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 4 days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and cultured for 4 or 8 days. 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 4 days 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 4 days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P < 0.05) but no changes in Bax expression were observed after 4 days of DMBA exposures. In contrast, after 8 days, 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.

Polycyclic Aromatic Hydrocarbons Cause Follicle Atresia and Apoptosis in Mouse Ovarian Follicles Cultured in Vitro that can be Reduced with the Activator of PI3K/Akt Pathway, 740Y-P

“…An experiment [13] on rats shows that prenatal expoused to BaP leads offsprings premature ovarian failure and ovarian tumorigenesis. A Study concerning the effect of PAH on follicular growth [14] showed that mice exposed to cigarette smoke could reduce antral follicles, therefore leading to appropriate preovulatory follicles decreased before ovulation. Relevant epidemiological studies [15,16] also showed that smoking women of childbearing age were usually accompanied with a low rate of pregnancy or a high rate of abortion and/or infertility.…”

Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice

“…High fat diet fed mice have increased levels of apoptosis in cumulus oocyte complexes and granulosa cells (Wu et al , 2010) and reduced cardiac Cx expression in female rats (Aubin et al , 2010), potentially supporting that obesity alters CX protein abundance. Additionally, DMBA altered Cx gene expression in neonatal ovaries (Ganesan and Keating, 2014), however, the effect of DMBA on Cx gene expression in adult mouse ovaries has not yet been studied. Thus, the impacts of both DMBA exposure and progressive obesity on ovarian Cx mRNA and protein abundance were investigated in ovaries from adult mice.…”

“…Protein was isolated from whole ovaries (n = 4–5) by homogenization in tissue lysis buffer containing protease and phosphatase inhibitors as previously described (Ganesan and Keating, 2014). Briefly, homogenized samples were placed on ice for 30 min, followed by two rounds of centrifugation at 10,000 rpm for 15 min and protein concentration was measured using a standard BCA protocol.…”

“…CX proteins are targeted by chemicals including retinoids, carotinoids, chemotherapeutic agents (Trosko and Ruch, 2002); Upham and Trosko, 2009; King and Bertram, 2005), cigarette components (Upham et al , 1996; Tai et al , 2007; Upham et al , 2008; Upham and Trosko, 2009); McKarns et al , 2000) and polycyclic aromatic hydrocarbons including 7,12-dimethylbenz[a]anthracene (DMBA) (Sharovskaya et al , 2006; Ganesan and Keating, 2014; (Trosko, 1989). DMBA, liberated from burning of organic matter incineration, destroys all type of follicles in the ovaries of exposed mice and rats, leading to ovarian failure (Gelboin, 1980; Mattison and Schulman, 1980).…”

“…DMBA destroys follicles by inducing apoptosis through increased expression of pro-apoptotic BAX and activation of the executioner protein caspase 3 (Tilly et al , 1991; Tsai-Turton et al , 2007). Additionally, DMBA altered CX protein expression when neonatal cultured rat ovaries were exposed in vitro (Ganesan and Keating, 2014). …”