Study On The Roles Of Deacetylase SIRT6 During Porcine Oocyte Meiotic Maturation

The quality of mammalian oocytes is critical for fertilization and embryo development,which directly impacts the production efficiency of livestock and the sustainable develepment of animal husbandry.The maturation of oocytes undergoes a series of sophisticated and complicated biological events,including germinal vesicle breakdown,spindle assembly,chromosome alignment and polar body extrusion.Multiple molecules coordinate to exert their functions during this process,and any defect may lead to the failure of development.However,the functions of many molecules in the process of oocyte development are not fully clarified.Thus,it is necessary to further investigate these molecules to reveal their roles during oocyte maturation,providing a comprehensive understanding of the regulatory mechanisms of oocyte development,and new ideas for improvement of oocyte quality and livestock reproduction.SIRT6 is a member of the Sirtuins family,the homolog of yeast Sir2,in mammals.It plays important roles in many physiological processes,such as inflammation,aging and cancer.It has been shown that SIRT6 participates in DNA repair and telomere maintenance to delay aging.However,the function of SIRT6 during mammalian oocyte maturation has not been fully determined.Therefore,in this study,we applied porcine oocyte as a research model and used the specific inhibitor of SIRT6 to explore its roles and possible mechanisms in the regulation of oocyte meiotic maturation,which could provide a theoretical basis for improving the in vitro maturation oocytes and the reproductive performance of livestock.The specific experimental content and results are shown as follows:Experiment 1: SIRT6 modulates the spindle assembly to promote porcine oocyte nuclear maturationIn this experiment,we added SIRT6-specific inhibitor during in vitro culture to explore the role of SIRT6 during oocyte meiotic nuclear maturation.The results showed that the expasion level of cumulus cells and the rate of polar body extrusion were significantly reduced in SIRT6-inhibited porcine oocytes.Investigation of related indicators during oocyte nuclear maturation by immunofluorescence and immunoblotting,we revealed that SIRT6 inhibition continuously activated spindle assembly checkpoint(SAC),which led to the oocyte meiotic failure.In addition,SIRT6 inhibition caused a remarkable increase in the abnormal rate of spindle assembly and chromosome alignment,which may be the main reason why SAC is presisently activated at M I stage.We next examined the microtubules dynamics as it is usually related to the spindle assembly.Both immunofluorescence and immunoblotting results validated that SIRT6 inhibition prominently decreased the acetylation level of α-tubulin in porcine oocytes.Altogther,our data indicate that SIRT6 promotes oocyte nuclear maturation by regulating microtuble dynamics and spindle assembly.Experiment 2: SIRT6 regulates the porcine oocyte cytoplasmic maturationOocyte maturation includes nuclear maturation and cytoplasmic maturation.Therefore,in this experiment,we further investigated the related indicators during oocyte cytoplasmic maturation,including the dynamic changes of some cytoskeleton and organelles.Actin is an important component of cytoskeleton,which plays a vital role in maintaining cell structure and cortical polarization.Immunofluorescence and quantitative analysis showed that SIRT6 inhibition led to a significant decrease in the fluorescence intensity of actin with a discontinuous distribution.In addition,the correct distribution of cortical granules is a key indicator of oocyte cytoplasmic maturation.We found that SIRT6 inhibition resulted in the mis-localization of cortical particles with faded signals.Ovastacin,a core component of cortical granules,is responsible for removal of the sperm binding site in the zona pellucida(ZP)to prevent polyspermy.Similarly,ovastacin displayed an abnormal distribution pattern with reduced fluorescence signals,which might impair the fertilization potential of oocytes.In summary,SIRT6 may participate in the cytoplasmic maturation of porcine oocytes by regulating actin cytoskeleton and cortical granules.Experiment 3: SIRT6 participates in the regulation of oxidative stress,DNA damage repair and apoptosis in porcine oocytesTo further explore the regulatory mechanism of SIRT6 during porcine oocyte maturation,we performed transcriptome analysis.The data showed that the transcription levels of genes related to oocyte meiosis,oxidative phosphorylation and cellular senescence had a significant change in SIRT6-inhibited oocytes.This verified our above observations about the meiotic defects caused by SIRT6 inhibition.In addition,the pathways of oxidative phosphorylation and cellular senescence indicate that dysfunction of SIRT6 might induce oxidative stress and apoptosis in porcine oocytes.Therefore,we further assessed the levels of reactive oxygen species(ROS)and DNA damage,as well as the occurrence of apoptosis.The results revealed that the levels of ROS,DNA damage and apoptosis in SIRT6-inhibited oocytes were significantly higher than those in control oocytes.Therfore,these observations indicate that the meiotic defects present in SIRT6-inhibitied oocytes may be caused by the excessive ROS-induced DNA damage and apoptosis.In conclusion,our study demonstrates that SIRT6 is an essential regulatory factor that promotes in vitro maturation of porcine oocytes by maintaining redox homeostasis.