3.1. Vitamin C reduces meiotic defects in MC-LR-exposed oocytes
To explore the toxic effects of MC-LR during maturation oocytes were cultured with varying concentrations of MC-LR (0 µM, 20 µM, 40 µM, 80 µM, and 120 µM) for 44 h in vitro. The proportion of polar body extrusion (PBE) and COC viability in the control and MC-LR-exposed oocytes is shown in Fig. 1. Almost all of the cumulus cells surrounding oocytes in the control group were fully expanded, while those in the MC-LR-exposed group exhibited poor expansion of COCs (Fig. 1A). Moreover, the majority of oocytes in the control group reached the meiosis II (MII) stage after 44 h of culture, and demonstrated extrusion of the polar body, but exposure to MC-LR significantly reduced the PBE rate (control: 64.52 ± 3.11%, n = 131; 20 µM: 60.10 ± 8.71%, n = 108, p > 0.05; 40 µM: 50.18 ± 4.36%, n = 153, p < 0.05; 80 µM: 39.15 ± 5.39%, n = 152, p < 0.01; 120 µM: 29.63 ± 2.32%, n = 148, p < 0.001; Fig. 1B). The concentration of 80 µM MC-LR was chosen for further studies because this not only caused obvious meiotic defects, but also allowed a proportion of oocytes to develop to the MII stage for further investigation.
To investigate whether VC can alleviate meiotic arrest caused by MC-LR, VC was supplemented to the IVM culture medium containing 80 µM MC-LR. We found that 100 µM VC significantly increased the rate of PBE in MC-LR exposure oocytes compared with MC-LR alone (61.21 ± 2.17%, n = 150, VS 41.42 ± 4.7%, n = 145, p < 0.01) but the higher concentration of 500 µM VC did not show the same effect (39.93 ± 2.68%, n = 115, p > 0.05) (Fig. 1A and C). Thus, the concentration of 100 µM VC was chosen for further study. These results suggested that MC-LR exposure inhibited porcine oocyte maturation in a dose-dependent manner, but VC can protect oocytes against meiotic defects caused by MC-LR exposure.
|
Figure 1. Vitamin C (100 µm) alleviates the meiotic defects in MC-LR-exposed oocytes. (A) Representative images of cumulus expansion and polar body extrusion (PBE) in the control, MC-LR-exposed and VC-rescued groups. Bar = 150 µm (i); 100 µm (ii); 20 µm (iii). (B) The rate of PBE was compared in control and different concentrations of MC-LR-exposed groups (20 µM, 40 µM, 80 µM and 120 µM) after being cultured for 44 h in vitro. (C)The rate of PBE was recorded in control and different concentrations of VC-supplemented groups (100 µM and 500 µM) after culture for 44 h with 80 µM MC-LR in vitro. ns: no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. |
3.2. VC alleviates spindle defects in MC-LR-exposed oocytes
Given that spindle formation is critical for PBE, we next examined spindle dynamics after MC-LR exposure. The results of immunofluorescence are shown in Fig. 2. Most oocytes in the control group exhibited regular spindle morphology and good chromosome alignment on the equatorial plate. In contrast, spindle formation was severely disrupted, and the chromosomes were disorganized, in the MC-LR-exposed group. Quantitative analysis showed that MC-LR-exposed oocytes exhibited a significantly higher proportion of aberrant spindles than control oocytes (77.58 ± 7.23%, n = 116, VS 16.57 ± 4.05%, n = 106, p < 0.01) (Fig. 2B). However, supplementation of VC decreased the proportion of abnormal spindles caused by MC-LR exposure (35.27 ± 4.05%, n = 104, VS 77.58 ± 7.23%, n = 116, p < 0.01), indicating that VC can restore spindle defects in MC-LR-exposed oocytes.
|
Figure 2. Vitamin C protects MC-LR-exposed oocytes from spindle defects. (A) Representative images of spindle morphology and chromosome alignment in the control, MC-LR-exposed and VC-rescued groups. Bar = 5 µm. (B) The rate of aberrant spindles in the control, MC-LR-exposed and VC-rescued groups. **p < 0.01. |
3.3. VC restores α-tubulin acetylation level in MC-LR-exposed oocytes
We next examined the level of acetylated α-tubulin, through immunofluorescence, because this post-translational modification is critical for the maintenance of stable microtubules in both mitotic and meiotic cells. As shown in Fig. 3, acetylated tubulin levels were significantly lower in MC-LR-exposed oocytes compared with control oocytes. Furthermore, VC supplementation significantly increased the level of acetylated α-tubulin in MC-LR-exposed oocytes. Quantitative analysis of the fluorescence intensity of acetylated α-tubulin validated these qualitative findings (MC-LR-exposed: 4.29 ± 0.79, n = 60, VS Control: 15.60 ± 1.71, n = 60, p < 0.001; VC supplementation: 8.64 ± 0.92, n = 60, VS MC-LR-exposed: 4.29 ± 0.79, n = 60, p < 0.01). (Fig. 3B). These results suggest that MC-LR may disorder spindle assembly by downregulating tubulin acetylation and that the presence of vitamin C can partly prevent these aberrations in oocyte development.
|
Figure 3. Vitamin C increases the level of acetylation of α-tubulin in MC-LR-exposed oocytes. (A) Representative images of α-tubulin in the control, MC-LR-exposed and VC-rescued groups. Bar = 5 µm. (B) Quantitative analysis of the fluorescence intensity of acetylated α-tubulin in the control, MC-LR-exposed and VC-rescued groups. **p < 0.01, ***p < 0.001. |
3.4. Vitamin C improves actin dynamics of MC-LR-exposed oocytes
Because actin filaments are the main driving force for asymmetric division in mammalian oocytes, we next examined the actin dynamics in both control oocytes and MC-LR-exposed oocytes. Phalloidin was used to label F-actin, and the results are shown in Fig. 4. In the control group, the actin filaments in most oocytes were evenly distributed on the plasma membrane and showed a strong immunofluorescent signal. However, in most MC-LR-exposed oocytes, the actin signal was so significantly reduced it was almost undetectable (Fig. 4A and B). Quantitative analysis of the actin fluorescence intensity (Fig. 4C) also showed a significant decrease in MC-LR-exposed oocytes, compared with the control group (1.26 ± 0.34, n = 60, VS 60.24 ± 8.20, n = 60, p < 0.001) and this was partially ameliorated by the co-supplementation of VC (20.30 ± 3.74, n = 60) (Fig. 4A and C). Moreover, the proportion of mislocalized actin was significantly increased in MC-LR-exposed oocytes (MC-LR-exposed: 79.58 ± 2.00%, n = 96, VS Control: 22.93 ± 4.34%, n = 100, p < 0.001) and co-supplementation with VC supplementation significantly reduced actin abnormalities caused by MC-LR exposure (VC supplement: 49.27 ± 3.59%, n = 110, VS MC-LR-exposed: 79.58 ± 2.00%, n = 96, p < 0.01) (Fig. 4A and B). These results show that VC is able to partially protect porcine oocytes from actin damage caused by MC-LR exposure.
|
Figure 4. Vitamin C improves the actin dynamics of MC-LR-exposed oocytes. (A) Representative images of actin distribution in the control, MC-LR-exposed and VC-rescued groups. Bar = 20 µm. (B) The rate of mislocalization of actin. (C) Quantitative analysis of the fluorescence intensity of actin in the control, MC-LR-exposed and VC-rescued groups. **p < 0.01, ***p < 0.001. |
3.5. Vitamin C reduces mitochondrial abnormalities in MC-LR-exposed oocytes
Mitochondria are essential for oocyte maturation. Abnormal mitochondria lead to a decrease in oocyte quality and prevent embryonic development[33]. To determine whether MC-LR exposure caused abnormal mitochondria, we used MitoTracker Red CMXRos to label mitochondria, and the results are shown in Fig. 5. In control oocytes, the mitochondria signals were mainly seen in the subcortical regions around lipid droplets. The exposure of oocytes to MC-LR resulted in an abnormal pattern of mitochondrial distribution (Fig. 5A). Quantitative fluorescence intensity analysis showed that the mitochondrial signals were reduced in MC-LR-exposed oocytes compared with the control group (7.18 ± 0.74, n = 60, VS 19.06 ± 0.98, n = 60, p < 0.001) (Fig. 5B). Supplementation of VC to MC-LR-exposed oocytes caused the distribution of mitochondria in these samples to appear similar to the control group and increased the fluorescent intensity of the mitochondrial signal compared with MC-LR-exposed oocytes which did not receive VC supplementation (11.50 ± 0.80, n = 60, VS 7.18 ± 0.74, n = 60, p < 0.001) (Fig. 5). These results suggest that VC can protect oocytes from mitochondria damage caused by MC-LR exposure.
|
Figure 5. Vitamin C protects MC-LR-exposed oocytes from mitochondrial damage. (A) Representative images of mitochondria distribution in the control, MC-LR-exposed and VC-rescued groups. Bar = 20 µm. (B) Quantitative analysis of the fluorescence intensity of mitochondria. ***p < 0.001. |
3.6. Vitamin C restores abnormal epigenetic alterations in MC-LR -exposed oocytes
Histone methylation modification is a pivotal epigenetic modification that is critical for the regulation of gene expression and gene silencing. Disruption of histone modifications in the oocyte can lead to meiotic arrest[[34].. The level of histone H3 lysine 4 di-methylation (H3K4me2), which is associated with active transcription, was studied to assess potential epigenetic modifications in MC-LR -exposed oocytes. As shown in Fig. 6, the fluorescence intensities of H3K4me2 were significantly reduced in MC-LR-exposed oocytes compared with the control group. However, VC supplementation alleviated this defect to some extent (Fig. 6A). Quantitative analysis also confirmed this (MC-LR-exposed: 6.012 ± 0.69, n = 60, VS Control: 21.53 ± 0.94, n = 60, p < 0.001; VC supplement: 16.14 ± 1.61, n = 60, VS MC-LR-exposed: 6.012 ± 0.69, n = 60, p < 0.001). (Fig. 6B) These results demonstrate that VC can protect oocytes from abnormal epigenetic alterations caused by MC-LR exposure.
|
Figure 6. Vitamin C can protect oocytes from abnormal epigenetic alterations caused by MC-LR exposure. (A) Representative images of H3K4me2 in the control, MC-LR-exposed, and VC-rescued groups. Oocytes were stained with rabbit polyclonal anti-H3K4me2 antibody to visualize H3K4me2 (green) and counterstained with Hoechst 33342 to visualize chromosomes (blue). Bar = 5 µm. (B) Quantitative analysis of the fluorescence intensity of H3K4me2. ***p < 0.001. |
3.7. VC decreases oxidative stress and alleviates DNA damage in MC-LR -exposed oocytes
MC-LR induces cytotoxicity via oxidative stress in many kinds of cells, including tissues of the ovary[20, 35]. To investigate whether MC-LR was inhibiting oocyte maturation via oxidative stress, we used DCFH staining to compare the ROS levels between control and MC-LR-exposed oocytes. MC-LR exposure resulted in increased ROS levels compared with oocytes in the control group (42.06 ± 5.09, n = 60, VS 2.73 ± 0.46, n = 60, p < 0.001; Fig. 7A) and VC significantly reduced the excessive ROS present in MC-LR-exposed oocytes (10.28 ± 1.16, n = 60, VS 42.06 ± 5.09, n = 60, p < 0.001) (Fig. 7A and B). These results suggest that VC decreased oxidative stress caused by MC-LR exposure during oocyte maturation.
Because oxidative stress can damage DNA, and MC-LR has been reported to inhibit DNA repair[36], we examined DNA damage by γ-H2A.X staining. Higher levels of γ-H2A.X signal were found in MC-LR-exposed oocytes compared with the control group, whereas VC supplement significantly reduced the γ-H2A.X signal (MC-LR-exposed: 32.21 ± 2.84, n = 60, VS Control: 5.38 ± 0.66, n = 60, p < 0.001; VC supplement: 12.53 ± 0.81, n = 60, VS MC-LR-exposed: 32.21 ± 2.84, n = 60, p < 0.001; Fig. 7C and D). These results suggest that VC can protect oocytes from DNA damage caused by MC-LR exposure.
|
Figure 7. VC decreases oxidative stress and alleviates DNA damage in MC-LR -exposed oocytes. (A) Representative images of ROS level in the control, MC-LR-exposed and VC-rescued groups. Bar = 20 µm. (B) Quantitative analysis of the fluorescence intensity of ROS. ***p < 0.001. (C) Immunofluorescent staining of γH2A.X showing DNA damage in control, MC-LR-exposed and VC-rescued groups. Bar = 5 µm. (D) Quantitative analysis of the fluorescence intensity of γH2A.X. ***p < 0.001. |