Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins

We studied the subcellular localization of two major secretory products of adult rat submandibular gland (RSMG), blood group A-reactive mucin glycoprotein and glutamine/glutamic acid-rich protein (GRP), by light and electron microscopic immunocytochemistry. The structure of the major neutral oligosaccharide of the mucin was shown to be: GalNAc alpha 1,3(Fuc alpha 1,2)Gal beta 1,3GalNAc. A mouse monoclonal antibody (1F9) with specificity for blood group A determinants was prepared against the mucin. The antibody recognized a single band of approximately 114 KD on Western blots of RSMG extract. A previously characterized monoclonal antibody (59) against GRP (Mirels et al.: J Biol Chem 262: 7289, 1987) reacted with a doublet of 45-50 KD on Western blots of extraparotid saliva. Immunofluorescence and immunoperoxidase staining of cryostat sections of RSMG with anti-mucin antibodies and anti-GRP antibodies revealed reactivity in acinar cells of the gland. No specific labeling was seen in duct cells of RSMG or in mucous acinar cells of the adjacent sublingual gland. Post-embedding immunogold labeling of thin sections of glutaraldehyde-fixed RSMG with anti-mucin showed strong labeling of the Golgi apparatus and secretory granules of acinar cells. Gold particles were seen mainly over electron-lucent areas of the granules. No labeling occurred over the endoplasmic reticulum. The labeling pattern with the anti-GRP antibodies was similar, except that both electron-dense and -lucent areas of the granules were labeled, and the endoplasmic reticulum was reactive. Double labeling with two different sizes of gold particles showed that both mucin and GRP co-localized in the same granules. Pre-absorption of the antibodies with their respective antigens eliminated immunolabeling of the acinar cells. These antibodies will be useful in studies of cell differentiation in RSMG and of synthesis, processing, and packaging of RSMG secretory products.     

Purification and characterization of a bovine pregnancy-associated glycoprotein

A 67000 Mr bovine pregnancy-associated glycoprotein (bPAG) has been isolated from fetal cotyledons and purified to homogeneity by HPLC. The purification was monitored by a double immunodiffusion test and by RIA in conjunction with an antiserum raised against a crude fraction of placenta-specific antigens. The molecular weight of bPAG was estimated to be 67000 by SDS-PAGE. The isoelectric points (pI) of the four isoforms, determined by high-resolution analytical electrofocusing in polyacrylamide gel, were 4.4, 4.6, 5.2, and 5.4. The carbohydrate content of the bPAG consisted of approximately 10.02 +/- 1.09% neutral sugar and variant amounts of sialic acid (from 0.29 +/- 0.06% in the most basic isoform to 2.1 +/- 0.31% in the most acidic isoform). A specific antiserum was raised against the purified bPAG. A specific RIA showed that the bPAG was antigenically unrelated to BSA, alphafetoprotein (AFP), and human schwangerschafts-spezifischen (pregnancy-specific) beta 1 glycoprotein (SP1). According to some characteristics (e.g. the molecular weight), the purified bPAG may correspond to a form of the pregnancy-specific protein B previously described by Sasser and colleagues (Biol Reprod 1986; 35:936-942).     

Light and electron microscopic immunolocalization of two nuclear antigens in the liver of Pleurodeles waltl using monoclonal antibodies

The distribution of 2 nuclear antigens in the interphase nuclei of liver of Pleurodeles waltl was determined with the help of monoclonal antibodies, using immunofluorescence for light microscopy and indirect immunoperoxidase and immunogold labeling procedures for electron microscopic localization. The antibodies C36/1 and A33/22 label antigens with relative molecular masses of 270 kDa and 80 kDa, and isoelectric points of 7.0 and 6.4, respectively. The liver of urodels is characterized by the presence of a peripheral layer of hematopoietic cells around the parenchymatous tissue formed by typical hepatocytes. The antibody C36/1 labels the nuclei of both types of cells, whereas the antibody A33/22 labels the nuclei of hepatocytes but not those of the peripheral hematopoietic cells. With both these antibodies, labeling, whenever observed, is restricted to fibrillar structures in the interchromatin space, i.e., to peri- and inter-chromatin fibrils; condensed chromatin, nucleoli, and nuclear envelope are not labeled.     

Light and electron microscopic studies of intramitochondrial bodies in bovine adrenocortical cells by proteolytic digestion.

The nature of the intramitochondrial bodies in bovine adrenocortical cells was investigated both light and electron microscopically, by applying enzymatic digestion on paraffin and epon sections. The result that these bodies were extracted completely either by pepsin or by trypsin strengthened the validity of the previous conclusion that their nature is proteinaceous.     

Light and electron microscopic investigations of six types of glandular cells of the bovine adenohypophysis

Summary Twelve bovine adenohypophyses were prepared for light and electron microscopy of the cell types of pars distalis. Correlation between the light and electron microscopy was effected by use of alternate thin and thick sections. Cytological changes in the experimental animals were used as criteria for the identification of six different types of secretory cells.Two types of acidophils, alpha and epsilon cells, are recognized in peripheral area of the pars distalis by light and electron microscopy. The alpha cells contain orangeophilic secretory granules of a maximum diameter of 400–450 m and correspond to ordinary acidophils (STH cells). The second type, epsilon cells, contains larger, fuchsinophilic granules of 600 to 900 m in diameter, increase in number and granulation after pregnancy and thyroidectomy, and are thought to be prolactin cells (LTH cells).Two types of amphophils, zeta and delta 1 cells, were found in the central area of the pars distalis. The zeta cells contain smaller numbers of amphophilic, cored granules (200 m maximum diameter) and based on the comparison with literature on other species of animals, are designated as ACTH cells. The delta 1 cells are round or oval and contain very dense, spherical granules (250–300 m) which are stained red or reddish purple with PAS, aldehyde thionin and PAS-methyl blue methods. They show extreme enlargement and bizarre cytoplasmic appearance after castration and are designated tentatively as LH gonadotrophs or LH cells.Two types of basophils, beta and delta 2 cells, were also identified by correlative light and electron microscopy. The beta cells are polygonal in outline, distributed exclusively in the zona tuberalis and contain large, less dense secretory granules (300–400 m) which are stained selectively with Gomori's aldehyde fuchsin. After thyroidectomy, they lose their secretory granules and are transformed into large, vacuolated thyroidectomy cells. They are therefore, identified as thyrotrophs or TSH cells. The delta 2 cells are round, oval or polygonal in shape and contain basophilic granules ranging from 220 to 300 m in diameter. They show extreme enlargement and vacuolization due to the dilation of endoplasmic reticulum, after castration, and are designated tentatively as FSH gonadotrophs or FSH cells.The investigation reported herein was supported by a Scientific Research Grant (No. 291049) from the Ministry of Education of Japan.     

Light and electron microscopic studies of intramitochondrial bodies in bovine adrenocortical cells by proteolytic digestion

Summary The nature of the intramitochondrial bodies in bovine adrenocortical cells was investigated both light and electron microscopically, by applying enzymatic digestion on paraffin and epon sections. The result that these bodies were extracted completely either by pepsin or by trypsin strengthened the validity of the previous conclusion that their nature is proteinaceous.This work was supported by a Scientific Research Grant, No. 144017, from the Ministry of Education of Japan to Prof. M. Yasuda     

Light and electron microscopical immunolocalization of neuropeptide Y-containing nerves in the hamster gallbladder

Neuropeptide Y (NPY)-containing nerve structures were detected in the Syrian hamster gallbladder by using peroxidase-antiperoxidase immunohistochemical staining and immunoelectron microscopic techniques. In addition, Alcian blue-P.A.S. was used to differentiate two types of surface cells in the epithelium of the gallbladder: the columnar cells and the tuft cells. NPY-immunoreactive varicose nerve fibers were detected in the adventitial layer of the arterioles, precapillary arterioles and around axial venules of mucosal folds; they were not observed in the capillary beds. Since NPY is associated with norepinephrine (NE) in the sympathetic nerves, it could prolong the adrenergic vasoconstrictive effects. We propose that NPY and NE are simultaneously released to restrict the blood flow in the subepithelial capillary beds by increasing vascular resistance thereby perturbing the absorption of water and electrolytes.     

Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis.

A sensitive and specific double-antibody RIA for a bovine pregnancy-associated glycoprotein (bPAG) is described. The limit of detection was 0.2 ng/ml. The assay was specific for bPAG in that pituitary and placental gonadotropic hormones and other placental or serum proteins assayed in serial dilutions did not cross-react. The RIA allowed measurement of bPAG in placental extracts, fetal serum, fetal fluids, and serum or plasma of pregnant cows. About 20% of unbred heifers and nonpregnant cows had detectable levels ranging from 0.30 +/- 0.09 to 0.50 +/- 0.17 ng/ml (mean +/- SD), and 15% of bull sera showed higher concentrations (3.01 +/- 1.73 ng/ml) of bPAG or bPAG-like protein. Variations among animals was observed in fetal serum bPAG concentrations. Bovine PAG was detected in maternal peripheral blood at Day 22 of pregnancy (mean +/- SD, 0.38 +/- 0.13 ng/ml) in some animals and at Day 30 in all pregnant cows. Peripheral serum bPAG levels increased progressively to 3.60 +/- 1.73 ng/ml (mean +/- SD) at Day 30 of pregnancy, to 24.53 +/- 8.81 ng/ml at Day 120, and to 1551.91 +/- 589.68 ng/ml at Day 270. Peak concentration of bPAG was 2462.42 +/- 1017.88 ng/ml and it occurred 1-5 days prior to parturition. After delivery, bPAG concentrations decreased steadily to 499.63 +/- 267.20 ng/ml at Day 14 postpartum (pp), 10.12 +/- 7.84 ng/ml at Day 60 pp, and 1.44 +/- 1.08 ng/ml at Day 90 pp. The undetectable concentration (less than 0.20 ng/ml) was reached by Day 100 +/- 20 pp. An investigation undertaken in Holstein heifers, Holstein cows, and Hereford cows used as recipients for purebred Holstein embryos supplied evidence of the influence of breed of recipient and sex of fetuses on peripheral concentrations of bPAG. A herd of 430 Holstein-Friesian heifers that had received transferred embryos were bled at Day 35 postestrus (pe) for measurement of bPAG. The bPAG was detected in 287 of 430 serum samples analyzed. By rectal palpation performed at Day 45 pe, 267 heifers with detectable levels of bPAG at Day 35 pe were confirmed to be pregnant as were 3 of 143 heifers previously diagnosed as not pregnant by RIA. These results suggest that detection of this placental-specific antigen in the serum could be used as a specific serological method for early pregnancy diagnosis in cattle from 28 days after breeding.     

Elastica-positive material in the atrial endocardium. Light and electron microscopic identification

The elastic layer of the endocardium is studied in various laboratory animals (mouse, rat, rabbit, cat, and dog) and in man. Coarse elastica-positive fibers form a tightly woven layer in the endocardium of the left atrium; the elastic layer consists of loosely arranged delicate fibers in the endocardium of the right atrium. Electron microscopy shows the elastic material to consist of homogeneous elastin (E) and of elastic fiber microfibrils (EFM). Elastic material in the endocardium of the left atrium is mainly formed of E with few EFM present. By contrast, the portion of EFM predominated that of E in elastic fibers from the right atrium, where some elastica-positive fibers even appear as pure bundles of microfibrils. This was also observed in human material obtained from aged individuals (8th decennium). It is concluded that EFM are not only progenitors of E but represent an independent fibrous component of the connective tissue.     

Light and electron microscopic studies of the ventricular wall

Summary Electron microscopic data confirm the results gained with rapid Golgi preparations of adult rodent brains that tanycytes occur in clusters along the lateral wall of the third ventricle. The cytoplasmic matrix of these cells is considerably denser than that of typical ependymal cells. They have filaments and microtubules throughout their cytoplasm along with mitochondria and polysomes. At the surface is a compact group of microvilli which suggest that tanycytes might selectively absorb material from the ventricle.The tanycytes are segregated from neuropil by other tanycyte processes, by neighboring ependymal cells and by astrocytes. Yet there are gaps in this sheath. At these points tanycytes either abut upon or surround nonglial components of the neural fabric.Their cytological features and relations with the neuropil suggest that tanycytes selectively absorb material from the ventricle and release it along the basal process, primarily affecting those segments of neurons immediately adjacent to the tanycyte.Supported by: NINDS Grants 5 R01 NS 09001-02 NEUA, 5T01 NB 5309, and GM 00958, and by the Eleanor Roosevelt Cancer Foundation Research Institute.Acknowledgements: This work was initiated in the Anatomy Department of the Harvard Medical School with facilities provided by Prof. S. L. Palay (U.S. Public Health Service Grant No. NB 05591). Dr. R. B. Wuerker kindly and patiently provided the instruction and orientation to electron microscopy. The major portion of the study was completed in the Neurology Department of the University of Utah with the extremely competent, challenging assistance of Dee Lerdahl, Nina Belgarian, Keith Johnson and Lynn Kendricks.     

Calbindin-D immunolocalization in developing chick thyroid: a light and electron microscopic study

Antiserum to calbindin-D, a 28 KD vitamin D-dependent calcium binding protein, was used to localize the protein immunocytochemically in developing chick thyroid by both light and electron microscopy. The protein first appeared in future follicular cells of developing thyroid tissue from 8-day-old embryos. The number of calbindin-D-containing cells increased rapidly to a near-plateau level at day 10; this concentration was sustained until day 15, and then declined to an undetectable level just before hatching. The protein was distributed throughout organelle-free areas of the follicular cell cytoplasm and extended into the nucleus; it was not present in the follicular colloid. Comparison of the time course of changes in calbindin-D content with known differentiative changes taking place in follicular cells suggests that the protein may function in some yet to be determined mechanism related to normal development of the thyroid.     

Light and electron microscopic studies of crayfish hemocytes

Using light and electron microscopy, three hemocyte types are described in the hemolymph of the crayfish. The coagulocyte comprises 65% of the total hemocyte number and contains medium-sized cytoplasmic granules, abundant dilated rough endoplasmic reticulum, and a highly developed Golgi complex. It rapidly undergoes cytolysis in vitro and participates in coagulation by releasing the contents of its granules to the hemolymph. The granulocyte comprises 31% of the total hemocyte number and is capable of phagocytosis. It contains large, irregularly shaped cytoplasmic granules, a moderately developed Golgi complex, and moderate amounts of non-dilated rough endoplasmic reticulum. During coagulation in vitro, the cell attaches and spreads onto the substratum; this is followed by a slow intracellular granule breakdown and cytolysis. The amebocyte comprises 4% of the total hemocyte number and it is also capable of phagocytosis. It possesses small cytoplasmic granules, many vacuoles, a moderately developed Golgi complex, and large amounts of smooth endoplasmic reticulum. It is distinguished from the other two cell types by being stable and motile in vitro.     

Light and electron microscopic observations on hydrocortisone-induced cleft palate in hamsters.

Palatal histogenesis in hydrocortisone-treated hamster fetuses was studied by both light and electron microscopy. At an early stage in the hydrocortisone-affected fetuses, when the palatal shelves hung vertically on either side of the tongue, necortic changes could be seen in some of the basal epithelial cells which lay adjacent to the fragmented basal lamina. The normal looking cells lay on an intact basal lamina and were attached to the contiguous necrotic cells by desmosomes. With horizontal reorientation of the palatal shelves and their approach to the midline, cellular necrosis and fragmentation of the basal lamina increased. When compared with normal cells, the hydrocortisone-affected ones were seen to be lighter, to contain fewer ribosomes and no lysosomes. At a later stage, when midline palatal fusion was lacking, the epithelium underwent stratification and keratinization while the necrotic debris was removed by mesenchymal macrophages. It appears that the normal process of protein synthesis is inhibited following hydrocortisone administration and that this, in turn, during palatogenesis, disrupts normal cellular differentiation and the integrity of the basal lamina, which are associated with the production of a cleft palate.