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. 2022 Oct 12;27(20):6830.
doi: 10.3390/molecules27206830.

Supplementation of SDF1 during Pig Oocyte In Vitro Maturation Improves Subsequent Embryo Development

Huaxing Zhao  1   2   3 , Yazheng Dong  1   2   3 , Yuxing Zhang  1   2   3 , Xiao Wu  1   2   3 , Xianjun Zhang  1   2   3 , Yalin Liang  1   2   3 , Yanan Li  1   2   3 , Fang Zeng  4 , Junsong Shi  2   5 , Rong Zhou  5 , Linjun Hong  1   2   3 , Gengyuan Cai  1   2   3 , Zhenfang Wu  1   2   3   6 , Zicong Li  1   2   3   6
Affiliations

Supplementation of SDF1 during Pig Oocyte In Vitro Maturation Improves Subsequent Embryo Development

Huaxing Zhao et al. Molecules. .

Abstract

The quality of in vitro matured oocytes is inferior to that of in vivo matured oocytes, which translates to low developmental capacity of embryos derived from in vitro matured oocytes. The developmental potential of in vitro matured oocytes is usually impaired due to oxidative stress. Stromal cell-derived factor-l (SDF1) can reduce oxidative stress and inhibit apoptosis. The aim of this study was to investigate the effects of SDF1 supplementation during pig oocyte in vitro maturation (IVM) on subsequent embryo development, and to explore the acting mechanisms of SDF1 in pig oocytes. We found that the IVM medium containing 20 ng/mL SDF1 improved the maturation rate of pig oocytes, as well as the cleavage rate and blastocyst rate of embryos generated by somatic cell nuclear transfer, in vitro fertilization, and parthenogenesis. Supplementation of 20 ng/mL SDF1 during IVM decreased the ROS level, increased the mitochondrial membrane potential, and altered the expression of apoptosis-related genes in the pig oocytes. The porcine oocyte transcriptomic data showed that SDF1 addition during IVM altered the expression of genes enriched in the purine metabolism and TNF signaling pathways. SDF1 supplementation during pig oocyte IVM also upregulated the mRNA and protein levels of YY1 and TET1, two critical factors for oocyte development. In conclusion, supplementation of SDF1 during pig oocyte IVM reduces oxidative stress, changes expression of genes involved in regulating apoptosis and oocyte growth, and enhances the ability of in vitro matured pig oocytes to support subsequent embryo development. Our findings provide a theoretical basis and a new method for improving the developmental potential of pig in vitro matured oocytes.

Keywords: IVM; SDF1; embryo development; oocyte quality; pig.

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Conflict of interest statement

All authors declare they have no conflict of interest that could inappropriately influence or be perceived to influence the submitted work.

Figures

Figure 1
Figure 1
The SDF1 concentration in porcine oocyte IVM medium and follicular fluid. Values are presented as mean ± SD. Values labeled with different letters differ at p < 0.05.
Figure 2
Figure 2
Effects of supplementation of SDF1 during pig oocyte IVM on the total cell number per blastocyst of SCNT (A), PA (B), and IVF (C) embryos. “*” represents p < 0.05.
Figure 3
Figure 3
SDF1 supplementation alleviates oxidative stress in the porcine oocytes during IVM. (A) Representative images of the control and SDF1-treated oocytes incubated with H2DCFDA showing ROS production. (B) Representative images of the control and SDF1-treated oocytes incubated with JC-1 for ∆Ψm measurements. (C) ROS levels in the indicated groups were calculated in terms of the intensity of green fluorescence. (D) ∆Ψm in the indicated groups was calculated as the ratio of red/green fluorescence intensity. Values are presented as mean ± SD. “*” represents p < 0.05.
Figure 4
Figure 4
Expression of apoptosis and oxidative stress-related genes in control and SDF1-treated pig oocytes. (A) Relative mRNA expression levels of apoptosis and oxidative stress-related genes in porcine oocytes. BAX (B) and BCL2 (D) protein levels in pig oocytes. Quantification of BAX (C) and BCL2 (E) proteins in pig oocytes. “*” represents p < 0.05.
Figure 5
Figure 5
(A) Principal component analysis (PCA) of sequenced pig matured oocytes in vitro samples from control and 20 ng/mL SDF1-treated groups and (B) volcano plot of differentially expressed genes between two groups.
Figure 6
Figure 6
Verification of randomly selected RNA-seq-identified DEGs by qPCR.
Figure 7
Figure 7
Functional enrichment analysis of DEGs between control and 20 ng/mL SDF1-treated groups. (A) The GO term enrichment analysis of DEGs. Different colors of nodes refer to the functional annotation of ontologies, the gray fonts represent non-significant terms, and the red fonts represent DEGs in significantly enriched GO terms. (B) The up-regulated and down-regulated DEGs in significantly enriched GO terms. (C) The KEGG term enrichment analysis of DEGs.
Figure 8
Figure 8
Comparison of TET1 and YY1 protein levels between the 20 ng/mL SDF1-treated and control porcine oocyte. Detection of TET1 (A) and YY1 (C) proteins in matured oocytes by immunofluorescence staining. Quantification of TET1 (B) and YY1 (D) proteins measured by immunofluorescence staining. (E) Detection of TET1 and YY1 proteins in matured oocytes by western blotting. (F) Quantification of TET1 and YY1 proteins measured by western blotting. “*” represents p < 0.05.
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