Cullins are the most important substrates of Nedd8 to date. (SAC). Accordingly, we identified early mitosis inhibitor 1 (Emi1), a key inhibitor of anaphase-promoting complex/cyclosome (APC/CFzr1), as a substrate of neddylation-mediated protein degradation. Thus, our study uncovered an unknown role of neddylation in female germ cells and suggests that proper neddylation is essential for oocyte maturation. ?0.05. Results Expression and subcellular localization of nedd8 during mouse oocyte meiosis To investigate the function of Nedd8 during oocyte meiosis, we first examined its expression level at each stage of oocyte maturation by dot blotting. Oocytes were collected after culture for 0, 2, 6, 8, and 12?h, corresponding to GV, GVBD, Pro-MI, MI, and MII stages, respectively. As shown in Physique 1a, Nedd8 was constantly expressed from GV to MII stages ( ?0.05; Physique 1a). Furthermore, we examined the subcellular localization of Nedd8 by immunofluorescence staining. Results showed that in GV stage, Nedd8 mainly accumulated in the nucleus (Physique 1b), whereas during GVBD and Pro-MI stages, it was localized to the cytoplasm (Physique 1c-d). During the following MI and MII stages when spindles had been already assembled, Nedd8 was found to be distributed around the spindle (Physique 1e-f). These results suggested that proteins involved in MI and MII stages might exhibit Nedd8 modifications and that the neddylation pathway might play a potential role in oocyte maturation. Open in a separate window Physique 1. Expression and subcellular localization of Nedd8 during mouse oocyte meiosis. (a) Mouse oocytes were collected after culture for 0, 2, 6, 8, and 12?h, corresponding Berberine Sulfate to germinal vesicle (GV), GV breakdown (GVBD), prophase of metaphase I (pro-MI), metaphase I (MI) and metaphase II (MII) stages, respectively. Whole lysates from 30 oocytes were loaded in each lane for dot blotting. Nedd8 levels were decided using an anti-Nedd8 antibody. The relative staining intensity of Nedd8 was assessed by densitometry in the histogram. Error bars represent the standard deviation. (b-f) Oocytes of different stages were collected for immunofluorescence staining. Blue: DNA; Green: -tubulin; Red: Nedd8. Scale bar, 10 m. Inhibition of neddylation causes MI arrest and spindle disorder during oocyte maturation To further investigate the role of neddylation during oocyte meiosis, we used MLN4924, a first-in-class NAE1 inhibitor [24], to disrupt the Nedd8 pathway. Mouse oocytes were cultured with gradient concentrations (0, 0.1 M, 0.5 M, 1 M and 5 M) of MLN4924 for 12?h. Results showed that oocytes exhibited MI arrest and the polar body exclusion (PBE) rate dose-dependently decreased when the MLN4924 concentration was higher than 0.5 M (47.75??5.17%, 0.5 M MLN4924 vs 80.49??2.58%, control, Rabbit polyclonal to Fyn.Fyn a tyrosine kinase of the Src family.Implicated in the control of cell growth.Plays a role in the regulation of intracellular calcium levels.Required in brain development and mature brain function with important roles in the regulation of axon growth, axon guidance, and neurite extension. ?0.01; 8.52??1.11%, Berberine Sulfate 1 M MLN4924 vs 80.49??2.58%, control, ?0.001; Physique 2a-b). The inhibitory effect reached the maximum at 1 M MLN4924, which Berberine Sulfate is similar to that of 5 M MLN4924 (8.52??1.11%, 1 M MLN4924 vs 7.11??2.05%, 5 M MLN4924, ?0.05; Physique 2b). Accordingly, the expression level of Nedd8 significantly decreased at the doses of 0.5 M (0.56??0.02 vs 1.03??0.05, ?0.001), 1 M (0.29??0.01 vs 1.03??0.05, ?0.001; Physique 2c-d). Therefore, 1 M MLN4924 was selected for subsequent study. To explore the subcellular oocyte phenotype after inhibition of neddylation, immunofluorescent staining was performed. Results revealed that in the MLN4924-treated group, the oocytes were arrested at the MI stage. Meanwhile, the spindle could not form bipolar structures and chromosomes could not segregate in the MI stage (Physique 2e). Open in a separate window Physique 2. Inhibition of neddylation causes oocyte MI arrest. (a) Images of oocytes in the control group and MLN4924-treated groups. Oocytes were cultured in medium with different concentrations of MLN4924 (0.1 M, 0.5 M, 1 M and 5 M) or without MLN4924. Scale bar, 100 m. (b) Germinal vesicle breakdown (GVBD) rate and polar.