Autophagy is required for proper meiosis of porcine oocytes maturing in vitro

Abstract

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components; however, the contribution of autophagy during meiosis has not been studied in porcine oocytes maturing in vitro. In this study, we observed that the autophagy-related gene, LC3, was expressed in porcine oocytes during maturation for 44 h in vitro. Knockdown of the autophagy-related gene, BECN1, reduced both BECN1 and LC3 protein expression levels. Moreover, BECN1 knockdown and treatment with the autophagy inhibitor, LY294002, during maturation of porcine oocytes in vitro impaired polar body extrusion, disturbed mitochondrial function, triggered the DNA damage response, and induced early apoptosis in porcine oocytes. Autophagy inhibition during oocyte maturation also impaired the further developmental potential of porcine oocytes. These results indicate that autophagy is required for the in vitro maturation of porcine oocytes.

Figure 1

Localization and expression of autophagy related gene in GV (0 h) and MII (44 h) stage oocytes. (A) Relative mRNA expression levels of LC3 and BECN1 at the GV and MII stage oocytes analyzed by qRT PCR. mRNA expression at the GV stage was arbitrarily set as onefold. Fold differences in the mRNA expression from equivalent numbers of GV and MII stage embryos are shown after normalisation against the internal standard GAPDH. Data are presented as the mean ± SEM. ^*P < 0.05. (B) Oocytes at the GV and MII stage were immunolabeled with anti-LC3 antibody and Hoechst33342 to visualize the localization of LC3 in porcine oocytes. The MII polar body is indicated by a white arrow. In the enlarged panel (indicated by the white box) dots represent the localization of LC3. Scale bar = 20 μm. (C) Quantification of LC3 dots in oocytes. Each value represents the mean ± SEM.

Figure 2

BECN1 dsRNA inhibits extrusion of the first polar body. (A–C) Knockdown of endogenous BECN1 mRNA and protein expression after BECN1 dsRNA injection was verified by qRT-PCR (A) and immunofluorescent staining (B,C). BECN1 mRNA and protein expression were significantly decreased after dsRNA injection. In the enlarged panel (indicated by the white box) dots represent the localization of LC3 labelled protein, BECN1 labelled protein. Note that the colocalisation of LC3 and BECN1. Scale bar = 20 μm. Each value represents the mean ± SEM. ^*P < 0.01. (D) Effect of BECN1 knockdown on the rate of oocyte polar body extrusion after 44 h of in vitro culture. The MII polar body is indicated by a white arrow. Scale bar = 20 μm. Each value represents the mean ± SEM. ^*P < 0.05.

Figure 3

Effects of autophagy inhibition on porcine oocyte maturation. (A,B) LC3 and BECN1 protein expression after 1 μM LY294002 treatment was determined by immunofluorescent staining. BECN1 and LC3 protein expression were significantly decreased after LY294002 treatment. In the enlarged panel (indicated by the white box) dots represent the localization of LC3 labelled protein, BECN1 labelled protein. Scale bar = 20 μm. Each value represents the mean ± SEM. *P < 0.01. (C) Effect of LY294002 treatment on the rate of oocyte polar body extrusion after 44 h of in vitro culture. The MII polar body is indicated by a white arrow. Scale bar = 20 μm. Each value represents the mean ± SEM. ^*P < 0.05.

Figure 4

Western blotting assay for conversion of LC3-I (cytosolic) to LC3-II (autophagosome bound). (A) LC3- I (lane 1), LC3-II (lane 2) and GAPDH (lane 3) in untreated MII (Control), dsBECN1-injected (dsBECN1) and LY294002 treated (LY294002) oocytes. Graphs (B,C) show LC3-II quantification by western blotting in oocytes in different groups. While B shows LC3-II contents (LC3-II/GAPDH ratio), (C) shows the LC3-II/ LC3- I ratio. ^*P < 0.05.

Figure 5

DNA damage and apoptosis of oocytes after dsBECN1 injection or autophagy inhibitor treatment. (A) Localization of γH2A.X in the nuclei of oocytes and early apoptosis in the membrane of oocytes by performing annexin-V staining. The percentage of γH2A.X positive and annexin-V positive oocytes significantly increased after injection of dsBECN1 or LY294002 treatment. Blue, DNA; red, γH2A.X; green, Annexin-V. The MII polar body is indicated by a white arrow. Scale bar = 20 μm. (B) Quantification of γH2A.X positive oocytes. Each value represents the mean ± SEM. *P < 0.01. (C) The percentage of annexin-V-positive oocytes. Each value represents the mean ± SEM. ^*P < 0.01. (D) DNA damage in oocytes was assessed by performing the comet assay. Control oocytes showed slight DNA damage, whereas dsBECN1-injected or LY294002-treated oocytes showed notable DNA damage. Scale bar = 20 μm. (E) Fold changes in tail moment and length in oocytes. Each value represents the mean ± SEM. ^*P < 0.01.

Figure 6

Mitochondrial potential in oocytes after dsBECN1 injection or autophagy inhibitor treatment. (A) JC-1 staining of dsBECN1-injected or LY294002-treated oocytes. ΔΨm was significantly lower in dsBECN1-injected or LY294002-treated oocytes than in control oocytes. Scale bar = 50 μm. (B) Fluorescence intensity of JC-1 in oocytes. Each value represents the mean ± SEM. ^*P < 0.01.

Figure 7

ROS content in oocytes after dsBECN1 injection or autophagy inhibitor treatment. (A) ROS in MI oocytes stained with DCDHF (green). Scale bar = 20 μm. (B) Relative fluorescence intensity of ROS. (C) ROS in MII oocytes stained with DCDHF (green). The MII polar body is indicated by a white arrow. Scale bar = 20 μm. (D) Relative fluorescence intensity of ROS. Control data values were arbitrarily set at 1. Values represent mean ± SEM from at least three separate experiments. ^*P < 0.01.

Figure 8

Preimplantation development after dsBECN1 injection or autophagy inhibitor treatment during IVM. (A) dsBECN1-injection or LY294002 treatment during oocyte maturation decreased the developmental potency of oocytes after parthenogenetic activation. Scale bar = 100 μm. (B) Embryonic development rates of control and dsBECN1-injected or LY294002-treated oocytes compared with control oocytes. ^*P < 0.05, ^**P < 0.01.