Effects of Endoplasmic Reticulum Stress Inhibitor Treatment during the Micromanipulation of Somatic Cell Nuclear Transfer in Porcine Oocytes

Abstract

We examined the effects of endoplasmic reticulum (ER) stress inhibitor treatment during the micromanipulation of porcine somatic cell nuclear transfer (SCNT) on the in vitro development of SCNT embryos. ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxycholic acid (TUDCA; 100 μM) were added to the micromanipulation medium and holding medium. The expression of X-box binding protein 1 ( Xbp1 ), ER-stress-associated genes, and apoptotic genes in SCNT embryos was confirmed at the one-cell and blastocyst stages. Levels of Xbp1 splicing and expression of ER-stress-associated genes in SCNT embryos at the one-cell stage decreased significantly with TUDCA treatment ( p <0.05). The expression of ER-stress-associated genes also decreased slightly with the addition of both salubrinal and TUDCA (Sal+TUD). The expression levels of caspase-3 and Bcl2-associated Xprotein ( Bax ) mRNA were also significantly lower in the TUDCA and Sal+TUD treatments ( p <0.05). At the blastocyst stage, there were no differences in levels of Xbp1 splicing, and transcription of ER-stress-associated genes and apoptosis genes between control and treatment groups. However, the blastocyst formation rate (20.2%) and mean blastocyst cell number (63.0±7.2) were significantly higher ( p <0.05) for embryos in the TUDCA treatment compared with those for control (12.6% and 41.7±3.1, respectively). These results indicate that the addition of ER-stress inhibitors, especially TUDCA, during micromanipulation can inhibit cellular damage and enhance in vitro development of SCNT embryos by reducing stress levels in the ER.

Endoplasmic Reticulum (ER) Stress Inhibitor or Antioxidant Treatments during Micromanipulation Can Inhibit Both ER and Oxidative Stresses in Porcine SCNT Embryos

“…Several studies have shown that ER stress is inhibited when TUDCA is treated to embryo or cell in culture period (Zhang et al, 2012a,b;Yoon et al, 2014;Lin et al, 2015, Mochizuki et al, 2018. In addition, our previous study showed that when TUDCA is treated during the micromanipulation and activation processes of the SCNT embryo, Xbp1s and ER stress-associated genes are significantly reduced, thereby increasing the development and quality of SCNT embryos (Park et al, 2019). This study substantiates our previous result.…”

“…There was no significant decrease in the splicing of Xbp1 in combination with ER stress inhibitor and antioxidant. Previous study showed that the combination of ER stress inhibitor TUDCA and salubrinal did not significantly decrease the Xbp1s and ER stress-associated gene transcription levels, suggesting that simultaneous treatment of both agents would have had an excessive effect on ER (Park et al, 2019). From this point of view, a similar tendency would have been observed when the ER stress inhibitor and antioxidant were simultaneously treated.…”

“…For the treatment groups, 100 μM TUDCA (Park et al, 2019), 100 μM Vit. C (Bae et al, 2012), or both TUDCA and Vit.…”

“…In general, ER stress inhibitor and antioxidant are treated during the maturation period of oocytes or culture period of embryos (Yoon et al, 2014;Mallol et al, 2015;Lin et al, 2015Lin et al, , 2016. In contrast, previous studies showed that development and quality of SCNT embryos were improved by treating ER stress inhibitors only during microinjection and activation of SCNT, inhibiting ER stress generation during SCNT, and reducing apoptosis (Park et al, 2017;Park et al, 2019). In addition, the ROS and apoptosis were reduced when antioxidant was treated during micromanipulation, fusion and activation of SCNT process (Bae et al, 2012(Bae et al, , 2015.…”

Inhibiting P2Y12 in Macrophages Induces Endoplasmic Reticulum Stress and Promotes an Anti-Tumoral Phenotype

“…In the conditions where Tudca was added, macrophages had also been pre-incubated with 100 µM Tudca, prior to adding HepG2 cells. This concentration has previously been shown to effectively inhibit ER stress in different cell types [ 55 , 56 ]…”