Interaction of rabbit sperm and egg

Summary The localization of proteoglycans in the predentin of the rat incisor was investigated by ultrastructural histochemistry. Ruthenium red stained the cell coat of the odontoblasts as well as intracellular vesicles. There was also a staining of the extracellular matrix, but not of collagen fibers in the predentin. Treatment with the enzyme hyaluronidase prior to staining with ruthenium red abolished the staining of the vesicles and the extracellular matrix but not that of the cell coat. Bismuth nitrate and phosphotungstic acid gave similar staining of odontoblast vesicles and extracellular matrix. It is likely that the stained structures contain proteoglycans. The importance of these proteoglycans and their ultrastructural localization are discussed in relation to intracellular transport and the calcification process.     

Pinocytosis as a select marker of ramified microglia in vivo and in vitro

Based on previous observations in tissue culture, we investigated pinocytotic activity as a potential cell marker for brain microglia. This functional activity was assessed in three different preparations derived from rat: primary cultures of mixed cerebral cortical cells, tissue slabs of whole cerebrum, and cultures of isolated or enriched microglial cells. Each preparation was incubated with the fluorescent dye lucifer yellow as a soluble tracer and then processed for light microscopy. Under the conditions utilized, ramified microglia specifically exhibited differentially high pinocytotic labeling in all cases; the dye was mainly localized within the cell somata, where it was sequestered in pinocytotic vesicles. In each preparation, the identity of the labeled cell population was confirmed as microglia through immunohistochemical staining with the monoclonal antibody (MAb) OX-42, a specific microglial marker. Therefore, pinocytotic labeling is proposed as a select cell marker for microglia, which may be extremely useful in the identification and study of ramified microglial cells.     

Ultrastructural evidence for dendritic release of acetylcholinesterase in the rat substantia nigra

Morphological evidence for dendritic secretion of acetylcholinesterase (AChE) in rat substantia nigra--a physiologically known phenomenon--was searched by means of a modified cytochemical method devised for fine localization of AChE activity at the electron microscopic level. DAB precipitate was observed in cluster of small vesicles in contact with the plasma membrane and in the extracellular space in the vicinity of the vesicles. Single coated or uncoated large vesicles filled with stained material were found in the cytoplasm of the dendrites at distance from or in contact with the plasma membrane. Immunoperoxidase staining with specific anti-serum against rat AChE gave similar localization of AChE. These results suggest that AChE is released from the dendrites of the nigral neurons by a process of vesicular exocytosis and captured by endocytosis. The relation of this process to a putative release from the smooth endoplasmic reticulum remains to be elucidated.     

Further studies on the ultrastructural localization of a magnesium dependant neutral ATPase in arteries

Summary A further analysis of the ultrastructural localization of a Mg⁺⁺ dependant neutral ATPase in arteries (thoracic aorta and basilar artery) has been performed in light of recent findings concerning the use of differential fixation and pitfalls in the standard Wachstein-Meisel (W-M) technique. The localization of reaction product was documented following fixation in 5% and 10% formaldehyde and 5% glutaraldehyde, and following incubation in the standard W-M media with ATP, AMP, -glycerophosphate as substrates. These results were compared to the localization using a modified W-M medium with ATP in which the lead ion concentration was reduced to 1.8 mM. Using the standard W-M procedure, formalin fixation gave a more intense but also a more diffuse (both intra- and extracellular) precipitate of reaction product than glutaraldehyde. The localization to cell structure remained the same in both cases, namely to the outer cell membrane, within its invaginations and in pinocytotic vesicles of both endothelial and smooth muscle cells. Following incubation in a medium with lower lead ion concentration, less extracellular precipitate was found and following glutaraldehyde fixation, very sparse precipitate of reaction product was localized to the cell membrane and its invaginations, often on the cytoplasmic side. The reduction of extracellular precipitate following pre-incubation in 5 mM cystein was believed to be due to inhibition of an unspecific alkaline phosphatase and phosphomonoesterase which had diffused out of the cell following fixation. Cysteine had no effect on the ATPase of the vascular wall. The significance of these results was discussed in light of previous studies on blood vessels and newer insights into this technique.     

Membrane-associated carbonic anhydrase activity in the kidney of CA II-deficient mice.

Carbonic anhydrase II-deficient mice offer a possibility to study the localization along the nephron of membrane-associated carbonic anhydrase (CA) activity without interference from the cytoplasmic enzyme. We studied the localization of CA in kidneys from CA II-deficient and control mice by immunocytochemistry (CA II) and histochemistry. Cytoplasmic staining was found in convoluted proximal tubule, thick limb of Henle, and principal and intercalated cells of collecting duct in the control animals but was absent in the CA II-deficient mice. In cells with cytoplasmic staining the cell nuclei were stained. Intense histochemical activity was associated with apical and basolateral membranes of convoluted proximal tubule, first part of thin limb, thick limb, and basolateral membranes of late distal tubule. In collecting ducts of control animals, the basolateral cell membranes of intercalated cells were the only clearly stained membranes. In CA II-deficient animals one type of intercalated cell was stained most intensely at the apical membranes and another only at the basolateral. We suggest that the former corresponds to Type A intercalated cells secreting H+ ions to the luminal side and the latter to Type B cells secreting H+ ions to the basolateral side.     

Golden hamster myoid cells during active and inactive states of spermatogenesis: correlation of testosterone levels with structure

Myoid cells were examined quantitatively in adult golden hamsters with active spermatogenesis and compared with hamsters in which the testes were regressed due to a modification in the light-dark cycle. A detailed morphometric study was undertaken utilizing animals previously examined. The cell-surface area and volumes of most organelles were not significantly different in animals which were gonadally active as compared with regressed animals. A slight, but significant, increase in nuclear volume (31%) and a slight, but significant, decrease (28%) in cell volume were recorded for regressed animals. The total volume of pinocytotic vesicles was increased dramatically (approximately threefold) in active animals in comparison with inactive animals (P less than 0.01), indicating that an increase in non-specific transport across the myoid cell is associated with spermatogenic activity. Intravascularly injected horseradish peroxidase was capable of entering pinocytotic vesicles in both active and inactive animals. Plasma luteinizing hormone (LH) as well as plasma and testicular testosterone levels were weakly (r = 0.64, 0.68, and 0.65, respectively), but significantly (P less than 0.05), correlated with cell size. Plasma and testicular testosterone were correlated with the total volume of pinocytotic vesicles (r = 0.74 and 0.68, respectively). The data indicate that although the rat myoid cell possesses receptors for testosterone, there are few structural manifestations of the hamster myoid cell that correlate well with testosterone levels. Thus, the hamster myoid cell differs from two other hormone-responsive somatic cells in the testis, the Sertoli cell and the Leydig cell, that show dramatic structural alterations with changes in gonadal activity and striking correlations of structural features with functional measures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of a new method of antibody-enzyme conjugation with maleimide derivative for immunohistochemistry: hepatocellular production, interstitial tissue distribution, and renal cell reabsorption of plasma albumin in guinea pig

A new sophisticated method for enzyme-antibody conjugation developed in quantitative solid-phase enzyme immunoassay was revealed to be an applicable method for immunohistochemistry; one that offered several advantages over present methods. Using a new maleimide derivative as a coupling reagent, monomeric conjugate of horseradish peroxidase and Fab' antibody was easily prepared with high efficiency and reproducibility. Nonspecific staining was greatly reduced in the presence of this monomeric conjugate. Since the enzyme activity and antigen-binding activity were well preserved in the conjugate, the reaction was strong enough to analyze the antigen localization in intracellular organelles or in interstitial tissue space by both light and electron microscopy. The fate of plasma albumin was investigated in liver, skin, and kidney using the new method with rabbit anti-guinea pig albumin antibody, and satisfactory results were obtained. In the liver, the reaction products were observed in the rough and smooth endoplasmic reticulum and the Golgi apparatus in hepatocytes, which confirmed the synthesis of plasma albumin in hepatocytes of guinea pig. In a study of the distribution of albumin, reaction products were seen in the intercellular space of the epidermis, along the basement membranes of epidermis and of proximal convoluted tubules in kidney, and among the collagen fibers in interstitial tissue, particularly at papillary dermis, suggesting the wide distribution of plasma albumin in interstitial extravascular tissue spaces. In addition, positive reaction was obtained in the apical vesicles and the lysosomes of the proximal convoluted tubules and in the pinocytotic vesicles of the basal cells of epidermis, suggesting the reabsorption and destruction of albumin in the kidney and the skin.     

Antidiuretic hormone response in the amphibian urinary bladder: time course of cytochalasin-induced vacuole formation, an ultrastructural study employing ruthenium red

Cytochalasin is known to inhibit the antidiuretic hormone-induced hydro-osmotic response (bulk water flow) in the amphibian urinary bladder without altering hormone-stimulated diffusional water permeability or short-circuit current. In addition, histological studies have shown that the mold metabolite induces the formation of large intracellular vacuoles or lakes in the epithelial cells. We report here a transmission electron microscopic time-course study which indicates that during the early phases of the ADH response cytochalasin causes the formation of numerous multivesicular bodies or aggregates derived from individual basolateral pinocytotic vesicles. Because of their apparent hypertonic nature, the vesicles, as well as the vesicular aggregates, accumulate water during hormone-stimulated hydro-osmotic flow. As a result, the multivesicular bodies dilate and fuse to form the large intracellular lakes characteristic of cytochalasin treatment in the presence of both an applied osmotic gradient and vasopressin. In the presence of mucosal ruthenium red, the luminal glycocalyx was heavily stained with this tracer. At no time, however, even in the presence of hormone, was there any evidence for the uptake of this dye at the apical epithelial border. In the presence of serosal ruthenium red, the lateral intercellular spaces, basolateral pinocytotic vesicles, basal lamina, and collagen, as well as other subepithelial structures, were ruthenium positive. With cytochalasin D, vasopressin, and serosal ruthenium red, both the pinocytotic vesicles and the multivesicular bodies demonstrated an apparent membrane associated ruthenium positive coat. The tracer data indicates that the basolateral pinocytotic vesicles, increased by the presence of hormone, are indeed endocytotic in nature. The mucopolysaccharide coat associated with these structures may be involved in ionic and/or fluid transport.     

A highly fluorescent simultaneous azo dye technique for demonstration of nonspecific alkaline phosphatase activity.

We describe a fluorescent histochemical technique for detection of nonspecific alkaline phosphatase (APase) in cells. The technique utilizes standard azo dye chemistry with naphthol AS-MX phosphate as substrate and fast red TR as the diazonium salt. The reaction product is a highly fluorescent red precipitate. Pre-implantation mouse embryos were used to establish optimal fixation and staining protocols and the specificity and sensitivity of the method. Fixation was in 4% paraformaldehyde for 1 hr, as glutaraldehyde induced autofluorescence of the cells. Maximal discriminable staining was detected after 15-20 min in the stain solution. The stain solution itself proved to be non-fluorescent, thus allowing visual observation of the progress of the staining reaction by fluorescence microscopy in its presence. To test the specificity of this fluorescent APase stain, a variety of cell types of known APase reactivity were stained by this protocol. Mouse lymphocytes and STO fibroblasts were negative, whereas F9 teratocarcinoma cells, intestinal epithelial cells, and rat fetal primordial germ cells were all found to be highly positive for APase activity, in agreement with published results on APase localization in these cells.     

Structural modulation of the surface and cytoplasm of oocytes during vitellogenesis in the lobster,Homarus americanus. An electron microscope-protein tracer study

The present investigation describes the ultrastructural changes which occur at the surface and in the cytoplasm of developing oocytes of the lobster, Homarus americanus, during vitellogenesis. The immature oocytes showed no surface specializations of the oolemma and no pinocytotic activity was observed. Horseradish peroxidase (HRP) tracer studies showed penetration of the tracer into the perivitelline space, but no uptake by the oocytes. The surfaces of oocytes examined during vitellogenesis, when yolk protein accumulation was maximal, exhibited numerous microvilli that projected into the perivitelline space, often appearing to be embedded in the follicular cell mass. In addition, the plasma membrane of vitellogenic oocytes contained many pinocytotic pits frequently situated at the bases of microvilli. The perivitelline space was engorged with electrondense material which appeared similar to that contained in pinocytotic structures of the oocytes. Vitellogenic oocytes incubated in HRP showed uptake of tracer reaction product by the coated pits and vesicles of the oolemma. Aggregation and subsequent fusion of these vesicles into large multivesicular bodies of ingested material were also observed in vitellogenic oocytes. Animals artificially induced to undergo vitellogenesis exhibited modulations of oocyte ultrastructure similar to those of normal vitellogenesis, notably, pinocytotic incorporation of extra-oocytic material and hypertrophy of oocyte surface microvilli. This study supports the hypothesis for a dual source of yolk protein in the American lobster.     

Trypanosoma cruzi: ultrastructural, cytochemical and freeze-fracture studies of protein uptake.

Epimastigotes and trypomastigotes of Trypanosoma cruzi, obtained from liquid cultures, have vesicles and multivesicular structures in their cytoplasm. Horseradish peroxidase (HRP) was used as a tracer to study the uptake of protein by these two forms. In epimastogotes HRP is ingested by a process of pinocytosis which occurs through the cytostome. Trypomastigotes do not have a cytostome, and pinocytosis occurs through the flagellar pocket region. The pinocytotic vesicles can fuse with each other to form large multivesicular structures that are more abundant in epimastigotes than in trypomastigotes. The cell membrane as well as the membranes of the pinocytotic vesicles and the large multivesicular structure have carbohydrates, as detected by the periodic acid-thiosemicarbazide-silver proteinate technique. Intramembranous particles were observed by using the freeze-fracture technique. The cell membrane has many particles, whereas the membranes of the vesicles and multivesicular structure have few or no particles.     

Rat skeletal muscle membrane associated carbonic anhydrase is 39-kDa, glycosylated, GPI-anchored CA IV.

Sarcolemmal membrane vesicle preparations from white and red muscles of rat were found to contain a carbonic anhydrase which was indistinguishable from carbonic anhydrase IV from rat lung. This isozyme appears to account for all of the carbonic anhydrase activity in the sarcolemmal vesicle preparations. Digestion of 39-kDa CA IV with endoglycosidase F reduced the Mr to 36 kDa, suggesting that it contains one N-linked oligosaccharide. Treatment of sarcolemmal vesicles with phosphatidylinositol-specific phospholipase C released all of the activity, indicating that the enzyme is anchored to membranes by a phosphatidylinositol-glycan linkage. White muscle sarcoplasmic reticulum vesicles also contain a small amount of 39-kDa CA IV-type enzyme. A 52-kDa polypeptide in sarcoplasmic reticulum membranes cross-reacts with anti-human CA II and anti-rat CA II antisera, but does not bind to the sulfonamide affinity column. This cross-reacting polypeptide has no detectable CA activity.     

Pinocytosis in the root cells of a salt-accumulating halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis> and its possible involvement in chloride transport

Ultrastructure of root cells in salt-accumulating halophyte Suaeda altissima (L.) Pall. was examined with transmission electron microscopy. Plants were grown hydroponically on nutrient media containing 3, 50, 250, and 500 mM NaCl. Some plants were exposed to hypersomotic salt shock by an abrupt increase in NaCl concentration from 50 to 400 mM. Growing S. altissima plants at high NaCl concentrations induced the formation of type 1 pinocytotic structures in root cells. Type 1 structures appeared as pinocytotic invaginations of two membranes, the plasmalemma and tonoplast. These invaginations into vacuoles gave rise to freely ‘floating’ multivesicular bodies (MVB) enclosed by a double membrane layer. The pinocytotic invaginations and MVB contained the plasmalemma-derived vesicles and membranes of endosome origin. The hyperosmotic salt shock led to formation of type 2 and type 3 pinocytotic structures. The type 2 structures were formed as pinocytotic invaginations of the tonoplast and gave rise to MVB in vacuoles. Unlike type 1 MVB, the type 2 MVB had only one enclosing membrane, the tonoplast. The type 3 structures appeared as the plasmalemma-derived vesicles located in the periplasmic space. The cytochemical electron-microscopy method was applied to determine the intracellular Cl^? localization. This method, based on sedimentation of electron-dense AgCl granules in tissues treated with silver nitrate, showed that the pinocytotic structures of all types contain Cl^? ions. The presence of Cl^? in pinocytotic structures implies the involvement of these structures in Cl^? transport between the apoplast, cytoplasm, and the vacuole.     

Ultrahistochemical localization of adenylate cyclase activity in the electric organ ofTorpedo marmorata

Summary The lead pyrophosphate precipitation technique was used to visualize adenylate cyclase activity with the electron microscope in unfixed electric organ and synapto-somes ofTorpedo marmorata, with special attention to presynaptic membranes. Specificity of the deposition of reaction product was ensured by using 5′-adenylyl imidodiphosphate as substrate and 5′-guanylyl imidodiphosphate and sodium fluoride as activators. Under suitable conditions a reaction product was deposited on the Schwann cell, on presynaptic vesicles, on the inner side of membranes of cisternae and on glycogen granules of the presynaptic region of the endplate. In some cases, a precipitate was also found on postsynaptic membranes of the synaptic cleft and on mitochondria. In isolated synaptosomes localization of the reaction product was identical with that of minced tissue. However, most strikingly, on presynaptic membranes no precipitate was ever found, neither in pieces of electric organ nor in isolated synaptosomes. Furthermore, the extended membrane system of the postsynaptic region of the electroplax remained always free of leed pyrophosphate precipitate.     

Histochemical demonstration of rat liver glycogen phosphorylase activity with iron (Fe++)

Summary A new histochemical method for light microscopic demonstration of liver glycogen phosphorylase activity has been introduced in this study.The method demonstrates phosphorylase activity by precipitating phosphate ions, liberated in the reaction catalyzed by the enzyme, with Fe⁺⁺ present in the incubating medium. The precipitate is visualized as ferrous sulphide.The new glycogen, formed in the same reaction, can also be demonstrated in this method after staining with iodine.The lobular localization of the reaction products obtained according to this method in the liver, corresponds well to that obtained according to earlier methods for the demonstration of only new-formed glycogen.     

Cytochemical localization of plasma membrane ATPase activity in plant cells

Summary The cytochemical localization of ATPase activity has been investigated in maize root cells using both lead and cerium-based capture methods. With both methods, staining at the plasma membrane was observed in all cells of the root, although the precipitate obtained with cerium was more uniform and granular than that with lead. Controls using no substrate or no magnesium, -glycerophosphate to replace ATP, vanadate or boiled tissue generally showed little or no staining. However, biochemical studies on purified plasma membrane fractions showed that ATPase activity was markedly inhibited by fixation, particularly by glutaraldehyde, and also by lead and cerium ions. Non-enzymic hydrolysis of ATP by cerium was greater than that by lead. The value and limitations of these procedures for the localization of plasma membrane H⁺-ATPase activity are summarized in relation to previous criticisms of these methods.Abbreviations DTT dithiothreitol - EDTA ethylene diaminetetraacetic acid - GP B-glycerophosphate - PCMBS p-chloromercuribenzene sulphonic acid - PMSF phenylmethylsulphonyl fluoride     

Ultrastructural localisation of alkaline phosphatase in the intertubular tissue of the testis in the domestic fowl

Alkaline phosphatase activity in the intertubular tissue of the testes of the domestic fowl was examined using an ultracytochemical technique based on the lead capture method. In the interstitial tissue, the Leydig cells, transitional cells and the fibroblasts displayed enzyme activity on their cell membranes. Vacuoles located in the transitional cells were lined by reaction products of enzyme activity, whereas the vacuoles representing extracted lipid droplets and present mainly in the Leydig cells were free of enzyme activity. In the peritubular tissue the cell processes of fibroblasts showed enzyme activity on the cell membranes and in pinocytotic vesicles. Cell processes lying adjacent to blood vessels showed pronounced activity. In the blood vessel itself some activity was present in the basement membrane and the endothelium. The surface of the red blood cell showed moderate activity. The possible role of alkaline phosphatase in the transfer of hormone from the Leydig cells to the seminiferous tubules and from the seminiferous tubules to the interstitium is discussed. The myoid cells and their processes were devoid of enzyme activity.     

A comparison of the fine-structural localization of nucleoside phosphatase activity in large intracranial blood vessels and the thoracic aorta of rabbits

Summary Nucleoside phosphatase activity was localized in rabbit intracranial blood vessels, namely cerebral and basilar arteries and veins, and in the thoracic aorta using the electron microscope. In the intracranial vessels the same ultrastructural localization of reaction product was found when ATP or ADP was used as substrate in a modified Wachstein-Meisel procedure using Mg ions as the enzyme activator. No reaction product was seen when using AMP or -glycero-phosphate as substrates, or in controls without any substrate. Reaction product was sparsely localized within cell membrane invaginations on all sides of endothelial and, to a lesser extent, of smooth muscle cells. Pinocytotic vesicles occasionally contained reaction product. The greatest amount of lead phosphate (reaction product) precipitate was found in the basement membranes of both endothelial and smooth muscle cells, particularly intense in the former case. A diffuse precipitate of reaction product was observed in the cytoplasm of adventitial fibroblasts.The thoracic aorta demonstrated the same localization with the following exceptions: generally greater concentrations of reaction product were found using ATP as substrate, than in the corresponding intracranial vessels; interendothelial gaps and the cell membrane invaginations of these gaps were completely filled with reaction product; there was no specific localization of reaction product in the basement membranes; and reaction product could also be demonstrated using AMP and glycero-phosphate as substrates. Acid phosphatase activity was localized in lysosomes in endothelial and smooth muscle cells from both types of vessels. The differences in enzyme localization between aorta and intracranial vessels were discussed, particularly in light of the differences in nucleoside phosphatase activity and transport functions between brain and somatic capillaries.     

Microfilament bundles and the control of pinocytotic vesicle distribution at the surfaces of normal and transformed fibroblasts

The surfaces of human WI38 and hamster embryo fibroblasts, and those of their SV40-transformed derivatives (WI38-VA13, wt/HEF, and ts A28/HEF), were freeze-etched and thin-sectioned in situ, to study the effects of transformation-related changes in microfilament bundles on plasma membrane architecture. Quantitative electron microscopic analysis showed that pinocytotic vesicles on the non-transformed cell surface frequently (48–63% of the samples) were arranged in ordered linear or fusiform aggregates delineated by longitudinal surface ridges and furrows. These longitudinal structures appeared to result from interactions between microfilament bundles and the plasmalemma. Transformed cell surfaces did not have many longitudinal microfilament-bundle impressions, and most of their pinocytotic vesicle aggregates (70–80%) were irregular in shape and composed of randomly scattered vesicles. Microfilament bundles present in both types of transformants were reduced significantly in thickness (p < 0.001). These changes were temperature sensitive in the ts A28/HEF system. The data strongly suggest that the distribution of pinocytotic vesicles at the fibroblast surface is influenced by the disposition of cortical microfilament bundles, and that this control mechanism is disrupted during cellular transformation.     

Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease

The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with A beta in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to A beta in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular A beta released by the regulated secretory pathway, but CA074Me had no effect on A beta released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal beta-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate beta-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as beta-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce A beta in AD.