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. 2021 Jul 6:9:708980.
doi: 10.3389/fcell.2021.708980. eCollection 2021.

Hexestrol Deteriorates Oocyte Quality via Perturbation of Mitochondrial Dynamics and Function

Dong Niu  1 , Kun-Lin Chen  1 , Yi Wang  1 , Xiao-Qing Li  1 , Lu Liu  1 , Xiang Ma  1 , Xing Duan  1
Affiliations

Hexestrol Deteriorates Oocyte Quality via Perturbation of Mitochondrial Dynamics and Function

Dong Niu et al. Front Cell Dev Biol. .

Abstract

Hexestrol (HES) is a synthetic non-steroidal estrogen that was widely used illegally to boost the growth rate in livestock production and aquaculture. HES can also be transferred to humans from treated animals and the environment. HES has been shown to have an adverse effect on ovarian function and oogenesis, but the potential mechanism has not been clearly defined. To understand the potential mechanisms regarding how HES affect female ovarian function, we assessed oocyte quality by examining the critical events during oocyte maturation. We found that HES has an adverse effect on oocyte quality, indicated by the decreased capacity of oocyte maturation and early embryo development competency. Specifically, HES-exposed oocytes exhibited aberrant microtubule nucleation and spindle assembly, resulting in meiotic arrest. In addition, HES exposure disrupted mitochondrial distribution and the balance of mitochondrial fission and fusion, leading to aberrant mitochondrial membrane potential and accumulation of reactive oxygen species. Lastly, we found that HES exposure can increase cytosolic Ca2+ levels and induce DNA damage and early apoptosis. In summary, these results demonstrate that mitochondrial dysfunction and perturbation of normal mitochondrial fission and fusion dynamics could be major causes of reduced oocyte quality after HES exposure.

Keywords: apoptosis; hexestrol; mitochondria; oocyte maturation; ovary; spindle.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
HES affects mouse ovary weight and morphology. (A) Compared with control group, body weight was slightly though not significantly increased. (B) Ovary weight was significantly reduced after exposure to HES. **P = 0.0074. Data are represented as mean ± SD from at least three independent experiments. (C) Representative H&E histochemical section images of ovaries from control and HES-exposed groups. Scale bar, 500 μm.
FIGURE 2
FIGURE 2
HES impairs oocyte maturation and early embryonic development. (A) Representative images of germinal vesicle breakdown oocytes in control and HES-exposed oocytes. Scale bar, 100 μm. (B) Percentage of germinal vesicle breakdown was quantified in control and in oocytes exposed to different concentrations of HES (50, 80, and 100 μM). ns, P > 0.05, and *P < 0.05. (C) Representative images of first polar body extruded-oocytes from control and HES-exposed groups. Scale bar, 100 μm. (D) Rate of polar body extrusion was recorded in control and HES-exposed groups. ***P < 0.001. (E) Representative images of fertilized eggs in control and HES-exposed group. Scale bar, 100 μm. (F) Early embryonic development was recorded in control and HES-exposed group. ***P < 0.001. Data are represented as mean ± SD from at least three independent experiments.
FIGURE 3
FIGURE 3
HES disturbs MT assembly and coalescence of MTOCs in MI spindle poles. P < 0.001. (A) Representative images of TPX2 localization in control and HES-exposed oocytes. Scale bar, 20 μm. (B) Spindle area was quantified in control and HES-exposed oocytes. ***P < 0.001. (C) Ratio of MT fluorescent intensity in spindle and cytoplasm was quantified in control and HES-exposed oocytes. ***P < 0.001. (D) Ratio of TPX2/spindle fluorescent intensity was recorded in control and HES-exposed oocytes. *P < 0.05 and ***P < 0.001. (E) Representative images of spindle morphologies and pericentrin localization in control and HES-exposed oocytes. Scale bar, 20 μm. (F,G) Number and area of pericentrin foci were quantified in control and HES-exposed oocytes. ***P < 0.001. Data are represented as mean ± SD from at least three independent experiments.
FIGURE 4
FIGURE 4
HES affects mitochondrial function in oocytes. (A) Mitochondria distribution was examined in control and HES-exposed oocytes. Scale bar, 20 μm. (B) Fluorescent intensity of Mito-Tracker was quantified in control and HES-exposed oocytes. ***P < 0.001. (C) Representative images of JC-1 kit staining in control and HES-exposed oocytes. Scale bar, 20 μm. (D) Mitochondrial membrane potential was recorded after HES exposure (Aggregate/Monomer). ***P < 0.001. (E) Representative images of ROS levels in control and HES-exposed oocytes. Scale bar, 20 μm. (F) Fluorescent intensity of ROS was analyzed in control and HES-exposed oocytes. ***P < 0.001. Data are represented as mean ± SD from at least three independent experiments.
FIGURE 5
FIGURE 5
HES perturbs mitochondrial fission and fusion process in oocytes. (A) Representative images of Drp1 localization in control and HES-exposed oocytes. Scale bar, 20 μm. (B) Fluorescent intensity of Drp1 was quantified after HES exposure. ***P < 0.001. (C) Representative images of phosphorylated-Drp1 localization in control and HES-exposed oocytes. Scale bar, 20 μm. (D) Fluorescent intensity of p-Drp1 was recorded after HES exposure. ***P < 0.001. (E) Drp1, p-Drp1, and MFN1 protein levels were examined by using Western blot. Data are represented as mean ± SD from at least three independent experiments. (F) The protein levels of Drp1, p-Drp1, and MFN1 were quantified by Image J.
FIGURE 6
FIGURE 6
HES increases intracellular calcium level and DNA damage to induce oocyte apoptosis. (A) Representative images of Ca2+ levels in control and HES-exposed oocytes. Scale bar, 20 μm. (B) Fluorescent intensity of Ca2+ was quantified in control and HES-exposed oocytes. ***P < 0.001. (C) Representative images of γH2AX localization in control and HES-exposed oocytes. Scale bar, 20 μm. (D) Fluorescent intensity of γH2AX was recorded in control and HES-exposed oocytes. ***P < 0.001. (E) Representative images of apoptotic oocytes in control and HES-exposed oocytes. Scale bar, 20 μm. (F) Fluorescent intensity of annexin-V was quantified in control and HES-exposed oocytes. ***P < 0.001. Data are represented as mean ± SD from at least three independent experiments.
FIGURE 7
FIGURE 7
Potential mechanism of abnormal meiotic progress induced by HES exposure.
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