The Synthesis Of Melatonin And Its Regulation Mechanism In Ovine Ovary

Melatonin is a hormone mainly synthesized in the pineal gland.As a potent antioxidant,melatonin removes free radicals from the body and protects the body from oxidative damage.In addition to regulating the hypothalamic pituitary affecting mammalian reproductive physiology,melatonin is directly involved in the regulation of ovarian function.The expression of melatonin membrane receptors MT1 and MT2has been detected in the mammalian reproductive system,and it has been found that the ovary itself is capable of synthesizing melatonin,suggesting that melatonin is involved in the regulation of ovarian function.However,it is unclear whether the synthesis of melatonin in the ovary and the expression of membrane receptors are regulated by other factors.In this study,ovine ovary was used as the research object.Firstly,the experimental techniques such as qPCR,western blotting and immunohistochemistry were used to analyze melatonin-synthesizing enzymes（HIOMT and AANAT）and membrane receptors（MT1 and MT2）expression and distribution characteristics in the corpus luteum（CL）of different stages of the estrous cycle and the cumulus–oocyte complexes（COCs）of different size follicles.Secondly,the effects of progesterone（P₄）,estradiol（E₂）and its receptor inhibitors on the expression of melatonin-related proteins in luteal cells and COCs were analyzed by in vitro culture of ovine luteal cells and COCs.Finally,the effect of melatonin on the proteome level in granulosa cells cultured in vitro was analyzed by Lable-free non-quantitative proteomics method,and the molecular mechanism of melatonin regulating the ovarian function of ovine was further elucidated.The main results of this study are as follows:1.In this experiment,qPCR,western blotting,immunohistochemistry,and ELISA were used to analyze the quantitative and localized expression of MT1,MT2,AANAT,and HIOMT in the CL of different stages of the estrous cycle in ovine,and the changes of melatonin levels in the CL.The results showed that MT1,MT2,AANAT,and HIOMT were highly expressed in middle stage CLs（days 6–12）compared to early（days 1–5）and late stages of CLs（days 13–18）.At the same time,melatonin levels in in middle stage CLs were significantly higher than in the early and late stages of CLs.The above results not only indicate that the ovine CL can synthesize melatonin,but also indicate that the CL is one of the targets of melatonin,suggesting that melatonin plays an important role in the development and maintenance of the CL.2.The luteal cells from middle phase CL were cultured,and the luteal cells were treated with different concentrations of P₄ and progesterone receptor（PGR）antagonist mifepristone（RU486）.By qPCR and western blotting,it was found that 10^-9 and 10^-8M P₄ could promote the expression of MT1 and MT2 in luteal cells.However,compared with 10^-8 M P₄ treatment group,10^-7 M P₄ inhibited the expression of MT1and MT2 in luteal cells.At the same time,RU486 also significantly inhibited the expression of MT1 and MT2 in luteal cells,and higher levels of P₄(10^-7 M)significantly inhibited the expression of PGR in luteal cells.This indicates that P₄promotes the expression of MT1 and MT2 in luteal cells through its nuclear receptor pathway.However,higher levels of P₄ blocked the expression of MT1 and MT2 by inhibiting the expression of PGR.3.In this experiment,the expressions of MT1,MT2,AANAT,and HIOMT in COCs of different size follicles and the changes of melatonin levels in different sizes of follicular fluid（FF）were analyzed by qPCR,western blotting,immunohistochemistry,and ELISA.The results showed that the expression levels of MT1,MT2,AANAT,and HIOMT in the COCs of small follicles were significantly higher than those in the large follicles.However,melatonin levels in large follicle FF were significantly higher than in small follicle FF.It not only indicated that melatonin participates in the regulation of follicles and oocytes through autocrine or paracrine pathways;it also indicated that the ovine COCs itself can synthesize and secrete melatonin,and in large follicles,the melatonin secreted by the COCs may mot be the main source of melatonin in the FF.4.The ovine COCs was cultured in vitro and treated with E₂ and the estrogen receptor（ER）inhibitor ICI182780.By qPCR and western blotting,it was found that E₂ decreased the expression of MT1,MT2,AANAT,and HIOMT as well as the secretion of melatonin in the COCs,but when ICI182780 was added,the expression of AANAT,HIOMT,MT1,MT2,as well as melatonin production were recovered.The above results indicated that E₂ is involved in the regulation of melatonin synthesis and membrane receptor expression in the COCs through the ER pathway.5.Differential proteins in ovine ovarian granulosa cells after treatment with 10^-9M melatonin were identified by Lable-free non-labeled quantitative proteomics analysis.A total of 64 differential proteins were found by biological analysis.Among the granulosa cells treated with melatonin,18 proteins were up-regulated and 6 were down-regulated.In addition,a total of 34 proteins were not expressed in the melatonin-treated group but were expressed in the control group,while 6 proteins were expressed in the melatonin-treated group and not in the control group.The expression of five differential proteins SOD1,EGFR,CAV1,FADD and COLIA1 was verified by western blotting.The results showed that the expression trend of selected proteins was basically consistent with the Lable-free identification results,indicating that the results obtained by Lable-free were reliable.Combined with bioinformatics analysis,melatonin is involved in the regulation of ovarian granulosa cell function mainly through anti-oxidant,anti-apoptotic and inflammatory responses.In summary,melatonin-related proteins and melatonin levels were expressed in the CL of different stages and the COCs of different size follicles of ovine.And the expression of melatonin-related proteins and the synthesis of melatonin are regulated by E2 and P4;in addition,melatonin can participate in the regulation of ovarian granulosa cell function in ovine through various pathways such as anti-oxidation,anti-apoptosis and inflammatory reaction.This result provides basic data for further research on the regulation mechanism of ovine reproductive mechanism.