This study was conducted to investigate the effect of activation method on the endoplasmic reticulum (ER) stress induction, apoptosis and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by four activation methods; 1) electric stimulus (ES) (E), 2) ES+10 μM Ca-ionophore (A23187) treatment (EC), 3) ES+2 mM 6-dimethylaminopurine (6-DMAP) treatment (ED), or 4) ES+A23187 and 6-DMAP treatments (ECD). Parthenogenetic embryos were sampled to analyze x-box binding protein 1 (Xbp1) mRNA, ER stress-associated genes and apoptosis genes at 3 h after ES and the 1-cell and blastocyst stages. In the EC group, the band intensity of spliced Xbp1 (Xbp1s) mRNA was higher than those of the other groups at the 3 h and 1-cell stage, and higher than that of the E group at the blastocyst stage. Four ER stress-associated genes were expressed at the highest level in the EC group and weakly expressed in the ED group at 3 h after activation. However, most of the genes were highly expressed at the 1-cell and blastocyst stages with some variation in the EC and ECD groups. Expression of Bcl-2-associated X protein (Bax) and caspase-3 mRNA was significantly higher in the EC group than in the other groups at all development stages. The developmental rates to the blastocyst stage were higher in the ED and ECD groups than in the E and EC groups. These results suggest that the intracellular ER stress of parthenogenetic porcine embryos is affected by the activation method and subsequently lead to the apoptosis of embryos.

This study was conducted to examine the effects of activation methods on the ER stress induction and subsequent apoptosis and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by four activation methods; 1) electric stimulus(ES) with two DC pulses of 1.25 kV/cm, for 30 ㎲ (E), 2) ES + 10 μM Ca-ionophore (A23187) treatment for 5 min (EC), 3) ES + 2 mM 6-dimethylaminopurine treatment for 3 h (ED), or 4) ES + A23187 + 6-DMAP (ECD). After activation, parthenogenetic embryos were in vitro cultured in PZM-3 medium and sampled to analyze the x-box binding protein 1 (Xbp1) mRNA, ER stress-associated genes and apoptotic genes at 3 h post ES and the 1-cell and blastocyst stages. The un-spliced and spliced x-box binding protein 1 (Xbp1) mRNA were confirmed by RT-PCR. Also ER stress-associated genes, such as the C/EBP homologous protein (CHOP), binding protein (BiP), activating transcription factor 4 (ATF4) and glucose-regulated protein 94 (GRP94), and apoptotic genes were analyzed by real-time quantitative RT-PCR (RT-qPCR). The band intensities of spliced Xbp1 (Xbp1s) mRNA was higher in the EC group than other three groups at 3 h and the 1-cell stage, while it was higher in the ED groups compared with E group at the blastocyst stage. Four ER stress-associated genes were showed the highest expression in the EC group and weakly expressed in the ED group at 3 h. However, most of those genes were highly expressed in EC and ECD groups at the 1-cell and blastocyst stages with some variation. The expressions of Bcl-2-associated X protein (Bax) and caspase-3 mRNAs were significantly higher in EC group than other three groups at all stages. The developmental rate to the blastocyst stage was higher (p<0.05) in ED and ECD groups (32.1±3.8 to 34.6±2.2%) than that of E group (26.1±3.9%). These results suggest that the intracellular ER stress of parthenogenetic porcine embryos is affected by activation method and subsequently lead to the apoptosis of embryos.

We investigate the effect of endoplasmic reticulum (ER) stress inhibitor treatment during parthenogenetic activation of oocytes on the ER stress generation, apoptosis, and in vitro development of parthenogenetic porcine embryos. Porcine in vitro matured oocytes were activated by 1) electric stimulus (E) or 2) E+10 μM Ca-ionophore (A23187) treatment (EC). Oocytes were then treated by ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxychloic acid (TUDCA, 100 μM) for 3 h prior to in vitro culture. Parthenogenetic embryos were sampled to analyze ER stress and apoptosis at the 1-cell and blastocyst stages. The x-box binding protein 1 (Xbp1) mRNA and ER stress-associated genes were analyzed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. At the 1-cell stage, although no difference was observed in Xbp1 splicing among treatments, BiP transcription level in the E group was significantly reduced by salubrinal treatment, and GRP94 and ATF4 transcription levels in EC group were significantly reduced by all treatments (p<0.05) compared to control. In the EC group, both apoptotic genes were reduced by ER stress inhibitor treatments compared to control (p<0.05) except Caspase-3 gene by TUDCA treatment. These results suggest that the treatment of ER stress inhibitor during parthenogenetic activation can reduce ER stress, and thereby reduce apoptosis and promote in vitro development of porcine parthenogenetic embryos.