Dioxin perturbs, in a dose- and time-dependent fashion, steroid secretion, and induces apoptosis of human luteinized granulosa cells.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is the most toxic congener of a large class of environmental pollutants. Several studies have shown that TCDD exposure reduced fecundity and ovulatory rate in rats and increased the incidence of endometriosis in monkeys. Recent work suggests that TCDD's endocrine-disrupting effects are, at least in part, caused by a direct action at the ovary. Although the factors involved in TCDD-induced toxicity are still under investigation, several studies have shown that TCDD induces programmed cell death, or apoptosis, in various tissues and may act in a similar fashion in the ovary. In the present study, we set out to evaluate the in vitro effects of TCDD on steroid secretion, specifically estradiol-17beta (E2) and progesterone, by human luteinized granulosa cells (LGC), and to further determine whether TCDD is capable of inducing apoptosis in this cell type. Human LGC were obtained from women participating in an in vitro fertilization program. Medium, with or without three different concentrations of TCDD and substrates [androstenedione (A4) or pregnenolone], was added to each culture. The media were collected at 4, 8, 12, 24, 36, and 48 h and were assayed by RIA. At 24 and 48 h, the LGC were fixed for assessment of DNA fragmentation via an in situ immunofluorescence technique. Transmission electron microscopy was also performed on LGC after 24 and 48 h with TCDD. TCDD, at all concentrations tested (3.1 pM, 3.1 nM, and 3.1 microM), significantly reduced E2 accumulation in the media at 8, 12, and 24 h, compared with controls. At 36 and 48 h, TCDD treatment (at 3.1 microM) caused a significant increase in E2, compared with controls. The effect of TCDD on E2 was abolished with the addition of A4. TCDD treatment did not alter progesterone accumulation. Apoptosis increased at 24 h with 3.1 microM TCDD, with no apparent effect at 3.1 nM. By 48 h, however, TCDD increased apoptosis in a dose-dependent manner. Transmission electron microscopy showed ultrastructural differences in LGC with 3.1 microM TCDD at 24 and 48 h. Collectively, the results of the present study suggest that TCDD perturbs E2 secretion by depletion of A4 precursor and increases apoptotic cell death of human LGC in a dose- and time-dependent fashion.