(Granulosa) Cells

0013-7227/98/$03.00/0 Endocrinology Copyright © 1998 by The Endocrine Society

Vol. 139, No. 8 Printed in U.S.A.

Luteinizing Hormone (LH) Stimulates Both Intracellular Calcium Ion ([Ca21]i) Mobilization and Transmembrane Cation Influx in Single Ovarian (Granulosa) Cells: Recruitment as a Cellular Mechanism of LH-[Ca21]i Dose Response* JORGE A. FLORES, CLAUDIA AGUIRRE, OM P. SHARMA†, JOHANNES D. VELDHUIS

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Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health Sciences Center, and National Science Foundation Center for Biology Timing, Charlottesville, Virginia 22908 ABSTRACT The gonadotropic hormones, LH and FSH, activate adenylyl cyclase in their respective target cells and thereby initiate many biochemical responses. In addition to stimulating cAMP production, both LH and FSH promote agonist-specific increases in the cytoplasmic concentration of free calcium ions ([Ca21]i) in gonadal cells. Here, we have applied single cell fluorescence video microscopy with the Ca21sensitive dye fura-2 to investigate the mechanism(s) by which LH induces a rise in the [Ca21]i in individual (swine) granulosa cells collected from single Graafian follicles. Stimulation with LH induced a rapid onset, biphasic, spike- and plateau-like [Ca21]i signal in responsive granulosa cells. The cellular mechanisms mediating this biphasic LH-stimulated increase in [Ca21]i were examined by external Ca21 removal and via the manganese (Mn21) quench technique, which showed that LH triggers initial intracellular Ca21 mobilization followed by delayed transmembrane Ca21 influx. Single cell Ca21 assessment of the LH dose-response mechanism(s) revealed that higher concentrations of LH progressively recruit a larger number of responding individual granulosa cells. Further analyses disclosed a marked [Ca21]i response heterogeneity among individual granulosa cells harvested from the same Graafian follicle. In addition, the percentage of cells responding to LH [but not to an alternative putative agonist of the phospholipase C (PLC) pathway, viz. endothelin-1] with

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ENE transfection experiments have recently documented the ability of a single receptor to activate dual intracellular signaling pathways after interacting with its cognate ligand. For example, the human TSH, hCG/LH, and PTH/PTH-related peptide receptors expressed in monkey kidney (COS) cells all mediate single ligand stimulation of both adenylyl cyclase and phospholipase C (PLC) (1, 2). Intracellular cAMP and soluble inositol phosphates are also generated by activation of the porcine calcitonin receptor (3), Received January 28, 1998. Address all correspondence and requests for reprints to: Dr. Jorge A. Flores, Biology Department, West Virginia University, Brooks Hall, P.O. Box 6057, Morgantown, West Virginia 26506-6057. * This work was supported in part by NIH Grant R01-HD-16806 (to J.D.V.), a supplement to this NICHHD grant (to J.A.F.), NIH P-30 Reproduction Center Grant 1-P30-HD-28934, and the National Science Foundation Center for Biological Timing (to J.A.F., C.A., J.D.V., and O.S.), and the Hospital Clinico de la Universidad de Chile (to C.A.). † Present address: Organon, Inc., West Orange, New Jersey 07052.

a biphasic [Ca21]i rise increased with maturational development of the follicle. Pretreatment of granulosa cells with a specific PLC inhibitor, U-73122 (but not with its inactive congener U-73343), significantly reduced the percentage of cells responding to a LH challenge from 78% to 25% (P , 0.0001) and prolonged the time required to achieve a half-maximal value of the [Ca21]i transient, viz. from 22 6 1.5 sec (n 5 27 cells) to 39 6 7.2 sec (n 5 12 cells; P 5 0.002). In cell population studies, LH stimulated in a concentration- and timedependent manner the accumulation of inositol phosphate in porcine granulosa cells. In summary, the present single cell investigations in mature granulosa cells demonstrate that LH drives initial intracellular Ca21 mobilization followed by transmembrane divalent cation influx. The PLC inhibitor U-73122 antagonizes this action of LH. By analyzing [Ca2]i responses in individual living granulosa cells, we further show that, despite within-follicle diversity, the LH dose biphasic [Ca21]i response arises via the recruitment of a larger number of responding gonadal cells rather than by increased [Ca21]i signal amplitude. Finally, the percentage of individual LH (but not endothelin-1)-responding granulosa cells increases with follicular maturation. Collectively, these data highlight the potential importance of the LH-stimulatable, PLC-transduced [Ca21]i signaling mechanism in the later stages of granulosa cell differentiation. (Endocrinology 139: 3606 –3612, 1998)

the murine LH receptor (4), and the rat LH receptor expressed in heterologous host cells (5). However, gene transfection experiments in nonnative cell populations do not establish whether and how specific signaling mechanisms are implemented by native receptors within the physiological milieu of the individual homologous (native) cell. Indeed, in the particular case of LH, reports documenting PLC activation by this gonadotropin in untransformed ovarian cells are limited and controversial. Although some investigations have reported that LH does not affect inositol phosphate accumulation in rat and pig granulosa (6) and luteal cells (7), other studies have described LH-stimulated inositol phosphate accumulation in rat granulosa cells (8, 9), and bovine (10) and porcine luteal cells (11). Here we implement monitoring of cytoplasmic concentration of free calcium ions ([Ca21]i) with high temporal resolution by semiquantitative fluorescence video microscopy in single (swine) granulosa cells to examine the mech-

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LH STIMULATES [Ca21]i DOSE RESPONSE

anisms of LH’s stimulation of [Ca21]i responses within individual homologous target (granulosa) cells expressing native LH receptors. We show that LH’s activation of the [Ca21]i signaling pathway is marked by significant cell to cell heterogeneity. Mechanistic investigations using the manganese quench technique further reveal that the LH-stimulated biphasic [Ca21]i signal arises by way of both intracellular Ca21 mobilization and transmembrane cation uptake. Moreover, we observe that the mechanism subserving the LH dose response of [Ca21]i in single ovarian cells embodies progressive recruitment of responding granulosa cells at higher LH concentrations. The percentage of responding cells is also higher for granulosa cells harvested from more mature follicles. Lastly, we show that a specific PLC inhibitor antagonizes LH-driven [Ca21]i signal generation in individual granulosa cells, thus supporting the relevance of activation of PLC by LH in [Ca21]i signaling in mature granulosa cells. Materials and Methods Ovine LH (NIDDK oLH-26) and FSH (NIDDK oFSH-17) were provided by the Hormone Distribution Office, National Pituitary Agency, NIDDK, NIH (Bethesda, MD). The LH stock solution was prepared in neutral saline at a concentration of 300 mm and stored at 270 C. The biological activity of this stock solution corresponds to 23 U NIH LH-S1. The PLC inhibitor U-73122 and its inactive control U-73343 were purchased from Research Biochemical International (Natick, MA), made in dimethylsulfoxide (DMSO) stock solutions (2.2 mm), and stored at 270 C. Fura-2/AM was supplied by Calbiochem (San Diego, CA). L-myo[1,2-3H(N)]inositol was purchased from New England Nuclear-DuPont (Boston, MA). Poly-l-lysine, BSA (Cohen’s fraction V), porcine insulin, DMSO, and poly-l-lysine were purchased from Sigma Chemical Co. (St. Louis, MO). Eagle’s MEM, medium 199, FCS, and penicillin-streptomycin were obtained from Life Technologies (Grand Island, NY).

Effects of LH on [Ca21]i Calcium measurements. Granulosa cells were collected as described previously (12) from immature (,70 kg) swine ovaries into HEPES-buffered MEM from individually dissected follicles measuring 1.0 or 5.0 mm in diameter. Cells were counted in a hemocytometer, and cell density was adjusted to 1 3 105 cells/ml by adding bicarbonate-buffered MEM supplemented with 0.1% FCS. This initial concentration of FCS in the MEM allowed granulosa cell attachment to the microscope slides. A 60-ml aliquot of the granulosa cell suspension was applied to a poly-llysine-coated microscope slide with a Cunningham chamber (13, 14). The Cunningham chambers were maintained overnight in a humidified incubator (37 C, 95% air-5% CO2). The following day the cells were used for calcium measurements. The medium in these experiments contained 127 mm NaCl, 5 mm KCl, 1.8 mm CaCl2, 2 mm MgCl2, 5 mm KHPO4, 5 mm NaHCO3, 10 mm HEPES, 10 mm glucose, and 0.1% BSA, pH 7.4. Manganese-containing medium was prepared by replacing CaCl2 with MnCl2 (1.8 mm). Granulosa cells were loaded with 2 mm fura-2/AM in experimental medium (without hormones) for 20 min at 37 C. The cells were then washed with experimental medium and incubated for an additional 20 min at 37 C to allow cytoplasmic deesterification of the fura-2/AM dye. After dye loading, the Cunningham chamber was placed on the stage of a Zeiss Axioplan microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with epifluorescence illumination. All experiments were performed at room temperature (22–23 C). The excitation light was supplied by a high pressure xenon arc UV lamp, and the excitation wavelengths were selected by 360- and 380-nm filters (2 nm half-bandwidth; Corion, Hollinston, MA) mounted in a rotating filter wheel (MAC 2000, Ludl Electronic Products, Hawthorne, NY) between the UV lamp and the microscope. Fluorescence emission was collected via the objective (UV-F, Nikon, Melville, NY; 320) and passed through a barrier filter (490 – 600 nm transmission) to the face of a silicon-intensified target camera (series 68, DAGE-MTI, Michigan City, IN). The resultant video

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signal was stored on broadcast quality tape at 33 frames/sec (U-matic Vo-5600, Sony Corp., Tokyo, Japan). LH-stimulated [Ca21]i responses. The ability of different LH concentrations (1.0, 10, 100, 1,000, and 10,000 pm) to stimulate a rise in [Ca21]i in individual granulosa cells was tested as previously described (12). The granulosa cells were collected from individual, well vascularized follicles at least 5.0 mm in diameter, except where indicated otherwise. Each LH concentration was evaluated in three slides per experimental day. In other experiments, the same cell was exposed to progressively increasing concentrations of LH or repetitively to the same concentration of LH. At least six independent experiments were performed to address each question using granulosa cells from a different follicle collected on a different day. To test whether LH’s ability to stimulate a transient rise in [Ca21]i was a function of follicular development, the responsiveness to a 10-nm LH challenge was investigated in individual granulosa cells collected from different sized follicles (1 vs. 5 mm diameter). Each cell was also exposed to 10 nm endothelin-1 (ET-1). ET-1 served as a positive control, which has been previously shown to stimulate a transient rise in [Ca21]i in swine granulosa cells (12). The ET-1 challenge was performed before or after the LH challenge. Results were independent of order. This protocol was performed in six independent experiments. As activation of PLC-linked receptors can mobilize both intracellular and external sources of Ca21 (15–21), we investigated the Ca21 source(s) for the LH-stimulated biphasic rise in [Ca21]i by removal of extracellular Ca21 before or immediately after a 10-nm LH challenge. However, the relative contribution and timing of intracellular Ca21 discharge and transmembrane Ca21 entry cannot be distinguished with these protocols (20). Thus, we used the Mn21 quench technique of Merritt et al. to identify transmembrane cation uptake (22). In various nonexcitable cells, extracellular Mn21 can serve as a surrogate cation for Ca21 entry (22). When Mn21 enters the cell, it binds to fura-2 and quenches the fluorescence signal (20, 22). Agonist-stimulated fluorescence signal quenching of fura-2-loaded cells in the presence of extracellular Mn21 is thus evidence of agonist-stimulated influx of extracellular divalent cations. Fura-2 fluorescence intensity was measured at two excitation wavelengths, 360 nm (Ca21 independent) and 380 nm (Ca21 dependent), to allow sequential monitoring of changes in [Ca21]i (380 nm) and Mn21 influx (360 nm). Approximately 15–25 sec after initiation of a recording, experimental Ca21-containing medium was replaced with Mn21-containing medium to record the basal rate of Mn21 influx. Subsequently, LH-stimulated Ca21 mobilization and Mn21 influx were studied by delivering 10 nm LH prepared in Mn21 medium. The specificity of LH-induced Mn21 influx was assessed by testing the ability of a second delivery of Mn21-containing but LH-deficient medium to affect the basal rate of Mn21 influx. At least 20 cells in three independent experiments were studied with this protocol. To directly test whether the ability of LH to stimulate a transient rise in [Ca21]i was mediated by PLC, the responsiveness of [Ca21]i to a 200-nm LH challenge was investigated in individual granulosa cells pretreated with U-73122, a PLC inhibitor, or its inactive congener, U-73343. In this protocol, we also tested the responsiveness to a 10-mm ET-1 challenge (12). Cells were pretreated with 10 mm of the PLC inhibitor U-73122, its inactive analog U-73343 (23), or vehicle [0.44% (vol/ vol) DMSO] for 2 min. Subsequently, the cells were exposed to 200 nm LH or 10 mm ET-1 in the continuing presence of the respective pretreatment compound for an additional 2 min. At the end of each treatment the calcium ionophore, ionomycin (10 mm), was delivered to verify cellular [Ca21]i responsiveness. Two or three slides of granulosa cells, allowed to anchored to slides as described above and in the presence of estradiol (0.5 mg/ml) for 48 h, were used per treatment in each of 4 independent experiments with separate batches of ovaries. A total of 210 and 221 cells were studied using LH or ET-1, respectively. Cells were classified as responding with an increase in [Ca21]i if the Fo (initial fluorescence emission intensity at each wavelength/Fi (fluorescence at time i) ratio was greater than baseline 1 3sd. The percentage of cells responding and the times required to achieve the half-maximal [Ca21]i value (t1/2) were calculated for each treatment. These data are presented as the mean 6 sem For statistical analysis, data were transformed to natural logarithm values and subjected to ANOVA followed by the Tukey highest significant difference multiple comparison test.

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Analysis. The video-recorded fluorescence intensity signal was captured and digitized using software (RADTIME) run on a QX-7 image analysis system (Quantex Corp., Sunnyvale, CA), which creates an ASCII file of the mean radiance values observed every 30 msec within the cell of interest. The ASCII file is exported to a spreadsheet program (Lotus 1–2-3, version 2.0, Lotus Development Corp.), where the 380 and 360 nm fluorescence values are converted to ratio values using the equation R 5 Fo/Fi. The data in graphic form represent the plot of the converted fluorescence ratio (y-axis) over time (x-axis).

Effect of LH on inositol phosphate accumulation Swine ovaries were obtained from a local abattoir, and granulosa cells were harvested and pooled from 3- to 5-mm (diameter) Graafian follicles by fine needle aspiration. To allow cell anchorage, monolayer cultures were initially established in medium 199 (which contains 0.05 mg/liter inositol) supplemented with 3% FCS, ovine FSH (100 ng/ml), and porcine insulin (3 mg/ml) at a plating density of 1 3 107 viable cells/35-mm tissue culture dish. These culture conditions promote LH responsiveness as defined by LH stimulation of progesterone biosynthesis and cAMP accumulation. After 48 h, the incubation medium was replaced by serum-free medium 199 supplemented with 0.1% BSA. After 24 h of serum-free culture, granulosa cells were equilibrium labeled with [3H]myo-inositol L-myo-[1,2-3H(N)]inositol by exposing them to medium 199 containing 0.1% BSA and 2.5 mCi/ml [3H]myo-inositol (60 Ci/mm) for 18 h. Thereafter, monolayers were washed twice in medium 199 with 0.1% BSA and incubated for 1 h in the presence of 10 mm unlabeled inositol. During the last 15 min of this “cold chase” period, 30 ml of a 0.5-m stock LiCl solution was added to each dish (final concentration, 10 mm). At designated intervals, medium alone (control) or LH (final concentration, 10 nm) was added in 80-ml aliquots. The reaction was stopped by addition of ice-cold chloroform-methanol (1:2, vol/vol). Cells were immediately scraped from the culture dishes maintained on ice, and centrifuged in biovials. [3H]Inositol phosphates were separated by anion exchange chromatography, as described previously (23a). [3H]Inositol mono-, bis-, and triphosphate ([3H]IP1, [3H]IP2, and [3H]IP3) were sequentially eluted into scintillation vials with 6 ml 0.2, 0.4, and 0.8 m ammonium formate containing 0.1 m formic acid, respectively. Ten milliliters of scintillation solution were added to each vial, and the radioactivity was determined in a liquid scintillation counter.

Results LH-Stimulated in [Ca21]i responses in single granulosa cells

There was significant [Ca21]i response heterogeneity among individual granulosa cells collected from the same follicle with respect to the effective LH concentration required to elicit an increase in [Ca21]i. LH concentrations of 10 pm or greater were able to stimulate a rise in [Ca21]i in a subpopulation of granulosa cells. The stimulated increases in [Ca21]i observed at low LH concentrations (10 pm) were typically of lower amplitude and slower onset than [Ca21]i changes elicited at higher LH concentrations (Fig. 1). There was a consistent enhancement in the number (percentage) of responding cells in the presence of increasing LH concentrations. For example, when 10 granulosa cells in a given microscopic field were challenged with 1 pm LH, no responders were observed. When the same cells were exposed to higher LH concentrations (10, 100, and 1000 pm, we observed 2, 3, and up to 5 cells that responded with a spike and plateau rise in [Ca21]i. Similar observations were made when the order of LH doses was reassigned in 6 different experiments using granulosa cells collected from different follicles (44 cells total). Augmentation of the number of LH-responsive cells at higher LH concentrations was not observed if the cells were challenged serially with the same (1.0 nm) LH

FIG. 1. Nature of LH’s concentration-dependent stimulation of increased [Ca21]i in single swine granulosa cells. Ovarian cells were collected from individual ovarian follicles of 5.0 mm in diameter and cultured overnight in Cunningham chambers built on poly-L-lysinecoated slides. Cells were loaded with the fluorescent dye, fura-2/AM, to monitor the actions of LH on [Ca21]i recorded serially at 30-msec intervals (see Materials and Methods). Representative temporal responses induced by 10 pM, 100 pM, 1 nM, 10 nM LH in individual granulosa cells are shown. The fluorescence intensity was recorded at an excitation wavelength of 380 nm. The box represents the time during which LH was present. Data are representative of 94 cells studied in 6 separate experiments.

concentration (40 cells total). Figure 1 also shows the [Ca21]i responses elicited in single granulosa cells exposed to LH concentrations greater than 100 pm. This stimulus evoked the rapid (within 10 –30 sec) onset of a single spike-like rise in [Ca21]i, which was followed by a more sustained plateau-like elevation of [Ca21]i. We next examined the influence of follicle diameter on granulosa cell responsiveness to LH and ET-1. In studies using 5.0-mm follicles, 43 6 14% of cells responded to LH with a spike and plateau pattern of [Ca21]i increase (Fig. 2). In contrast, only 4 6 1.5% cells from 1.0-mm follicles responded similarly (6 experiments, total of 175 cells). The percentages of granulosa cells that responded to ET-1 in 5.0and 1.0-mm follicles were not different (33 6 8.5% and 32 6 11%, respectively; 6 experiments, 95 cells). In addition, LH and ET-1 delivered sequentially 10 min apart in either order increased [Ca21]i in the same individual granulosa cell that was ET-1 responsive (8 experiments, 30 cells). The initial spike-like rise in [Ca21]i in response to a LH stimulus was still observed in the absence of extracellular


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FIG. 2. Effect of Graafian follicle diameter on the percentage of granulosa cells able to respond to LH (10 nM) and ET-1 (10 nM) with a biphasic rise in the [Ca21]i. Data are from a total of 175 cells studied in 8 different experiments.

Ca21 (Fig. 3, upper panel). However, the apparent plateau increase in [Ca21]i after the [Ca21]i spike was inhibited or abolished by removal of extracellular Ca21 (Fig. 3). In the presence of extracellular Ca21, the peak of the [Ca21]i rise averaged a 2.9 6 0.6-fold increase over basal (assuming basal 5 1.0), and then maintained a value that was 1.6 6 0.1-fold basal for 2.5 min (n 5 40 cells in 6 independent experiments). In the absence of extracellular Ca21, only the fold increase in the plateau-like [Ca21]i increase sustained after the spike declined significantly (1.2 6 0.1; P , 0.05, by Student’s two-tailed unpaired t test; n 5 26 cells in 4 independent experiments). Similar reductions in the plateau [Ca21]i level were observed when extracellular Ca21 was removed during the postspike interval (n 5 24 cells in 4 independent experiments). Twenty of 40 granulosa cells (50%) stimulated with 10 nm LH clearly had an increased rate of Mn21 entry over that observed in their respective basal states (Fig. 4). In all 20 cells, LH-stimulated Mn21-induced quenching of fura-2 fluorescence was always preceded by the spike-like increase in intracellular (mobilized) Ca21. There was a subpopulation of granulosa cells (20 cells) in which no increased rate of Mn21 quench was detected within 2.5 min after the spike of mobilized calcium induced by the 10-nm LH challenge (not shown). After 48 h of granulosa cell culture as described above with estradiol (0.5 mg/ml), stimulation with 200 nm LH elicited a biphasic increase in [Ca21]i in 78% of granulosa cells (Fig. 5). Similarly, we found that 91% of granulosa cells responded with a rapid biphasic increase in [Ca21]i to a 10-mm ET-1 challenge. Pretreatment of granulosa cells with 10 mm U-73343 (inactive congener) or vehicle did not significantly affect the percentage of cells responding to either LH or ET-1.

FIG. 3. Removal of extracellular Ca21 attenuates the plateau phase of increased [Ca21]i after the spike-like response to LH’s stimulation of single swine granulosa cells. Cells were collected and prepared as described in Fig. 1. Available extracellular Ca21 was removed by adding 2.5 mM EGTA to the experimental medium before (top) or after (bottom) the LH (1.0 nM) stimulus. These results were reproduced in a total of 90 cells in 6 independent experiments.

On the other hand, the number of responding cells was significantly reduced to 25% in the presence of 10 mm U-73122 (P , 0.001) in LH-stimulated cells and to 36% (P , 0.001) in ET-1 stimulated cells. Furthermore, granulosa cells responding to LH or ET-1 in the U-73122 pretreatment group took a significantly longer time to achieve half-maximal [Ca21]i value (t1/2). LH- plus U-73343-treated cells exhibited a t1/2 of 21.7 6 1.5 sec (n 5 27), and LH plus U-73122-treated cell a t1/2 of 39.1 6 7.2 sec (n 5 12; P 5 0.002; see Fig. 5). Similarly, in ET-1-stimulated cells, the t1/2 of 13.9 6 2.6 sec (n 5 34) for U-73343 cotreatment vs. the t1/2 of 43.6 6 14 sec (n 5 7; P , 0.002) for U-73122 cotreatment indicated a greater latency to half-maximal [Ca21]i increases during PLC inhibitor treatment. Effect of LH on inositol phosphate accumulation

The ability of LH to activate hydrolysis of prelabeled phophatidylinositol bisphosphate in cultured granulosa cells was evident by the rapid appearance of radiolabeled watersoluble inositol phosphates as a function of both LH con-


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FIG. 4. Manganese quench technique showing that extracellular cation influx contributes to the delayed LH-stimulated plateau-like [Ca21]i rise in single swine granulosa cells. Fura-2 fluorescence emission was monitored at two excitation wavelengths, 360 nm (Ca21 independent) and 380 nm (Ca21 dependent) sequentially. Dual wavelength excitation allows one to monitor changes in [Ca21]i and Mn21 influx. Approximately 15–25 sec after initiating fluorescence recording, experimental Ca21-containing medium was exchanged for Mn21containing medium to record the basal rate of Mn21 quenching of fura-2. Subsequently, LH-stimulated [Ca21]i mobilization and Mn21 influx were studied by delivering 10 nM LH prepared in Mn21-containing medium. These results were reproduced in a total of 20 (of 40) cells in 3 independent experiments.

centration and time (Fig. 6). Half-maximal stimulation of inositol phosphate production was observed at a LH concentration of 1.0 nm. A maximally stimulating concentration of LH (10 nm) induced a rapid increase in the accumulation of inositol phosphates recognized within 30 sec. In contrast, inositol phosphate accumulation in the control (mediumtreated) cells remained unchanged throughout the experiment. Discussion

The present experiments combined population and single cell observations in well differentiated ovarian granulosa cells to delineate how LH activates intracellular Ca21 second messenger signaling. Our findings implicate phosphodiesterase, PLC, mediation based on the present observations that 1) physiological LH concentrations stimulate the rapid liberation of inositol phosphates in (pig) granulosa cell populations; and 2) LH generates a biphasic spike- and plateaulike [Ca21]i signal in single granulosa cells, which can be abolished by a specific PLC inhibitor, but not its inactive congener. Moreover, assuming that the native LH receptor in granulosa cells mediates ligand coupling to PLC, the LH concentrations (10 pm to 10 nm) needed to elicit PLC activation in vivo fall within the physiological range of gonadotropin concentrations attained basally and during the spon-

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FIG. 5. Effects of U-73122, a PLC inhibitor, on LH-stimulated [Ca21]i mobilization in granulosa cells. Granulosa cells were harvested and prepared for [Ca21]i measurements in single cells as described in Materials and Methods. Cells were pretreated with 10 mM of the PLC inhibitor U-73122, its inactive analog U-73343, or vehicle [0.44% (vol/vol) DMSO] for 2 min. Then, granulosa cells were exposed to 200 nM LH or 10 mM ET-1 in the continuing presence of the respective pretreatment agent for an additional 2 min. In those cells responding to LH or ET-1, the time required to achieve the half-maximal value (t1/2) was calculated. In A (LH-stimulated cells) and C (ET-1 stimulated cells), granulosa cells were classified as responding with an increase in [Ca21]i if the Fo/Fi ratio was greater than baseline 1 3SD. In B and D, the time required to achieve the half-maximal [Ca21]i value (t1/2) was calculated for each treatment. Data are presented as the mean 6 SEM. Statistical significance is designated by an asterisk (ANOVA followed by Tukey’s highest significant difference multiple comparison test). Data are from a total of 210 (LH-stimulated) and 221 (ET-1 stimulated) granulosa cells evaluated in 4 independent experiments.

taneous proestrous LH surge in vivo, e.g. approximately 25 nm, as estimated in the rat. By single cell fluorescence video microscopy, we could demonstrate granulosa cell to cell heterogeneity in the actions of a given dose of LH in inducing a rapid onset biphasic rise in [Ca21]i. Of physiological interest, the fraction of individual granulosa cells responding with a spike and plateau [Ca21]i signal was significantly higher when granulosa cells were collected from more developmentally mature follicles. This finding is in agreement with the reported developmental expression of the LH receptor in populations of more highly differentiated granulosa cells (24), with recent studies using staged avian Graafian follicles (25), and with greater LH receptor messenger RNA expression in large preovulatory follicles (26). Moreover, transfection experiments have demonstrated that LH receptor signaling via PLC is dependent on receptor density (27). Of interest, we did not observe heightened ligand-induced [Ca21]i signaling with greater follicular maturation for the alternative PLC agonist, ET-1. As ET-1-mediated [Ca21]i signaling was preserved in the same individual granulosa cell that was unresponsive to LH,


FIG. 6. Concentration- and time-dependent stimulation by LH of water-soluble inositol phosphate accumulation in porcine granulosa cell populations. Monolayer cultures of (swine) granulosa cells were prelabeled with [3H]myo-inositol for 18 h and exposed to a 1-h pulse chase with unlabeled myo-inositol before administration of different concentrations of LH (top panel). In other experiments, granulosa cells were stimulated with one concentration of LH (10 nM) for the indicated times. The liberation of inositol phosphates was assessed by anion exchange chromatography. Data are the mean 6 SD (n 5 3 replicates). Similar results were obtained in one (top panel) and two (lower panel) additional experiments with different batches of granulosa cells.

we infer that single cell unresponsiveness to LH is not due to cellular phosphatidylinositol substrate or PLC enzymic deficiency, endoplasmic IP3 receptor insensitivity, or depletion of intracellular Ca21 stores. Accordingly, other factors, such as variations in the cellular expression or activation of functional G protein-coupled LH receptors, may offer a basis for granulosa cell to cell heterogeneity in [Ca21]i signaling. Using a single cell experimental strategy, we could investigate the relationship between the LH concentration and the [Ca21]i signaling response in the individual target (ovarian) cell bearing native LH receptors. We discovered that lower

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concentrations of LH elicit [Ca21]i signals of typically lower amplitude and delayed onset in granulosa cells, which may reflect an apparently incremental (quantal) nature of IP3mediated intracellular Ca21 mobilization via activation of IP3-sensitive Ca21 channels. For example, submaximal IP3 concentrations generated by low LH concentrations may only activate release of Ca21 from localized intracellular stores; on the other hand, stimulation of granulosa cells with higher LH concentrations may initiate more generalized propagation of Ca21 release, as has been inferred for intact and permeabilized cells (28). Indeed, Ca21 release by hormonal stimulation or IP3 delivery to permeabilized cells or membrane-reconstituted IP3 receptors appears to be a quantal rather than a continuous process (28, 29). In addition to the qualitative differences in the time course of [Ca21]i responses to varying concentrations of LH, our single granulosa cell studies reveal that the most significant mechanism by which higher concentrations of LH elicit a greater overall population response is by cell recruitment. In particular, granulosa cells within any given follicle were heterogeneous with respect to the threshold concentration of LH required to elicit a spike- and plateau-like [Ca21]i signal. Thus, higher LH concentrations progressively exceed the threshold requirements of a larger fraction of the ovarian (granulosa) cell population. Accordingly, the number of granulosa cells responding, rather than the magnitude of the individual cell’s response, primarily governs the population [Ca21]i response to any given LH concentration. A similar cellular dose-response mechanism has been recognized recently for the stimulation by FSH and ET-1 of [Ca21]i rises in (rat) Sertoli cells (30, 31), but not, to our knowledge, previously for LH actions. The present study demonstrates that the LH-stimulated biphasic [Ca21]i signal in granulosa cells involves both intracellular Ca21 mobilization and transmembrane cation influx. This inference is consistent with the idea that the native LH receptor of differentiated granulosa cells is coupled to PLC, as are other receptors that typically evoke these [Ca21]i dynamics (32–34). We used extracellular Ca21 depletion and Mn21 quench experiments to show that LH-stimulated Ca21 influx across the plasma membrane occurs only after the LH-stimulated mobilization of intracellular Ca21 stores. Initial agonist-mediated [Ca21]i mobilization followed by Mn21 entry has been observed in other cells with PLC-mediated second messenger signaling, e.g. human umbilical vein endothelial cells (33), and is compatible with the so-called capacitance model of Ca21 influx. This model asserts that the IP3-driven initial discharge of an internal Ca21 store establishes the basis for secondary (delayed) Ca21 entry (34). Interestingly, some granulosa cells (viz. ;50%) exhibited an apparently typical rapid initial spike pattern of intracellular Ca21 mobilization without evident Mn21 influx. This suggests that at the single cell level, Ca21 release from IP3 stores is not always coupled to full activation of capacitative Ca21 influx and/or that effective [Ca21]i depletion is not achieved uniformly by a single IP3-stimulated [Ca21]i spike-like response. Additional biochemical requirements may exist in granulosa and other cells before the IP3 signal and/or other mediators can bring about delayed Ca21 influx. This disparity has also been reported in rat basophilic leukemia cells,


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where sustained Ca21 release evoked by IP3 failed to activate capacitative Ca21 entry (35). Also, in some nonexcitable cells, depletion of the IP3-sensitive Ca21 stores does not result in capacitative Ca21 influx (36, 37). In summary, the present investigations of the mechanisms of actions of LH in motivating [Ca21]i signaling in single untransformed granulosa cells support the idea that native LH receptors are coupled to PLC. Based on single cell analysis, we infer further that LH stimulates a biphasic rise in [Ca21]i by way of immediate intracellular Ca21 mobilization and delayed transmembrane [Ca21]i influx. Stimulation of individual granulosa cells reveals significant cell to cell heterogeneity in [Ca21]i responses to fixed or increasing concentrations of LH. Moreover, higher concentrations of LH elicit a greater population [Ca21]i response in granulosa cells by recruiting a larger number of responding target cells without markedly altering the intensity of the second messenger Ca21 signal in any given granulosa cell. Acknowledgments We are grateful to Dr. Denis Leong for providing microscopy facilities for a portion of this work, to Dr. H. Pumarino, Director of Endocrinology Section (Hospital Elimeo de la Universidad de Chile), for support of Dr. Aguirre, and to Patsy Craig for expert preparation of the manuscript.

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