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. 2024 Jul;66(4):726-739.
doi: 10.5187/jast.2024.e68. Epub 2024 Jul 31.

Lysophosphatidic acid improves development of porcine somatic cell nuclear transfer embryos

Ling Sun  1   2 , Tao Lin  1   2 , Jae Eun Lee  2 , So Yeon Kim  2 , Ying Bai  1 , Dong Il Jin  2
Affiliations

Lysophosphatidic acid improves development of porcine somatic cell nuclear transfer embryos

Ling Sun et al. J Anim Sci Technol. 2024 Jul.

Abstract

This study was conducted to investigate whether lysophosphatidic acid (LPA) could improve the development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT-derived embryos were cultured in chemically defined polyvinyl alcohol (PVA)-based porcine zygote medium (PZM)-4 without or with LPA, and the development, cell proliferation potential, apoptosis, and expression levels of pluripotent markers were evaluated. LPA significantly increased the rates of cleavage and blastocyst formation compared to those seen in the LPA un-treatment (control) group. The expression levels of embryonic development-related genes (IGF2R, PCNA and CDH1) were higher (p < 0.05) in the LPA treatment group than in the control group. LPA significantly increased the numbers of total, inner cell mass and EdU (5-ethynyl-2'-deoxyuridine)-positive cells in porcine SCNT blastocysts compared to those seen in the control group. TUNEL assay showed that LPA significantly reduced the apoptosis rate in porcine SCNT-derived embryos; this was confirmed by decreases (p < 0.05) in the expression levels of pro-apoptotic genes, BAX and CASP3, and an increase (p < 0.05) in the expression level of the anti-apoptotic gene, BCL2L1. In addition, LPA significantly increased Oct4 expression at the gene and protein levels. Together, our data suggest that LPA improves the quality and development of porcine SCNT-derived embryos by reducing apoptosis and enhancing cell proliferation and pluripotency.

Keywords: Apoptosis; Cell proliferation; Lysophosphatidic acid; Oct4; Somatic cell nuclear transfer.

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Conflict of interest statement

No potential conflict of interest relevant to this article was reported.

Figures

Fig. 1.
Fig. 1.. Effect of LPA on the development of porcine SCNT-derived embryos.
(A) The influence of different concentrations (0 μM, n=121; 1 μM, n=123; 10 μM, n=121; 20 μM, n=121; 50 μM, n=119) of LPA on porcine SCNT-derived blastocyst formation. 10 μM LPA was used in the next study, because 10 μM LPA is the optimal concentration for porcine SCNT-derived embryo development. (B) Images of porcine SCNT blastocysts after LPA treatment. (C) The development of porcine SCNT-derived embryos cultured with LPA (10 μM). Experiment was conducted at least thrice, and a total of 137 (Control) and 146 (LPA group) embryos were used for the assay, respectively. (D) The expression levels of embryonic development-related genes in SCNT-derived embryos cultured with LPA. Different letters indicate a significant difference (p < 0.05). Scale bar = 200 μm in (B). LPA, lysophosphatidic acid; SCNT, somatic cell nuclear transfer.
Fig. 2.
Fig. 2.. Effect of LPA on the cell proliferation potential in porcine SCNT-derived blastocysts.
(A) Differential staining of porcine SCNT blastocysts. Blue (Hoechst 33342) and pink (Hoechst 33342 plus PI) show inner cell mass (ICM) and trophoblast (TE) cells, respectively. (B) Numbers of total cells (TC), ICM cells, TE cells and the ratio of ICM cells to TE cells in porcine SCNT-derived blastocysts. Experiment was conducted at least thrice, and a total of 19 (Control) and 21 (LPA group) embryos were used for the assay. (C) Porcine SCNT-derived blastocysts were stained with EdU to detect DNA replication (green), and nuclei were stained with DAPI (blue). (D) The numbers of EdU-positive cells and the ratio of EdU-positive cells to TC. Experiment was conducted at least thrice, and a total of 23 (Control) and 24 (LPA group) embryos were used for the assay. Different letters indicate a significant difference (p < 0.05). Scale bar = 200 μm in (A) and 100 μm in (C). LPA, lysophosphatidic acid; SCNT, somatic cell nuclear transfer.
Fig. 3.
Fig. 3.. Effect of LPA on ROS level, mitochondrial membrane potential and apoptosis in porcine SCNT-derived blastocysts.
(A) ROS levels in porcine SCNT-derived embryos exposed to LPA treatment. Experiment was conducted at least thrice, and a total of 41 (Control) and 47 (LPA group) embryos were used for the assay. (B) and (C) Fluorescence pixel ratio (red/green) and representative images of JC-1 staining (red, J-aggregates; green, J-monomers) in porcine SCNT embryos. Experiment was conducted at least thrice, and a total of 18 (Control) and 26 (LPA group) embryos were used for the assay. (D) Images of apoptotic cells in porcine SCNT blastocysts evaluated by TUNEL assays. Red indicates apoptotic cells and blue indicates nuclei. (E) Apoptosis rates (TUNEL-positive cell number/total cell number) in porcine SCNT-derived blastocysts. Experiment was conducted at least thrice, and a total of 21 (Control) and 24 (LPA group) embryos were used for the assay. (F) The expression levels of apoptosis-related genes (BAX, CASP3 and BCL2L1) in SCNT blastocysts. Different letters indicate a significant difference (p < 0.05). Scale bars = 100 μm in (C) and (D). LPA, lysophosphatidic acid; SCNT, somatic cell nuclear transfer; ROS, reactive oxygen species.
Fig. 4.
Fig. 4.. Effect of LPA on the expression levels of pluripotency markers in porcine SCNT-derived blastocysts.
(A) The expression of Oct4 protein in porcine SCNT-derived blastocysts. (B) Quantification of Oct4 expression. Experiment was conducted at least thrice, and a total of 18 (Control) and 22 (LPA group) embryos were used for the assay. (C) The gene expression levels of Oct4, Sox2 and Nanog. Different letters indicate a significant difference (p < 0.05). Scale bar = 100 μm in (A). LPA, lysophosphatidic acid; SCNT, somatic cell nuclear transfer.
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