The addition of DDE to human granulosa cells caused [Ca2+]cyt elevations, which were detected as increases in the baseline 340:380 nm ratios, and oscillations, which were recorded as increased frequency of peak ratios. In previous studies with porcine granulosa cells, the major effect of DDE was an inhibtion of progesterone production. However, the concentrations of DDE used in those studies were much greater than those normally found in human tissues. At lower doses, comparable to those used in the present study, neither inhibitory nor stimulatory effects on progesterone production were observed. The consistency in Ca2+ response to DDE in the present study and the apparent dose-dependent effect of 0.1-1.0 ^g/ml DDE provide substantial evidence that DDE does stimulate [Ca2+]cyt elevations and intake from the exterior of the cell 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. 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. In addition, there was no change in cell shape (such as membrane blebs), which occurs in cytotoxic endothelial cells. 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. 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. Because the areas of detection of [Ca2+]y 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.
For a positive control for [Ca2+]cyt in human granulosa cells, we used ATP, which regulates a variety of biological processes and elicits [Ca2+] changes in human gran-ulosa-lutein cells. ATP colocalizes with neurotransmitters at concentrations >100 mM and is coreleased with both adrenaline and acetylcholine. Extracellular ATP can reach the granulosa cells through the wide sympathetic innervation of the ovary. Although we found stimulation of [Ca2+]cyt at 300 ^M 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 pur-inoreceptors and the oscillatory response in [Ca2+]cyt in the human granulosa cell, as previously reported.
We previously observed a synergistic effect of DDE on FSH stimulation of aromatase activity in human granulosa cells, and the calmodulin system and [Ca2+]cyt play a major role in granulosa cell steroidogenesis. 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 cal-cium-calmodulin system seemed to play an important role in FSH-stimulated steroidogenesis, or that in porcine granulosa cells, where Ca2+ modulated the FSH stimulation of steroidogenesis. 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. The Ca2+ changes were dependent on extracellular Ca2+. By contrast, using single human granulosa cells Lee et al. were not able to demonstrate any effect of FSH (2-4 ^g/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. 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. By contrast, the response of the granulosa-lutein cells to DDE was homogeneous, further supporting a nongen-omic 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.