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. 2023 Oct 20;12:RP88523. doi: 10.7554/eLife.88523

Figure 6. Evaluation of C-type natriuretic peptide (CNP) supplementation on mitophagy activity of aged oocytes.

Figure 6.

(A) Representative images of mitochondria morphology and structure in young, aged, and aged + CNP oocytes by TEM. (B) Accumulation of mitochondria damage in young, aged, and aged + CNP oocytes. Under TEM images, percentages of damaged mitochondria per area (500 nm × 500 nm) were shown. At least four visions were chosen and mitochondria were counted by two individuals. (C) Representative images of mitochondria reactive oxygen species (ROS) stained with MitoSOX in young, aged, aged + CNP oocytes. Scale bar, 20 μm. (D) Fluorescence intensity of MitoSOX signals was measured in young aged, aged + CNP oocytes. (E) Western blots of P62（62 kDa）, LC3-I/II (14-16 kDa), PINK1（60 kDa）, and Parkin（50 kDa） in young, aged, and aged + CNP oocytes. GAPDH (37 kDa) was used as internal control. (F–I) Relative gray value of proteins detected with western blots compared with controls. (J) Oocyte cAMP concentrations were measured in young, aged, and aged + CNP mice. (K) Representative images at day 0, day 2, day 4, and day 6 of cultured preantral follicles with or without CNP or CNP + H89 treatment. Scale bar = 50 μm. (L) Diameters of preantral follicles with or without CNP or CNP + H89 treatment from day 0 to day 6. Six independent culture experiments were performed. (M) Western blots of PINK1 and Parkin in aged, aged + CNP, and aged + CNP + H89-treated oocytes. GAPDH was used as internal control. (N–O) Relative gray value of proteins detected with western blots compared with controls. (P) Double immunofluorescence staining of Parkin and TOMM20. The mitochondria outer membrane protein TOMM20 was performed to reveal the translocation of PRKN proteins on mitochondria. Red, PRKN; green, TOMM20; blue, DNA was labeled with Hoechst 33342. Bar: 20 μm. (Q) The colocalization of Parkin and TOMM20 in oocytes from aged, aged + CNP, and aged + CNP + H89-treated mice was compared. Pearson’s R shows the results of co-location analysis.