Ultrastructural localization of substance P immunoreactivity in the ventral horn of the rat spinal cord

Summary At the light microscope level, the minute concentrations of substance P (SP) in rat spinal ventral horn can be visualized best by amplification with the double bridge PAP method of Vacca et al. (1975; 1980) in 5 m paraffin tissue sections. Morphologically, the immunoreactive sites resemble punctate bodies. They occur in close apposition with the large ventral horn cells and their associated neuronal processes. By the Sternberger PAP procedure, we now describe these punctate bodies at the electron microscope level. Ultrastructurally, they appear as tiny boutons (terminal and preterminal) and small unmyelinated processes. The boutons and processes typically contain one to several immunolabeled dense core vesicles among many immunolabeled clear vesicles. They range in size near the limit of resolution of the light microscope (LM), thereby justifying further the use of LM amplification staining by the double bridge method. The immunolabeled boutons often synapse with large smooth dendrites (which may originate from motoneurons) by asymmetrical or symmetrical synaptic densities. Their synaptic densities appear immunostained as well. The data support the view that the electrophysiological action of SP in the ventral horn occurs in part by synaptic action along the processes of the ventral horn cells. Other mechanisms of action are considered for the peptide as well. Additional types of membrane specializations (synaptoid junctions) and SP neural circuits are described below.The work, presented at the Histochemical Society's 29 Annual Meeting in Vancouver, B.C. April 1–2, 1978, was partially supported by CCHD 10-12-04-3600-67 (LLV)     

Ultrastructural localization of substance P immunoreactivity in the ventral horn of the rat spinal cord

At the light microscope level, the minute concentrations of substance P (SP) in rat spinal ventral horn can be visualized best by amplification with the double bridge PAP method of Vacca et al. (1975; 1980) in 5 microns paraffin tissue sections. Morphologically, the immunoreactive sites resemble punctate bodies. They occur in close apposition with the large ventral horn cells and their associated neuronal processes. By the Sternberger PAP procedure, we now describe these punctate bodies at the electron microscope level. Ultrastructurally, they appear as tiny boutons (terminal and preterminal) and small unmyelinated processes. The boutons and processes typically contain one to several immunolabeled dense core vesicles among many immunolabeled clear vesicles. They range in size near the limit of resolution of the light microscope (LM), thereby justifying further the use of LM amplification staining by the double bridge method. The immunolabeled boutons often synapse with large smooth dendrites (which may originate from motoneurons) by asymmetrical or symmetrical synaptic densities. Their synaptic densities appear immunostained as well. The data support the view that the electrophysiological action of SP in the ventral horn occurs in part by synaptic action along the processes of the ventral horn cells. Other mechanisms of action are considered for the peptide as well. Additional types of membrane specializations (synaptoid junctions) and SP neural circuits are described below.     

Progesterone dependent uptake of uteroglobin by rabbit endometrium

Summary Unlabeled or³H-labeled UGL, isolated from rabbit lungs, was injected intraluminally into the uteri of ovariectomized low dose progesterone substituted and unsubstituted animals as well as into mated animals on the fifth day of pregnancy. It was offered to the endometrium, in this last case, in competition with endogenously synthesized UGL.³H-labeled IgG served as a control. The animals were killed 24 h after the experiment. Immunohistograms and autoradiograms showed that UGL was taken up by the whole endometrium of the ovariectomized animals but only when progesterone was present. In the preimplantation uterus, uptake was limited to clusters of smaller cells accumulated in the lumen adjacent to the epithelium, whereas IgG remained within the uterine lumen, forming a dense layer on the endometrial surface. A preliminary report of parts of this investigation has been presented at the 15th EDBO International Embryological Conference, June 21–25, 1982, in Strasbourg (France) (Abstract 187)     

Progesterone dependent uptake of uteroglobin by rabbit endometrium

Unlabeled or 3H-labeled UGL, isolated from rabbit lungs, was injected intraluminally into the uteri of ovariectomized low dose progesterone substituted and unsubstituted animals as well as into mated animals on the fifth day of pregnancy. It was offered to the endometrium, in this last case, in competition with endogenously synthesized UGL. 3H-labeled IgG served as a control. The animals were killed 24 h after the experiment. Immunohistograms and autoradiograms showed that UGL was taken up by the whole endometrium of the ovariectomized animals but only when progesterone was present. In the preimplantation uterus, uptake was limited to clusters of smaller cells accumulated in the lumen adjacent to the epithelium, whereas IgG remained within the uterine lumen, forming a dense layer on the endometrial surface.     

Ultrastructural immunocytochemical localization of fibronectin in the developing rat lung

In a previous study changes in the macrodistribution of fibronectin during rat-lung development were examined. Using the peroxidase-antiperoxidase immunocytochemical technique, we have demonstrated the presence of fibronectin in embryonic, neonatal, and adult rat lung at the ultrastructural level. In the embryo, fibronectin is found both in an intra- and extracellular association with isolated pneumoblasts, and in a periodic distribution along the basal lamina. The neonate displays fibronectin in an intracellular association with early type-I cells and on their basal and luminal surfaces, but not in association with type-II cells. Neonatal basal lamina is diffusely labeled by anti-fibronectin antiserum. Fibronectin in adult tissue is found both intracellularly and on the basal and luminal surfaces of type-I cells but not in type-II cells. The basal lamina and interstitial connective tissue are slightly or non-reactive. These observations confirm and extend our initial suggestion that fibronectin is involved in rat-lung development.     

Uptake of tritiated uteroglobin by the endometrium of the rabbit during peri-implantation

Summary Uteroglobin, labelled with N-succinimidyl-(2–3³H)-propionate, was applied in vivo for 3 h to pregnant rabbit uteri 7 and 9 days after mating. Light- and electronmicroscopic autoradiographs showed that the endometrial epithelium, both ciliated and non-ciliated cells, is able to take up³H-uteroglobin, however, with differing intensity. Large areas of labelling were found in the luminal epithelium, whereas the glandular epithelium contained fewer silver grains. Moreover, intensively labelled single cells or symplasms occurred in both luminal and glandular epithelium. They were identified as degenerating or dead cells. After internalization by pinocytosis or phagocytosis, the tritiated uteroglobin was observed in multivesicular bodies or in lysosomes with floccular content. Later, radioactivity was either found within residual bodies or distributed throughout the entire epithelium and the subepithelial stroma, i.e., the silver grains could no longer be assigned to specific cell organelles.     

Cell-free synthesis of preuteroglobin and processing to uteroglobin by polysomes from rabbit endometrium

Polysomes from rabbit endometrium, incubated in a cell-free system in the presence of [35S]methionine, completed polypeptide chains pre-initiated in vivo. Polypeptide chains were analyzed by SDS-gel electrophoresis after immunoprecipitation with anti-uteroglobin. Endometrial polysomes, freed of membranes by detergent treatment, synthesized both mature uteroglobin and preuteroglobin not yet cleaved in vivo. Membrane-bound polysomes synthesized exclusively mature uteroglobin, indicating the processing of preuteroglobin by the membranes bound to the ribosomes.     

Ultrastructural features of the ventral prostate epithelial cells in the Australian plains rat, Pseudomys australis

The fine structure of epithelial cells of the small ventral prostate of Pseudomys australis males was studied. The cells were sometimes binucleated, exhibited relatively little granular endoplasmic reticulum, generally few secretory granules (probably reflecting a low proteinaceous secretory activity), but had abundant agranular endoplasmic reticulum (AER), similar to that in steroidogenic cells. Some of the mitochondria also showed tubular cristae. Furthermore, most cells had some irregular dense bodies in the cytoplasm which may have developed, by a gradual transformation process, from the membranes of AER, mitochondria and other organelles; they could be the product of degenerative changes in these organelles. These findings indicate a significant difference in the structure of these cells from those present in the ventral prostate of the hopping mouse, Notomys alexis. It is speculated that this gland secretes relatively little protein but perhaps more lipid or cholesterol-derived substance.     

Glucocorticoid and developmental regulation of uteroglobin synthesis in rabbit lung.

The synthesis of uteroglobin in rabbit lung was studied after the administration of glucocorticoids to intact adult animals as well as during the late stages of rabbit development. The synthesis of uteroglobin was compared with levels of translatable uteroglobin mRNA in the lung. Uteroglobin synthesis was determined both by incorporation of [25S]methionine into the protein by lung explants incubated in vitro and by radioimmunoassay measurements of uteroglobin concentration in lung. Lung poly(A)-containing mRNA, isolated by oligo(dT)--cellulose chromatography, was translated in cell-free systems and the activity of uteroglobin mRNA was determined after immunoprecipitation. Dexamethasone administration increased about 2-fold the synthesis of lung uteroglobin compared with the controls. The effect of cortisol was more moderate. Both glucocorticoids did not affect the degradation rate of lung uteroglobin, but produced increases in the translatable levels of uteroglobin mRNA parallel to those observed for uteroglobin synthesis. During the late stages of rabbit development, both the synthesis of lung uteroglobin and the translatable levels of its mRNA increase in parallel about 12-fold in a biphasic fashion. A first increase occurred between 2 days before and 2 days after birth. Starting at 5 days of age, there was a second increase in both parameters, which at 12 days of age reached values close to those observed in adult rabbits. Our results suggest that the rate of lung uteroglobin synthesis could be mainly determined by the translatable levels of its mRNA.     

5alpha-Dihydrotestosterone binding protein in rat ventral prostate; purification, nuclear incorporation, and subnuclear localization.

Treatment of cytosol from the rat ventral prostate with cold acetone (-20 degrees C) evoked a 8 approximately 10-fold increase in the binding capacity with 5alpha-dihydrotestosterone (DHT). Starting from the extract of acetone-dried prostate cytosol, some 400 approximately 600-fold purification of the DHT-binding protein complex was acieved by (NH4)2504 fractionation, DEAE-cellulose chromatography and gel- filtration with Sephadex G-200. The purified 3H-DHT-binding protein complex was incorporated into the nuclei from the ventral prostate in a temperature dependent manner. The similar incorporation was also observed in nuclei from the liver and the kidney...     

Ultrastructural localization of Na+,K+-ATPase in rat and rabbit kidney medulla

Na+,K+-ATPase was localized at the ultrastructural level in rat and rabbit kidney medulla. The cytochemical method for the K+-dependent phosphatase component of the enzyme, using p-nitrophenylphosphate (NPP) as substrate, was employed to demonstrate the distribution of Na+, K+- ATPase in tissue-chopped sections from kidneys perfusion-fixed with 1% paraformaldehyde-0.25% glutaraldehyde. In other outer medulla of rat kidney, ascending thick limbs (MATL) were sites of intense K+-dependent NPPase (K+-NPPase) activity, whereas descending thick limbs and collecting tubules were barely reactive. Although descending thin limbs (DTL) of short loop nephrons were unstained, DTL from long loop nephrons in outer medulla were sites of moderate K+-NPPase activity. In rat inner medulla, DTL and ascending thin limbs (ATL) were unreactive for K+-NPPase. In rabbit medulla, only MATL were sites of significant K+-NPPase activity. The specificity of the cytochemical localization of Na+,K+-ATPase at reactive sites in rat and rabbit kidney medulla was demonstrated by K+-dependence of reaction product deposition, localization of reaction product (precipitated phosphate hydrolyzed from NPP) to the cytoplasmic side of basolateral plasma membranes, insensitivity of the reaction to inhibitors of nonspecific alkaline phosphatase, and, in the glycoside-sensitive rabbit kidney, substantial inhibition of staining by ouabain. The observed pattern of distribution of the sodium transport enzyme in kidney medulla is particularly relevant to current models for urine concentration. The presence of substantial Na+,K+-ATPase in MATL is consistent with the putative role of this segment as the driving force for the countercurrent multiplication system in the outer medulla. The absence of significant activity in inner medullary ATL and DTL, however, implies that interstitial solute accumulation in this region probably occurs by passive processes. The localization of significant Na+,K+-ATPase in outer medullary DTL of long loop nephrons in the rat suggests that solute addition in this segment may occur in part by an active salt secretory mechanism that could ultimately contribute to the generation of inner medullary interstitial hypertonicity and urine concentration.     

Ultrastructural and kinetic studies on uteroglobin secretion in the uterus and oviduct of the pseudopregnant rabbit

The intracellular localization of uteroglobin, a progesterone-induced protein, was studied in uterus and oviduct by means of immunoelectron microscopy with the protein A-gold technique. In the uterus, uteroglobin was synthesized in the columnar epithelium of the endometrium where most of the cells were immunoreactive. The protein was localized mainly in small secretory granules which were seen in the process of release into the uterine lumen. The luminal microvilli were also heavily stained. In the oviduct, the secretory cells contained large immunoreactive granules at the apical zone, some of which were observed while discharging into the lumen. Within these secretory granules, uteroglobin accumulated particularly in lens-shaped patches at the periphery of the granules. In vitro kinetic studies on the secretion of newly synthesized uteroglobin indicated that the ability to store uteroglobin is greater in the oviduct than in the uterus; however, the rate of uteroglobin secretion is greater in the uterus than in the oviduct. Thus, there appears to be a good correlation between the microscopic and the functional observations.     

Immunocytochemical localization and lectin-binding properties of the 22 kDa secretory protein from rat ventral prostate

The distribution of the 22 kDa secretory protein from rat ventral prostate was studied by light and electron microscopic immunocytochemistry. An anti-22 kDa protein antiserum was raised in rabbits and its specificity was tested by Western blotting. With the immunofluorescence technique, the 22 kDa protein was detected in the luminal secretions and intracellular apical granules of the ventral prostate. No reaction was observed in the seminal vesicle or dorsolateral prostate. After castration, no intracellular immunoreactivity was detected in ventral prostate, although positively labeled secretory material was retained within the acinar lumen. Restoration of normal intracellular staining pattern was incomplete after 5 daily testosterone injections. At the ultrastructural level, labeling was confined to apical secretory granules and condensing vacuoles. The 22 kDa protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose was shown to bind intensely to wheat germ agglutinin (WGA) but only faintly to Concanavalin A. This protein was thus demonstrated to contain N-acetylglucosamine residues. Accordingly, on tissue sections, WGA reacted intensely with condensing vacuoles and secretory granules.     

Glucocorticoid-dependent uteroglobin synthesis and uteroglobulin mRNA levels in rabbit lung explants cultured in vitro

In rabbit lung explants cultured in vitro in a synthetic medium, the synthesis of the protein uteroglobin decayed progressively becoming virtually undetectable between 24-48 h of culture. Addition of glucocorticoids to the medium maintained the synthesis of uteroglobin. This glucocorticoid effect was dose-dependent with optima at about 0.1 microM and 1 microM for dexamethasone and cortisol respectively. Estradiol, progesterone, triiodothyronine, insulin or 10% calf serum added to the medium were ineffective in maintaining uteroglobin synthesis. Actinomycin D (10 micrograms/ml) added to the medium inhibited the effect of cortisol on uteroglobin synthesis. After 24 h of culture, both the relative levels of uteroglobin mRNA, measured by molecular hybridization, and uteroglobin synthesis were correlatively higher (up to 10-fold) in glucocorticoid-treated than in control explants.     

Ultrastructural localization of carbonyl reductase in mouse lung

Summary The immunocytochemical localization of tetrameric carbonyl reductase in the mouse lung was determined by an electron-microscopical immunogold procedure using monospecific antibodies against the enzyme. The labelling of carbonyl reductase was observed within the mitochondria of the ciliated and non-ciliated cells of the bronchioles and the type II alveolar pneumocytes, and the density of labelling in the non-ciliated cells was higher than those in the other cells. No significant labelling was detected over other compartments of the epithelial cells. The labelling was undetectable in the type I alveolar cells, alveolar macrophages and connective tissue cells of the lung. These results clearly indicate the localization of carbonyl reductase to the mitochondrial matrix of these epithelial cells, of which the non-ciliated bronchiolar cells contained particularly high amounts of the enzyme.