Effcts Of Melatonin On Delaying The Ovarian Aging In Kunming Mice

Ovarian aging is accompanied by a significant decline in ovarian follicle pool and oocyte reserve,as well as an increased number of low-quality oocytes.Oxidative stress and mitochondrial dysfunction are the two major issues in the aged ovaries.Melatonin（N-acetyl-5-methoxytryptamine）is an endogenously produced indoleamine that can modulate many important physiological reactions in the body.Administration of melatonin,whose pineal secretion decreases with age,was highly effectively at preventing age-dependent oxidative damage and mitochondrial functional impairment of various tissues and cells.The study showed that melatonin would be readily bioavailable to the ovary and could directly affect ovarian function after oral ingestion.Thus,the use of melatonin to improve ovarian aging seems feasible.1.Young Kunming females（aged 2–3 months）were fed with melatonin added to drinking water for 12 months and the protective effects of melatonin against ovarian aging have been explored.Compared with the vehicle,the plasma levels remained higher in melatonin-treated group.In mice at the age of 14–16 month,the average number of pups in the melatonin treated groups was significantly higher than from age-matched mice not receiving melatonin.However,it is notable that there was an increased pregnancy failure in the aged female mice following successful mating regardless of melatonin treatment.The quantity of follicles continued to decline with age,but ovarian follicles were higher in mice treated with melatonin than that of in vehicle.Meanwhile,the number of atretic follicles was significantly lower in mice treated with melatonin.The oocyte number was counted and found that the mean numbers of both ovarian germinal vesicle（GV）oocytes and ovulated metaphase II（MII）oocytes were higher in mice treated with melatonin than in age-matched untreated mice.We further evaluated the quality of oocyte and found that oocytes from mice treated with melatonin showed higher in vitro maturation（IVM）rate when compared with the vehicle.Mice treated with melatonin exhibited a higher frequency of normal spindle morphology than age-matched untreated control.The pronuclear formation was delayed in oocytes from aged females,which was attenuated by treatment with melatonin.Moreover,melatonin also improved the early embryonic development of oocytes from aged females,as shown by increased Oct4-positive cells and reduced frequency of apoptosis in blastocysts.The data indicated that melatonin could delay the ovarian aging in Kunming mice.2.Ovarian oxidative stress during aging process not only impairs follicle development but also affects oocyte quality,which reduces the ratio of oocyte maturation,fertilization,embryo quality,and pregnancy.The ovaries were collected to explore the effects of melatonin on ultrastructure,oxidative damage,antioxidant enzymes activities,and expressions of antioxidant genes.The observation results with transmission electronic microscopy indicated that administration of melatonin alleviated the injury of mitochondria in ovaries of aged females.Melatonin mediated the activities of antioxidant enzymes and the expressions of antioxidant genes in aged ovaries,thus decreased the reactive oxygen species（ROS）level,malonaldehyde（MDA）and protein carbonyl contents,and also delayed the erosion of telomere.Additionally,melatonin treatment significantly suppressed the expressions of Bax and Cleaved caspase-3 in aged ovaries,and increased the expression of Bcl-2 when compared with those of the vehicle.The data indicated that melatonin attenuates the oxidative damage of aged ovaries.3.Mitochondria are the primary energy generators in the body and are also the major source and target of free radicals.In particular,mitochondrial dysfunction is considered a major factor contributing to the aging process.Long-term treatment with melatonin prevented aging-associated damage in the ovarian mitochondria,as assessed by the decreased production of mitochondrial ROS,the recovery of glutathione（GSH）levels and the reduction of 8-hydroxydeoxyguanosine（8-OHdG）content.In the ovarian mitochondria from female mice treated with melatonin,the Adenosine Triphosphate（ATP）content was significantly higher than that in age-matched untreated females.Furthermore,we measured the activities of complexes I-IV of mitochondrial electron transport chain（ETC）.Though aging did not affect the activity of complex IV,there was a significant decrease in activities of complex I,II and III in aged ovaries compared with those of in young group.However,administration of melatonin resulted in a remarkable augmentation of the ETC activities in ovarian mitochondria,as compared with the age-matched mice without treatment.Reports indicate that insufficient mtDNA levels and ATP contents could be directly associated with diminished oocyte competence during aging.The data showed that the average mtDNA copy numbers in MII oocytes from melatonin-treated mice were significantly higher compared with the age-matched mice without treatment.Mice treated with melatonin significantly increased the ATP levels both in GV and MII oocytes.Furthermore,melatonin treatment reduced the ratio of oocytes with abnormal distribution of mitochondria,which was increased in aged ovaries.There was a positive correlation between mtDNA heterogeneity and age.Thus,we performed the denaturing high performance liquid chromatography（DHPLC）and found an obvious increase of mtDNA heterogeneity in D-loop region in the oocytes from aged females.However,treatment with melatonin decreased the frequency of mtDNA heteroplasmy in D-loop region.The data indicated that melatonin improves ovarian mitochondrial function during aging process.4.To explore the protective mechanism of melatonin,primary granulosa cells were isolated and incubated with H₂O₂ to mimic oxidative stress and mitochondrial dysfunction in vivo.In vitro studies confirmed that melatonin attenuates H₂O₂-induced oxidative stress and mitochondrial dysfunction in granulosa cells.The ChIP assay indicated that melatonin treatment promoted the nuclear translocation of FOXO3A,thus increasing the antioxidant genes expression of SOD2 and CAT.Knockdown of Adenosine Monophosphate Activated Protein Kinase（AMPK）using specific siRNA blocked the nuclear translocation of FOXO3A and attenuated the protective effects of melatonin.Importantly,compared with the age-matched untreated control,melatonin treatment also increased the expression of p-AMPK in aged mouse ovaries.5.We found the elevated ROS level,reduced GSH/GSSG ratio,abnormal zona hardening,and mitochondrial dysfunction indicated by reduced mitochondrial membrane potential（MMP）,mtDNA copy number,and ATP level during the oocyte aging process in vitro,accompanied by a decreased oocyte quality and developmental competence.However,these adverse effects are markedly abolished by incubating oocytes with 10^-33 M melatonin.In addition,we also observed that the expression of Sirt3 was decreased at both the transcriptional and translational levels in MII oocytes aged in vitro.Moreover,the acetylation level of SOD2（K68）was increased during the oocyte aging process in vitro.However,the beneficial effects of melatonin on the delay of postovulatory aged MII oocytes were blocked when 3-TYP was used（Sirt3 inhibitor）.In conclusion,long-term melatonin treatment exerts protection against ovarian aging through mitigating oxidative stress and maintaining mitochondrial function,and these beneficial effects may contribute to the activation of AMPK signaling pathway.Furthermore,melatonin delays the postovulatory MII oocyte aging via regulating Sirt3-SOD2 pathway.