Mono-(2-ethylhexyl) phthalate induces oxidative stress and inhibits growth of mouse ovarian antral follicles

Abstract

Mono-(2-ethylhexyl) phthalate (MEHP) is the active metabolite of the most commonly used plasticizer, di-(2-ethylhexyl) phthalate, and is considered to be a reproductive toxicant. However, little is known about the effects of MEHP on ovarian antral follicles. Thus, the present study tested the hypothesis that MEHP inhibits follicle growth via oxidative stress pathways. The data indicate that MEHP increases reactive oxygen species (ROS) levels and inhibits follicle growth in antral follicles, whereas N-acetylcysteine (NAC; an antioxidant) restores ROS levels to control levels and rescues follicles from MEHP-induced inhibition of follicle growth. To further analyze the mechanism by which MEHP induces oxidative stress and inhibits follicle growth, the expression and activities of various key antioxidant enzymes (copper/zinc superoxide dismutase [SOD1], glutathione peroxidase [GPX], and catalase [CAT]) and the expression of key cell-cycle regulators (Ccnd2, Ccne1, and Cdk4) and apoptotic regulators (Bcl-2 and Bax) were compared in control and MEHP-treated follicles. The data indicate that MEHP inhibits the expression and activities of SOD1 and GPX; does not inhibit Cat expression; inhibits the expression of Ccnd2, Ccne1, Cdk4, and Bcl-2; but increases the expression of Bax compared to controls. Furthermore, NAC blocks these toxic effects of MEHP. Collectively, these data suggest that MEHP induces oxidative stress by disrupting the activities of antioxidant enzymes. This may lead to decreased expression of cell-cycle regulators and antiapoptotic regulators and increased expression of proapoptotic factors, which then may lead to inhibition of follicle growth.

FIG. 1

Effect of MEHP and NAC on ROS levels in antral follicles. Antral follicles were exposed to DMSO or MEHP (0.1–100 μg/ml) with or without NAC (0.5–1 mM) for 96 h in vitro and subjected to in vitro ROS assays to measure ROS levels. The levels of ROS were normalized to protein level in each sample and reported as relative fold-change compared to DMSO controls. All data represent the mean ± SEM from three independent experiments (n = 35 follicles/treatment/experiment). Bars with different letters are significantly different from each other (P ≤ 0.05).

FIG. 2

Effect of MEHP exposure on antral follicle growth. Antral follicles were cultured in the presence of DMSO or MEHP (0.1–100 μg/ml) for 96 h. Growth of follicles was monitored during culture and reported as percentage change over time. The graph represents the mean ± SEM from at least three separate experiments. Lines with an asterisk (*) are significantly different from DMSO controls at selected time points (n = 10–16 follicles/treatment/experiment; P ≤ 0.05).

FIG. 3

Effect of MEHP exposure on the expression of cell-cycle regulators and apoptotic genes. After exposure of antral follicles to DMSO controls or MEHP (1 μg/ml) for 24–96 h in vitro, the follicles were collected and subjected to qPCR analysis for the expression profiles of Ccnd2, Ccne1, and Cdk4 (A) and the expression profiles of Bcl-2 and Bax (B). All values were normalized to Actb as loading control and reported as relative fold-change compared to DMSO levels. The graph represents the mean ± SEM from at least three separate experiments. Lines with an asterisk (*) are significantly different from DMSO controls at selected time points (n = 10–16 follicles/treatment/experiment; P ≤ 0.05).

FIG. 4

Effect of MEHP and NAC cotreatment on antral follicle growth. Antral follicles were cultured in the presence of DMSO or MEHP (0.1–100 μg/ml) with or without NAC (0.5–1 mM) for 96 h. Growth of follicles was reported as percentage change at 96 h. Bars with different letters are significantly different from each other (n = 10–16 follicles/treatment/experiment; P ≤ 0.05). Data on the graph represent the mean ± SEM from at least three separate experiments.

FIG. 5

Effect of MEHP and NAC cotreatment on cell-cycle regulators and apoptotic genes. Antral follicles were exposed to DMSO or MEHP (10 and 100 μg/ml) with or without NAC (1 mM) for 96 h and then subjected to qPCR analysis for Ccnd2, Ccne1, and Cdk4 (A) or Bcl-2 and Bax (B) mRNA expression levels. All values were normalized to Actb as loading control and reported as relative fold-change compared to DMSO levels. Data on the graph represent the mean ± SEM from at least three separate experiments. Bars with an asterisk (*) are significantly different from DMSO controls (n = 10–16 follicles/treatment/experiment; P ≤ 0.05).

FIG. 6

Effect of MEHP and NAC cotreatment on Sod1, Gpx, and Cat mRNA expression levels. After exposure of antral follicles to DMSO or MEHP (0.1–100 μg/ml) for 96 h in vitro, the follicles were collected and subjected to qPCR analysis for Sod1 (A), Gpx (B), and Cat (C) mRNA expression levels. In addition, antral follicles were exposed to DMSO or MEHP (100 μg/ml) with or without NAC (1 mM) for 96 h and subjected to qPCR to measure the mRNA expression levels of Sod1 and Gpx (D). All values were normalized to Actb as a loading control and reported as relative fold-change compared to DMSO controls. Data on the graph represent the mean ± SEM from at least three separate experiments. An asterisk (*) indicates a significant difference from the DMSO controls (n = 10–16 follicles/treatment/experiment; P ≤ 0.05).

FIG. 7

Effect of MEHP and NAC on SOD1, GPX, and CAT activities in antral follicles. A) Antral follicles were exposed in vitro to DMSO or MEHP (0.1–100 μg/ml) for 72 or 96 h and then subjected to specific assays to measure the enzyme activities of SOD1, GPX, and CAT. All values were normalized to protein level as a loading control and reported as relative fold-change compared to DMSO controls. Data on the graph represent the mean ± SEM from at least three separate experiments. An asterisk (*) indicates a significant difference from DMSO controls (n = 24 follicles/treatment/experiment; P ≤ 0.05). B and C) Antral follicles were cultured in the presence of DMSO or MEHP (10 and 100 μg/ml) with or without NAC (0.5–1 mM) for 96 h. After culture, the follicles were collected and subjected to specific activity assays for SOD1 activity (B) and GPX activity (C). The activities of SOD1 and GPX were normalized to protein level as a loading control. All values are reported as relative fold-change compared to DMSO controls. Data on the graph represent the mean ± SEM from at least three separate experiments. Bars with different letters are significantly different from each other (n = 16–24 follicles/treatment/experiment; P ≤ 0.05).