Synergistic effects between FSH and 1,1-dichloro-2,2-bis(P-chlorophenyl)ethylene (P,P'-DDE) on human granulosa cell aromatase activity

Edward V. Younglai1, Alison C. Holloway, Gareth E. Lim and Warren G. Foster

Department of Obstetrics & Gynaecology, Reproductive Biology Division, McMaster University, Health Sciences Centre, Hamilton, Ontario, Canada

1 To whom correspondence should be addressed at: Department of Obstetrics & Gynaecology, McMaster University Medical Centre, 1200 Main Street West, Hamilton, Ontario, Canada, L8N 3Z5. e-mail: younglai{at}mcmaster.ca


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: 1,1-Dichloro-2,2-bis(P-chlorophenyl)ethylene (P,P'-DDE, DDE), a metabolite of DDT, is a persistent hormonally active environmental toxicant which has been found in human serum and follicular fluid. The objective of this study was to investigate the interaction between FSH and ppDDE on aromatase activity in primary cultures of human granulosa cells. METHODS: Granulosa cells were obtained at the time of oocyte retrieval for IVF procedures and cultured in defined medium containing FSH and environmentally relevant concentrations of DDE. Aromatase activity was measured by incubating the cells with 1{beta}-[3H]androstenedione and measuring the release of 3H2O. RESULTS: The granulosa cell response to FSH was highly dependent on the basal level of aromatase activity (r = –0.703, P = 0.001, n = 17) with the highest activity occurring at low basal levels of aromatase activity. Enzyme activity was significantly stimulated at 100 ng DDE/ml. A synergistic effect on aromatizing activity was observed when cells were co-cultured with DDE and FSH. CONCLUSIONS: Concentrations of DDE similar to those present in human follicular fluid enhance basal and FSH-stimulated granulosa cell aromatizing enzyme activity.

Key words: aromatase/DDE/FSH/synergism


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pesticides are high-volume, widely used, environmental chemicals which have been thought to have a role in many adverse human health outcomes such as carcinogenesis, neurotoxicity and reproductive and developmental effects (Nilsson, 2000Go; Turusov et al., 2002Go). A major group of pesticides is the organochlorine pesticides which are detectable in air, dust, ground water, food and body tissues of humans (Baukloh et al., 1985Go; Jarrell et al., 1993Go; Schecter et al., 1994Go; Dewailly et al., 1996Go). One well-known organochlorine pesticide, DDT (1,1-dichlorodiphenyltrichloroethane), used for the control of mosquitoes, has been banned in North America since 1972, but is still being used in some countries such as Mexico (Ayotte et al., 2001Go) and therefore will continue to enter the environment through long range transport. The metabolite of DDT, 1,1-dichloro-2,2-bis(P-chlorophenyl)ethylene (DDE), persists in the environment and is consistently found in reproductive fluids of women requesting IVF in Canada (Jarrell et al., 1993Go) and in serum and follicular fluid of women from other parts of the world (Baukloh et al., 1985Go). DDE has also been detected in breast milk (Dewailly et al., 1996Go), in cervical mucus secretions (Wagner et al, 1990Go) and in amniotic fluid (Foster et al., 2000Go). The consequences of DDE exposure on human reproductive health are poorly understood.

In women undergoing IVF procedures, there is a negative correlation between high levels of DDE in follicular fluid and fertilization (Younglai et al., 2002Go) with high levels associated with failed fertilization. It is not known whether DDE affects fertilization directly but it has been shown to alter steroid production in granulosa cells. Thus, in porcine granulosa cells progesterone production is inhibited in the presence of high concentrations of DDE (Crellin et al., 1999Go, 2001; Chedrese and Feyles, 2001Go). DDE has also been shown to increase aromatase expression and activity in hepatic microsomes (You et al., 2001Go). The aim of this study therefore was to determine whether DDE can affect the production of estrogens in human granulosa cells via alterations in the activity of the aromatase enzyme system.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell preparation
Ethical approval was obtained from the institutional research ethics board for this work. 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, Canada; 0.5 mg per day for 10–14 days) and recombinant FSH (12–85 ampoules, 75 IU per ampoule Gonal F; Serono Canada Ltd, 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. Cells and fluid were centrifuged for 5 min at 320 g. The supernatant was removed and 9 ml sterile distilled water was added to the pellet to lyse the red blood cells. After 20 s, 1 ml 10xconcentrated phosphate-buffered saline (PBS) was added to stop the reaction. Cells were pelleted by centrifugation for 5 min at 320 g and resuspended in 3 ml of plating medium (Dulbecco’s modified Eagle’s medium:Ham’s F-12 containing 10% fetal bovine serum (Gibco, Life Technologies, Canada), 100 IU/ml penicillin, 0.1 mg/ml streptomycin and 0.25 µg/ml amphotericin B (Sigma–Aldrich, USA). Unlysed red blood cells were removed by layering the cell suspension over 3 ml Ficoll-Paque Plus (Amersham Biosciences, Canada) centrifuging for 10 min at 400 g and removing the cells at the media–Ficoll interface. Cells were then pelleted at 725 g for 10 min, resuspended in medium and seeded into 48-well tissue culture plates (BD Biosciences, Canada) at a density of 100 000 cells per well in 0.5 ml medium. Cells from 51 patients were used in this study.

Reagents and chemicals
Human pituitary FSH, EC-232-662-3, Sigma lot 092K1105, 7000 IU IRP 68/140 per mg and DDE [1,1-dichloro-2,2-bis(P-chlorophenyl) ethylene] were purchased from Sigma–Aldrich Chemicals. Radioactive 1{beta}-[3H]androstenedione was obtained from Perkin Elmer Life Technologies, USA. All tissue culture supplies were purchased from Life Technologies.

Cell culture
After culturing for 72 h, medium was replaced with varying concentrations of FSH for 24 h to obtain a dose–response curve. Media were then removed and aromatase activity was measured by a modification of the tritiated water formation assay previously described (Younglai and Jarrell, 1983Go). Briefly, the cells were incubated for 4 h with 0.5 ml medium containing 2.5 µCi/ml 1{beta}-[3H]androstenedione. At the end of this incubation, 300 µl of the media was removed, treated with 300 µl dextran-coated charcoal (250 mg/ml charcoal, EM Science, 2.5 mg/ml dextran, Pharmacia, Uppsala, in PBS) for 2 h at 4°C. The samples were then centrifuged for 20 min at 2500 g to remove the charcoal and the radioactivity in the supernatant as 3H2O was determined. Cells from 17 patients were used for this experiment.

In the second series of experiments, cells were treated with DDE at concentrations ranging from 1 ng/ml to 1 µg/ml for 24 h. These concentrations cover the range of concentrations of DDE reported to be present in human follicular fluid and serum (Jarrell et al., 1993Go). In a third experiment, DDE (100 ng/ml, a concentration representative of that found in human follicular fluid) and human FSH (35 mIU/ml, which was reflective of the normal mid-cycle peak of FSH (Hall et al., 1992Go), were added separately and together to cells from 10 patients. Each treatment was done in quadruplicate for every patient.

Following the removal of supernatant for aromatase activity, the cells were dissolved in 1 mol/l NaOH for protein determination by the method of Bradford (1976Go). Enzyme activity was then expressed in terms of tritiated water formed per mg protein in each well. To correct for variations in basal level of activity, changes observed were expressed as a percentage change over control wells for each patient with the control being set at 100%.

Statistical analysis
Data were analysed by one-way analysis of variance (ANOVA) and tested for homogeneity of variance and normality using Sigma Stat (SPSS, USA). Where data failed the normality test and/or the equal variance test, data were analysed using Kruskal–Wallis one-way ANOVA on ranks. Where significance was indicated (P < 0.05), the data were compared to the control group using appropriate post-hoc comparisons, either Tukey’s or Dunn’s ({alpha} = 0.05). For the dose–response of DDE, the data were further analysed with Dunnett’s multiple comparisons (P < 0.05).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As shown in Table I, no significant change was detected in aromatizing enzyme activity of primary cultures of granulosa cells from eight patients. Since the enzyme activity seemed to be related to basal activity, cells from nine more patients were studied. Figure 1 shows that there was a significant negative correlation between FSH stimulation of aromatase activity and basal enzyme activity when a concentration of 35 mIU FSH/ml was used (r = –0.703, P = 0.001, n = 17). The aromatase activity in response to FSH was normalized and expressed as a percentage of the control or basal activity. The basal activity shown on the x-axis in Figure 1 is the mass of tritiated water formed per 4 h per mg protein from 1{beta}-[3H]androstenedione converted to estrogen.


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Table I. Dose–response of granulosa cell aromatase activity to FSH (n = 8)
 


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Figure 1. Relationship between basal aromatase activity of granulosa cells measured as the release of 3H2O per mg protein, and stimulation with 35 mIU/ml FSH. Activity was normalized to the control which was set at 100%. FSH stimulation of aromatase activity was higher when the basal activity was low. r = –0.703, P = 0.001, n = 17 patients.

 
Similarly, there was a negative correlation between the magnitude of stimulation observed after treatment with 100 ng/ml DDE and the basal aromatase activity in cell cultures from 24 patients (r = –0.429, P = 0.0363, data not shown). Figure 2 shows the aromatase activity in response to increasing concentrations of DDE from 1 ng/ml to 1 µg/ml, i.e. 1, 5, 10, 50, 100, 500 and 1000 ng. A significant effect of DDE treatment on aromatase activity was found (H = 15.397, P = 0.031, ANOVA). Further analysis showed that 100 ng DDE/ml gave a 35% stimulation of aromatizing enzyme activity (Dunnett’s multiple comparisons, P < 0.05).



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Figure 2. Relationship between aromatase activity of granulosa cells and various concentrations of 1,1-dichloro-2,2-bis (P-chlorophenyl)ethylene (DDE): 1, 5, 10, 50 100, 500 and 1000 ng/ml. Aromatase activity was normalized and expressed as a percentage of control in response to increasing concentrations of DDE. Analysis of variance: P = 0.03, n = 24 patients.

 
The effects of a combination of DDE and FSH were then examined. As shown in Figure 3, when cells were co-cultured with this mixture the mean increase was 38.8% compared to 15.2% with DDE alone or 6.8% with FSH (ANOVA, H = 8.679, P = 0.034, n = 10). The effect of co-culturing with the mixture represented a 2-fold increase over the total effect of DDE and FSH added separately.



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Figure 3. The effects of FSH and 1,1-dichloro-2,2-bis (P-chlorophenyl)ethylene (DDE), alone and in combination, on aromatase activity of granulosa cells in culture. Activity was 2-fold greater than the sum of stimulations by either FSH or DDE alone, P = 0.034, n = 10 patients.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is known that the granulosa cell under the influence of FSH can aromatize androgens to estrogens (Hillier et al., 1994Go). While it is apparent from our studies that FSH did stimulate the aromatase enzyme system, the level of stimulation was not consistent among patients. This marked variation in aromatase activity in response to FSH was highly dependent on the basal level of activity and therefore generation of dose–response curves for FSH on many patients would result in wide standard errors. The inverse relationship between basal aromatase and the magnitude of response to FSH has not hitherto been reported. All of the patients studied were treated with GnRH antagonists followed by a highly purified form of FSH. It is therefore possible that many FSH receptors on the granulosa cells were occupied, leading to a lower response. An alternative hypothesis would be that locally produced inhibin induced by FSH may inhibit aromatase through a paracrine mechanism as has been reported in bovine granulosa cells in vitro (Jimenez-Krassel et al., 2003Go).

In earlier studies from our laboratory (Lobb et al., 1998Go), granulosa cells from patients treated with Pergonal (menopausal gonadotrophins, Serono Canada Ltd), a mixture of FSH and LH, or a pure form of FSH, Metrodin (Serono Canada Ltd), showed marked differences in the production of progesterone, and even more so compared to patients who ovulate normally (Lobb and Younglai, 2001Go). However, it has been reported recently that a 2-fold increase in aromatase activity could be observed in granulosa cells obtained from assisted reproduction patients in the presence of 5 ng/ml FSH (Whitehead and Lacey, 2003Go). The treatment protocols for the patients were not provided. With granulosa cells from patients treated with Pergonal or Metrodin, Yie et al. (1995Go) also observed that FSH at 20 ng/ml could stimulate aromatase activity but there was a seasonal variation in response. The doses of FSH used are all similar to that used in the current study. Cells from normally ovulating women do respond to FSH by producing more estrogen (Hillier et al., 1981Go). However, in none of the studies described was an attempt made to perform a dose–response. Our study highlights the need to evaluate stimulation protocols when using cells from ovarian stimulation patients.

The most significant finding of this study is the synergistic effect of co-incubation with FSH and DDE on aromatase activity of human granulosa cells. This is in contrast to the effects of FSH and DDE alone on aromatase activity where no significant stimulation was observed. This can be explained by the high variability in response depending on the basal enzyme activity. This variability makes the observation of the synergistic effect of the combined treatment even more convincing. Such a synergistic effect of FSH and an environmental toxicant has not hitherto been reported to our knowledge. Since the estradiol:testosterone ratio is strictly controlled during follicular development (Xia and Younglai, 2000Go) and estradiol plays a pivotal role in oocyte maturation (Tesarik and Mendoza, 1997Go) the synergistic effect of DDE and FSH raises the question as to whether enhanced estradiol production in vivo induced by the effect of endocrine disrupting chemicals on aromatase activity may accelerate the maturation of oocytes and thereby contribute to infertility in patients exposed to these substances. There is a close regulatory association between the oocyte and granulosa cells during follicular development (Buccione et al., 1990Go), and, because the estradiol:testosterone ratio (Xia and Younglai, 2000Go) in human follicular fluid affects the maturity of the enclosed oocytes, these data suggest that DDE may affect fertility via alterations in the steroidogenic pathway.

Potentiation of progesterone production in the presence of DDE (10–100 ng DDE/ml) and cholera toxin has been reported in swine granulosa cells (Crellin et al., 1999Go) and it was suggested that the DDE was acting through the protein kinase A activator. Modulation of the cytochrome P450 side chain cleavage enzyme (P450scc) gene was implicated since its expression increased in the presence of 1 and 10 ng/ml DDE but not at 100 ng/ml. Whether a similar situation exists with the human granulosa cell P450 aromatase system in the presence of DDE is not known. With rat granulosa cells DDT was found to stimulate basal progesterone production but not FSH stimulation of progesterone (Nejaty et al., 2001Go). Species variation must therefore be considered in any interpretation of results.

It is known that DDE is ubiquitous and is found in various tissues of humans throughout the world (Baukloh et al., 1985Go; Jarrell et al., 1993Go; Schecter et al., 1994Go; Dewailly et al., 1996Go; Turusov et al., 2002Go). The association of high levels of follicular fluid DDE and failed fertilization (Younglai et al., 2002Go), serum DDE and abnormal sperm characteristics (Hauser et al., 2002Go) and the pivotal role of estradiol in oocyte maturation (Tesarik and Mendoza, 1997Go) prompted us to hypothesize that DDE might be inhibitory to the aromatase enzyme system leading to lowered estradiol production. No inhibition of aromatizing activity was observed at any concentration of DDE tested, thereby disproving the hypothesis. The stimulatory effect of DDE on aromatase activity was not as dramatic as that when used in combination with FSH. Using a different endpoint, Chedrese’s group has also shown that DDE can stimulate progesterone production in porcine granulosa cells (Chedrese and Feyles, 2001Go; Crellin et al., 2001Go)

There are several mechanisms by which DDE could be acting as an endocrine disruptor (Cooper and Kavlock, 1997Go): through the steroidogenic pathway, as we and others (Crellin et al., 2001Go) have suggested, through receptor-mediated changes in protein synthesis (Kelce et al., 1995Go), as androgens or estrogens (Andersen et al., 2002Go) and by altering the flux of ions across the membrane (Kolaja and Hinton, 1977Go). The latter mechanism is possible since a calcium-activated potassium channel has been identified in the ovary (Kunz et al., 2002Go) and a calcium calmodulin system has been shown to be involved in FSH-stimulated steroidogenesis in rats and pigs (Carnegie and Tsang, 1983Go; Flores et al., 1992Go; Jayes et al., 2000Go). However, with human granulosa cells, FSH did not change calcium fluxes in single cells (Lee et al., 2002Go). It is possible that the conditions or stimulation protocols may be masking the calcium effect in human cells.

An increase in aromatase activity by DDE alone or in combination with FSH can result in an increase in locally produced estradiol which can have a proliferative paracrine effect on surrounding granulosa cells (Richards, 1980Go) or on folliculogenesis itself (Drummond and Findlay, 1999Go). In addition, estradiol may enhance progesterone synthesis through facilitation with FSH and LH (Richards et al., 1979Go). All of these downstream effects of DDE cannot be ruled out.

In conclusion, we have demonstrated that DDE at concentrations found in human tissues can enhance FSH stimulation of aromatase activity in human granulosa cells. While it is tempting to speculate that this effect might be beneficial to the oocyte since estradiol seems to play an important role in its maturation, it could also be detrimental in producing too much estradiol early in folliculogenesis and accelerate oocyte maturation, so that asynchrony in cytoplasmic–nuclear maturation results in failed fertilization. It is also possible that this DDE stimulation may exert a paracrine effect on granulosa cell proliferation and progesterone production.


    Acknowledgements
 
This study was supported by Canadian Institutes for Health Research. The authors gratefully acknowledge the assistance of M.S.Neal, the clinicians and nursing staff at the Centre for Reproductive Care, Hamilton Health Sciences for the provision of cells.


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Submitted on June 24, 2003; accepted on March 10, 2004.