Effects Of Salidroside On Porcine Oocyte Maturation In Vitro And Preimplantation Embryonic Development

The in vitro culture system of porcine oocyte maturation and early embryo provides a theoretical basis for basic biological research.Although an in vitro culture system has been successfully established decades ago,the in vitro culture system in porcine species is not yet perfect,and the quality of in vitro embryos is comparable to`that of embryos produced in vivo due to oxidative stress caused by elevated oxygen levels.Moreover,the function of mitochondria in different stages of oocyte and embryo development affects the quality of oocyte and embryo development.A large number of studies have shown that abortion is related to mitochondrial dysfunction in blastocysts.Salidroside（SAL）has a molecular formula of C₁₄H₂₀O₇,has multiple hydroxyl groups,and mainly exists in the roots of Rhodiola rosea.It has many biological and pharmacological properties,such as anti-cancer,anti-oxidation,anti-aging,anti-lipid metabolism disorder,and has a protective effect on mitochondria,but there is no research report on the mechanism of SAL’s influence on oocyte and early embryo development.Therefore,in order to improve the culture system of porcine oocytes and in vitro embryos,this study explored the mechanism of the effect of adding SAL in vitro on the development of porcine oocytes and early embryos.Experiment 1:Supplement SAL in porcine oocyte maturation medium,and analyze the mechanism of its influence on porcine oocyte maturation in vitro.Mature MII stage oocytes were obtained after 44 h of SAL-treated dosing culture during in vitro maturation（IVM）culture,and the following experiments were performed.The experimental results showed that compared with the control group,the SAL group increased the oocyte maturation rate,and the blastocyst rate of parthenogenetic embryos in the SAL treatment group increased from 38.89%±5.38%to 52.14%±7.32%（P<0.05）.The m RNA expression and MAPK phosphorylation level of C-MOS,mitogen-activated protein kinase（MEK）and extracellular signal-regulated kinase 1/2（ERK1/2）in the MAPK pathway of the SAL treatment group increased significantly,and oocytes decreased significantly.The m RNA expression of mitosis genes（CDK1,Cyclin B and GDP-1）also increased significantly.And after the oocytes were treated with SAL,the expression of apoptosis-related genes（Caspase-3,BAX）decreased,the expression of anti-apoptotic genes（BCL-2）increased,and the expression ofγH2AX protein decreased significantly,indicating that SAL promoted Oocyte nuclei mature.In addition,SAL decreased the level of reactive oxygen species（ROS）in oocytes,increased the level of GSH in oocytes,and increased the m RNA expression of oxidative stress-related genes SOD,CAT,and GPX.After adding SAL,mitochondrial membrane potential（MMP）,ATP level,relative m RNA expression of mitochondria-related genes（PGC-1,TFAM）and mitochondrial DNA copy number were all increased.In order to further analyze the protective effect of SAL on oocytes,it was found that SAL significantly up-regulated the expression of AMPK gene.And because cumulus cells are closely related to oocyte maturation,the experimental results showed that SAL significantly increased the diffusion area of cumulus cells,decreased the ROS level of cumulus cells and increased the mitochondrial copy number,indicating that SAL promoted oocyte cytoplasmic maturation.After parthenogenetic activation（PA）and nuclear transfer（SCNT）blastocysts,not only the rate of blastocysts increased significantly,but also the number of blastocyst cells increased significantly.For parthenogenetic embryos,the expression levels of COX2and pluripotency genes（OCT4,NANOG,SOX2,and CDX2）related to blastocyst formation were increased in the SAL treatment group.EDU staining of blastocysts showed that the number of proliferating cells increased,and TUNEL staining showed that apoptosis Decreased number of dead cells.Experiment 2:Adding SAL during in vitro development（IVC）of early pig embryos,and analyzing the effect on early embryos.The results showed that the addition of SAL during in vitro culture enhanced the developmental ability of PA and SCNT in porcine embryos.Compared with the control group,the blastocyst rate of parthenogenetic embryos in the SAL group increased from 27.02%±7.13%to 47.19±7.76%（P<0.01）,and the blastocyst rate of nuclear transfer embryos increased from 24.66%±1.51%to 31.95%±2.15%（P<0.05）.In addition,SAL decreased embryo ROS levels,increased intraembryonic GSH levels,and slowed hydrogen peroxide-induced ROS levels.In addition,adding SAL can increase MMP,ATP levels and the relative m RNA expression of mitochondria-related genes（PGC-1,TFAM）at the 4-cell stage;SAL can significantly improve the gene and protein expression of mitochondria-based apoptosis and oxidative stress pathways（BAX,BCL-2,Nrf2,HO-1）,totipotent genes（OCT4,SOX2）,the expression of blastocyst generation-related gene COX2,and the proliferation of blastocyst cells increased,while the expression of apoptosis gene Caspase9 increased.The expression level ofγH2AX protein decreased significantly.SAL significantly increased the cell number of nuclear transfer blastocysts and the expression of blastocyst-related gene COX2 and down-regulated the expression of apoptosis gene Caspase9.These results indicated that SAL improved the nucleoplasmic maturation of oocytes and the developmental ability of subsequent embryos by regulating mitochondria,MAPK phosphorylation,and oxidative stress during in vitro maturation of porcine oocytes.And the addition of SAL in IVC had a beneficial effect on the development of early pig embryos,and the use of SAL proposed a new optimization scheme for in vitro embryo production.