Effect of Endoplasmic Reticulum (ER) Stress Inhibitor Treatment during Parthenogenetic Activation on the Apoptosis and In Vitro Development of Parthenogenetic Porcine Embryos

Abstract

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.

High Temperature Disrupts Organelle Distribution and Functions Affecting Meiotic Maturation in Porcine Oocytes

“…Transcription Polymerase Chain ReactionmRNA was extracted from 35 COCs after 24 h of culture in IVM medium using a Dynabeads mRNA Direct Kit (61,012; Thermo Fisher Scientific, Waltham, MA, United States), and cDNA was synthesized using the First Strand Synthesis Kit (cat# 6210; LeGene, San Diego, CA, United States) in accordance with the manufacturer's instructions. The primer sequences for amplification of cDNA were the same as those used in previous studies(Han et al, 2016;Park et al, 2018). qRT-PCR was performed using a WizPure qPCR Master (W1731-8; Wizbio Solutions, Seongnam, South Korea) according to the manufacturer's instructions, on a QuantStudio ™ six Flex Real-Time PCR System (Applied Biosystems, Waltham, MA, United States).…”