Localization of luteinizing hormone β-mRNA by in situ hybridization in the sheep pars tuberalis

Summary The localization of luteinizing hormone beta (LH)-mRNA was studied by in situ hybridization in the pars tuberalis of sheep using a homologous sheep double-stranded ³²P-or ³⁵S-cDNA. The labelled cDNA probe detected one mRNA sequence in the pars tuberalis by Northern blot analysis; this sequence was similar to that detected in the pituitary. In situ, the labelling of LH-mRNA in the horizontal and sagittal tissue sections was found throughout the pars tuberalis. This labelling was prevented by adding an excess of cold probe or treating the sections by ribonuclease before in situ hybridization. Controls showed a labelling in the pars distalis, but not in the median eminence, hypothalamus, cerebral cortex and liver sections. Double labelling by using a specific LH-antiserum indicated that the labelling of LH-mRNA appeared more intense in LH-containing cells that were found only in the ventral part of the pars tuberalis. These results suggest that the entire pars tuberalis is able to produce the LH subunit, but that the level of translation greatly varies according to the location of the cells.     

Immunohistochemical characterization of nurse cells in normal human thymus

Summary Lymphoepithelial complexes known as thymic nurse cells (TNC) have been isolated and described in the thymus of several animal species including man. Most of the investigations on TNC have been carried out in enzymatically digested thymuses in which TNC were isolated by differential sedimentation. In the present study we demonstrate TNC in immunohistochemically stained sections of human thymus as ring-shaped cells completely enclosing thymocytes and localized not only in the cortex, but also at the corticomedullary junction where they have not been previously described. TNC expressed epithelial markers [low and high molecular weight keratins identified by 35H11 and 34E12 monoclonal antibodies, a cortical antigen shared with neuroectodermal neoplasms recognized by the GE2 monoclonal antibody, and tissue polypeptide antigen (TPA:B₁)], class II histocompatibility antigens (HLA-DR), and thymosin ₁. Double staining experiments with the nuclear proliferation-associated antigen Ki-67 and the cortical epithelium marker GE2 showed that most thymocytes enclosed in these cortical TNC were not proliferating. The antigens expressed by TNC indicate that not only cortical, but also medullary epithelial cells are part of the TNC system. The possible role of TNC in the education and maturation of thymocytes is discussed.     

Detection of mRNA and hnRNA using a digoxigenin labelled cDNA probe byin situ hybridization on frozen tissue sections

Summary A full-length cDNA clone encoding the constant region of T cell receptor chain was labelled by random priming DNA with digoxigenin-dUTP. The probe was used to estimate the relative amount of the receptor chain mRNA byin situ hybridization on frozen sections from human thymus and lymph nodes. The hybridization was visualized in blue using an anti-digoxigenin antibody conjugated with alkaline phosphatase and a subsequent enzyme-catalysed colour reaction. The distributions of the signal in tissue sections were as expected. Moreover, labelled cells showed hybrids both in the cytoplasm and in the nucleus, and strongly and weakly stained cells were clearly distinguishable. The results indicate that this method ofin situ hybridization should be useful in the detection of specific mRNA in frozen sections.     

The proliferation of adrenal medullary cells in newborn and adult mice

Summary The proliferative activity of newborn and adult mouse adrenal medullary cells was determined with light and electron microscopic autoradiography. The H³ thymidine labelling index of 2 weeks old mice adrenal medullary cells was about 9.4 % and declined to less than 1 % in adult mice. In electron microscopic autoradiography labelled norepinephrine as well as epinephrine cells could be seen. Only in 1 and 2 weeks old mice some morphologically undifferentiated cells were visible. In formaldehyde induced fluorescence combined with light microscopic autoradiography the fluorescence intensities of labelled and unlabelled medullary cells were measured. On average the fluorescence intensity of labelled cells was lower than that of unlabelled cells. The differences could be explained by a higher number of autoradiographic silver grains laying on the cytoplasm of labelled cells. These results give evidence that fully differentiated adrenal medullary cells are capable of division.This study was supported by Jubiläumsfonds der Österreichischen Nationalbank grant No. 818     

Quantitation of movement of the phosphoryl group during catalytic transfer in the arginine kinase reaction: 31P relaxation measurements on enzyme-bound equilibrium mixtures

³¹P nuclear spin relaxation measurements have been made on enzyme-bound equilibrium mixtures of lobster-muscle arginine kinase in the presence of substituent activating paramagnetic cation Co(II) (in place of Mg(II)), i.e., on samples in which the reaction, ECoATParginine ECoADPP-arginine, is in progress. The results have been analyzed on the basis of a previously published theory (Nageswara Rao, B.D. (1995) J. Magn. Reson., B108, 289–293) to determine the structural changes in the reaction complex accompanying phosphoryl transfer. The analysis enables the determination of the change in the Co(II)-³¹P (-P(ATP)) vector as the transferable phosphoryl group moves over and attaches to arginine to form P-arginine. It is shown that the Co(II)-³¹P distance of 3.0 Å, representing direct coordination of Co(II) to -P(ATP), changes to 4.0 Å when P-arginine is formed in the enzyme-bound reaction complex. This elongation of the Co(II)-³¹P vector implies an excursion of at least 1.0 Å for the itinerant phosphoryl group on the surface of the enzyme.     

Biphasic Modulation of GABAA Receptor Binding by Steroids Suggests Functional Correlates

Neuroactive steroids and other positive modulators of GABA_A receptors showed regional variation in both the efficacy and potency for modulation of [³⁵S]TBPS binding to rat brain membrane homogenates, with biphasic concentration-dependence. GABA present in the binding assays prevented the enhancement phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GABA_A receptors, expressed in insect cell line Sf9 using baculovirus, enhancement by steroids of [³⁵S]TBPS binding was sensitive to the presence of the 2 subunit and the nature of the subunit (122S > 12, 62, 622S, and 62). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are consistent with a three-state model in which the receptor mono-liganded by GABA or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for TBPS.     

Quantitative radioautographic study of intracellular localization of calmodulin antagonist,W-7, in Chinese-hamster ovary cells

Summary The purpose of the present study was to analyse quantitatively the localization of calmodulin antagonist, n-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) in CHO-Kl cells. The cultured CHO-Kl cells were labelled with 1 (16.7 M), 2 (33.4 M), 5 (83.5 M) and 10 Ci/ml (167 M) tritiated W-7. Some cells were preincubated in 10, 50 and 100 M unlabelled W-7 for 30 min and then labelled with 2 or 5 Ci/ml tritiated W-7 for 1 h. The cells were doubly fixed in glutaraldehyde and osmium-tetroxide solution, and embedded in Epon. For light-microscopic radioautography, 2 m-thick sections were wet mounted with radioautographic emulsion and exposed for 1 month. The radioautograms showed that large numbers of silver grains were mainly localized in the cytoplasm as well as in the nucleus. Quantitative analysis demonstrated that, in both the cytoplasm and nucleus, the number of silver grains was dependent on the concentration of the administered tritiated W-7 and the number was dramatically decreased by the pretreatment of unlabelled W-7. These results show that, in CHO-Kl cells, the W-7 binding sites are saturable. It is concluded that W-7 may get into CHO-Kl cells and be bound to a specific protein that may be calmodulin protein.     

Pattern of secretion in thymic epithelial cells: Ultrastructural studies of the effect of blockage at various levels

Summary The observation of secretory phenomena in mouse thymic epithelial cells is disappointing since no real secretion image is found. An adequate technique for such a study is to block the secretion pathway and to observe by electron microscopy cells accumulating secretory products. For this purpose, we used three means of blocking secretion: Firstly, since the thymic epithelial cell is regulated by a feedback phenomenon, secretion was blocked by antibodies against thymulin, one of the hormones secreted by these cells. Secondly, colchicine was used to modify the intracellular transport of the secretory product. In both of these types of experiments, electron microscopy showed a great increase in the number of clear vacuoles and their granular contents in epithelial cells. In a third series of experiments, we used monensin at a concentration that blocks the intracellular transport of secretory proteins at the various levels of the Golgi apparatus. In this series, only an increased number of vacuoles was observed, but they appeared devoid of all granular content. It can be concluded that in the thymic epithelial cell, a discrete system of secretion directs the passage of the product, originating in the cisternae of the endoplasmic reticulum, into clear vacuoles, the terminal element of the cellular secretory apparatus.     

Genetic organization of the ovine MHC class II region

To study the class II genes of the major histocompatibility region of the sheep genome, human HLA class II genes corresponding to the known subregions in man (DR, DQ, DP, DO, and DZ) were used for Southern hybridization analysis of sheep DNA and to probe a sheep genomic library. Hybridizing bands were noted for all probes except DP. DQ and and DR appear to be present as multicopy genes, while DR-, DZ-, , and DO -like genes appear to be single copy. All bands detected with the DP probe were also detectable with other chain probes. From eight -bacteriophage clones of a sheep genomic library nine distinct class II genes were identified. These genes were characterized by differential hybridization analysis and restriction mapping. Two genes were DR -like, three DQ-like and four DQ -like. The extensive cross-hybridization observed with chain probes was not seen with chain probes. The results of this study suggest that the major histocompatibility complex class II region of the sheep has a similar genetic organization to that of man, with the provisional exception of the DP subregion.Abbreviation used in this paper OLA ovine major histocompatibility complex     

Co-localization of vimentin and cytokeratins in M-cells of rabbit gut-associated lymphoid tissue (GALT)

Summary The occurrence of cytokeratins, vimentin, and desmin in the dome epithelia and adjacent non-dome epithelia in four locations of gut-associated lymphoid tissues (GALT) of adult and newborn rabbits (Peyer's patches, sacculus rotundus, caecal lymphoid patches and appendix) was studied with monoclonal antibodies, using the indirect immunoperoxidase technique. In all locations investigated in adult animals, antibodies specific for vimentin labelled (1) M-cells, which engulf intraepithelial lymphocytes, (2) columnar epithelial cells at the base of the domes lacking an apparent contact with lymphocytes (immature M-cells), and (3) flat cells, which lie in the lamina propria under the dome epithelium, and which line the basal lamina with thin cytoplasmic processes. In newborn rabbits, columnar epithelial cells resembling the immature M-cells of adults were selectively stained with vimentin antibodies. In M-cells, the strongest immunoreactivity was present in the perinuclear region and close to the pocket membrane, whereas the most apical and most basal parts of the cytoplasm showed no vimentin-immunoreactivity. Enterocytes in the dome epithelium and in the non-dome epithelium were vimentin-negative. M-cells and enterocytes bound antibodies against cytokeratin peptides 18 and 19 in adults and newborn animals. Compared with enterocytes, M-cells showed less intense staining for cytokeratins. Dome epithelia and no-dome epithelia did not contain desmin-immunoreactive cells. The results suggest that vimentin is a sensitive marker for M-cells in rabbit GALT.     

Combined use of radioimagers and radioactive 3′OH DNA nick end labelling to quantify apoptosis in cell lines and tissue sections: applications to virus-induced apoptosis

DNA fragmentation is a key feature of the degradation phase of apoptosis. In this work we have developed an assay, based on radioimager (-IMAGER and -IMAGER) quantification of radioactive nick end labelling (RANEL), which is quantitative, rapid and sensitive to study in vitro and in vivo induced apoptosis. To establish the technique, in vitro apoptosis of T cell lines was induced by stimulation of the Fas receptor; cells were labelled using TdT-mediated [-³³P] dCTP nick end labelling, after which then radioactivity was quantified using a -IMAGER. We have also shown that the RANEL method can be applied to the quantification and visualisation, by -IMAGER analysis, of liver tissue sections from mouse Fas-induced fulminant hepatitis or from Dengue-1 virus infected individuals. Finally, this system has also been used to detect apoptosis induced by rabies virus in Jurkat T cells. These data have established a large field of application for the RANEL assay.     

Thymic nurse cells: morphological study during their isolation from murine thymus

Summary Thymic nurse cells (TNC), which are multicellular complexes composed of epithelial cells and thymocytes, were obtained from C3H-mice thymuses. They were described by means of light and electron microscopy. The morphology of epithelial cells forming isolated TNC compared to that of small tissue fragments obtained by enzymatic digestion revealed that TNC could be derived from all parts of the thymus: cortex, corticomedullary junction and medulla, the cortex being their principal source. This variety of origin, the presence of several epithelial cells inside a single TNC, the presence of non-lymphoid cells, and the various locations of eleaved desmosomes confirmed that their aspect in vitro as round and sealed structures can be considered to be an artifact due to the isolation technique used. Indeed, during this procedure, they are formed by a process of wrapping of the epithelial cytoplasm around the tightly associated thymocytes. All three epithelial cell types: cortical reticular cells, medullary reticular cells, and medullary globular cells can form TNC.A portion of this work was presented at the first Thymus Workshop. Rolduc, Netherlands, April, 1988     

The subcellular distribution of transferrin in rat choroid plexus studied with immunogold labelling of ultracryosections

Summary Choroid plexus epithelium from third ventricle choroid plexus of 2–3-week-old rats was examined for transferrin-like immunoreactivity. In 5 µm paraffin sections most epithelial cells exhibited a pronounced immunoperoxidase staining for transferrin. The ultrastructure of the epithelium in question was examined by conventional electron microscopy. Immunolabelling of ultracryosections with IgG-gold, protein-A gold or protein-A gold—antiprotein-A protein-A gold showed an intense labelling of the basal extracellular space. The lateral intercellular space and the luminal surface showed a more variable labelling; no labelling of the tight junction zone was seen. Intracellularly a distinct labelling of the uptake and disposal pathway (the endosomal—lysosomal system) was observed, but also the synthetic machinery (rough endoplasmic reticulum, stacked Golgi membranes) showed a characteristic labelling. Thus it seems likely that both uptake and synthesis of transferrin occur in choroid plexus epithelial cells.     

Synthesis and secretion of caseins by the mouse mammary gland: Production and characterization of new polyclonal antibodies

Polyclonal antibodies to mouse - and /-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both - and /-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse -lactalbumin. Residual amounts of caseins were located also in the apical surface of epithelial cells surrounding the ducal lumen of virgin mammary gland sections. In contrast to the significant level of -casein in the milk, the amount of this protein compared to - or -caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.     

Lectin binding sites inParamecium tetraurelia cells

Summary Though all three lectins tested (ConA, RCA II, WGA) bound to the entire cell membrane, none bound selectively to the docking site of secretory organelles (trichocysts); the same results were achieved with FITC-conjugates, or, on the EM level, with peroxidase- or gold-labeling. Only WGA triggered the release of trichocysts and none of the lectins tested inhibited AED-induced synchronous exocytosis.When exocytosis was triggered synchronously in the presence of any of these three lectins (FITC-conjugates), the resulting ghosts trapped the FITC-lectins and the cell surface was immediately afterwards studded with regularly spaced dots (corresponding to the ghosts located on the regularly spaced exocytosis sites). These disappeared within about 10 min from the cell surface (thus reflecting ghost internalization with a half life of 3 min) and fluorescent label was then found in 6–10 vacuoles, which are several m in diameter, stain for acid phosphatase and, on the EM level, contain numerous membrane fragments (other-wise not found in this form in digesting vacuoles). We conclude that synchronous massive exocytosis involves lysosomal breakdown rather than reutilization of internalized trichocyst membranes and that these contain lectin binding sites (given the fact free fluorescent probes did not efficiently stain ghosts).Trichocyst contents were analyzed for their lectin binding capacity in situ and on polyacrylamide gels. RCA II yielded intense staining (particularly of tips), while ConA (fluorescence concentrated over bodies) and WGA yielded less staining of trichocyst contents on the light and electron microscopic level. Only ConA- and WGA-staining was inhibitable by an excess of specific sugars, while RCA II binding was not. ConA binding was also confirmed on polyacrylamide gels which also allowed us to assess the rather low degree of glycosylation (1% by comparison with known glycoprotein standards) of the main trichocyst proteins contained in their expandable matrix.Since RCA II binding could be due to its own glycosylation residues we looked for an endogenous lectin. The conjecture was substantiated by the binding of FITC-lactose-albumin (inhibitable by a mixture of glucose-galactose). This preliminary new finding may be important for the elucidation of trichocyst function.Abbreviations AED aminoethyldextran - BSE backscatter electrons - ConA Concanavalin A - DAB 3,3-diaminobenzidine - EM electron microscope - FITC fluorescein-isothiocyanate - kD kiloDalton - ME mercaptoethanol - MIP membrane-intercalate particle - M_r apparent molecular weight - PAGE polyacrylamide-gel-electrophoresis - PAS periodic acid Schiff - pI isoelectric point - POX peroxidase - RCA II Ricinus communis agglutinin II - SDS sodium dodecylsulphate - SEM scanning electron microscope - WGA wheat germ agglutinin     

Galectin-1 is expressed by thymic epithelial cells in myasthenia gravis

Galectin-1, a member of a family of carbohydrate binding proteins, is synthesized by thymic epithelial cells in normal juvenile thymus, and mediates adhesion of immature T cells to thymic epithelium. Because cell adhesion molecules are proposed to play a role in the thymic hyperplasia and neoplasia seen in the autoimmune disease myasthenia gravis, we examined the expression of galectin-1 in myasthenic thymi. We detected abundant galectin-1 expression in thymic epithelial cells in 27 hyperplastic and neoplastic thymi from patients with myasthenia gravis. Primary cultures of neoplastic epithelial cells from a thymoma continued to express galectin-1, and bound immature T cells; T cell binding was inhibited by the addition of the -galactosides lactose and thiodigalactoside, suggesting that galectin-1 on the thymoma cells and a saccharide ligand on the T cells participated in cell-cell adhesion. Expression of galectin-1 by thymic epithelial cells may play a role in the thymic pathology seen in myasthenia gravis.     

Histochemical demonstration of Δ5-3 ß-OHD activity in the granulosa cells of ovarian follicles of immature and mature mice correlated with some ultrastructural observations

Summary A systematic study of ⁵-3-hydroxysteroid dehydrogenase in the granulosa cells of immature and mature mice was made. The histochemical results were compared with the ultrastructural findings on the same cells in an attempt to determine whether the granulosa cells are capable of a steroidogenic role.In newborn and immature mice the granulosa cells of a great amount of follicles demonstrated a moderate or strong histochemical activity. In mature mice the granulosa cells demonstrated a weak or moderate activity normally only in preovulatory follicles and in some other atretic follicles. The granulosa cells of the normal developing follicles did not show such activity. In addition the histological control of numerous parallel sections demonstrated particularly in immature ovaries the presence of a great amount of atretic follicles.In the cytoplasm of the granulosa cells of the follicles in immature ovaries only clusters of lipid droplets with ribosomes were noted; while in the preovulatory follicles of mature animals there started to appear mitochondria with tubular cristae, smooth membranes of the endoplasmic reticulum and irregular lipid droplets. In the obviously atretic follicles several granulosa cells as well as theca interna cells showed numerous lipid droplets and ribosomes together with different degenerating organelles. The granulosa cells of the normal developing follicles showed a well developed Golgi complex, granular endoplasmic reticulum and free ribosomes.The histochemical and ultrastructural findings suggest a steroidogenic role of the granulosa cells only in the larger preovulatory follicles (probably related to an early luteinization of this layer) but this role was not demonstrated in the same cells in normal developing follicles.In addition, since an histochemical positivity was demonstrated also in the granulosa cells of some obviously atretic follicles, it is possible that many of the follicles having granulosa cells filled with lipid droplets and attached ribosomes and histochemically positive might be, in the immature ovaries, in a very precocious stage of atresia.It is to precise for these cells whether a cytoplasm with these two strictly correlated components (lipid droplets and attached ribosomes) and showing an histochemical positivity could carry-on all the biochemical steps involved in steroid biosynthesis or only has only a temporary capability to produce some precursors of steroids.The present results were partially presented to the 56^ème Congrès de l'Association des Anatomistes (Nantes, 4–8/4/1971) and to the 66° Verhandlungen der Anatomischen Gesellschaft (Zagreb, 2/4/1971).     

Heterogeneity of epithelial cells in the human thymus

Summary To evaluate interrelationships among epithelial cells, and between morphology and function in the microenvironment, we studied the ultrastructural morphology of epithelial cells in sections of human thymus from donors aged 2 months to 31 years. Six types of epithelial cells were observed: subcapsular-perivascular (type 1); pale (type 2); intermediate (type 3); dark (type 4); undifferentiated (type 5); and large-medullary (type 6). Cells of types 2, 3 and 4 were found throughout the organ. The type-2 to -4 epithelial cells may represent various stages in a differentiation process. In this, type-2 cells are very active and type-4 cells are possibly degenerating elements. Type-4 cells can also contribute to Hassall's corpuscles. Type-5 cells were located mainly in the cortico-medullary region and showed the morphological characteristics of undifferentiated elements. Type-6 cells were located exclusively in the medulla and displayed characteristics of cellular activity. Small Hassall's corpuscles consisted of type-6 epithelial cells; in larger corpuscles many nuclei of type-6 cells were found. Cells of types 2 and 6 contained tubular structures (diameter approximately 20 nm).Concerning the function of thymus epithelial cells, the features associated with protein synthesis observed in cellular types 2 and 6 make them likely candidates for humoral factor-producing and/or secreting elements. In addition, type-2 and -3 cells in the cortex appear to contribute to a special pattern of epithelium-lymphocyte interaction (thymic nurse cells), as demonstrated by the intracytoplasmic location of lymphocytes in the epithelial cells. The various steps in intrathymic T-cell maturation occur at locations in a microenvironment composed of morphologically distinct epithelial cells.     

Optimal techniques for the immunocytochemical demonstration of beta-thromboglobulin, platelet factor 4, and fibrinogen in the alpha granules of unstimulated platelets

Summary The distribution of -thromboglobulin, platelet factor 4, and fibrinogen in unstimulated platelets was investigated by several immunocytochemical techniques. All three substances were found to be localized in the majority of platelet alpha granules either by immunoperoxidase methods on saponin-treated platelets or by colloidal gold immunoconjugates on frozen thin sections. The optimal conditions for preparing and fixing platelets for immunocytochemistry were also determined. Platelets obtained from blood dripped directly into fixative or anticoagulated blood were compared systematically with respect to shape. Temperature was found to be the most important variable. Immediately fixed platelets were generally disc-shaped, regardless of the temperature of the fixative. Reducing the temperature of blood (stored with anticoagulant) before fixation resulted in more swollen and fewer disc-shaped platelets. However, if the blood was mixed with an anticoagulant and maintained at 37° C for 1 h before fixation, the same number of disc-shaped platelets were present as in samples from blood fixed immediately. The intracellular localization of -thromboglobulin, platelet factor 4, and fibrinogen was consistent regardless of platelet preparatory procedure, but several technical problems were encountered with respect to plasma membrane labelling when control experiments were analysed. Immediately fixed, non-permeabilized platelet plasma membranes were always labelled, no matter which control substances or immunoperoxidase markers were used. However, when platelets were washed by centrifugation, the plasma membranes were negative. Exposure to saponin markedly diminished labelling of the plasma membranes. Optimal techniques for the immunocytochemical demonstration of these alpha granule proteins in platelets are presented in this report.This paper is based in part on theHistochemical Journal Lecture for 1983 given by Dr Bainton at a Symposium on Haematological Cytochemistry in Cambridge on 29 September 1983 at the invitation of the Royal Microscopical Society.