Figure 7. Experiments using TNF-α-knockdown CCs and the COCs from TNF-α-knockout (TNF-α-/-) mice. In panels (A) to (D), CCs were transfected with negative control siRNA (NC) or siRNAs (siR) 1, 2 or 3 against TNF-α before culture in CZB containing 200-µM H2O2 and CM collection. In panels (A) and (B), DOs recovered 13 h post hCG injection were cultured in the CM for 12 h before ethanol treatment for activation (A) or culture in CZB for different times for fragmentation observation (B). In panels (C) and (D), concentrations of sTNF-α or sFasL in the CM were measured by ELISA, respectively. In panels (E) and (F), COCs recovered 13 h post hCG injection from wild-type C57BL/6J mice and TNF-α-/- mice were cultured for 12 h in CZB medium before examination for ethanol activation (E) or for fragmentation (F). To observe fragmentation, the COCs were freed of CCs and the resulting DOs were cultured in CZB for different times before examination for fragmentation. To examine activation or fragmentation, each treatment was repeated 3-5 times with each replicate including about 30 oocytes. For measurement of sTNF-α or sFasL in CM, each treatment was repeated 3 times with each replicate including 100 µl of CM from one culture well. a-e: Values with a different letter above bars differ significantly (P < 0.05).