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. 2023 Jan;56(1):e13335.
doi: 10.1111/cpr.13335. Epub 2022 Sep 20.

Exposure to phenanthrene affects oocyte meiosis by inducing mitochondrial dysfunction and endoplasmic reticulum stress

Yi Wang  1 , Si-Hong Li  1 , Shu-Jie Yang  1 , Xiao-Qing Li  1 , Lu Liu  1 , Xiang Ma  1 , Dong Niu  1 , Xing Duan  1
Affiliations

Exposure to phenanthrene affects oocyte meiosis by inducing mitochondrial dysfunction and endoplasmic reticulum stress

Yi Wang et al. Cell Prolif. 2023 Jan.

Abstract

Objectives: Phenanthrene (PHE) is one of the most abundant polycyclic aromatic hydrocarbons (PAHs), which is a widespread environmental contaminant. Various studies showed that PHE has adverse impacts on animals and human health. It has been shown that PHE exposure induced follicular atresia and endocrine dyscrasia in female mice. However, the potential mechanism regarding how PHE affects female reproductive system especially the oocyte quality has not been elucidated.

Methods and results: In this study, we set up PHE exposure model and found that PHE exposure compromised oocytes maturation competence by inhibiting spindle assembly and chromosomes alignment. Moreover, PHE exposure induced mitochondrial dysfunction and endoplasmic reticulum (ER) stress, leading to increased reactive oxygen species (ROS) and aberrant calcium levels in cytoplasm, eventually induced oxidative stress and DNA damage in oocytes. Furthermore, we found that oral administration of PHE caused the occurrence of oxidative stress and apoptosis in female ovary. In addition, the oocyte exhibited aberrant spindle morphology and failure of actin cap formation in metaphase II oocytes.

Conclusions: Taken together, our study demonstrated that mitochondrial dysfunction and ER stress-induced oxidative stress and DNA damage are the major cause of poor oocyte quality after PHE exposure.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Figures

FIGURE 1
FIGURE 1
Phenanthrene (PHE) exposure impairs oocyte maturation. (A) Representative images of germinal vesicle breakdown and polar body extrusion oocytes in control and PHE‐exposed oocytes (arrowheads: the oocytes underwent germinal vesicle breakdown (GVBD) and polar body extrusion in control group, but failed GVBD and polar body extrusion in PHE‐treated group). Scale bar, 100 μm. (B) Percentage of GVBD was quantified in control and oocytes exposed to different concentrations of PHE (200, 300 and 400 μM). (C) Representative images of first polar body extruded‐oocytes from control and PHA‐exposed groups. Scale bar, 100 μm. Data are represented as mean ± SD from at least three independent experiments. ns p > 0.05, *p < 0.05 and **p < 0.01
FIGURE 2
FIGURE 2
Phenanthrene (PHE) exposure disturbs MI spindle assembly and chromosome alignment. (A) Representative images of spindle morphologies in control and PHE‐treated group. Scale bar, 20 μm. (B) Spindle area was quantified in control and PHE‐exposed oocytes. (C) The percentage of aberrant spindle morphology was quantified after PHE treatment. (D,E) Quantification of chromosomes distance in control and PHE‐exposed oocytes. (F) Representative images of TPX2 in control and PHE‐treated groups. Scale bar, 20 μm. (G) Normalized fluorescent intensity of TPX2 in control and PHE‐treated groups. Each point in the histogram represents the number of oocytes. Data are represented as mean ± SD from at least three independent experiments. **p < 0.01, ***p < 0.001
FIGURE 3
FIGURE 3
PHE exposure affects mitochondrial function in oocytes. (A) Representative images of JC‐1 kit staining in control and PHE‐exposed oocytes. Scale bar, 100 μm. (B) Mitochondrial membrane potential was recorded after PHE‐exposed (Aggregate/Monomer). (C) Representative images of cytosolic Ca2+ levels in oocytes after PHE treatment. Scale bar, 100 μm. (D) Fluorescent intensity of Ca2+ levels were quantified in control and PHE‐treated groups. Each point in the histogram represents the number of oocytes. Data are represented as mean ± SD from at least three independent experiments. ***p < 0.001
FIGURE 4
FIGURE 4
PHE exposure causes aberrant mitochondrial dynamics and endoplasmic reticulum stress. (A) Representative images of DRP1 in control and PHE ‐treated groups. Scale bar, 20 μm. (B) Normalized fluorescent intensity of DRP1 in control and PHE‐treated groups. (C) The relative mRNA expression of Fis1, DRP1 and MFN2 compared with the control group. (D) Representative images of ER‐tracker in control and PHE‐treated groups. Scale bar, 20 μm. (E) Normalized fluorescent intensity of ER‐tracker in control and PHE‐treated groups. (F) The relative mRNA expression of Chop, ATF4 and GRP78 compared with the control group. Each point in the histogram represents the number of oocytes. Data are represented as mean ± SD from at least three independent experiments. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001
FIGURE 5
FIGURE 5
PHE exposure induces oxidative stress and DNA damage. (A) Representative images of DCFH‐DA staining in control and PHE‐treated groups. Scale bar, 100 μm. (B) Fluorescent intensity of ROS was analysed in control and PHE‐exposed oocytes. (C) Representative images of γH2AX staining in control and PHE‐treated groups. Scale bar, 10 μm. (D) Fluorescent intensify of γH2AX was quantified in control and PHE‐treated groups. Each point in the histogram represents the number of oocytes. Data are represented as mean ± SD from at least three independent experiments. ***p < 0.001
FIGURE 6
FIGURE 6
PHE causes oxidative stress and apoptosis in ovarian tissues and disturbs MII oocytes quality. (A) Schematic of the oral gavage administration of PHE and the effects on the ovaries. (B) Weight of ovarian in control and PHE‐treated groups. (C) The relative mRNA expression of Sod 1, Bax and Bcl 2 in control and PHE‐treated groups. (D) Representative images of spindle morphologies in control and PHE‐treatment oocytes. Scale bar, 20 μm. (E) The rate of aberrant spindle was recorded in control and PHE‐treatment oocytes. (F) Representative images of actin on the cortical and the fluorescence intensity curves in control and treatment oocytes. Scale bar, 20 μm. (G) Fluorescent intensity of cortical actin was quantified in control and PHE treatment oocytes. (H) Cortical area actin fluorescence intensity curve was recorded in control and PHE‐treatment oocytes. (I) The rate of abnormal formation of actin cap was quantified in control and PHE treatment oocytes. Each point in the histogram represents the number of oocytes. Data are represented as mean ± SD from at least three independent experiments. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001
FIGURE 7
FIGURE 7
Mechanism of abnormal meiotic progression caused by PHE exposure. PHE exposure caused mitochondrial dysfunction by inducing the imbalance of mitochondrial dynamics and ER stress, which further resulted in the occurrence of DNA damage and reduced oocyte quality
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