Roles Of SIRTUINS In Mammalian Oocyte Meiosis

As everyone knows,there is a much delay of child-bearing age with the increased phenomenon of delayed marriage.Increased age of female is negatively related to fertility and oocyte quality.So how to improve the female oocyte quality especially the elder ones is a big challenge for all researchers.Along with the improvement of living standard,the prevalence of obesity is becoming more and more serious.It has caused obesity related medical problems such as the effect of obesity on human reproduction.There has been plenty of evidence that female has a harmful effect on maternal fertility and pregnancy.Obese women take longer to conceive,even if cycling regularly,and have a high risk of miscarriage and defect of offspring.It is of great significance to reduce the effect of obesity on fertility.Sirtuins protein are a group of NAD+ dependent histone deacetylase,including homology Sirtl-Sirt7 in mammalian were originally found in yeast.The family is highly conserved from bacteria to complex eukaryotes.Studies have confirmed that Sirtuin has a wide range of physiological functions and plays an important role in the regulation of aging and longevity.In this study,we used the female aging mice as a model to elaborate and improve the elderly female egg quality from the molecular level;We also used HFD mice as a model to study the mechanism of Sirt3 in maternal obesity in oocyte and provided a new idea for sloving the increasingly serious problem of obesity.In addition,there is little report about Sirtuin on porcine oocyte maturation.In the study we used Sirtuin specific inhibitor NAM and Sirtinol to study Sirtuin effect in porcine oocyte meiosis.Experiment 1.Sirt2 functions in spindle organization and chromosomealignment in mouse oocyte meiosis.Micro injection of Sirt2 morpholino(Sirt2 morpholino Sirt2-MO)and Sirt2mRNA,western blot,immunofluorescence staining were used in this experiment to clarify the role of Sirt2 in mouse meiotic division.Here,by confocal scanning,our results showed that Sirt2 is distributing uniformly in the cytoplasm of the oocyte during GV;Sirt2 gradually gathers in the spindle region with the process of meiosis,suggesting that Sirt2 may be related to the formation or assembly of spindle.In mouse oocyte,specific knockout of Sirt2 will affect the oocytes first polar body(PB1)emission(p<0.05);we find that specific depletion of Sirt2 in mouse oocytes results in spindle defects and chromosome disorganization(p<0.05),with impaired microtubule-kinetochore interaction.Moreover,knockdown and overexpression experiments reveal that Sirt2 modulates the acetylation status of histone H4K16 and a-tubulin in oocytes,which may in part mediate the defective phenotypes described above by influencing microtubule dynamics and kinetochore function.Finally we find lower Sirt2 protein level in oocytes from aged mice by immunoblotting and that maternal age-associated meiotic defects can be ameliorated through overexpression of Sirt2(p<0.05),providing support for the hypothesis that decreased Sirt2 is one of a number of factors contributing to oocyte age dependent deficits.In summary,our data indicate a role for Sirt2 during oocyte meiosis and uncover a striking beneficial effect of increased Sirt2 expression on aged oocytes.Experiment 2.Sirt3 prevents maternal obesity associated oxidative stress and meiotic defects in mouse oocytes.Maternal obese environment has been reported to induce oxidative stress and meiotic defects in oocytes.However the underlying molecular mechanism remains unclear.Here,using mice fed a high fat diet(HFD)as an obesity model,we first detected enhanced reactive oxygen species(ROS)content and reduced Sirt3 expression in HFD oocytes.We further observed that specific depletion of Sirt3 in control oocytes elevates ROS levels while Sirt3 overexpression attenuates ROS production in HFD oocytes,with significant suppression of spindle disorganization and chromosome misalignment phenotypes that have been reported in the obesity model.Candidate screening revealed that the acetylation status of lysine 68 on superoxide dismutase(SOD2K68)is dependent on Sirt3 in oocytes,and acetylation-mimetic mutant SOD2K68Q results in almost threefold increase in intracellular ROS.Moreover,we found that acetylation levels of SOD2K68 are increased by～80%in HFD oocytes and importantly,that the non-acetylatable-mimetic mutant SOD2K68R is capable of partially rescuing their deficient phenotypes.Together,our data identify Sirt3 as an important player in modulating ROS homeostasis during oocyte development,and indicate that Sirt3-dependent deacetylation of SOD2 plays a protective role against oxidative stress and meiotic defects in oocytes under maternal obese conditions.Experiment 3.Sirtuin inhibition adversely affects porcine oocyte meiosis.Here,we employed Sirtuin inhibitors,nicotinamide(NAM)and Sirtinol,to investigate their effects on porcine oocyte maturation.The rate of meiotic maturation of oocytes decreased after treatment with NAM and Sirtinol and was accompanied by a failure of cumulus expansion.It suggests that Sirtuin is involved in porcine oocyte meiosis.Moreover,NAM and sirtinol treatment disrupted oocyte polarity and inhibited the formation of the actin cap and cortical granule-free domain,and induced spindle abnormalities and chromosome misalignment.These results suggest that Sirtuins are involved in establishing oocyte polarity and organizing meiotic structures during porcine oocyte maturation.