Rotenone causes mitochondrial dysfunction and prevents maturation in porcine oocytes

Abstract

Rotenone is a commonly used insecticidal chemical in agriculture and it is an inhibitor of mitochondrial complex Ⅰ. Previous studies have found that rotenone induces the production of reactive oxygen species (ROS) by inhibiting electron transport in the mitochondria of somatic and germ cells. However, there is little precise information on the effects of rotenone exposure in porcine oocytes during in vitro maturation, and the mechanisms underlying these effects have not been determined. The Cumulus-oocyte complexes were supplemented with different concentrations of rotenone to elucidate the effects of rotenone exposure on the meiotic maturation of porcine oocytes during in vitro maturation for about 48 hours. First, we found that the maturation rate and expansion of cumulus cells were significantly reduced in the 3 and 5 μM rotenone-treated groups. Subsequently, the concentration of rotenone was determined to be 3 μM. Also, immunofluorescence, western blotting, and image quantification analyses were performed to test the rotenone exposure on the meiotic maturation, total and mitochondrial ROS, mitochondrial function and biogenesis, mitophagy and apoptosis in porcine oocytes. Further experiments showed that rotenone treatment induced mitochondrial dysfunction and failure of mitochondrial biogenesis by repressing the level of SIRT1 during in vitro maturation of porcine oocytes. In addition, rotenone treatment reduced the ratio of active mitochondria to total mitochondria, increased ROS production, and decreased ATP production. The levels of LC3 and active-caspase 3 were significantly increased by rotenone treatment, indicating that mitochondrial dysfunction induced by rotenone increased mitophagy but eventually led to apoptosis. Collectively, these results suggest that rotenone interferes with porcine oocyte maturation by inhibiting mitochondrial function.

Fig 1. Rotenone reduces the maturation capability of porcine oocytes.

(A) COC morphology after incubation for 44 h. Scale bar represents 100 μm. (B) The ratio of cumulus cells expansion (%) according to grade (μm); (C) Relative mean COC diameter; (D) Maturation rate (%) in Con. (n = 5,052), Rot 2μM (n = 257), Rot 3μM (n = 6,029), Rot 5μM group (n = 2,845), respectively. (E) The ratio of arrest stage in Con. (n = 57) and rotenone Rot 3μM (n = 120). Con., control group. Rot., rotenone-exposed group. *(P < 0.05), **(P < 0.01), ***(P < 0.001) vs control group.

Fig 2. Rotenone induces oxidative stress in porcine oocytes.

(A and B) Total ROS and total GSH levels in Con. and Rot. Group (n = 30). Scale bars represent 100 μm. Green = ROS, blue = GSH. (C) Immunofluorescence images of MitoSOX and TOM20. Red = MitoSOX, green = TOM20, blue = DNA. (D) The relative intensity of MitoSOX expression of oocytes in the Con., Rot. and positive control (H₂O₂) of MitoSOX Group (n = 15). The oocytes were treated with 200 μM of H₂O₂ as a positive control of MitoSOX. Scale bar represents 20 μm. Con., control group. Rot., 3μM rotenone-exposed group. Negative Con., only the secondary antibody was stained in mature oocytes as a negative control. *(P < 0.05), ****(P < 0.0001).

Fig 3. Rotenone induces mitochondrial dysfunction in porcine oocytes.

(A) Relative fluorescence intensity of Mito-Tracker and TOM20 in porcine oocytes after rotenone treatment. Red = Mito-Tracker, green = TOM20, blue = DNA. Scale bars represent 20 μm. (B) Ratio of Mito-Tracker/TOM20, representing the number of functional mitochondria and total mitochondria, respectively (n = 30). (C) Relative ATP production in control and rotenone-treated oocytes (n = 16). Con., control group. Rot., 3μM rotenone-exposed group. Negative Con., only the secondary antibody was stained in mature oocytes as a negative control. **(P < 0.01), ***(P < 0.001).

Fig 4. Rotenone disrupts mitochondrial biogenesis in porcine oocytes.

(A) Relative mitochondrial DNA copy number of oocytes in the control and rotenone-treated groups (n = 3). (B) Representative images of TOM20 intensity in the control and rotenone-treated groups. Green = TOM20, blue = DNA. Scale bar represents 20 μm. (C) Relative fluorescence intensity of TOM20 in the control and rotenone-treated groups (n = 20) (D) Representative images of SIRT1 intensity in the control and rotenone-treated groups. Green = SIRT1, blue = DNA. Scale bar represents 20 μm. (E) Relative fluorescence intensity of SIRT1 in the control and rotenone-treated groups (n = 30). (F) Western blot of SIRT1 (120 kDa) protein expression in porcine oocytes after rotenone treatment. (G) Relative intensity analysis for SIRT1 (n = 3). Con., control group. Rot., 3μM rotenone-exposed group. Negative Con., only the secondary antibody was stained in mature oocytes as a negative control. *(P < 0.05), **(P < 0.01), ***(P < 0.001).

Fig 5. Rotenone induces mitophagy in porcine oocytes.

(A) Fluorescence images of PINK1 and TOM20 in porcine oocytes after rotenone treatment. Red = PINK1, green = TOM20, blue = DNA. Scale bars represent 20 μm. (B) Relative fluorescence intensity of PINK1 in the control and rotenone-treated groups (n = 30). (C) Colocalization graphs of PINK1 and TOM20 in the control and rotenone-treated groups. x-axis: PINK1; y-axis: TOM20. (D) Pearson value of colocalization of PINK1 and TOM20 in the control and rotenone-treated groups, which could indicate damaged mitochondria (n = 35). (E) Images of LC3 fluorescence in porcine oocytes of the control and rotenone-treated groups. Green = TOM20, red = LC3, blue = DNA, yellow = intersection of green and red. Scale bars represents 20 μm. (F) Relative fluorescence intensity of LC3 on the intersection of green and red in the control and rotenone-treated groups (n = 28). (G) Colocalization graphs of LC3 and TOM20 in the control and rotenone-treated groups. x-axis: LC3; y-axis: TOM20. (H) Images of ubiquitin fluorescence in porcine oocytes of the control and rotenone-treated groups. Green = ubiquitin, red = TOM20, blue = DNA, yellow = intersection of green and red. Scale bars represents 20 μm. (I) Relative fluorescence intensity of ubiquitin on the intersection of green and red in the control and rotenone-treated groups (n = 24). (J) Western blot of monomeric ubiquitin (7 kDa) expression in porcine oocytes after rotenone treatment. (K) Band intensity analysis for ubiquitin (n = 3). Con., control group. Rot., 3μM rotenone-exposed group. Negative Con., only the secondary antibody was stained in mature oocytes as a negative control. *(P < 0.05), **(P < 0.01).

Fig 6. Rotenone leads to apoptosis in porcine oocytes.

(A) Fluorescence images of cytochrome C and Mito-Tracker in porcine oocytes after rotenone treatment. Green = cytochrome C, red = Mito-Tracker, blue = DNA. Scale bars represent 20 μm. (B) Colocalization graphs of cytochrome C and Mito-Tracker in the control and rotenone-treated groups. x-axis: MitoTracker; y-axis: TOM20. (C) Pearson value of colocalization of cytochrome C and Mito-Tracker in the control and rotenone-treated groups, which could indicate activation of apoptosis (n = 50). (D) Representative images of caspase3 intensity in the positive control (DTT), control and rotenone-treated groups. Green = caspase3, blue = DNA. Scale bar represents 20 μm. (E) Relative fluorescence intensity of caspase3 in the control and rotenone-treated groups (n = 19). Con., control group. Rot., 3μM rotenone-exposed group. Negative Con., only the secondary antibody was stained in mature oocytes as a negative control. *(P < 0.05), ***(P < 0.001) ****(P < 0.0001).

Fig 7. Rotenone exposure disturbs meiotic maturation by inducing mitochondrial dysfunction in porcine oocytes.

Rotenone exposure causes mitochondrial dysfunction, and damaged mitochondria inhibit mitochondrial biosynthesis. Damaged mitochondria trigger mitophagy, and mitophagy can mitigate mitochondrial damage. Damaged mitochondria induce ROS, eventually leading to apoptosis.