The effect of dichlorodiphenyldichloroethylene (DDE) on steroidogenesis in granulosa cells
Crellin, Natasha Kay
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The insecticide dichlorodiphenyltrichloroethane (DDT) and its major metabolite p,p'- dichlorodiphenyldichloroethylene (DDE) are endocrine disrupters. The DDT metabolite p,p'-DDE has been found contaminating human tissues and follicular fluid around the world, due to its persistence and continued use. The focus of this research was to investigate the effects of DDE on progesterone synthesis in a stable porcine granulosa cell line, JC-410, and in primary cultures of porcine granulosa cells. Further objectives of this research were to validate the JC-410 cell line as an in vitro model for the study of endocrine disrupters on ovarian steroidogenesis, and to elucidate the mechanism of action of DDE. Low concentrations of DDE, 0.1-100 ng/ml, did not affect basal progesterone synthesis in the JC-410 cells. However, 10 ng/ml DDE increased 8-Br-cAMP- and cholera toxin (CT)-stimulated progesterone synthesis 0.4-0.7-fold over the levels observed with 8-Br-cAMP or CT alone. This effect was confirmed in primary cultures of porcine granulosa cells. Neither basal nor CT-stimulated cAMP levels were changed by 0.1-100 ng/ml DDE. In the JC-410 cells, 1 and 10 ng/ml DDE increased CT stimulated cytochrome P450-cholesterol side chain cleavage (P450scc) mRNA levels 0.3- and 0.4-fold over the values observed with CT alone. High concentrations of DDE, 3000 and 10000 ng/ml, reduced basal progesterone synthesis in the JC-410 cells 0.51- and 0.75-fold, respectively. As well, DDE, 300-10000 ng/ml, blocked the CT-induced stimulation of progesterone synthesis. High concentrations of DDE, 3000 and 10000 ng/ml, decreased basal cAMP generation 0.33- and 0.64-fold, respectively. The addition of DDE 300, 3000, and 10000 ng/ml decreased CT-stimulated cAMP levels by 0.16, 0.32, and 0.48-fold respectively, as compared to CT alone. Basal expression of the P450scc gene was decreased 0.61- and 0.82-fold by 3000 and 10000 ng/ml DDE, respectively. Addition of DDE, 3000 and 10000 ng/ml, blocked the CT-induced stimulation of P450scc gene expression. Cellular protein levels were increased 0.35- and 0.42-fold, respectively, by 3000 and 10000 ng/ml DDE. It was concluded that DDE, at environmentally relevant concentrations, modulates granulosa cell steroidogenesis in a dose dependent manner, through two different mechanisms of action. At low concentrations, DDE potentiates the conversion of cholesterol to pregnenolone induced by PKA activators, thus stimulating progesterone production. At high concentrations, DDE inhibits basal and stimulated cAMP generation and P450scc gene expression, thus decreasing progesterone production. Based on these observations, it is possible that DDE could disrupt gonadotropin control of ovarian steroidogenesis, thus representing a threat to human reproductive health and fertility.