Microinjection of Mitochondria Derived from Serum-Starved Somatic Cells Affects Parthenogenetic Development of Murine and Bovine Oocytes.

In somatic cell nuclear transfer protocols, a small amount of donor cell mitochondria is transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model has demonstrated that transferred mitochondria affected parthenogenetic development of oocytes. In many protocols, donor cells are subjected to serum-starvation prior to nuclear transfer. Mitochondrial viability is dramatically affected by cell culture conditions and serum starvation induced an increase in mitochondrial viability in confluent cells. This study was conducted to examine whether donor mitochondria derived from serum-starved cells affected the development of reconstructed embryos using murine and bovine parthenogenetic models. Murine oocytes were collected from B6D2F1 mice. Mitochondria were isolated by differential centrifugation from primary cells (ear of M. spretus mice) at confluency (non-starved), and after serum starvation for either 1 or 2 weeks. Approximately 1-2 pl of the mitochondrial suspension were injected into oocytes using a Piezo micromanipulator. Surviving oocytes were activated by 10 mM SrCl2 and 5 µg/ml cytochalasin B for 5 hrs; then cultured in modified CZB medium for 4 days. Bovine oocytes were obtained from ovaries stored at 15°C for overnight, and matured for 24 hrs. Mitochondria were isolated from fibroblast cells (ear of Japanese Black cattle) at confluency or after serum-starvation for one week. After mitochondrial microinjection, surviving oocytes were activated by a single DC pulse of 57 V/mm for 50 µsec, treated by 0.4 mg/ml 6DMAP for 5 hrs, and then, cultured in IVD101 medium for 7 days. All murine oocytes injected mitochondria with control or 1 or 2 week serum-starved mitochondria [morula formation (MF): 66%, 48%, 44%; blastocyst formation (BF): 44%, 37%, 34%, respectively] showed delayed development when compared to oocytes injected with buffer or uninjected (MF: 91%, 92%; BF: 70%, 89%; P < 0.01). Thus, the oocytes injected serum-starved mitochondria showed lower rates of MF (48%, 44%) when compared to oocytes injected non-starved mitochondria (66%, P < 0.01), and were not different by period (1 week or 2 weeks) of serum starvation (P > 0.05). The bovine oocytes injected serum-starved mitochondria showed lower rates of MF (38%) when compared to oocytes injected with buffer or uninjected (57%, 53%; P < 0.05), and of BF (14%)when compared to uninjected (25%; P < 0.05). The developmental rates between non- and serum starvation were not significantly different (MF: 50%, 38%; BF: 19%, 14%, respectively; P > 0.05). The overall results demonstrate that injection of serum-starved mitochondria influenced parthenogenetic development of both murine and bovine oocytes. Our results suggest that the somatic mitochondria introduction accompanying nuclei has the capacity to affect reconstructed embryo development, especially when using serum-starved cells as donor cells.