| The quality of mammalian oocytes determines the potential of subsequent embryo development and is the key prerequisite for modern animal reproduction techniques such as in vitro maturation,in vitro fertilization,embryo transfer and animal cloning.The influence factors of oocyte quality mainly include the individual's genetic factors and external environmental factors.Of them,environmental factors are more complex and usually act on the oocytes through the follicle microenvironment.An important reason why female fertility drops sharply with age is that the changes of the follicle microenvironment caused by aging reduce the quality of oocytes.In addition,the normally matured oocytes without timely fertilization will result in another form of aging,that is,post-ovulatory aging,which seriously affects the rate of in vitro fertilization and in vitro embryo production.Even if they can complete the fertilization,aged oocytes are usually accompanied by the high frequency of aneuploidy and arrest at some stages of early embryonic development,resulting in the embryonic lethality,miscarriage and birth defects.Because oocyte aging is a complex process,there are many hypotheses about the mechanism of aging-induced aneuploid eggs,among which the oxygen free radical-mitochondrial damage hypothesis is widely accepted.The hypothesis claims that if the rate of production of reactive oxygen species(ROS)during oocyte aging exceeds the removal rate of the intracellular antioxidant system,ROS will continuously accumulate within oocytes and cause the varying degrees of oxidation damage to the oocytes through the highly oxidized free radicals,consequently leading to the defective meiotic progression and aneuploidy.Previous studies have reported that antioxidants can alleviate the aging process of oocytes by removing ROS to improve their quality,but the underlying molecular mechanisms and related signaling pathways are not fully known.The answers to these questions are of great significance to reveal the correlation among oocyte aging,occurrence of aneuploidy and antioxidation,providing the theoretical basis for the application of antioxidants in improving the fertility of female animals.The present study consists of two parts.The first part is a literature review which mainly introduces the background of the research.The second part is the experimental studies which are divided into two chapters based on the the work and data.The main research content shows as follows:Experiment 1.Coenzyme Q10 ameliorates the quality of postovulatory-aged oocytesPrevious studies have shown that postovulatory aging seriously impairs oocyte quality and affects subsequent fertilization as well as early embryo development,which has become one of the most important issues in limiting in vitro fertilization and in vitro embryo production.At present,the mechanisms of compromised oocyte quality caused by postovulatory aging has not been fully understood,and the application of antioxidants to prevent and improve aged oocyte quality needs further investigation.In this study,coenzyme Q10(CoQ10)was supplemented during the in vitro culture of postovulatory aged oocytes to examine the importance of oxidation and antioxidation balance to the quality of oocytes,and to dissect the mechanisms regarding how CoQ10 improves the poor oocyte quality and abnormal fertilization caused by the postovulatory aging.By immunofluorescence staining,immunoblotting,mitochondrial live staining,sperm-egg binding and in vitro fertilization,we found that CoQ10 alleviated the meiotic defects in postovulatory aged oocytes,including disrupted spindle assembly,incorrect chromosome alignment,aberrant formation of actin cap,and abnormal distribution of mitochondria and cortical granules.In addition,CoQ10 prevented the premature loss of sperm binding site ZP2 by inhibiting the release of the cortical granule component ovastacin before fertilization,thereby ensuring the sperm-binding ability of postovulatory aged oocytes.CoQ10 also maintained the sperm-egg fusion ability of postovulatory aged oocytes by protecting the correct distribution of the sperm fusion protein Juno,thereby improving its fertilization ability.Lastly,we further investigated its mechanism and found that CoQ10 reduced ROS levels in aged oocytes,thus alleviating DNA damage and reducing the incidence of early apoptosis.In summary,the presented data indicate that postovulatory aging seriously weakens the maturation and fertilization potential of oocytes by disrupting the assembly and distribution of organelles,as well as the dynamics of fertilization regulators.CoQ10 can effectively eliminate the excessive ROS and improve the quality of postovulatory aged oocytes.Experiment 2.Insufficiency of melatonin in follicular fluid is a reversible cause for advanced maternal age-related aneuploidy in oocytesMelatonin is a natural hormone primarily secreted by the pineal gland which regulates the circadian rhythms.In the meantime,as a robust antioxidant,melatonin readily scavenges the most toxic free radicals to prevent the deterioration of cells.Notably,melatonin exists in the follicular fluid produced by the ovarian and granulosa cells in addition to possibly taking it up from the blood.Therefore,melatonin plays an important role in the regulation of follicle development and ovarian function in different mammalian species.It has been reported that melatonin levels in follicular fluid are highly correlated with the ovarian reserve and in vitro fertilization outcomes.However,the relationship between the endogenous level of melatonin and advanced maternal age-related decline of oocyte quality remains elusive.To explore the correlation between melatonin levels in the follicular fluid in maternally aged animals and generation of aneuploidy oocytes,44-48 week old mice were used as the aging model.The effects of maternal aging on the ROS level,DNA damage level,early apoptosis incidence,oocyte meiotic progression,sp indle/chro mo so me structure,and chromosome euploidy were examined by the immunofluorescence staining,quantitative fluorescence measurement,and chromosome spreading.In addition,RNA sequencing analysis revealed that the maternal aging had a great effect on the gene expression in the oxidative phosphorylation and oocyte meiosis pathways,which was consistent with the phenotype in aged oocytes observed above.More importantly,no matter supplemention of melatonin to the culture medium in vitro or administration of melatonin in vivo,it could effectively improve the low quality of aged oocytes and reduce the incidence of oocyte aneuploidy.Specifically,melatonin restored the changes of gene expression in the oxidative phosphorylation and meiosis pathways in aged oocytes,corrected the abnormalities in the meiotic procession,recovered the spindle assembly failure and chromosome misalignment,thereby reducing the occurrence of aneuploidy.Collectively,this paper demonstrates that the level of melatonin in follicular fluid is one of the key factors determining the quality of oocytes in maternally aged mice,and restoration of melatonin level can reverse the production of aneuploid eggs caused by aging.This study reveals the direct relationship between melatonin amount and the quality of oocytes in aged female animals,providing strong scientific evidence for the application of melatonin to improve female animal fertility. |
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