Figure 5. Rad51 RNAi treatment resulted in the removal of mitochondria by mitophagy.

(A) The mRNA expression profiles of autophagy-related genes were examined by RT-PCR. The expression levels of Atg5, Atg12 and Becn1 were slightly reduced, while the expression levels of Map1lc3a and Map1lc3b were increased in Rad51-silenced MI oocytes. H1foo was used as an internal control. IBMX 8 h → Plain 16 h, Rad51-silenced oocytes cultured in IBMX supplement for 8 hours followed by 16 hours of culture in plain M16 medium. (B) Immunofluorescence staining of Beclin1 in MI oocytes. Beclin1 is a marker protein for autophagy initiation. The oocytes were fixed in 4% paraformaldehyde and then stained with an antibody against Beclin1 (green). DNA was counterstained with DAPI (blue). Beclin1 expression is up-regulated in Rad51-silenced oocytes. The scale bars indicate 20 μm. (C) Rad51-silenced MI oocytes displayed increases in the size and number of Beclin1 puncta. The data are the numbers of Beclin1 puncta and are presented as the mean ± SEM. The asterisk represents statistical significance at p < 0.001 determined by a paired Student’s t-test. (D) Representative images showing the formation of Map1lc3b puncta. Map11c3b is a marker protein for mitophagy. Oocytes were fixed in 4% paraformaldehyde and then stained with DAPI (DNA, blue), MitoTracker (mitochondria, red) and anti-Map1lc3b antibody (green) and imaged using the LSCM. The 10 mM 3-MA was used to block autophagy. 3-MA-, oocytes were cultured without 3-MA addition; 3-MA+, oocytes were cultured with 3-MA. The scale bars indicate 20 μm. (E) Silencing of Rad51 expression resulted in the increased formation of Map1lc3b puncta. The average number of Map1lc3b puncta per oocyte was measured in MI oocytes after treatment of GFP (a–h) or Rad51 (i–p) RNAi and cultured in medium supplemented with (e–h, m–p) or without (a–d, i–l) 10 mM 3-MA. The data are presented as the mean ± SEM. Different letters (a~d) indicate significant differences at p < 0.001.