Toxic effects of atrazine on porcine oocytes and possible mechanisms of action

Abstract

Because atrazine is a widely used herbicide, its adverse effects on the reproductive system have been extensively researched. In this study, we investigated the effects of atrazine exposure on porcine oocyte maturation and the possible mechanisms. Our results showed that the rates of oocyte maturation significantly decreased after treatment with 200 μM atrazine in vitro. Atrazine treatment resulted in abnormal spindle morphology but did not affect actin distribution. Atrazine exposure not only triggered a DNA damage response but also decreased MPF levels in porcine oocytes. Our results also revealed that atrazine worsened porcine oocyte quality by causing excessive accumulation of superoxide radicals, increasing cathepsin B activity, and decreasing the GSH level and mitochondrial membrane potential. Furthermore, atrazine decreased developmental competence of porcine oocytes up to the blastocyst stage and changed some properties: cell numbers, apoptosis, and related gene expression levels. Collectively, our results indicate that porcine oocyte maturation is defective after atrazine treatment at least through disruption of spindle morphology, MPF activity, and mitochondrial function and via induction of DNA damage, which probably reduces developmental competence.

Fig 1. Effects of various concentrations of atrazine on IVM of porcine oocytes.

(A) Expansion of the peripheral layers of cumulus cells. (B) The polar body extrusion rate was significantly reduced after atrazine treatment. Control: a normal IVM medium; 50–500 μM: different concentrations of atrazine. Differences between bars superscripted with different letters (a, b, or c within the same graph) are statistically significant (p < 0.05). Values are shown as mean ± standard deviation from three independent experiments. The number of oocytes observed in each experimental group is displayed in the bar.

Fig 2. Atrazine causes oocyte spindle abnormalities during porcine oocyte maturation.

(A) Spindle formation after atrazine treatment. (B) Actin signals after atrazine treatment. (C) Rate of abnormal spindle formation was significantly increased. (D) Relative actin fluorescence signals in the membrane and in the cytoplasm of meiosis I (MI) oocytes. Differences between bars superscripted with different letters (a or b within the same graph) are statistically significant (p < 0.05). The number of oocytes observed in each experimental group is displayed in the bar. Values are presented as mean ± standard deviation from three independent experiments. Blue, chromatin DNA; green, tubulin; red, actin.

Fig 3. Damaging effects of atrazine on DNA at the GVBD stage.

(A) DNA damage levels (as indicated by H2A.X fluorescence intensity). (B) Number of γH2AX foci in different groups. Differences between bars superscripted with different letters (a or b within the same graph) are statistically significant (p < 0.05). Values are presented as mean ± standard deviation from three independent experiments. Blue, chromatin DNA; green, H2A.X signals.

Fig 4. Effects of atrazine on maternal gene expression and MPF activity.

(A) Expression levels of the maternal transcripts of cdc2 and cyclin B1. (B) MPF activity in MII oocytes, after maturation of porcine COCs in the presence of 200 μM atrazine. Differences between bars superscripted with different letters (a or b within the same graph) are statistically significant (p < 0.05). Values are presented as mean ± standard deviation from three independent experiments.

Fig 5. Effects of atrazine on intracellular levels of ROS, GSH and cathepsin B activity.

(A) MII oocytes maturated in the normal IVM medium or the medium supplemented with 200 μM atrazine were stained with H2DCFDA to evaluate ROS levels. (B) Effects of 200 μM atrazine supplementation during IVM on intracellular ROS levels in mature oocytes. (C) MII oocytes were stained with CMF2HC (Cell Tracker Blue) to assess GSH levels. (D) GSH levels in mature oocytes. (E) MII oocytes were stained with Magic red cathepsin B. (F) Cathepsin B levels in mature oocytes. Differences between bars superscripted with different letters (a or b within same graph) are statistically significant (p <0.05). The experiment was repeated three times.

Fig 6. Effects of atrazine on mitochondrial membrane potential (Δφm).

(A) The membrane potential was calculated as the ratio of red fluorescence, green fluorescence, and merged data. (B) Fluorescence emitted by each MII oocyte was analyzed by means of the ImageJ software (red/green). Differences between bars superscripted with different letters (a or b within same graph) are statistically significant (p < 0.05). The experiment was repeated three times.

Fig 7. Effects of atrazine administered during IVM on subsequent early embryonic development.

(A) Blastocysts when COCs were cultivated in the normal IVM medium or medium supplemented with 200 μM atrazine. (B) The blastocyst rate in two groups. (C) Immunofluorescent staining of apoptotic cells in blastocysts from control and 200 μM atrazine-treated groups (×400). (D) Apoptotic cell rates per blastocyst in different groups. (E) Bcl-2, Bax, and Casp3 mRNA amounts in 7-day porcine blastocysts in controls and groups treated with 200 μM atrazine during IVM. (F) Immunofluorescent staining of BrdU in blastocysts from control and 200 μM atrazine-treated groups (×400). (G) Percentages of proliferating cells in different groups. Values are presented as mean ± standard deviation. The numbers of embryos examined in each experimental group are shown in the bars. Differences between bars superscripted with different letters (a or b) are statistically significant (p < 0.05).