Mechanism Involvement Of Melatonin And BMP-6 In The Regulation Of Ovine Luteinization Granulosa Cells

Luteinization of granulosa cells before ovulation is a necessary condition for follicular ovulation.Ovarian diseases such as persistent follicles in female animals are often accompanied by failure of granulosa cell luteinization process and failure to differentiate into luteal cells.Bone Morphogenetic Protein-6（BMP-6）inhibits the synthesis of progesterone before follicular rupture,thereby inhibiting the luteinization of granulosa cells.Melatonin（Mel）promotes progesterone synthesis in granulosa cells and participates in various physiological regulatory effects,including antioxidant,anti-inflammatory,and differentiation.However,it is unclear whether BMP-6 affects the luteinization process of sheep follicular granulosa cells through receptors BMPRIB and BMPRII in sheep ovaries,and whether Mel and BMP-6 participate in regulating the internal environment of sheep luteinized granulosa cells,thereby affecting the reproductive mechanism of ovulation.Therefore,in this study,mature follicular granulosa cells from sheep were used as the research object.Enzyme-linked immunosorbent assay（ELISA）,real-time fluorescence quantitative PCR（RT q PCR）,Western blotting,immunohistochemistry（IHC）,tissue and cell immune fluorescence（IHF）were used to explore the physiological roles of Mel and BMP-6 in luteinized granulosa cells of sheep follicles.The results are as follows:1.Expression patterns of BMP-6 and its main membrane receptors BMPRIB and BMPRII in sheep folliclesFirstly,the expression patterns of BMP-6 and its main membrane receptors BMPRIB and BMPRII in sheep follicles were verified,which is the basis for the physiological function of BMP-6.The distribution of BMP-6 and its main membrane receptors BMPRIB and BMPRII in small,medium,and large follicles of sheep was detected using IHC and IFC,and the results showed the expression of BMPRIB and BMPRII in granulosa cells.The expression patterns of BMP-6,BMPRIB,and BMPRII were further detected by RT q PCR and Western blotting methods.The results showed that BMP-6,BMPRIB,and BMPRII were distributed in sheep granulosa cells of large,medium,and small follicles,and were most significantly expressed in granulosa cells of large follicles.The above results indicate that sheep granulosa cells are also one of the target cells for BMP-6 to function,indicating that BMP-6 is involved in the physiological regulation of sheep follicles.2.Establishment,identification,and related research of luteinized granulosa cells from sheep follicles in vitroTo verify the regulation of Mel and BMP-6 on luteinized granulosa cells,mature granulosa cells were co treated with forskolin and insulin growth factor（IGF）to establish a sheep follicular luteinized granulosa cell model in vitro.The identification results of ELISA,IHF,RT q PCR,and Western blotting showed that the cells exhibited characteristic secretion of progesterone,expression of synthase,and absence of granulosa cell marker protein FSHR,indicating the successful establishment of the luteinized granulosa cell model.Subsequently,Mel membrane receptors MT1 and MT2,as well as BMP-6 membrane receptors BMPRIB and BMPRII,were detected to be highly expressed in LGCs,indicating their role in sheep luteinized granulosa cells.3.Effects of melatonin and rb BMP-6 on progesterone content,progesterone related synthase and mitochondrial membrane potential in LGCsAfter gradient concentration and time treatment,the secretion of progesterone and the expression of its synthase were detected using molecular experiments to determine the optimal time and concentration of rb BMP-6 treatment in vitro.Finally,the effects of rb BMP-6 and melatonin on LGCs were studied through co treatment.The results of molecular experiments and JC-1 experiments showed that melatonin not only relieved the ability of rb BMP-6 to inhibit progesterone synthesis in LGCs,They can also maintain mitochondrial homeostasis by protecting the mitochondrial membrane potential of LGCs,and inhibit the apoptosis of LGCs.4.Mechanism of melatonin and BMP-6 in sheep LGCs under internal environmental stressThe oxidative stress model and inflammation model of LGCs were established using H₂O₂and LPS to simulate the stressful environment of preovulatory follicles,respectively,and the regulatory effects of melatonin and rb BMP-6 on the oxidative stress and inflammation responses of LGCs induced by LPS and H₂O₂,respectively,were examined to investigate the mechanisms of their effects on the internal environment of LGCs in preovulatory follicles.The results showed that H₂O₂promoted the expression of apoptosis-related proteins by inhibiting the activation of ERK1/2 signaling,while LPS significantly activated the expression levels of Akt signaling and inflammatory factors in LGCs.rb BMP-6 further promoted the expression of H₂O₂-induced apoptotic proteins,while melatonin inhibited this effect.Secondly,rb BMP-6 also further promoted the expression of LPS-induced inflammatory factors,which melatonin rescinded.Further,after treatment of LGCs with luzindole（N-acetyl-2-benzyltryptamine）,a selective antagonist of melatonin membrane receptor MT1,and dorsomorphin,a receptor inhibitor of the BMP-6 receptor BMPRIB,in H₂O₂and LPS-treated groups,respectively,in vitro,MT1 was found to mediated the Mel/ERK1/2 and Mel/Akt pathways and inhibited the damaging effect of BMP-6 on LGCs after binding BMPRIB.While ERK1/2 inhibitor PD98059 and PI3K/Akt inhibitor LY294002 treated LGCs respectively,melatonin was found to undo the damaging effects of rb BMP-6 on LGCs in LPS and H₂O₂environments through MT1 activation ERK1/2 pathway or inhibition of PI3K/Akt phosphorylation.In a nutshell,BMP-6 and its receptor-related proteins are expressed in large,medium and small follicles of sheep.Melatonin via its high affinity receptor MT1 and BMP-6binded with BMPRIB are involved in regulating multiple physiological functions of LGCs.This experiments provides a basis for further exploring the effects of melatonin and BMP-6on the reproductive mechanisms and their regulation within the follicles during ovulation in females.