Ultrastructural localization of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium of rat

The effect of ovarian hormones on the activities of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium was studied in immature and ovariectomized rats, using ultracytochemical techniques. Comparative studies were done on normal rats at the luteal phase and on day 14 of pregnancy. Various vaginal cells show different degrees of response to progesterone and diethylstilbestrol (DES) with regard to glucose-6-phosphatase activity. Intense glucose-6-phosphatase activity was observed in the cisternae of granular endoplasmic reticulum (rER), Golgi saccules and vesicles, and nuclear envelope of both basal cells and stromal cells of progesterone treated rats, whereas in the basal cells and stromal cells of DES-treated and control animals the enzyme was totally lacking. Detectable glucose-6-phosphatase activity was also observed, however, in the rER cisternae and Golgi complex of keratohyalin-secreting squamous intermediate cells of the vaginal epithelium of DES-treated rats. Alkaline phosphatase was also found on the limiting membranes of secretory granules of mucocytes in animals at the luteal phase and during pregnancy. DES and progesterone in the doses used did not affect alkaline phosphatase activity in the rat vagina. Overall, progesterone enhances glucose-6-phosphatase activity in basal cells of the rat vagina prior to completion of mucification. Alkaline phosphatase was found in all cells involved in mucin secretion.     

Ultrastructural localization of alkaline phosphatase in the cyanobacteriaCoccochloris peniocytis andAnabaena cylindrica

Summary Histochemical techniques applied at the ultrastructural level have established the periplasmic space as the site of cell bound alkaline phosphatase activity inAnabaena cylindrica andCoccochloris peniocytis. For localization of activity unfixed cells were reacted with calcium nitrate, which acts as the initial capture reagent. After this deposition, the cells were suspended in 2% lead nitrate to convert the calcium phosphate to more electron dense lead phosphate. The majority of cell bound activity appeared to be associated with layer 3 of the cell wall. InA. cylindrica a secondary site of cell bound activity appeared to be in the sheath. Placement in a phosphate free medium caused a substantial increase in the enzyme activity ofA. cylindrica while the activity present in log phase cells ofC. peniocytis was similar to that found in phosphate starved cells.C. peniocytis also secretes the enzyme into the surrounding medium.     

Ultrastructural localization of alkaline phosphatase activity in the intestine of developingRana Catesbeiana

Summary Alkaline phosphatase activity has been localized at the light and electron microscopic levels in the intestine of developing frog,Rana catesbeiana. The intensity of the histochemical reactivity decreases along the intestinal tract. The intracellular localization of the enzymatic activity shows continuous series of organelles loaded with the reaction product from the Golgi zones to the brush border. These results are in agreement with the biochemical observations made on the same material.This work was supported by grants from the France-Quebec agreements (J. Hourdry) and from the Medical Research Council of Canada (J.S. Hugon)     

Ultrastructural localization of alkaline phosphatase in the hypertrophic chondrocyte of the frog

Summary The ultrastructural localization of alkaline phosphatase was studied in the hypertrophic chondrocyte of the frog (Rana temporaria) by incubating sections of glutaraldehyde fixed tissue in a medium containing sodium glycerophosphate and calcium chloride. Control specimens were incubated in substrate free medium.Alkaline phosphatase (orthophosphoric monoester phosphohydrolase) is a hight molecular weight glycoprotein that hydrolyses phosphorylated metabolites much as acid phosphatase does except that its action is optimal at an alkaline pH.The results of this investigation showed that alkaline phosphatase activity was present within the cytoplasm and around the plasma membrane of frog hypertrophic chondrocytes. Although only a small proportion of frog hypertrophic chondrocytes demonstrated enzyme activity, there was evidence that this was concentrated within Golgi lamellae and vesicles leaving other organelles unreactive. The finding of alkaline phosphatase activity within Golgi lamellae of hypertrophic chondrocytes is regarded as unusual although positive reactions within chondrocyte lysosomes have previously been reported (Doty and Schofield, 1976).     

Ultrastructural localization of alkaline phosphatase in the blue-green bacterium Plectonema boryanum.

Histochemical techniques applied at the ultrastructural level have clearly established the periplasmic space as the site of alkaline phosphatase activity in Plectonema boryanum. Considerable enzyme activity is found after the organism is placed in a phosphate-free medium for 5 days. This activity is found only in the cellular fraction of the culture with no activity present in the culture medium. Localization of the site of enzyme activity in cells was investigated by a modification of the method of Costerton. Unfixed cells were reacted with calcium nitrate, which acts as the initial capture reagent. After this deposition, the cells were suspended in 2% lead nitrate to convert the calcium phosphate to more electron-dense lead phosphate. The majority of activity appears associated with layer 3 (periplasmic space) of the cell wall.     

Ultrastructural localization of alkaline phosphatases in rat incisor odontoblasts.

The localization of alkaline phosphatases in dentinogenically active rat incisor odontoblasts was studied by means of subcellular fractionation and electron microscopical histochemistry. Subcellular fractionation revealed the predominant phosphatase activity to be present in the microsome fraction and to a lesser extent in the mitochondrial fraction. Adenosine triphosphate degrading enzyme activity was determined in the presence or absence of (+/-)-6(m-bromophenyl)-5, 6-dihydroimidazo(le) (2,1-b) thiazole oxalate (R 8231). Before the histochemical study, the effects on phosphatase activities by aldehyde fixation were studied by biochemical assay. A method of fixation for optimal preservation of phosphatase activity is presented. Phosphatase electron microscopic histochemistry was performed by using ATP as a substrate and with or without addition of the inhibitor R 82319 Precipitates were seen in the membranes of vesicles present in the odontoblast process and the Golgi region. When there were signs of insufficient fixation, precipitates were also seen in the outer membranes of mitochondria. No phosphatase activity was seen in the cell membrane. ATP degrading enzyme activities mediated by nonspecific alkaline phosphatase (APase) and Ca2+ -adenosine triphosphatase thus have the same morphological localization. This close association is consistent with earlier biochemical studies.     

Ultrastructural localization of alkaline phosphatase in the hypertrophic chondrocyte of the frog.

The ultrastructural localization of alkaline phosphatase was studied in the hypertrophic chondrocyte of the frog (Rana temporaria) by incubating sections of glutaraldehyde fixed tissue in a medium containing sodium beta glycerophosphate and calcium chloride. Control specimens were incubated in substrate free medium. Alkaline phosphatase (orthophosphoric monoester phosphohydrolase) is a high molecular weight glycoprotein that hydrolyses phosphorylated metabolites much as acid phosphatase does except that its action is optimal at an alkaline pH. The results of this investigation showed that alkaline phosphatase activity was present within the cytoplasm and around the plasma membrane of frog hypertrophic chondrocytes. Although only a small proportion of frog hypertrophic chondrocytes demonstrated enzyme activity, there was evidence that this was concentrated within Golgi lamellae and vesicles leaving other organelles unreactive. The finding of alkaline phosphatase activity within Golgi lamellae of hypertrophic chondrocytes is regarded as unusual although postitive reactions within chondrocyte lysosomes have previously been reported (Doty and Schofield, 1976).     

Characterization and localization of alkaline phosphatase activity in rat testes

Alkaline phosphatase activity in extracts of testes of sexually immature (13 days old) and sexually mature rats has been characterized by its heat sensitivity, the extent of inhibition by homoarginine and phenylalanine, and by polyacrylamide gel electrophoresis. The testicular enzyme appears to be a liver-bone-kidney-type alkaline phosphatase. There are no significant differences in the properties of the enzyme from animals of these two ages. Spermatocytes and early spermatids contain very little alkaline phosphatase activity; the specific activity of a nonflagellate germinal cell suspension is only 1/20th that of the whole testis. Since the constant level of activity in immature and mature animals is not consistent with the enzyme activity being present only in late spermatids, we conclude that the majority of the testicular enzyme is present in nongerminal cells. The presence of alkaline phosphatase in plasma membrane purified from testes of adult rats was demonstrated.     

Ultrastructural localization of acid phosphatase in the neurohypophysis of the rat

Summary In the neurohypophysis of the rat, acid phosphatase is located in the lysosomes of pituicytes. These organelles contain lipid droplets which increase in number and size and distend them so that they lose their usual aspect while preserving their phosphatase activity.     

Ultrastructural localization of alkaline phosphatase activity in the intestine of developing Rana catesbeiana.

Alkaline phosphatase activity has been localized at the light and electron microscopic levels in the intestine of developing frog, Rana catesbeiana. The intensity of the histochemical reactivity decreases along the intestinal tract. The intracellular localization of the enzymatic activity shows continuous series of organelles loaded with the reaction product from the Golgi zones to the brush border. These results are in agreement with the biochemical observations made on the same material.     

Fine structural localization of alkaline phosphatase in rat ovum during cleavage

On a submicroscopic level alkaline phosphatase activity was demonstrated by cytochemical methods in all stages of segmenting rat ova under survey, i.e. in the unfertilized and fertilized ovum, in the two-, four- and eight-cell stages and in the blastocyst. The reaction product was present in some cytoplasmic organelles as well as on cell membranes. A considerable number of cytoplasmic organelles with alkaline phosphatase activity was found in all stages from the one-cell up to the eight-cell stage. The reaction product was deposited in the tubules and vesicles of the smooth endoplasmic reticulum, in the nuclear envelope and in the Golgi complex as well. Some multivesicular bodies, autophagic vacuoles and majority of residual bodies out of the secondary lysosomes showed enzymatic activity. In the multicellular stages no significant differences were observed between the individual blastomeres in the incidence and distribution of the alkaline phosphatase activity. On the blastocyst-stage was found a low incidence of enzymatically active cytoplasmic organelles. Alkaline phosphatase activity was demonstrated in some minute vesicles below the cell membrane and in some secondary lysosomes. No essential differences were found between the cells of the embryoblast and the cells of the trophoblast in the incidence of enzymatically active structures. In the one-cell stage the activity of alkaline phosphatase was present on the cell membrane only sporadically, in the two- and four-cell stages enzymatic activity was found in this localization in a third of all specimen. In the eight-cell stage alkaline phosphatase activity was demonstrated on the cell membranes of all blastomeres. In the blastocyst the reaction product was deposited regularly on the membranes of the trophoblastic cells turned towards the zone pellucida, frequently on membranes of mutual tactile cells of the trophoblast and the embryoblast and only sporadically on cell membranes limiting the blastocyst cavity.     

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes

Summary The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytesin vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell—cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.     

Ultrastructural localization of alkaline phosphatase in the eggs of Hydatigera taeniaeformis (Taenia taeniaeformis)

Freshly shed gravid proglottids from a three-month-old infection of Hydatigera taeniaeformis collected from the faecal droppings of infected cats were used for this study. They were treated for transmission electron microscopy (TEM) followed by incubation using the lead precipitate method. Control sections were incubated in a substrate-free medium, a substrate medium containing 1.0 mM sodium fluoride (NaF) (an inhibitor), and the last sections were denatured at 90 degrees C for 1 min prior to incubation. Intensive alkaline phosphatase activity in the embryophoric blocks and the outer embryophoric membrane was revealed. The reaction products were also indicated in the oncospheral membrane. However, no enzyme activity was seen in any other part of the egg. The enzyme was also absent in the control sections. The presence of alkaline phosphatase activity in the outer embryophoric and oncospheral membranes suggested that this enzyme may be involved in carbohydrate metabolism and nutritional absorption, and also may play a role in the transport of nutrients and other substances from the adult to the developing embryo, respectively.     

Ultrastructural localization of acid phosphatase in the posterior pituitary of the dehydrated rat

Summary Acid phosphatase (AcPase) was localized by means of electron-microscopic histochemistry and estimated biochemically in the posterior pituitary of rats deprived of water, given 2% NaCl ad libitum, or given tap water ad libitum over 6 days. Autophagic vacuoles, some of which gave a positive AcPase reaction, often contained neurosecretory granules (NSG) in nerve endings of control animals on tap water. Nerve endings of water-deprived or salt-treated rats were depleted of NSG, but frequently contained dense membranous residual bodies, some of which appeared to enclose microvesicles. Smooth endoplasmic reticulum located in axons and terminals appears to be a source of hydrolytic enzymes for neurohypophysial lysosomes. The total amount of AcPase per posterior lobe increased progressively to 40% above control levels after 6 days of water deprivation or salt administration, and this increase may reflect accelerated production of neuronal components in neurohypophysial cells whose secretory rate has been stimulated by elevated body osmolarity.Supported by the Medical Research Council of Canada.Medical Research Associate of the Medical Research Council of Canada.     

Ultrastructural localization of alkaline phosphatase activity in the normal and osteochondrotic joint cartilage of growing pigs

The present study aimed to describe the ultrastructural localization of alkaline phosphatase (AP) activity in articular-epiphyseal growth cartilage of the commercial pig and the minipig of wild hog ancestry, comparing areas with a normal endochondral ossification with those where the calcification of the matrix is insufficient, as in osteochondrotic cartilage. Intense AP activity was primarily present in the cytoplasm, the plasmalemmae, the long cellular processes and the matrix vesicles budding off from proliferative and hypertrophic chondrocytes in those areas of cartilage where normal calcification appeared. In the osteochondrotic cartilage, the only detectable AP activity was restricted to a few morphologically viable hypertrophic cells in the surroundings of the lesion. The lack of AP activity could partially explain the insufficient calcification of the osteochondrotic cartilage.