The Role And Related Mechanism Investigations Of Autophagy In Ovarian Function Decline With Age

Objective The present study aims to explore the age-related autophagy alternations in ovarian tissues,and the correlations between ovarian autophagy and ovarian deserve in the part one.The second part of our study further investigates the regulatory effect and the involving mechanisms of autophagy on ovarian reserve and function,and explores the possibility of autophagy intervention delaying ovarian aging.Methods（1）Ovarian and serum samples from females and mice of different ages were collected and tested to evaluate their ovarian reserve,function and autophagy change.（2）Correlation analysis between ovarian autophagic proteins and serum hormones,ovarian AMH proteins of female mice were conducted using Pearson/Spearman correlation analysis to further detect the exact significance and the correlation coefficient.（3）Ovarian functional indicators including estrous cycle,ovarian hormone secretion,and follicular development were evaluated in young female mice after 3-MA administration.（4）Biological functional alternations of murine primary granular cells and developmental potentials of murine oocytes were tested after 3-MA in vitro exposure.（5）Last but not the least,ovarian functional indicators including estrous cycle,ovarian hormone secretion,and follicle development were evaluated in young female mice after rapamycin administration.Results（1）Significantly decreased serum AMH levels were recognized in females aged above 40,serum E₂ was reduced but serum FSH was elevated in the same female population.（2）In female ovaries,P62 was macroscopically expressed in follicle units and ovarian stroma.Reduced ovarian autophagy were recognized in females aged before 50,afterwards,an inverse tendency was observed.（3）The expressions of ovarian P62 protein were negatively correlated with serum AMH in females.The expressions of ovarian LC3II/I were positively correlated with serum E₂ in females.（4）Murine ovarian volume consecutively increased before 36 weeks.Abundant ZP3-positive signals of healthy follicles were detected in murine ovaries before 24 weeks.Consistently decreased primordial follicles but increased atretic follicles were noticed in murine ovaries with increasing weeks.Ovarian AMH proteins increased before 12 weeks,and gradually reduced afterwards.Serum AMH reached climax at 12 weeks and significantly decreased afterwards.Serum E₂ were reduced in female mice after 24 weeks and serum FSH remarkably increased after 12 weeks.（5）P62-positive stainings were observed in follicle units of fertile mice and ovarian stroma of aged ovaries after 24 weeks.Autophagosomes and autophagolysosomes were decreased in murine ovaries with age（primarily in ovarian granular cells）.Downregulation of autophagic components were discovered in murine ovaries of 6 weeks to 24 weeks on transcriptional level.However,a compensation was noticed during 36 weeks.In translation level,murine autophay were compensated after 36 weeks.（6）The expressions of ovarian P62 protein were negatively correlated with ovarian AMH proteins in female mice.（7）3-MA displayed systemic toxicity and ovarian toxicity in young female mice.（8）Inhibition of autophagy disrupted ovarian hormones scretion accelerated depletion of ovarian reserve in young female mice.（9）Transcriptional sequencing indicated autophagy modulated ovarian steroid hormone biosynthesis.（10）Autophagy regulated hormone synthesis through modulation of multiplication capacity of granular cells in vtro.（11）Transcriptional sequencing indicated autophagy modulated follicle recruitment,development and fertilization.（12）Inhibition of autophagy interfered oocyte fertilization.（13）Autophagy regulated in vitro maturation and meiosis of oocytes through modulation of mitochondrial function.（14）Rapamycin is relatively safe to old female mice.（15）Activation of autophagy improved ovarian hormones secretion and decreased atretic follicles in old female mice.Conclusions Ovarian autophagy was located in follicle units and ovarian stroma,and displayed a downwards tendency with increasing age in females and female mice.Ovarian autophagy significantly correlated with ovarian reserve and function.Inhibition of autophagy disrupted ovarian hormones scretion,and accelerated depletion of ovarian reserve of young female mice,the involving mechanisms included regulation of ovarian hormone synthesis through modulation of multiplication capacity of granular cells,and modulation oocyte quality,development and fertilization through regulation of mitochondrial function.Activation of autophagy improved ovarian hormones secretion and decreased decreased atretic follicles in old female mice,indicated that autophagy intervention is expected to be a promising strategy delaying age-related ovarian function decline.PART I Alternations of Autophagy in Age-Related Ovarian Function Decline,and Correlations Analysis between Autophagy and Ovarian FunctionObjective So far,the alternations of autophagy in age-related ovarian function decline and the role of autophagy in regulating ovarian function remain unclear.The present study aims to explore the age-related autophagy alternations in ovarian tissues,and the correlations between ovarian autophagy and ovarian deserve,function in human and mice,and to investigate the macroscopic and microscopic location of autophagy in ovarian tissues.Methods Ovarian and serum samples from females and mice of different ages were collected and tested to evaluate their ovarian reserve,function and autophagy change.（1）Superfluous ovaries of female population with normal ovarian function aged 18 to 30,30 to 40,40 to 50,and 50 to 60 were obtained from operating rooms（3 ovaries in each age group,12 ovaries in total）.（2）LC3II/I,P62,Beclin1,and ATG3 protein expressions were quantitatively analyzed using WB.P62 immunohistochemical staining was applied to detect the macroscopic location of autophagy proteins in female ovaries.（3）Serum AMH,E2,and FSH in corresponding females were tested by clinical laboratory to provide preliminary evaluation of female ovarian function.（4）One hundred and eighty female C57BL/6 mice aged 1-week,6-week,12-week,24-week,36-week and 48-week were sacrificed at diestrus,and mice ovaries of different-week groups were collected（30 mice in each week group,180 mice in total）.（5）Transcriptional and translational levels of autophagy-related molecules in different-week murine ovaries were determined using Rt-q PCR and WB.（6）P62 immunohistochemical staining and transmission electron microscope image were both applied to locate autophagy in murine ovaries macroscropically and microscopically.（7）Ovarian reserve and function of different-week female mice were comprehensively analyzed based on their ovarian reserve and ovarian endocrine function.The former was generally evaluated using H&E staining,follicle counting,ZP3 immunofluorescence,serum AMH,and ovarian AMH protein WB.The later was quantitatively measured by serum E2,and FSH ELISA.（8）Correlation analysis between ovarian LC3II/I,P62 proteins and serum AMH,E2,and FSH levels（ovarian AMH proteins in female mice）were conducted using Pearson/Spearman correlation analysis to further detect the exact significance and the correlation coefficient.Results（1）The age-related alternations of ovarian reserve and ovarian function in females: Significantly decreased serum AMH levels were recognized in females aged above 40,serum E2 was reduced but serum FSH was elevated in the same female population.（2）The locations and expressions of autophagy molecules in ovaries of different-age females: In female ovaries,P62 was macroscopically expressed in follicle units and ovarian stroma.Developing follicles decreased in female ovaries of advancing age,and there was barely visible P62-positive staining in female ovaries after 40 years old.Reduced ovarian LC3II/I,Beclin1,and ATG3 but up-regulated P62 protein expressions were recognized in females aged before 50,afterwards,an inverse tendency was observed.（3）The correlation analysis between autophagy and ovarian reserve,function in females: The expressions of ovarian P62 protein were negatively correlated with serum AMH in females.The expressions of ovarian LC3II/I were positively correlated with serum E2 in females.There were no significant correlations between ovarian P62,LC3II/I and other serum hormones.（4）The age-related alternations of murine ovarian reserve and ovarian function: Murine ovarian volume consecutively increased before 36 weeks.Abundant ZP3-positive signals of healthy follicles were detected in murine ovaries before 24 weeks,such signals significantly decreased in 36-week and 48-week murine ovaries.Consistently decreased primordial follicles but increased atretic follicles were noticed in murine ovaries with increasing weeks.Ovarian AMH proteins increased before 12 weeks,and gradually reduced afterwards.Serum AMH reached climax at 12 weeks and significantly decreased afterwards.Serum E2 were reduced in female mice after 24 weeks and serum FSH remarkably increased after 12 weeks.（4）The locations and expressions of autophagy molecules in ovaries of different-week mice: P62-positive stainings were observed in follicle units of fertile mice,but were primarily detected in ovarian stroma of aged ovaries after 24 weeks.Abundant autophagosomes as well as autophagolysosomes were captured in ovaries of 1-week and 6-week female mice under transmission electron microscope（primarily in ovarian granular cells,a small fraction of them were detected in interstitial cells rich in lipid droplets）.Both autophagosomes and autophagolysosomes were reduced with increasing weeks,barely detected in 48-week ovaries.Downregulation of LC3 B,Beclin1,and Atg7 m RNA were discovered in murine ovaries of 6 weeks to 24 weeks.However,a slight upregulation of the above 3 genes was noticed during 36 weeks.In translation level,LC3II/I was consistently decreased,and the expressions of ovarian P62 were first reduced before 36 weeks but conversely modulated afterwards.（5）The correlation analysis of autophagy and ovarian function in females mice: The expressions of ovarian P62 protein exhibited no signigicant correlations with other indicators,but were negatively correlated with ovarian AMH proteins in female mice.Ovarian LC3II/I displayed no significant correlations with ovarian AMH proteins and serum hormones.Conclusions Female ovarian autophagy was located in follicle units and ovarian stroma,and displayed a downwards tendency with increasing age.Continuing decline of autophagy was noted in female ovaries before 50 years old,a compensatory enhancement of ovarian autophagy took effect ever since.In females,autophagy substrate protein P62 was negatively correlated with serum AMH,and ovarian LC3II/I was positively correlated with serum E2.Murine ovarian autophagy was located in follicle units and ovarian stroma as well.P62-positive staining gradually transferred from follicle units to ovarian stroma since 24 weeks in female mice.Murine ovarian autophagy displayed a downwards tendency with increasing weeks.In mice,ovarian autophagy reduced from 6 week to 24 week,autophagy compensation intiated during 36 weeks.Autophagy substrate protein P62 was negatively correlated with ovarian AMH proteins of female mice.PART II The Regulatory Effect of Autophagy on Ovarian Reserve and Function and Related Mechanism InvestigationObjective Preliminary results in part one indicated that autophagy was correlated with ovarian reserve and function,but the regulatory effect of autophagy intervention on ovarian reserve and function remains unclear.The following part of our study amis to investigate the regulatory effect and the involving mechanisms of autophagy on ovarian reserve and function,and to explore the possibility of autophagy intervention delaying ovarian aging.Methods（1）One hundred 12-week female C57BL/6 mice were randomly allocated to receive introperitoneal injection of 3-MA（10mg/kg）or 3-MA-vehicle（Vehicle young,V-Y）daily（50 mice in each group）.Meanwhile,another 50 36-week female C57BL/6 mice were intrapertonelly injected with 3-MA-vehicle（Vehicle old,V-O）.（2）Sixty 36-week female C57BL/6 mice were randomly allocated into Rapamycin（Rapa）group or Rapamycinvehicle（Rapamycin-vehicle,V）group,animals were intrapertonelly injected with Rapamycin（2mg/kg）or corresponding vehicle accordingly once a day（30 mice in each group）.The drug administration lasted for 2 weeks.Animals were weighed weekly before,during,and after the drug aniministration（if not being secraficed）.Drug safety,animal modeling and mice ovarian function were assessed.（3）Five animals of each group（15 animals in total）were randomly chosen to record estrus cycle fluctuations using vaginal smear.Fifteen animals in 3-MA,V-Y,and V-O groups（45 animals in total）were prepared for IVF experiments.（4）Other animals were sacrificed at diestrus to collect hearts,livers,spleens,lungs,kidneys,uteri and ovaries.Before sacrificing,individual blood samples were obtained to evaluate serum AMH and E2.（5）Eight ovaries of each group（24 ovaries in total）were randomly chosen,4 of which were processed to ovarian sections for ovarian follicles counting and immunofluorescence detection,the left 4 ovaries were captured by transmission electron microscope to record autophagosomes.（6）Another 3 ovaries from 3-MA and V-Y groups（6 ovaries in total）were sent for transcriptome sequencing to explore the involving mechanisms of autophagy-modulated ovarian reserve and function.WB and Rt-q PCR were conducted to determine the relative expressions of autophagy molecules,pro-follicle-development components,and steroid-hormone-synthesis-related molecules in ovaries following different drug regimens.（7）Primary murine granular cells were obtained from 30 3-week C57BL/6 mice and randomly allocated into 3MA5-GC group,3MA10-GC group,and NC-GC group.After adherence,culture medium of both groups was replaced with fresh medium containing 3-MA（5m M）,3-MA（10m M）or not.Twenty-four hours later,the granular cells with different treatments were harvested to evaluate alternations of cell apoptosis,multiplication capacity and steroid-hormone-synthesis-related molecules.（8）Oocytes were obtained from 30 12-week C57BL/6 mice received ovulation induction and randomly allocated into 3MA-O group and NC-O group.The ovulated oocytes were cultured in M2 medium containg 3-MA（5m M）or not according to their groups.Fifteen hours later,the oocytes were harvested to assess their developing potential and mitochonrial function.Results（1）Safety evaluation of autophagy inhibitor 3-MA on young female mice: 3-MAtreated mice experienced a significant weight loss and ovarian index decline,serum ALT,AST,and BUN were also increased after 3-MA administration.（2）Inhibition of autophagy disrupted ovarian hormones scretion in young female mice: 3-MA administration resulted in a remarkable reduction of LC3II/I,Beclin1,and ATG3 proteins and an elevation of P62 proteins in 12-week murine ovaries.Barely detectable autophagosomes were captured in ovaries under transmission electron microscope after 3-MA treatment.Increased irregular estrus cycle rates,prolonged estrus cycles,and even cycle arrests were identified in 3-MA treated mice.（3）Inhibition of autophagy accelerated depletion of ovarian reserve in young female mice: Dramatically decreased serum AMH was also discovered in 3-MA group.Significantly decreased primordial follicles but increased primary follicles were recorded in 3-MA-treated animals.A slight increase of secondary follicles and antral follicles were also noted in 3-MA-treated ovaries.（4）Autophagy modulated ovarian steroid hormone biosynthesis: To rank the differentially expressed genes according to their fold changes between 3-MA and V-Y ovaries,the top 10 differentially expressed genes downregulated via autophagy inhibition were primarily correlated with steroid hormone systhesis.GO analysis also suggested that autophagy intervention affected steroid hormones metabolism.Rt-q PCR data indicated 3-MA treatment was associated with a downregulation of steroidhormone-systhesis-related genes Cyp17a1,Cyp19a1 and Hsd17b1.（5）Autophagy regulated hormone synthesis through modulation of multiplication capacity of granular cells: Compared with NC-GC group,granular cells after 3-MA treatment displayed slightly intensified staining of TUNEL but significantly attenuated staining of Ed U.Autophagyinhibited granular cells also showed dose-dependent decline of cell viability along with downregulation of PCNA on transcriptional and translational levels.Pro-apoptosis component BAX was merely transcriptionally upregulated in 3MA10-GC group.Decreased expression of Hsd3b1,Hsd17b1,and Cyp17a1 were also noted in granular cells after in vitro autophagy inhibition.（6）Autophagy modulated follicle recruitment,development and fertilization: Heatmap of differentially expressed genes between 3-MA and V-Y ovaries indicated that 3-MA signigicantly enhanced the expressions of Zp2,Gdf9,Padi6,Nlrp14,Igf1,and Mt1.The top 10 differentially expressed genes upregulated by autophagy inhibition were mainly related to ovarian follicle recruitment,development,and fertilization.GO analysis further identified that autophagy intervention affected expressions of ovarian genes related to reproduction,and meiosis.Rt-q PCR data indicated 3-MA treatment was associated with an upregulation of follicle-development-related genes Gdf9,and Nobox,as well as pro-fertilizaiton genes Zp1,Zp2,and Zp3.WB further confirmed enhanced profollicle-development GDF9 and BMP15 expressions at translation level and increased phospholylation level of rps6（a downstream effector of typical follicle-development pathway PI3K-AKT）after 3-MA medication.Intensive ZP3 immunofluroscence signals were observed in 3-MA-treated female mice ovaries.（7）Inhibition of autophagy interfered oocyte fertilization: 3-MA-treated animals were correlated with lower fertilization rates and blastocyts rates when compared with V-Y animals（or even V-O animals）.（8）Autophagy regulated in vitro maturation and meiosis of oocytes through modulation of mitochondrial function: Compared with NC-O group,oocytes received 3-MA administration had fewer first polar bodys extruded.Immunofluorescence stainings of spindle and TOM20 in 3MAO group were weaker and irregular.Attenuated fluorescent staining of mitoctracker,decreased mitochondrial membrane potential and reduced ATP synthesis were noted in 3-MA treated oocytes.（9）Safety evaluation of autophagy activator Rapamycin on old female mice: Rapamycin displayed no significant influence on mice weight and organ indices.（10）Activation of autophagy improved ovarian hormones secretion in old female mice:Rapamycin administration led to a significant uprelation of LC3II/I,Beclin1,and ATG3 proteins and downregulation of P62 proteins in 36-week murine ovaries.Increased number of autophagosomes in ovaries were observed under transmission electron microscope after Rapamycin treatment.We recognized elevated serum E2 but decreased FSH in Rapamycintreated female mice.（11）Activation of autophagy increased ovarian reserve in old female mice: Significantly increased serum AMH was identified in Rapamycin-treated female mice.Reduced atretic follicles but increased primary and secondary follicles were noted in mice following Rapamycin administration.Pro-follicle-development proteins GDF9 and BMP15 were significantly upregulated in Rapamycin-treated murine ovaries.Conclusions Inhibition of autophagy disrupted ovarian hormones scretion,and accelerated depletion of ovarian reserve of young female mice,the involving mechanisms included regulation of follicle recruitment,development,fertilization,and steroid hormone systhesis.Autophagy regulated ovarian hormone synthesis through modulation of multiplication capacity of granular cells.Autophagy modulated oocyte quality,subsequent development and fertilization through regulation of mitochondrial function.Activation of autophagy improved ovarian hormones secretion and increased ovarian reserve in old female mice,indicated that autophagy intervention is expected to be a promising strategy delaying agerelated ovarian function decline.