Melatonin Delays The Reproductive Aging Of Mice By Inhibiting Follicular Reserve Loss

Ovarian aging refers to the physiological process of active loss of ovarian function in female animals before the body ages.It is a kind of gain-of-function mutation,which can effectively avoid the physiological burden and life threat to the mother caused by older pregnancy.The depletion of the follicular reserve in the ovary is the root cause of ovarian aging.Follicle reserve includes both the static follicle reserve composed of primordial follicles and the dynamic follicle reserve composed of early growing follicles.The activation of primordial follicles and early follicular growth not only provide a source of follicles for females'periodic reproductive activities,but more importantly,their rate and scale directly determine the rate of ovarian aging.Melatonin?Melatonin,MLT?is an important antioxidant hormone secreted by the pineal gland.Studies in recent years have shown that long-term intake of melatonin can effectively delay ovarian aging in experimental animals,but the specific mechanism remains to be studied in depth.Previous studies have confirmed that exogenous MLT intake can effectively delay ovarian aging,but it is not clear whether endogenous melatonin also plays the same role in the ovarian aging process.In addition,melatonin is not only an antioxidant,many functions have been given in the course of millions of years of evolution.Therefore,it is not clear whether there are other unrevealed ways in which melatonin can delay ovarian aging besides its antioxidant effect.In this study,q-PCR,immunofluorescence,immunohistochemistry,high performance liquid chromatography,RNA-Seq,gene knockout and other techniques were used to systematically evaluate the effect of endogenous melatonin on ovarian aging,and further study the potential mechanism of melatonin in delaying ovarian aging.The main results are as follows:?1?Melatonin synthesis rate-limiting enzyme SNAT is located in primordial follicles and early follicles.Granulosa cells isolated from early follicles can synthesize melatonin.In addition,the expression levels of Fshr and Lhcgr in the ovary gradually increased with the growth of early follicles,while the expression level of Snat gradually decreased,and the level of melatonin in the ovary also decreased.?2?After adding 10^-7 and 10^-8 mol/L of melatonin to the culture medium in vitro,the number of activated follicles was significantly reduced,and the proportion of atretic follicles in the ovaries was also decreased,but the difference was not significant?P=0.086?.After the addition of 10^-7mol/L melatonin,the proportion of 5a follicles decreased,but the difference was not significant?P=0.092?,and the number of atresia follicles did not change significantly after the addition of melatonin.?3?At the activation stage of primordial follicles,the activation rate of primordial follicles was significantly decreased after injection of melatonin at 1mg/kg and 15mg/kg?P<0.01?.WB results showed that melatonin treatment did not affect the phosphorylation levels of downstream proteins Rps6kb1 and Eif4ebp1 in the m TOR signaling pathway,but the phosphorylation levels of Akt were significantly decreased?P<0.05?.Analysis of Foxo3 immunofluorescence sections showed that melatonin treatment significantly reduced the nucleation rate of Foxo3 and inhibited the activation of primordial follicles.?4?In early stages of follicular growth after injection of melatonin in the body,in the experimental group 5 b follicle,6 follicular atresia follicles and lower?P<0.05?,the proportion of the follicle development related genes the expression of Fshr and Lhcgr levels?P<0.05?,the superovulation treatment after ovulation number after mating with nature embryo number plant were significantly lower?P<0.05?;A total of 598differentially expressed genes were screened out by RNA-SEQ in the ovary of PD17 mice.GO annotation found that the differentially expressed genes were mainly enriched in biological processes related to cell proliferation and tissue development,such as"Negative regulation of cell proliferation"and"Blood Vessel Development signaling".Immunohistochemistry and fluorescence quantitative results showed that the level of proliferation marker PCNA decreased.KEGG analysis showed that melatonin affected several signaling pathways related to follicular development,such as the"PI3K-Akt signaling pathway"and"MAPK signaling Pathway",and WB results confirmed that the PI3K-Akt signaling pathway was inhibited.?5?After Snat gene knockout in mice,follicular activation and atremia were significantly increased in ovaries?P<0.05?,apoptosis and follicular atremia were increased,and Foxo3 transport to cytoplasm was increased.MLT supplementation could effectively alleviate the above abnormal phenotypes.There was no significant difference in the litter size of the Snat knockout mice in early adulthood.At 5-6 months of age and8-9 months of age,the litter size of the knockout mice was significantly lower than that of the wild type,and the number of follicles growing in the ovaries was significantly reduced by Snat knockout?P<0.05?.The expression levels of the ovarian oocyte marker Gdf9,Nobox and Zp3 in the Snat knockout mice were significantly lower than that of the wild type?P<0.05?.?6?Long-term intake of melatonin at the experimental dose?15mg/kg?had no significant effect on the daily weight gain,organ index,oestrous cycle,mating time,pregnancy time,delivery time,body temperature and other reproductive rhythm and health indicators of adult mice,but it reduced w-LCC,W-MCC and other indicators reflecting the level of immune cells.The above results confirmed that endogenous melatonin was involved in the regulation of ovarian aging process and that melatonin could delay ovarian aging directly by inhibiting the loss of follicular reserve in the ovary.