Immunocytochemical localization of aminopeptidase N on the cell surface of isolated porcine thyroid follicles

Summary The ultrastructural location of aminopeptidase N on the cell surface of isolated porcine thyroid follicle cells was studied with immunocytochemistry using antibodies against intestinal aminopeptidase N and protein A-colloidal gold. Gold particles, indicating immunoreactivity, were selectively attached to the apical cell surface. Occasionally, there was a sparse labelling of the basal cell surface. In follicles kept at 4° C most gold particles at the apical cell surface appeared as clusters, with each gold particle situated at a constant distance of about 20 nm from the membrane surface. The gold particles were concentrated on the membranes of microvilli, in comparison to the smooth (intermicrovillar) portions of the apical plasma membrane. In follicles incubated at 37° C for 5–180 min gold particles were slowly internalized by predominantly smooth-surfaced micropinocytic vesicles and subsequently appeared in colloid droplets and lysosomes. Gold particles were not observed in Golgi cisternae. TSH did not appear to influence the rate of internalization. TSH-induced pseudopods were unlabelled.Our electron-microscopic observations confirm previous immunofluorescence-microscopic evidence that aminopeptidase N is selectively expressed in the apical plasma membrane domain in the thyroid follicle cell. Furthermore, aminopeptidase N appears to be distributed in microdomains within the apical plasma membrane. Earlier indications of molecular differences between the pseudopod membrane and the apical plasma membrane proper are further emphasized.This study was supported by Grant No 12X-537 from the Swedish Medical Research Council     

Immunocytochemical localization of insulin- and somatostatin-like material in human breast tumors

Four types of human breast lesions and C3H mouse mammary adenocarcinomas (type A) were examined for the immunocytochemical localization of cells containing hormone-like substances. Insulin- or somatostatin-like immunoreactive material was observed in scattered single cells and nests of tumor cells in seven of eight infiltrating duct carcinomas, and in the majority of tumor cells from an anaplastic carcinoma. A few somatostatin-immunoreactive cells were observed in only one of seven fibroadenomas studied. No immunoreactive cells were observed in mouse adenocarcinomas or in human breast dysplasias. These results suggest that cells with hormone-like immunoreactivity may be a common feature in two types of malignant human breast tumors.     

Immunocytochemical localization of interferons in human trophoblast populations.

It is known that Interferon (IFN) is present in normal body fluids and tissues during pregnancy. Using an immunohistochemical technique and a panel of monoclonal antibodies we have localized IFN-alpha, -beta and -gamma directly on formalin-fixed paraffin-embedded normal human placentae at different stages of pregnancy and in the hydatidiform mole. The results show that IFNs is mostly localized in villous syncytiotrophoblast and in extravillous interstitial-trophoblast. No reactivity was observed in villous cytotrophoblast or in cytotrophoblast cell columns. The most intense staining was observed for IFN-alpha and -beta, while IFN-gamma was rather weak. There is then a gradual diminution in IFN reactivity with increasing gestation age being almost imperceptible at term. These results suggest that IFN may deploy antiviral, immunomodulator and differentiation activities during normal human pregnancy.     

Immunocytochemical localization of lactoferrin in human neutrophils

Summary The subcellular localization of lactoferrin in human neutrophils was studied by an electron-microscopic immunoperoxidase method. This molecule was detected in small granules of blood polymorphonuclear leukocytes. A morphometrical analysis showed that there was no significant difference in the mean size between lactoferrin-positive and myeloperoxidase-negative granules. In contrast, the mean size of myeloperoxidase-positive granules was significantly larger than that of lactoferrin-positive granules. This indicates that lactoferrin is contained in the myeloperoxidase-negative, secondary, granules of human neutrophils. In immature bone marrow mononuclear neutrophils, lactoferrin was present in cytoplasmic granules of somewhat larger size than lactoferrin-positive granules of polymorphonuclear leucocytes. A morphometrical study showed that the mean size of lactoferrin-positive granules was significantly greater in immature bone marrow cells than in polymorphonuclear leucocytes. This indicates that lactoferrin-positive granules decrease in size as the cells mature. Besides cytoplasmic granules, lactoferrin was demonstrated in the Golgi complex and a part of the rough endoplasmic reticulum of immature bone marrow neutrophils, probably myelocytes and early metamyelocytes. These results show that lactoferrin is synthesized and packed into secondary granules in immature bone marrow neutrophils and therefore that the secondary granules are a type of secretory granule.     

Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme.

Aminopeptidase P (EC 3.4.11.9) was solubilized from pig kidney membranes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and then purified by a combination of anion-exchange and hydrophobic-interaction chromatographies. Contaminating peptidase activities were removed by selective affinity chromatography. The purified enzyme was apparently homogeneous on SDS/PAGE with an Mr of 91,000. Enzymic deglycosylation revealed that aminopeptidase P is a glycoprotein, with up to 25% by weight of the protein being due to the presence of N-linked sugars. The phospholipase-solubilized aminopeptidase P was recognized by an antiserum to the cross-reacting determinant (CRD) characteristic of the glycosyl-phosphatidylinositol anchor. This recognition was abolished by mild acid treatment or deamination with HNO2, indicating that the CRD was due exclusively to the inositol 1,2-cyclic phosphate ring epitope generated by the action of PI-PLC. The activity of aminopeptidase P was inhibited by chelating agents and was stimulated by Mn2+ or Co2+ ions, confirming the metallo-enzyme nature of this peptidase. Selective inhibitors of other aminopeptidases (actinonin, amastatin, bestatin and puromycin) had little or no inhibitory effect.     

Subcellular fractionation and subcellular localization of aminopeptidase N in the rabbit enterocytes

Summary A fast and easy procedure is proposed for preparing concomitantly from the same sample of intestinal mucosa of A⁺ rabbits, four fractions high enriched in the brush-border and basolateral plasma membrane domains, rough endoplasmic reticulum, and smooth endoplasmic reticulum plus Golgi apparatus membranes, respectively. This is the first time the technique of flow fluorometry has been applied to characterize the brush-border and basolateral membrane fractions using polyclonal or monoclonal antibodies against antigens common to or specific for these two plasma membrane domains. This technique definitely proves the presence of aminopeptidase in at least 60% of the basolateral membrane vesicles, where its level is about 4.5% of that in the brush-border membrane vesicles. The endoglycosidase H-sensitive intermediate of glycosylation of aminopeptidase N in the steady state is accumulated in both the rough and smooth endoplasmic reticulum membranes. Although the rough membrane is more extensive it contains only about 40% of this transient form.     

Immunocytochemical localization of elastase 1 in human pancreas

By light and electron microscopic immunocytochemistry the distribution is described of human pancreatic elastase 1 (E1) during ontogenesis, in adults, in cases of acute and chronic panceatitis, acute pancreatic ischaemia as well as pancreatic tumours. E1-positive cells were first detected in ductal sprouts in the 14th gestational week. Complete acini expressing E1 could be found from the 17th to the 20th week of gestation onwards. Scattered distinct E1-positive epithelia could be found in the ducts of fetal and adult pancreas. By immunoelectron microscopy, E1 was localized in rough endoplasmic reticulum, condensing vacuoles, zymogen granules of acinar epithelia and in acinar lumina. E1 appeared to be distributed homogeneously in zymogen granules. As specific markers of acinar cells, both monoclonal antibodies under study identified heterotopic pancreatic acini in peribiliar glands of the liver and also helped to visualize different damage patterns in pancreatitis. The acinar epithelia surrounding acute lipolytic necroses initially reacted more intensely with the E1-antibodies than undamaged pancreatic tissue. In acute ischaemia, acinar cells which are dissociated from intercalated ducts lost their immunocytochemical reactivity for E1. Pancreatic parenchyma involved in advanced acute pancreatitis as well as in chronic inflammation was detected only weakly by both E1-antibodies. However, atrophic lobules in post-inflammatory scars were stained more intensely by the E1-antibodies than normal parenchyma. Pancreatic tumours (adenomas, adenocarcinomas, solid-cystic tumours and islet cell tumours) were not labelled by these antibodies.     

Immunocytochemical localization of testosterone in human hepatocellular carcinoma

Summary A study of 10 hepatoma patients was carried out for the detection and localization of oestrogen, progesterone, testosterone, and their corresponding binding sites in liver tumour cells using immunoperoxidase and immunofluorescent techniques on histopathological sections and cultured monolayers. None of the hepatomas was positive for progesterone and its receptor; one was positive for oestrogen and its receptor; five were positive for testosterone and its receptor. Oestrogen, testosterone and their respective receptors localized predominantly to the cytoplasm of hepatoma cells. The results obtained in this investigation reveal an association between testosterone and human liver cancer which may be useful in the selection and monitoring of liver cancer patients to future hormonal therapy.     

Immunomicroscopic localization of aminopeptidase N in the pig enterocyte. Implications for the route of intracellular transport

The subcellular localization of aminopeptidase N (EC 3.4.11.2) in the pig enterocyte was investigated by immunofluorescence and immunoelectron microscopy (immunogold staining). By indirect immunofluorescence on either frozen or paraffin-embedded sections, a very intense staining in the microvillar membrane and a weak intracellular staining was demonstrated. No staining was detected in the basolateral membrane. Likewise, the immunogold labelling on Epon-embedded sections was concentrated in the microvillar membrane, whereas the basolateral membrane did not contain significant amounts of labelling. Labelling was demonstrated in the Golgi apparatus and in a minor fraction of the intracellular smooth vesicles positioned between the Golgi apparatus and the microvillar membrane. These observations are compatible with the view that newly synthesized aminopeptidase N is delivered directly to the microvillar membrane by smooth vesicles having a diameter about 70 to 100 nm and does not pass the basolateral membrane on its way to the brush border membrane.     

Immunocytochemical localization of beta-N-acetyl glucosaminidase in human reproductive organs

Immunocytochemical localization of hexosaminidase activity in human males revealed that the enzyme activity is localized mainly in the Sertoli cells and interstitial tissue of the testis and in the columnar cells of the epididymis. In seminal vesicles, activity was observed around the glandular epithelium in the form of fine granules.     

The human aminopeptidase N gene: isolation,chromosome localization,and DNA polymorphism analysis

Summary DNA encoding the human aminopeptidase N (EC 3.4.11.2) gene (PEPN) was first isolated using rat cDNA probes and then used in Southern analysis of DNA from mouse-human somatic cell hybrids to assign this gene to the long-arm region (q11-qter) of human chromosome 15. This human genomic DNA probe detects a frequent DraIII polymorphism that is a useful marker for human chromosome 15.     

Immunocytochemical localization of type 5 17beta-hydroxysteroid dehydrogenase in human reproductive tissues.

17beta-hydroxysteroid dehydrogenase (17beta-HSD) controls the last step in the formation of all androgens and all estrogens. At least six 17beta-HSD isoenzymes have been identified. The recently cloned Type 5 17beta-HSD transforms 4-dione into testosterone. To gain a better understanding of the role of this enzyme in reproductive tissues, we immunocytochemically localized the enzyme in human male and female reproductive organs. In the ovary of adult premenopausal women (25-40 years of age), immunostaining was found in corpus luteum cells. In the uterus, staining was detected only in the epithelial cells of the endometrium. Immunolabeling was also detected in the mammary gland, a positive reaction being detected in epithelial cells of acini and intralobular ducts as well as in the surrounding stromal cells. In the testis, strong staining was seen in the Leydig cells, and a weak but specific reaction was occasionally detected in Sertoli and germ cells. In the prostate, specific labeling was observed in alveoli and stromal fibroblasts. In alveoli, all the basal cells were generally labeled, whereas the luminal cells exhibited variations in immunoreactivity. In all the reproductive organs examined, specific staining was routinely detected in the walls of blood vessels, including the endothelial cells. These results indicate a cell-specific localization of Type 5 17beta-HSD in the different human reproductive organs, thus suggesting new mechanisms of local androgen and estrogen formation that may play an important physiological role.     

Immunocytochemical localization of oxytocin and neurophysin in human corpora lutea

Corpora lutea, corpora albicantia, and ovarian stroma from normal human premenopausal ovaries were examined for the presence of oxytocin and neurophysin by using highly specific antisera and peroxidase-antiperoxidase light-microscopic immunohistochemistry. Oxytocin and neurophysin immunoreactivity was found in some but not all cells of the corpora lutea obtained on days 19 to 24 of the menstrual cycle. Stromal tissue and corpora albicantia did not give a positive reaction for either of these peptides, and negative results were also obtained with corpora lutea of mid- and term-pregnancy and preovulatory follicles. Specificity of the immunohistochemical reaction was confirmed by immunoabsorption tests. The specific localization of immunoreactive oxytocin and neurophysin in corpora lutea of the human menstrual cycle directly demonstrates the presence of oxytocin- and neurophysin-positive cells within the human corpus luteum.     

Immunocytochemical localization of PTHrP in human and rat salivary glands

Parathyroid hormone-related protein (PTHrP) was isolated from tumours and is thought to represent the main factor responsible for humoral hypercalcaemia, which accompanies neoplastic diseases. At present, the protein is known to reside in multiple tissues and organs of both humans and animals. Our study was aimed at demonstrating the presence of PTHrP in normal salivary glands (parotid and submandibular) of rats and humans. Application of immunocytochemical techniques permitted to document the presence of PTHrP in the human and in the rat salivary glands. In all cases, an intense reaction was observed in intra- and interlobular ducts. In rat salivary glands, PTHrP was also present in cells of mucous acini. In our opinion, the presence of PTHrP in the ducts indicates participation of the protein in electrolyte transport across the epithelial cells. The positive reaction noted in mucous acini of rat salivary glands may indicate accessory role of PTHrP in the secretory processes in the glands.     

Immunocytochemical localization of DJ-1 in human male reproductive tissue

DJ-1 was identified as an activated ras-dependent oncogene product, and was also found to be an infertility-related protein (contraception-associated protein 1; CAP 1) that was reduced in rat spermatozoa treated with ornidazole, one of the endocrine disrupting substances that causes reversible infertility in rats. CAP 1 is present in spermatozoa but is not detectable in the epididymal fluid of fertile rats and appears to be shed from sperm during treatment with ornidazole. To determine the functions of DJ-1 in the human reproductive system as a target protein of endocrine active substances, we identified the localization of DJ-1 in human testis, epididymis, ejaculated spermatozoa, and seminal plasma. DJ-1 was present in cells existing in the seminiferous tubules and Leydig cells. Some strong expressions were observed in Leydig cells and Sertoli cells, suggesting a relation with spermatogenesis via androgen receptor (AR). In ejaculated spermatozoa, DJ-1 existed on the surface of the posterior part of head and the anterior part of the midpiece. DJ-1 was also present on sperm flagella when the antibody penetrated the plasma membrane, suggesting that there are two putative roles in fertilization, one is binding to the egg, and the other is flagella movement. In contrast to previous findings, we detected DJ-1 in seminal plasma of fertile men. These results demonstrate that DJ-1 in human seminal plasma is not only from spermatozoa but also from the testis and epididymis. It is suggested that DJ-1 may play an important and as yet uncharacterized role in spermatogenesis and fertilization in humans.     

Immunocytochemical localization of renin in mouse kidney.

The distribution of renin in mouse kidney was examined in immunohistochemical studies by using an antiserum against pure mouse submaxillary renin and the peroxidase-antiperoxidase (PAP) technique. At antibody dilutions from 1:10(4) to 1:10(6), renin was found in high concentrations in the epitheloid cells of the vasa afferentia and, in lower concentrations, in the wall of some of the vasa efferentia. Renin was also detected in most of the interlobular arteries. Mesangial cells and Goormaghtigh cells were always free of specific staining. At high antiserum concentrations (i.e., dilutions from 1:10(2) to 1:10(4)) specific reaction product was also observed in the apical part of proximal tubule cells. This staining may represent filtered and pinocytozed renin.     

Immunocytochemical localization of the Ca2+-ATPase polypeptide in human platelets

Specific polyclonal antibodies raised against purified human platelet Ca2+-ATPase were used with protein A-gold immunocytochemistry to localize this protein in human platelets. Immunolabeling specifically detected Ca2+-ATPase over the surface connected membrane system (SCS) in sections of paraformaldehyde-fixed, Lowicryl-embedded platelets. The maximum density of label, determined by quantitative morphometric techniques, was observed over electron-dense regions within the SCS which may represent specialized structures for uptake and release of Ca2+. Less intense immunolabeling was observed over cytosol and may represent localization over the dense tubular system (DTS) which was not readily visualized under the processing procedures employed.     

受精蛋白β在人精子表面的免疫组织化学定位

Fertilin is a kind of sperm plasma membrane protein that mimics snake venom protein. It belongs to the ADAMs family of surface proteins that contain a disintegrin and a metalloprotease domain. Fertilin functions in the sperm-egg binding process by connecting the sperm to the egg plasma membrane via a binding site in the disintegrin domain of fertilin beta (HF93). Its localization on the sperm is in the change. In this study, the monoclonal antibody against human fertilin beta was prepared and used to analyze the localization of fertilin beta on capacitated and acrosome-reacted sperm by immunofluorescence and immunoelectron microscopy techniques. The results were as follows: (1) fertilin beta became restricted to the anterior head during the course of capacitation. (2) During the course of acrosome reaction, the expression and localization of fertilin beta changed immensely on the anterior head and restricted to the lateral of posterior head at last. The restrictions of fertilin beta to the anterior head of capacitated sperm of human beings indicated that fertilin beta may be involved in the binding the sperm to the epithelial cells of the oviduct; the restrictions of fertilin beta to the posterior head domain of acrosome-reacted sperm implied its function in sperm-egg binding and fusion.     

Immunocytochemical localization of neuropeptide Y (NPY) in the human hypothalamus

Summary In order to study the distribution of neuropeptide Y-like immunoreactivity in the human hypothalamus, an immunocytochemical localization of this peptide was performed. Using antibodies developed against synthetic porcine neuropeptide Y (NPY), we have been able to localize immunoreactivity in neuronal cell bodies located exclusively in the infundibular nucleus. Immunostained fibers were found in several regions in the hypothalamus with a high concentration in the periventricular areas. Fibers were also found in the neurovascular zone of the median eminence, the pituitary stalk and the posterior pituitary. These results suggest that immunoreactive material related to porcine NPY is present in the human hypothalamus, with a distribution similar to that observed in the rat.     

Immunocytochemical localization of cathepsin D in the rat osteoclast.

We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 micron) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections. At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsin-negative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface. These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.