Mitochondria are highly dynamic organelles that undergo constant fusion/fission as well as activities orchestrated by large dynamin-related GTPases. These dynamic mitochondrial processes influence mitochondrial morphology, size and function. Therefore, this study was conducted to evaluate the effects of mitochondrial fission inhibitor, mdivi-1, on developmental competence and mitochondrial function of porcine embryos and primary cells. Presumptive porcine embryos were cultured in PZM-3 medium supplemented with mdivi-1 (0, 10 and 50 μM) for 6 days. Porcine fibroblast cells were cultured in growth medium with mdivi-1 (0 and 50 μM) for 2 days. Our results showed that the rate of blastocyst production and cell growth in the mdivi-1 (50 μM) treated group was lower than that of the control group (P < 0.05). Moreover, loss of mitochondrial membrane potential in the mdivi-1 (50 μM) treated group was increased relative to the control group (P < 0.05). Subsequent evaluation
revealed that the intracellular levels of reactive oxygen species (ROS) and the apoptotic index were increased by mdivi-1 (50 μM) treatment (P < 0.05). Finally, the expression of mitochondrial fission-related protein (Drp 1) was lower in the embryos and cells in the mdivi-1-treated group than the control group. Taken together, these results indicate that mdivi-1 treatment may inhibit developmental competence and mitochondrial function in porcine embryos and primary cells.
γ-tocotrienol (GTT), an isomer of vitamin E, has been the subject of increasing interest due to its strong anti-oxidant effects. Therefore, in this study, the effects of GTT on blastocyst development, expression levels of reactive oxygen species (ROS) and apoptotic index were investigated in preimplantation porcine embryos. After in vitro maturation and fertilisation, porcine embryos were cultured for 6 days in porcine zygote medium 3 supplemented with or without GTT (200μM) under oxidative stress conditions (200μM hydrogen peroxide (H2O2)). Blastocyst development was significantly improved in the GTT-treated group when compared with the H2O2-treated group (P<0.05). Subsequent evaluation of the intracellular levels of ROS and numbers of apoptotic nuclei in GTT-treated blastocysts revealed that ROS levels of GTT-treated porcine blastocysts were decreased (P<0.05) and the numbers of apoptotic nuclei were reduced by GTT treatment in porcine embryos. Moreover, the total cell numbers of blastocysts were significantly increased in the GTT-treated group relative to the untreated group under H2O2-induced oxidative stress (P<0.05). The expression levels of apoptosis-related genes (BCL-XL, BAX) in GTT-treated blastocysts were then investigated using real-time reverse transcription polymerase chain reaction. Expression of the anti-apoptotic BCL-XL gene was shown to be increased in the GTT-treated blastocyst group, whereas expression of the pro-apoptotic BAX gene was decreased. Taken together, these results suggest that GTT (200μM) under H2O2-induced oxidative stress, thereby improving the developmental competence of porcine embryos via modulation of intracellular levels of ROS and the apoptotic index during the preimplantation stage.
Bovine somatic cell nuclear transfer (SCNT) is an important and powerful tool for basic
research and biomedical and agricultural applications, however, the efficiency of SCNT has
remained extremely low. In this study, we investigated the effects of cathepsin B
inhibitor (E-64) supplementation of culture medium on in vitro
development of bovine SCNT embryos. We initially used three concentrations of E-64 (0.1,
0.5, 1.0 μm), among which 0.5 μm resulted in the highest rate of blastocysts production after in
vitro fertilization (IVF), and was therefore used for further experiments.
Blastocyst development of SCNT embryos in the E-64 treatment group also increased relative
to the control. Moreover, the cryosurvival rates of IVF and SCNT blastocysts were
increased in E-64 treatment groups when compared with the control. On the other hand, we
found that IVF and SCNT blastocysts derived from E-64-treated groups had increased total
cell numbers and decreased apoptotic nuclei. Furthermore, assessment of the expression of
apoptosis-related genes (Bax and Bcl-xL) in bovine IVF and SCNT blastocysts treated with
E-64 by real-time RT-PCR analysis revealed suppressed expression of the pro-apoptotic gene
Bax and stimulated expression of the anti-apoptotic gene Bcl-xL. Taken together, these
finding indicate that addition of E-64 to embryo culture medium may have important
implications for improving developmental competence and preimplantation quality in bovine
IVF and SCNT embryos.
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