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. 2021 Mar;25(1):43-53.
doi: 10.12717/DR.2021.25.1.43. Epub 2021 Mar 31.

Development of Porcine Somatic Cell Nuclear Transfer Embryos Following Treatment Time of Endoplasmic Reticulum Stress Inhibitor

Mi-Jeong Kim  1 , Bae-Dong Jung  1 , Choon-Keun Park  2 , Hee-Tae Cheong  1
Affiliations

Development of Porcine Somatic Cell Nuclear Transfer Embryos Following Treatment Time of Endoplasmic Reticulum Stress Inhibitor

Mi-Jeong Kim et al. Dev Reprod. 2021 Mar.

Abstract

We examine the effect of endoplasmic reticulum (ER) stress inhibitor treatment time on the in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT embryos were classified by four groups following treatment time of ER stress inhibitor, tauroursodeoxycholic acid (TUDCA; 100 μM); 1) non-treatment group (control), 2) treatment during micromanipulation process and for 3 h after fusion (NT+3 h group), 3) treatment only during in vitro culture after fusion (IVC group), and 4) treatment during micromanipulation process and in vitro culture (NT+IVC group). SCNT embryos were cultured for six days to examine the X-box binding protein 1 (Xbp1) splicing levels, the expression levels of ER stress-associated genes, oxidative stress-related genes, and apoptosis-related genes in blastocysts, and in vitro development. There was no significant difference in Xbp1 splicing level among all groups. Reduced expression of some ER stress-associated genes was observed in the treatment groups. The oxidative stress and apoptosis-related genes were significantly lower in all treatment groups than control (p<0.05). Although blastocyst development rates were not different among all groups (17.5% to 21.7%), the average cell number in blastocysts increased significantly in NT+3 h (48.5±2.3) and NT+IVC (47.7±2.4) groups compared to those of control and IVC groups (p<0.05). The result of this study suggests that the treatment of ER stress inhibitor on SCNT embryos from the micromanipulation process can improve the reprogramming efficiency of SCNT embryos by inhibiting the ER and oxidative stresses that may occur early in the SCNT process.

Keywords: Endoplasmic reticulum (ER) stress inhibitor; Endoplasmic reticulum stress; In vitro development; Pig; Somatic cell nuclear transfer.

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Conflict of interest statement

The authors declare no potential conflict of interest.

Figures

Fig. 1.
Fig. 1.. Splicing level of Xbp1 mRNA in SCNT blastocysts.
The mRNAs of unspliced Xbp1 (Xbp1u) and spliced Xbp1 (Xbp1s) were detected by semi-qPCR (upper panel) and band intensities (means±SEM) were measured densitometrically (lower panel). SCNT, somatic cell nuclear transfer. Xbp1, X-box binding protein 1.
Fig. 2.
Fig. 2.. ER stress-associated gene expression in SCNT blastocysts.
ER stress-associated genes (BiP, GRP94, ATF4, and CHOP) were quantified by RT-qPCR (means±SEM). a,b Values with different letters are significantly different (p<0.05). ER, endoplasmic reticulum; SCNT, somatic cell nuclear transfer.
Fig. 3.
Fig. 3.. Oxidative stress-related gene expression in SCNT blastocysts.
Oxidative stress-related genes (Nrf2 and HO-1) were quantified by RT-qPCR (means±SEM). a,b Values with different letters are significantly different (p<0.05). SCNT, somatic cell nuclear transfer.
Fig. 4.
Fig. 4.. Apoptotic gene expression in SCNT blastocysts.
Apoptotic genes (Bax and caspase3) were quantified by RT-qPCR (means±SEM). a,b Values with different letters are significantly different (p<0.05). SCNT, somatic cell nuclear transfer.
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