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Ovary |
Department of Obstetrics & Gynecology,3 Reproductive Biology Division,
the Department of Medicine,4 McMaster University, Health Sciences Centre, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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calcium, estradiol, follicle-stimulating hormone, granulosa cells, human chorionic gonadotropin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There are several mechanisms by which DDE could be acting as an endocrine disruptor: through the steroidogenic pathway [8], through receptor-mediated changes in protein synthesis [9], as antiandrogens or estrogens [10, 11], as inhibitors of synthesis of other hormones [12], and by altering the flux of ions across the membrane, as occurs in egg shells [13]. DDE causes the thinning of egg shells in birds probably because of an inhibition of Ca2+-dependent adenosine triphosphatase, calcium binding protein, and carbonic anhydrase [14]. DDT at concentrations of 64 and 128 µM inhibited ATP-induced Ca2+ uptake in bovine oviductal cells [15]. Calcium has been implicated in the mechanism of action of FSH in stimulating progesterone formation in rat [16] and porcine [17] granulosa cells, and hCG is a stimulator of Ca2+ uptake in human granulosa cells [18]. The inhibitory effects of DDT on Ca2+ uptake in bovine oviductal cells [15] and the effects of its metabolite DDE on thinning of egg shells [14] led us to hypothesize that DDE inhibits Ca2+ uptake in human granulosa cells. The aim of this study was to determine whether DDE can alter intracellular Ca2+ concentrations in human granulosa cells and affect the Ca2+ response to FSH and hCG.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human pituitary FSH (EC-232-662-3, lot 092K1105, 7000 IU IRP 68/ 140 per mg), ionomycin, ATP, and DDE were purchased from Sigma- Aldrich Chemicals (Oakville, ON, Canada). Human chorionic gonadotropin (lot B18856, 3050 IU/mg, WHO 1st IRP 75/551) was purchased from Calbiochem (La Jolla, CA). All tissue culture supplies were purchased from Gibco Life Technologies (Burlington, ON, Canada). Fura-2 acetoxymethyl ester was obtained from Molecular Probes (Eugene, OR). The gonadotropins were dissolved in normal saline before use, and DDE was dissolved in dimethyl sulfoxide (DMSO).
Cell Preparation
Approval for this work was obtained from the institutional research ethics board. Informed consent was obtained from each patient at the time of oocyte retrieval for IVF. Human granulosa cells were obtained at the time of oocyte retrieval from patients having IVF treatment at Hamilton Health Sciences Centre for Reproductive Care. Patients were treated with a long luteal protocol of GnRH agonist (Lupron; Abbott Laboratories, Montreal, QC, Canada; 0.5 mg/day for 10–14 days) and recombinant FSH (12–85 ampules, 75 IU/ampule Gonal F; Serono Canada, Ltd., Oakville, ON, Canada) followed by hCG (Profasi; Serono). After removal of oocyte- cumulus complexes, the remaining follicular aspirates were transported to the research laboratory in polypropylene tubes. The cells and fluid were centrifuged for 4 min at 175 x g, the supernatant was removed, and 9 ml sterile distilled water was added to the granulosa cell pellet to lyse the red blood cells. After 20 sec, 1 ml 10x concentrated PBS was added to stop the reaction. The cells were pelleted by centrifugation for 4 min at 175 x g and resuspended in 3 ml of plating medium, which consisted of minimum essential medium containing 10% calf serum (Gibco Life Technologies), 100 units/ml penicillin, 0.1 mg/ml streptomycin, 200 mM L-glutamine, 10 mM nonessential amino acids, 50 µl gentamicin, and 2.5 µl amphotericin B (Sigma-Aldrich, St. Louis, MO). The granulosa cells were then plated at a concentration of 100–200 000 cells/dish on coverslips glued to the bottom of 35-mm Falcon culture dishes. After 2 days in culture, the granulosa-lutein cells (hereinafter referred to as granulosa cells) were labeled with 8 µM Fura-2 acetoxymethyl ester for about 30 min. The medium used for analyzing Ca2+ changes was 10 mM Hepes (pH 7.4) containing 126 mM NaCl, 6 mM KCl, 10 mM glucose, 1.5 mM CaCl2, and 0.3 mM MgCl2. One run of each culture dish typically took about 20–30 min, and each experiment was performed in quadruplicate. At least 12 cells from a total of three separate patients on different days were monitored for each test substance.
Digital Fluorescence Ratio Calcium Imaging
Changes in Ca2+ concentration in the cytosol ([Ca2+]cyt) were measured using a dynamic digital Ca2+ imaging system (Image-I/FL; Universal Imaging Corporation, Downington, PA) with a lamp (HBO 100 W/DC; Zeiss, Thornwood, NY) coupled to an inverted microscope (IM 35; Zeiss) with a 100x oil immersion lens and a numerical aperture of 1.25, as previously described [19]. A filter wheel held filters at 340 and 380 nm, which alternated, and images were captured on the first and second quadrant of the monitor screen. The ratio between these two wavelengths (340:380 nm) was displayed on the third quadrant, and the time event of the ratio changes at selected regions of the cells was displayed on the fourth quadrant. Emitted fluorescence was detected with a 540-nm filter. Images were integrated and collected with a camera (TM-720; Pulnix, Alexandria, VA; maximal at 3 sec/frame) initially at a speed of 15 sec/frame. When there were no [Ca2+]cyt oscillations or when a rapid transient elevation in [Ca2+]cyt occurred, the speed was slowed to 30 sec/frame to conserve digital memory space for data collection in experiments running longer than 30 min. The optical part of the microfluorometric system was custom- modified to reduce photobleaching, and each measurement was made in the dark [19]. Exposure to light was minimized to further reduce photobleaching, and background values were preset by the defocusing technique. Prior to each set of experiments, the pseudocolor grading was calibrated against the in situ Fura-2 fluorescence ratio for maximum in the presence of 10 µM ionomycin (a calcium ionophore) and for minimum in the presence of 5 mM EGTA (2.5 times more concentrated than the Ca2+ concentration in the cell medium). None of the drugs used in this work caused quenching of the fluorescence spectrum of Fura-2. Calibration of the flourescence signal at the end of each experiment with excess EGTA and ionomycin confirmed the reciprocal change of the intensities at 340 and 380 nm with the change of [Ca2+]cyt. Because the relative [Ca2+]cyt as reflected by the 340:380 nm ratio is not dependent on changes of absolute intensity at each wavelength, a noticeable loss of Fura-2, which sometimes occurred when [Ca2+]cyt rose too high, did not affect the ratio image of the cells. The software for the imaging processing converted the fluorescence data obtained at 340 and 380 nm to 340:380 nm ratios pixel by pixel, and the ratios were expressed by the corresponding pseudocolors. Further conversion from the ratio values to the Ca2+ concentration values was not attempted because of intrinsic problems in the estimation of absolute [Ca2+]cyt [20].
In all cases, granulosa cells were always exposed to 1–2 mM Ca2+ except for the experiments requiring Ca2+-free conditions, where the medium was replaced with Ca2+-free isotonic physiological medium containing 0.1 mM EGTA immediately prior to measurement. Although distilled and deionized water were used for the preparation of solutions, contaminating Ca2+ from containers and other chemicals may contribute up to 10 µM Ca2+. Therefore, 0.1 mM EGTA was always included in the Ca2+- free medium. No changes in Ca2+ uptake could be elicited with up to 0.5% DMSO in the medium, the maximum concentration used in all these studies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1 shows the [Ca2+]cyt changes that occurred when the granulosa cells were exposed to an environmentally relevant dose of DDE (100 ng/ml). This figure is representative of three separate experiments using cells from a single patient in each experiment. Six probes were placed, two on each of three cells, with one near the nucleus and the other near the cell membrane. An increase in [Ca2+]cyt above the baseline was observed in all the cells, and the changes became oscillatory. The changes in [Ca2+]cyt were more prominent in the peripheral regions of the cell, represented by the purple, blue, and green probes, rather than in the central nuclear region, as seen in the time frame. These [Ca2+]cyt changes could be terminated with the addition of 2.5 mM EGTA, which caused the [Ca2+]cyt to return to baseline levels.
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When the concentration of DDE was increased to 1 µg/ ml, the changes induced in [Ca2+]cyt were more pronounced (Fig. 2). The response was biphasic, and elevation started near the cell membrane and progressed to the nuclear region. All monitored cells showed similar biphasic responses to DDE, with a sharp increase in [Ca2+]cyt followed by much larger increases over 10 min before the addition of EGTA. A few cells in the field did not show any change in fluorescence intensity.
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To determine whether the [Ca2+]cyt changes were due to uptake from the extracellular medium or to release from intracellular stores, cells were cultured in Ca2+-free medium. In Ca2+-free medium, 1 µg/ml DDE induced [Ca2+]cyt changes, but these changes were smaller than those observed with the standard medium (Fig. 3). When the concentration of Ca2+ in the medium was adjusted to 2.5 mM, there was a 3- to 8-fold increase in [Ca2+]cyt, with some oscillatory pattern. One probe (yellow) placed near the nucleus had a smaller response.
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Pharmacological effects are normally transient as long as the agent remains in contact with the tissue. Therefore, in an attempt to remove the effects of DDE, cells were washed thoroughly with medium and then observed. Washing did not remove the [Ca2+]cyt oscillations, but addition of EGTA immediately depressed the [Ca2+]cyt to below the resting level (Fig. 4). Not all cells responded in an identical manner to DDE; some cells showed responses of lower magnitude. Preincubation of cells for 48 h with 0.1 µg/ml DDE suppressed the normal sharp increase in [Ca2+]cyt and Ca2+ oscillations, i.e., cells preincubated with DDE over 48 h maintained their normally low [Ca2+]cyt (data not shown).
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Effect of the Agonist ATP
Because ATP increases [Ca2+]cyt, experiments were conducted to confirm this action as a positive control. ATP was added in increments to the medium such that the final cumulative dose was 300 µM. At the lower concentrations, small but distinct oscillations were observed (Fig. 5). Only when the cumulative dose of ATP reached 300 µM was there a marked increase in [Ca2+]cyt. A transient increase in [Ca2+]cyt was seen in all four monitored cells soon after the cumulative dose reached 300 µM ATP. The response was greater in three of the four monitored cells.
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This response to ATP was more evident in the next series of experiments, when the initial concentration of ATP was increased to 100 µM and the cumulative dose of ATP increased to 200 µM and finally to 300 µM (Fig. 6). In this series of experiments, marked elevations and oscillations were observed, with a suggestion of a dose response. However, in the presence of both ATP and DDE there was a significant and sustained increase in [Ca2+]cyt and oscillations, with one cell having a response that was higher than the detection limit. All monitored cells showed an identical pattern of effects but with different magnitudes.
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This potentiating effect of DDE on ATP-induced [Ca2+]cyt oscillations was more clearly seen when the order of adding ATP and DDE was reversed. When an environmentally relevant concentration of 100 ng/ml DDE was added, there was a small increase in [Ca2+]cyt and oscillations were also evident (Fig. 7). With the subsequent addition of increasing concentrations of ATP from 100 to 300 µM, there was a marked concentration-related increase in [Ca2+]cyt and oscillations in three of the four cells monitored. The fourth monitored cell showed [Ca2+]cyt changes with ATP that were of the same magnitude and frequency as those induced with DDE. After the addition of EGTA, the [Ca2+]cyt dropped below the baseline of cells before the addition of DDE and ATP.
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Effects of FSH and hCG
Because hCG and FSH induce Ca2+ changes in granulosa cells [16–18], these gonadotropins (which are the normal tropic hormones for the ovary) could also be used as positive controls. However, the Ca2+ response of granulosa cells to FSH stimulation was not as consistent as the response to DDE. Although minor oscillations could be seen over the period of observation with 17.5 mU/ml FSH, a more prolonged effect was not observed. Increasing the dose to 175 mU/ml FSH induced a sharp transient increase in [Ca2+]cyt followed by low-magnitude oscillations higher than the resting level (Fig. 8). This effect was observed in three of seven experiments. One probe (yellow) was directly on a nucleus, and the change in ratio at the time of the transient peak was not as pronounced as were those at the periphery.
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The results of adding hCG and DDE to granulosa cells are shown in Figure 9. Six probes were placed on various sites of the seven cells. Human chorionic gonadotropin at 50 IU/ml caused an elevation of [Ca2+]cyt and oscillations in many of the monitored cells. No marked synergism or additive effects were observed when DDE at an environmentally relevant dose was added, although there was a greater increase over hCG in one of probed areas. All cells responded to the ionophore ionomycin and to EGTA, even those that failed to respond to hCG.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fact that [Ca2+]cyt elevations can still be detected in Ca2+-free medium indicates that Ca2+ is also being released from intracellular stores. The suppressive effect of 48 h of preincubation with DDE on the subsequent response to DDE exposure indicates that the cells can adapt to exposure to environmental toxicants by evoking alternative mechanisms for maintaining the normal resting intracellular [Ca2+]cyt. The DMSO used for dissolving DDE had no effect on [Ca2+]cyt changes, indicating that the effects seen were due to DDE. All these observations point to a novel mechanism of action of DDE that has not been reported previously. DDE has been shown to be a potent androgen receptor antagonist [10] and to induce hepatic cytochrome P-450 monooxygenases [23] and hepatic aromatases [24]. Its effects on steroidogenic enzymes have already been alluded to [9, 21, 22]. The effect on intracellular Ca2+ changes in human granulosa cells represents another potentially important mechanism of action that can be attributed to DDE; these effects are seen at concentrations well below the no-observable-adverse-effect level, at levels normally found in human tissues.
Because DDT has estrogenic properties [25] and DDE has been reported to increase aromatase activity [24], a similar mechanism of action may occur in any estrogen-responsive tissue. In pig and hen granulosa cells [26], estradiol in the range of 10–10–10–6 M evoked a rapid transient 4- to 8-fold increase in intracellular calcium ion concentrations ([<011>Ca2+]i) that was not suppressed in the presence of 2 mM EGTA or by pretreatment with Ca2+ channel blockers. The effect could be blocked with inhibitors of inositol phospholipid hydrolysis but not with inhibitors of RNA or protein synthesis. From these results, the researchers concluded that Ca2+ was released from intracellular stores through a phophoinositide breakdown pathway via activation of a cell surface receptor in pig and chicken granulosa cells but not in immature rat granulosa cells. By contrast, DDE in Ca2+ medium, at levels normally present in human reproductive tissues, caused a small (2-fold; Fig. 3) but significant increase in human granulosa cell [Ca2+]cyt that could be prevented with EGTA. This increase was 3- to 8- fold in the presence of extracellular Ca2+. We also observed frequent oscillations in [Ca2+]cyt activity in the presence of DDE. Thus, the pattern of [Ca2+]cyt changes induced by DDE represent a unique observation. The rapid response of the chicken granulosa cells to estradiol [26] was attributed to a nongenomic membrane action of this estrogen. The similarity of the effect of DDE to that of estradiol is strongly suggestive of an identical mechanism of action.
The probe for detection of Ca2+ changes in individual cells covered an area with a diameter of five pixels. Up to nine probes have been used to follow the progression of Ca2+ changes through the cell membrane to the nucleus [27]. Thus, changes can be detected adjacent to the cell membrane, over intracellular organelles, or in the nucleus. Using this imaging approach, we found that DDE induces major changes in the peripheral regions of the cell, as seen in the greater Ca2+ oscillation near the cell membrane (Fig. 1). Changes in Ca2+ in the nuclear area were secondary, as seen when the probes were placed over the nucleus. This finding is in contrast to that for vascular endothelial cells, where perturbation of cell Ca2+ was more pronounced in the nuclear region than in the cytosol [27]. In addition, there was no change in cell shape (such as membrane blebs), which occurs in cytotoxic endothelial cells [27]. Not all cells responded to DDE in a similar manner, and some cells did not respond at all. Other researchers have shown that the distribution of luteinizing hormone receptors in the granulosa cells of the follicle is markedly different depending on the proximity to the oocyte [28]. In addition, there are small and large cells in the corpus luteum that develop from the granulosa theca, and these cells interact to produce progesterone at twice the amount released by each cell type alone [29]. Because the areas of detection of [<011>Ca2+]i were the equivalent of five pixels per cell, the possibility for detecting gradient changes within the cell makes this study unique and not comparable to previous studies using single cells [18, 30–32].
For a positive control for [Ca2+]cyt in human granulosa cells, we used ATP, which regulates a variety of biological processes [33] and elicits [Ca2+]i changes in human granulosa-lutein cells [32]. ATP colocalizes with neurotransmitters at concentrations >100 mM and is coreleased with both adrenaline and acetylcholine [33, 34]. Extracellular ATP can reach the granulosa cells through the wide sympathetic innervation of the ovary [35]. Although we found stimulation of [Ca2+]cyt at 300 µM ATP (Fig. 5), this effect was potentiated in the presence of DDE (Figs. 6 and 7). Such a potentiation of ATP has not be described before and begs the question as to whether the presence of ATP in the ovary can potentiate the effects of DDE, which is found in the follicular fluids of a majority of women attempting IVF. The results of this study also confirm the existence of purinoreceptors and the oscillatory response in [Ca2+]cyt in the human granulosa cell, as previously reported [31, 32].
We previously observed a synergistic effect of DDE on FSH stimulation of aromatase activity in human granulosa cells [36], and the calmodulin system and [Ca2+]cyt play a major role in granulosa cell steroidogenesis [16, 17, 30]. We therefore postulated that DDE and FSH could be acting in a similar manner to alter Ca2+ fluxes. Coincubation of granulosa cells with both FSH and DDE failed to elicit any potentiation (data not shown) of [Ca2+]cyt increases, as seen with ATP (Figs. 6 and 7). However, in three of seven experiments we observed a prominent transient peak in [Ca2+]cyt that could not be sustained (Fig. 8). This finding is in contrast to that in rat granulosa cells, where the calcium-calmodulin system seemed to play an important role in FSH-stimulated steroidogenesis [16], or that in porcine granulosa cells, where Ca2+ modulated the FSH stimulation of steroidogenesis [17]. FSH evoked marked Ca2+ elevation and sustained oscillations in single porcine granulosa cells, leading the researchers to suggest that this pathway is equally important in steroidogenesis [30]. The Ca2+ changes were dependent on extracellular Ca2+. By contrast, using single human granulosa cells Lee et al. [18] were not able to demonstrate any effect of FSH (2–4 µg/ml) on Ca2+ elevation. A transient increase within a cellular compartment where our probes were placed would not be detected in a whole cell. Our results are similar to those of Lee et al. in that FSH failed to evoke major oscillations and increases in [Ca2+]cyt in human granulosa cells, but our results differ in the detection of small oscillations and a transient spike in about half of the experiments.
The effects of hCG on granulosa cells were similar to those observed by Lee et al [18]. No marked additive or synergistic effect could be detected when DDE was mixed with the hCG. The heterogeneous response to both hCG and FSH corroborates previous observations on the heterogeneity of gonadotropin receptor effects on granulosa cells [28]. By contrast, the response of the granulosa-lutein cells to DDE was homogeneous, further supporting a nongenomic mechanism of action of DDE.
We have demonstrated for the first time (using a dynamic digital Ca2+ imaging system) that DDE can stimulate Ca2+ mobilization in human granulosa cells from both intra- and extracellular sources, that DDE has a nongenomic membrane mechanism of action and can potentiate ATP effects on [Ca2+]cyt elevations, and that FSH at physiological concentrations can induce a transient peak in [Ca2+]cyt. We are currently investigating the nature of the intracellular Ca2+ stores and the plasmalemmal Ca2+ channels of the human granulosa cell.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Edward Younglai, Department of Obstetrics & Gynecology, McMaster University Medical Centre, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5. FAX: 905 524 2911; younglai{at}mcmaster.ca
Received: 5 December 2003.
First decision: 5 January 2004.
Accepted: 2 February 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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