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Endocrinology Vol. 141, No. 6 2220-2228
Copyright © 2000 by The Endocrine Society

ARTICLES

Luteinizing Hormone (LH) Drives Diverse Intracellular Calcium Second Messenger Signals in Isolated Porcine Ovarian Thecal Cells: Preferential Recruitment of Intracellular Ca2+ Oscillatory Cells by Higher Concentrations of LH1

C. Aguirre2, F. C. L. Jayes and J. D. Veldhuis

Division of Endocrinology, Department of Internal Medicine, National Science Foundation Center for Biological Timing, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Dr. J. D. Veldhuis, Division of Endocrinology, Department of Internal Medicine, Box 202, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: jdv{at}virginia.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study examines Ca2+ second messenger signaling driven by LH in isolated porcine thecal cells. To this end, we implemented semiquantitative fluorescent (fura-2) videomicroscopic imaging of single thecal cells in vitro. Stimulation of 388 cells with LH (5 µg/ml) elicited an intracellular Ca2+ ([Ca2+]i) signal in 85 ± 5.3% of individual thecal cells (n = 11 experiments). Among 337 LH-responsive cells, we identified four predominant temporal modes of [Ca2+]i signaling: 1) [Ca2+]i oscillations with periodicities of 0.5 to 4.5 min-1 (63 ± 4.5%), 2) a [Ca2+]i spike followed by a sustained plateau (17 ± 2.6%), 3) a [Ca2+]i spike only (5.8 ± 2.6%); and 4) a [Ca2+]i plateau only (3.8 ± 1.5%). The prevalence, but not the amplitude or frequency, of LH-induced [Ca2+]i oscillations in thecal cells was dependent on the agonist concentration. Reduced availability of extracellular Ca2+ induced by treatment with EGTA or cobaltous chloride did not block the initiation, but reversibly abolished ongoing [Ca2+]i oscillations (72% of cells) or increased the mean [Ca2+]i interspike periodicity from 1.09 ± 0.16 to 0.59 ± 0.07 min-1 (P < 0.05). Putative phospholipase C inhibition with U-73122 (10 µM) also abolished or frequency-damped LH-driven [Ca2+]i oscillations in 95 ± 4.7% of cells. [Ca2+]i oscillations in thecal cells were not abrogated by overnight pretreatment with pertussis toxin. We conclude that 1) thecal cells (unlike earlier findings in granulosa cells) manifest a diverse array of [Ca2+]i signaling responses to LH at the single cell level; 2) LH can dose dependently recruit an increasing number of individually [Ca2+]i oscillating thecal cells; 3) extracellular Ca2+ is required for LH to sustain (but not initiate) frequent and high amplitude [Ca2+] oscillations in thecal cells; and 4) these signaling actions of LH are mediated via phospholipase C, but not a pertussis-toxin sensitive mechanism. Accordingly, the present data extend the apparent complexity of LH-induced [Ca2+]i second messenger signaling and identify at the single cell level LH’s dose-responsive drive of [Ca2+]i oscillations in gonadal cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAJORITY OF studies of gonadotropin-induced second messenger signaling to date have focused on the cAMP signal (1, 2, 3). However, FSH also evokes a monophasic slow onset intracellular Ca2+ ([Ca2+]i) signaling response in (testis) Sertoli and (ovarian) granulosa cells by activating Ca2+ influx (4, 5, 6). FSH-stimulated Ca2+ entry in granulosa cells is linked functionally to induction of cholesterol side-chain cleavage cytochrome P450 gene expression (7). In contrast, LH elicits a temporally biphasic, rapid-onset [Ca2+]i signal, prompt release of water-soluble inositol phosphates and diacylglycerol, and membrane translocation of protein kinase C in granulosa-luteal cells (8, 9, 10, 11, 12). Analogously, hCG and LH reportedly stimulate either a slow-onset (2–3 min) and prolonged (15-min) Ca2+ signal or no [Ca2+]i signal, respectively, in adult rat Leydig cells (13, 14). Although ovarian thecal cells are also crucial gonadal androgen-secreting targets of LH action, to our knowledge there is no consensus regarding the nature or mechanisms of LH-driven Ca2+ second messenger signaling in mammalian thecal cells. Such knowledge is potentially quite significant, as much of thecal cell pathobiology remains largely unexplained (15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Accordingly, the present analysis examines the nature, scope, and preliminary mechanisms of LH-directed [Ca2+]i signaling in (porcine) thecal cells at the single cell level. We thereby identify remarkable cell-cell heterogeneity of [Ca2+]i signaling modes and unmask a dose dependency of LH’s stimulation of [Ca2+]i oscillations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and hormones
LH (NIDDK oLH-26) and FSH (NIDDK oFSH-20) were provided by the Hormone Distribution Office, National Pituitary Agency, NIAMDD, NIH (Bethesda, MD). Porcine endothelin-1 (ET-1) was purchased from Bachem (Torrance, CA), and BSA (fraction V), porcine insulin, ionomycin, dimethylsulfoxide (DMSO), EGTA, cobalt chloride, collagenase (type IV), and deoxyribonuclease I were obtained from Sigma (St. Louis, MO). Culture medium and supplements were obtained from Life Technologies, Inc. (Grand Island, NY). Fura-2/acetoxymethyl ester (fura-2/AM) was purchased from Calbiochem (San Diego, CA). The phospholipase C (PLC) inhibitor U-73122 and its congener U-73343 were purchased from Research Biochemicals International (Natick, MA), dissolved in DMSO, and diluted in vehicle to 10 µM (0.4%, vol/vol, final concentration of DMSO) immediately before use. Purified pertussis toxin (PT) was a gift from Dr. Erik Hewlett (Department of Pharmacology, University of Virginia, Charlottesville, VA).

Thecal cell culture
Ovaries from prepubertal (50–65 kg) swine were obtained from a local abattoir. Thecal cells were isolated from small antral follicles (3–5 mm in diameter) by collagenase/deoxyribonuclease digestion of residual follicle linings after mechanical removal of granulosa cells, as described by May et al. (25). This method provides a thecal cell preparation with limited granulosa cell contamination. To further reduce any fibroblast contamination, 10 x 106 cells were cultured/10-cm dish (Corning, Inc., Corning, NY) in Eagle’s MEM supplemented with 3% (vol/vol) FBS, porcine insulin (3 µg/ml), and antibiotics for 2 h. The dish was then agitated to dislodge thecal cells, which were replated on glass chamber slides (Nalge Nunc International, Naperville, IL) at a density of 105 cells/ml. Cells were allowed to anchor for 48 h at 37 C in supplemented medium (above) in a humidified incubator (95% air-5% CO2), before culture medium was replaced with serum-free Eagle’s MEM containing 0.1% BSA (wt/vol), porcine insulin (3 µg/ml), and antibiotics. Incubations were continued at 37 C for an additional 14–16 h before imaging.

Measurement of serial changes in [Ca2+]i
A Cunningham chamber was built on each slide, and medium was replaced with so-called S-medium, a defined, phenol red-free, balanced salt solution consisting of 127 mM NaCl, 5 mM KCl, 2 mM MgCl, 1.8 mM CaCl2, 0.5 mM KH2PO4, 5 mM NaHCO3, 10 mM HEPES, 10 mM glucose, and 0.1% BSA, pH 7.4 (4, 26). Slides were preincubated with the Ca2+ indicator dye, fura-2/AM (3 µM), at 37 C for 20 min, washed with S-medium to remove unincorporated dye and incubated for an additional 20 min to allow deesterification of fura-2/AM.

[Ca2+]i responses were monitored serially at room temperature (22–23 C) using a Carl Zeiss Axioplan epifluorescence microscope (New York, NY) equipped with a fluor x20 objective. The UV excitation wavelengths were selected by narrow bandpass filters at 360 or 380 nm (Corion, Holliston, MA). Broadband fluorescence emission was monitored continuously via a silicon-intensified target camera (SIT-68 DAGE-MTI, Inc., Michigan, IN). Video images were stored on 0.75-in. broadcast quality videotape (30 frames/sec) for later analysis (below).

Cells were exposed to S-medium (balanced salt solution vehicle, above), then to FSH (20 ng/ml) for 4 min to identify contaminating granulosa cells, and thereafter to LH and/or the specific intervention described in each experiment. At the end of each study, we delivered the Ca2+ ionophore, ionomycin (10 µM), to verify fluorescence recordings. FSH-responsive cells (presumptively granulosa cells, 19 ± 1.2% of all imaged cells) were excluded from further analysis. To monitor nonspecific changes in fluorescence (e.g. due to photobleaching and/or dye leakage), short sequences of video images were recorded at 360-nm excitation wavelength (Ca2+ insensitive) at the beginning, during, and end of each experiment (26).

Image analysis
The recorded video signal was captured and digitized using software (Radtime) run on a QX-7 image analysis system (Quantex Corp., Sunnyvale, CA). Fluorescence intensity values after 380 nm excitation were converted to relative values using the equation R = Fo/Fi, where Fo is the initial emission intensity, and Fi is fluorescence at time i (4).

Statistical analysis
Cells were classified as responsive to an intervention, when Fo/Fi rose more than 3 SD above the prestimulus baseline (mean of 30-sec recordings) within 4 min. The (total) percentage of cells responding to any given stimulus and the (sub) percentage of LH-responsive cells showing any given response pattern were calculated. Graphs and tables show the mean ± SEM for at least 3 (and up to 11) independent experiments, each performed using a different batch of thecal cells, with 1–4 slides/intervention.

The percent response was calculated within each independent experiment, and data are summarized as the average ± SEM of replicate experiments. Data in the LH dose studies and the Co2+ inhibitor studies were analyzed by the {chi}2 test using the total numbers of cells in a certain response category pooled across all replicate experiments. The PLC inhibitor data were analyzed using a 3 x 3 contingency table to compare numbers of responding cells among the three different response categories and across the three intervention groups. Individual P values were calculated after z-score (standardized normal deviate) transformation. Significance was inferred at a protected P value of 0.01 to allow for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LH-induced [Ca2+]i responses in single thecal cells
After exposure to LH (5 µg/ml), a global mean (± SEM) of 85 ± 5.3% of porcine thecal cells (337 of 388 cells tested in 11 separate experiments) responded with a significant increase in [Ca2+]i. We identified four distinct and predominant temporal patterns of LH-driven [Ca2+]i signals (Fig. 1Go): [Ca2+]i oscillations (63 ± 4.5%), a [Ca2+]i spike followed by a sustained plateau (17 ± 2.6%), a [Ca2+]i spike only (5.8 ± 2.6%), and a [Ca2+]i plateau only (3.8 ± 1.5%). The [Ca2+]i signal was not readily categorizable in 10 ± 3.4% of cells. [Ca2+]i oscillations, the predominant signaling pattern, were maintained for at least three cycles and typically for 10–30 min after a single application of LH (30 min longest time tested; Fig. 1AGo). The periodicity of [Ca2+]i oscillations varied among different LH-responsive cells, but not within individual cells, namely, from 0.5–4.5 min-1 (absolute range).



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  		Figure 1. Illustrative [Ca2+]i signals in single porcine theca cells stimulated by LH (5 µg/ml). LH was delivered at 5 min. The {uparrow} indicates addition of ionomycin (10 µM) at the end of the experiment [see Materials and Methods]. Data are relative fluorescence intensities monitored every 30 msec during excitation at 380 nm.

 
LH concentration-dependent effects
Exposure of single thecal cells to 0.2, 1, or 10 µg/ml LH (a total of 87, 92, and 114 cells studied, respectively) evoked similar percentages of total [Ca2+]i-responding cells compared with 5 µg/ml LH (Fig. 2Go, inset). Whereas stimulation with 1 and 10 µg/ml LH elicited a similar distribution of [Ca2+]i response patterns, the lowest concentration of LH tested (0.2 µg/ml) significantly reduced the occurrence of [Ca2+]i oscillations to 37 ± 14% (P <= 0.0001) and increased the nonreadily categorizable responses (Fig. 2Go, other) to 24 ± 4% (P <= 0.0001). The latter signals principally consisted of delayed, transient, and low amplitude rises in [Ca2+]i. We detected no correlation between the concentration of LH and the frequency or amplitude of [Ca2+]i oscillations.



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  		Figure 2. Distribution of four predominant [Ca2+]i signal types among 573 single theca cells stimulated by different concentrations of LH. "Other" refers to [Ca2+]i changes that could not be readily categorized. Inset, Percentage of LH-responsive cells.

 
Alternative nongonadotropin agonist
We also evaluated the effects of ET-1 (10 µM; n = 107 cells). In four separate experiments 94 ± 3.6% of cells responded to ET-1. Subanalysis showed that 77 ± 4.2% of LH-responsive cells exhibited a spike-plateau (Fig. 3Go), and 21 ± 3.5% exhibited an oscillatory-like response. ET-1-induced oscillations were characterized by slower rises and troughs that did not return to baseline.



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  		Figure 3. Illustrative action of ET-1 (10 µM) on [Ca2+]i signaling by individual thecal cells. Exposure to ET-1 is indicated by the bar above the plot. The arrow indicates application of ionomycin (10 µM). A, Spike-plateau signal type. B, Oscillation signal type.

 
Role of extracellular Ca2+ in [Ca2+]i oscillations
To assess the dependence of LH-triggered [Ca2+]i oscillations on extracellular Ca2+, perfusate Ca2+ (1.8 mM) was chelated with 3 mM EGTA, or cellular Ca2+ entry was antagonized with 100 µM cobalt chloride (which nonspecifically blocks cellular uptake of divalent cations). Agents were added during the ongoing [Ca2+]i oscillatory phase or before the LH stimulus (below).

Effect of EGTA on LH-induced [Ca2+]i oscillations
Stimulation of porcine thecal cells (77 cells; 3 separate experiments) with LH (5 µg/ml) yielded 95 ± 4% total responding cells, 72 ± 3% of which showed [Ca2+]i oscillations. Considering delivery of the LH stimulus as time zero, the bathing medium was replaced with vehicle containing LH and EGTA 6.5 min later. Cells were monitored for another 7 min. Delayed EGTA addition rapidly abolished LH-induced [Ca2+]i oscillations in 38 of 53 oscillating cells (Fig. 4AGo) or only reduced their frequencies (15 of 53 cells; Fig. 4BGo).



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  		Figure 4. Representative plots showing the effects of delayed addition of EGTA to chelate extracellular Ca2+ during ongoing LH-induced [Ca2+]i oscillations in single thecal cells. Exposure to LH or LH and EGTA is indicated by corresponding bars above the plots. A, EGTA abolished [Ca2+]i oscillations in 72% of thecal cells. B, EGTA reduced the frequency of [Ca2+]i oscillations in 28% of cells.

 
In further studies, thecal cells (231 cells in 5 separate experiments) were preexposed to S-medium containing EGTA for 30–60 sec before stimulation with LH in the continuing presence of EGTA. Under these conditions, 92 ± 5.2% of thecal cells responded with an initial increase in [Ca2+]i. The presence of EGTA did not alter the distribution of temporal response patterns compared with cells stimulated with LH only (n = 170 cells; Fig. 5Go), but caused a progressive decrease in the amplitude and frequency of [Ca2+]i oscillations within 4–6 min (Fig. 6Go). These suppressive effects were fully reversible, as replacement of medium with EGTA-free S-medium restored [Ca2+]i oscillations promptly (Fig. 6Go).



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  		Figure 5. Distribution of [Ca2+]i signaling responses among 378 LH-responsive single thecal cells. Cells were treated with LH (5 µg/ml) only (n = 162) or were preexposed to EGTA (3 mM) for 30–60 sec before stimulation with LH in the continuing presence of EGTA (n = 216). Cells were monitored for 6–10 min after the addition of LH. Inset, Percentage of LH-responsive cells.

 


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  		Figure 6. Representative [Ca2+]i signaling response by a single porcine thecal cell to a LH stimulus (5 µg/ml) delivered in the continued presence of 3 mM EGTA and subsequent replacement with EGTA-free vehicle. Bars indicate the duration of treatment.

 
Effect of cobalt chloride on [Ca2+]i oscillations
In 3 other separate experiments (209 cells), LH initiated an increase in [Ca2+]i in 90 ± 3.7% of all cells, and 77 ± 3.1% of these [Ca2+]i responses were oscillatory. When medium was replaced with vehicle containing LH and CoCl2 approximately 5 min after delivery of LH, oscillations were not affected in 16 ± 6.0%, were quickly abolished in 29 ± 6.9%, and were maintained at lower frequencies in 55 ± 1.0% of thecal cells (Fig. 7Go, A–C). In the last case, the oscillation periodicity was reduced from 1.1 ± 0.16 to 0.59 ± 0.07 min-1 (P < 0.05, by paired t test).



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  		Figure 7. Impact of delayed addition of CoCl2 (100 µM) on ongoing LH-induced [Ca2+]i oscillations in single thecal cells. A, No effect on [Ca2+]i oscillations was observed in 16% of cells. B, [Ca2+]i oscillations were abolished in 29% of cells. C, The frequency of [Ca2+]i oscillations was reduced in 55% of cells.

 
Preincubation with CoCl2 for 3 min before LH stimulus (120 cells in 3 separate experiments) did not reduce the total percentage of [Ca2+]i-responsive cells, i.e. 86 ± 5.1% of cells. However, only 43 ± 6.5% of responding cells exhibited an oscillatory [Ca2+]i pattern (P <= 0.0001 vs. LH alone, by {chi}2 test; Fig. 8Go). There was a reciprocal increase in the percentage of cells responding with nonoscillatory patterns (Fig. 8Go).



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  		Figure 8. Distribution of [Ca2+]i signal types among single thecal cells stimulated by LH (5 µg/ml) alone or after preincubation with CoCl2 for 3 min and then stimulated simultaneously with CoCl2 (100 µM) and LH. Cells were monitored for 5 min thereafter. *, P <= 0.001; **, P <= 0.0001 (vs. LH only). Inset, Percentage of LH-responsive thecal cells.

 
Effect of pertussis toxin (PT) pretreatment on LH-stimulated [Ca2+]i oscillations
To determine whether a PT-sensitive G protein mediates LH-induced [Ca2+]i oscillations, thecal cells were pretreated with 500 ng/ml PT for 14–16 h before stimulation with LH (5 µg/ml). This PT exposure regimen causes ADP-ribosylation of an inhibitory guanine nucleotide-binding protein in pig granulosa cells (27). However, prior exposure to PT did not alter the distribution of [Ca2+]i signal patterns in LH-stimulated thecal cells (Table 1Go).


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  		Table 1. Effect of pertussis toxin pretreatment (PT) on LH-stimulated [Ca2+]i signals in porcine thecal cells

 
Effect of PLC inhibitor U-73122 on LH-induced [Ca2+]i oscillations
To investigate whether LH-evoked [Ca2+]i oscillations require activation of PLC, we tested the effects of the PLC inhibitor U-73122 (10 µM) on LH-stimulated [Ca2+]i signaling (8). In 3 separate experiments (total of 282 cells), when U73122 was added 5 min after the LH stimulus, [Ca2+]i oscillations ceased (Fig. 9CGo), continued at lower frequencies (Fig. 9BGo), or occasionally remained unaltered (Fig. 9AGo). After delayed addition of U-73122, only 5% of ongoing [Ca2+]i oscillations continued unaltered (Fig. 10AGo). In addition, a higher percentage of cells exposed to U-73122 (49 ± 10%) discontinued [Ca2+]i oscillations (P <= 0.0001 vs. vehicle, 13 ± 9%; Fig. 10CGo).



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  		Figure 9. Delayed treatment of individual thecal cells with control solvent, U73122, an active phospholipase C inhibitor, or U73343, a less active congener, affected ongoing LH-induced [Ca2+]i oscillation in three possible ways. A, Oscillations continued unaltered. B, Oscillations continued at reduced frequency. C, Oscillations ceased. The distribution of treatment effects is shown in Fig. 10Go. The last [Ca2+]i rise at the arrow reflects the addition of ionomycin.

 


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  		Figure 10. Percentage distributions (mean ± SEM) of LH-induced [Ca2+]i responses in thecal cells exposed to control vehicle (DMSO), U-73343 (10 µM; a sparingly active PLC inhibitor), or U-73122 (10 µM; an active phospholipase C antagonist). **, P <= 10-4; ***, P <= 10-5 (vs. DMSO). Representative examples of response categories are shown in Fig. 9Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Whereas much of clinical thecal cell pathobiology remains unexplained, this knowledge deficit may in part reflect an unexplored diversity of (gonadotropin-dependent) regulatory mechanisms operating in this target cell population. To this end, we sought to extend knowledge of the cellular mechanisms of LH-driven second messenger signaling in thecal cells by examining non-cAMP (Ca2+) signaling. We pursued this analysis at the single cell level to assess the possible cell-cell heterogeneity of [Ca2+]i signaling responses and to inferentially reconstruct the nature of LH’s population-based dose-responsive actions. These experiments revealed a remarkably consistent (85% of thecal cells) ability of LH to trigger activation of one or more distinct time patterns of [Ca2+]i signaling, viz., recurrent [Ca2+]i oscillations, a biphasic spike and plateau response, [Ca2+]i spike only, and plateau only responses. Moreover, we could establish that a higher LH concentration specifically increases the number of [Ca2+]i oscillating cells while reducing the prevalence of plateau or spike and plateau only responses.

The most prevalent [Ca2+]i signal type induced by LH in thecal cells consisted of sustained and extracellular Ca2+-dependent [Ca2+]i oscillations (persisting for up to 30 min, the longest time tested). Unlike [Ca2+]i oscillatory activity in some other endocrine and many nonendocrine cells (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38), the agonist concentration did not control the amplitude or frequency of [Ca2+]i oscillations. Instead, a novel finding in thecal cells was the ability of increasing LH doses to govern the percentage of Ca2+ oscillation-responsive target cells maintaining typical periodicities of 0.5–4.5 min-1. The latter range further illustrates the prominent between-cell variability in the frequency of [Ca2+]i transients elicited by the same LH concentration. In this endocrine cell, the [Ca2+]i periodicity remained quite stable in any single LH-responsive cell, perhaps denoting consistency of [Ca2+]i dynamics within individual, but not among different, thecal cells. Other studies suggest that LH receptor density might play a role in the relative ability to stimulate PLC over adenylyl cyclase activity, but we are unaware of any further evidence linking receptor concentration to the subtype of [Ca2+]i signal generated by LH (39). Whether oscillatory [Ca2+]i frequencies govern secretory granule exocytosis, mitochondrial NADPH metabolism, and/or specific gene expression in thecal cells, as suggested recently for some other steroidogenic tissues, is not yet known (29, 40, 41, 42, 43, 44, 45, 46, 47, 48).

The prominent temporal diversity of the [Ca2+] signaling modes elicited by LH among porcine thecal cells contrasts with that of granulosa cells in the same species (8). Indeed, no [Ca2+]i oscillations have been reported in the latter cell type. This cell-dependent signaling selectivity suggests that the target cell micromilieu conditions the precise nature of the [Ca2+]i second messenger signal driven by any given agonist. The biochemical basis for such a distinction has not been elucidated.

The mechanisms of LH-driven [Ca2+]i signaling in thecal cells involved both a rapid-onset release of Ca2+ from internal stores (spike phase) and delayed influx of extracellular Ca2+ (ongoing oscillatory and sustained plateau phases). These Ca2+ sources inferred in primary cultures of porcine thecal cells resemble those also identified for the murine LH receptor transfected in Xenopus oocytes (49). An extracellular source of Ca2+ is also crucial to maintain agonist-driven [Ca2+]i oscillations in some nonendocrine and certain other nonexcitable endocrine cells; e.g. ATP in porcine aortic smooth muscle cells, phenylephrine in canine pulmonary artery smooth muscle cells, histamine in human umbilical-vein endothelial cells, and ACTH in adrenal zona-fasciculata cells (32, 35, 50, 51, 52). However, in one study, angiotensin II maintained short term [Ca2+]i oscillations without extracellular Ca2+ in systemic arterial smooth muscle cells, albeit not in adrenal zona glomerulosa cells (35). This apparent difference might reflect unequal intracellular Ca2+ pool sizes in the two cell types and/or other cell-specific factors.

A specific phospholipase C inhibitor, U-73122 (but not its congener U-73343), significantly antagonized LH’s ability to initiate or sustain oscillatory [Ca2+]i signals (50, 53). Agonist stimulation of PLC activity also can mediate [Ca2+]i oscillations in some nongonadal cells without or with PT sensitivity. In thecal cells, a PT-sensitive GTP-binding protein does not appear to be involved in LH-stimulated [Ca2+]i oscillations. This particular attribute also characterizes the spike and plateau [Ca2+]i responses of granulosa-luteal cells to LH, albeit not those to ET-1 (8, 54).

The literature provides few and controversial reports of specific calcium signals in primary Leydig cell cultures. We are aware of two single cell (fura-2) studies in adult rat Leydig cells. In one, no [Ca2+]i changes could be identified in response to LH action, whereas ET-1 was effective in 30% of Leydig cells (14). In another analysis, hCG induced a slow-onset (2–3 min) and sustained (15-min) monophasic [Ca2+]i plateau that was dependent on extracellular Ca2+ (13). To our knowledge, [Ca2+]i oscillatory activity has not been reported in this target cell under LH action. However, several independent laboratories have observed that LH/hCG can consistently stimulate Ca2+-activated Cl- or K+ conductance channels (39, 49, 55, 56, 57). The precise relationship, if any, between the latter nonvoltage-dependent ion channel activity and [Ca2+]i oscillations and/or [Ca2+]i spike and plateau signals generated in the ovarian homologue, the thecal cell, is not known. Functionally, Ca2+-dependent PKC activation may participate in desensitization of rat and mouse Leydig cells to LH actions (56).

In addition to unmasking a remarkable thecal cell to cell diversity of [Ca2+]i signaling modes, analysis at the single cell level elucidated a mechanism of LH’s dose responsiveness. Indeed, to our knowledge the ability of higher pituitary hormone concentrations to recruit an increasing percentage of [Ca2+]i oscillatory cells has not been recognized in granulosa or Sertoli cells or described in the corpus luteum, steroidogenic cells of the adrenal gland, placenta, or testis. Thus, we do not know whether this is a unique mechanism mediating pituitary trophic hormone dose responsiveness on target steroidogenic cell populations. This mechanism might become pertinent in thecal cells driven by high LH concentrations during the preovulatory LH surge and thereby confer additional thecal vs. granulosa cell specificity of LH’s actions at this time. Moreover, whereas higher concentrations of LH are required to activate PLC-dependent [Ca2+]i transients compared with adenylyl cyclase in various transfected and native LH receptor-bearing cells (39, 49, 58), the biochemical mechanisms by which escalating LH concentrations preferentially recruit oscillatory (rather than purely spike, plateau only, or spike and plateau) [Ca2+]i signals is not known. The present model of highly consistent LH concentration-dependent actions on second messenger [Ca2+]i signal oscillations should provide a useful basis for further exploring such signal-specific control by a single agonist.


   Acknowledgments
 
We thank Patsy Craig for her skillful preparation of the manuscript.


   Footnotes
 
1 This work was supported in part by NIH Training Grant in Reproductive Neuroendocrinology T32-DK-07646, National Research Scientist Award 1-F32-HD-08284–01, NIH Grants HD-16393 and HD-16806, NIH Grant P30-HD-28934 (Center for Cellular and Molecular Reproduction), and the NIH U-54 Specialized Cooperative Centers Program in Reproductive Research (NICHD HD-96–008). Back

2 Current address: Abbott Laboratories de Chile Ltd., Avenue El Salto 5380, Huechuraba, Santiago, Chile. Back

Received November 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Marsh JM 1975 The role of cyclic AMP in gonadal function. In: Greengard P, Robison GA (eds) Advances in Cyclic Nucleotide Research. Raven Press, New York, pp 137–167
  2. Leung PCK, Armstrong DT 1980 Interactions of steroids and gonadotropins in the control of steroidogenesis in the ovarian follicle. Annu Rev Physiol 42:71–92[CrossRef][Medline]
  3. Urban RJ, Veldhuis JD 1992 Endocrine control of steroidogenesis in granulosa cells. In: Milligan SR (eds) Oxford Reviews of Reproductive Biology. Oxford University Press, New York, vol 14:226–262
  4. Flores JA, Veldhuis JD, Leong DA 1990 Follicle stimulating hormone evokes an increase in intracellular free calcium ion concentrations in single ovarian (granulosa) cells. Endocrinology 127:3172–3179[Abstract/Free Full Text]
  5. Flores JA, Leong DA, Veldhuis JD 1992 Is the calcium signal induced by FSH in swine granulosa cells mediated by adenosine 3',5'-cyclic monophosphate-dependent protein kinase A? Endocrinology 130:1862–1866[Abstract/Free Full Text]
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