Effect ofd-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

Summary Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [³H]thymidine uptake, is very low. Inl-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured ind-valine medium was significantly lower than that of cells cultured inl-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured ind-valine medium was significantly higher than that of cells cultured inl-valine medium. Cytosine arabinoside decreased the number of labeled cells in bothl-valine andd-valine cultures. From these results, it appears thatd-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures. This research was sponsored by grants from the National Institute of Child Health and Human Development, Bethesda, MD (HD-03820, HD-11816, HD-05797), and the Mellon Foundation.     

Culture of bovine mammary epithelial cells in D-valine modified medium: selective removal of contaminating fibroblasts

Bovine mammary epithelial cells were cultured in nutrient medium containing D-valine substituted for L-valine in an attempt to control fibroblast overgrowth. Contamination of epithelial cell cultures with fibroblasts was prevented in cultures maintained in D-valine but not in control cultures containing L-valine. Microscopic examination of cells cultured in the D-valine medium demonstrated similar morphology as controls after 27 days in culture. Concentration of alpha-lactalbumin in the D-valine medium was similar to levels found in control medium. Results suggest D-valine modified medium controls fibroblast overgrowth of cell cultures, but doesn't impair epithelial cell morphology or function.     

In vitro analysis of proliferating epithelial cell populations from the mouse mammary gland: Fibroblast-free growth and serial passage

Summary Normal and neoplastic mouse mammary epithelial cells were cultured in nutrient medium containing D-valine substituted for L-valine. Fibroblast overgrowth was prevented and epithelial cell functions and morphology were retained in cultures maintained in, D-valine medium up to 2 months. A nonenzymatic technique was devised to dissociate epithelial cell monolayers. The combined use of this dissociation buffer and D-valine nutrient medium made it possible to passage serially normal and neoplastic mammary epithelial cells. Normal cells were derived from mammary glands of animals stimulated with exogenous hormones for various periods. The period of in vivo hormonal stimulation influenced the ability of normal mammary epithelial cells to attach and proliferate in primary and serially passaged cultures. A greater proportion of cells derived from glands following 2 to 4 weeks of hormonal stimulation were recovered after replating and showed higher labeling indices during serial passage than cells from unstimulated or 5- to 7-week stimulated groups. This investigation was supported by Grant No. CA 05388 from the National Cancer Institute and by Cancer Research Funds of the University of California.     

Rat epididymal epithelial cells and 17beta-estradiol synthesis under hCG stimulation in vitro

Epithelial cells of human and animal epididymis display features of steroidogenic cells. Rat epididymal epithelial cells in vitro produce androgens which are converted to 17beta-estradiol, and released into the medium. The regulation of the epididymal steroidogenesis is not fully understood but it could be expected that it remains under LH influence. In previous study we observed that the morphology of rat epididymal epithelial cells in vitro was affected by hCG and the increase of amount of lipid droplets, glycogen and PAS-positive substances was observed. The present studies show the organelles which take part in synthesis of steroids in rat epididymal epithelial cells in vitro and the effect of hCG on E2 synthesis. The cells were cultured in the medium with/without DHT and without DHT in supplementation with hCG. After hCG stimulation the amount of an active mitochondria were increased when compared to the amount of mitochondria in the epididymal epithelial cells cultured without DHT. Ultrastructure of the cells was similar to the cells cultured with DHT, while the cytoplasm of the cells cultured without DHT was disorganized. The synthesis of 17beta-estradiol was stimulated by hCG, that exerted its effect through LH/hCG receptors, localized in the epididymal epithelial cells.     

Structural and functional aspects of cultured epididymal epithelial cells isolated from pubertal rats

Acidic epididymal glycoprotein (AEG) and androgen-binding protein (ABP) antisera were used to study functional activities of primary cell cultures of the epididymal epithelium of 20--23-day-old rats. Extensive AEG immunoreactivity was associated with almost all epithelial cells of the distal caput, corpus and cauda epididymidis. ABP immunoreactivity was solely confined to some epithelial cells of the caput epididymidis. AEG and ABP immunoreactive cells were identified as principal cells. Morphological studies of enzymically dispersed aggregates of the epididymal epithelial cells showed that stromal cells were satisfactorily removed and that cell aggregates consisted of a predominant population for cells displaying the morphological characteristics of principal cells. Scanning and transmission electron microscopic studies of cultured epididymal epithelial cells in monolayers demonstrated that microvilli and pit-like invaginations of the cell surface were preserved during the first 7--10 days of culture and then gradually disappeared. Other characteristic subcellular structures such as Golgi apparatus and rough endoplasmic reticulum cisterna were preserved. Electrophoretic analysis of [35S]methionine-labelled secretory polypeptides released by epididymal epithelial cells into the culture medium demonstrated a distinct protein band pattern which differed from that observed in the medium of cultured rat Sertoli cells. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually immature rats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell function.     

Selective isolation and culture of a proliferating epithelial cell population from the hamster trachea

A reliable cell isolation technique was developed to allow the cultivation of cells from the hamster respiratory tract. Repeated thermolysin treatments and gradient centrifugation yielded a cellculture completely free from contamination by fibroblasts. Viable cells could be isolated from as little tissue as a single hamster trachea, but in vitro proliferation occurred only if the hamster was less than 4 months of age. The cultured cells could be repeatedly passaged and subcultured for weeks by employing normal tissue culture techniques. Morphologically, the monolayers appeared to be a homogeneous population of epithelial cells, and successful cloning of freshly isolated single cells resulted in apparently identical cultures. The epiethelial origin of these cells was also suggested by continued growth in minimum essential medium with D-valine substituted for L-valine. The relative ease with which this cell type can be isolated, cultured, and manipulated in vitro should encourage its application as a model of the respiratory epithelium.     

Inhibition of proliferation of contaminating fibroblasts by D-valine in cultures of smooth muscle cells from human myometrium

Replacement of L-valine with D-valine in a standard culture medium can selectively inhibit fibroblast proliferation. The aim of the present study was to investigate whether human myometrial cells cultured with D-valine instead of L-valine can survive and express their characteristics. Cultured cells (95-98%) maintain expression of the intermediate filament desmin, which is the specific marker for mature muscle cells. By transmission electron microscopy, the cells showed the general morphology of smooth muscle cells in culture. Oxytocin in serum-free culture medium at 37 degrees C (5 min) caused a concentration-dependent increase in cellular Na and total Ca, and a decrease in K content as determined by X-ray microanalysis. The percentage of cells cultured with D-valine responding to oxytocin stimulation was larger than that of cells cultured with L-valine, suggesting less contamination of smooth muscle cells by fibroblasts in the presence of D-valine. As shown by measurements with fura-2, D-valine-cultured cells retained the characteristic increase in intracellular free Ca2+ ions after oxytocin stimulation.     

Glycosphingolipid patterns in primary mouse kidney cultures

Primary kidney cultures from C57BL/6J mice, 6 weeks of age or older, were produced using D-valine medium to select for epithelial cell growth. After allowing the cells to attach and proliferate for 1 week following plating, medium was changed once per week. Cells formed nearly confluent monolayers during the second week of culture. The cultured cells contained all of the glycosphingolipids seen in the adult kidney, analyzed by high performance liquid chromatography as their perbenzoyl derivatives. Glucosylceramide, however, was highly predominant in the cultured cells, whereas dihexosyl- and trihexosylceramides predominate in the intact kidney. Sex differences in glycolipid contents found in the intact kidney were also apparent in these cultured cells: The concentration of neutral glycolipids, in general, was higher in male cells than in those derived from females, and the male-specific glycolipid nonhydroxy fatty acid digalactosylceramide was high in male cells but very low in female cells. Neutral glycosphingolipids were labeled in 2-week-old cultures using [3H]palmitate. The [3H]palmitate was incorporated into all of the glycolipids within 2 hr of labeling. Hence, adult mouse kidney cells in D-valine medium retain their differentiated characteristics for a sufficient period of time to allow investigation of glycolipid syntheses in monolayer cultures of epithelial cells derived from this organ.     

Glycosidases in cultured rat epididymal cells: enzyme activity, synthesis and secretion

During transit through the epididymis, spermatozoa acquire fertilizing the cell surface exhibits an altered glycoprotein pattern. Epididymal cells and their secretions contribute to these sperm-surface changes. To examine this process, epithelial cells from rat caput and cauda epididymidis were cultured and examined for the synthesis, processing and secretion of two glycoprotein-modifying enzymes, beta-galactosidase and beta-glucuronidase. Cells were cultured four days, incubated with D-2-[3H] mannose and L-[35S] methionine, and placed in isotope-free media. Levels of both cellular and secreted beta-galactosidase and beta-glucuronidase were determined by immunoprecipitation of cell homogenates or medium, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and scintillation counting of bands. During a 1-h pulse, both caput and cauda cells synthesize two precursor forms of beta-galactosidase (Mr = 84,000 and 87,000), which are processed to the mature (Mr = 63,000) enzyme during a 24-h chase. Caput cells release a high molecular weight (HMW) form (Mr = 90-100,000) and mature beta-galactosidase into the media, but not the Mr = 84-87,000 precursor. On the other hand, cauda cells release mostly mature beta-galactosidase. Ratios of radiolabeled mannose/methionine demonstrate a 7-fold greater mannose content in the cellular precursor of beta-galactosidase than in total protein. Another glycosidase, beta-glucuronidase, is synthesized as a Mr = 78,000-precursor which is processed to the mature Mr = 72,000 form. Medium in which caput and cauda cells were cultured contains both mature enzyme and a Mr = 94,000 form, but no 78,000-precursor form. Ratios of radiolabeled mannose/methionine in the cellular precursor of beta-glucuronidase are 2-fold greater than ratios in the total glycoprotein. Secretion is the major pathway of turnover for several epididymal glycosidases, since more than 50% of the total is secreted/day. These results indicate that cultured epithelial cells from the epididymis synthesize glycosidases and that processing and release differ, depending on the enzyme and the epididymal segment from which the epithelial cells were isolated.     

Isolation, culture and characteristics of epididymal epithelial cells from adult cats

A tissue-culture system in which cells retain defined ultrastructural and functional characteristics was established to provide a basis for functional investigations of the epididymal duct in the cat. A widely used culture protocol for rat epididymal epithelium was used as a starting point and subsequently modified. The cellular population of the cat's epididymal epithelium was isolated by successive collagenase and trypsin digestion. A high yield of isolated cells obtained with good viability, were cultured in DMEM/F12 medium supplemented with foetal bovine serum, in absence or in presence of additional dihydrotestosterone (1 nM). The plated primary cultures reached confluence within 5-8 days, producing a monolayer of cohesive cells. Samples taken after 6 days in culture were processed for transmission and scanning electron microscopies. Immunocytochemical staining was used to estimate the purity of the epithelial cell population in the monolayers. The cell cultures displayed several functional traits of in vivo epithelia, including [35S] hypotaurine and [35S] taurine production. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually mature cats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell functions, metabolic activities and their regulation in cats.     

Possible involvement of microtubules and microfilaments of the epididymal epithelial cells in 17beta-estradiol synthesis

The rat epididymal epithelial cells revealed features of steroidogenic cells and released 17beta-estradiol (E2) into the culture medium. In steroidogenic cells, elements of the cytoskeleton due to their influence on organelle distribution are implicated in the regulation of steroidogenesis. In the present study, the morphology of cultured epididymal epithelial cells in light, scanning and transmission electron microscopes was evaluated. The organization of microtubules and microfilaments revealed by fluorescence microscopy, and the concentration of E2 in cultured medium were also studied. The epididymal epithelial cells were cultured in different conditions: in the medium with or without exogenous testosterone (T) and in the co-culture with Leydig cells as a source of androgens. The cells in co-culture located close to Leydig cells were rich in glycogen, PAS-positive substances and lipid droplets, in higher amount than the cells cultured with addition of exogenous testosterone. Stress fibers and microtubules of epididymal epithelial cells cultured with exogenous T and in co-culture with Leydig cells presented typical structure, and numerous granular protrusions appeared on the surface of the cells. Disorganization of microtubules and shortening of stress fibers as well as the smooth cell surface deprived of granular protrusions were observed in the epididymal epithelial cells cultured without T. Change of the cytoskeleton organization caused by the absence of androgen in culture medium resulted in an increased E2 secretion.     

Evidence for epithelial-mesenchymal interactions mediating glucocorticoid effects in developing chick liver

When trypsin-dissociated liver cells from 17-day chick embryos were grown in regular minimum essential medium, mixed hepatocyte-fibroblast cultures resulted. When D-valine was substituted for L-valine in this medium, fibroblast growth was suppressed, leaving virtually pure hepatocyte cultures. Tyrosine aminotransferase activity is induced by cortisol in mixed cultures. No induction of enzyme activity is observed with cortisol exposure to hepatocytes, grown in D-valine. However, when cortisol-containing medium is conditioned by pre-incubation with mixed cells and then transferred to hepatocytes, tyrosine aminotransferase activity is induced. Enzyme activity is also induced in mixed cells incubated in D-valine medium in the presence of cortisol. It appears that a substance produced in the presence of fibroblasts exposed to cortisol is capable of inducing tyrosine aminotransferase activity in hepatocytes. This activity, which we have termed fibroblast hepatocyte factor, is heat stable, of low molecular weight, and antigenically different from fibroblast pneumonocyte factor, a factor similar to that produced by lung fibroblasts exposed to cortisol.     

Preliminary studies on the cultivation and characterization of mini-pig prostate epithelial cells

Mini-pig prostate epithelial cells exhibited the unique metabolic characteristics associated with the specialized function of production and secretion of high levels of citric acid. Epithelial cell suspensions from mini-pig prostate were successfully grown in primary and secondary cultures. The cultured epithelial cells exhibited rapid proliferation reaching confluency in approximately 6 days. Growth and proliferation of fibroblasts were markedly restricted by the dominance of epithelial cell growth. Confluent cultures could be maintained for approximately 6 weeks. The epithelial cells retained their polymorphic appearance in primary and secondary cultures and exhibited the characteristic formalin-resistant acid phosphatase reaction. Testosterone stimulated mitochondrial aspartate aminotransferase (mAAT) activity and citrate production by confluent epithelial cell cultures. These initial results indicate that cultured epithelial cells derived from mini-pig prostate might be an excellent model related to human for studies of prostate biology and hormonal regulation.     

D-valine as a selective agent for normal human and rodent epithelial cells in culture.

A nutrient medium has been developed to enable the growth of normal epithelial cells while selectively inhibiting fibroblast proliferation. In this medium, D-valine is substituted for L-valine; and only those cells containing D-amino acid oxidase can convert the D-amino acid into its essential L-enantiomer. The ability to select for cells with this enzyme has enabled us to maintain epithelial cell populations free from fibroblast overgrowth. The presence of D-amino acid oxidase has been histochemically confirmed in the epithelial cells selected from renal cell suspensions and explants. The ability to proliferate in the selective medium is transmitted to the clonal progeny of these cells. Moreover, epithelial cell proliferation of this medium indicates the presence of D-amino acid oxidase, which we have detected in tissues where it had not previously been reported-fetal human kidney, lung, and cord. Fibroblasts will not grow in the selective medium, but will proliferate normally if the product of the D-amino acid oxidase reaction is supplied.     

Steroidogenic characteristics of in vitro cultured epididymal epithelial cells of the rat

The paper presents the steroidogenic features of cultured epithelial cells of rat epididymis and their ability to synthesize steroid hormones. The cytoplasm of epididymal epithelial cells accumulated lipid droplets and contained active enzymes of steroidogenesis. Numerous mitochondria with lamellar cristae occurred near lipid droplets. Frequently, mitochondria formed a direct contact with lipid droplets and smooth endoplasmic reticulum. The hormone assay showed that the epididymal epithelial cells cultured without dihydrotestosterone synthesized and released the following steroids: dehydroepiandrosterone (DHEA), testosterone (T) and 17beta-estradiol (E). The levels of DHEA and T were very low. The concentration of E detected in media of cultured epididymal epithelial cells exceeded many times the concentration of E in control media. The cytoplasmic presence of organelles and enzymes that participated in the steroid synthesis indicated their similarity to steroidogenic cells. Epididymal epithelial cells were capable of moderate in vitro synthesis of androgens. It cannot be excluded that steroidogenesis in the cultured epididymal epithelial cells is maintained to sustain 17beta-estradiol synthesis pathways.     

In vitro culture of epithelial cells from the caput, corpus, and cauda epididymis of Sus domesticus

This work describes a protocol to culture epididymal epithelial cells from the caput, corpus, and cauda regions of Sus domesticus. Epididymal epithelial fragments were obtained by dissection and enzymatic digestion with collagenase. About 30 epididymal fragments from each epididymal region were cultured in 24-well culture plates with supplemented RPMI-1640 medium at 37 degrees C, 5% CO2 in air, and 100% humidity. A confluent monolayer of polygonal and tightly packed epithelioid cells from the three epididymal regions was obtained after 12-16 days in culture and maintained in vitro for more than 60 days. The proportion of epididymal epithelial cells in these cultures was assessed by immunofluorescent staining for cytokeratins. Throughout the 2 months of culture, about 80% of the cells were cytokeratin-positive. Electron microscopy observations indicated that cultured cells from caput, corpus, and cauda epididymal regions were tightly adhered to each other by junctional complexes and that stereocilia were present in their apical membranes. Moreover, the presence of an extensive rough endoplasmic reticulum, Golgi apparatus and numerous vesicles in the cytoplasm suggested that cultured cells maintained secretory and absorptive activities. These results show that the epididymal epithelial cells in culture from S. domesticus retain some fundamental features that characterize the epididymal epithelium in the intact organ. This system might be a valuable tool for studying the mechanism of sperm maturation in vitro, including epididymal cell secretions and the analysis of regional differences.     

Functional characterization of a conditionally immortalized mouse epididymis caput epithelial cell line MEPC5 using temperature-sensitive simian virus 40 large T-antigen

A conditionally immortalized epididymis caput cell line, MEPC5, was established by infecting primary cultured mouse epididymis caput cells with a temperature-sensitive simian virus 40 large T-antigen. At a permissive temperature of 33 degrees C, the large T-antigen was expressed and the cells grew continuously. However, the downregulation of T-antigen at a nonpermissive temperature of 39 degrees C and the upregulation of cell density at 33 degrees C were associated with growth arrest and the increased protein expression of p21(waf1), a cell cycle inhibitor. The cells expressed epididymal caput-expressed genes such as phosphatidylethanolamine binding protein, polyoma enhancer activator 3, ME1, sulfated glycoprotein-2 (SGP-2), androgen receptor, and retinoic acid receptor alpha. Interestingly, the expression levels of ME1 and SGP-2 were significantly elevated under the cell growth-restricted conditions. The established mouse epididymis caput epithelial cell line MEPC5 retains some characteristics of differentiated epididymis epithelial cells, and should prove an excellent model for studies of gene expression and the physiological functions of epididymis caput epithelial cells.     

gamma-glutamyl transpeptidase and related enzyme activities inthe reproductive system of the male rat.

The γ-glutamyl transpeptidase activity of the epididymis is much higher than that of the several other organs of the reproductive system of the male rat. The epididymal caput has much more activity than the epididymal cauda. Relatively low activity was found in spermatozoa. The enzyme is present in the epididymal fluid in a particulate form suggesting that it originates from membranes of epididymal epithelial cells. The epididymal caput exhibits high γ-glutamylcysteine synthetase activity indicating an active γ-glutamyl ycle in this tissue, which plays an important role in transport phenomena.