Histochemical investigation of the folliculogenesis in the ovary of the Mongolian gerbil (Meriones unguiculatus)

Summary The ovaries of 70 mature Mongolian gerbils (Meriones unguiculatus) were investigated morphologically and enzyme histochemically for the appearance of acid phosphatase, non-specific esterases, succinate dehydrogenase and thiamine pyrophosphatase. In the oocyte two particular enzyme active zones exist depending on the state of development. In young oocytes acid phosphatase, succinate dehydrogenase and thiamine pyrophosphatase can be found only in the perinuclear zone. From the late secondary follicle on, activity of acid phosphatase, succinate dehydrogenase and non-specific esterases can be detected only in a peripheral area of cytoplasm, whereas thiamine pyrophosphatase is present in the entire ooplasm.In the follicular epithelium a different pattern of enzyme distribution suggests a functional differentiation of the epithelial cells during folliculogenesis.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Enzymic markers of thyroid C cells in some rodents.

The paper provides comparative data of the localization of histochemical reactions demonstrating the activities of alpha-glycerophosphate and succinate dehydrogenases, acid phosphatase, non-specific esterases and non-specific acetylcholinesterase in the C cells of thyroids of 26 animals belonging to 5 rodent species. The family Muridae is represented by the Wistar albino rat and albino mouse, the family Microtidae by the bank vole Clethrionomys glareolus (Schreber 1780), the field vole Microtus agrestis L. 1761, and the pine vole Pitymys subterraneus De Selys-Longchamps 1825. The observed enzyme activity differences were most conspicuous on comparing the rat and mouse thyroids and in a much less degree the Microtidae thyroids. Among the histochemical reactions tested that for succinate dehydrogenase proved to be least effective as a C cell marker, alpha-glycerophosphate dehydrogenase being better, and acid phosphatase and non-specific esterases the best (not in the rat thyroid). The reaction for non-specific cholinesterase (with some limitations) gave satisfactory results in the C cells of all animal's thyroids. The present paper continues earlier studies [19] on the morphology of the C cells in thyroid glands of the rodents of the families Muridae and Microtidae and aims at supplementing them with histochemical data of enzymic activities. It deals with enzyme reactions that are employed as C cell markers in Mammals other than Rodents.     

Histochemical observations on the exoerythrocytic malaria parasite Plasmodium berghei in rat liver

Enzyme histochemical methods were performed on sporozoite infected liver tissue of rats in order to gain insight into the nutrition and metabolism of exoerythrocytic forms of Plasmodium berghei. The following enzymes were demonstrated in the hepatocytic stages of the parasites, obtained 41 and 48 h after inoculation of sporozoites: acid phosphatase, cytochrome oxidase, NADH-tetrazolium reductase, succinate dehydrogenase, NAD+ and NADP+ dependent isocitrate dehydrogenase, NADP+-dependent malate dehydrogenase, lactate dehydrogenases, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenases and alpha-glycerol-phosphate dehydrogenase. The results suggest that a conventional Embden-Meyerhoff pathway, pentose phosphate pathway and Krebs' citric acid cycle may in part be present in these exoerythrocytic parasites. Alkaline phosphatase, nucleoside polyphosphatase, 5' nucleotidase, glucose-6-phosphatase, alpha-glucan phosphorylase, NAD+ dependent malate dehydrogenase, amino-peptidase M and non-specific esterases were not detected by our techniques in the parasite. The enzyme distribution of this intrahepatocytic malaria parasite revealed by histochemistry is compared with the enzyme distribution in the other phases of the parasite's life cycle.     

Enzyme cytochemistry of the alveolar sacs and golgian-like cisternae in the ciliate Ichthyophthirius multifiliis

In the cell cortex of the parasitic ciliate Ichthyophthirius multifiliis different kinds of cisternae were observed: the alveolar sacs, thick membrane cisternae and the endoplasmic reticulum. The thick membrane cisternae possess coated dilated rims and sometimes could be observed close to the endoplasmic reticulum. Using cytochemical techniques acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in the thick membrane cisternae and in the alveolar sacs of trophozoites. In the endoplasmic reticulum acid phosphatase activity was not detected and only very small amounts of thiamine pyrophosphatase and nucleoside diphosphatase reaction product were observed. After exit from the host, a reduction in acid phosphatase activity was evident in the alveolar sacs. At theront stage acid phosphatase activity is absent from these structures. However, high thiamine pyrophosphatase and nucleoside diphosphatase activities remain in the alveolar sacs during the whole life cycle. On the other hand, acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in thick membrane cisternae of theronts. Based on the morphological aspects and enzymatic content the thick membrane cisternae of the cell cortex are designated as golgian-like cisternae. The cytochemical results point out a relationship between the alveolar sacs and the Golgi complex.     

Enzyme Cytochemistry of the Alveolar Sacs and Golgian-Like Cisternae in the Ciliate Ichthyophthirius multifiliis

In the cell cortex of the parasitic ciliate Ichthyophthirius multifiliis different kinds of cisternae were observed: the alveolar sacs, thick membrane cisternae and the endoplasmic reticulum. The thick membrane cisternae possess coated dilated rims and sometimes could be observed close to the endoplasmic reticulum. Using cytochemical techniques acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in the thick membrane cisternae and in the alveolar sacs of trosphozoites. In the endoplasmic reticulum acid phosphatase activity was not detected and only very small amounts of thiamine pyrophosphatase and nucleoside diphosphatase reaction product were observed. After exit from the host, a reduction in acid phosphatase activity was evident in the alveolar sacs. At theront stage acid phosphatase activity is absent from these structures. However, high thiamine pyrophosphatase and nucleoside diphosphatase activities remain in the alveolar sacs during the whole life cycle. On the other hand, acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in thick membrane cisternae of theronts. Based on the morphological aspects and enzymatic content the thick membrane cisternae of the cell cortex are designated as golgian-like cisternae. The cytochemical results point out a relationship between the alveolar sacs and the Golgi complex.     

A cytochemical profile of mucus-secreting,ciliated and subcolumnar basal cells of the human cerivical mucous membrane

Summary A range of enzymatic activities in cervical mucus-secreting, ciliated and subcolumnar basal cells were assessed using light and electron microscopic cytochemical techniques. Enzymes detected in all three cell types were those of the tricarboxylic acid cycle, pentose-phosphate and glycolytic pathways, other mitochondrial associated enzymes (NADH and NADPH dehydrogenase), acid phosphatase and non-specific esterase. Mucus-secreting and ciliated cells exhibited thiamine pyrophosphatase and 5 nucleotidase activities while leucine aminopeptidase was most convincingly demonstrated in mucus-secreting cells. Alkaline phosphatase, on the other hand, was detected only in mucus-secreting and subcolumnar basal cells. The profile of enzymatic activities in subcolumnar basal cells closely resembles that of mature lining cells and further supports the hypothesis that these cells differentiate into functioning columnar cells.     

Experimental protein malnutrition in primates: Cytochemical studies on the cerebellum of the squirrel monkey,Saimiri Sciureus

Synopsis The cerebellum of healthy and malnourished squirrel monkeys was studied histopathologically and cytochemically for a number of important enzymes such as phosphatases (acid and alkaline phosphatase, ATPase, thiamine pyrophosphatase), esterases (simple esterase and acetylcholinesterase), dehydrogenases (succinate, malate and isocitrate dehydrogenase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase), monoamine oxidase and phosphorylase. The Purkinje cells, stellate and basket cells were found to be more sensitive to protein malnutrition compared to the other types of cells in the cerebellum. An increase in the number of dark cells with large amounts of ribonucleoprotein complex in the Purkinje cell layer of the extremely malnourished animals sacrificed after 15 weeks on a low protein diet may be significant and may reflect either an abnormal metabolic process or an interruption in the axonal transport of RNA complex. This may also be directly related to a significant reduction in the level of oxidative enzymes, especially those of the tricarboxylic acid cycle, these being the main source of energy stored in ATP. At the same time the level of lysosomal enzymes, which are responsible for the catalysis of the different degradation reactions, is greatly increased and indicates cellular catabolism. The present investigations point to the probability that the neurons adapt to the changed environment by beginning to utilize structural proteins for their basic metabolism.     

Localization of some Hydrolases and Succinate Dehydrogenase in the Sporophyte--gametophyte Junction in Physcomitrium cyathicarpum Mitt.

Three groups of hydrolases, viz., acid and alkaline phophatasesand esterases and a respiratory enzyme, succinate dehydrogenase,have been localized in the zone constituting the sporophyte-gametophytejunction in the moss Physcomitrium cyathicarpum Mitt. (Funariaceae).Increased respiratory and phosphatase activities in the transfercells of the haustorial foot and vaginula implicate these cellsin active transport. Physcomitrium cyathicarpum Mitt., sporophyte-gametophyte junction, haustorial foot, hydrolases, succinate dehydrogenase, transfer cells, active transport     

Histochemistry of hydrolytic enzymes in resting submandibular glands of rabbits

Summary The distribution of several hydrolytic enzymes was investigated in rabbit submandibular glands at both the light and electron microscopical levels. Glands were fixed by either immersion or perfusion fixation with a variety of fixatives containing 1–2% glutaraldehyde and 2–4% formaldehyde in 0.1m cacodylate buffer at pH 7.2. Light microscopically, the acinar cells showed some staining for ATPase, acid phosphatase and nonspecific esterases but showed weak staining for thiamine pyrophosphatase. Acid phosphatase staining occurred most strongly in granular tubule cells. Staining for esteroproteases was confined to the periluminal rims of intercalary and striated ducts. Alkaline phosphatase was very sensitive to glutaraldehyde and was confined to myoepithelial cells.Electron microscopical observations revealed the presence of acid phosphatase reaction product in lysosomes, immature granules and in GERL-like structures, the last being much more conspicuous in the granular tubule cells. ATPase reaction product was localized to the basal and luminal plasma membranes and lumina of both acinar and granular tubule cells. The Golgi complex of these two types of cells exhibited only moderate amounts of reaction product for thiamine pyrophosphatase. Alkaline phosphatase activity, on the other hand, was exclusively localized to myoepithelial cells in their plasma membranes and sometimes in the nuclear envelope.     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Summary Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum and nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.     

Structure and histochemistry of the normal intestine of the fowl. I. The mature absorptive cell

Synopsis The fine structure and cytochemistry of the intestinal epithelial cell of the fowl have been investigated. The fine structure of the mature absorptive cell of the fowl duodenum was very similar to that described for man and other mammals. Minor differences were the thinner microvillous glycocalyx, the unusual length of the cells and their microvilli, and the wide distribution of lysosomal bodies. The membrane-associated enzymes alkaline phosphatase, ATPase (pH 7.2) and leucine naphthylamidase were mainly associated with the brush border; this organelle also gave positive reactions for mucopolysaccharides and phospholipids. No enzyme activities were found in the terminal web.The distribution of lysosomes between the terminal web and the Golgi apparatus was correlated with the granular localization of the lysosomal enzymes acid phosphatase, -glucuronidase and non-specific esterase. The mitochondrial enzyme succinate dehydrogenase was seen to be localized in rod-like dots which marked the distribution of mitochondria in the absorptive cell. The localization of mitochondrial ATPase (pH 9.4) was not clearly demonstrated because of diffusion artifacts. The region of the Golgi apparatus gave a strong reaction for thiamine pyrophosphatase, together with weak reactions for acid and alkaline phosphatases after extensive overincubation.The endoplasmic reticulum-associated enzymes glucose-6-phosphatase and nonspecific esterase were distributed throughout the absorptive cell, with a maximum activity apical to the Golgi apparatus. Additionally, the jejunal absorptive cells showed endoplasmic reticulum-as well as lysosomal-associated -glucuronidase.     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum an nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.     

Enzyme histochemical studies on the pathological changes in human Sertoli cells

Two forms of human Sertoli cell disorders were characterized enzyme histochemically from the testicular biopsy material of infertile and subfertile patients. Sertoli cell asthenia: a slight injury of the Sertoli cell with exfoliation of individual germ cells; marked by the rarefaction of reaction zones of thiamine pyrophosphatase (TPPase) and a decrease in lactate dehydrogenase (LDH). Sertoli cell insufficiency: severe Sertoli cell damage with the formation of a "puff" and a heavy exfoliation of germ cells (dislocation of Sertoli cell nucleus and cytoplasm along with the related germ cells into the lumen of seminiferous tubule); marked by a heterogeneous activity pattern of TPPase, the disappearance of LDH, maintenance of a slightly weakened activity of alkaline phosphatase, and an increase of acid phosphatase. In the case of Sertoli-cell-only syndrome, the high prismatic Sertoli cells showed strong acid phosphatase activity with scattered weak TPPase reaction, whereas the flat or cube-like Sertoli cells exhibited weak acid phosphatase activity with only one small round reaction zone of TPPase in each cell. In addition, the frequency of the occurrence of Sertoli cell asthenia, Sertoli cell insufficiency, and Sertoli-cell-only syndrome is reported, and its correlation with the andrological diseases discussed.     

Über die Entwicklung des Enzymmusters in den neurosekretorischen hypothalamischen Zentren der Ratte

Zusammenfassung Es wird die ontogenetische Entwicklung des Fermentmusters in den neurosekretorischen hypothalamischen Zentren der Ratte (N. paraventricularis und N. supraopticus) vom 1.–49. Tag post partum beschrieben und mit dem färberisch-lichtmikroskopischen Verhalten der Kerngebiete verglichen.Die noch sehr unreifen Ganglienzellen des neugeborenen Tieres enthalten bereits Neurosekret und zeigen eine kräftige Reaktion auf saure Phosphatase und Thiaminpyrophosphatase. Die übrigen Fermentreaktionen fallen noch schwach bis negativ aus. Morphologische Entwicklung und Ausbildung des Neurosekretbildes sind am Ende der 2. Lebenswoche abgeschlossen. NADH-und NADPH-Diaphorase, LDH, Esterasen und alkalische Phosphatase (Kapillardichte) erreichen ihre endgültige Aktivität am Ende der 3. Lebenswoche. Der charakteristische Ausfall der Reaktionen auf saure Phosphatase und Glucose-6-Phosphatdehydrogenase bildet sich im Laufe der 4. Lebenswoche heraus. Die Ursache der zeitlichen Differenzen zwischen den Terminplänen der Entwicklung von Neurosekretbild und Fermentmuster ist unklar.Das Fermentmuster der neurosekretorischen Zentren reifer Tiere zeichnet sich durch hohe Aktivitäten an saurer Phosphatase, Thiaminpyrophosphatase und Glucose-6-phosphat-dehydrogenase aus. Die Bedeutung dieser Enzyme für den spezifischen Stoffwechsel der neurosekretorischen Zellen wird diskutiert. Hinsichtlich der übrigen Fermentaktivitäten unterscheiden sie sich nicht von gewöhnlichen Nervenzellen.

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Summary The ontogenetic development of the enzyme pattern in rat neurosecretory hypothalamic centers (N. paraventricularis and N. supraopticus) from the 1st to the 49th day post partum is described. It is compared with the development as revealed by conventional staining and light microscopy.The still immature neurons of the newborn rat already contain neurosecretory substance and give strong reactions for acid phosphatase and thiamine pyrophosphatase. With other enzyme reactions, weak or negative results are obtained. The morphological development and accumulation of neurosecretory material is completed at the end of the 2nd week after birth. NADH and NADPH diaphorases, LDH, esterases and alkaline phosphatase, however, reach their definitive level of activity at the end of the 3rd week. The characteristic appearance of the acid phosphatase and glucose-6-phosphate dehydrogenase reactions emerges in the course of the 4th week. The cause for the time differences between the development of staining behaviour and enzyme pattern remains to be clarified.The enzyme pattern of the neurosecretory centers of adult animals is characterized by high activities of acid phosphatase, thiamine pyrophosphatase and glucose-6-phosphate dehydrogenase. The possible role of these enzymes in the specific metabolism of neurosecretory cells is discussed. The cells do not seem to differ, though, from normal nerve cells as to the remaining enzyme activities investigated.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Methodological problems in the histochemical demonstration of succinate semialdehyde dehydrogenase activity

Methodological aspects of the histochemical technique for the demonstration of succinate semialdehyde dehydrogenase activity (EC 1.2.1.24) (indicative of the degradative step of gamma-aminobutyric acid catabolism) have been analysed in rat Purkinje neurons, where gamma-aminobutyric acid has been shown to be a neurotransmitter, and in hepatocytes, where it is metabolized. During a histochemical incubation for the enzyme, artefacts of succinate dehydrogenase activity and the 'nothing dehydrogenase' reaction are produced. Inhibition of these artefacts by the addition of two inhibitors, malonate and p-hydroxybenzaldehyde, revealed specific reaction products. Formazan granules, which can be ascribed only to specific succinate semialdehyde dehydrogenase activity, are obtained by adding malonate to the incubation medium in order to inhibit both succinate dehydrogenase activity and nothing dehydrogenase. The formation of these granules is completely inhibited by p-hydroxybenzaldehyde, an inhibitor of succinate semialdehyde dehydrogenase activity. Different levels of succinate semialdehyde dehydrogenase activity were noted in Purkinje neurons. This activity was also found in hepatocytes, mostly in the portal area, but with a lesser degree of intensity and specificity. Indeed, non-specific formazan granules were still produced, because of the 'nothing dehydrogenase' reaction, even in the presence of malonate. Thus, a malonate-insensitive 'nothing dehydrogenase' reaction seems to be present in neural and hepatic tissues.     

Light and electron microscopic studies on the enzyme cytochemistry of leucocytes of eels, Anguilla species

The leucocytes of three anguillid eels were studied using enzyme cytochemistry. Leucocytes were stained for peroxidase, alkaline phosphatase, acid phosphatase, aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, lysozyme, a variety of non-specific esterases, chloroacetate esterase and two proteases. All cells were negative for aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, and β-galactosidase. Very few neutrophils, thought to be mature, and all eosinophils contained peroxidase-positive granules, and some monocytes showed very weak peroxidase staining. All leucocytes lacked alkaline phosphatase, but all cells except lymphocytes and thrombocytes of A. dieffenbachii contained acid phosphatase. Neutrophil acid phosphatase released into phagosomes was associated with Escherischia coli bacteriolysis. Neutrophils also secrete lysozyme and, with monocytes, produce and secrete a variety of esterases. The possible interaction of lysozyme, acid phosphatase and esterases in bacteriolysis is discussed.     

Salivary glands of the cat: A histochemical study

Synopsis The submandibular, sublingual and parotid glands of the cat have been studied. Mucosubstance histochemistry demonstrated acidic mucosubstances with varying properties in the acini. Thiamine pyrophosphatase and nucleoside diphosphatase reaction products were seen with a Golgi-like appearance in acinar cells. Granules of acid phosphatase, -glucuronidase and E600-resistant esterase reaction products, presumably representing lysosomal enzyme activities, were seen in acinar and ductal cells. Diffuse acid phosphatase and -glucuronidase reaction products were seen in central cells of the submandibular acini, and diffuse non-specific esterase reaction product was seen in acinar and ductal cells. Arylamidase reaction product was associated with some acinar cells. Reaction product from a peroxidase technique was seen in demilunar cells of the submandibular acini, in parts of the sublingual acini, in parotid acini, and in ductal cells. Cytochrome oxidase and succinate dehydrogenase reaction products were seen most strongly in striated ducts, whereas NADH- and NADPH-diaphorase reaction products were seen at a high level throughout the ducts.     

The trans-tubular network of the hepatocyte Golgi apparatus is part of the secretory pathway

We have recently demonstrated the presence of sialyltransferase and sialic acid in a trans-tubular network (TTN) continuous with trans Golgi apparatus cisternae of rat liver hepatocytes. Based on these findings, we concluded that this structure, which also exhibited thiamine pyrophosphatase and acid phosphatase activity, is an integral part of the Golgi apparatus and functions in sialylation. In the present study, by comparing the distribution of a major hepatocyte secretory product with that of sialyltransferase, we sought to determine whether the TTN is also part of the secretory pathway. Examination of adjacent serial thin sections labeled for albumin showed its presence throughout the TTN and simultaneously provided new details about the structural complexity of the TTN. Double-immunolabeling with protein A-gold allowed the direct demonstration of albumin throughout the sialyltransferase containing TTN. Additional double staining protocols (combination of preembedding enzyme cytochemistry with postembedding immunolabeling) revealed the presence of albumin in both the thiamine pyrophosphatase and acid phosphatase positive regions of the TTN. These data show that albumin, a nonglycosylated secretory protein, reaches the TTN where terminal glycosylation of glycoproteins occurs. Therefore, it appears that the TTN of rat hepatocytes which functions in terminal glycosylation is also part of the constitutive secretory pathway.     

Effect of thiamine phosphates on the activity of regulatory enzymes of the pyruvate dehydrogenase complex

The effect of thiamine triphosphate (ThTP) and thiamine diphosphate (ThDP) on the activity of rat liver pyruvate dehydrogenase complex regulatory enzymes (kinase and phosphatase) was studied in experiments with isolated enzyme preparations. It is shown that ThDP caused a pronounced activation of pyruvate dehydrogenase phosphatase (Ka is equal to 65.0 nM). ThTP inhibits phosphatase competitively against the substrate--the phosphorylated pyruvate dehydrogenase complex. The both thiamine phosphates inhibit the pyruvate dehydrogenase kinase activity almost similarly in concentrations exceeding 10 microM. The physiological significance of the antagonistic action of ThDP and ThTP on the pyruvate dehydrogenase phosphatase activity is discussed.     

体外冲击波碎石术对兔肝酶活性的影响

采用超微组织化学方法,观察了体外冲击波碎石术(ESWL)对家免肝脏酶活性的影响。实施 ESWL 后,肝细胞的琥珀酸脱氢酶(SDH)和毛细胆管壁上的碱性磷酸酶(ALP)、焦磷酸硫胺素酶(TPPase)反应活性减弱或消失。TPPase 从损伤的肝细胞高尔基体分泌面扁囊、溶酶体样小泡和毛细胆管内溢出,并伴有肝细胞面的质膜上出现了 TPPase 反应产物和形成膜包内凹小泡。结果提示 ESWL 可对肝细胞及毛细胆管的功能和结构有损伤作用。