Ovarian substance induces steroid production in cultured granulosa cells

Summary The rat ovary produces an apparently low molecular weight substance that mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induces temporal cell rounding and, later on, intensive progestin production. However, unlike FSH, OS does not induce accumulation of cyclic AMP (cAMP) in the granulosa cells. The ovarian factor cannot be cAMP as its action is not abolished by phosphodiesterase (PDE) treatment. Neither is it a possible PDE inhibitor, as it does not augment cAMP accumulation in granulosa cells or Friend erythroleukemic cells induced by FSH or PGE₁, respectively. The factor is still active after heating for 20 min at 90° C but is rapidly inactivated by alkali treatment. In addition, treatment with various proteases did not abolish the steroidogenic activity. These findings suggest a possible novel intraovarian regulator of the granulosa cell function. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by the United States-Israel Binational Science Foundation, Grant 2656/81. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Promotion of mitosis in cultured newt limb regenerates by a diffusible nerve factor

Summary Regeneration blastemata of adult newt forelimbs were cultured transfilter to dorsal root ganglia on extremely low porosity (0.05 μm) filters. Mitotic index profiles in these blastemata were compared with those obtained using filters of greater porosity (0.45 μm). In the above experiments nerves and blastema tissue were separated by 5 or 25 μm, i.e., the thickness of the respective filters. The results show that the transfilter mitogenic effect of the nerves was retained when the lower pore size filters were used. In addition, sensory ganglia grown at the bottom of a culture well, separated from the blastema explants by a distance of approximately 2 mm, were nevertheless able to promote blastema cell proliferation. The ganglia can thus be considered to be providing a “sustained conditioning” of the medium with neuromitogenic factor(s). This study also shows that nerves can promote blastema cell mitosis, although cell-to-cell contact between nerves and responding cells was precluded. This work was supported by National Science and Engineering Research Council (NSERC) Grants A6933 to M. Globus and A9753 to S. Vethamany-Globus.     

Guidance of cortical radial migration by gradient of diffusible factors

During the development of cerebral cortex, newborn pyramidal neurons originated from the ventricle wall migrate outwardly to the superficial layer of cortex under the guidance of radial glial filaments. Whether this radial migration of young neurons is guided by gradient of diffusible factors or simply driven by a mass action of newly generated neurons at the ventricular zone is entirely unknown, a potential guidance mechanism that has long been overlooked. Our recent study showed that a guidance molecule semaphorin-3A, which is expressed in descending gradient across cortical layers, may serve as a chemoattractive guidance signal for radial migration of newborn cortical neurons toward upper layers. We hypothesize the existence of four groups of extracellular factors that can guide the radial migration of young neurons: (1) attractive factors expressing in superficial layers of cortex, (2) repulsive factors enriched in the ventricular zone, (3) pro-migratory factors uniformly expressed in all cortical layers and (4) stop signals locally expressed in the outmost layer of cortex.Key words: radial migration, cortex, guidance, semaphorin, diffusible factors, growth coneThe mammalian cerebral cortex has the typical laminar structure, the formation of which is essential for neurons in each cortical layer to establish the specific input and output connections with other brain regions. The development of the cortical laminar structure is known to involve the well-coordinated radial migration of newborn pyramidal neurons during development.¹ After young neurons are generated from the ventricular zone (VZ) and subventricular zone (SVZ), they leave their birthplace and migrate along radial glial filaments toward the surface of cortical plate (CP), crossing existing cortical layers composed of earlier born neurons and eventually settling down beneath the marginal zone (MZ, layer I).^1–3 It is generally accepted that the adhesion between neurons and radial glial filaments provides the directionality for these young neurons, and the targeting of neurons to specific lamina was controlled by the selective detachment of migrating neurons from radial glial fibers upon reaching the designated cortical layer.^2,3 However, we believe that the radial glial fibers can only serve as the adhesive scaffold for migrating neurons and constrain their migration in the radial dimension; it remains an open question regarding the nature of the signals that cause newborn neurons to migrate consistently outward along the fiber rather than inward. Whether the radial migration of cortical neurons is guided by gradient of diffusible factors or simply driven by a mass action of newly generated neurons at the VZ is entirely unknown, a potential guidance mechanism that has long been overlooked.Recently we found that the radial migration of layer II/III cortical neurons during development is guided by an extracellular guidance molecule semaphorin-3A (Sema3A).⁴ We observed that Sema3A is expressed in a descending gradient across the cortical layers, whereas its receptor neuropilin-1 (NP1) is expressed at a high level in migrating neurons. By in utero electroporation, we were able to monitor the migration of a subpopulation of cortical neurons in their native environment and examine the effect of perturbing Sema3A signaling. We found that downregulation or conditional knockout of NP1 in young neurons impeded their radial migration with severe misorientation of affected neurons during their migration without altering their cell fate. Studies in cultured cortical slices further showed the requirement of the endogenous gradient of Sema3A for the proper migration of newborn neurons. Results from transwell chemotaxis assays in dissociated culture of newborn cortical neurons also supported the notion that Sema3A attracts the migration of these neurons through the receptor NP1. Thus, Sema3A may serve as a chemoattractive guidance signal for the radial migration of newborn cortical neurons toward upper layers. This is the first demonstration that radial migration of cortical neurons is guided by gradient of extracellular guidance factors. This study also suggests that guidance factors may guide the radial migration by their actions on the growth cone of the leading process of migrating neurons, via mechanisms similar to that found for their actions on axon guidance and dendritic orientation, followed by long-range cytoplasmic signaling that coordinates the forward motility of the entire neuron.⁵In this study, we have only observed an attractive effect of Sema3A in the radial migration of the layer II/III cortical neurons. However, to form the highly ordered laminar structure of the cortex, the entire process of neuronal migration is likely to depend on coordinated actions of multiple factors in the developing cortex, including other semaphorin family members and other guidance molecules, e.g., slits⁶ and ephrins,⁷ which are also expressed in the CP. We hypothesize that four groups of extracellular factors orchestrate to promote the proper radial migration and cortical lamination: (1) factors that are expressed in superficial layers of cortex and in a descending gradient, like Sema3A, may attract the upward migration of newborn neurons (attractive factors), (2) factors enriched in the VZ may exert repulsive action and help to “push” newborn neurons out of their birthplace (repulsive factors), (3) those factors widely expressed in all cortical layers may promote the motility of migrating neurons (pro-migratory factors) and (4) Some repulsive cues may be locally expressed in the superficial layer of cortex to prevent the over migration of neurons when they have arrived at the outmost layer (stop signal). Under the guidance of these four groups of factors, newborn neurons migrate all the way from VZ to the outmost layer of CP and then settle down. One of our recent tasks is to try to identify these four groups of factors.If the radial migration and cortical lamination are guided by diffusible factors, why is radial glial system necessary for this migration process? In other words, why earlier-born neurons in different layers cannot provide the supportive adhesion to young neurons during their radial migration? A potential explanation is that neurons in cortex undergo maturation after terminating their migration, accompanying with changes in their expression profiles of adhesion ligands, and become less and less supportive to the neuronal migration. In contrast, as a kind of cortical progenitor cells, radial glial cells maintain a relatively ‘young’ state and continue to express supportive adhesion ligands over a very long developmental stage. Thus, only the radial glial filament is capable of providing a bridge for newborn neurons to migrate over a very long distance across the non-permissive cell layers. In summary, we believe that during the cortical radial migration, signals from diffusible factors override the adhesive signal from radial glial fibers to promote the appropriate migration and placement of newborn neurons.? Open in a separate windowFigure 1A schematic diagram for the guidance of cortical radial migration by diffusible factors. (A) A model for the distribution of four groups of guidance factors in developing cortex. Radial glial filaments are shown in red, young neurons are in green. There may exist a descending gradient of attractive factors in upper cortical layers (yellow) and an ascending gradient of repulsive factors (blue) near the ventricular zone (VZ). Stop signals (purple) may come from the surface of cortex, and pro-migratory factors (dots) may be widely distributed. (B) Representative image of EGFP-labeled neurons migrating along radial glial filaments in the cortical tissue of E20 mouse. Sections were counterstained with DAPI (Red). Scale bar, 100 µm.     

Guidance of cortical radial migration by gradient of diffusible factors

During the development of cerebral cortex, newborn pyramidal neurons originated from the ventricle wall migrate outwardly to the superficial layer of cortex under the guidance of radial glial filaments. Whether this radial migration of young neurons is guided by gradient of diffusible factors or simply driven by a mass action of newly generated neurons at the ventricular zone is entirely unknown, a potential guidance mechanism that has long been overlooked. Our recent study showed that a guidance molecule semaphorin-3A, which is expressed in descending gradient across cortical layers, may serve as a chemoattractive guidance signal for radial migration of newborn cortical neurons toward upper layers. We hypothesize the existence of four groups of extracellular factors that can guide the radial migration of young neurons: (1) attractive factors expressing in superficial layers of cortex, (2) repulsive factors enriched in the ventricular zone, (3) pro-migratory factors uniformly expressed in all cortical layers, and (4) stop signals locally expressed in the outmost layer of cortex.     

A simple and efficient procedure for the rapid homogenization of cultured animal cells grown in monolayer

Cell swelling takes place rapidly when animal cells in monolayer culture are treated with hypotonic buffer in situ; scraping of the swollen cells causes virtually 100% cell lysis. Because this procedure avoids the use of scraping and centrifugation to collect the cells and the use of Dounce homogenizers for cell disruption, recovery of the cell extract is very high. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activities of cell extracts prepared by this method are virtually identical to those prepared by the conventional procedure involving Dounce homogenization.     

The influence of transcortin on adrenocorticotropin-stimulated corticosterone production in monolayer cultured rat adrenal cells

To verify the influence of the protein binding status of steroids adjacent to adrenal cells on steroidogenesis, the effect of transcortin, a specific binding protein of cortisol or corticosterone, on adrenocorticotropin (ACTH)-stimulated corticosterone production in monolayer cultured rat adrenal cells was studied. The transcortin in concentration of 5 x 10(-7) M was loaded with 0, 2.5, 5 and 10 pg/ml ACTH-(1-24), and the cells were incubated for 2 and 4 hours. Since molar concentrations of corticosterone produced in the medium were below the transcortin concentration at all levels of stimulation, protein-unbound corticosterone in the medium may have been largely reduced by the addition of transcortin. However, the total corticosterone production was not influenced by the transcortin added to the medium. It was speculated that protein-unbound steroid within the concentration range modulated by transcortin in the area surrounding the adrenal cells may not affect adrenal steroidogenesis.     

A novel diffusible substance can overcome the apparent AbrB repression of the Bacillus subtilis fatR promoter

In this work we present evidence for a novel diffusible extracellular factor that modulates gene expression in Bacillus subtilis. The factor was found when studying the regulation of the fatR-cyp102A3 operon. In a Spo0A(-) mutant expression of the fatR-cyp102A3 operon was almost abolished. The fatR-cyp102A3 expression defect of a Spo0A(-) mutant could be overcome either by a mutation in the abrB gene or by a diffusible substance excreted by wild-type, abrB mutant and abrB-spo0A double mutant strains.     

Extracellular metabolism-dependent uptake of lysolipids through cultured monolayer of differentiated Caco-2 cells

Glycerophospholipids are known to be hydrolyzed in the intestinal lumen into free fatty acids and lysophospholipids that are then absorbed by the intestinal epithelial cells. A monolayer of enterocyte-differentiated Caco-2 cell is often used to assess the intestinal bioavailability of nutrients. In this study, we examined how differentiated Caco-2 cells process lysoglycerolipids such as lysophosphatidylcholine (LPC). Our findings were twofold. (1) Caco-2 cells secreted both a lysophospholipase A-like enzyme and a glycerophosphocholine-phosphodiesterase enzyme into the apical, but not basolateral, lumen, suggesting that food-derived LPC is converted to a free fatty acid, sn-glycerol-3-phosphate, and choline through two sequential enzymatic reactions in humans. The release of the latter enzyme was differentiation-dependent. (2) Fatty acid-releasing activities toward exogenous fluorescent LPC, lysophosphatidic acid and monoacylglycerol were shown to be higher on the apical membranes of Caco-2 cells than on the basolateral membranes. These results suggest that human intestinal epithelial cells metabolize lysoglycerolipids by two distinct mechanisms involving secreted or apical-selective expression of metabolic enzymes.     

A cytolytic assay for the measurement of palytoxin based on a cultured monolayer cell line

A cytolytic assay that could detect palytoxin and its congeners has been developed by the use of an established cell line grown as monolayer to replace the current hemolytic method. We used MCF-7 cells and cytolysis was measured by the release of cytosolic lactate dehydrogenase (LDH) in the buffer added to treated cells (culture supernatant). A dose-dependent increase in LDH activity in culture supernatants was detected when MCF-7 cells were exposed to palytoxin and its analogue ostreocin D. The cytolytic response induced by palytoxin and ostreocin D was specific for this group of compounds, acting on Na+/K+-ATPase, as it was prevented when cells were preincubated with ouabain. The specificity of our assay for palytoxin and its congeners was confirmed by the finding that cytolysis was not detected when MCF-7 cells were exposed to unrelated toxins such as maitotoxin, tetrodotoxin, okadaic acid, and yessotoxin, even in the case of compounds that elicit cytotoxic responses under our experimental conditions. Using extracts from biological materials after spiking with the palytoxin standard, we found a good correlation between palytoxin levels measured by our cytolytic assay and the expected values. Our cytolytic assay detected palytoxin in naturally contaminated materials, but estimates were significantly higher than the palytoxin contents determined by LC-MS, indicating that naturally contaminated materials contain biologically active palytoxin congeners. We conclude that our cytolytic assay based on the use of MCF-7 cell monolayers is a viable alternative to animal-based methods for the determination of palytoxin and its congeners in contaminated materials.     

Transcellular transport of angiotensin II through a cultured arterial endothelial monolayer

We have studied the mechanisms of angiotensin II (A-II) transport through a cultured arterial endothelial cell monolayer. The transport of 125I-labeled A-II was inhibited by excess unlabeled A-II (50 microM) and [Sar1, Ile8]-A-II (50 microM), but was not inhibited by bradykinin (50 microM). The transport process was shown to be temperature dependent and was inhibited by 10 mM NaN3 plus 50 mM 2-deoxyglucose. Monensin (50 microM), an inhibitor of endocytotic trafficking, reduced the rate of transport of 125I-A-II. It is also shown that the specific pathway for A-II transport was unidirectional from the apical to the basolateral surface of the endothelial cell monolayer.     

Relationship between reaggregability of monolayer cultured chick embryonic liver cells and their surface properties

When liver cells dissociated from 10-day-old chick embryos were cultured as monolayers, the reaggregability of the harvested cells declines steeply with time of cultivation. Immunological and virological techniques were used to detect cell surface changes during monolayer cultivation. An early rapid increase in Forssman (F) antigen was demonstrated by the complement fixation test and the fluorescent antibody technique. An increase parallel to that of F antigen was also found, using the hemagglutination inhibition (HI) test, in the receptor activities of these cultured cells for Japanese encephalitis (JE) virus and influenza virus. The reaggregability recovered with receptor-destroying enzyme (RDE) from Vibrio chorelae. Therefore, we concluded that a cell surface change or aberration, recognized as an increase in the biologically identifiable determinant sites such as F antigen and virus receptors, was responsible for the loss of reaggregability. In contrast, we confirmed that the intracellular machinery required for JE virus multiplication never changed during protracted monolayer cultivation.     

Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts.

The methodology has been developed to measure Cl activity and transport in cultured cells grown on a monolayer using the entrapped Cl-sensitive fluorophore 6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ). The method was applied to a renal epithelial cell line, LLC-PKI, and a nonepithelial cell line, Swiss 3T3 fibroblasts. SPQ was nontoxic to cells when present for greater than h in the culture media. To load with SPQ (5 mM), cells were made transiently permeable by exposure to hypotonic buffer (150 mOsm, 4 min). Intracellular fluorescence was monitored continuously by epifluorescence microscopy using low illumination intensity at 360 +/- 5 nm excitation wavelength and photomultiplier detection at greater than 410 nm. Over 60 min at 37 degrees C, there was no photobleaching and less than 10% leakage of SPQ out of cells; intracellular SPQ fluorescence was uniform. SPQ fluorescence was calibrated against intracellular [Cl] using high K solutions containing the ionophores nigericin and tributyltin. The Stern-Volmer constant (Kq) for quenching of intracellular SPQ by Cl was 13 M-1 for fibroblasts and LLC-PKl cells. In the absence of Cl, SPQ lifetime was 26 ns in aqueous solution and 3.7 +/- 0.6 ns in cells, showing that the lower Kq in cells than in free solution (Kq = 118 M-1) was due to SPQ quenching by intracellular anions. To examine Cl transport mechanisms, the time course of intracellular [Cl] was measured in response to rapid Cl addition and removal in the presence of ion or pH gradients. In fibroblasts, three distinct Cl transporting systems were identified: a stilbeneinhibitable Cl/HCO3 exchanger, a furosemide-sensitive Na/K/2Cl cotransporter, and a Ca-regulated Cl conductance. These results establish a direct optical method to measure intracellular [Cl] continuously in cultured cells.     

A diffusible signal from arbuscular mycorrhizal fungi elicits a transient cytosolic calcium elevation in host plant cells

The implication of calcium as intracellular messenger in the arbuscular mycorrhizal (AM) symbiosis has not yet been directly demonstrated, although often envisaged. We used soybean (Glycine max) cell cultures stably expressing the bioluminescent Ca(2+) indicator aequorin to detect intracellular Ca(2+) changes in response to the culture medium of spores of Gigaspora margarita germinating in the absence of the plant partner. Rapid and transient elevations in cytosolic free Ca(2+) were recorded, indicating that diffusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca(2+)-mediated signaling. Similar responses were also triggered by two Glomus isolates. The fungal molecules active in generating the Ca(2+) transient were constitutively released in the medium, and the induced Ca(2+) signature was not modified by the coculture of germinating spores with plant cells. Even ungerminated spores were able to generate the signaling molecules, as proven when the germination was blocked by a low temperature. The fungal molecules were found to be stable to heat treatment, of small molecular mass (<3 kD), and, on the basis of extraction with an organic solvent, partially lipophilic. Evidence for the specificity of such an early fungal signal to the AM symbiosis is suggested by the lack of a Ca(2+) response in cultured cells of the nonhost plant Arabidopsis (Arabidopsis thaliana) and by the up-regulation in soybean cells of genes related to Medicago truncatula DMI1, DMI2, and DMI3 and considered essential for the establishment of the AM symbiosis.     

Molecular sieving characteristics of the cultured endothelial monolayer

We examined the selectivity of the bovine pulmonary artery endothelial monolayer in vitro to molecules of different sizes. The cultured bovine pulmonary endothelial monolayer was grown on a gelatinized filter and the transendothelial transport was studied by determining the permeability of molecules ranging from 182 to 340,000 daltons under diffusion conditions. The permeabilities across the cultured bovine endothelium were modeled according to cylindrical pore theory. The data were best fit by a two-pore model with radii 65 A and 304 A and a ratio of small to large pores of 160:1. The results indicate that the cultured endothelial monolayer is a selective barrier to molecules of different sizes and that the molecular selectivity is consistent with a diffusional pathway through endothelial pore equivalents. The cultured endothelial monolayer is a useful system for studying the permeability characteristics of the endothelial barrier.     

Microfluidic perfusion for regulating diffusible signaling in stem cells

Background

Autocrine & paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonic stem cell (ESC) pluripotency and lineage commitment. Although autocrine signaling via fibroblast growth factor-4 (FGF4) is known to be required in mouse ESC (mESC) neuroectodermal specification, the question of whether FGF4 autocrine signaling is sufficient, or whether other soluble ligands are also involved in fate specification, is unknown. The spatially confined and closed-loop nature of diffusible signaling makes its experimental control challenging; current experimental approaches typically require prior knowledge of the factor/receptor in order to modulate the loop. A new approach explored in this work is to leverage transport phenomena at cellular resolution to downregulate overall diffusible signaling through the physical removal of cell-secreted ligands.

Methodology/Principal Findings

We develop a multiplex microfluidic platform to continuously remove cell-secreted (autocrine\paracrine) factors to downregulate diffusible signaling. By comparing cell growth and differentiation in side-by-side chambers with or without added cell-secreted factors, we isolate the effects of diffusible signaling from artifacts such as shear, nutrient depletion, and microsystem effects, and find that cell-secreted growth factor(s) are required during neuroectodermal specification. Then we induce FGF4 signaling in minimal chemically defined medium (N2B27) and inhibit FGF signaling in fully supplemented differentiation medium with cell-secreted factors to determine that the non-FGF cell-secreted factors are required to promote growth of differentiating mESCs.

Conclusions/Significance

Our results demonstrate for the first time that flow can downregulate autocrine\paracrine signaling and examine sufficiency of extracellular factors. We show that autocrine\paracrine signaling drives neuroectodermal commitment of mESCs through both FGF4-dependent and -independent pathways. Overall, by uncovering autocrine\paracrine processes previously hidden in conventional culture systems, our results establish microfluidic perfusion as a technique to study and manipulate diffusible signaling in cell systems.     

Retraction of cultured endothelial cell monolayer by human breast cancer cells, MCF-7.

A human breast cancer cell line (MCF-7), when sealed on confluent bovine pulmonary aortic endothelial cell (CPAE) monolayers, induced morphological changes (retraction) in CPAE cells. The area of retraction depended on the incubation time and the number of MCF-7 cells, suggesting that MCF-7 cells had the capacity to retract CPAE cells. This capacity was reduced by 60% by pretreatment of MCF-7 cells with 17β-estradiol (E) and progesterone (Pg). The extent of retraction was not affected by the addition of various protease inhibitors. CPAE retraction was induced also by adding conditioned medium (CM) from the culture of MCF-7 cells. Considerably less activity was detected in the CM obtained from MCF-7 cells cultured in the presence of E and Pg. The retraction was reversed in 24 h by culturing the monolayer in fresh medium without CM and was not induced by trypsin treatment of the CM.