidase in foetal and neonatal rat tissues: Fluorescence microscopy image analysis

Summary To quantitate the developmental changes in selenium-dependent cellular glutathione peroxidase during the perinatal period, tissue sections from foetal (day 12 to day 22) and neonatal (day 6) rats were stained immunohistochemically using specific polyclonal antiserum. The intensity of the staining was quantified by fluorescence microscopy image analysis. There was a general trend of enriched glutathione peroxidase in the epithelial linings and metabolically active sites. Significant fluorescence was detected in cardiomyocytes, hepatocytes, renal tubular epithelium, bronchiolar epithelium and intestinal epithelium at day 15. The intensity increased in a stepwise manner therafter. The overall increase in the intensity of staining in the heart, liver, kidneys, lungs and intestine was 1.5-, 2.3-, 1.6-, 1.7- and 3.0-fold, respectively. The phase of most rapid increase occurred during the foetal period in the liver, intestine and heart. In the kidneys and lungs, glutathione peroxidase increased significantly during foetal life, and to a similar extent postnatally. These results suggest that the intracellular H₂O₂-scavenging system develops during the foetal period as an essential mechanism for living under atmospheric oxygen conditions. The late development observed in the kidneys and lungs is consistent with the relative biological immaturity of these organs in full-term neonates.     

Immunohistochemical localization of superoxide dismutases in fetal and neonatal rat tissues.

We investigated the developmental profile of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in tissue sections obtained from fetal (Day 12 to 21 of gestation) and neonatal (Day 0 and 6) rats. Tissues were stained immunohistochemically with specific antisera against the respective rat SODs. There was a general trend towards richness of SODs in the epithelial linings and metabolically active sites, although differential distribution between the two SODs also existed. At Day 12 of gestation, immunoreactivity for both SODs was detected in the cardiomyocytes but not in other tissues. Hepatocytes expressed CuZnSOD at Day 14 and MnSOD at Day 17. By Day 18 CuZnSOD was detected in the epithelial cells of the gastrointestinal tract, respiratory tract, pancreatic islets, kidneys, and adrenals. These tissues exhibited MnSOD staining at Day 19. CuZnSOD occurred in the epithelia of the thyroid, thymus, and salivary glands at Day 19, while MnSOD was seen at Day 21. The increase in intensity of the staining for SODs occurred no later than postnatal Day 0, indicating that most tissues accumulated SODs during late gestation. Breathing atmospheric oxygen during early extrauterine life did not appreciably intensify the SOD staining. These results suggest that perinatal increase in SODs occurs as a general mechanism of preparation for birth.     

Estrogen induction of ornithine aminotransferase in rat kidney slices

Immunologic titration of kidney extracts from control and estradiol-injected rats with anti-ornithine aminotransferase serum shows that the hormone induces the enzyme by increasing its concentration in the kidney. The induction has been studied in vitro with kidney slices. Several-fold increase in the rate of ¹⁴C-leucine incorporation into the enzyme by hexestrol has been demonstrated. In addition, certain antiestrogens also induce the enzyme synthesis in vitro. These results show that the induction of this enzyme in the kidney does not require the participation of an estrogen metabolite from another tissue.     

The regulation of ornithine aminotransferase synthesis by glucagon in the rat.

Ornithine aminotransferase (OAT) from rat liver mitochondria was purified to homogeneity. A monospecific antiserum against the enzyme protein was prepared in rabbits. Immunotitrations were performed on OAT present in crude mitochondrial extracts obtained from the livers and kidneys of rats in several hormonal and dietary states. No evidence was found for the existence of an immunologically reactive but enzymatically inactive form of OAT. The relative rate of enzyme synthesis in vivo was studied by pulselabeling rats with [4, 5-³H]leucine, isolating the enzyme protein by immunoprecipitation, and dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels. Nine hours after a single subcutaneous injection of a glucagon oil emulsion, a 3-fold increase in OAT activity and a 12-fold increase in the synthetic rate of the enzyme were observed. Serine dehydratase activity increased on a time-course very similar to that of OAT following glucagon injection. These increases occurred only on low (0–12.5%) protein diets. At higher levels of dietary protein (30% and up), no further stimulation of OAT synthesis by glucagon was observed. Administration of actinomycin D within the first 2 h after glucagon injection resulted in an inhibition of OAT induction. When the administration of the antibiotic was delayed until 4 h after glucagon, no inhibition of OAT induction was observed. Glucose repression of the glucagon induction of the enzyme in hepatic mitochondria was demonstrated to be the result of a rapid inhibition of OAT synthesis.     

Immunohistological localization of alpha 1-microglobulin in normal rat tissues

The tissue distribution of rat alpha 1-microglobulin (alpha 1-m) was studied by indirect immunofluorescence in various rat tissues using a polyvalent rabbit antiserum to the purified antigen and a monoclonal antibody (H23) to the human homologue, in parallel with a polyclonal anti-rat IgA antiserum. It was found that all tissues stained by anti-IgA were also alpha 1-m positive; these tissues included tissues of the stomach, duodenum, ileum, colon, pancreas, trachea, esophagus and jejunum. However, the observation that IgA plasma cells as well as secretory cells, while positively stained by anti-IgA, are alpha 1-m negative suggests that the association between IgA and alpha 1-m occurs at a postsecretory stage, after the IgA molecules have been transported across the epithelial cells. Additionally, hepatocytes were intensely stained by anti-alpha 1-m antibodies, indicating that the liver, as already suggested by metabolic studies on isolated guinea-pig liver explants, may be responsible for the synthesis of this protein. Among lymphoid tissues, an intense and homogeneous staining was observed in the thymus and the white pulp of the spleen. Sections of lymph nodes, however, showed differential staining; apart from a few isolated dendritic cells in the mantle region of the lymphoid follicles, the germinal centers and medullary cords showed no staining with anti-alpha 1-m antibodies. The paracortical cells, macrophages in the subcapsular sinus, and interfollicular lymphocytes showed intense cytoplasmic staining with anti-alpha 1-m antibodies. In other tissues, macrophages, monocytes, tissue histiocytes, and dendritic cells were alpha 1-m positive. Although they confirm the presence of alpha 1-m in the lymphoid tissues, as already reported in man, these results show that the protein is also present in hepatocytes and in exocrine fluids containing IgA. Since alpha 1-m, like secretory component, can bind to IgA to form stable complexes, these two heavily glycosylated proteins may have similar biologic properties.     

Purification of ornithine aminotransferase by immunoadsorption

Ornithine aminotransferase was purified by conventional biochemical methods from rat kidney, rat liver, and human liver. Affinity-purified antibodies raised to the rat kidney enzyme were used to produce an immunoadsorbent enabling a one-step purification of ornithine aminotransferase to be made from crude human liver extracts. The harsh chemical conditions often required to desorb immunoadsorbents were avoided by isolating antibodies with low functional affinity and employing an electrophoretic desorption method which allowed the enzyme activity to be retained. The close structural similarity between human and rat ornithine aminotransferase was demonstrated by immunodiffusion reactions. It was therefore possible to purify the enzyme from human liver using immobilized antibodies raised against rat kidney ornithine aminotransferase. Furthermore, desorption was more readily achieved due to the lower affinity for the human enzyme.