The Role Of Autophagy Defect In The Pathogenesis Of Premature Ovarian Insufficiency

Chapter ⅠThe role of autophagy defect in granulosa cell participating in the pathogenesis of premature ovarian insufficiencyBackgroundPremature ovarian insufficiency(POI)refers to the decline of female ovarian function before the age of 40,characterized by menstrual abnormalities(amenorrhoea oligomenorrhea or polymenorrhea)and abnormal hormone profile(high gonadotropin and low estrogen),as well.The diagnosis threshold of POI is FSH>25 U/L,which is helpful for the early identification and intervention of ovarian dysfunction.Not only can POI lead to infertility,but also cause chronic diseases secondarily such as cardiovascular disease,osteoporosis,which is detrimental to both physical and mental health of women.The causes of POI are complicated and highly heterogeneous,including genetic factors,iatrogenic factors,immune abnormalities,environmental factors.Autophagy is a kind of highly conservative self-degradation system in eukaryotic cells,with the use of lysosomal enzyme to degrade substrates such as damaged proteins and senescent organelles,which means a recycle of material and energy.In basal state,autophagy can maintain the cellular homeostasis of the material and energy to remove waste proteins and damaged organelles involved in cell differentiation,tissue development and immune response when cells subjected to various conditions of stress,such as insufficiency in energy,reactive oxygen species(ROS)product,invasion of pathogenic microorganism and genome damage,as a self-protective mechanism,autophagy can promote survival.avoid the occurrence of cell death.Follicle is the basic structure and functional unit of ovary,developmental disorders of ovarian oocyte and senescence of granulosa cell can lead to follicular atresia.Existing studies have shown that induction and regulation of autophagy are closely related to apoptosis of granulosa cells and oocytes,mediating the follicle atresia.Accumulated evidence proved that the precisely regulated autophagy process is plays an important role in the growth and differentiation of oocytes and granulosa cells.Therefore,the maintenance of autophagic homeostasis is vital for ovarian function.ObjectiveIn this study,the mRNA expression profile microarray was performed to screen out the down-regulated autophagy related genes in the granulosa cells of biochemical POI patients,which were verified in independent samples.In addition,in vitro and in vivo experiments were conducted to explore the changes in biological behavior and the mechanism caused by the autophagy defects in granulosa cells.The purpose of this study was to reveal the existence of autophagy defects in the granulosa cells of biochemical POI patients,as well as to clarify the role of autophagy in the pathogenesis of POI diseases through functional experiments.MethodsIn this study,we raised 34 patients with biochemical POI and 36 normal controls who visited the hospital due to fallopian tube abnormality or male factors from Reproductive Hospital Affiliated to Shandong University during September 2013 to September 2016.Granulosa cells were collected from the follicular fluid obtained in procedure of assisted reproductive technology,in order to extract the RNA and protein samples.mRNA expression microarray was performed to analyze the differentially expressed autophagy related genes in granulosa cells of 10 bPOI patients and 10 matched controls.The results of microarray were verified in an amplified number of independent samples by quantitative real-time PCR,meanwhile,the autophagy markers LC3B and p62 were detected at the protein level to evaluate the autophagy level of granulosa cells in POI patients by Western Blot.In vitro functional experiments,we selected two key autophagy related genes authenticated to be silenced by RNAi technology and used autophagy inhibitor Chloroqune to downregulate autophagy in granulosa cells.Following that,proliferation ability was detected in utilization of CCK-8 and EdU assays.The expression of differentiation markers was detected by quantitative real-time PCR and Western Blot detection,combining with detection of synthetic capability of estradiol(E2)to comparing the difference in differentiation between the two groups of granulosa cells.Meanwhile,DNA double-strain break(DSB)was induced in KGN cells by Etoposide(ETO).Then,the expression and clearance of y-H2AX(a marker of DNA damage)were detected by Western Blot.In vivo,inhibition of autophagy was conducted by intraperitoneal injection of chloroquine in mice.Changes in expression of differentiation markers of granulosa cells were detected by Western Blot,and the serum E2 levels were tested as well.After DSB induced by ETO.expression and location of y-H2AX were showed by Western Blot and immunofluorescence.ResultsⅠ.Autophagy defect in granulosa cells of biochemical POI(bPOI)patientsA total of 21 down-regulated autophagy related genes were detected in the granulosa cells of bPOI patients by mRNA microarray,Verification of 12 important autophagy related genes(A TG5、BECN1、SIRT1、TRIM1 3、VMP1、WDR45、PRKAA1、RB1CC1、SQSTM1,WAC、PIK3 C3,WIPI1)selected from the results of the microarray were performed among an expanded number of independent samples,the results showed that 6 of these genes(ATG5、BECNl1,SIRT1、TRIM13、VMP1、WDR450)were confirmed downregulated in granulosa cells of bPOI patients significantly,which was in accordance with the results of microarray.Meanwhile,the detection of autophagy markers by Western Blot showed decline in LC3B-II/LC3B-I and accumulation of p62.which indicated the level of autophagy was downregulated in the granulosa cells of bPOI.The data in this part showed that autophagy deficiency existed in granulosa cells of bPOI patients.Ⅱ.Autophagy defect leads to the disorder of differentiation in granulosa cellsTo explore the functional changes of granulosa cells caused by autophagy defect,\we knocked doxwn 2 key autophagy related genes A TG5 and BECN1 by RNAi,as well as treated KGN cells(a cell line of human ovarian granulosa cell)and mouse granulosa cells in vitro.After the inhibition of autophagy,no apparent changes in cell viability and proliferation were observed.However,following the autophagy inhibition in granulosa cells,the differentiation markers of granulosa cells CYP19A1 and FSHR were downregulated significantly,and the E2 product declined,which indicated a deficiency in differentiation in granulosa cells.We used cycloheximide(CHX)to inhibit synthesis of protein in granulosa cells,and we observed a deficient degradation of the inhibitory transcriptional factor WT1.Furthermore,overexpression of WT1 by adenovirus led to significant decline in both expression of differentiation markers CYP19A1 and FSHR and product ability of E2 in KGN cells.On the contrary,after silencing the expression of WT1 by RNAi,we observed an apparent increase in expression of CYP19A1 and FSHR and a significant elevation in E2 product,which indicated the regulatory role of WT1 in differentiation of granulosa cells.Additionally,after activating autophagy in KGN cells by rapamycin(Rap),the expression of CYP19A1 and FSHR was upregulated and the E2 product elevated,as well as the level of WT1 protein declined,which confirmed that autophagy influenced the differentiation of granulosa cells through the regulation of WT1 degradation.At last.we treated mice by intraperitoneal injection of chloroquine to inhibit autophagy in vivo,and the expression of ovarian granulosa cells specific differentiation markers aromatase and FSHR and serum E2 were profile downregulated,as well as accumulation of WT1 was detected.These results proved autophagy inhibition caused deficiency in differentiation in granulosa cells in vivo.Ⅲ,Autophagy defect affects DNA damage repair in granulosa cellsTo observe the effect of autophagy defect on DNA damage repair in granulosa cells.in vitro,after treatment of Etoposide(ETO)to induce DNA double-stranded break(DSB)in granulosa cells,the expression of DNA damage marker γ-H2AX accumulated and the delayed clearance repair were observed in the autophagic defect group,which indicated that the DNA damage was aggravated and repair was delayed in granulosa cell with autophagy defect.In vivo,after injection of chloroquine in mice,DSB was induced by ETO injection intraperitoneally,the expression of DNA damage marker γ-H2AX in mouse ovaries increased with time,and finally we observed a failure in clearance of γ-H2AX.The result of immunofluorescence showed thatγ-H2AX mainly expressed in granulosa cells,which is not only verified that the defects of autophagy cause DNA damage repair disorders granulosa cells in vivo,but also confirmed that granulosa cells are the most sensitive cell type to DNA damage in ovaries.In vitro,the results of cell immunofluorescence staining showed that after DNA damage,there was a reduce in the expression of homologous recombination repair(HR)markers RAD51 in granulosa cell with autophagy defect,and the expression of the non-homologous end connection(NHEJ)markers KU70 has no obvious difference compared with the control group,which indicated that DNA damage repair disorder in granulosa cells with autophagy defects mainly because of the impairment in the HR pathways rather than the NHEJ pathway caused by autophagy defect.Further mechanism studies found that,on the one hand,autophagy defects lead to increase in proteasome activity,and CHK1,an important molecule involved in homologous recombination repair,was excessively degraded by the proteasome system.On the other hand,autophagy defects cause aggregation of substrates p62.which hinders DNA damage-induced ubiquitination of histone H2A,resulting in impaired HR ability.Finally,EdU assay confirmed that the proliferation of autophagy defective granulosa cells was significantly decreased after DNA damage,while TUNEL staining and Western Blot results showed that DNA damage repair disorder did not lead to the occurrence of apoptosis in granulosa cells.ConclusionThis study confirmed the existence of autophagy defects in granulosa cells of biochemical POI patients.On the one hand,autophagy defect can lead to insufficient degradation of WT1 and thus hindered affect the differentiation process of granulosa cells.On the other hand.autophagy defects impair the ability of homologous recombination repair,thereby reducing the proliferation capacity of granulosa cells after DNA damage.Therefore,autophagy defects participate in the occurrence of POI di sease by affecting the differentiation of granulosa cells and the DNA damage repair Function.Chapter ⅡVariation analysis of autophagy related gene TMEM150B in patients with premature ovarian insufficiencyBackgroundThe causes of premature ovarian insufficiency(POI)are complicated and highly heterogeneous,and genetic defect is an important cause,accounting for approximately 20-25%of cases.Recent studies have shown that age at natural menopause,early menopause and POI share common genetic susceptibility loci,and genes related to age at natural menopause play an important role in the pathogenesis of POI.In European women,TMEM150B has been proved to be a new gene related to natural menopause significantly,early menopause and POI.The protein encoded by TMEM150B is involved in the regulation of autophagy and promotes autophagy to facilitate cell survival under stress.However,the role of this gene in the pathogenesis of POI remains unclear,and whether variants of TMEM150B gene contribute to the pathogenesis of POI in Chinese female patients needs to be determined.ObjectiveIn this study,we are dedicated to reveal the exact role of TMEM150B gene in the pathogenesis of POI in methods of the variation analysis of TMEM1S0B gene in Chinese POI patients,which is helpful to understand the pathogenesis of POI from a novel perspective of autophagy.MethodsCollected peripheral venous blood samples from 408 cases of Chinese han patients with non-syndromic POI in the Reproduction hospital affiliated to Shandong university during September 2014 to July 2016,and extracted genomic DNA from the samples above.After PCR amplification of 8 exons and exon-intron boundary of TMEM150B gene,the Sanger sequencing was performed,at the same time the intronic single nucleotide polymorphism(SNP)rs11668344 was detected,as well.Online software RegRNA was used to predict the combination of 3’UTR and microRNA.The data of normal control groups containing 260 cases of east Asian population was obtained from 1000 Genomes Project’(http://www.internationalgenome.org).Calculation of genotype frequencies and allele frequencies of these variations and statistical analysis was used to consider whether there is any difference in these frequencies between the POI patients and controls.ResultsNine mutations of TMEM150B gene were found in 408 POI patients,among which two novel mutations were located at 3’UTR,and none of them were predicted to affect the binding of microRNA.At the same time,6 known SNPs were found by sequencing,including two synonymous exotic SNPs(rs375066257,rs369826666),three missense exotic SNPs(rs774183872,rs199806152,rs7246479),and one intronic SNP(rs75629207).Compared with the control group,there was no significant difference in allele frequencies and genotype frequencies of the 5 exotic SNP(rs375066257,rs369826666,rs774183872,rs199806152,rs7246479).Although there was a significant difference in genotype frequency of the intronic SNP rs75629207,the long distance from exon-intron boundary made it impossibly affect mRNA splicing.Meanwhile,there was no significant difference in genotype frequency and allele frequency of intronic SNP rs11668344 between the POI patients and control groups.ConclusionThis study indicated that the perturbations in the TMEM150B gene may not be a common explanation for pathogenesis of POI in Chinese women.The exact role of variation of autophagy related genes playing in the pathogenic mechanism of POI needs to be further elucidated.