Effect of basal lamina of ovarian follicle on T- and L-type Ca2+ currents in differentiated granulosa cells

Elikplimi K. Asem1, Wuxuan Qin1, and Stanley G. Rane2

1 Department of Basic Medical Sciences, School of Veterinary Medicine, and 2 Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Patch clamp experiments were conducted to study the effects of basal lamina (basement membrane) of chicken ovarian follicle on membrane Ca2+ currents in differentiated chicken granulosa cells in a homologous system. The whole cell patch clamp technique was used to simultaneously monitor membrane capacitance (an indirect measure of total cell surface area) and currents flowing through voltage-dependent Ca2+ channels (using Ba2+ as the charge carrier). Membrane capacitance was smaller in cells incubated on intact basal lamina than in control cells (incubated on tissue culture-treated plastic substratum). Granulosa cells expressed both T- and L-type Ca2+ currents, and the amplitudes of the currents in cells incubated on intact basal lamina were significantly lower than those of control cells. Also, granulosa cells incubated on intact basal lamina were found to have significantly lower T- or L-type Ca2+ current densities than control cells. Intact basal lamina that had been stored for 12 mo produced effects on T- and L-type Ca2+ currents similar to those caused by freshly isolated basal lamina. The basal lamina was solubilized completely in one step and used to coat glass coverslips (uncoated glass coverslips served as controls). Granulosa cells incubated on coverslips precoated with solubilized basal lamina assumed spherical shape similar to those incubated on intact basal lamina. Similar to the observations made for intact basal lamina, the solubilized basal lamina suppressed T- and L-type Ca2+ currents in the differentiated granulosa cells. Moreover, fibronectin, laminin, and type IV collagen, obtained from commercial sources, attenuated T- and L-type Ca2+ currents in the differentiated granulosa cells. This interplay between basal lamina and Ca2+ currents may be one mechanism that subserves the effects of the matrix material on metabolic functions of granulosa cells.

basement membrane; extracellular matrix; calcium current; calcium channel; patch clamp; ovary; chicken; fibronectin; laminin; type IV collagen


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BASEMENT MEMBRANES (basal laminae) are specialized extracellular matrix sheets that provide mechanical support and important signals for growth and differentiation to cells with which they are associated (72). The components of basement membrane obtained from nonovarian sources regulated the morphology and steroidogenesis in granulosa cells from rat (1-3, 9, 28) and human (27) ovaries. Similarly, it was shown recently that basal lamina of the chicken ovarian follicle regulated cell shape and progesterone synthesis in chicken granulosa cells (5, 7). However, the signaling mechanisms involved in the actions of basement membrane (basal lamina) in granulosa cells are not fully known.

The results of studies with porcine (63), rat (22, 61), or chicken (6) granulosa cells showed that basal and gonadotropin-stimulated steroid hormone biosynthesis required the obligatory presence of Ca2+ in the external medium. In addition, external Ca2+ is required for steroid hormone-induced protein synthesis in avian granulosa cells (17). Experiments with radioisotopes demonstrated transmembrane Ca2+ exchange in porcine granulosa cells (62), and studies with Ca2+-sensitive fluorescent dyes revealed Ca2+ influx into rat (66), chicken (32, 49), and porcine (23-25) granulosa cells. Furthermore, experiments conducted with patch clamp techniques showed that avian and mammalian granulosa cells express Ca2+-specific channels (57) and Ca2+ currents (14, 38, 58, 65).

Because basement membrane/basal lamina is an important regulator of progesterone synthesis in granulosa cells (1-3, 5, 9) and because external Ca2+ is required for the regulation of metabolic functions in granulosa cells, it was hypothesized that basal lamina modulates the functions of Ca2+ channels in granulosa cells. To examine the effects of basal lamina on transmembrane Ca2+ transport in granulosa cells in a homologous system, it is necessary to obtain a pure preparation of intact basal lamina from the ovarian follicle. The avian ovarian follicle was used as a model system because its unique anatomic structure permitted the isolation of pure and intact basal lamina and granulosa cells. In the avian ovarian follicle, the granulosa layer (membrana granulosa) consists of a single layer of granulosa cells located between the basal lamina and the perivitelline layer (10, 52, 53), making possible the isolation of intact basal lamina in a hypotonic solution (7). In the present study, the effect of basal lamina (obtained from the largest preovulatory chicken follicle) on differentiated granulosa cells (isolated from the preovulatory follicle) was examined in a homologous system.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals. HEPES, collagenase type IV, soybean trypsin inhibitor, trypsin, BSA (fraction V), penicillin G, streptomycin, fungizone, aspartic acid, N-methyl-D-glucamine (NMDG), adenosine-triphosphate-magnesium (ATP-Mg), tetraethyl ammonium chloride (TEA), nifedipine, EGTA, mouse type IV collagen, mouse laminin, cycloheximide, actinomycin D, inorganic salts, and Trizma base were purchased from Sigma Chemical (St. Louis, MO). Medium 199 (M199), containing Hanks' salts, was from GIBCO-BRL (Grand Island, NY). Flunarizine was obtained from Calbiochem (San Diego, CA). Human cellular fibronectin was obtained from Upstate Biotechnology (Lake Placid, NY).

Animals. Single-Comb White Leghorn hens, obtained from Purdue University Poultry Research Farms (West Lafayette, IN) in their first year of reproductive activity, were caged individually in a windowless, air-conditioned room with a 14:10-h light-dark cycle. They had free access to a layer ration and tap water. The time of egg lay of each bird in the colony was noted daily to the nearest 30 min. The layers were injected with ketamine (50 mg/kg body wt) 10 min before hens were killed by cervical dislocation ~10-12 h before the expected time of ovulation of the largest preovulatory follicle (F1). The F1 was removed and placed in ice-cold Hanks' basic salt solution (HBSS) containing 140 mM NaCl, 5 mM KCl, 1.1 mM MgCl2, 2.5 mM CaCl2, 10 mM HEPES, and 5.6 mM glucose (pH 7.4). The theca and granulosa cell layers (membrana granulosa) were separated by the method of Gilbert et al. (29).

Isolation of intact basal lamina. Basal lamina was isolated as previously described (7). Briefly, the granulosa cell layer obtained from the F1 was placed in a hypotonic solution containing 10 mM Tris · HCl (pH 7.4), 0.5 mg/l leupeptin, 1 mM EDTA-Na2, 0.7 mg/l pepstatin, and 0.2 mM phenylmethylsulfonyl fluoride in a petri dish. The granulosa cells, sandwiched between the basal lamina and perivitelline layer, were lysed hyposmotically, and the basal lamina and perivitelline layer were separated. This basal lamina of avian ovarian follicle (BLAOF) preparation is intact and complete. The side of basal lamina that was in contact with granulosa cells in situ was designated the "granulosa side," and the side in contact with theca tissue was designated the "theca side."

Solubilization of the basal lamina. Basal laminae were solubilized in one step. They were placed in a microfuge tube, and solubilization buffer containing 6 M guanidine-HCl, 50 mM Tris · HCl (pH 7.4), and 5% beta -mercaptoethanol was added (100 µl per basal lamina). After shaking for 60 min at 4°C, the entire basal lamina was solubilized (referred to as "total fraction"). Exclusion of beta -mercaptoethanol from the solubilization buffer led to incomplete solubilization of the basal lamina (fragments remained). The solubilized material was placed in a 3-kDa cutoff dialysis membrane and dialyzed against 150 mM NaCl and 50 mM Tris · HCl (pH 7.4) at 4°C overnight; it turned cloudy because of precipitation of proteins.

Preparation of intact basal lamina-containing dishes for cell culture. For experiments designed to study the effects of intact basal lamina on membrane currents, pieces of the intact basal lamina (2-3 cm2) were spread in 35-mm tissue culture dishes (Corning, Corning, NY) and allowed to dry in a laminar flow hood. Blank tissue culture-treated plastic substratum (plastic substratum) served as control. Unless stated otherwise, the experiments were conducted with cells incubated on the granulosa side of the intact basal lamina. The intact basal lamina-containing dishes were either used immediately or wrapped in aluminium foil and stored at 4°C. Tissue isolation and preparation of culture dishes were carried out under sterile conditions.

Preparation of solubilized basal lamina-coated coverslips for cell culture. The solubilized basal lamina (total fraction) was diluted with deionized water, and aliquots of 100-200 µl containing 5-50 µg of protein were spread on a 12-mm-round glass coverslip (Warner Instrument, Hamden, CT) and allowed to dry under a tissue culture hood (precoated coverslips). Glass coverslips that received only vehicle served as controls. The precoated coverslips were either used immediately or wrapped in aluminium foil and stored at 4°C. Tissue isolation, solubilization, dialysis, and preparation of coverslips were carried out under sterile conditions.

Preparation of fibronectin-, laminin-, and type IV collagen-coated dishes for cell culture. Fibronectin, laminin, or collagen IV was diluted with deionized water, and aliquots of 1-2 ml containing 5-50 µg of protein were spread in Corning 32-mm culture dishes and allowed to dry under a tissue culture hood (precoated culture dishes). Culture dishes that received only vehicle served as controls.

Granulosa cell culture. Granulosa cells were dispersed in M199 containing 350 mg/l NaHCO3, 10 mM HEPES, collagenase (500,000 U/l), and trypsin inhibitor (200 mg/l) at pH 7.4 (51). Cell viability, determined by the trypan blue exclusion method, was routinely >95%. Granulosa cells were plated in intact basal lamina-containing dishes or in 35-mm dishes containing coverslips that were precoated with the total fraction of solubilized basal lamina. The cells were incubated for 1-24 h at 37°C in serum-free M199 containing 0.1% (wt/vol) BSA, 350 mg/l NaHCO3, and HEPES 10 mM (pH 7.4) (51).

Electrophysiology. To record Ca2+ currents, the pipette contained 140 mM NMDG, 140 mM aspartate, 5 mM EGTA, 2 mM ATP-Mg, 1 mM MgCl2, and 10 mM HEPES at pH 7.2; the bath solution contained 127 mM NMDG, 127 mM aspartate, 0.086 mM CaCl2, 1 mM MgCl2, 5 mM TEA, 10 mM barium acetate, 5.6 mM glucose, and 10 mM HEPES at pH 7.4. The conventional whole cell recording method was used in this study. Recording pipettes were fabricated from borosilicate filament glass (Warner Instrument). Electrode resistance was between 3 and 9 MOmega in the bath solution. Unless noted otherwise, the linear (leak) component of the total membrane current was subtracted by extrapolating the linear currents obtained during voltage steps in more negative potential regions (-100 to -80 mV), where no voltage-activated currents were seen. Current amplitudes were small enough that the series resistance error was <5 mV. Analog compensation was applied to attenuate capacitive current transients and to estimate cell capacitances, a measure of total cell plasma membrane area. Membrane currents were measured with an Axopatch 1-D patch clamp (Axon Instruments, Foster City, CA) and filtered at 1 kHz. The currents were digitized and stored directly to disk (DigiData 1200 Interface, Axon Instruments). Data analysis was performed with pClamp 6.0.3 software (Axon Instruments).

In the conventional whole cell mode, the membrane potential was held at -80 mV. Membrane currents were elicited by test pulses of 333 ms in duration from -100 mV to +80 mV in 20-mV steps. All experiments were conducted at room temperature (21-23°C). Cells were perfused such that complete bath exchange was accomplished within 1 min. All bath solutions were at pH 7.4. For experiments designed to study the effects of solubilized basal lamina, the granulosa cells incubated on coverslips were transferred to a perfusion chamber (Warner Instrument) containing bath solution (see Electrophysiology). The capacity of the chamber was 180 µl.

Morphometric analysis of cells. Light-microscopic images of granulosa cells were collected from at least five identical locations of each incubation well or coverslip on an inverted Nikon microscope (20× objective) and stored. The outlines of individual cells were traced, and the parameters of mean surface area covered by each cell, cell perimeter, and circularity were determined with Optimas 6.0 Software (Bothell, WA). Higher estimates of circularity (which is independent of size) were associated with greater irregularity of cell profile. A perfect circle has a circularity of 12.

Determination of cell viability. Granulosa cells were incubated in 24-well culture plates precoated with solubilized basal lamina. Uncoated wells served as controls. After the removal of the serum-free M199 incubation medium, the cells were detached with trypsin (0.4 mg/ml) containing Ca2+ and Mg2+-free HBSS. The cells were washed two times with Ca2+-Mg2+-free HBSS, and viability was estimated with the trypan blue (0.1%) exclusion technique.

Data analysis. The data were analyzed by ANOVA followed by the post hoc Tukey test to determine significant differences among treatment means. A two-tailed t-test was performed where applicable. Differences at P < 0.05 were considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Collagenase-dispersed differentiated granulosa cells obtained from the F1 of hen ovary were incubated in serum-free medium for 1-24 h on tissue culture-treated plastic substratum (plastic substratum). Patch clamp experiments were conducted in the conventional whole cell configuration, with conditions under which primarily Ca2+ currents can be monitored. With a holding potential of -80 mV and step commands between -100 and +80 mV, inward currents were recorded in the differentiated granulosa cells. In ~32% of the cells studied, a fast activating and inactivating inward (transient) current was recorded (Fig. 1). The current was activated at -40 mV and required 8-13 ms to reach peak amplitude. In 52% of cells, a slowly inactivating, long-lasting current was superimposed on the fast activating/inactivating one (Fig. 2); this current remained steady, not returning to basal levels within the duration of the test pulse. The remainder of the cells (~15%) did not express inward currents under the present experimental conditions.


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Fig. 1.   Functional expression of only a fast activating and inactivating (transient) whole cell Ca2+ current in differentiated granulosa cells incubated on plastic. Granulosa cells isolated from the largest preovulatory follicle of hen ovary were incubated on plastic in serum-free Medium 199 (M199) for 24 h. Voltage protocol is shown above tracings. A: fast activating/inactivating inward currents recorded when cell was stimulated from a holding potential of -80 mV to a test potential of -20 mV with 333-ms command pulse. B: cell was stimulated from a holding potential of -50 mV to test potential of -20 mV. C: cell was stimulated from a holding potential of -80 mV to test potential of -20 mV in the presence of flunarizine (20 µM).



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Fig. 2.   Functional expression of both fast and slowly inactivating whole cell Ca2+ currents in differentiated granulosa cells incubated on plastic. Granulosa cells isolated from the largest preovulatory follicle of hen ovary were incubated on plastic in serum-free M199 for 24 h. Voltage protocol is shown above tracings. A: fast and slowly inactivating inward currents recorded when cell was stimulated from a holding potential of -80 mV to a test potential of -20 mV with 330-ms command pulse. B: cell was stimulated from a holding potential of -50 mV to test potential of -20 mV. C: cell was stimulated from a holding potential of -80 mV to a test potential of -20 mV in the presence of nifedipine (5 µM).

Characterization of the Ca2+ currents. The types of Ca2+ currents expressed by the granulosa cells were determined by modifying the holding potential and applying known Ca2+ channel blockers. When the holding potential was changed from -80 to -50 mV, the amplitude of the fast activating/inactivating (transient) current decreased dramatically (Figs. 1B and 2B); this is an indication that it is a T-type Ca2+ current. Indeed, the application of the T-type Ca2+ channel blocker flunarizine (20 µM) suppressed the fast activating transient current (Fig. 1C), supporting the view that it is a T-type Ca2+ current. In cells that expressed both transient and long-lasting inward currents, flunarizine (20 µM) blocked the transient current component but not the long-lasting current. The remaining current (long-lasting current) had activation/inactivation kinetics that are reminiscent of the L-type Ca2+ current (see Fig. 2B). The long-lasting current was suppressed by the L-type Ca2+ channel blocker nifedipine (5 µM) (Fig. 2C), demonstrating that it is an L-type Ca2+ current. In additional experiments, nifedipine blocked the long-lasting current component (Fig. 3B) but not the fast current component that could be blocked by flunarizine (Fig. 3C). The peak of the transient current was regarded as the amplitude of T-type current. The amplitude of the L-type current was measured at the end of the step potential.


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Fig. 3.   Effects of Ca2+ channel blockers on the fast and slowly inactivating whole cell Ca2+ currents in differentiated granulosa cells incubated on plastic. Granulosa cells isolated from the largest preovulatory follicle (F1) of hen ovary were incubated on plastic in serum-free M199 for 24 h. Voltage protocol is shown above tracings. A: fast and slowly inactivating inward currents recorded when cell was stimulated from a holding potential of -80 mV to a test potential of -20 mV with 330-ms command pulse. B: cell was stimulated from a holding potential of -80 mV to a test potential of -20 mV in the presence of nifedipine (5 µM) alone. C: cell was stimulated from a holding potential of -80 mV to a test potential of -20 mV in the presence of nifedipine (5 µM) and flunarizine (20 µM).

The Ca2+ currents appeared to be expressed to the same extent in granulosa cells incubated on plastic and glass substrates. When the same batches of granulosa cell preparations were used, the density of T-type current was 4.92 ± 0.60 pA/pF (n = 11 cells) and 4.55 ± 0.64 pA/pF (n = 11 cells) for cells incubated on plastic and glass, respectively; similarly, the density of L-type current was 1.02 ± 0.26 (n = 11) and 1.09 ± 0.24 (n = 11) for cells incubated on plastic and glass.

Effect of intact basal lamina on T- and L-type Ca2+ currents and membrane capacitance. Differentiated granulosa cells incubated on intact basal lamina for 24 h in serum-free M199 assumed spherical shape, whereas those incubated on plastic substratum in control dishes became highly flattened (see Fig. 7). The T-type Ca2+ current was suppressed in differentiated granulosa cells incubated on basal lamina (Fig. 4B). Similarly, the L-type Ca2+ current was inhibited in cells incubated on basal lamina (Fig. 4E). The current-voltage relation of T- and L-type Ca2+ currents demonstrates a reduction in current amplitudes at several voltages (Fig. 4, C and F). The amplitude of the T-type Ca2+ current (Fig. 5A) or L-type Ca2+ current (Fig. 5D) was significantly (P < 0.05) greater for cells incubated on plastic than for cells incubated on basal lamina. When the T- and L-type Ca2+ current amplitudes were normalized to plasma membrane area, the current densities in granulosa cells incubated on basal lamina were significantly (P < 0.05) smaller than those of cells incubated on plastic (Fig. 5, B and E). Membrane capacitance, an indirect measure of total plasma membrane area (and directly proportional to membrane area), was significantly (P < 0.05) smaller for cells incubated on intact basal lamina than for the control cells incubated on plastic (Fig. 5C).


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Fig. 4.   Effect of freshly prepared intact basal lamina on T- and L-type Ca2+ currents in differentiated granulosa cells. Granulosa cells isolated from the F1 of hen ovary were incubated on plastic or intact basal lamina of the F1 of hen ovary in serum-free M199 for 24 h. A: Ca2+ currents recorded in granulosa cells incubated on plastic (control cell) with a holding potential of -80 mV, test potentials between -100 and +80 mV, and 333-ms command pulse. B: Ca2+ currents recorded in granulosa cells incubated on basal lamina with a holding potential of -80 mV and a test potential of -100 to +80 mV. C: current-voltage relationship of T-type Ca2+ currents. D: Ca2+ currents recorded in granulosa cells incubated on plastic (control cell) with a holding potential of -50 mV, test potentials between -60 and +80 mV, and a 333-ms command pulse. E: Ca2+ currents recorded in granulosa cells incubated on basal lamina with a holding potential of -50 mV and test potential of -60 to +80 mV. F: current-voltage relationship of L-type Ca2+ currents. Each point is the mean ± SE of 9 cells (for control) and of 9 cells (for basal lamina), respectively, from 3 separate experiments.



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Fig. 5.   Effect of freshly prepared intact basal lamina on cell membrane capacitance, amplitude, and density of whole cell current T- and L-type Ca2+ currents. Granulosa cells isolated from the F1 of hen ovary were incubated on plastic or intact basal lamina in serum-free M199 for 24 h. Cells were stimulated from a holding potential of -80 mV to a -20-mV test potential with 333-ms command pulse. A: T-type Ca2+ current amplitude. B: T-type Ca2+ current density (amplitude normalized to capacitance). C: membrane capacitance. D: L-type Ca2+ current amplitude. E: L-type Ca2+ current density (amplitude normalized to capacitance). Each point is the mean ± SE (n = 38 cells for control; n = 15 cells for basal lamina) from 5 separate experiments. *P < 0.05, significantly different from corresponding control value.

Time course of effect of intact basal lamina on T- and L-type Ca2+ currents. Both T- and L-type Ca2+ currents could be recorded within 1 h of incubation of differentiated granulosa cells in serum-free medium; the T- and L-type Ca2+ currents remained expressed throughout 24 h of incubation (Fig. 6). Basal lamina suppressed significantly (P < 0.05) both T- and L-type Ca2+ currents after 24-h incubation; however, it had marginal (but nonsignificant) inhibitory effects on the currents after 1-12 h of incubation (Fig. 6).


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Fig. 6.   Time course of functional expression of T- and L-type Ca2+ currents in differentiated granulosa cells. Granulosa cells isolated from the F1 of hen ovary were incubated on plastic or intact basal lamina in serum-free M199 for 1, 6, 12, or 24 h. Cells were stimulated from a holding potential of -80 mV to a -20-mV test potential with a 333-ms command pulse. A: T-type Ca2+ current density. B: L-type Ca2+ current density. Each point is the mean ± SE at 1 h (n = 18 control; 7 basal lamina), 6 h (n = 12 control; 10 basal lamina), 12 h (n = 9 control; 9 basal lamina), and 24 h (n = 38 control; 15 basal lamina) from 4 separate experiments. *P < 0.05 vs. respective control value; **P < 0.05 vs. control value at 1 h.

Time course of effect of intact basal lamina on cell shape. Time course experiments revealed that intact basal lamina caused granulosa cells to become rounded within 1 h of incubation in serum-free medium. By comparison, granulosa cells incubated on plastic became flat. The morphometric parameters of granulosa cells cultured on intact basal lamina or plastic are shown in Fig. 7; within 60 min of incubation, the mean area occupied by cells incubated directly on the basal lamina was significantly (P < 0.05) less than that of cells incubated on plastic (Fig. 7A). Similarly, the morphometric parameter of perimeter was significantly (P < 0.05) lower after 60 min of incubation on basal lamina (Fig. 7B). The circularity of cells was reduced (P < 0.05) by basal lamina after 24 h of incubation (Fig. 7C).


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Fig. 7.   Morphometric parameters of granulosa cells incubated on freshly prepared intact basal lamina. Differentiated granulosa cells isolated from the largest preovulatory chicken ovarian follicle were incubated in serum-free M199 for different times on plastic (control) or on intact basal lamina (basal lamina); areas occupied by cells (A), perimeter (B), and circularity of cells (C) were determined. Data are means ± SE of >= 60 cells from 1 experiment. *P < 0.05 vs. respective control value.

Influence of storage on the effect of intact basal lamina on membrane capacitance and T- and L-type Ca2+ currents. Membrane capacitance and T- and L-type Ca2+ currents were recorded in differentiated granulosa cells incubated in intact basal lamina-containing culture dishes that had been stored for 12 mo or longer; the results are shown in Fig. 8. The membrane capacitance of cells incubated on intact basal lamina stored for 12 mo was significantly (P < 0.05) smaller than that in the cells incubated on plastic (Fig. 8A). Also, the density of the T- and L-type Ca2+ currents in control cells was significantly (P < 0.05) larger than those of cells incubated on basal lamina stored for 12 mo (Fig. 8, A and B).


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Fig. 8.   Effect of intact basal lamina stored for 12 mo on membrane capacitance and densities of whole cell T- and L-type Ca2+ currents. Granulosa cells isolated from the F1 of hen ovary were incubated on plastic or intact basal lamina that had been stored at 4°C for 12 mo in serum-free M199 for 24 h. Cells were stimulated from a holding potential of -80 mV to a -20-mV test potential with a 333-ms command pulse. A: membrane capacitance. B: T-type Ca2+ current density. C: L-type Ca2+ current density. Each point is the mean ± SE (n = 20 cells for control; n = 13 cells for basal lamina) from 3 separate experiments. *P < 0.05, significantly different from respective control value.

Relation between membrane capacitance (cell shape) and T- and L-type Ca2+ currents. Because of the apparently consistent relationship between the expression of T- and L-type Ca2+ currents and membrane capacitance (cell shape) in response to basal lamina, correlative changes in these two parameters in individual cells were estimated. The amplitudes or densities of the Ca2+ currents of granulosa cells incubated on plastic and basal lamina were plotted vs. their respective membrane capacitances (Fig. 9). The slopes of the linear regressions were not different from zero (P > 0.05), demonstrating that no correlation existed between the amplitude or density of the T- and L-type Ca2+ currents and the membrane capacitance (cell shape) in granulosa cells incubated on plastic or on basal lamina. Moreover, the relative changes in amplitudes of the T- and L-type Ca2+ currents (50-80%) in cells incubated on basal lamina were between 2.7- and 4.2-fold greater than the change in membrane capacitance (19%) (see Fig. 5). This suggested that changes in membrane capacitance (associated with cell shape) alone cannot account for the observed effects of basal lamina on the T- and L-type Ca2+ currents.


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Fig. 9.   Relation between amplitude and density of T- and L-type Ca2+ currents and membrane capacitance of cells incubated on plastic or basal lamina. Granulosa cells isolated from the F1 of hen ovary were incubated on plastic or intact basal lamina in serum-free M199 for 24 h. Cells were stimulated from a holding potential of -80 mV to a -20-mV test potential with 333-ms command pulse. A: T-type Ca2+ current amplitude vs. membrane capacitance. B: T-type Ca2+ current density vs. membrane capacitance. C: L-type Ca2+ current amplitude vs. membrane capacitance. D: L-type Ca2+ current density vs. membrane capacitance. Each point is datum from a single cell from 3 separate experiments. The slope of the linear regression for cells incubated on either control or basal lamina was not significantly different from zero.

Effect of solubilized basal lamina on T- and L-type Ca2+ currents. In additional experiments, the effects of solubilized basal lamina on T- and L-type Ca2+ currents were assessed. Differentiated granulosa cells were incubated on coverslips precoated with solubilized basal lamina (5 µg/cm2) (see MATERIALS AND METHODS). Similar to observations made for granulosa cells incubated on intact basal lamina, granulosa cells incubated on coverslips precoated with solubilized basal lamina have reduced membrane capacitance and T- and L-type Ca2+ current density (Fig. 10).


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Fig. 10.   Effect of solubilized basal lamina on membrane capacitance and densities of whole cell T- and L-type Ca2+ currents. Granulosa cells isolated from the F1 of hen ovary were incubated on coverslips precoated with solubilized basal lamina (5 µg/cm2) in serum-free M199 for 24 h. Cells were stimulated from a holding potential of -80 mV to a -20-mV test potential with a 333-ms command pulse. A: membrane capacitance; B: T-type Ca2+ current density; C: L-type Ca2+ current density. Each point is the mean ± SE (n = 28 cells for control; n = 11 cells for basal lamina) from 3 separate experiments. *P < 0.05, significantly different from respective control value.

To determine whether the effects of basal lamina were caused by change in the viability of granulosa cells, the cells were incubated on plastic as well as in culture wells precoated with solubilized basal lamina. The viability of differentiated granulosa cells incubated for 24 h in serum-free medium on plastic (99.03 ± 0.7%, n = 3 wells) was not different (P > 0.05) from that of cells incubated on 7 µg/cm2 solubilized basal lamina (96.71 ± 3.11%, n = 3 wells).

Effects of fibronectin, laminin, or type IV collagen on T- and L-type Ca2+ currents. Experiments were conducted to determine the effects of extracellular matrix proteins that are components of basement membranes on T- and L-type Ca2+ currents. Differentiated granulosa cells were incubated in culture wells precoated with fibronectin (5 µg/cm2), laminin (2 µg/cm2), type IV collagen (5 µg/cm2), or solubilized basal lamina (5 µg/cm2). Similar to observations made for granulosa cells incubated on solubilized basal lamina, granulosa cells incubated on fibronectin, laminin, or collagen IV have reduced membrane capacitance and T- and L-type Ca2+ current density (Fig. 11).


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Fig. 11.   Effect of fibronectin, laminin, and type IV collagen on membrane capacitance and densities of whole cell T- and L-type Ca2+ currents. Granulosa cells isolated from the F1 of hen ovary were incubated in culture dishes precoated with commercially available fibronectin (5 µg/cm2), laminin (2 µg/cm2), type IV collagen (5 µg/cm2), or solubilized basal lamina (5 µg/cm2) in serum-free M199 for 24 h. Cells were stimulated from a holding potential of -80 mV to a -20-mV test potential with a 333-ms command pulse. A: T-type Ca2+ current density; B: L-type Ca2+ current density; C: membrane capacitance. Each point is the mean ± SE (n = 14 cells for control; n = 21 cells for fibronectin, n = 22 cells for laminin, n = 16 cells collagen IV, n = 21 cells for basal lamina) from 3 separate experiments. *P < 0.05, significantly different from control value.

Effects of cycloheximide and actinomycin D on the Ca2+ currents. The effects of the protein synthesis inhibitor cycloheximide and transcription inhibitor actinomycin D on the T- and L-type Ca2+ currents were tested. Differentiated granulosa cells were incubated on plastic and intact basal lamina for 24 h, and membrane currents were recorded. Cycloheximide or actinomycin D had no appreciable effect on the expression of the Ca2+ currents in granulosa cells incubated on plastic; similarly, the inhibitors had no significant effect on Ca2+ currents in granulosa cells incubated on basal lamina (Fig. 12).


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Fig. 12.   Effects of cycloheximide or actinomycin D on T- or L-type Ca2+ current density. Differentiated chicken granulosa cells were incubated on plastic or intact basal lamina in serum-free M199 for 24 h in the absence and presence of 5 µg/ml cycloheximide or 8 µg/ml actinomycin D. Membrane potential was held at -80 mV and stepped to -20 mV with a 333-ms command pulse. A: T-type current; B: L-type current. Each point is the mean ± SE (n = 15 cells for control, n = 10 cells for cycloheximide, N = 9 cells for actinomycin D, n = 16 cells for basal lamina, n = 10 cells for basal lamina plus cycloheximide, n = 9 cells for basal lamina plus actinomycin D) from 2 separate experiments. *P < 0.05, significantly different from control value; **P < 0.05, significantly different from cycloheximide alone; ***P < 0.05, significantly different from actinomycin D alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study show that basal lamina of the chicken ovarian follicle can regulate T- and L-type Ca2+ currents in differentiated chicken granulosa cells. The ability of the basal lamina to suppress Ca2+ currents was not affected by solubilization, because, similar to the intact form, the solubilized basal lamina (containing components with molecular mass >3 kDa) suppressed the T- and L-type Ca2+ currents in differentiated granulosa cells. The concentration of solubilized basal lamina used in the present study was shown to significantly regulate progesterone production in chicken granulosa cells (5). An interesting finding of the present study was that the storage of basal lamina for >= 12 mo at 4°C did not affect its ability to regulate the Ca2+ currents. In relation to this observation is the recent finding that basal lamina stored for >= 18 mo regulated the shape of granulosa cells to a similar extent as freshly prepared ones (7). The present results confirm an earlier report that chicken granulosa cells express T-and L-type voltage-dependent Ca2+ channels (58). The present results also confirm reports that chicken (14, 45, 57, 58, 65) and porcine (38) granulosa cells express whole cell T-type Ca2+ currents.

How did the basal lamina attenuate the expression of Ca2+ currents? The possibility remains that basal lamina inhibited the activation of existing Ca2+ channels; however, it is also possible that basal lamina reduced the number of Ca2+ channel proteins or suppressed the synthesis of Ca2+ channel proteins. The observation that the exposure of differentiated granulosa cells (incubated on plastic or basal lamina for 24 h) to cycloheximide or actinomycin D did not result in a change in the effects of basal lamina on the Ca2+ currents indicates, perhaps, that the actions of basal lamina monitored here did not directly involve gene transcription or protein synthesis. The mechanisms whereby basal lamina exerted its effects will be determined in the future.

The observations that known components of basement membranes, fibronectin, laminin, and type IV collagen suppressed both T- and L-type Ca2+ currents (in the present study) suggest that the actions of intact basal lamina are mediated in part by these matrix proteins. It is noteworthy that progesterone production by rat granulosa cells incubated in serum-free medium for 24 h in laminin-coated or fibronectin-coated wells decreased significantly (1). Moreover, steroidogenesis was suppressed in differentiated human granulosa cells cultured in laminin-coated wells (26). Other investigators have shown that extracellular matrix proteins, especially those of basement membrane origin, have been shown to regulate ion transport in various types of cells. For example, inward whole cell Na+ currents were significantly reduced in rat adult alveolar type II cells cultured on fibronectin-coated coverslips for 48 h (71). Also, rat adult alveolar type II cells cultured on human amniotic basement membrane had smaller short-circuit currents than cells cultured on collagen substrate (20). Furthermore, Matrigel (reconstituted basement membrane) modulated the K+ channel density in fetal rat alveolar type II cells (41).

Because granulosa cells are exposed to the basal lamina as a unit in vivo (but not individual components in isolation), the effect of basal lamina was examined as a unit (in the present study) with the presumption that the outcome would be the result of combined effects of different components of the matrix material. Although, in the present study, the nature and characteristics of the components of basal lamina that regulated the inward currents and their mechanisms of action are unknown, some of the components are likely to stimulate cellular processes that other components suppress, whereas some components would be without effect. Therefore, the actions of the individual components of basal lamina may be synergistic, additive, antagonistic, or neutral, as the case may be. It was shown recently that several components of basal lamina of chicken ovarian follicle reacted positively to antibodies raised against extracellular matrix proteins (type IV collagen, laminin, entactic, heparan sulfate proteoglycan, and fibronectin), growth factors [epidermal (EGF), platelet-derived (PDGF), basic and acidic fibroblast (FGF), insulin-like (IGF), transforming (TGF-alpha , TGF-beta )], cytokines, matrix metalloproteinases, and their tissue inhibitors (8). The possibility remained that these bioactive components of basal lamina acted concomitantly to regulate the Ca2+-carried inward currents in the present studies. Indeed, the actions of integrins (receptors of extracellular matrix proteins) may synergize or overlap with those of growth factors. [Integrins are a large family of heterodimeric transmembrane proteins with different alpha - and beta -subunits that function as extracellular matrix adhesion receptors (33)]. Integrins can activate signaling pathways known to be regulated by growth factors. For example, integrins have been shown to cooperate with growth factors to increase signaling for mitogenic processes (40, 48, 64). Similarly, growth factors EGF, PDGF-BB, or basic FGF cooperated with integrins (ligand occupied and aggregated) to enhance the transient activation of the extracellular signal-regulated kinase class of mitogen-activated protein (MAP) kinase (48). In addition, EGF, PDGF, or FGF also synergized with integrins (ligand occupied and aggregated) to enhance tyrosine phosphorylation of the growth factor receptors (48). Integrins activated Shc, which is known to link several growth factor-activated pathways (70). In certain cases, integrins and growth factors cooperated to increase cell proliferation (40, 48, 64). Integrin-mediated adhesion (cell anchorage) was shown to be necessary for growth factor (EGF, PDGF) activation of the MAP kinase cascade (40). In addition, integrin-mediated adhesion was required for PDGF-stimulated phosphatidylinositol bisphosphate hydrolysis (44). Integrins activated the Ras-Raf-MAP kinase pathway, protein kinase C, and phosphatidylinositol 3'-kinase (13, 16, 59).

Earlier studies showed that progesterone synthesis in granulosa cells of several species was reduced in Ca2+-deficient incubation media (6, 22, 61, 63), and a recent study demonstrated that the abundance of messenger RNA of cytochrome P450scc (the enzyme that catalyzes the metabolism of cholesterol to pregnenolone) in porcine granulosa cells was reduced in Ca2+-deficient medium (37). Therefore, one of the mechanisms by which Ca2+ modulates steroid hormone synthesis is the increase in the transactivation of the cytochrome P450scc gene (37). Because, in granulosa cells, the suppression of uptake of extracellular Ca2+ resulted in the attenuation of progesterone production (6, 22, 61, 63) and transcription of the cytochrome P450scc gene, and because basal lamina suppressed inward Ca2+ current in differentiated granulosa cells (in the present study), it would be expected that basal lamina would decrease progesterone production in differentiated chicken granulosa cells. Indeed, chicken ovarian basal lamina suppressed progesterone synthesis in differentiated chicken granulosa cells in vitro (7). It is noteworthy that the suppression of steroidogenesis in differentiated granulosa cells by solid basement membrane or extracellular matrices is not limited to avian granulosa cells. Notably, Aten et al. (9) observed that rat granulosa cells incubated on Matrigel matrix, basement membrane reconstituted from extracts of Engelbreth-Holm-Swarm tumor, produced less progesterone than those incubated on plastic. In addition, progesterone production by rat granulosa cells in laminin-coated wells decreased (1). In other studies, when human granulosa cells retrieved from patients undergoing in vitro fertilization were cultured in laminin-coated wells, steroidogenesis was suppressed (26). Similarly, human chorionic gonadotropin-induced progesterone synthesis was attenuated in laminin-coated dishes (26). Therefore, reports in the literature indicate that the incubation of granulosa cells in dishes precoated with reconstituted basement membrane, components of basement membranes, or basal lamina results in the reduction of steroid hormone synthesis. The relation between an extracellular matrix-induced rounded shape and steroidogenesis in granulosa cells was reported (for review see Ref. 2). It appears that the nature of the relation between basement membrane protein-induced rounded shape and steroid hormone synthesis is influenced by factors such as the state of granulosa cell differentiation and the form of the matrix protein (whether solid or liquid) among others (1, 2, 7, 9, 26).

In the present study, basal lamina prevented the spreading/stretching of granulosa cells and reduced their membrane capacitance (a measure of plasma membrane surface area). It was reported that chicken granulosa cells, especially the differentiated ones, synthesize and deposit large quantities of fibronectin in serum-free culture (4, 18). The morphology of granulosa cells incubated on tissue culture-treated plastic substratum was likely caused by integrin-mediated adhesion of cells to newly deposited (endogenous) extracellular matrix, and not by direct interaction with plastic. It is therefore possible that, in granulosa cells incubated on basal lamina, the interaction of cells with deposited endogenous extracellular matrix was prevented or attenuated, leading to the observed changes in cell adhesion, morphology, and function (6, 7, and present study). As such, the spreading on the tissue culture-treated plastic substratum probably reflects greater adhesion of granulosa cells to deposited extracellular matrix, such as fibronectin, than to cells incubated on basal lamina; that is, the granulosa cells spread less efficiently on basal lamina; consequently they became rounded (Ref. 7 and the present study).

It is known that mechanical force imposed on cells by stretch, gravity, hemodynamic (shear) stresses, and movement can influence cellular behavior and function. In tissue culture models, strong traction forces are exerted by cells (21, 31), and the spreading/stretching of cells can generate intracellular signals to regulate cell functions (15). The attachment of a cell to the extracellular matrix, via integrins, could also exert mechanical force (through tugging of the integrins) (45). This integrin-mediated force can be transmitted throughout a tensionally integrated cell and induce changes in chemical signaling, the organization of intracellular organelles, and reorganization (stiffness) of the cytoskeleton and nucleus, thereby regulating various physiological processes in the cell (34-36, 43, 60, 67-69). Thus the extracellular matrix (via local mechanical tension on cells) could regulate cell function by mechanically stabilizing the lattice of cytoskeleton and nucleus (43). Whether or not basal lamina can cause the reorganization of the cytoskeleton and intracellular organelles remains to be determined.

After the binding of extracellular matrix with integrin, a specialized cytoskeletal structure (known as focal adhesion complex) forms intracellularly at the site of integrin binding. The focal adhesion complex serves as a molecular bridge, and it mechanically couples integrins (integrin-extracellular matrix complex) to the actin cytoskeleton (11, 19, 47). This focal adhesion complex also participates in the direction of much of the signaling machinery of the cell (12, 16, 34, 35, 46, 54-56, 59). Therefore, focal adhesion complexes could integrate mechanical signals associated with changes in cell morphology with chemical signals generated by integrins and thereby regulate signaling downstream (36). Practically, any link that physically connects the transmembrane receptors to the cytoskeleton can mediate the transmission of mechanical force across the cell membrane. The focal adhesion complex can and does serve as a molecular link between the transmembrane integrins to the cytoskeleton (11). Integrins, therefore, could serve as mechanotransducers. It was shown that mechanical stretch activated stretch-sensitive Ca2+ channels that mediated the influx of extracellular Ca2+ in human umbilical endothelial cells (50), fibroblasts (30), and fish keratocytes (39). The roles of integrins in the actions of basal lamina in avian granulosa cells are yet to be determined.

In summary, the unique anatomic structure of the avian ovarian follicle enabled the isolation of pure and intact basal lamina (basement membrane) and its associated granulosa cells and provided the opportunity to study the effect of basal lamina on transmembrane ion currents in the granulosa cells in a homologous system. Both intact basal lamina and its solubilized form suppressed inward Ca2+ currents in differentiated granulosa cells. The results may explain (partly) the effect of basal lamina on the metabolic functions of differentiated avian granulosa cells.


    ACKNOWLEDGEMENTS

We thank Drs. Michael Kinch and Riyi Shi for their comments on the manuscript.


    FOOTNOTES

This work was supported by funds from Purdue University School of Veterinary Medicine. W. Qin was supported by a Purdue Research Foundation research assistantship.

Present address of S. G. Rane: Fujisawa Research Institute of America, Northwestern University/Evanston Research Park, 1801 Maple Ave., Evanston, IL 60201.

Address for reprint requests and other correspondence: E. K. Asem, Dept. of Basic Medical Sciences, School of Veterinary Medicine, Purdue Univ., 1246 Lynn Hall, West Lafayette, IN 47907-1246 (E-mail: asem{at}vet.purdue.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 June 2001; accepted in final form 30 August 2001.


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Am J Physiol Endocrinol Metab 282(1):E184-E196
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