The Sirtuin Family Genes Are Involved In Follicular Development And Its Related Molecular Mechanisms

BackgroundThe follicle is the basic structural and functional unit of the ovary.When a woman is born,a large number of dormant primordial follicles are stored in both ovaries.After entering adolescence,99%of the primordial follicles goes to atresia.Only less than 1%of the primordial follicles are stimulated by the mature hormone levels in the body and gradually develop.The follicular size grows bigger,and the follicular cells also proliferate from a single layer of flattened granulosa cells into multi-layered columnar shaped cells.Then they differentiate into functional outer theca cells and inner granulosa cells.With the growth and development of the follicles,the primary oocytes undergo meiosis,and the follicular cavity gradually forms.The primordial follicles grow successively into primary follicles,secondary follicles,Graafian follicles,mature follicles and finally ovulation happens.As women aging,the primordial follicle pool stored in the ovaries gradually shrinks.Primary ovarian insufficiency(POI)occurs when the primary follicles in the ovaries are depleted prematurely.Primary ovarian insufficiency refers to amenorrhea and less or no ovulation rate in women before the age of 40,accompanied by increased FSH and reduced estrogen level,which eventually leads to fertility problems.Therefore,maintaining primordial follicular pool is important for extending female reproductive life.Primordial follicle activation lays down female reproductive functions and is regulated by a variety of cellular signaling pathways,such as the Hippo pathway,PI3K-PTEN-AKT-FOXO3 pathway,etc.,which have not been clarified so far.Sirtuin,is a highly conservative class of NAD+-dependent deacetylases with a total of 7 subtypes,widely expressed in tissues and organs,involved in the regulatory processes of physiological events including cell aging,metabolism,apoptosis,cell cycle,cell survival and genomic stability.The Sirtuin family has seven members which differs in their subcellular localization and protein structural specificity.Among them,Sirtuinl,Sirtuin2,Sirtuin6,and Sirtuin7 are mainly expressed in the nucleus,while Sirtuin3,Sirtuin4,and Sirtuin5 are expressed in the mitochondrion.Studies have shown that the Sirtuin family is closely related to ovarian follicular development and oocyte maturation in the female reproductive system.In the obese rat model,Sirtuin1 can improve the loss of primordial follicles and follicular atresia induced by a high-calorie diet,while over-expression of Sirtuinl in a mouse model can mimic the primordial follicles protection induced by a calorie restriction diet.Therefore,Sirtuin1 may be involved in maintaining ovarian reservation as well as primordial follicle pool.In view of this,this study took postnatal mouse ovaries as the research object to explore the expression and localization of sirtuin family genes in follicles at various developmental stages.The regulatory role and molecular mechanism of Sirtuinl in early follicular development were also emphasized.Objective:Explore the differential expression patterns and subcellular localization of the seven isoforms of Sirtuin genes during ovarian follicular development;clarify the effects of Sirtuinl on early follicular development;study the effects and mechanisms of Sirtuinl on granulosa cells proliferation and apoptosis;explore the regulation of Sirtuinl on mitochondrial function of granulosa cells.Methods:In this study,the relative expression levels of Sirtuin family isoforms in ovaries at different developmental stages in neonatal mice at different days were clarified by using real time-PCR;IHC technology was used to detect subcellular localization and expression pattern of Sirtuinl,Sirtuin4,and Sirtuin6 in mouse follicles at all stages.Sirtuinl adenovirus overexpression system and HE staining were used to verify the effect of Sirtuinl on early follicular development.Sirtuinl adenovirus overexpression system and Sirtuinl silencing by siRNA transfection were used to study the effect of Sirtuinl on granulosa cell proliferation and apoptosis.Real-time PCR and Western Blot were used to investigate the potential mechanism of Sirtuinl on mitochondrial function of granulosa cells.Results:1.The seven isoforms of Sirtuin gene are differently expressed during ovarian follicular development.The mRNA expression of Sirtuinl,Sirtuin4 and Sirtuin6 gradually decreased with follicular development.Sirtuin5 expression first increased and then decreased,reaching a peak in the secondary follicular phase.Compared with each subtype:Sirtuin2 is expressed at a higher level in all developing stages of ovarian follicles;Sirtuin7 maintains a high level of expression in all follicular development stages other than the primordial follicle stage;Sirtuin6 is highly expressed only in the primordial follicle stage.2.The results of immunohistochemistry showed that Sirtuinl was concentratedly expressed in the cytoplasm and nucleus of granulosa cells in early follicles,but hardly expressed in oocytes and ovarian stroma.Sirtuin4 and Sirtuin6 are expressed in the cytoplasm of granulosa cells and oocytes,and are strongly expressed in the corpus luteum.3.The ovaries of different ages were cultured by the in vitro culture system of mouse ovaries.Ovaries were infected by adenovirus to over-express Sirtuin1.The results showed that the over-expression of Sirtuinl inhibited the transformation process from primordial follicles to primary follicles,as well as from primary follicles to secondary follicles.This suggests that Sirtuinl retards early follicular development.4.Interference with the expression level of Sirtuinl significantly affects the proliferation of granulosa cells,which may be related to the change in the expression level of CCN2.5.Interference with the expression level of Sirtuinl significantly affects apoptosis of granulosa cells and KGN cells,which may be related to the change in the relative expression level of AMPK/PI3K/Aktl/caspase.6.The change of Sirtuinl expression level in KGN affected the expression of Mfn2(mitochondrial fusion protein 2)and mitochondrial autophagy-related genes Atg5,Beclinl,and LC3B,suggesting that Sirtuinl may affect follicular development by affecting mitochondrial function of granulosa cells.Conclusion:Sirtuin family genes have different expression patterns duing follicular development,and the expression levels of Sirtuinl,Sirtuin4 and Sirtuin6 gradually decrease with the development of ovarian follicles.Overexpression of Sirtuinl delayed the transition from primordial follicles to primary follicles,as well as from primary follicles to secondary follicles,suggesting that Sirtuinl may be involved in the regulation of early follicular development.The regulation of Sirtuinl on early follicular development may be related to the effect of Sirtuinl on granulosa cell proliferation,and the interference of Sirtuinl expression level changes the expression of mitochondrial fusion protein and autophagy-related protein,suggesting that the effect of Sirtuinl on follicular development may be realized by affecting the mitochondrial function of granulosa cell.