Acute 7,12-dimethylbenz[a]anthracene exposure causes differential concentration-dependent follicle depletion and gene expression in neonatal rat ovaries

Abstract

Chronic exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA), generated during combustion of organic matter including cigarette smoke, depletes all ovarian follicle types in the mouse and rat, and in vitro models mimic this effect. To investigate the mechanisms involved in follicular depletion during acute DMBA exposure, two concentrations of DMBA at which follicle depletion has (75 nM) and has not (12.5 nM) been observed were investigated. Postnatal day four F344 rat ovaries were maintained in culture for four days before a single exposure to vehicle control (1% DMSO; CT) or DMBA (12 nM; low-concentration or 75 nM; high-concentration). After four or eight additional days of culture, DMBA-induced follicle depletion was evaluated via follicle enumeration. Relative to control, DMBA did not affect follicle numbers after 4 days of exposure, but induced large primary follicle loss at both concentrations after 8 days; while, the low-concentration DMBA also caused secondary follicle depletion. Neither concentration affected primordial or small primary follicle number. RNA was isolated and quantitative RT-PCR performed prior to follicle loss to measure mRNA levels of genes involved in xenobiotic metabolism (Cyp2e1, Gstmu, Gstpi, Ephx1), autophagy (Atg7, Becn1), oxidative stress response (Sod1, Sod2) and the phosphatidylinositol 3-kinase (PI3K) pathway (Kitlg, cKit, Akt1) 1, 2 and 4 days after exposure. With the exception of Atg7 and cKit, DMBA increased (P < 0.05) expression of all genes investigated. Also, BECN1 and pAKT(Thr308) protein levels were increased while cKIT was decreased by DMBA exposure. Taken together, these results suggest an increase in DMBA bioactivation, add to the mechanistic understanding of DMBA-induced ovotoxicity and raise concern regarding female low concentration DMBA exposures.

Keywords: DMBA; Follicle; Ovary.

Figure 1. Impact of single DMBA exposure on follicle morphology and number

PND4 rat ovaries were cultured for 4 d in control media and thereafter exposed once to 1% DMSO (vehicle control) or 12.5 nM DMBA or 75 nM DMBA. Following four additional days of culture, follicles were classified and counted: (A) Primordial Follicles; (B) Small Primary Follicles; (C) Large Primary Follicles; (D) Secondary Follicles. Values represent mean ± SE total follicles counted/ovary, n=5. ^# = P < 0.1.

Figure 2. Effect of single DMBA exposure on follicle morphology and number

PND4 rat ovaries were cultured for 4 d in control media and thereafter exposed once to (A) 1% DMSO (vehicle control) or (B) 12.5 nM DMBA or (C) 75 nM DMBA. D depicts DMBA-induced shrunken oocytes indicated by the open arrow. Following eight additional days of culture, follicles were classified and counted: (E) Primordial Follicles; (F) Small Primary Follicles; (G) Large Primary Follicles; (H) Secondary Follicles. Values (E–H) represent mean ± SE total follicles counted/ovary, n=5; * = different from control in each follicle type, P < 0.05.

Figure 3. Effect of DMBA on ovarian expression of chemical biotransformation genes

PND4 rat ovaries were cultured for 4 d in control media and thereafter exposed to a single 1% DMSO (vehicle control) or DMBA (12.5 nM or 75 nM). Following 1, 2 or 4 additional days of culture, mRNA was isolated and (A) Cyp2e1, (B) Gstp, (C) Gstm, and (D) Ephx1 levels evaluated by quantitative RT-PCR. Values represent fold-change ± SEM relative to a control value of 1, normalized to Gapdh. * = different from control, P < 0.05; ^# = P < 0.1.

Figure 4. Effect of DMBA on ovarian expression of reactive oxygen species metabolism genes

PND4 rat ovaries were cultured for 4 d in control media and thereafter exposed to a single 1% DMSO (vehicle control) or DMBA (12.5 nM or 75 nM). Following 1, 2 or 4 additional days of culture, mRNA was isolated and (A) Sod1 or (B) Sod2 levels evaluated by quantitative RT-PCR. Values represent fold-change ± SEM relative to a control value of 1, normalized to Gapdh. * = different from control, P < 0.05; ^# = P < 0.1. ^## = P = 0.1.

Figure 5. Effect of DMBA on ovarian expression of autophagy genes

PND4 rat ovaries were cultured for 4 d in control media and thereafter exposed to a single 1% DMSO (vehicle control) or DMBA (12.5 nM or 75 nM). Following 1, 2 or 4 additional days of culture, mRNA was isolated and (A) Atg7 or (B) Becn1 levels evaluated by quantitative RT-PCR. Values represent fold-change ± SEM relative to a control value of 1, normalized to Gapdh. * = different from control, P < 0.05; ^# = P < 0.1.

Figure 6. Effect of DMBA on ovarian expression of autophagy genes

PND4 rat ovaries were cultured for 4 d in control media and thereafter exposed to a single 1% DMSO (vehicle control) or DMBA (12.5 nM or 75 nM). Following 1, 2 or 4 additional days of culture, mRNA was isolated and (A) Kitlg or (B) cKit or (C) Akt1 levels evaluated by quantitative RT-PCR. Values represent fold-change ± SEM relative to a control value of 1, normalized to Gapdh. * = different from control, P < 0.05; ^# = P < 0.1.

Figure 7. Localization and effect of DMBA on ovarian EPHX1, BECN1, cKIT or pAKT^Thr308 protein

PND4 rat ovaries were cultured for 4 d in control media and thereafter exposed to a single (A) 1% DMSO (vehicle control) (B) 12.5 nM DMBA or (C) 75 nM DMBA. Following four additional days of culture, ovaries were fixed in formalin, and immunofluorescence staining was performed using a primary antibody directed against (A–C) EPHX1, (E–G) BECN1, (I–K) cKIT or pAKT^Thr308. EPHX1 is represented in green and the Hoechst nuclear stain is in blue. BECN1, cKIT and pAKT^Thr308 are represented in red and the Hoechst nuclear stain is in green. Quantification of (D) EPHX1, (H) BECN1, (L) cKIT or (P) pAKT^Thr308 was performed and * = different from control, P < 0.05; ^# = P < 0.1.