The Effect And Mechanism Of SIRT2 In Maturation And Aging Of Bovine Oocyte

Sirtuins(SIRT1-7)family,as extremely important factors in cell metabolism and stress response,are increasingly concerned by researchers on their reproductive roles.Previous evidences have shown that SIRT2 plays an important role in somatic cell mitosis,energy metabolism and aging,while there are fewer reports about the link between SIRT2 and oocyte maturation,aging,and the mechanism of SIRT2-mediated action is even less clear.In this study,the bovine oocytes,ovarian cumulus cells and granulosa cells were employed to analyze the localization of SIRT2 in bovine ovary tissue and oocyte subcellular,its expression in the process of egg maturation,embryo development and aging;reveal the effects of SIRT2 on oocyte maturation,aging;determine the roles of SIRT2 on cumulus-oocyte gap junction communication and steroid hormone synthesis,and SIRT2-mediated molecular pathways.This study attempted to systematically clarify the molecular mechanism of SIRT2-mediated bovine oocyte maturation and aging,and might provide scientific basis for improving oocyte quality and delaying oocyte aging.The main results and conclusions of this study are as follows:1.SIRT1-7 were observed in bovine oocytes,and the expression levels of SIRT1 and SIRT2 were the highest than those of other Sirtuins.Except for SIRT7,the m RNA expression level of Sirtuins family was gradually decreased in a time-dependent manner during the aging process of bovine oocytes.Especially after 48 hours of aging in vitro,the m RNA levels of SIRT1,SIRT2 and SIRT5 in bovine oocytes were significantly lower than those of fresh mature oocytes.Further experiment showed that SIRT2 was highly expressed in ovarian granulosa cells,oocytes,cumulus cells,and SIRT2 was located in the cytoplasm and nucleus of bovine oocytes.The expression level of SIRT2 was the highest in the meiosis stage of bovine oocytes,and gradually down-regulated until the blastocyst stage after maturation.These results suggest that SIRT2 may be involved in the maturation and aging of bovine oocytes.2.Treatment with 5 ?M Sir Real2(SIRT2 specific inhibitor)almost completely inhibited the deacetylase activity of SIRT2,without affecting the viability of bovine oocytes in vitro maturation.SIRT2 inactivation significantly inhibited oocyte nuclear maturation and embryonic development,while SIRT1 inactivation had no significant effect on nuclear maturation.Confocal scanning and quantitative analysis revealed that SIRT2 inactivation significantly increased spindle/chromosomal defects in bovine oocytes by the upregulating of acetylated ?-tubulin and H4K16.More importantly,SIRT2 inactivation blocked cytoplasmic maturation through disrupting the redistribution of cortical granules,endoplasmic reticulum and mitochondria during bovine oocyte maturation.Meanwhile,SIRT2 inactivation resulted in increased intracellular ROS,decreased ATP production,and low mitochondrial membrane potential,thereby causing mitochondrial dysfunction.Further analysis showed that SIRT2 inactivation disrupted mitochondrial biosynthesis and dynamics via down-regulating TFAM and Mfn2 expression and up-regulating DRP1 expression.Moreover,SIRT2 inactivation inhibited Fox O3 a nuclear translocation by increasing the acetylation level of Fox O3 a,thereby down-regulating the expression of Fox O3a-dependent antioxidant genes(such as SOD2 and Cat).These findings demonstrate that SIRT2-mediated deacetylase activity is necessary for oocyte nuclear and cytoplasmic maturation.3.Inhibition of SIRT2 activity closed the gap junction communication between the oocyte and cumulus cells during in vitro maturation.SIRT2 inactivation promoted Cx43 phosphorylation,and upregulated the MEK/MEK signaling pathway.Interestingly,SIRT2 inactivation-mediated Cx43 phosphorylation was abolished by MEK1/2 antagonist.Meanwhile,the acetylation level of MEK1/2 was increased by SIRT2 inactivation,indicating that SIRT2 regulates Cx43 phosphorylation through MEK/ERK signals.Functionally,downregulating the MEK/ERK signal partially relieved the closure of gap junction communication caused by SIRT2 inactivation.Further analysis showed that SIRT2 inactivation prevented the localization of Cx43 on the cumulus cell membrane and oocyte zona pellucida by up-regulating Cx43 acetylation levels.These evidences suggest that SIRT2 depends on the down-regulating of phosphorylation and acetylation of Cx43 to promote bovine cumulus-oocyte gap junction communication.4.The inactivation of SIRT2 significantly inhibited the secretion of estradiol and testosterone,while promoted the secretion of progesterone in bovine ovarian granulosa cells.Quantitative analysis of gene expression showed that SIRT2 inactivation significantly down-regulated ESRs and AR expression,and up-regulated PGR expression.SIRT2 inactivation or SIRT2 knock-down significantly inhibited the protein levels of PPAR?,LXR?,LXR?,whereas increased the protein level of PPAR?.Co-immunoprecipitation analysis revealed that SIRT2 knock-down significantly increased acetylated PPAR?,but did not affect acetylated PPAR?,indicating that SIRT2 regulates the expression of PPAR? through its deacetylase pathway.Further studies had shown that PPAR activation or PPAR inactivation could mimic the inhibitory effects of SIRT2 knock-down on LXR? expression and LXR?/RXR dimer formation.Similar to SIRT2-KD interfering with steroid hormone synthesis homeostasis,PPAR? activation or PPAR? inactivation down-regulated estradiol,testosterone synthesis,and up-regulated progesterone synthesis.It was note that LXR? activation recovered the effects of SIRT2 knock-down,PPAR? activation and PPAR? inhibition on the secretion of steroid hormone.Furthermore,LXR? activation could also abolish the effects of SIRT2 knock-down,PPAR? activation and PPAR? inactivation on the down-regulation of St AR,CYP17 and aromatase expression,and the up-regulation of CYP11A1 expression.These results suggest that SIRT2 relies on LXR? to promote the expression of estradiol biosynthesis enzymes,and inhibit the expression of progesterone synthase,thereby mediating the secretion of steroid hormones.5.Inhibition of SIRT2 activity significantly increased the activation rate,cytoplasmic fragmentation,and spindle defects in bovine oocytes,thereby accelerating the aging process of bovine oocytes.SIRT2 inactivate caused to higher ROS levels,abnormal mitochondrial distribution,low ATP production,and loss of mitochondrial membrane potential,thereby increasing oxidative stress and mitochondrial dysfunction in aging oocytes.As the aging of bovine oocytes,SIRT2 expression was also significantly decreased.Meanwhile,down-regulation of SIRT2 increased the autophagy in bovine oocytes.Annexin-V staining revealed that the aging was accompanied with apoptosis,and SIRT2 inhibition increased the apoptosis rate in aged oocytes.Further study showed that SIRT2 inhibition or abnormal up-regulation of autophagy significantly increased caspase-3 activity in aging oocytes,while the down-regulation of autophagy could block the promotion of caspase-3 activity caused by SIRT2 inactivation.The results indicate the down-regulating of SIRT2 is a key mechanism underlying of cellular aging.In summary,SIRT2 directly regulated spindle formation,chromosome alignment,mitochondrial function and antioxidant system through acetylation of target proteins such as ?-tubulin,H4K16,TFAM and Fox O3 a,thereby promoting nuclear and cytoplasmic maturation during the meiotic process of bovine oocytes.Furthermore,SIRT2 stimulated the gap junction communication in cumulus-oocyte complex by regulating the acetylation and phosphorylation of Cx43,and induced estradiol secretion of ovarian granulosa cells through the PPARs/LXR? pathway,thereby participating in cytoplasmic maturation of bovine oocytes.In addition,the down-regulation of SIRT2 caused by aging led to spindle defects,oxidative stress,and mitochondrial dysfunction,thereby accelerating the aging process,and activating autophagy-mediated bovine oocyte death.This study attempted to elucidate the biological functions of SIRT2 in the maturation and aging of bovine oocytes.