OBJECTIVE: In our previous papers, we demonstrated that TCDD and PCBs (both coplanar PCB 126 and non-coplanar PCB 153) caused the decreased estradiol secretion in cultured pig ovarian follicles. However, the mechanism of action is not clearly understood. Moreover, to our knowledge, the expression of AhR in pig follicular cells was not described yet. METHODS: In order to elucidate if TCDD, PCB 126, and PCB 153 can disrupt ovarian steroidogenesis via AhR-dependent mechanism, granulosa and theca cells were isolated from pig ovarian small follicles and subsequently treated with 32 pg/ml of TCDD, 100 ng/ml of PCB 153, 100 pg/ml of PCB 126 in an in vitro culture. After 3-hour exposure to the chemicals, cells were prepared for immunohistochemical analysis or collected for immunobloting, and the effects of TCDD and PCBs on the levels of AhR protein were studied. RESULTS: Under basal conditions, AhR staining showed a diffuse cytoplasmic pattern of distribution as well in granulosa and theca cells. However, immunocytochemical labeling of AhR in theca cells was less evident and only few cells displayed cytoplasmic staining. Immunoblotts prepared from granulosa and theca cell lysates detected a strong band of 120 kDa indicating AhR protein expression. The level of AhR expression detected by Western blot analysis was higher in granulosa cells than in theca cells. Intensive cytoplasmic and nuclear labeling corresponding to AhR protein in TCDD and PCB 126 treated granulosa cells was observed. In PCB 153 treated cells, the level of AhR staining was comparable with control. Western blot analysis showed a strong band in TCDD-treated grnulosa cells and only slightly differences compared with control in those exposed to PCB 126. The effect of both PCBs on immunocytochemical AhR staining in theca cells was less evident. Immunoblott analysis did not reveal any substantial differences between the control and TCDD- and PCBs-treated theca cells. CONCLUSIONS: This study has shown different AhR expression in porcine theca and granulosa cells, in respect of both its intracellular localization and cellular distribution. Both, dioxin and dioxin-like PCB activated AhRs in granulosa but not in theca cells. Differences in AhR-responsiveness of granulosa and theca cells to TCDD and dioxin-like PCB 126 are probably connected with the differences of these cells in estradiol secretion and different proliferative potential.
OBJECTIVE: In our previous papers, we demonstrated that TCDD and PCBs (both coplanar PCB 126 and non-coplanar PCB 153) caused the decreased estradiol secretion in cultured pig ovarian follicles. However, the mechanism of action is not clearly understood. Moreover, to our knowledge, the expression of AhR in pig follicular cells was not described yet. METHODS: In order to elucidate if TCDD, PCB 126, and PCB 153 can disrupt ovarian steroidogenesis via AhR-dependent mechanism, granulosa and theca cells were isolated from pig ovarian small follicles and subsequently treated with 32 pg/ml of TCDD, 100 ng/ml of PCB 153, 100 pg/ml of PCB 126 in an in vitro culture. After 3-hour exposure to the chemicals, cells were prepared for immunohistochemical analysis or collected for immunobloting, and the effects of TCDD and PCBs on the levels of AhR protein were studied. RESULTS: Under basal conditions, AhR staining showed a diffuse cytoplasmic pattern of distribution as well in granulosa and theca cells. However, immunocytochemical labeling of AhR in theca cells was less evident and only few cells displayed cytoplasmic staining. Immunoblotts prepared from granulosa and theca cell lysates detected a strong band of 120 kDa indicating AhR protein expression. The level of AhR expression detected by Western blot analysis was higher in granulosa cells than in theca cells. Intensive cytoplasmic and nuclear labeling corresponding to AhR protein in TCDD and PCB 126 treated granulosa cells was observed. In PCB 153 treated cells, the level of AhR staining was comparable with control. Western blot analysis showed a strong band in TCDD-treated grnulosa cells and only slightly differences compared with control in those exposed to PCB 126. The effect of both PCBs on immunocytochemical AhR staining in theca cells was less evident. Immunoblott analysis did not reveal any substantial differences between the control and TCDD- and PCBs-treated theca cells. CONCLUSIONS: This study has shown different AhR expression in porcine theca and granulosa cells, in respect of both its intracellular localization and cellular distribution. Both, dioxin and dioxin-like PCB activated AhRs in granulosa but not in theca cells. Differences in AhR-responsiveness of granulosa and theca cells to TCDD and dioxin-like PCB 126 are probably connected with the differences of these cells in estradiol secretion and different proliferative potential.