Effect Of Mogroside Ⅴ In Modulating Oxidative Stress In Porcine Oocyte

It has been reported that numerous factors,such as environmental pollution,malnutrition,aging and others contribute to the accumulation of reactive oxygen species（ROS）in oocytes,thereby resulting in the oxidative stress-induced deterioration of oocyte quality and female fertility.Therefore,understanding oxidative stress effectively and developing useful strategies to improve the livestock antioxidant capacities,will refine in vitro maturation（IⅤM）system,further elevate the rate of oocyte utilization,and then give guiding significance for the improvement of reproductive efficiency and the increase of livestock farm revenue.Mogroside Ⅴ（MⅤ）,one of the main mogrosides isolated from the fruits of Siraitia grosvenorii,has been shown to exert antioxidant capacity.However,the exact roles of MⅤ in modulating oxidative stress in oocytes and in alleviating female infertility remain yet to be elucidated.In this study,we used porcine oocytes as the research object,and performed IⅤM,immunofluorescence,single-cell transcriptome sequencing and other techniques to investigate the effects and potential mechanism of MⅤ in oxidative stress-induced oocyte meiosis.The detail research contents and results are as follows:1.MⅤ rescued impaired maturation of porcine oocytes induced by hydrogen peroxide treatment.We first explored the effects of different concentrations of hydrogen peroxide（H₂O₂）treatment and MⅤ supplementation on oocyte maturation.We used the first polar body excretion rate as a marker of oocyte maturation.Our results showed that the limited cumulus expansion and significantly reduced maturation rate of oocytes after 200μM H₂O₂ treatment,and 25μM MⅤ supplementation ameliorated these phenomena.We further found that MⅤ supplementation promoted the oocyte cytoplasmic maturation through preventing cortical granules from the aberrant distribution,and drove the nuclear maturation by maintaining the cytoskeleton structure.Overall,our experiments suggested that MⅤ supplementation could dramatically improve the oocyte maturation disorder induced by oxidative stress.2.Identification of target effector molecules of oxidative stress in porcine oocytesTo further explore the exact effects of H₂O₂ treatment on porcine oocyte maturation,we performed single-cell transcriptome sequencing of control and H₂O₂-treated MetaphaseⅡ（MⅡ）stage oocytes to putative downstream effectors.Our heatmap and volcano plot data results founded that H₂O₂-treated oocytes produced 200 differentially expressed genes（DEGs）compared to controls,including 93 down-regulated and 107 up-regulated.The Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis suggested that DEGs were significantly enriched in pathways involved in oocyte meiosis and oxidative phosphorylation.Meanwhile,our Gene Ontology（GO）analysis data showed that H₂O₂ treatment in oocytes resulted in significantly abnormal expression of genes related to mitochondrial,respiratory chain and cell cycle regulation.3.MⅤ alleviated the impairment of oocyte mitochondrial dysfunction induced by oxidative stress.The enrichment of DEGs in the oxidative phosphorylation pathway in transcriptome data suggested that oxidative stress and MⅤ may mainly affect oocyte maturation by affecting mitochondrial function.Thus,we evaluated the effects of MⅤ supplementation on the mitochondrial distribution pattern and membrane potential in H₂O₂-treated oocytes,revealing that MⅤ supplementation reduced the excessive ROS induced by the mitochondrial dysfunction,and consequently suppressed the early apoptosis.In conclusion,our study demonstrated that MⅤ supplementation is an effective approach to ameliorate the oxidative stress-induced meiotic defects via recovering the mitochondrial integrity in porcine oocytes.