Cell-free mitochondrial DNA increases granulosa cell apoptosis and reduces aged oocyte blastocyst development in the mouse.

Cell-free mitochondrial DNA (cf-mtDNA) released into the extracellular environment can cause cellular inflammatory responses and damage. Here, we investigated the effects of cf-mtDNA on mouse ovarian granulosa cell function and on the developmental competence of oocytes matured in vitro. Granulosa cells in the cf-mtDNA treatment group had a lower ATP content (P < 0.05), a higher apoptotic cell percentage (P < 0.01), and higher mRNA and protein levels of apoptosis-related factors than the control group (P < 0.01). TLR9, NF-кB p65 and MAPK p38 expression levels in granulosa cells were significantly increased in the cf-mtDNA treatment group (P < 0.05). The blastocyst formation rate of aged mice oocytes matured in vitro decreased significantly (P < 0.05) when cf-mtDNA was added to the media, compared with the control. However, the oocytes from young mice were not affected. Our results suggest that cf-mtDNA may impair granulosa cell function and induce granulosa cell apoptosis, subsequently decreasing blastocyst development in aged oocytes. This role of cf-mtDNA may be associated with the binding to TLR9 and the activation of NF-кB p65 and MAPK p38 signaling pathways.