Intermittent Fasting Improves Oocyte Quality In Aged Or Obese Mice

The primordial follicle pool is established in the fetal period or postnatal in female,and the number of oocytes does not increase thereafter.Thus,the quality of oocytes largely determines the reproductive potential of female animals.However,aging or obesity can lead to a decrease in oocyte quality and reproductive potential.Due to the particularity of the ovary,it tends to senescence earlier than other organs and tissues,which is characterized by the gradual depletion of oocyte reserves,deterioration of oocyte organelle function,excessive accumulation of oxygen free radicals,oxidative damage,abnormal spindle assembly,and increased chromosomal mismatching rate,resulting in a decline in oocyte quality.Studies have shown that intermittent fasting(IF)can improve glucose metabolism regulation,mitochondrial biogenesis and function,reduce the generation of oxygen free radicals,and enhance antioxidant and anti-stress ability,thereby contributing to improve health.Whether IF attenuates the deterioration of oocyte quality caused by aging or obesity?The aim of the study was to explore the effects and molecular mechanisms of IF for alleviating the decline of oocyte quality and reproductive ability in female mice caused by aging or obesity.The main research contents and research results obtained are as follows:(1)The natural aging female mice(58-60 weeks old)were subjected to an IF program of fasting every other day(24 hours ad libitum/24 hours fasting)for 30 days,to investigate whether IF could alleviate the decline of oocyte quality and reproductive performance in naturally aging females.①The estrous cycle was monitored in young mice(6-8 weeks old),naturally aging mice,and IF-treated naturally aging mice through staining analysis of vaginal exfoliated cell smears.Results showed that the estrous cycle of naturally aging mice was disrupted,but after IF treatment,the phenomenon of estrous cycle disruption was partially restored.②Ovarian histological analysis showed that the number of follicles in the ovaries of naturally aging mice was significantly less than that of young mice,whereas IF treatment significantly increased the number of primordial,primary,and antral follicles in the ovaries of naturally aging mice.③ Analysis of in vivo matured oocytes revealed that IF increased ovulation number(n=0.0126),first polar body(PB1)excretion rate(n=0.0466),and reduced oocyte fragmentation rate(n=0.0456)in naturally aging mice.④IF treatment also increased germinal vesicle breakdown(GVBD)(n=0.0134)and PB1 excretion rate(n=0.0021)of oocytes matured in vitro in naturally aging mice.⑤Immunofluorescence staining and fluorescent probe staining analysis in live cells found that IF effectively restored oocyte spindle assembly(n=0.0121),chromosome segregation,and distribution of mitochondria(n=0.0012),golgi apparatus(n=0.0252),endoplasmic reticulum(n=0.0195),and cortical granules(n=0.0305),while increasing mitochondrial membrane potential(n=0.0452)and ATP levels(n=0.0012),and reducing reactive oxygen species(ROS)accumulation(n<0.0001),DNA damage(n=0.0317),and apoptosis(n<0.0001).These results suggest that IF helps to relieve oxidative stress and promote the nuclear and cytoplasmic maturation of natural aging mouse oocytes.⑥ Through in vitro fertilization(IVF),it was found that IF treatment effectively increased the in vitro fertilization rate and in vitro blastocyst development rate of natural aging mouse oocytes.⑦The pregnancy rate of aging mice treated with IF did not increase significantly(n=0.4584),but the average litter size was significantly increased(n=0.0431).⑧The results of oocyte transcriptome sequencing showed that IF treatment improved several signaling pathways that are beneficial to the maturation and development of oocytes,such as restoring the SIRT1/ERK1/2 signaling pathway and increasing the level of SIRT1 pathway activator NAD+.Further treatment of mice with the SIRT1 inhibitor EX527 revealed that the beneficial effects of IF on activation of ERK1/2 phosphorylation,ROS scavenging,and resistance to oxidative damage were completely abolished.In summary,IF can improve the antioxidant defense,eliminate excessive ROS accumulation,inhibit DNA damage and apoptosis in naturally aging mouse oocytes,and improve oocyte quality through the NAD+/SIRT1/ERK1/2 signaling pathway.Therefore,IF is a promising approach to protect oocytes from aging-induced oxidative damage and contribute to partial recovery of reproductive outcomes in naturally aging female animals.(2)Obesity is another important factor causing oocyte quality damage in female animals.This study aimed to analyze whether IF could improve the oocyte quality and offspring growth and development in high fat diet(HFD)-induced obese mice.The obese mice underwent 30 days of alternate-day fasting(24 hours ad libitum/24 hours fasting)to study the regulatory effects of IF on the reproductive ability and oocyte quality of obese female mice.①Histological examination of ovarian tissue sections stained with H.E.revealed a significant reduction in the number of follicles in obese mice compared to those on a normal diet(ND)at different stages of development.However,HFD mice treated with IF(HFD+IF)showed a significant increase in the number of primordial,secondary,preantral,and antral follicles.②Mature oocytes collected in vivo from HFD+IF mice had significantly restored ovulation number(n=0.0492)and PB1 excretion rate(n=0.0015),and significantly reduced oocyte fragmentation rate(n=0.0260)compared with HFD mice.③The results of oocyte in vitro maturation indicated that IF treatment effectively restored the in vitro maturation potential of obese mouse oocytes,significantly increasing the incidence of GVBD(n=0.0441)and PB1 excretion rate(n=0.0004)of oocytes in HFD mice.④Immunofluorescence staining and live cell fluorescence probe staining analysis showed that IF restored the spindle assembly(n=0.0316),chromosome separation(n=0.0316),and mitochondrial distribution(n=0.0451)of oocytes in HFD mice.IF also increased the mitochondrial membrane potential(n=0.0208)and ATP levels(n<0.0001),maintained communication between the cumulus cells and oocytes,reduced ROS accumulation(n=0.0210),DNA damage(n<0.0001),and apoptosis(n<0.0001),thereby alleviating oxidative stress damage,promoting communication between cumulus cells and oocytes,and improving oocyte quality in obese mice.⑤ The fertilization and early embryo development competence were analyzed by IVF test,and it was found that IF treatment effectively increased the fertilization rate and blastocyst development rate of obese mouse oocytes.⑥The results of natural mating experiments showed that IF treatment increased the pregnancy rate,litter size(n=0.0234),head-rump length(n=0.0113),and birth weight(n=0.0040)of HFD mice.⑦Transcriptome sequencing analysis of ND,HFD,and HFD+IF-treated mouse oocytes revealed that IF treatment improved signaling pathways that favor mitochondrial oxidative phosphorylation and regulation of mRNA translation.⑧The study demonstrated that IF treatment helps to restore translational activity in oocytes,as demonstrated by L-Homopropargylglycine(HPG)staining(n<0.0001).⑨Through transcriptome data analysis,qRT-PCR,and Western blot detection,it was proven that the RNA-binding protein LSM14B may mediate the regulation of IF on oocyte mRNA translation.⑩Knockdown of LSM14B protein in ND oocytes by siRNA fully reproduced the phenotypes associated with meiosis defect and decreased translational activity observed in HFD oocytes.?Injection of in vitro transcribed Lsm14b mRNA into HFD mouse oocytes to express exogenous LSM14B protein rescued the meiosis-deficient phenotype and translational activity of HFD mouse oocytes.In conclusion,IF rescues the overall translation level of oocytes by increasing the expression of LSM14B protein in HFD mice,and improves the antioxidant defense ability of oocytes in obese mice by improving the mitochondrial oxidative phosphorylation function,thereby improving the maturation quality of oocytes.The present studies indicate that IF enhances oocyte maturation quality,fertilization ability,and embryonic development ability in natural aging or high-fat diet-induced obese mice.This highlights the potential and molecular mechanisms of IF as a dietary intervention to rescue the reproductive defects of female animals caused by natural aging or obese,and thereby providing a new solution for maintaining reproductive capacity.