Disruption of 3D MCF-12A Breast Cell Cultures by Estrogens – An In Vitro Model for ER-Mediated Changes Indicative of Hormonal Carcinogenesis

Introduction

Estrogens regulate the proliferation of normal and neoplastic breast epithelium. Although the intracellular mechanisms of estrogens in the breast are largely understood, little is known about how they induce changes in the structure of the mammary epithelium, which are characteristic of breast cancer. In vitro three dimensional (3D) cultures of immortalised breast epithelial cells recapitulate features of the breast epithelium in vivo, including formation of growth arrested acini with hollow lumen and basement membrane. This model can also reproduce features of malignant transformation and breast cancer, such as increased cellular proliferation and filling of the lumen. However, a system where a connection between estrogen receptor (ER) activation and disruption of acini formation can be studied to elucidate the role of estrogens is still missing.

Methods/Principal Findings

We describe an in vitro 3D model for breast glandular structure development, using breast epithelial MCF-12A cells cultured in a reconstituted basement membrane matrix. These cells are estrogen receptor (ER)α, ERβ and G-protein coupled estrogen receptor 1 (GPER) competent, allowing the investigation of the effects of estrogens on mammary gland formation and disruption. Under normal conditions, MCF-12A cells formed organised acini, with deposition of basement membrane and hollow lumen. However, treatment with 17β-estradiol, and the exogenous estrogens bisphenol A and propylparaben resulted in deformed acini and filling of the acinar lumen. When these chemicals were combined with ER and GPER inhibitors (ICI 182,780 and G-15, respectively), the deformed acini recovered normal features, such as a spheroid shape, proliferative arrest and luminal clearing, suggesting a role for the ER and GPER in the estrogenic disruption of acinar formation.

Conclusion

This new model offers the opportunity to better understand the role of the ER and GPER in the morphogenesis of breast glandular structure as well as the events implicated in breast cancer initiation and progression.     

Evolution of metaplastic squamous cells of alveolar walls in pulmonary fibrosis produced by paraquat

Sequential histologic, ultrastructural, immunohistochemical and morphometric studies were made of the evolutional changes of metaplastic and regenerating alveolar epithelial cells in monkeys from 3 days to 8 weeks after paraquat administration. In the early proliferative phase, many alveoli were lined by single-layered and stratified squamous epithelium and bronchiolized epithelium (i.e., presumably derived from bronchi and bronchioles). The regenerating epithelial cells had well developed bundles of actin-like filaments, which were arranged parallel to the basal surfaces of the cells and were associated with zonulae adherentes; these cells also had intermediate filaments and some desmosomes, but lacked basement membranes, hemidesmosomes and anchoring fibrils. They covered either denuded, wavy and disrupted original epithelial basement membranes or areas of developing intraalveolar fibrosis. In zones of squamous epithelial cell metaplasia associated with intraalveolar fibrosis, fibronexus-like structures appeared to be responsible for the initial adhesion of the cells to the underlying connective tissue. In later phases, single-layered and stratified squamous epithelial cells disappeared, and only bronchiolized epithelial cells, with hemidesmosomes and anchoring fibrils on their basal surfaces, were found in fibrotic alveoli. Although bronchiolized and squamous metaplastic epithelial cells are generally thought to be formed as late events in pulmonary damage, such cells play an important role in early, temporary repair of damaged alveoli.     

Increased proliferation and altered growth factor dependence of human mammary epithelial cells overexpressing the Gab2 docking protein

The docking protein Gab2 is a proto-oncogene product that is overexpressed in primary breast cancers. To determine the functional consequences of Gab2 overexpression, we utilized the immortalized human mammary epithelial cell line MCF-10A. In monolayer culture, expression of Gab2 at levels comparable with those detected in human breast cancer cells accelerated epidermal growth factor (EGF)-induced cell cycle progression and was associated with increased basal Stat5 tyrosine phosphorylation and enhanced and/or more sustained EGF-induced Erk and Akt activation. Three-dimensional Matrigel culture of MCF-10A cells resulted in the formation of polarized, growth-arrested acini with hollow lumina. Under these conditions, Gab2 increased cell proliferation during morphogenesis, leading to significantly larger acini, an effect dependent on Gab2 binding to Grb2 and Shp2 and enhanced by recruitment of the p85 subunit of phosphatidylinositol 3-kinase. Pharmacological inhibition of MEK revealed that, in addition to direct activation of phosphatidylinositol 3-kinase, increased Erk signaling also contributed to Gab2-mediated enhancement of acinar size. In addition, Gab2 overcame the proliferative suppression that normally occurs in late stage cultures and conferred independence of the morphogenetic program from exogenous EGF. Finally, higher levels of Gab2 expression led to the formation of large disorganized structures with defective luminal clearance. These findings support a role for Gab2 in mammary tumorigenesis.     

DNA methylation control of tissue polarity and cellular differentiation in the mammary epithelium

Alterations in gene expression accompany cell-type-specific differentiation. In complex systems where functional differentiation depends on the organization of specific cell types into highly specialized structures (tissue morphogenesis), it is not known how epigenetic mechanisms that control gene expression influence this stepwise differentiation process. We have investigated the effect of DNA methylation, a major epigenetic pathway of gene silencing, on the regulation of mammary acinar differentiation. Our in vitro model of differentiation encompasses human mammary epithelial cells that form polarized and hollow tissue structures (acini) when cultured in the presence of basement membrane components. We found that acinar morphogenesis was accompanied with chromatin remodeling, as shown by alterations in histone 4 acetylation, heterochromatin 1 protein, and histone 3 methylated on lysine 9, and with an increase in expression of MeCP2, a mediator of DNA-methylation-induced gene silencing. DNA hypomethylation induced by treatment with 5-aza-2' deoxycytidine during acinar differentiation essentially prevented the formation of apical tissue polarity. This treatment also induced the expression of CK19, a marker of cells that are in a transitional differentiation stage. These results suggest that DNA methylation is a mechanism by which mammary epithelial differentiation is coordinated both at the tissue and cellular levels.     

The lipid kinase PI4KIIIβ and the eEF1A2 oncogene co-operate to disrupt three-dimensional in vitro acinar morphogenesis

The study of in vitro morphogenesis is fundamental to understanding neoplasia since the dysregulation of morphogenic pathways that create multi-cellular organisms is a common hallmark of oncogenesis. The in vitro culture of human breast epithelial cells on reconstituted basement membranes recapitulate some features of in vivo breast development, including the formation of a three-dimensional structure termed an acinus. Importantly, the capacity to disrupt in vitro acinar morphogenesis is a common property of human breast oncogenes. In this report, we find that phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar devlopment. Furthermore, expression of an oncogenic PI4KIIIβ activator, eEF1A2 (eukaryotic elongation factor 1 alpha 2), phenocopies the PI4KIIIβ multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters the localization of PI(4)P and PI(4,5)P₂ within acini, indicating the importance of these lipids in acinar development. Our work shows that PI4KIIIβ, eEF1A2 and PI(4)P likely play an important role in mammary neoplasia and acinar development.     

Bim regulation of lumen formation in cultured mammary epithelial acini is targeted by oncogenes

Epithelial cells organize into cyst-like structures that contain a spherical monolayer of cells that enclose a central lumen. Using a three-dimensional basement membrane culture model in which mammary epithelial cells form hollow, acinus-like structures, we previously demonstrated that lumen formation is achieved, in part, through apoptosis of centrally localized cells. We demonstrate that the proapoptotic protein Bim may selectively trigger apoptosis of the centrally localized acinar cells, leading to temporally controlled lumen formation. Bim is not detectable during early stages of three-dimensional mammary acinar morphogenesis and is then highly upregulated in all cells of acini, coincident with detection of apoptosis in the centrally localized acinar cells. Inhibition of Bim expression by RNA interference transiently blocks luminal apoptosis and delays lumen formation. Oncogenes that induce acinar luminal filling, such as ErbB2 and v-Src, suppress expression of Bim through a pathway dependent on Erk-mitogen-activated protein kinase; however, HPV 16 E7, an oncogene that stimulates cell proliferation but not luminal filling, is unable to reduce Bim expression. Thus, Bim is a critical regulator of luminal apoptosis during mammary acinar morphogenesis in vitro and may be an important target of oncogenes that disrupt glandular epithelial architecture.     

Increasingly transformed MCF-10A cells have a progressively tumor-like phenotype in three-dimensional basement membrane culture

Background  

MCF-10A cells are near diploid and normal human mammary epithelial cells. In three-dimensional reconstituted basement membrane culture, they undergo a well-defined program of proliferation, differentiation, and growth arrest, forming acinar structures that recapitulate many aspects of mammary architecture in vivo. The pre-malignant MCF-10AT cells and malignant MCF-10CA1a lines were sequentially derived from the MCF-10A parental cell line first by expression of a constitutively active T24 H-Ras generating the MCF-10AT cell line. This was followed by repeated selection for increasingly aggressive tumor formation from cells recovered from xenograft tumors in immuno-compromised mice, generating the MCF-10CA1a cell line. When inoculated subcutaneously into the flanks of immuno-compromised mice, MCF-10AT cells occasionally form tumors, whereas MCF-10CA1a cells invariably form tumors with a shorter latency than MCF-10AT derived tumors.     

Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. An ultrastructural study

Calcium modulation of keratinocyte growth in culture was studied by both transmission (TEM) and scanning electron microscopy (SEM). Under standard culture conditions (1.2-1.8 mM calcium), cells were connected by desmosomes and stratified to 4-6 cell layers. Many aspects of in vitro epidermal maturation were analogous to the in vivo process, with formation of keratohyalin granules, loss of nuclei, formation of cornified envelopes and shedding of cornified cells containing keratin filaments. When the medium calcium concentration was lowered to 0.02-0.1 mM, the pattern of keratinocyte growth was strikingly changed. Cells grew as a monolayer with no desmosomal connections and proliferated rapidly, shedding largely non-cornified cells into the medium. Large bundles of keratin filaments were concentrated in the perinuclear cytoplasm. The elevation of extracellular calcium to 1.2 mM induced low calcium keratinocytes to stratify, keratinize and cornify in a manner analogous to that seen when plated in standard calcium medium. The earliest calcium-induced ultrastructural change was the asymmetric formation of desmosomes between adjacent cells. Desmosomal plaques with associated tonofilaments were observed 5 min after calcium addition; symmetric desmosomes were formed within 1-2 h. This system is presented as a useful model for the study of the regulation of desmosome assembly and disassembly.     

The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini

The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.     

Identification of the cytoskeletal proteins in lens-forming cells, a special epithelioid cell type

Proteins of contractile and cytoskeletal elements have been studied in bovine lens-forming cells growing in culture as well as in bovine and murine lenses grown in situ by immunofluorescence microscopy using antibodies to the following proteins: actin, myosin, tropomyosin, α-actinin, tubulin, prekeratin, vimentin, and desmin. Lens-forming cells contain actin, myosin, tropomyosin, and α-actinin which in cells grown in culture are enriched in typical cable-like structures, i.e. microfilament bundles. Antibodies to tubulin stain normal, predominantly radial arrays of microtubules. In the epithelioid lens-forming cells of both monolayer cultures grown in vitro and lens tissue grown in situ intermediate-sized filaments of the vimentin type are abundant, whereas filaments containing prekeratin-like proteins (‘cytokeratins’) and desmin filaments have not been found. The absence of cytokeratin proteins observed by immunological methods is supported by gel electrophoretic analyses of cytoskeletal proteins, which show the prominence of vimentin and the absence of detectable amounts of cytokeratins and desmin. This also correlates with electron microscopic observations that typical desmosomes and tonofilament bundles are absent in lens-forming cells, as opposed to a high density of vimentin filaments. Our observations show that the epithelioid lens-forming cells have normal arrays of (i) microfilament bundles containing proteins of contractile structures; (ii) microtubules; and (iii) vimentin filaments, but differ from most true epithelial cells by the absence of cytokeratins, tonofilaments and typical desmosomes. The question of their relationship to other epithelial tissues is discussed in relation to lens differentiation during embryogenesis. We conclude that the lens-forming cells either represent an example of cell differentiation of non-epithelial cells to epithelioid morphology, or represent a special pathway of epithelial differentiation characterized by the absence of cytokeratin filaments and desmosomes. Thus two classes of tissue with epithelia-like morphology can be distinguished: those epithelia which contain desmosomes and cytokeratin filaments and those epithelioid tissues which do not contain these structures but are rich in vimentin filaments (lens cells, germ epithelium of testis, endothelium).     

Characterization of a myoepithelial cell line derived from a neonatal rat mammary gland

A clonal, myoepithelial-like cell line has been obtained from a primary culture established from the mammary gland of a 7-d-old rat. In a number of respects, this cell line, termed Rama 401, resembles the myoepithelial cells of the mammary gland, especially when grown on floating collagen gels. The cells grow as multilayers on the gel surface and form branching structures that do not appear to contain a lumen. They are rather elongated, with irregular-shaped, flattened nuclei that contain large amounts of peripheral chromatin. Elongated processes project from the cell surface and numerous membrane pinocytotic vesicles can be seen. The cytoplasm is filled with linear arrays of 5- to 7-nm filaments with occasional dense foci. Cell junctions with associated 8- to 11-nm tonofilaments are also observed. Immunofluorescence techniques reveal actin and myosin filaments and also intermediate filaments of both prekeratin and vimentin types. Rama 401 cells secrete an amorphous material that, when an immunoperoxidase technique is used, stains with antibodies to basement membrane-specific type IV collagen. Localized densities of the cell membrane, which resemble hemidesmosomes, are located adjacent to these extracellular deposits. Immunofluorescence staining and immunoprecipitation techniques reveal that the cells also synthesize two other basement membrane proteins, laminin and fibronectin. The type IV collagen consists of two chains with molecular weights of 195,000 and 185,000.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and alpha 2 beta 1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize alpha 2 beta 1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Novel egg white-based 3-D cell culture system

Although three dimensional (3-D) cell culture systems have numerous advantages over traditional monolayer culture, the currently available 3-D cell culture media are cost-prohibitive for regular use by the majority of research laboratories. Here we show a simple system based on avian egg white that supports growth of cells in 3-D, at a significantly decreased cost. Specifically, we show that growth of immortalized human breast epithelial cells (MCF10A) in egg white-based medium results in formation of acini with hollow lumens, apoptotic clearance of the cells in the lumen, and apicobasal polarization comparable to what has been described using established 3-D culture media such as reconstituted basement membrane preparations (BM). There was no significant difference in MCF10A proliferation and acinar size between egg white and BM. We also cultured different established cell lines, oncogene-transformed MCF10A, and mouse mammary epithelial cells in egg white and BM, and observed similar morphology. In summary, our data convincingly argue that egg white can be used as a suitable alternative model for 3-D cell culture studies. We strongly believe that this simple and inexpensive method should allow researchers to perform 3-D cell culture experiments on a regular basis, and result in a dramatic increase of use of the 3-D cell culture in research. Thus, this finding lays the foundation for significantly increased, cost-effective use of 3-D cultures in cell biology.     

SWI/SNF chromatin remodeling enzyme ATPases promote cell proliferation in normal mammary epithelial cells

The ATPase subunits of the SWI/SNF chromatin remodeling enzymes, Brahma (BRM) and Brahma‐related gene 1 (BRG1), can induce cell cycle arrest in BRM and BRG1 deficient tumor cell lines, and mice heterozygous for Brg1 are pre‐disposed to breast tumors, implicating loss of BRG1 as a mechanism for unregulated cell proliferation. To test the hypothesis that loss of BRG1 can contribute to breast cancer, we utilized RNA interference to reduce the amounts of BRM or BRG1 protein in the nonmalignant mammary epithelial cell line, MCF‐10A. When grown in reconstituted basement membrane (rBM), these cells develop into acini that resemble the lobes of normal breast tissue. Contrary to expectations, knockdown of either BRM or BRG1 resulted in an inhibition of cell proliferation in monolayer cultures. This inhibition was strikingly enhanced in three‐dimensional rBM culture, although some BRM‐depleted cells were later able to resume proliferation. Cells did not arrest in any specific stage of the cell cycle; instead, the cell cycle length increased by approximately 50%. Thus, SWI/SNF ATPases promote cell cycle progression in nonmalignant mammary epithelial cells. J. Cell. Physiol. 223:667–678, 2010. © 2010 Wiley‐Liss, Inc.     

Epidermal growth factor (EGF)-induced morphological changes in the basement membrane of chick embryonic skin

Summary The effect of epidermal growth factor (EGF) on the basement membrane structure of chick embryonic skin cultured in a chemically defined medium (BGJb) containing 20 mM hydrocortisone, and EGF at 10, 50, or 100 ng/ml supplemented with 5% delipidized fetal calf serum, was examined by electron microscopy. During development of the epidermis in vitro, EGF (100 ng/ml) caused striking changes to occur in the basement membrane structure and in the keratinization process. The basement membrane frequently became discontinuous with many gaps apparent in section, and occasionally became folded following detachment from the basal surface of the epidermis and protruded into the underlying dermis. In the basal and intermediate cells of EGF-treated epidermis, tonofilament bundles were decreased in number, while desmosomes and hemidesmosomes revealed no significant changes in morphology.     

ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini

Both ErbB1 and ErbB2 are overexpressed or amplified in breast tumours. To examine the effects of activating ErbB receptors in a context that mimics polarized epithelial cells in vivo, we activated ErbB1 and ErbB2 homodimers in preformed, growth-arrested mammary acini cultured in three-dimensional basement membrane gels. Activation of ErbB2, but not that of ErbB1, led to a reinitiation of cell proliferation and altered the properties of mammary acinar structures. These altered structures share several properties with early-stage tumours, including a loss of proliferative suppression, an absence of lumen, retention of the basement membrane and a lack of invasive properties. ErbB2 activation also disrupted tight junctions and the cell polarity of polarized epithelia, whereas ErbB1 activation did not have any effect. Our results indicate that ErbB receptors differ in their ability to induce early stages of mammary carcinogenesis in vitro and this three-dimensional model system can reveal biological activities of oncogenes that cannot be examined in vitro in standard transformation assays.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize α2β1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Intermediate-sized filaments of the prekeratin type in myoepithelial cells

Myoepithelial cells from mammary glands, the modified sweat glands of bovine muzzle, and salivary glands have been studied by electron microscopy and by immunofluorescence microscopy in frozen sections in an attempt to further characterize the type of intermediate-sized filaments present in these cells. Electron microscopy has shown that all myoepithelial cells contain extensive meshworks of intermediate-sized (7--11-nm) filaments, many of which are anchored at typical desmosomes or hemidesmosomes. The intermediate-sized filaments are also intimately associated with masses of contractile elements, identified as bundles of typical 5--6-nm microfilaments and with characteristically spaced dense bodies. This organization resembles that described for various smooth muscle cells. In immunofluorescence microscopy, using antibodies specific for the various classes of intermediate-sized filaments, the myoepithelial cells are strongly decorated by antibodies to prekeratin. They are not specifically stained by antibodies to vimentin, which stain mesenchymal cells, nor by antibodies to chick gizzard desmin, which decorate fibrils in smooth muscle Z bands and intercalated disks in skeletal and cardiac muscle of mammals. Myoepithelial cells are also strongly stained by antibodies to actin. The observations show (a) that the epithelial character, as indicated by the presence of intermediate-sized filaments of the prekeratin type, is maintained in the differentiated contractile myoepithelial cell, and (b) that desmin and desmin-containing filaments are not generally associated with musclelike cell specialization for contraction but are specific to myogenic differentiation. The data also suggest that in myoepithelial cells prekeratin filaments are arranged--and might function--in a manner similar to the desmin filaments in smooth muscle cells.     

Ultrastructural aspects of cat submandibular glands

Submandibular glands of five adult female cats were examined by conventional electron microscopic techniques. All gland acini are mucous secreting and each acinus is capped with mucous secreting demilunar cells. Secretory product of demilunar cells is more electron lucent than that of acinar cells. The demilunes show intercellular tissue spaces and intercellular canaliculi whereas similar specializations are absent between acinar cells. Mitochondria and arrays of granular endoplasmic reticulum are more numerous in demilunar cells than in acinar cells. In acinar and demilunar cells secretory droplets first appear as enlarged Golgi saccules which subsequently become closely related to cisternae of the granular endoplasmic reticulum. Filamentous structures, interpreted as mucin molecules, are present in secretory droplets of acinar cells. Intercalated ducts are short, consisting of several junctional cells between acini and striated ducts. Striated ducts are long and tortuous and contain light cells, dark cells and basal cells. Light cells contain numerous membrane bound granules in their distal ends whereas dark cells show electron lucent vesicles in the same position. Basal cells contain a paucity of organelles and membrane plications but exhibit hemidesmosomes along their basal plasma membranes. Myoepithelial cells are abundant in relation to acinar and demilunar cells. Nerve terminals are present in some instances between acinar cells or between acinar and myoepithelial cells.