J Vet Sci.  2013 Sep;14(3):235-240. 10.4142/jvs.2013.14.3.235.

Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos

• Hwang IS
• Bae HK
• Cheong HT

• KMID: 1705552
• DOI: http://doi.org/10.4142/jvs.2013.14.3.235

Abstract

The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H2O2 and .OH radical levels, mitochondrial morphology and membrane potential (DeltaPsi), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H2O2 (35.6 +/- 1.1 pixels/embryo) and .OH radical levels (44.6 +/- 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 +/- 1.5 and 23.8 +/- 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The DeltaPsi of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 +/- 0.04 vs. 1.21 +/- 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 +/- 26.4 microm vs. 425.6 +/- 25.0 microm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.

Keyword

cattle; cellular damage; DNA fragmentation; ROS generation; somatic cell nuclear transfer

MeSH Terms

Animals
*Apoptosis
Caspase 3/metabolism
Cattle
Colorimetry/veterinary
Comet Assay/veterinary
*DNA Damage
DNA, Mitochondrial/*genetics/metabolism
Embryo Transfer/veterinary
Embryo, Mammalian/*cytology/embryology
Fertilization in Vitro/veterinary
In Situ Nick-End Labeling/veterinary
Membrane Potential, Mitochondrial
Microscopy, Confocal/veterinary
Microscopy, Fluorescence/veterinary
Mitochondria/*metabolism
Nuclear Transfer Techniques/*veterinary
Reactive Oxygen Species/*metabolism
Caspase 3
DNA, Mitochondrial
Reactive Oxygen Species

Figure

• 

  Fig. 1 Levels of reactive oxygen species (ROS) in bovine somatic cell nuclear transfer (SCNT) and in vitro fertilized (IVF) embryos. Five replicates were performed (n = 50~55 embryos in each group). The SCNT and IVF embryos were analyzed on the same day, enabling direct comparisons between SCNT and IVF groups. Data are presented as the mean ± SEM (bars). *Significantly different from IVF within each treatment (p < 0.05).

• 

  Fig. 2 Fluorescence microscopic evidence of mitochondrial and DNA damage in one-cell stage IVF (A, C, E and G) and SCNT (B, D, F and H) embryos. (A and B) MitoTracker Red staining images of embryos. (C and D) JC-1 stained embryos. (E and F) TUNEL images of embryos. (G and H) Comet images of fragmented DNA migration of embryos. Scale bars = 50 µm.

• 

  Fig. 3 Mitochondrial membrane potential (ΔΨ) of IVF and SCNT embryos (n = 40 ~ 50 in each group). Data are presented as the mean ± SEM (bars). *Significantly different from IVF (p < 0.05).

• 

  Fig. 4 Tail moment length of IVF (n = 37) and SCNT (n = 38) embryos. Data are presented as the mean ± SEM (bars). *Significantly different from IVF (p < 0.05).

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