Quantitation and localization of acinar cell-specific mucin in submandibular glands of mice during postnatal development

Summary In this study, antiserum to acinar cell-specific mucin was utilized to determine whether mucin could be detected in the mouse submandibular gland prior to cytodifferentiation of acinar cells. Results from radioimmunoassay indicated that mucin occurs in submandibular glands from newborn mice, i.e., before the appearance of mature acinar cells. Additionally, mucin quantitated in various stages of development was found to be antigenically identical to adult mucin. After sections of glands were treated with immunohistochemical reagents, we observed that the mature acinar cell-specific mucin was present in secretory terminal-tubule cells and in proacinar cells of newborn animals. The present findings suggest that in young animals, the proacinar cells are an immediate precursor of acinar cells and that the secretory terminal-tubule cells may represent an earlier stage in development of acinar cells. In adult female glands, mucin was also detected in the granular intercalating-duct cells. This latter observation is consistent with the hypothesis that these cells are an intermediate in the acinar cell replacement process.     

Growth factor regulation of the amylase promoter in a differentiating salivary acinar cell line

Salivary glands contain two major epithelial cell types: acinar cells which produce the primary salivary secretion, including amylase, and ductal cells which reabsorb electrolytes but also secrete kallikrein. Here we investigated salivary acinar cell differentiation in vitro using the activity of the salivary amylase and tissue kallikrein promoters as markers of acinar cell and ductal cell differentiation, respectively. Each of the promoter sequences was cloned into a replication-deficient adenoviral vector containing the luciferase reporter gene. Previous studies showed that a human submandibular gland cell line (HSG) differentiated into acinar cells when cultured on a reconstituted basement membrane matrix (Matrigel). The luciferase activity of the amylase promoter vector (AdAMY-luc) was low in HSG cells cultured on plastic, where they grow as an epithelial monolayer. The promoter activity increased approximately tenfold when HSG cells were cultured on Matrigel and developed an acinar phenotype. Under the same conditions, the luciferase activity of the kallikrein promoter (AdKALL-luc) was not induced. Because HSG cells demonstrate acinar cell morphology, but not amylase gene expression, when cultured on laminin-1, certain soluble components of Matrigel were tested for their ability to induce the amylase promoter during in vitro differentiation of acinar cells. We find that epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), which are present in the basement membrane, and hepatocyte growth factor (HGF) increase activity of the amylase promoter. Other basement membrane-derived growth factors such as TGF-beta, basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PGDF), as well as tumor necrosis factor (TNF-alpha), keratinocyte growth factor (KGH), nerve growth factor (NGF) and interferon gamma (IFN-gamma) were inactive. This system will be further exploited to study the mechanisms by which extracellular matrix molecules and growth factors regulate salivary acinar cell differentiation.     

Human salivary gland acinar cells spontaneously form three-dimensional structures and change the protein expression patterns

Applying tissue engineering principles to design an auto-secretory device is a potential solution for patients suffering loss of salivary gland function. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of reports on the in vitro cell biology and physiology of human salivary gland cells. This study used a low-calcium culture system to selectively cultivate human parotid gland acinar (PGAC) cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α-amylase) and function-related proteins (aquaporin-3, aquaporin-5, and ZO-1). Notably, when the cells reached confluence, three-dimensional (3D) cell aggregates were observed in crowded regions. These self-formed cell spheres were termed post-confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function-related proteins compared to the two-dimensional (2D) cells. Translocation of aquoporin-3 from cytosolic to alongside the cell boundaries, and of ZO-1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form PCSs without the help of 3D scaffolds and retain certain differentiation and polarity. This phenomenon implies that it is possible to introduce 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering.     

Determinants of nerve cell patterns during development: a review

The aim of the work reviewed is to define some of the mechanisms which are implicated in the control of neural cell pattern formation in the developing central nervous system. This question was examined by studies of brain embryonic development in normal and reeler mutant mice, which are characterized by profuse architectonic anomalies. The adult reeler phenotype is characterized by extreme abnormalities of cell positioning in the telencephalic and cerebellar cortices as well as by distinct architectonic anomalies in non-cortical structures such as the inferior olive, the facial nerve nucleus and other brainstem nuclei. Studies of the embryonic development of these structures reveal that neurons are generated at the normal time and migrate along normal pathways. Moreover, the processes of directional axonal growth, differentiation of class-specific features of neurons and glia, and synaptogenesis appear unaffected by the reeler mutation. In all instances, however, the early cell patterns formed by reeler neurons is consistently less regular than in normal embryos. These data indicate that brain architectonics does not exclusively result from the maturation of cells, neurites and connections, but is also contingent upon a specific stabilization of early neurons at the end of migration. One may infer that the presence of a normal allele at the reeler locus is necessary for this stabilization to occur normally, or that it is submitted to genetic control. Although the factor(s) responsible for the stable configuration of neural cell patterns are still unknown, several hypotheses can be considered. There is ample evidence against the role of diffusible factors, mesodermal components and afferent fiber systems. So far, most data point to the importance of cell-cell interactions which can be of three types: homophilic (neuronal-neuronal), heterophilic (neuronal-glial), or both. The cell-interaction mechanism could have been acquired during brain evolution of the mammalian lineage and the reeler gene could act by perturbing, directly or indirectly, these cell interactions. A better definition of the mechanisms responsible for the organization of nerve cell patterns is central to our understanding of brain development in normal as well as in pathological states. By following the example of recent successful research on invertebrate brain development, we believe that the genetic approach to this important question is a valuable one.     

Immunolocalization of a spectrin-like protein (fodrin) in pancreatic acinar cells

A spectrin-like protein (fodrin) was localized in porcine pancreas using an immunoperoxidase procedure with antibodies raised against erythrocyte spectrin. Fodrin was primarily associated with the cell plasma membrane although some was also detectable in the cytoplasm of the acinar cells. The membrane labelling of the acinar cells was uneven such that the lateral and basal membranes were strongly labelled by anti-spectrin antibodies whereas the apical membranes were poorly labelled. The implications of the results to secretion and to the occurrence of specific membrane domains are discussed.     

Periodicity of cell attachment patterns during Escherichia coli biofilm development

The complex architecture of bacterial biofilms inevitably raises the question of their design. Microstructure of developing Escherichia coli biofilms was analyzed under static and laminar flow conditions. Cell attachment during early biofilm formation exhibited periodic density patterns that persisted during development. Several models for the origination of biofilm microstructure are considered, including an activator-inhibitor or Turing model.     

Immunolocalization of basic fibroblast growth factor during chicken cardiac development

Basic fibroblast growth factor (bFGF) has been identified in cultured cardiac myocytes as well as in myocardial tissue of both embryonic and adult organisms; bFGF has also been demonstrated to regulate proliferation and differentiation of these cells in culture. Such studies suggest a possible role for bFGF in cardiac myogenesis. In vitro studies using cultured endothelial and neuronal cells also suggest that myocyte-derived bFGF may be involved in the regulation of vascularization and/or innervation of the developing heart. We have generated a spatial and temporal map for bFGF in the developing chick heart using immunohistochemical techniques and our monospecific polyclonal rabbit antihuman bFGF IgG. A progressive decrease in bFGF expression was seen in the highly trabeculated region of the ventricular myocardium, relative to the myocardium directly underlying the epicardial tissue, with increasing developmental age. bFGF expression was limited to the cytoplasm of cardiac myocytes; neither vascular endothelium nor smooth muscle contained anti-bFGF immunoreactive material. A correlation between the temporal and spatial pattern of bFGF expression seen here, with the pattern of myocyte proliferation and differentiation reported by others, suggests a role for bFGF in the autocrine regulation of myocyte proliferation and differentiation.     

Meiosis and retrotransposon silencing during germ cell development in mice

In mammals, germ cells derive from the pluripotent cells that are present early in embryogenesis, and then differentiate into male sperm or female eggs as development proceeds. Fusion between an egg and a sperm at fertilization allows genetic information from both parents to be transmitted to the next generation, and produces a pluripotent zygote to initiate the next round of embryogenesis. Meiosis is a central event in this self-perpetuating cycle that creates genetic diversity by generating new combinations of existing genetic alleles, and halves the number of chromosomes in the developing male and female germ cells to allow chromosome number to be maintained through successive generations. The developing germ cells also help to maintain genetic and chromosomal stability through the generations by protecting the genome from excessive de novo mutation. Several mouse mutants have recently been characterised whose germ cells exhibit defects in silencing the potentially mutagenic endogenous retroviruses and other retrotransposons that are prevalent in mammalian genomes, and these germ cells also exhibit defects in progression through meiosis. Here we review how mouse germ cells develop and proceed through meiosis, how mouse germ cells silence endogenous retroviruses and other retrotransposons, and discuss why silencing of endogenous retroviruses and other retrotransposons may be required for meiotic progression in mice.     

Changes in numbers of pancreatic acinar cell nuclei and in DNA content during raw soya flour feeding in mice

Nuclei of pancreatic cells were isolated by trypsin-detergent digestion of fresh tissue and stained with propidium iodide, and nuclear DNA was measured by flow cytometry. Samples were isolated from mice fed either chow or raw soya flour (RSF) for periods ranging from 1 day to 48 weeks, beginning at 4 weeks of age. In chow-fed mice, the pancreas contained about 80% diploid (2N) and 20% tetraploid (4N) cells at the start of the study, but tetraploidy gradually increased to about 40% 2 weeks later (6 weeks of age) and remained at this level from that time onwards. Low levels of octaploid nuclei (8N) were also present in some animals after 2 weeks. In RSF-fed mice, about 20% tetraploid nuclei were also present for 1 and 2 days after starting RSF, but by 4 days tetraploidy had increased significantly to 40% and by 14 days had further increased to 50%. This level was significantly higher than that seen in chow-fed animals and was maintained for up to 48 weeks. Significantly higher numbers of octaploid nuclei were also present in the RSF-fed animals. In both chow- and RSF-fed mice, most cells were mononuclear, averaging 70% in chow-fed and 64% in RSF-fed animals. This difference was significant. This study shows that the mouse pancreas differs from the rat pancreas in the absence of a large population of binucleate acinar cells and the presence of considerable nuclear tetraploidy. Raw soya flour feeding leads to significant changes in these features, but in this species these changes do not appear to predispose to neoplasia.     

Loss of hyperpolarization-activated Cl(-) current in salivary acinar cells from Clcn2 knockout mice

ClC-2 is localized to the apical membranes of secretory epithelia where it has been hypothesized to play a role in fluid secretion. Although ClC-2 is clearly the inwardly rectifying anion channel in several tissues, the molecular identity of the hyperpolarization-activated Cl(-) current in other organs, including the salivary gland, is currently unknown. To determine the nature of the hyperpolarization-activated Cl(-) current and to examine the role of ClC-2 in salivary gland function, a mouse line containing a targeted disruption of the Clcn2 gene was generated. The resulting homozygous Clcn2(-/-) mice lacked detectable hyperpolarization-activated chloride currents in parotid acinar cells and, as described previously, displayed postnatal degeneration of the retina and testis. The magnitude and biophysical characteristics of the volume- and calcium-activated chloride currents in these cells were unaffected by the absence of ClC-2. Although ClC-2 appears to contribute to fluid secretion in some cell types, both the initial and sustained salivary flow rates were normal in Clcn2(-/-) mice following in vivo stimulation with pilocarpine, a cholinergic agonist. In addition, the electrolytes and protein contents of the mature secretions were normal. Because ClC-2 has been postulated to contribute to cell volume control, we also examined regulatory volume decrease following cell swelling. However, parotid acinar cells from Clcn2(-/-) mice recovered volume with similar efficiency to wild-type littermates. These data demonstrate that ClC-2 is the hyperpolarization-activated Cl(-) channel in salivary acinar cells but is not essential for maximum chloride flux during stimulated secretion of saliva or acinar cell volume regulation.     

Immunolocalization of cytochrome P450 aromatase in rat testis during postnatal development

Aromatization of androgens into estrogens in rat testis is catalyzed by the microsomal enzyme cytochrome P450 aromatase. In this work, aromatase cellular site was investigated in prepuberal, peripuberal and postpuberal testis, from 10-, 21- and 60-day-old rats respectively. Paraffin-embedded testis sections were processed for P450arom immunostaining using a rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase. Next, biotinylated anti-rabbit IgG was applied, followed by ABC/HRP/complex amplification with diaminobenzidine as chromogen. Prepuberal testis sections showed a strong immunoreactivity of aromatase in Sertoli cell cytoplasm while interstitial cells were immunonegative. In peripuberal testis sections, cytoplasmic immunoreaction was weak in Sertoli cells, but it was strong in spermatocytes and sporadic in Leydig cells. Postpuberal testis sections displayed a moderate aromatase immunoexpression in spermatocytes while a strong immunostaining was observed in round and elongated spermatids, as well as in Leydig cells. These results indicate a different age-dependence of aromatase localization in rat testicular cells during gonadal development. In particular, inside the seminiferous tubules, the aromatization site moves from Sertoli cells to late germ cells, suggesting a proliferative role of aromatase in prepuberal testis and its subsequent involvement in meiotic and post-meiotic germ cell maturation.     

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.     

Responses of salivary acinar cells to intracellular alkalinization

Responses of rat submandibular acini to intracellular alkalinization were investigated. Intracellular alkalinization was induced by addition of NH⁴Cl or methylamines, or by prepulse with Na butyrate. Only partial recovery occurred following Na butyrate prepulse or methylated amine addition, but full recovery was observed following addition of NH⁴Cl. The latter recovery was DIDS and dimethylamiloride-insensitive but was inhibited by bumetanide or high [K⁺] and stimulated in Na⁺ free buffer and by ouabain. Acetylcholine stimulated recovery from NH⁴Cl- or Na butyrate pre-pulse-induced alkalinization and reduced the extent of alkalinization induced by methylated amines. Acetylcholine-stimulated recovery from NH⁴Cl-induced alkalinization was mimicked by substance P or ionomycin and was partially Ca²⁺-dependent. This stimulated recovery was bumetanide-insensitive but was partially sensitive to charybdotoxin. Taken together, these data indicate that in unstimulated cells, recovery from alkalinization induced by NH⁴Cl occurs by bumetanide-sensitive transport of the NH⁴⁺ ion, that DIDS-inhibitable anion transport contributes little to this recovery, and that acetylcholine and other Ca²⁺-elevating agents accelerate recovery from NH⁴Cl-induced alkaline challenge by a mechanism insensitive to bumetanide, DIDS, ouabain, and dimethylamiloride but sensitive to extracellular Ca²⁺ and to charybdotoxin. Partial recovery from alkaline challenge can also occur in the absence of NH⁴⁺ ions, and acetylcholine also stimulates this mode of recovery. Together, these data suggest that these cells have little intrinsic ability to recover from intracellular alkalinization and that the NH⁴⁺ ion may be a surrogate for K⁺ in at least two ion transport pathways. © 1994 wiley-Liss, Inc.     

Involvement of thrombopoietin in acinar cell necrosis in l-arginine-induced acute pancreatitis in mice

Thrombopoietin (TPO) plays an important role in injuries of different tissues. However, the role of TPO in acute pancreatitis (AP) is not yet known. The aim of the study was to determine the involvement of TPO in AP. Serum TPO was assayed in necrotizing pancreatitis induced by l-arginine in mice. Recombinant TPO and anti-TPO antibody were given to mice with necrotizing pancreatitis. Amylase, lipase, lactate dehydrogenase, myeloperoxidase activity and pancreatic water content were assayed in serum and tissue samples. Pancreas and lung tissue samples were also collected for histological evaluation. Immunohistochemistry of amylase α and PCNA were applied for the study of acinar regeneration and TUNEL assay for the detection of apoptosis in the pancreas. Increased levels of serum TPO were found in necrotizing pancreatitis. After TPO administration, more severe acinar necrosis was found and blockade of TPO reduced the acinar necrosis in this AP model. Acinar regeneration and apoptosis in the pancreas were affected by TPO and antibody treatment in necrotizing pancreatitis. The severity of pancreatitis-associated lung injury was worsened after TPO treatment, but attenuated after Anti-TPO antibody treatment. In conclusion, serum TPO is up-regulated in the necrotizing pancreatitis induced by l-arginine in mice and may be a risk factor for the pancreatic acinar necrosis in AP. As a pro-necrotic factor, blockade of TPO can attenuate the acinar necrosis in AP and may be a possible therapeutic intervention for AP.     

Changes in lectin binding patterns of Leydig cells during fetal and postnatal development in mice

Summary Changes in the lectin binding of mouse Leydig cells during fetal and postnatal development were examined by light- and electron-microscopy using eight different biotinylated lectins (ConA, WGA, RCA-I, UEA-I, GS-I, PNA, SBA and GS-II). At the light-microscopic level, ConA, WGA, RCA-I, UEA-I and GS-I showed the same binding pattern in which all five lectins bound to the plasma membrane and cytoplasm of Leydig cells from the 13th day post coitum (p.c.) to the 8th postnatal week. PNA, SBA and GS-II reactions were positive in the plasma membrane and cytoplasm of Leydig cells from the 13th day p.c. to 15th day post partum (p.p.) but disappeared completely by day 20. At the electron-microscopic level, gold particles representing the GS-I or GS-II binding sites were distributed primarily along the cell surface membrane, including that of microvilli, as well as in the cytoplasm. These results indicate that certain glycoconjugates bearingD-galactose,N-acetyl-D-galactosamine, andN-acetyl-D-glucosamine residues are expressed on the cell surface and in the cytoplasm of Leydig cells during the period from the 13th day p.c. to around the 20th day p.p. The results suggest that these glycoconjugates might play some role in modulating hormone-receptor interaction in the Leydig cells before the 20th day. Furthermore, these results may indicate that sugar residues expressed on the cell surface and in the cytoplasm of Leydig cells are different from those in the fetal-neonatal and adult phases.