| BackgroundPOI(premature ovarian insufficiency.POI)is a term to define the women younger than 40 years of age who present with amenorrhea lasting more than 4 months and hypoestrogenic-hypergonadotropic serum profile(follicle stimulating hormone(FSH)levels≥40mIU/mL on two occasions).This disorder is characterized by anovulation,amenorrhea,sex steroid deficiency,and infertility.The most common presentation is secondary amenorr-hea and the main consequences are infertility and psychological stress.Most of the women with this disorder do not have menstrual history,specific of POI development,but infertility associated with the diagnosis is the most problematic aspect of the disease.However,POI is an important cause of secondary amenorrhea and infertility,some patients may continue to ovulate and conceive.The disease affects 2-5%of general population.Follicle depletion and follicle dysfunction are two main etiological mechanisms.Follicle depletion can be the consequence of a primitive reduced pool of oocytes or an accelerated follicular atresia.The etiology of the disease varies in different patients:chromosomal/genetic abnormalities,metabolic/enzymatic factors,autoimmunity,infections,environmental toxins,and iatrogenic influences involve in development of the disease.Nevertheless,the precise cause is undetermined in a significant portion of patients.These cases were classified as idiopathic.Molecular basis of ovarian folliculogensis and etiopathogenesis of POI,a common cause of infertility in women,characterized by amenorrhea,hypoestrogenism,and elevated gonadotropin levels in women under the age of 40 are not fully understood.The reproductive lifespan of women and most other mammals is ultimately dependent on the size of the ovarian primordial follicle pool,which is generally established during embryonic development or after birth.To produce mature oocytes for fertilization,primordial follicles are recruited from the reservoir of dormant follicles in the growing follicle pool through a process termed follicular activation,and subsequently undergo a series of development steps.During this entire process,a large number of follicles undergo apoptotic death(atresia)if not selected for further growth.Menopause or ovarian failure occurs when the primordial follicle pool is exhausted.POI,also known as premature menopause,is characterized by cessation of ovarian function,amenorrhea,hypoestrogenism,and elevated gonadotropin levels in women under 40 years of age,affecting 1-5%and 0.1%of women under the ages of 40 and 30,respectively.A disastrous consequence of POI is loss of fertility,which is due to the absence of follicles in most cases,and in others,the inability of the remaining follicles to respond to hormonal stimulation.Nevertheless,the detailed etiology of POI remains to be established,and the factors and mechanisms involved in regulating the processes of primordial follicle activation,follicle growth,and follicle survival or atresia are not completely understood at present.Mechanistic taget of rapamycin(mTOR)is a highly conserved Ser/Thr protein kinase that forms two distinct functional complexes termed mTOR complexl(mTORC1)and mTORC2.mTORC1,essentially comprising mTOR,mLST8,and rapamycin-sensitive adaptor protein of mTOR(Raptor),regulates cell growth,proliferation and metabolism.Activation of mTORC1 promotes the phosphoryltion of two downstream targets,p70 ribosomal S6 kinase-1(S6K1)and eukaryotic initiation factor 4E binding protein-1(4E-BP1),stimulationg ribosome biogenesis and protein synthesis.However,mTORC2 is insensitive to rapamycin.The essential core of mTORC2 comprises mTOR,mSIN1,mLST8,and the rapamycin-insensitive subunit,Rictor.mTORC2 regulats the activity of Akt and PKC and controls cell survival and cytoskeletal organization.Incontrast to mTORC1,or which many upstream signals and cellular functions have been defined,relatively little is known about mTORC2 biology.PI3K signaling is significant in folliculogenesis processes,including follicle activation,development,and survival.As a downstream target of PI3K,mTORC1 is involved in the regulation of folliculogenesis.Stimulation of mTORC1 leads to over activation of the entire pool of primordial follicles,causing POI and infertility.However,the roles of Rictor/mTORC2 in folliculogenesis are currently unknown.Mechanistic target of rapamycin complex2(mTORC2)is emerging as a cental regulator of cell metabolism,proliferation,and survival.However,it’s role in folliculogenesis and POI has not been reported.Here,we showed that the signaling activity of mTORC2 is inhibited in a 4-vinylcyclohexene diepoxide(VCD)-induced POI mouse model.Notably,mice with oocyte-specific ablation of Rictor,a key component of mRORC2,demonstrated POI phenotypes,including massive follicular death,excessive loss of functional ovarian follicles,abnormal gonadal hormone secretion,and consequently,secondary subfertility in conditional knock-out(cKO)mice.Furthermore,reduced levels of Ser-473-phosphorylated Akt and Ser-253-phosphorylated Foxo3a and elevated pro-apoptotic proteins,Bad,Bax,and cleaved poly ADP-ribose polymerase(PARP),were observed in cKO mice,replicating the signaling alterations in 4-VCD-treated ovaries.These results indicate a critical role of the Rictor/mTORC2/Akt/Foxo3a pro-survival signaling axis in folliculogenesis.Interestingly,loss of maternal Rictor did not cause obvious developmental defects in embryos or placentas from cKO mice.suggesting that maternal Rictor is dispensable for preimplantation embryonic development.Our results collectively indicate key roles of Rictor/mTORC2 in folliculogenesis,follicle survival,and female fertility and support the utility of oocyte-specific Rictor knock-out mice as a novel model for POI.In this study,we demonstrated that Rictor activity and downstream mTORC2 signaling are suppressed in ovaries treated with the ovotoxicant,4-VCD,an occupational chemical shown to cause POI in human and animal models by accelerating the apoptotic process of atresia.Additionally,induction of oocyte-specific ablation of Rictor with the Cre-loxp system suing Zp3-Cre and Rictor loxp/loxp mice resulted in POI phenotypes.Our findings support acritical role of Rictor/mTORC2 infollicle survival and oogenesis.Materials and methods1.We constructed the mice with oocyte-specific deletion of Rictor by the application of Cre-loxP system.In order to know the genotype of mice by using PCR and agarose gel electrophoresis.In order to identify the knockdown effect by Western blot and immunohistochemistry.2.Analysis of effect of Rictor/mTORC2 on the number of mice follicle and follicle classification and follicle morphology and structure by HE staining,immunohistochemistry and immunofluorescence.3.Detection of follicle apoptosis by TUNEL assay in Rictor/mTORC2 of mice.4.Detection of the effect of Rictor/mTORC2 on the changes of serum hormone in mice by blood biochemical analysis.5.Detection of the effect of Rictor/mTORC2 on the development of embryo by the observation the number of and the status of the pups per litter,and on the morphology and structure,spongiotrophoblastlayer and labyrinthlayer of placenta by HE staining,analysis the placental lactogen-1 by immune-histochemistry.6.Detection of the effect and mechanism of Rictor/mTORC2 on the follicle cell and POI by immunofluorescence technique,Western blot technique,Pulldown and immunoprecipitation.Results1.4-VCD Destroys Early-stage Follicles and Suppress the Rictor/mTORC2 Signaling PathwayTo assess the role of mTORC2 in POI the activity of Rictor,a central component of the mTORC2 signaling pathway,was finely monitored in 4-VCD-treated ovaries.As expected,15 days of VCD treatment reduced the number of primordial follicles by 45%and primary follicles by 55%that of control mice,respectively,with a weak toxic effect on follicles of higher grade.Interestingly,the level of phosphor-Rictor(p-Rictor,Thr-1135),an inhibitory modifier of Rictor,was up-regulated in VCD-treated ovaries.Rictor is a core component of mTORC2 that promotes cell survival through phosphorylating and activating Akt in various contexts,inturn,phosphorylating and inhibiting Foxo3a,a pro-apoptotic transcription factor.As expected,expression of p-Akt(Ser-473)and p-Foxo3a(Ser-253)was reduced in VCD-treated ovaries.Simulataneously,the levels of Bad,Bax,and cleaved PARP,three typical pro-apoptotic proteins,were elevated.Serum-and glucocorticoid-regulated kinase 1(SGKI),another mTORC2 substrate,was not affected because the phosphorylation of N-Myc downstream-regulated 1(NDRG1)at Thr-346,are adout for SGK1 activity,was comparable between the control and VCD-treated mice.In addition,we observed an increase in Thr-389-phosphorylated S6K1.These results provide in vivo evidence supporting the previous finding that S6K1 mediates direct phosphorylation and inhibition Rictor at Thr-1135,acting as a novel mTORC1-dependent inhibitory feedback loop on mTORC2.Thus,phosphorylation and inactivation of Rictor appear to be significantly involved in VCD-induced follicular apoptosis.2.Oocyte-specific Deletion of Rictor Replicates the Effect of VCD on OocytesTo identify the role of Rictor/mTORC2 in follicle survival,we generated mice with oocyte-specific deletion of Rictor using the Cre-loxp system.As expected,Rictor expression was observed in ovarian follicles of control littermates,but not those of Cko mice,suggesting complete Cte-mediated recombination of the floxed Rictor gene.A decrease in p-Akt(Ser-473)was evident,attributable to the loss of Rictor.Interetingly,we observed specific reduction of p-S6(Ser-235/Ser-236),a downstream signal/target of mTORC1,particularly in the corpus luteum.This result was in line with the previous finding that mTORC1 is activated by mTORC2 via inhibition of its negative regulator,tuberous sclerosis complex 1/2(TSC 1/2),throught Akt.Next,we monitored the expression of downstream signals/targets of Rictor/mTORC2.Loss of Rictor led to decreased p-Foxo3a(Ser-253)and increased Bad,Bax,and cleaved PARP levels.These changes in signaling molecules were confirmed via Western blot analysis.Thus,oocyte-specific ablation on of Rictor duplicates the effects of VCD on the underlying signaling pathway controlling follicular survival in the ovary.Our results collectively reveal a central role of Rictor in the signaling axis of mTORC2/Akt/Foxo3a/pro-apoptotic proteins for regulation of follicular survival.3.Loss of Rictor in Oocytes Causes Progressively Extensive Cell Apoptosis and Follicle Loss.Next,we focused on the follicle number in cKO mice.cKO mice developed normally but had smaller ovaries than control mice,with a ovary weight decreased to 45%of the control mice at the age of 8 months.Consistent with enhanced expression of pro-apoptotic proteins,significantly fewer healthy follicles and less corpus luteum and more atretic follicles were morphologically observed in the ovaries of cKO mice.Notably,follicles were rarely found in cKO mice at the 8 months.To precisely assess follicle loss caused by Rictor deletion,the number of follicles at each stage of five sections from each ovary was classified and counted.When compared with control mice,we observed 40%loss of healthy follicles and 50%decrease in corpus luteum in 6-month-old cKO mice.In 8-month-old cKO mice,a 67%loss of healthy follicles and a 71%decrease in corpus luteum were observed,suggesting a progressive follicle loss after Rictor deletion in oocytes.Corpus luteum develops from follicular granulose cells after ovulation.Less corpus luteum indicates lower levels of ovulation or availability of a smaller dominant follicle reserve for ovulation,which maybe attributed to excessive follicle loss or arrest of development from small to mature follicles.In this regard,it is reasonable to belive that excessive follicle loss is one of the critical causes because more atretic follicles were detected in cKO mice.To further determine at which follicular stage follicle loss occurs,the number of follicles at each stage was analyzed.Although cKO mice had fewer small follicles(including primordial and primary follicles)than control mice,this difference was not statistically significant.It has been show that ZP3-Cre-mediated gene deletion occurs at the primary and/or later follicular stages,and thus deprivation of Rictor in oocytes did not cause significant primordial follicle loss.Indeed,massive follicle loss in cKO mice occurred at large and mature follicles.Consistently,acceleration of cell apoptosis was observed in large and mature follicles in 8-month-old cKO mice,in line with the result of elevated pro-apoptotic protein levels in cKO mice.These results in dicate that deletion of Rictor in oocytes enhanced cell apoptosis and follicle loss,and thus decreased follicular reserve available for ovulation.4.Abnormal Hormone Secretion in cKO MiceAs a gonad,the ovary is largely regulated by gonadotropin hormones(FSH and LH).and inturn,produces other gonadal hormones(E2 and P4).Accordingly,we assessed the hormone parameters in cKO mice.Both FSH and LH levels in cKO mice were higher,although not to assignificant extent,when compared with those in control mice.Considering the existence of follicular development arrest and deficient ovulation,these changes may represent certain positive feedback responses because FSH is responsible for follicular development and LH is responsible for ovulation.Incontrast,both estradiol and progesterone secretion were significantly decreased in cKO mice,especially progesterone with a reduction of almost 50%that of the control evel.As an important secretory source for estradiol and the exclusive source for progesterone,less functional corpus luteum in cKO mice consequently produced insufficient hormones,partly leading to subfertility in cKO mice.5.Maternal Rictor Is Dispensable for Embryonic DevelopmentBecause recombination mediated by Zp3-Cre specifically occurs in oocytes,it is not only an excellent tool to understand the physiological roles of target genes during folliculogenesis and oogenesis,but also widely used to explore maternal gene effects during embryonic development.Notably,a recent study on mullelic disruption of the Rictor gene revealed that mTORC2 is essential for fetal growth and viability.To further determine whether maternal Rictor is necessary for embryonic development,sexually mature female cKO(Zp3-Cre +,Rictor loxp/loxp;loss of Rictor in ovulated oocytes)and control(Zp3-Cre-,Rictorloxp/loxp;normal ovulated oocytes)mice were crossed with wild-type C57BL/6J males with proven fertility.In this mating scheme,embryos derived from the oocytes of cKO mice were devoid of maternal Rictor,giving rise to embryos with the genotype-/Rictor.However,we failed to observe visible physical or histological differences between embryos of control and cKO mice.Although cKO mice had lower numbers of embryos at each scheduled time point,the total number of embryos within either the control or the cKO group was relatively stable throughout the duration of observation,indicating that maternal Rictor deficiency is acceptable for normal embryonic development.This assumption was supported by the finding that the average mass of embryos at each time point is comparable between control and cKO mice.In addition,histological data revealed no obvious morphological or structural abnormalities in E16 placentas from cKO mice,and placental lactogen 1(PL-1),a marker for trophoblast giant cells,exhibited similar expression and distribution patterns to those of control mice.Although cKO mice were susceptible to delivery of dead pups,no physical visible developmental defects were observed in the pups.However,specific histological differences were evident between the uteri of control and cKO mice,with smaller cavities and fewer secretory glands in cKO mice,which possibly led to abortion or dystocia.These results support theory that paternal Rictor alone is sufficient for embryonic development.Taken together,we showed that the signaling activity of mTORC2 is inhibited in a 4-vinylcyclohexene diepoxide(VCD)-induced POI mouse model.Notably,mice with oocyte-specific ablation of Rictor,a key component of mTORC2,demonstrated POI phenotypes,including massive follicular death,excessive loss of functional ovarian follicles,abnormal gonadal hormone secretion,and consequently,secondary subfertility in conditional knock-out(cKO)mice.Furthermore,reduced levels of Ser-473-phosphorylated Akt and Ser-253-phosphorylated Foxo3a and elevated pro-apoptotic proteins,Bad,Bax.and cleaved poly ADP-ribose polymerase(PARP),were observed in cKO mice,replicating the signaling alterations in 4-VCD-treated ovaries.These results indicate a critical role of the Rictor/mTORC2/Akt/Foxo3a pro-survival signaling axis in folliculogenesis.Interestingly,loss of maternal Rictor did not cause obvious developmental defects in embryos or placentas from cKO mice,suggesting that maternal Rictor is dispensable for preimplantation embryonic development.Our results collectively indicate key roles of Rictor/mTORC2 in folliculogenesis.follicle survival,and female fertility and support the utility of oocyte-specific Rictor knock-out mice as a novel model for POI.Conclusion1.Our result indicate that maternal Rictor is not required for preimplantation embryonic development.2.Rictor/mTORC2 is necessary for the oocytes and follicular development and maturation,down-regulation of Rictor/mTORC2 lead to the POI.3.Disruption of Rictor in oocytes causes early depletion of functional ovarian follicles,excessive follicular atresia,aberrant gonadal hormone secretion,and secondary subfertility in cKO mice,reminiscent progressive of POI phenotypes.4.Rictor/mTORC2 plays a critical role in folliculogenesis,follicle survival,and femal fertility and that it is inactivation in oocytes causes POI.5.The oocyte-specific deletion of Rictor in mice can be induced as a novel model for the study of the POI.Our results collectively indicate key roles of Rictor/mTORC2 in folliculogenesis,follicle survival,and female fertility and support the utility of oocyte-specific Rictor knock-out mice as a novel model for POI. |
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