Mechanism Of NAD~+ In Delaying Ovarian Aging Through Improving Mitochondrial Function

BackgroundFemale fertility gradually declines with age.Although there are many factors that can lead to a decline in fertility,ovarian ageing plays a very important role.Importantly deterioration of the ovarian reserve and the oocyte quality and quantity are the main cause of ovarian ageing.Mitochondria is the power factory in cells which is essential to produce energy for maintaining cell life activities and tissue metabolism.Meanwhile mitochondria can also effect ovarian reserve and oocyte quality.Mitochondria can be directly affected in the aging process.Mitochondria participate in the general aging process and the functions including production of energy or regulating various cell signaling pathways that gradually degrade with ageing.Decreased mitochondrial function during aging will increase the level of reactive oxygen species(ROS)in oocytes which may further damage mitochondria.It can cause abnormal DNA double-strand break(DSB)repair in oocytes.And abnormal meiosis leads to a decrease in the quality of oocytes,which further affects fertilization and embryo development.The depletion of follicles in the ageing process of reproductive system is caused by the apoptosis of oocytes and cells surrounding them and mitochondria plays a key role in this follicular atresia through the function in cell survival and apoptosis.On the other hand,the initial size of the follicle pool seems to be determined in a variety of ways during the embryonic period,including mitochondrial biogenesis.Nicotinamide adenine dinucleotide(NAD+)is a cofactor for key enzymes in metabolic pathways such as glycolysis,tricarboxylic acid cycle,oxidative phosphorylation,etc.It participates in a variety of redox reactions in cells,maintains mitochondrial function for metabolism,build new cells,resist free radicals and DNA damage,ect.Nicotinamide ribose(NR)is the precursor of NAD+,which can be converted into NAD+in the body.Several studies have shown that the content of NAD+ in other tissues will decrease with aging,and NR supplementation can increase the content of NAD+in organs and improve the mitochondrial function energy metabolism.ObjectiveThis project aims to study the improvement effect of nicotinamide adenine dinucleotide(NAD+)on the decline of female fertility caused by aging,explore its potential mechanism of delaying ovarian aging,and provide a reliable theory for the clinical application of nicotinamide adenine dinucleotide(NAD+).Method1.2-month-old and 8-months-old female C57/BL6 mice were obtained from Beijing Vital River Experimental Animals Centre.Randomly divide 8-month-old female mice into two groups.One group is provided with ordinary drinking water for the control group,and the other supplement Nicotinamide Riboside(NR)in drinking watern for 4 months.2.After 4 months treatment,using cycling assay and ultra-high performance liquid chromatography mass spectrometry assay(UHPLC-MS)to measure NAD+level in ovary.3.Compare the changes in fertility and ovarian reserve of mice between different groups by recording the litter size after mating trials and counting the number of follicles at different stage using hematoxylin-eosin staining.4.The oocyte morphology and immunofluorescence staining were used to compare the quality of oocytes between different groups.5.The morphology of mitochondria in the cell was observed by transmission electron microscopy of oocytes,the proportion of mitochondrial morphology in different groups was counted,the distribution of mitochondria and mitochondrial membrane potential of oocytes were detected by immunofluorescence staining,and mitochondrial copy number of oocytes was performed by RT-PCR.Using RT-PCR and western blot to detect ovarian mitochondrial related genes and protein expression.Ultra-high performance liquid chromatography mass spectrometry assay(UHPLC-MS)was used to detect the key metabolites of TCA cycle,glycolysis pathway and oxidative phosphorylation pathway between different groups.Results1.NR supplementation can prevent age-related decline in ovarian NAD+levels2.NR supplementation can improve ovarian function and ovarian reserve in aging mice3.NR supplementation can increase the number of oocytes after superovulation,improve the quality of oocytes,and fertility4.NR supplementation can improve the function of mitochondria in oocytes and ovariesConclusionOvarian NAD+level gradually decreases with ageing.Supplementation of NAD+ precursor nicotinamide ribose(NR)can increase ovarian NAD+;content.Increased NAD+ level can improve ovarian reserve,increase oocytes number after superovulation and increase litter size.NR supplementation can also reduce the ROS level and abnormal spindle ratio in ageing oocytes,increase mitochondrial membrane potential,reduce mitochondrial aggregation,and improve ovarian mitochondrial energy metabolism.