Immunocytochemistry of the striated rootlets associated with solitary cilia in human oviductal secretory cells

The human oviduct epithelium is a simple columnar structure that consists primarily of ciliated and secretory cells. Solitary cilia usually extend from the apical cell surface of secretory cells. By injecting crude preparations of striated rootlets into rats, we successfully obtained six monoclonal antibodies (R38, R67, R95, R149, R155, R213) that commonly labeled ciliary rootlets. Using these antibodies, proteins of 205-215 kDa were identified by immunoblotting. Using a clone, R67, we investigated the morphology of the striated rootlets associated with solitary cilia by immunocytochemistry. It was found that the shapes of the rootlets were not simple but varied considerably. The rootlets had branched, radiated, arched, and looped shapes. This is the first report of the rootlets having a variety of shapes. The 205- to 215-kDa antigens identified by the six different antibodies were mostly localized to dark bands of striations, suggesting that they are constitutive components of dark striations of the rootlet.     

Evolutionary conservation of an epitope associated with striated rootlets in different epithelial ciliated cells.

In ciliated cells of metazoa, striated rootlets associated with basal bodies anchor the ciliary apparatus to the cytoskeleton. We have used here a monoclonal antibody against a 175 kDa protein associated with the striated rootlets of quail ciliated cells, to study ciliated cells of different species. In mussel gill epithelium the antibody recognized a protein of 92 kDa which shows a periodic distribution along the striated rootlets. In frog ciliated palate epithelium, two different rootlets are associated with basal bodies, both are decorated and only one protein of 48 kDa is recognized on immunoblot. The antigen is arranged in a helix around the striated rootlets. In rabbit ciliated oviduct epithelium, we detected the presence of very small and thin rootlets which are weakly labeled. We have shown that an epitope associated with the striated rootlets is preserved through evolution although the molecular weight of the peptide varies. We have also observed the appearance of this epitope on protein associated with junctional complexes in rabbit and cytoskeleton component in quail oviduct.     

Cytodifferentiation of striated duct cells and secretory cells of the convoluted granular tubules of the rat submandibular gland.

The structural and functional development of the striated ducts and convoluted granular tubules (CGT) of the rat submandibular gland (SMG) were studied by electron microscopy and alkaline protease chemistry. Development of the SMG was followed from 14 days of gestation through 30 weeks of age. The specialized morphology of the basal aspect of the striated duct cells arises from cellular extensions which are first seen at 20 days of gestation. These processes elongate and intertwine with similar processes from adjacent cells, and as the cells enlarge the processes are compressed together giving the appearance of "infolding" of the basal plasma membrane. Mitochondria migrate to the basal part of the cell and are seen in close relationship to the cellular extensions throughout the development of these cells. Development of the striated duct is complete by one week after birth. The CGT develop from the proximal portions of intralobular striated ducts. At one week after birth, cells of the proximal striated duct demonstrate apical vacuoles. By two weeks after birth these vacuoles are replaced by distinct zymogen-like granules. There is a progressive accumulation of large numbers of secretory granules in the CGT cells as the animals age. However, rough endoplasmic reticulum is a relatively inconspicuous cellular component throughout development. The accumulation of alkaline protease activity in the gland closely parallels the pattern of granule accumulation.     

Morphofunctional characteristics of the striated centrosome rootlets of cultured embryonic pig kidney cells

The analysis of the centrosome structure in PK-cells has shown that the striated rootlets occur in 25-30% of the cells. Most frequently, the centrosome contains 1-2 rootlets, oriented either to the active or to the nonactive centriole. Occasionally, they occur in both the centrioles simultaneously. The presence of striated rootlets does not correlate with the presence of a primary cilium in the centrosome: there occur centrosomes with the primary cilium but without striated rootlets, and conversely: with striated rootlets but without the primary cilium.     

Scanning electron microscopy of human submandibular gland

The fracture surface of human submandibular gland analyzed by scanning electron microscopy is studied here. Acini showed spherical granules of 0.7 +/- 0.28 micron diameter, their most distinctive feature. Some empty, septate cavities found contiguous to serous acini were considered to be mucous acini. Striated ducts had a circular lumen, with microvilli forming prominences. Blebs, some intact and others ruptured, were interpreted as apocrine secretion. The 'separating zone' of the striated cells was distinguishable from the rest of the cell because the structure of the cell was granular whereas the 'separating zone' was fibrillar.     

Prenatal development of the human submandibular gland

The prenatal development of the human submandibular gland has been investigated in 26 fetuses from the 10th week of gestation to full term. At 10-12 weeks, the glandular elements (primitive ducts and acini) were immature and surrounded by a loose mesenchyme. The acinar cell population increased gradually till the age of 32 weeks, and the rate of increase was diminished thereafter. At 16 weeks, intercalated and striated ducts were distinguished and their number increased till the age of 32 weeks when their number seemed to be stabilized. The development of the granular convoluted tubule cells from the proximal segments of striated ducts occupied the later stages of development. They appeared around the age of 20 weeks and proceeded till full term. At birth, the gland appeared devoid of mucous acini and fat cells and the secretory end-pieces were of the serous type. During the second trimester, periodic acid-Schiff- and alcian blue-positive secretory materials appeared in the epithelial cells of both ducts and acini, and in their lumina. This secretory activity was transitory and disappeared around the age of 28 weeks. The possible function of these secretory products is discussed.     

Label-retaining cells in the rat submandibular gland.

To identify stem cells in salivary glands, label-retaining cells (LRCs) were established in rat submandibular glands. Developing and regenerating glands were labeled with bromodeoxyuridine (BrdU). To cause gland regeneration, the glands were injured by duct obstruction. BrdU LRCs were observed in all the parenchymal structures except for the acinus of the glands labeled during regeneration. Among these LRCs, a few, but not many, expressed neither keratin18 (K18; an acinar/duct cell marker) nor alpha-smooth muscle actin (alphaSMA; a myoepithelial cell marker), and thus were putative stem cells. These (K18 and alphaSMA)(neg) LRCs were invariably observed in the intercalated duct and the excretory duct. In the intercalated duct, they were at the proximal end bordering the acinus (the neck of the intercalated duct). Next, to test the above identification, gland extirpation experiments were performed. LRCs were established by labeling developing glands with iododeoxyuridine (IdU) in place of BrdU. Removal of one submandibular gland forced the IdU-LRCs in the remaining gland to divide. They were labeled with chlorodeoxyuridine (CldU). The (K18 and alphaSMA)(neg) LRCs in the neck of the intercalated duct and in the excretory duct did not change in number or in IdU label. The CldU label appeared in these cells and then disappeared. These results indicate that the (K18 and alphaSMA)(neg) LRCs have divided asymmetrically and are thus considered salivary gland stem cells.     

Substructure of solitary cilia in mouse kidney

Summary Recent scanning electron microscopic studies confirm the presence of solitary cilia on most epithelial cells along the mammalian nephron and collecting ducts.By transmission electron microscopy we have found that the axonemata of such cilia consist of a maximal number of 9 doublet and no singlet filaments. 10% of the cross-sectioned cilia contain 9 doublets arranged in a peripheral ring (9+0 pattern). 30 % of the cross-sections contain 8 or 7 doublets in peripheral ring and 1 or 2 doublets in the central region (8+1 and 7+2 patterns). Serial sections and goniometer tilt reveal the central doublets to originate as dislodged peripheral doublets. 60% of the sectioned cilia contain filament numbers between 8 and 4. In patterns of 5 and 4 filaments single microtubules predominate.The functional significance of these atypical cilia is discussed.We are indebted to Prof. B. Afzelius and Prof. Th. Brun for valuable information and discussions during this work. The technical assistance of Miss K. Weltzin, Mr. E. Erichsen, Mr. R. Jensen and Mr. J. Røli is greatly appreciated     

Bovine submandibular gland extracts interact with the human hemostasis factors.

Glycoconjugates were extracted from the bovine submandibular gland and their anticoagulant activity was tested on the blood coagulation of human beings. The methods employed demonstrated that the thromboelastographic parameters were only modified by the highest concentration of glycoconjugates. Activated Partial Thromboplastin Time (aPTT) and Thrombin Time (TT) were affected by dose-coupling response. Prothrombin Time (PT) and Reptilase Time (RT) were not modified.     

Cytomorphological study on human submandibular gland following treatment with secretagogue drugs

Using specimens of human submandibular glands, we have investigated in vitro the morphological modifications induced by clozapine, a dibenzodiazepine derivative that is used in psychotic patients and that provokes hypersalivation, a side-effect of therapy. The effects of the drug, used alone or in combination with carbachol, have been compared with those observed after treatment with drugs acting on specific receptors. To quantify the response to stimulation, we have calculated (with statistical methods) the number of microvilli and microbuds (corresponding to pits seen in images obtained by transmission electron microscopy) per square micrometre of the cytoplasmic surface of the intercellular canaliculi luminal membrane in images obtained by high-resolution scanning electron microscopy. Clozapine, when directly acting on human submandibular specimens, induces a small secretory response in serous cells; this is partially decreased by muscarinic and adrenergic antagonists and by combined incubation with carbachol, thus confirming its behaviour as a partial agonist to muscarinic receptors. We also suggests that the drug acts on the nerve terminals contained within the glandular specimens.This work was funded by MIUR (Italian Ministry for University and Research) and COFIN.     

Subcellular localization of epidermal growth factor in human submandibular gland

Epidermal growth factor in human submandibular gland was localized at the subcellular level by means of an immunogold staining method. Labelling was observed in serous acini and ducts. In the acini, gold particles were found within secretory granules, indicating that the growth factor is released into the saliva through granule exocytosis. In the ductal system, the most intense reactivity was revealed in the principal cells of striated ducts. In these cells, an abundant population of small cytoplasmic vesicles was specifically stained. Immunoreactive vesicles were found both apically and basally, suggesting that ductal cells can release their products not only into the saliva but also into the interstitium.     

Proteomic analysis of human transplanted submandibular gland in patients with epiphora after transplantation

Submandibular gland autotransplantation is effective for treating severe dry eye syndrome. However, more than 40% of patients show epiphora within 3-6 months after treatment. The mechanism underlying the hypersecretion in epiphora remains to be elucidated for developing novel interventions. Since salivary gland secretion is dependent on a variety of proteins, we analyzed the changes in protein expression in transplanted glands of epiphora patients with 2-D gel electrophoresis and electrospray ionization quadrupole/time-of-flight mass spectrometry and evaluated their possible roles in epiphora. There were 23 proteins that showed altered expression in the glands of epiphora patients, 15 being up-expressed and 8 being down-expressed. The expression of secretory proteins was decreased in these glands, including alpha-amylase, cystatin S, SA, and SN. In contrast, cytoskeletal proteins were all up-regulated, including actin and vimentin. Immunofluorescence revealed that the intensity ratio of F-actin in apical and lateral cytoplasm to total F-actin in acini was decreased in the glands of epiphora patients. Carbachol stimulation induced a similar redistribution of F-actin in the control glands. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was increased in both carbachol-stimulated and epiphora glands. Preincubation of submandibular glands with ERK1/2 inhibitors PD98059 or U0126 inhibited carbachol-induced F-actin redistribution. These results indicated that differentially expressed proteins participated in the hypersecretion of transplanted submandibular glands and the redistribution of F-actin might be involved in this hypersecretion in an ERK1/2-dependent manner.     

Depolymerization of microtubules by colcemid induces the formation of elongated and centriole-nonassociated striated rootlets in PtK(2) cells

Striated rootlets are cross-banded structures associated with the basal body, which extends the cilium. To determine whether microtubule dynamics influence the shape and distribution of striated rootlets, we have depolymerized the microtubules by colcemid and observed the rootlets by the immunohistochemical technique with the R4109 antibody that specifically reacts with a 195-kDa protein in the rootlets in PtK(2) cells. In control interphase cells, striated rootlets were observed in various profiles such as fibrillar, branched, or looped shapes and were associated with a pair of centrioles. Treatment with colcemid (0.1 micro g/ml or more) resulted in the elongation and/or structural complication of the centriole-associated rootlets and the organization of intracytoplasmic free rootlets. These changes appeared 6 h after colcemid treatment and became more prominent with time. The changes were reversible and almost disappeared 2 h after removal of the drug. Immunoelectron microscopy confirmed that the R4109 antibody decorated both centriole-associated rootlets and free rootlets. These findings indicate functional relationships between cytoplasmic microtubules and striated rootlets and the existence of rootlet-nucleating factors in the cytoplasm, in addition to centrioles.     

Proteomic analysis of secretion from human transplanted submandibular gland replacing lacrimal gland with severe keratoconjunctivitis sicca

Purpose: Proteomic analysis of secretions from transplanted or non-transplanted submandibular glands in patients with severe keratoconjunctivitis sicca and tears from normal eyes. Experimental design: Secretions from submandibular glands transplanted to replace lacrimal glands and non-transplanted submandibular glands were collected at 1year from 5 patients with severe keratoconjunctivitis sicca undergoing transplantation, and tears were collected from 3 normal subjects. 2-D electrophoresis (2-DE), then mass spectrometry was used to identify proteins. Western blot analysis was used to confirm protein expression. Results: We identified 34 and 11 distinct proteins in the saliva from transplanted submandibular glands and tears, respectively. The saliva from transplanted submandibular glands contained almost all the proteins abundant in tear fluid. The functions of identified proteins in the saliva from transplanted submandibular gland were mainly immune response and anti-bacterial. In total, 7 proteins showed differential expression between the saliva of transplanted and non-transplanted submandibular glands. The upregulation of short palate, lung and nasal epithelium carcinoma-associated protein 2 and carbonic anhydrase VI was confirmed by Western blot analysis. Conclusions: Identified proteins in saliva from transplanted submandibular glands may protect ocular structures. These findings can help in understanding the functional status of transplanted submandibular glands.     

Proteomic analysis of secretion from human transplanted submandibular gland replacing lacrimal gland with severe keratoconjunctivitis sicca

Purpose: Proteomic analysis of secretions from transplanted or non-transplanted submandibular glands in patients with severe keratoconjunctivitis sicca and tears from normal eyes. Experimental design: Secretions from submandibular glands transplanted to replace lacrimal glands and non-transplanted submandibular glands were collected at 1 year from 5 patients with severe keratoconjunctivitis sicca undergoing transplantation, and tears were collected from 3 normal subjects. 2-D electrophoresis (2-DE), then mass spectrometry was used to identify proteins. Western blot analysis was used to confirm protein expression. Results: We identified 34 and 11 distinct proteins in the saliva from transplanted submandibular glands and tears, respectively. The saliva from transplanted submandibular glands contained almost all the proteins abundant in tear fluid. The functions of identified proteins in the saliva from transplanted submandibular gland were mainly immune response and anti-bacterial. In total, 7 proteins showed differential expression between the saliva of transplanted and non-transplanted submandibular glands. The upregulation of short palate, lung and nasal epithelium carcinoma-associated protein 2 and carbonic anhydrase VI was confirmed by Western blot analysis. Conclusions: Identified proteins in saliva from transplanted submandibular glands may protect ocular structures. These findings can help in understanding the functional status of transplanted submandibular glands.