RESULTS

Ca2+ Changes in Response to DDE

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.

When the concentration of DDE was increased to 1 ^g/ 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.

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 ^g/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.

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 ^g/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).

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 ^M. At the lower concentrations, small but distinct oscillations were observed (Fig. 5). Only when the cumulative dose of ATP reached 300 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 ATP. The response was greater in three of the four monitored cells.

This response to ATP was more evident in the next series of experiments, when the initial concentration of ATP was increased to 100 and the cumulative dose of ATP increased to 200 and finally to 300 (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.

This potentiating effect of DDE on ATP-induced [Ca2+Lyt 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 ^M, 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.

Effects of FSH and hCG

Because hCG and FSH induce Ca2+ changes in granulosa cells, 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.

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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FIG. 1. Effect of 100 ng/ml DDE on [Ca2+]cyt changes in primary cultures of human granulosa cells. Two probes were used for each of the three cells, one in the central nuclear region (red, yellow, and orange) and one near the cell membrane (purple, green, and blue). Note the sequential increases in [Ca2+]cyt oscillations over time. The different color tracings in the time profile denote change of fluorescence ratio at the location of each probe in single cells (2 days after culture), identified by the dots with corresponding colors in the square frame numbered 1. The ratio image in each frame was taken at the time identified by the numbers corresponding to the image numbers. The ticks along the time profile indicate the time when a chemical was added. At 3 min, DDE was added followed at 35 min by 2.5 mM EGTA. Most of the color changes took place at the periphery of the cells. Chelation of Ca2+ with EGTA caused an immediate drop in ratio. All cells showed responses to DDE albeit at different magnitudes, which may reflect the positioning of the probes on different parts of the cell. Over the 38 min, the baseline level of [Ca2+]cyt rose followed by increased frequency of peak ratios or oscillations. Note the absence of changes over the nucleus (red, yellow, and orange probes) or of changes in cell shape.
FIG. 2. Effect of 1 ^g/ml DDE on [Ca2+]cyt changes in primary cultures of human granulosa cells. The labeling is similar to that in Figure 1. Each numbered frame refers to the time when the ratio image was taken. DDE was added at 2 min, and EGTA was added at 10.5 min. Four probes were placed outside the nucleus on four different cells. Note the biphasic response in [Ca2+]cyt increase and oscillations. All four probed cells showed similar responses. The second set of 340-nm and 380-nm images reveal no significant loss of fluorescence intensity over the course of the experiment.

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FIG. 3. Effects of 1 ^g/ml DDE on intracellular Ca2 + changes in Ca2+-free medium. The labeling is the same as that in Figure 1. Cells were incubated in Ca2+-free medium and treated with DDE at 3 min. At 14 min, 2.5 mM Ca2 + solution was added, and EGTA was added at 20.6 min. Four probes were placed on four different cells. Note the rapid biphasic response in [Ca2+]cyt changes following the addition of Ca2+ to the medium and the return to resting level after addition of EGTA. The different magnitudes of response reflect changes within the cell where the probes were placed. The second set of 340-nm and 380-nm frames emphasizes the lack of dye leakage.
FIG. 4. Effect of washing with buffer on the response of human granulosa cells to DDE. Only the ratio images at the different time points are shown. Cells were treated with 0.1^g/ml DDE at 7 min, with 0.5 ^g/ml DDE at 25 min, and with 1 |xg/ ml DDE at 35 min. At 48 min, cells were washed three times with isotonic buffer. Four probes were placed on four cells. The [Ca2+]cyt oscillations remained elevated after washing and were reduced only after addition of EGTA at 62 min, which reduced the oscillations to a level lower than the resting level. All cells showed [Ca2+]cyt oscillations and higher levels than baseline.

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FIG. 5. Effect of the agonist ATP on [Ca2+]cyt changes in primary cultures of human granulosa cells. Probes were placed on four cells. Images at 340 and 380 nm and ratios are depicted. Cells were treated with 10 ^M ATP at 3 min, 100 ^M ATP at 7 min, 200 ^M ATP at 14.5 min, and 300 ^M ATP (cumulatively added) at 19.8 min. EGTA was added at 25.8 min. Note the sharp transient increase in [Ca2+]cyt oscillations at 300 ^M ATP and the smaller but distinct oscillations at the lower concentrations of ATP. As [Ca2+]cyt rises very rapidly it sometimes cause a leak of the Fura-2 dye, resulting in a loss of images at both 340 nm and 380 nm (frames 5 and 6). The images returned when the light intensity at 380 nm was adjusted to its maximum (last frame next to the time profile).
FIG. 6. Effect of treatment of cultured human granulosa cells with a combination of ATP and DDE. The labeling is similar to that in Figure 1. At 5 min, 100 ^M ATP was added, followed at 9 min by another 100 ^M ATP (total 200 ^M) and at 16 min by more ATP, for a cumulative concentration of 300 ^M; ATP. Then at 19 min, 1000 ng/ml DDE was added, and at 29 min 2.5 mM EGTA was added. Note the sequential increases in [Ca2+]cyt oscillations and the potentiating effect of DDE on ATP stimulation.

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FIG. 7. The effect of pretreatment of human granulosa cells with 100 ng/ml DDE followed by ATP. The labeling is similar to that in Figure 1. DDE was added at 3 min, and small [Ca2+]cyt oscillations were observed. At 17 min, 100 ^M ATP was added, followed by a cumulative dose of 300 ^M ATP at 26 min and EGTA at 40 min. Note the potentiation of [Ca2+]cyt increases and oscillations in three of the four probed cells and the return to below baseline after addition of EGTA.
FIG. 8. Effects of FSH on [Ca2+]cyt in cultured human granulosa cells. This figure is a representative positive response in three of seven experiments. Four probes were placed on four cells. FSH (17.5 mU/ml) was added at 3 min, and small [Ca2+]cyt oscillations were observed. At 15 min, 175 mU/ml FSH was added, resulting in a sharp transient peak (about 30 sec) in [Ca2+]cyt oscillations followed by smaller oscillations.

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FIG. 9. Effects of hCG on [Ca2+]cyt in cultured human granulosa cells. The images at 340 nm and 380 nm are shown at the identical time frames as the ratios of frames 1 and 4. Six probes were placed. The second set of 340- and 380-nm images reveal no signifciant loss of fluorescence intensity over the entire course of the measurements. Human chorionic gonadotropin (50 IU/ml) was added at 8 min, followed by 100 ng/ml DDE at 27 min, 3 ^M ionomycin at 35 min, 10 ^M ionomycin at 38 min, and 5 mM EGTA at 44 min. Note the [Ca2+]cyt oscillations in some of the cells, all of which responded to the ionophore and EGTA.