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MICROINJECTION OF MITOCHONDRIAL FRACTIONS INTO FVB OOCYTES INFLUENCES IN VITRO SURVIVAL RATES.
Acton, Beth1, Jurisicova, Andrea 1,2, Ahmady, Ali1, Xu, Jing1, Perez, Gloria2, Casper, Robert1, 1 2
ABSTRACT- Recent experiments support the hypothesis that mitochondria may be the cytoplasmic agents that protect against apoptosis in oocytes and during embryo development. The objective of the study was to isolate mitochondria from stem cells and to determine if microinjection of these organelles into murine FVB oocytes, which are prone to fragmentation, could change in vitro survival rates. We postulated that injection of healthy mitochondria would improve survival while injection of damaged mitochondria would increase cell death. Both damaged and undamaged populations of mitochondria were isolated from ES cells (RI). The presence of an enriched mitochondrial fraction was confirmed through electron microscopy. Mitochondria were microinjected within one hour of collection (both damaged and undamaged) or after aging at room temperature for two hours. The mitochondria were microinjected into murine FVB oocytes obtained 23 hours after gonadotropin administration. Non-injected as well as sham injected oocytes served as controls. All oocytes were scored for the presence or absence of fragmentation 24 hours following injection as well as for the percent (by volume) of fragmentation. The oocytes microinjected with healthy mitochondria demonstrated a lower rate of fragmentation (22.6%) than both the non-injected control group (35.7%) and the sham injected control group (43.8%). With injection of healthy mitochondria, those oocytes that did fragment had a lower extent of fragmentation (18.1%) than seen in the sham injected controls (21.4%). Oocytes that were injected with damaged mitochondria displayed the highest fragmentation rate (58.3%) and fragmented to the highest degree (29%). Injection of healthy mitochondria appears to improve in vitro survival rates and to lessen the degree of fragmentation in FVB oocytes. In contrast, injection of damaged or aged mitochondria increased in vitro cell death and the degree of fragmentation in the oocytes. It is known that aged oocytes undergo spontaneous activation in culture followed by cellular fragmentation. Our results indicate that increasing the number of healthy mitochondria suppresses fragmentation and promotes cellular cleavage.
KEY WORDS: mitochondria, apoptosis, oocyte, embryo development
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