Figure 2.

Blocking the autophagy in the granulosa cell decreased the quality of oocyte. (A) The sketch of experimental design for co-cultures. (B) Western blot analysis of autophagy related protein expression in the cells subjected to 3-MA and CQ treatment. Each lane corresponds to an independent biological sample. Normalized quantification of mean gray intensity was determined from 3 separate experiments. (C) Representative images of matured oocytes cultured in vitro from control (n = 30), 3-MA (n = 35), and CQ (n = 35) treatment cumulus granulosa cells. The rate of PB1 was recorded in three independent biological replicates. Scale bar: 80 mm. (D, E, and F) Representative images of cortical granule (CG) distribution (D), meiotic spindle and chromosomes (E), and autophagosomes (F) attachment in control, 3-MA, and CQ treatment groups. Oocytes were immunoassayed with indicated antibody to show the CG, spindles, and LC3-II, respectively. The rate of mis-localized CGs (n = 11) (D), the thickness of spindle middle plate (n = 11). C indicates maximal span of chromosomes; S indicates maximal spindle lengths (E), and the fluorescence intensity of LC3-II (n = 8) (F) were recorded in control, 3-MA, and CQ treated groups. Scale bar: 20 μm. Data in (D), (E) and (F) are presented as mean percentage (mean ± SEM) of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired two-tailed Student’s t test.