Arginine Regulates Zygotic Genome Activation in Porcine Embryos Under Nutrition Restriction

Abstract

Arginine has a positive effect on pre-implantation development in pigs. However, the exact mechanism by which arginine promotes embryonic development is undefined. Here, single-cell RNA sequencing technology was applied to porcine in vivo pre-implantation embryos from the zygote to morula stage, it was found that that the expression of arginine metabolism-related genes clearly changed from the 2-cell stage to the 4-cell stage, when zygotic genome activation (ZGA) occurs in porcine embryos. Further analysis showed that arginine metabolism-related genes are significantly correlated with key ZGA genes. To determine the function of arginine in porcine embryos during ZGA, the in vitro fertilization embryos were cultured in PZM-3 medium (0.12 mM arginine, Control group), a modified PZM-3 medium (0 mM arginine, Block group) and a modified PZM-3 medium supplemented with arginine (0.12 mM arginine, Block + Arg group). The results showed that the 4-cell arrest rate was significantly increased in the Block group compared to the Control group (P < 0.05). The 4-cell arrest rate in the Block + Arg group was significantly decreased than that in the Block group (P < 0.05). Meanwhile, the expression of ZGA marker genes and SIRT1 protein in 4-cell embryos was significantly decreased in the Block group compared to the Control group, and their expression was significantly increased in the Block + Arg group. In addition, we observed that the glutathione (GSH), ATP levels, and lipid droplet contents were significantly increased, and the reactive oxygen species (ROS) level was decreased in the Block + Arg group compared to the Block group. Compared with Control group, spermine content in culture medium and the mRNA expression of ornithine decarboxylase1 (ODC1) of embryos in the Block group were significantly decreased (P < 0.05), and those in the Block + Arg group were significantly increased compared with the Block group (P < 0.05). Moreover, when difluoromethylornithine (an inhibitor of ODC1) was added to the modified PZM-3 medium supplemented with arginine, the effect of arginine on ZGA was inhibited. In summary, our findings demonstrated that arginine may regulate ZGA under nutrition restriction in porcine embryos by promoting polyamine synthesis.

Keywords: arginine; embryonic development; energy metabolism; oxidative stress; polyamine; porcine; zygotic genome activation.

Figure 1

Gene expression landscape of porcine pre-implantation development. (A) Microscopic imaging of porcine pre-implantation embryos in vivo (from left to right: zygote, 2-cell, 4-cell, 8-cell, and morula). (B) PCA of the transcriptome of single blastomeres from five stages of an in vivo porcine pre-implantation embryo. Dots of the same color represent blastomeres from different embryos at the same developmental stage. (C) Histogram showing upregulated and downregulated genes between two adjacent stages. (D) Results of qRT-PCR analysis of the expression of key zygotic genes. n = 3; different letters (a, b) indicate significant differences (P = 0.05). (E) Results of qRT-PCR analysis of the expression of arginine metabolism-related genes. n = 3; different letters (a, b) indicate significant differences (P = 0.05). (F) Arginine metabolism in porcine pre-implantation embryos. Arginine metabolism-related pathways from the KEGG database are shown in hierarchical clusters at five embryonic developmental stages.

Figure 2

Correlation analysis of arginine metabolism and ZGA. (A) This scatter plot shows the KEGG pathways that were significantly enriched among the differentially upregulated genes between 4-cell and 2-cell stage (3,226). (B) Heatmap of correlation of arginine metabolism-related genes and key ZGA genes. Red represents a positive correlation, and black represents a negative correlation. The closer the R value is to 1, the darker the color. (C) Scatter plot of the correlation between ODC1 gene and EIF1A gene expression. (D) Scatter plot of the correlation between NOS3 gene and EIF1A gene expression.

Figure 3

Effects of arginine on the 4-cell block and ZGA under nutrition restriction. (A) Developmental status of embryos cultured in different media. Embryos cultured in PZM-3 medium as a Control group at (a) 48 h (4-cell) and (d) 72 h (morula). Embryos cultured in mPZM-3 medium as a Block group at (b) 48 h (4-cell) and (e) 72 h (4-cell block). Embryos cultured in mPZM-3 medium supplemented with arginine as the Block + Arg group at (c) 48 h (4-cell) and (f) 72 h (morula). (B) The 4-cell arrest rate at 72 h in the Control, Block and Block + Arg groups. (C) Representative epifluorescence microscopic images of SIRT1 in the Control, Block, and Block + Arg groups. Bar = 50 μm. (D) Expression of ZGA genes measured by qRT-PCR. EIF1A: eukaryotic translation initiation factor 1A; DPPA2: developmental pluripotency associated gene 2; ZSCAN4: zinc finger and SCAN domain containing 4. n =3; different letters (a, b) indicate significant differences (P = 0.05). (E) Results of the statistical analysis of embryonic SIRT1 intensity in the Control, Block, and Block + Arg groups. Control: n = 40; Block: n = 40; Block + Arg: n = 40.

Figure 4

Effects of arginine on the metabolism status of embryos during ZGA under nutrition restriction. (A) The 4-cell embryos were stained with DCFH-DA to measure the intracellular levels of ROS in the Control, Block and Block + Arg groups. Bar = 100 μm. (B) The 4-cell embryos were stained with Cell Tracker Blue to measure the intracellular levels of GSH in the Control, Block and Block + Arg groups. Bar = 100 μm. (C) ATP level of 4-cell embryos stained with BODIPY-ATP in the Control, Block and Block + Arg groups. Bar = 50 μm. (D) Images of 4-cell embryos obtained by epifluorescence microscopy. Lipid droplets were stained with the lipophilic dye BODIPY 493/503 in the Control, Block and Block + Arg groups. Bar = 50 μm. (E–H) Results of the statistical analysis of embryonic ROS, GSH, ATP and lipid droplet intensity in the Control, Block and Block + Arg groups. Control: n = 30; Block: n = 30; Block+Arg: n = 30. Different letters denote significant differences (P < 0.05).

Figure 5

Analysis of the arginine metabolic pathway during ZGA under nutrition restriction. (A) Representative images of DAF-FM fluorescence in the Control, Block and Block + Arg groups. Nitric oxide production viewed by using DAF-FM staining of 4-cell embryos. Bar = 50 μm. (B) Results of the statistical analysis of embryonic NO intensity in the Control, Block and Block + Arg groups. Control: n = 30; Block: n = 30; Block + Arg: n = 30. (C) Expression of the NOS gene measured by qRT-PCR. NOS: nitric oxide synthase. (D) Results of the statistical analysis of spermine content in the culture medium at 48 h after IVC in the Control, Block, Block + Arg and Block + Arg + DFMO groups. Control: n = 4; Block: n = 4; Block+Arg: n = 4; Block + Arg + DFMO: n = 4. (E) Expression of the ODC1 gene measured by qRT-PCR. ODC1: ornithine decarboxylase 1. n =3; different letters (a, b) indicate significant differences (P = 0.05).

Figure 6

The effect of arginine on ZGA via ODC1 under nutrition restriction. (A) Representative fluorescence images of SIRT1 in the Block + Arg and Block + Arg + DFMO groups. Bar = 50 μm. (B) Expression of ZGA genes measured by qRT-PCR in the Block + Arg and Block + Arg + DFMO groups. n =3; different letters (a, b) indicate significant differences (P = 0.05). (C) Results of the statistical analysis of whole-cell SIRT1 intensity in the Block + Arg and Block + Arg + DFMO groups. Block + Arg: n = 30; Block + Arg + DFMO: n = 30. (D) The 4-cell embryos were stained with DCFH-DA to measure the ROS levels in the Block + Arg and Block + Arg + DFMO groups. Bar = 100 μm. (E) Results of the statistical analysis of embryonic ROS intensity in the Block + Arg and Block + Arg + DFMO groups. Block + Arg: n = 30; Block + Arg + DFMO: n = 30. (F) The 4-cell embryos were stained with Cell Tracker Blue to measure the GSH levels in the the Block + Arg and Block + Arg + DFMO groups. Bar = 100 μm. (G) Results of the statistical analysis of embryonic GSH intensity in the Block + Arg and Block + Arg + DFMO groups. Block + Arg: n = 30; Block + Arg + DFMO: n = 30. (H) The 4-cell embryos were stained with BODIPY-ATP to measure the ATP levels in the Block + Arg and Block + Arg + DFMO groups. Bar = 50 μm. (I) Results of the statistical analysis of embryonic ATP intensity in the Block + Arg and Block + Arg + DFMO groups. Block + Arg: n = 30; Block + Arg + DFMO: n = 30. (J) The 4-cell embryos were stained with the lipophilic dye BODIPY 493/503 to measure Lipid droplets in the Block + Arg and Block + Arg + DFMO groups. Bar = 50 μm. (K) Results of the statistical analysis of embryonic Lipid droplets intensity in the Block + Arg and Block + Arg + DFMO groups. Block + Arg: n = 30; Block + Arg + DFMO: n = 30. Different letters denote significant difference (P < 0.05).