Prenatal development and histochemical characteristics of gastrointestinal mucins in sheep fetuses

The object of this study was to describe the prenatal development and histochemical properties of mucins in the sheep gastrointestinal tract. To determine changes in the mucin profile, the sections were stained with specific histochemical stains for carbohydrates. While neutral and mixed mucins were observed in the superficial epithelial cells of the abomasal pyloric region, acidic mucins were detected in the secretory ducts and corpus of the glands. Acidic mucins consisted predominantly of sialomucins. In the duodenal villi, the number of goblet cells containing neutral mucins increased toward the end of gestation, whereas Brunner's glands contained acidic mucins until the 95th day of gestation and both acidic and neutral mucins thereafter. The jejunal goblet cells contained either acidic, neutral, or mixed mucins. Goblet cells containing acidic mucins, which were mainly localized to the ileal crypts and villi, mostly contained sulfated mucins. While villi were observed in the proximal colon until the 115th day of gestation, later the typical crypt structure emerged. During the period in which the villi were found in the proximal colon, the goblet cells containing sulphomucins were predominant, whereas the goblet cells containing sialomucins were predominant after the typical crypt structure was formed. In conclusion, gastrointestinal mucins may be involved in the formation of meconium during the prenatal period, and acidic mucins may contribute to the strength of the intestinal barrier against pathogens and digestive enzymes, as the barrier is not fully functional after birth.     

Quantification of increased MUC5AC expression in airway mucus of smoker using an automated image-based approach

Microscopic analysis of mucus quantity and composition is crucial in research and diagnostics on muco-obstructive diseases. Currently used image-based methods are unable to extract concrete numeric values of mucosal proteins, especially on the expression of the key mucosal proteins MUC5AC and MUC5B. Since their levels increase under pathologic conditions such as extensive exposure to cigarette smoke, it is imperative to quantify them to improve treatment strategies of pulmonary diseases. This study presents a simple, image-based, and high-processing computational method that allows determining the ratio of MUC5AC and MUC5B within the overall airway mucus while providing information on their spatial distribution. The presented pipeline was optimized for automated downstream analysis using a combination of bright field and immunofluorescence imaging suitable for tracheal and bronchial tissue samples, and air–liquid interface (ALI) cell cultures. To validate our approach, we compared tracheal tissue and ALI cell cultures of isolated primary normal human bronchial epithelial cells derived from smokers and nonsmokers. Our data indicated 18-fold higher levels of MUC5AC in submucosal glands of smokers covering about 8% of mucosal areas compared to <1% in nonsmoking individuals, confirming results of previous studies. We further identified a subpopulation of nonsmokers with slightly elevated glandular MUC5AC levels suggesting moderate exposure to second-hand smoke or fine particulate air pollution. Overall, this study demonstrates a novel, user-friendly and freely available tool for digital pathology and the analysis of therapeutic interventions tested in ALI cell cultures.     

Histology of the stomach in the Caucasian squirrel,a scanning electron microscope and immunohistochemical study

This study was performed to investigate the gross anatomy, histochemistry, and immunohistochemistry of the stomach in the Caucasian squirrel. A total of five male squirrels were included. The obtained results showed that the shape of the stomach was acutely crescent and consisted of four regions namely; cardiac, fundus, body, and pylorus. The stomach glandular and its wall was consisted of four layers; serosa, muscularis, submucosa, and mucosa. All part of the mucosa was covered by a simple columnar epithelium. Furthermore, gastric glands were of the simple tubular type. The mucous cells of the fundic and body regions were PAS-positive. In the lower third of the gastric glands, parietal, and chief cells were identified. The number of chief cells was increased toward the pyloric area. Scanning electron microscopy showed that the fundus region contained many plicae and grooves and each plica carried numerous glands opening. In the body region, cells were polygonal and formed network-like structures. In the pyloric region, a mucosal ridge between the body and antrum and a thick mucosal crest on the lesser curvature were identified. Immunohistochemical staining showed that Sox9 was strongly expressed in the fundic region, however, its expression in the body and pylorus was moderate to weak. In conclusion, there are some similarities and differences between the stomach of the Caucasian squirrel and other mammals including rodents. The obtained results may enhance our basic knowledge regarding the feeding habits and digestive system histophysiology of this species.     

Stem cells in the development and differentiation of the human adrenal glands

There are no studies on stem cells (SCs) and development and differentiation (DD) of the human adrenal glands. The SCs in DD of the adrenal glands were herein investigated histochemically and immunohistochemically in 18 human embryonic adrenal glands at gestational week (GW) 7–40. At 7 GW, the adrenal glands were present, and at 7 GW, numerous embryonic SCs (ESCs) are seen to create the adrenal cortex. The ESCs were composed exclusively of small cells with hyperchromatic nuclei without nucleoli. The ESCs were positive for neural cell adhesion molecule, KIT, neuron‐specific enolase, platelet‐derived growth factor receptor‐α, synaptophysin, and MET. They were negative for other SC antigens, including chromogranin, ErbB2, and bcl‐2. They were also negative for lineage antigens, including cytokeratin (CK)7, CK8, CK18, and CK19, carcinoembryonic antigen, carbohydrate antigen 19‐9, epithelial membrane antigen, HepPar1, mucin core apoprotein (MUC)1, MUC2, MUC5AC, and MUC6, and cluster differentiation (CD)3, CD45, CD20, CD34, and CD31. The Ki‐67 labeling index (LI) was high (Ki‐67 LI = around 20%). α‐Fetoprotein was positive in the ESCs and adrenal cells. The ESC was first seen in the periphery of the adrenal cortex at 7–10 GW. The ESC migrates into the inner part of the adrenal cortex. Huge islands of ESC were present near the adrenal, and they appeared to provide the ESC of the adrenal. At 16 GW, adrenal medulla appeared, and the adrenal ESCs were present in the periphery or the cortex, in the cortical parenchyma, corticomedullary junctions, and in the medulla. The adrenal essential architecture was established around 20 GW; however, there were still ESCs. At term, there are a few ESCs. These data suggest that the adrenal glands were created by ESCs. Microsc. Res. Tech., 78:59–64, 2015. © 2014 Wiley Periodicals, Inc.     

Immunocytological detection of salivary mucins (MUC5B) on the mucosal pellicle lining human epithelial buccal cells

The mucosal pellicle is defined as the protein film adsorbed onto oral mucosa. This study aimed at characterizing the ultrastructure of human epithelial buccal cells and localizing salivary mucins MUC5B, a major constituent of the mucosal pellicle. Cells were sampled from the buccal surface and prepared for Transmission Electron Microscopy using high‐pressure freezing/cryosubstitution followed by immunogold labelling of MUC5B. Morphologically, cells were visualized as typical cells of the superficial layer of a squamous nonkeratinized epithelium with a partly degraded plasma membrane. The outer surface of the plasma membrane was lined with a biological material of medium electron density. MUC5B were detected in the extracellular space, and particularly in the vicinity of the plasma membrane, sometimes onto fibrils protruding from the membrane. This area was, therefore, considered as constituting the mucosal pellicle, which appeared as a mixed film of both salivary and epithelial components. The distribution of gold particles suggested that the surface of the pellicle was not uniform, and that the film thickness could reach up to 100 nm. This work showed the feasibility of visualizing and characterizing the mucosal pellicle directly on human epithelial buccal cells sampled in a noninvasive manner. Microsc. Res. Tech. 77:453–457, 2014. © 2014 Wiley Periodicals, Inc.     

Mucin coatings suppress neutrophil adhesion to a polymeric model biomaterial

Following our recent study on the fractionation, characterization, and model adsorption of mucins derived from bovine salivary glands (BSM), porcine stomach scrapings (PGM), and human whole saliva (MG1), we here present a microscopic evaluation of the interactions between mucin-coated substrates and human neutrophils. Our results show that surface-coating with BSM, PGM, and MG1 can be effectively used to suppress the adhesion of neutrophils to a polymeric model biomaterial (Thermanox). Neutrophil morphologies found on Thermanox substrates coated with mucins resemble those observed for nonactivated neutrophils found in circulation. Notably, low neutrophil adhesion can be obtained at a significantly lower coating concentration (0.125 mg/mL) for the compositionally complex MG1 mucin than for the relatively pure BSM and PGM mucins (1 mg/mL). Furthermore, since coating at a low BSM and PGM concentration (0.25 mg/mL) results in higher cell counts and more spread cells than in the high-concentration case, we suggest that dense mucin surface packing is critical for good coating performance. In conclusion, the present study demonstrates how mucins from three different sources, of different compositional and structural status, efficiently can be used to suppress neutrophil adhesion and activation. This finding makes them potent candidates for use as biomaterial coatings.     

Huge clusters of embryonic stem cells in human embryos: A morphologic study

Background: Nothing is known about huge clusters (HC) of embryonic stem cells (ESC) in human fetal organs (HFO). Aim: To know the status of HC‐ESC in HFO. Methods: Morphology and immunohistochemistry (IHC) in 32 HFO of 7–40 gestational weeks (GW). Results: HC‐ESC were seen in many HFO including central nervous system, spinal cords, spine, soft tissue, bone, skin, thyroid, lung, liver, pancreas, gall bladder, extrahepatic bile duct, adrenal, kidney, bladder, foregut, midgut, hindgut, female and male genital organs, and neurons. HC‐ESC's were composed of two populations depending on constituting cells. One were large cells with ample acidophilic cytoplasms with vesicular nuclei and nucleoli. The other were small cells with scant cytoplasm with hyperchromatic nuclei without nucleoli, resembling lymphocytes. The HC‐ESC were frequently showed neuronal differentiation. HC‐ESC were positive for NCAM, synaptophysin, NSE, chromogranin, PDGFRA, AFP, ErbB2, bcl‐2, KIT, MET. They were negative for CD45, CD3, CD20, EMA, CEA, CA19‐9, cytokeratin (CK) 7, CK8, CK18, CK19, MUC1, MUC2, MUC5AC, and MUC6. The mean Ki‐67 labeling index (LI) was 13% ± 7%. HC‐ESC showed a little glycogen but lacked mucins. These HC‐ESC were seen in 7–25 GW, and they were rarely seen in 26–40 GW. Conclusions: The morphology, IHC, and ontogeny of HC‐ESC were described. Microsc. Res. Tech. 77:825–831, 2014. © 2014 Wiley Periodicals, Inc.     

The pars intercerebralis of the locust brain: a developmental and comparative study.

The anterior midline of the brain, also known as the pars intercerebralis, contains the largest collection of neurosecretory cells in the central nervous system of the grasshopper. In this study, we use immunocytochemical, intracellular staining, and histological methods to establish the ontogenies of the various cell types in the brain midline, and show how these cells contribute to the pars intercerebralis of the adult brain. We show that the adult pars intercerebralis develops from three distinct embryonic cell groups: (1) the median neurosecretory cells, which derive from a subset of neuroblasts in the protocerebral hemispheres, and which project axons to the corpora cardiaca; (2) the paired primary commissure pioneers, which derive directly from the mesectoderm of the dorsal median domain and whose axons project to the ventral nerve cord via the midline tract; and (3) the six progeny of the median precursor in the dorsal median domain, which share a common axonal projection with the primary commissure pioneers. Since the adult pars intercerebralis is a fusion product of these independent cellular components, it can only be understood in terms of its origins in the embryonic brain. When the expression pattern of the TERM-1 antigen is compared in subsets of median neurosecretory cells in a wide range of insect orders, the results suggests a common organizational Bauplan for the pars intercerebralis. This hypothesis is supported by the identification of putative homologs of the grasshopper primary commissure pioneers in all these insects.     

A histochemical approach to glycan diversity in the urothelium of pig urinary bladder

Intracellular glycans in the urothelium of urinary bladder of 10 adult male Landrace pigs were characterized in situ by immunohistochemical detection of Muc1 mucin by anti MUC1 from rabbit, conventional histochemical techniques (Periodic‐Acid Schiff, Alcian Blue pH 2.5, High‐Iron Diamine), and binding with 13 lectins (PNA, DBA, RCA‐I, WGA, SBA, BSI‐B4, ConA, AAA, UEA‐I, LTA, LFA, MAA‐II, SNA) combined with chemical and enzymatic pre‐treatments (β‐elimination, desulfation and neuraminidase) to gather reference data for this model animal. Muc1 mucin was detected in the secreting granules of superficial cells and the underlying layer of intermediate cells. The secreting granules in both intermediate cells and superficial cells were rich in carbohydrates, with the oligosaccharidic chains mostly O‐linked to proteins. Glycoproteins were prevailing over glycosaminoglycans (GAGs). In both superficial and intermediate cells sulfated and/or sialylated glycans were present, sulfation decreasing in the deeper layers. Lectin‐binding detected presence of terminal sialic acid linked mostly in α2,6 to GalNAc, Gal terminal or subterminal to sulfates, GalNAc, GlcNAc, and Fuc, mostly linked in α1,6, α1,3 α1,4 and α1,2 to GlcNAc or Gal, but not to lactosamine chains. Except for fucosylation, the oligosaccharidic chains in the glycoproteins of the urothelium of pig urinary bladder were similar to those linked to human MUC1, which is fundamental in cell adhesion and immunological processes in the urothelium. The co‐distribution of Muc1 and saccharidic residues suggests that many of them are linked to the glycoprotein.     

Imaging of intracellular spherical lamellar structures and tissue gross morphology by a focused ion beam/scanning electron microscope (FIB/SEM)

We report the use of a focused ion beam/scanning electron microscope (FIB/SEM) for simultaneous investigation of digestive gland epithelium gross morphology and ultrastructure of multilamellar intracellular structures. Digestive glands of a terrestrial isopod (Porcellio scaber, Isopoda, Crustacea) were examined by FIB/SEM and by transmission electron microscopy (TEM). The results obtained by FIB/SEM and by TEM are comparable and complementary. The FIB/SEM shows the same ultrastructural complexity of multilamellar intracellular structures as indicated by TEM. The term lamellar bodies was used for the multillamellar structures in the digestive glands of P. scaber due to their structural similarity to the lamellar bodies found in vertebrate lungs. Lamellar bodies in digestive glands of different animals vary in their abundance, and number as well as the thickness of concentric lamellae per lamellar body. FIB/SEM revealed a connection between digestive gland gross morphological features and the structure of lamellar bodies. Serial slicing and imaging of cells enables easy identification of the contact between a lamellar body and a lipid droplet. There are frequent reports of multilamellar intracellular structures in different vertebrate as well as invertebrate cells, but laminated cellular structures are still poorly known. The FIB/SEM can significantly contribute to the structural knowledge and is always recommended when a link between gross morphology and ultrastructure is investigated, especially when cells or cellular inclusions have a dynamic nature due to normal, stressed or pathological conditions.     

Morphological tools to evaluate the digestory apparatus in rocky cavy (Kerodon rupestris)

This research describes for the first time the complete morphology of the digestive apparatus of rock cavies. Dissection, light microscopy, and scanning electron microscopy were performed. The oral cavity has: the hard palate without palatine wrinkles and the soft palate; the tongue composed by striated musculature, with presence of vallate, foliated, and fungiform papillae with taste buds and filiform papillae with mechanical function; and, 20 teeth of the hypsodonts type. Esophagus, stomach, small intestine (duodenum, jejunum, and ileum), and the large intestine (cecum, colon, and rectum) are found. The anus is present at the end of the alimentary channel. Organs of digestive tube are composed by four tunics: mucosa, submucosa, muscular, and serosa. The duodenum, jejunum and ileum have villi. Jejunum, ileum and cecum present Lieberkühn crypts. The cecum has mucous glands. Colon and rectum are folded and have goblet cells. Anus presents sebaceous glands. As associated glands it is found the liver with six lobes and gallbladder; a lobulated pancreas; and a pair of each major salivary gland (parotid, mandibular, and sublingual). Parotid glands have serous acini and mandibular and sublingual glands have mucous acini. Pancreas has adenomers. The liver has hepatocytes and portal vein, hepatic artery, and bile duct (portal triad), separated by sinusoids. It is concluded that the digestive apparatus of the rock cavy has variations in the dentition, lingual papillae, and acini of the salivary glands when compared to other rodents. Other variations refer to the well‐developed cecum characteristic of herbivorous behavior.     

Morphology and lectin‐binding sites of pyloric caeca epithelium in normal and GnRH‐treated Atlantic bluefin tuna (Thunnus thynnus) Linnaeus 1758

Mucosal epithelium of pyloric caeca was studied in normal and in GnRH‐treated Atlantic bluefin tuna Thunnus thynnus L., using morphological analysis, conventional and lectin glycohistochemistry. The lining epithelium consisted of columnar (absorptive) cells, goblet cells and intraepithelial leucocytes. The epithelium from normal animals was significantly taller than GnRH‐treated samples. Conventional histochemistry displayed the same staining pattern in normal and hormone‐treated specimens which showed a mixture of neutral and sulphated acidic glycoconjugates in the luminal surface and goblet cells, and neutral glycans in apical granules of enterocytes. Lectin histochemistry revealed a different glycoconjugate pattern in normal and GnRH‐treated tunas. In normal specimens the luminal surface expressed sialoglycoconjugates which bound MAL II, SNA, KOH‐sialidase‐PNA, KOH‐sialidase‐SBA as well as asialoglycans stained with HPA, SBA, GSA I‐B₄, LTA. N‐linked glycans were highlighted by Con A and KOH‐sialidase‐WGA. In GnRH‐treated tunas the luminal surface did not react with SNA, SBA and LTA. The columnar cells of normal tunas bound KOH‐sialisase‐PNA in the apical region, KOH‐sialidase‐PNA, KOH‐sialidase‐DBA, HPA, SBA, KOH‐sialidase‐SBA and KOH‐sialidase‐WGA in apical granules, GSA I‐B₄ and LTA in the supranuclear region. GnRH‐treated specimens showed some columnar cells that stained with KOH‐sialidase‐WGA in the apical granules and with GSA I‐B₄ in the supranuclear region. The goblet cells of normal animals produced mucins positive to PNA, HPA, KOH‐sialidase‐DBA, SBA, GSA II. The latter three binding sites lacked in GnRH‐treated tunas. The results suggest that the mucosal epithelium of Thunnus thynnus L. pyloric caeca expresses a complex glycan pattern that is affected by GnRH‐treatment. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

MRT letter: A novel tegumental gland in female imagoes of the neotropical termite Cornitermes cumulans (Isoptera,termitidae, syntermitinae)

In general, the exocrine glands of social insects are structures involved in the chemical communication associated with social life. Here, we report the discovery of an unknown tegumental gland that is present in the female imagoes of Cornitermes cumulans and occurs next to the well‐developed tergal glands that have previously been described. The tegumental glands release their secretion in the intersegmental membrane and are composed of bicellular units, a secretory cell and a canal cell, that are closely located to the epidermal cells in the inferior part of the eighth and ninth tergites. The ultrastructure of the glandular cells showed abundant smooth endoplasmic reticulum, suggesting that the secretion may be pheromonal, although its function is still unknown. These exocrine structures are facing the tergal glands, and we hypothesized that they act synergistically with the tergal glands to generate short‐range attraction during tandem behavior. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

Epidermal glands in the abdomen of a basal ant Dinoponera lucida (Formicidae: Ponerinae)

The basal ant Dinoponera (Hymenoptera: Ponerinae) has lost the morphologic queen caste so that all females may be potential reproductive individuals, and the nestmate recognition results from cuticular hydrocarbons cues. However, data about the origin of that substance in Ponerinae ants are scarce. This study reports the occurrence of epidermal glands in the abdomen of the ant Dinoponera lucida. In this ant, the epidermis of the abdominal sternites has tall cells with well-developed nucleus contrasting with flattened and collapsed epidermis in the tergites, suggesting a glandular function in the epidermis of the sternites. The possible role of the glandular epidermis in the synthesis of cuticular hydrocarbons for the nestmate recognition is discussed.     

Effect of a 2-month light cycle regimen on testicular parameters of adult Ile-de-France rams.

This experiment was conducted in Ile-de-France adult rams to examine the target point of a 2-month light cycle regimen on seminiferous tubule functions, on intertubular compartment and on Leydig cell parameters. Eight rams were subjected to a 2-month light cycle regimen and were compared to sexually active or inactive rams. In light-treated rams, testis weight was maintained equal or was higher than that of sexually active rams. Both tubular and intertubular tissues were found significantly higher in light-treated than in sexually active rams. The mean ratio of basement membrane area of the seminiferous tubules per Sertoli cells and the daily productions of A1 spermatogonia and of leptotene primary spermatocytes were significantly increased in light-treated rams as compared with sexually active or inactive rams. Meanwhile, the dairy productions of diplotene primary spermatocytes, of round spermatids, of spermatozoa and of the rete testis fluid were not significantly increased in light-treated as compared with sexually active rams but significantly greater than those of sexually inactive rams. Total volume, total numbers, and individual volumes of Leydig cells were at least equal or higher in light-treated than in sexually active rams.     

Light and transmission electron microscopic structure of skin glands and dermal scales of a caecilian amphibian Gegeneophis ramaswamii,with a note on antimicrobial property of skin gland secretion

Amphibian skin secretions contain a variety of bioactive compounds that are involved in diverse roles such as communication, homeostasis, defence against predators, pathogens, and so on. Especially, the caecilian amphibians possess numerous cutaneous glands that produce the secretory material, which facilitate survival in their harsh subterranean environment. Inspite of the fact that India has a fairly abundant distribution of caecilian amphibians, there has hardly been any study on their skin and its secretion. Herein, we describe, using light microscopy and electron microscopy, two types of dermal glands, mucous and granular, in Gegeneophis ramaswamii. The mucous glands are filled with mucous materials. The mucous‐producing cells are located near the periphery. The granular glands are surrounded by myoepithelial cells. A large number of granules of different sizes are present in the lumen of the granular gland. The granule‐producing cells are present near the myoepithelial lining of the gland. There are small flat disk‐like dermal scales in pockets in the transverse ridges of the posterior region of the body. Each pocket contains 1–4 scales of various sizes. Scanning electron microscopic (SEM) study of the skin surface showed numerous funnel‐shaped glandular openings. The antibacterial activity of the skin secretions was revealed in the test against Escherichia coli, Klebsiella pneumoniae, and Aeromonas hydrophila, all gram‐negative bacteria. SEM analyses confirm the membrane damage in bacterial cells on exposure to skin secretions of G. ramaswamii.     

Ultrastructural study of the intracellular behavior of four mineral elements in the lactating mammary gland cells: study using conventional transmission electron microscopy

The effects of parenteral injection of aluminum, indium, gadolinium, or terbium in rats have been previously studied in several organs such as the liver, the kidneys, etc., but never in mammary glands. In this work, we have attempted to study the subcellular localization of these elements after their intraperitoneal administration. Their subsequent effects in the lactating mammary gland cells have also been studied. Our results using conventional transmission electron microscopy have shown that the lysosomes of the mammary glandular epithelial cells are the intracellular site of accumulation of the studied elements. Our results have also show intracellular deteriorations such as an expanded ergastoplasm and altered mitochondria after intraperitoneal injection of aluminum and indium.