Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

Abstract

7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles in a concentration-dependent manner. The impact of DMBA on connexin (CX) proteins that mediate communication between follicular cell types along with pro-apoptotic factors p53 and Bax were investigated. Postnatal day (PND) 4 Fisher 344 rat ovaries were cultured for 4days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5nM or 75nM) and cultured for 4 or 8days. RT-PCR was performed to quantify Cx37, Cx43, p53 and Bax mRNA level. Western blotting and immunofluorescence staining were performed to determine CX37 or CX43 level and/or localization. Cx37 mRNA and protein increased (P<0.05) at 4days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P<0.05) but CX43 protein increased (P<0.05) at 4days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P<0.05) but no changes in Bax expression were observed after 4days of DMBA exposures. In contrast, after 8days, DMBA decreased Cx37 and Cx43 mRNA and protein but increased both p53 and Bax mRNA levels. CX43 protein was located between granulosa cells, while CX37 was located at the oocyte cell surface of all follicle stages. These findings support that DMBA exposure impacts ovarian Cx37 and Cx43 mRNA and protein prior to both observed changes in pro-apoptotic p53 and Bax and follicle loss. It is possible that such interference in follicular cell communication is detrimental to follicle viability, and may play a role in DMBA-induced follicular atresia.