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Rapid effects of pesticides on human granulosa-lutein cells

Department of Obstetrics and Gynecology, Reproductive Biology Division, McMaster University, Health Sciences Centre, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5

Correspondence should be addressed to E Younglai; Email: younglai{at}mcmaster.ca

	Abstract

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Following our previous demonstration that p,p'-DDE (dichlorodiphenylchloroethylene), at environmentally relevant concentrations, can rapidly increase intracellular calcium [Ca²⁺]_i concentrations in human granulosa-lutein cells, we examined whether other pesticides, such as Kepone, o,p-DDE and methoxychlor, have similar effects. Cultured human granulosa-lutein cells were loaded with Fura-2 AM, and changes in [Ca²⁺]_i concentrations within small areas of single cells were studied with a dynamic digital Ca²⁺ imaging system. Kepone, at concentrations of 0.2–2 nmol/ml, consistently increased [Ca²⁺]_i concentrations 2–6 times higher than baseline values within minutes of exposure. Methoxychlor at concentrations of 2.8–280 nmol/ml failed to alter [Ca²⁺]_i levels consistently in cells from 10 patients. However, at 0.28 and 1.4 nmol/ml, increases in [Ca²⁺]_i concentrations could be elicited by methoxychlor. The isomer o,p-DDE at 3 nmol/ml increased [Ca²⁺]_i in granulosa cells of 11/20 patients. Pertussis toxin treatment inhibited the [Ca²⁺]_i increases induced by estradiol, p,p'-DDE, o,p-DDE and methoxychlor, but not by Kepone or progesterone, indicating that Kepone and progesterone may act through an insensitive G protein-coupled receptor. The [Ca²⁺]_i increases induced by Kepone also occurred in Ca²⁺-free medium, suggesting that [Ca²⁺]_i mobilization occurred from the smooth endoplasmic reticulum. Thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca²⁺ pump, also stimulated [Ca²⁺]_i increases but did not inhibit the Ca²⁺ response to all the pesticides. These results demonstrate that pesticides can have a rapid effect on human granulosa-lutein cells, and a nongenomic mechanism of action is suggested

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Calcium (Ca²⁺) is an important intracellular messenger controlling many cellular processes, rapid highly localized Ca²⁺ spikes controlling fast responses and repetitive global Ca²⁺ transits or waves regulating slower processes (Berridge et al. 2000, Bootman et al. 2001). The concentration of Ca²⁺ within the cell is controlled by two general mechanisms: (i) entry from extracellular fluid by voltage-operated channels or (ii) release from endoplasmic reticulum by capacitative entry, which activates the store-operated channel permitting Ca²⁺ influx from the extracellular fluid. An increase in intracellular calcium [Ca²⁺]_i uptake is one of the earliest events of nongenomic action of steroids (Falkenstein et al. 2000). Such ligands act through G-protein receptors stimulating the formation of inositol trisphosphate (IP₃), which in turn activates the endoplasmic reticulum Ca²⁺ pump (Petersen et al. 2005). Luteinizing human granulosa cells respond to androstenedione with a rapid increase in [Ca²⁺]_i effected by mobilization of Ca²⁺ stores from the endoplasmic reticulum and by Ca²⁺ influx from the extracellular fluid through the voltage-dependent Ca²⁺ channel (Machelon et al. 1998). Human granulosa cells are therefore known to respond to steroids in a nongenomic manner.

Studies on the rapid nongenomic effects of pesticides are very limited. Kepone, an insecticide with a long half-life, has been extensively studied since the ‘Kepone episode’ in Virginia (Guzelian 1982). It was found to inhibit gonadotropin-stimulated 11-ketotestosterone production by non-genomic action in Atlantic croaker testes (Loomis & Thomas 2000). Kepone, as well as o,p-DDT, p,p'-DDT and methoxychlor, blocked the progestogen-induced stimulation of sperm motility in Atlantic croaker sperm (Thomas & Doughty 2004). The chlorinated insecticide o,p-DDT was found to inhibit L-type Ca²⁺ channels in vascular smooth muscle cells and evoke a rapid endothelium-independent relaxation of the coronary vasculature similar to that induced by estradiol (Ruehlmann et al. 1998). It also mimicked estradiol modulation of [Ca²⁺]_i changes in pancreatic ß cells (Nadal et al. 2000). o,p-DDD was also found to increase [Ca²⁺]_i in myometrial smooth muscle cells (Juberg et al. 1995), and o,p- DDE stimulated [Ca²⁺]_i uptake and prolactin release in GH3/B6 pituitary tumor cells (Watson et al. 2005, Wozniak et al. 2005). These studies suggest that insecticides can have rapid nongenomic effects.

Dichlorodiphenylchloroethylene ( p,p'-DDE), the metabolite of DDT (dichlorodiphenylchloroethane), is one of the most abundant persistent metabolites of insecticides found in the environment. The highest concentrations have been reported for human endometrium (median, 4.7 µg/kg wet weight) and body fat (median, 446 µg/kg wet weight), and it was the most often detected environmental toxicant in human tissues (Schaefer et al. 2000). It is also present in human cervical fluid (Wagner et al. 1990) and follicular fluid (Younglai et al. 2002) at concentrations of 60–1200 ng/ml. Cord blood of arctic Innuit has 0.33 µg/l of p,p'-DDE, levels which have been shown to kill human embryonic fibroblasts in culture (Simonetti et al. 2001). Methoxychlor, which was developed to replace DDT, is more labile, but also has adverse effects on reproduction (Cummings 1997). While most studies to date have focused on the genomic effects of these environmental toxicants (Younglai et al. 2005a), it is becoming clear that they may also have other effects at the membrane level.

O,p-DDE, the isomer of p,p'-DDE, has a high binding affinity for the membrane estradiol receptor of SKBR3 breast cancer cells and mimicks the action of estradiol (Thomas et al. 2005). Dieldrin, endosulfan and o,p-DDE at low concentrations increased calcium [Ca²⁺]_i concentrations and prolactin secretion in a pituitary tumor cell line (Watson et al. 2005, Wozniak et al. 2005) and rapidly activated the extracellular regulated kinases (Bulayeva & Watson 2004). These nongenomic effects have been extended in our laboratory to human granulosa-lutein cells, where p,p'-DDE was found to increase [Ca²⁺]_i concentrations rapidly (Younglai et al. 2004). In view of the critical role Ca²⁺ plays in cell proliferation (Brini & Carafoli 2000, Bootman et al. 2001), we examined the role of other environmental toxicants, such as o,p-DDE, methoxychlor and Kepone, on [Ca²⁺]_i in cultured human granulosa-lutein cells. These contaminants are persistent and have been reported to compete with natural ligands for membranes of gonadal tissue in marine animals (Thomas & Doughty 2004). Human beings are also exposed to 0.1–4 ppb/day of methoxychlor via the diet (Agency for Toxic Substances and Disease Registry 2002; US Department of Health and Human Services, Public Health Service, Atlanta, GA, USA). We focused on [Ca²⁺]_i changes, since this is an easily measured indicator of the rapid action of an agonist.

	Materials and Methods

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Approval was obtained from the institutional research ethics board for this work. Patients signed an informed consent form to allow use of their granulosa cells for research. Patients were treated with a long luteal protocol of gonadotropin-releasing hormone (GnRH) agonist (Lupron; Abbott Laboratories, Montreal, Canada; 0.5 mg per day for 10–14 days) and recombinant follicle-stimulating hormone (FSH) (Gonal F; Serono Canada, Oakville, ON, Canada; 12–85 ampoules, 75 IU per ampoule) followed by human chorionic gonadotropin (hCG) (Profasi; Serono). As a condition of use of these cells, patients were anonymized. After removal of oocyte–cumulus complexes, the remaining follicular aspirates were combined and transported to the research laboratory in polypropylene tubes, and the granulosa cells isolated and cultured. Methods of isolation and conditions of culture have been described previously (Younglai et al. 2004), except for the Lab-Tek two-chamber slides (glass cover slip on the underside) with a capacity of 3 ml per chamber. After 3–7 days in culture, areas containing small luteinized cells, characterized by the cytoplasmic nuclear ratio, were chosen for imaging. Cells were generally used after 2–3 days in culture. Chemicals were added as a threefold concentrated solution to achieve the final desired concentration.

Chemicals and reagents
Estradiol and progesterone were purchased from Steraloids, Newport, RI, USA. Fluvestrant (ICI 182,780) was obtained from Tocris Cookson, Ellisville, MO, USA. Fura-2 acetoxymethyl ester was obtained from Molecular Probes, Portland, OR, USA. Kepone (chlordecone) and o,p-DDE were purchased from AccuStandard, New Haven, CT, USA. All other chemicals were purchased from Sigma-Aldrich Chemicals, Oakville, Canada. All tissue culture supplies and growth factors were purchased from Life Technologies, Burlington, Canada. The steroids and pesticides were dissolved in dimethyl sufoxide (DMSO). The final concentration of DMSO never exceeded 0.5%, since this was previously shown to be optimum (Younglai et al. 2004, 2005b).

Incubation medium conditions for imaging
Granulosa-lutein cells were exposed to 1–2 µmol Ca²⁺/ml except for the experiments requiring Ca²⁺-free conditions, where the medium was replaced by the Ca²⁺-free isotonic physiologic medium containing 0.1 µmol EGTA/ml immediately prior to the measurement. Although distilled and deionized water was used for the preparation of solutions, contaminating Ca²⁺ from containers and other chemicals may contribute up to 10 nmol Ca²⁺ /ml.

Digital dynamic fluorescence ratio measurements
Changes in Ca²⁺ concentration were measured after loading the plated cells with the dye Fura 2-AM, as previously described (Younglai et al. 2004), with a dynamic digital Ca²⁺ imaging system (Image-I/FL, Universal Imaging Corporation, Downington, PA, USA) with a Zeiss lamp (XBO 100 W/DC) coupled to a Zeiss inverted microscope (Zeiss IM 35) with a 100 x oil immersion lens and a numerical aperture of 1.25. Images were integrated and collected by a Pulnix camera (TM-720, maximal at 3 s/frame) initially at a speed of 15 s/frame. In general, 1–2 digital probes, covering an area of five pixels each on the monitor, were placed on the image of each cell, usually near the plasma membrane or over the nuclear region. At least two cells per field were chosen. Changes in fluorescence ratio were recorded as colored tracings from each corresponding probe and the data stored. Since the probes covered small areas of interest within the cell, quantitation of calcium changes was not attempted. Images were saved and in the event some areas of interest showed oversaturation of color during processing the sequences were rerun with new areas of interest. Experiments were performed on 2–5 cells per treatment. At least three independent experiments were performed to address each question, using granulosa cells from a different patient recruited on a different day. Representative patterns of response are shown in the figures. In some instances, the positions of the probing windows were changed from the original placements to capture the spatial changes in [Ca²⁺]_i concentrations.

	Results

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Effects of pesticides, estradiol and progesterone
Fig. 1 shows the Ca²⁺ response of granulosa-lutein cells to the pesticides as well as estradiol and progesterone. In Fig. 1A, two probing windows were placed on each of two separate cells. Addition of p,p'-DDE, 3 nmol/ml, caused an immediate increase in [Ca²⁺]_i concentrations above the baseline in both cells, the largest increase occurring near the cell membrane (red square and red tracing). Addition of 3 nmol/ml estradiol also increased [Ca²⁺]_i concentrations, and the experiment was then terminated with EGTA. Figure 1B shows that p,p'-DDE again caused an immediate increase in [Ca²⁺]_i and did not prevent another [Ca²⁺]_i increase in the presence of 3 nmol/ml progesterone. Results with p,p'-DDE and steroids were similar to those previously reported (Younglai et al. 2004, 2005b) and served as positive controls. Both o,p-DDE and Kepone had effects similar to those of p,p'-DDE (Fig. 1C–F), but the [Ca²⁺]_i oscillations were not as obvious. Cells from 4/8 patients responded to 3 nmol/ml o,p-DDE. Fig. 1E and F represent experiments where the [Ca²⁺]_i responses were of low amplitude and short duration. In 11 experiments with Kepone, the [Ca²⁺]_i response was rapid and elevated, necessitating the addition of EGTA to reduce the response. Fourteen other experiments with Kepone concentrations of 0.2–3 nmol/ml showed a consistent increase in [Ca²⁺]_i concentrations that was 2–6 times higher than baseline values within minutes of exposure. As shown in Fig. 1G, methoxychlor increased [Ca²⁺]_i concentrations about twofold at concentrations of 0.28 and 1.4 nmol/ml (6/12 experiments), but above 2.8 nmol/ml, no effect could be elicited in 10 experiments (Fig. 1H). None of the pesticides had an effect on the subsequent [Ca²⁺]_i response to estradiol or progesterone.

View larger version (61K): [in this window] [in a new window]   Figure 1 Effects of pesticides, progesterone and estradiol, 3 nmol/ml each, except for Kepone (1 nmol/ml) and methoxychlor (1 nmol/ml for panel G and 3 nmol/ml for panel H), on [Ca2+]i changes in granulosa-lutein cells. Digital probes were placed on individual cells, and each tracing in the time profile denotes changes of fluorescence ratio at the location of a probe in a single cell, identified by the small squares in the insert. The ticks along the time profile indicate the time when a chemical is added. Each figure represents response of cells from at least three different experiments. When [Ca2+]i concentrations were high, EGTA, 2 mM, was added to chelate all the Ca2+ ions. When [Ca2+]i had returned to baseline, 1 µM ionomycin was added to check the integrity of the cell membrane. Cells in all experiments responded to the pesticides as well as to progesterone and estradiol, except for methoxychlor. The insert in the figure shows the location of the probing windows on the cells at the start of the experiment. Probes were color coded to match the time profile of changes in 340/380 nm ratio during the course of each experiment. P4: progesterone; E2: estradiol; DDE: p,p'-dichlorodiphenychloroethylene; opD: o,p-dichlorodiphenylchloroethylene; Kep: Kepone; MXC: methoxychlor; Ion: ionomycin.

View larger version (27K): [in this window] [in a new window]   Figure 2 Effects of 3 nmol/ml o,p-DDE and 1 nmol/ml each of Kepone (Kep) and methoxychlor (MXC) in Ca2+-free medium (containing 0.1 mM EGTA) and after addition of 2 mM extracellular Ca2+. Each graph represents cells from three different experiments. The solid line in each graph represents the period when extracellular Ca2+ was removed with EGTA. The protocol was identical to that of Fig. 1. Note the rapid increase in [Ca2+]i in the absence of extracellular Ca2+ when Kepone was added but the lack of response to o,p-DDE and methoxychlor. Panel A represents four experiments, panel B three experiments, and panels C and D three experiments each. Addition of extracellular Ca2+ caused increases in [Ca2+]i in the presence of all the pesticides. Abbreviations are the same as in Fig. 1.

View larger version (58K): [in this window] [in a new window]   Figure 3 Effects of 2 µM thapsigargin (TSG) followed by pesticides or steroids on [Ca2+]i uptake in granulosa-lutein cells. Panel A represents 18 experiments. Note the sustained increase of [Ca2+]i in the presence of TSG. The concentrations of steroids and pesticides used were the same as those in Fig. 2. TSG inhibited the increase in [Ca2+]i concentrations induced by estradiol and progesterone (B: seven experiments, C: four experiments, D: three experiments), but not those of the pesticides (E–H: three experiments each). Other abbreviations are the same as in Fig. 1.

View larger version (70K): [in this window] [in a new window]   Figure 4 Effects of cyclopiazonic acid (CPA) (1 µM) followed by 1–3 nmol/ml pesticides or steroids on [Ca2+]i uptake in granulosa-lutein cells. Each panel represents four experiments. Increases in [Ca2+]i concentrations were not as high as observed in the presence of either steroids or pesticides alone. Other abbreviations are the same as in Fig. 1.

View larger version (61K): [in this window] [in a new window]   Figure 5 Effects of adding 1 nmol/ml methoxychlor or 3 nmol/ml p,p'-DDE, estradiol or progesterone before exposure to 2 µM thapsigargin or 1 µM cyclopiazonic acid (CPA) on [Ca2+]i uptake in granulosa-lutein cells. Increases in [Ca2+]i concentrations were observed in all instances. Note the short-duration response to the lower concentration of methoxychlor. Abbreviations are the same as in previous figures. Kepone at 1 nmol/ml and o,p-DDE at 3 nmol/ml were also tested, and the results were similar. A total of 32 experiments were performed with the steroids or pesticides added prior to thapsigargin or CPA. The different panels represent [Ca2+]i responses obtained.

View larger version (71K): [in this window] [in a new window]   Figure 6 Effects of pretreatment of granulosa-lutein cells with 100 ng/ml pertussis toxin for 24 h prior to exposure to 1 nmol/ml methoxychlor or Kepone and 3 nmol/ml estradiol, progesterone or both isomers of DDE. Each panel represents three experiments each. The pertussis toxin remained in the media throughout stimulation and recordings. Except for progesterone and Kepone (A and D), the [Ca2+]i response to exposure by p,p'-DDE, o,p-DDE, estradiol and methoxychlor was inhibited (B, C, E and F). Abbreviations are the same as in previous figures.

	Discussion

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Nongenomic effects of pesticides on human reproductive tissues have not been adequately studied to date. Human cell lines have provided a model to conduct such studies. For example, the GH3/B6 pituitary cell line was shown to respond to picomolar and nanomolar concentrations of o,p-DDE with an increase in [Ca²⁺]_i concentrations and prolactin release (Wozniak et al. 2005). The human breast cancer cell line, SKBR3, which lacks nuclear receptors, responded to 0.1–1 µM o,p-DDE similarly to estradiol, and evidence was provided of a mechanism via a G-protein-coupled receptor on the cell membranes (Thomas et al. 2005). Interestingly, the estradiol antagonists, tamoxifen and ICI 182780, also acted at the cell membranes in this system.

The rapid effects of insecticides mediated by binding to membrane receptors have been demonstrated in other non-mammalian species. For example, Kepone and o,p- DDD bind to the plasma membrane receptor for the progestational maturation-inducing steroid in ovaries of spotted sea trout (Das & Thomas 1999). Kepone can also inhibit the gonadotropin-stimulated androgen production in testicular tissue from Atlantic croaker by an estrogen membrane receptor (Thomas et al. 1998). The nongenomic action of a progestin on sperm motility in Atlantic croaker can be partially or completely blocked by o,p'-DDT, Kepone, chlordane, methoxychlor and p,p'-DDT at concentrations of 0.01–10 µM (Thomas & Doughty 2004). This effect may be reversed with 10-fold higher concentration of the progestin, confirming previous findings that Kepone and o,p-DDE can displace [³H]-17,20ß,21-trihydroxy-4-pregne-3-one from its membrane receptor in sperm from Atlantic croaker (Loomis & Thomas 2000). It would appear from the data in Fig. 1 that the treatment of human granulosa-lutein cells with pesticides did not have an inhibitory effect on the subsequent [Ca²⁺]_i response to estradiol or progesterone. This would argue against competition for the same membrane receptors if this were the preferred mechanism of action. Radiolabel binding studies with purified membrane preparations will be required to answer this question.

Environmental toxicants have usually been shown to have effects at the nuclear level, p,p'-DDE having antiandrogenic effects (Kelce & Wilson 1997, Gray et al. 2001). The prostatic cell line (PALM) has been reported to respond to the organochlorine pesticides DDT, o,p- DDT, chlordane, aldrin, dieldrin, endrin, endosulfan and methoxychlor, which compete with the nuclear androgen receptor for the synthetic androgen compound, R1881 (Lemaire et al. 2004). The human hepatoma cell line HepG2, transiently transfected with the human androgen receptor and an androgen-responsive reporter, has also been used to show that o,p-DDT, o,p-DDE, o,p-DDD, p,p'-DDT, p,p'-DDE and p,p'-DDD all behave as antagonists at concentrations above 10^–6 M (Maness et al. 1998). p,p'-DDE has some agonist activity at 10^–5 M. Methoxychlor is weakly antagonistic, but its metabolite is 10-fold more potent. Methoxychlor also competes with estradiol for binding to estrogen receptors in MCF-7 cells; the relative binding affinity was 0.04% for o,p'-DDT and 0.004% for methoxychlor (vom Saal et al. 1995). It also induces premature nuclear expression of the estrogen receptor gene in the neonatal uterine epithelium of BALB/c mice (Eroschenko et al. 1996).

The lack of effect of methoxychlor at concentrations greater than 2 nmol/ml is surprising, since it is generally found to be a weak estrogen with a binding affinity of 0.004% compared with 100% with estradiol (vom Saal et al. 1995). It was this observation that led to the choice of the initial concentrations of methoxychlor. In cell-based assays, methoxychlor is also weakly estrogenic (Andersen et al. 2002). In classical pharmacologic studies, adverse effects are measured in terms of dose response and sometimes immediate changes. However, in studying the effects of environmental toxicants and particularly endocrine disrupters, the classical dose response is not always applicable since effects may be observed at extremely low concentrations and none at higher concentrations (Krimsky 2001). The inverse U-shaped dose response observed with methoxychlor may be an example of such a nonclassical dose response.

The changes in Ca²⁺ fluxes induced by the pesticides may affect the calcium-binding proteins and gene expression. For example, methoxychlor and DDT increase cellular calcium uptake and downregulate the expression of the trophoblast-specific, human, calcium-binding protein in trophoblast cells (Derfoul et al. 2003). These insecticides also inhibit cell proliferation, induce apoptosis and suppress expression of several trophoblast differentiation marker genes. Methoxychlor was also shown to induce follicular atresia in mice through higher Bax expression (Borgeest et al. 2004, Miller et al. 2005), whereas, in mouse ovarian surface epithelial cells, it increases the cell-cycle regulators, cyclinD2 and cdk4, and Bcl, and inhibits apoptosis (Symonds et al. 2005). It would be interesting to follow the expression of Bcl-2 and Bax genes after exposure of human granulosa-lutein cells to the pesticides.

The [Ca²⁺]_i changes induced by the pesticides in human granulosa-lutein cells may also be involved in opening of the Ca²⁺-activated K⁺ channel (BK_Ca). Kunz et al.(2002) have shown that oxytocin, estradiol and progesterone, which are produced on stimulation of human granulosa-lutein cells by hCG, induce increases in [Ca²⁺]_i levels. They concluded that BK_Ca channel activity in granulosa cells is mediated by components of the intraovarian signaling system, thereby interlinking a systemic hormonal and a local neuroendocrine system in control of steroidogenesis. FSH can also induce an increase in [Ca²⁺]_i concentrations in single granulosa cells (Flores et al. 1990). Changes in [Ca²⁺]_i have also been associated with antimitogenic activity of progesterone in rat granulosa cells (Peluso et al. 2002). Since the pesticides seem to act via mechanisms similar to those of estradiol and progesterone, it is possible that they may involve the BK_Ca channel as well as antimitogenic activities.

The effects of the insecticides on [Ca²⁺]_i concentrations in Ca²⁺-free media were varied. The rapid elevation in [Ca²⁺]_i induced by Kepone was unexpected, suggesting that at some stages in granulosa cell differentiation, cells may be very susceptible to the effects of Kepone. This conclusion was suggested by the time profiles in Figs 1C and D, and 2A and B, in which the [Ca²⁺]_i -induced peaks were of low amplitude. This pattern of response was similar to that of p,p-DDE, suggesting that Kepone may be acting at the same sites as estradiol. However, the further addition of Kepone after replenishment of extracellular Ca²⁺ is similar to the effects of progesterone on granulosa-lutein cells (Younglai et al. 2005b) and may indicate that Ca²⁺ is required for stabilization of the membranes before Kepone can act. The lack of effect of o,p-DDE and methoxychlor in Ca²⁺-free media indicates that these insecticides cannot mobilize Ca²⁺ from the smooth endoplasmic reticulum.

The most variable [Ca²⁺]_i responses were observed with o,p-DDE and methoxychlor, no effect occurring in a large percentage of experiments. Since each experiment was done on an average of three cells per culture chamber with a plating density of 100 000 cells per ml, it is possible that the chosen cells lacked binding proteins for the pesticides. Because of the difficulty of retrieving stimulation and follicular response data on each patient, we could not relate the lack of [Ca²⁺]_i responses to patients considered to be ‘poor’ responders, who comprise 9–24% of patients treated by in vitro fertilization (Keay et al. 1997).

Data on the inhibitors of the endoplasmic reticulum calcium pump indicate that in the granulosa-lutein cells the initial increase in [Ca²⁺]_i concentrations may be the result of release from the endoplasmic reticulum stores. Thapsigargin inhibits the endoplasmic reticulum pump independently of production of IP₃ or activation of protein kinase C, and has no effect on the plasma membrane Ca²⁺-ATPase (Thastrup et al. 1990). Cyclopiazonic acid also inhibits both Ca²⁺ uptake and Ca²⁺-dependent ATPase activity of skeletal sarcoplasmic reticulum (Seidler et al. 1989). Thapsigargin had two different Ca²⁺ response patterns in human granulosa-lutein cells. A sustained elevation for over 15 min and a shorter one of under 5 min. A similar response pattern has been described for thapsigargin in chicken granulosa cell cultures (Morley et al. 1992). The significance of these prolonged effects are unknown, but it has been postulated that the temporal and spatial pattern of response can trigger individual events and generate global waves that can spread throughout the cell (Berridge 2005). Cyclopiazonic acid had small amplitude effects on [Ca²⁺]_i concentrations in granulosa-lutein cells. Neither inhibitor prevented the increase in [Ca²⁺]_i concentrations induced with all the pesticides tested, suggesting that these pesticides can overide the endoplasmic reticulum calcium pump and act directly at the plasma membrane pump. The inhibition of the calcium effect of estradiol and progesterone by thapsigargin is unexpected and suggests that the plasma membrane pump is very efficient in maintaining intracellular calcium. On the other hand, the prolonged increase in [Ca²⁺]_i concentrations may lead to mucalpain and caspase-12 activation, thereby causing apoptosis, as seen in breast cancer cells (Sergeev 2005).

The main channels responsible for the release of Ca²⁺ from internal stores are the IP₃ and ryanodine receptors on the endoplasmic reticulum (Berridge 2004, 2005). The mitochondria also play a role in cellular Ca²⁺ homeostasis and function (Leo et al. 2005). Abnormally high elevations in [Ca²⁺]_i can lead to increases in ATP formation, with consequent loss of cytochrome c and the onset of apoptosis (Leo et al. 2005). ATP has been shown to cause apoptosis preceded by cytoplasmic blebbing in human granulosa-lutein cells (Park et al. 2003). Although we have also shown that ATP can induce [Ca²⁺]_i elevations (Younglai et al. 2004), the imaging system shuts down if the [Ca²⁺]_i concentrations rise above a 340/380 nm ratio of 5. Therefore, we believe that the [Ca²⁺]_i levels induced by the pesticides represent physiologic responses, except where the [Ca²⁺]_i response to Kepone exceeds the recording capacity of the imaging system.

Pertussis toxin inactivates sensitive G-proteins by ADP ribosylation of the -subunit, which includes members of the G_i and G_o family (Bristol & Rhee 1994). These G-proteins are subdivided according to their sensitivity to pertussis toxin (Simon et al. 1991). Our results suggest that estradiol, p,p'-DDE, o,p-DDE and methoxychlor act through a pertussis toxin-sensitive G-protein, since pre-treatment with the toxin inhibited the [Ca²⁺]_i response to stimulation by these agents. Estradiol and androstenedione act via a pertussis toxin-sensitive G-protein in porcine granulosa cells (Lieberherr et al. 1999). On the other hand, the [Ca²⁺]_i response to progesterone and Kepone was not inhibited, suggesting that these two compounds acted through a pertussis toxin-insensitive G-protein. These latter results are similar to the progesterone effects on [Ca²⁺]_i and IP₃ formation in luteinized porcine granulosa cells, where a pertussis toxin-insensitive G-protein is also involved (Machelon et al. 1996). The progesterone acts in a nongenomic manner in the luteinized porcine granulosa cells and the source of Ca²⁺ for the increased [Ca²⁺]_i concentrations depends on the stage of luteinization. Our results therefore suggest that pesticides can act via a G-protein-coupled receptor mechanism, as has been previously demonstrated for o,p-DDE in the SKBR3 human breast cancer cell line (Thomas et al. 2005). It is possible that the differences in [Ca²⁺]_i response to the pesticides may be related to the chemical structure of the organochlorines (two diphenyls in the DDEs and methoxychlor) and cyclooctane/cyclopentane rings in Kepone.

In conclusion, our data suggest that insecticides have an additional mechanism of action that involves a G-protein-coupled membrane receptor in evoking a rapid response in elevating intracellular Ca²⁺ from the endoplasmic reticulum or extracellular sources, thereby activating additional pathways in cell physiology. Like ovarian sex hormones such as estradiol and progesterone, which can have both genomic and nongenomic effects, the pesticides must be considered in this new light.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
This work was supported by the Canadian Institutes of Health Research. Dr Y J Wu is a Visiting Scholar from the Department of Obstetrics and Gynecology, Second Hospital of Hebei Medical University, China. We thank Michael Neal and the physicians of the Centre for Reproductive Care, Hamilton Health Sciences, for providing the granulosa cells. We are grateful to Dr C-Y Kwan of the Department of Medicine for providing access to the calcium imaging system and for consultation. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 20 August 2005
First decision 19 September 2005
Revised manuscript received 24 October 2005
Accepted 29 November 2005

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Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 

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