Development Of Porcine Parthenotes Research Articles

Kaempferol attenuates mitochondrial dysfunction and oxidative stress induced by H2O2 during porcine embryonic development

Kaempferol (3,4′,5,7-tetrahydroxyflavone, KAE) is one of the most commonly occurring dietary flavonols. The biological and pharmacological effects of kaempferol depend upon whether it acts as an antioxidant, anti-inflammatory, or anticancer agent. The present study explored the influence of KAE supplementation on in vitro damage to porcine oocytes and its underlying mechanisms. Different concentrations of KAE (0.05, 0.1, 0.5, 1 μM) were added to porcine zygote medium 5 during in vitro culture. The results showed that supplementation with 0.1 μM KAE significantly increased the blastocyst formation rate. Blastocyst formation and quality were significantly increased in the 200 μM H2O2 treatment group following addition of 0.1 μM KAE. KAE prevented the H2O2-induced compromise of mitochondrial membrane potential and reactive oxygen species generation. Furthermore, the extent of autophagy and DNA damage in the blastocysts was attenuated by supplementation with KAE in the H2O2 induced oxidative injury group compared to that observed in controls. These results suggest that KAE has beneficial effects on the development of porcine parthenotes by attenuating oxidative stress and increasing mitochondrial function.

72 Protective Effects of C-Phycocyanin on the Developmental Competence of Porcine Parthenotes

C-Phycocyanin (CP) is a biliprotein enriched in blue-green algae that is known to possess antioxidant, anti-apoptosis, anti-inflammatory, and radical-scavenging properties in somatic cells. However, the protective effect of CP on porcine embryo developmental competence in vitro remains unclear. In the present study, we investigated the effect of CP on the development of porcine early embryos as well as its underlying mechanisms exposing them to H2O2 to induce oxidative stress. The levels of reactive oxygen species, mitochondrial membrane potential, apoptosis, DNA damage, and autophagy in the blastocysts were observed by staining with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), 5,5′,6,6’-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate (dUTP) nick-end labelling (TUNEL), anti-cytochrome c, and anti-γH2A.X (Ser139), respectively. Colocalization assay of mitochondria and cytochrome c of blastocysts were staining with MitoTracker Red CMXRos and anti-cytochrome c. All data were subjected to one-way ANOVA. Different concentrations of CP (1, 2, 5, 8, 10 µg mL−1) were added to porcine zygote medium 5 (PZM-5, l-glutamine concentration of PZM-3 was modified from 1 to 2 mM) during in vitro culture. The results showed that 5 µg mL−1 CP significantly increased blastocyst formation (62.5 ± 2.1 v. 52.7 ± 2.4; P < 0.05) and hatching rate (10.9 ± 1.9 v. 36.6 ± 5.2; P < 0.05) compared with controls. Blastocyst formation (53.1 ± 2.3 v. 40.1 ± 2.3; P < 0.05) and quality were significantly increased in the 50 µM H2O2 treatment group following 5 µg mL−1 CP addition. C-Phycocyanin prevented the H2O2-induced compromise of mitochondrial membrane potential, release of cytochrome c from the mitochondria, and generation of reactive oxygen species. Furthermore, apoptosis, DNA damage level, and autophagy in the blastocysts were attenuated by supplementation of CP in the H2O2-induced oxidative injury group compared with that in controls. These results suggest that CP has beneficial effects on the development of porcine parthenotes by attenuating mitochondrial dysfunction and oxidative stress.

C-Phycocyanin protects against mitochondrial dysfunction and oxidative stress in parthenogenetic porcine embryos

C-Phycocyanin (CP) is a biliprotein enriched in blue-green algae that is known to possess antioxidant, anti-apoptosis, anti-inflammatory, and radical-scavenging properties in somatic cells. However, the protective effect of CP on porcine embryo developmental competence in vitro remains unclear. In the present study, we investigated the effect of CP on the development of early porcine embryos as well as its underlying mechanisms. Different concentrations of CP (2, 5, 8, 10 μg/mL) were added to porcine zygote medium 5 during in vitro culture. The results showed that 5 μg/mL CP significantly increased blastocyst formation and hatching rate. Blastocyst formation and quality were significantly increased in the 50 μM H2O2 treatment group following 5 μg/mL CP addition. CP prevented the H2O2-induced compromise of mitochondrial membrane potential, release of cytochrome c from the mitochondria, and reactive oxygen species generation. Furthermore, apoptosis, DNA damage level, and autophagy in the blastocysts were attenuated by supplementation of CP in the H2O2-induced oxidative injury group compared to in controls. These results suggest that CP has beneficial effects on the development of porcine parthenotes by attenuating mitochondrial dysfunction and oxidative stress.

Inhibition of the Arp2/3 complex impairs early embryo development of porcine parthenotes

ABSTRACTThe Arp2/3 complex, which nucleates actin filaments, comprises a stable assembly of seven-protein subunits including two actin-related proteins (Arp2 and Arp3). Previous work showed that Arp2/3 binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. In the present study, we show that the Arp2/3 complex is critical for cytokinesis during early embryonic development in porcine parthenotes. The Arp2/3 complex is concentrated at the cortex of each cell at the 1-, 2-, and 4-cell stages, and at the periphery at the morula stage. The amount of Arp2/3 significantly decreased at the blastocyst stage in parthenogenetically activated porcine embryos. Inhibition of the Arp2/3 complex in the pig embryos by the Arp2/3-specific inhibitor CK666 resulted in abnormal cell division, a decrease in developmental rate and total cell numbers, and an increase in the ratio of trophectoderm cell number to inner cell mass number in blastocyst-stage embryos. In addition, 4-cell stage embryos subjected to CK666 treatment exhibited significantly decreased expression of ZGA genes (Pou5f1, Sox2, and Nanog), suggesting that the Arp2/3 complex plays an important role in early porcine embryo development. Thus, our data demonstrate that the Arp2/3 complex is required for early embryonic development in pigs and appears to regulate the expression of pluripotency genes.

Effects of epigallocatechin-3-gallate on the developmental competence of parthenogenetic embryos in the pig

This study was conducted to examine the effects of (-)-epigallocatechin gallate (EGCG) supplementation on the developmental competence and quality of parthenogenetic porcine embryos during culture. Parthenogenetic embryos derived from in vitro matured oocytes were cultured for eight days in a modified North Carolina State University (NCSU)-37 solution supplemented with EGCG at different concentrations (0, 1, 5, 10 and 50 µM). Supplementation of 1 and 5 µM EGCG during in vitro culture of embryos showed no significant influence on the rate of cleavage or that of blastocyst formation or on the total cell number and DNA fragmentation indices of blastocysts when compared to those of a control group. However, when 10 and 50 µM EGCG were supplemented into the culture medium, the cleavage rates were significantly lower than those of the other groups. No embryo developed to the blastocyst stage. Results suggest that treatment with low EGCG during in vitro culture has no influence on the developmental competence of porcine embryos but the presence of high concentrations of EGCG is apparently harmful for in vitro development of porcine parthenotes.

195 SODIUM SELENITE INCREASES DEVELOPMENTAL ABILITY AND DECREASES APOPTOSIS IN PORCINE PARTHENOTES

Selenium is an essential component of serum required for cell proliferation and growth. It has also been shown to have a beneficial effect on in vitro maturation of oocytes. This study examined the development and apoptosis of porcine parthenotes treated with sodium selenite (SS). Immature cumulus–oocyte complexes were cultured in TCM-199 supplemented with 25 mM NaHCO3, 10% (v/v) porcine follicular fluid, 0.57 mM cysteine, 0.22 µg mL−1 sodium pyruvate, 25 µg mL−1 gentamycin sulfate, 0.5 µg mL−1 Follitropin V, 10 ng mL−1 epidermal growth factor, and 1 µg mL−1 estradiol-17β for 42–44 h. The matured oocytes were activated by electro-stimulation (single electrical pulse of 1.36 kV cm−1 for 30 µs) and treated with 10 µg mL−1 cytochalasin B for 4 h. Activated oocytes were cultured to the blastocyst stage in NCSU23 supplemented with 0.4% polyvinyl alcohol in the presence or absence of 25 ng mL−1 SS for 7 days at 39°C in a humidified atmosphere of 5% CO2 in air. Inner cell mass (ICM) and apoptosis rates in blastocysts were evaluated by differential double staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. In addition, relative abundance of Bax/BclXL transcripts was quantified by real-time qRT-PCR. The activity of caspase-3 and MAP kinase in blastocysts was examined by western blotting. The cleavage rate, blastocyst rate, total cell number, apoptosis ratio, and ICM ratio of parthenotes in NCSU23 + PVA without/with SS is shown in Table 1. Expression level of Bax/BclXL genes in blastocysts decreased 0.4 ± 0.1-fold in the presence of SS. Furthermore, activity of caspase-3 in blastocysts decreased and activity of MAP kinase in blastocysts increased with SS. Therefore, our data suggest that SS may play a critical role in increasing the development of porcine parthenotes and in reducing their apoptosis. Table 1.Developmental ability and apoptosis of porcine parthenotes cultured in the presence or absence of sodium selenite This work was supported by the Research Project on the Production of Bio-organs (No. 200503030202), Ministry of Agriculture and Forestry, Republic of Korea.

Mouse granulocyte–macrophage colony-stimulating factor enhances viability of porcine embryos in defined culture conditions

The aim of this study was to determine the effects of mouse granulocyte–macrophage colony-stimulating factor (mGM-CSF) on development of porcine parthenotes and nuclear transferred embryos, and on their expression of implantation-related genes. In the presence of bovine serum albumin, mGM-CSF did not increase the percentage of oocytes that developed to the blastocyst stage and at day 7 did not increase cell numbers of embryos. Addition of 2 ng/ml GM-CSF to protein-free culture medium significantly increased the compaction and blastocoel formation of 1- to 2-cell parthenotes developing in vitro. However, total cell numbers were not increased when they were cultured in the presence of GM-CSF. Semi-quantitative reverse transcriptase polymerase chain reaction revealed that mGM-CSF enhances mRNA expression of the leukemia inhibitory factor receptor, but does not influence interleukin-6 or sodium/glucose co-transporter protein gene expression in blastocyst stage parthenotes. These results suggest that mGM-CSF may enhance viability of porcine embryos developing in vitro in a defined culture medium.