| Oxidative stress is one of the important factors affecting the development of mammalian follicles.Oxidative stress is mainly caused by the continuous increase of reactive oxygen species（ROS）,which is considered to be one of the main reasons for the damage of follicular granulosa cells and eventually apoptosis.Current studies have shown that apoptosis of granulosa cells is the initiating factor leading to follicular atresia,and atresia is mainly the process of granulosa cell apoptosis.In mammalian ovaries,the follicular blood vessels are distributed in theca interna.Due to the avascular environment formed in follicular,the granulosa cells are under hypoxic conditions and accompanied by the production of ROS,which has a negative impact on the development of follicles.The previous research of our group found that the hypoxic environment in vitro can induce the apoptosis of porcine follicular granulosa cells,which confirmed this conclusion.Melatonin（N-acetyl-5-methoxytryptamine,melatonin）is mainly an indole hormone secreted by the pineal gland of animals.Melatonin is a powerful antioxidant and exists in the follicles.Melatonin has been proven to be a powerful antioxidant found in follicles.During the development of the follicle,as the follicle cavity continues to grow,the concentration of melatonin is also increasing,which suggests that melatonin plays an important role in the development of the follicle.Under the premise of these research backgrounds,we hypothesized:Although there are a large number of follicles atresia,why some follicles develop to ovulation?What function does melatonin play in the follicle?We speculate that melatonin can scavenge ROS generated in hypoxic environment,alleviate the oxidative stress level of granular cells,and prevent granular cells from apoptosis.In order to verify the above conjecture,we cultured porcine granulosa cells in vitro,and added exogenous melatonin under hypoxic conditions to detect the effect of melatonin on the apoptosis of follicular granulosa cells in a hypoxic environment,and further explore the mechanism of action of melatonin.The test results are as follows:1.Melatonin can alleviate the apoptosis of granular cells caused by hypoxia.In order to detect the effect of melatonin on the apoptosis of granulosa cells induced by hypoxia,the primary granulosa cells isolated from porcine follicles were cultured in an incubator under normoxia or hypoxia（containing 1%O2）.After 12 hours of treatment,the total protein of cells was extracted,and the level of hypoxia inducible factor-1α（HIF-1α）was detected by Western blot.This result showed that HIF-1αincreased significantly in the hypoxia group,indicating that the hypoxia model was successfully established.Subsequently,different concentrations of melatonin were added to granular cells cultured under normoxia and hypoxia to detect cell viability and apoptosis-related protein Cleaved-Caspase 3.The test results showed that the hypoxic environment significantly reduced the cell viability of granular cells and promoted the expression of the apoptotic protein Cleaved-Caspase 3.The concentration of melatonin with 10-9,10-7 and 10-5 mol/L can rescue granular cells from apoptosis.In order to verify this result,melatonin with 10-7mol/L was selected in this study,the apoptosis rate of normoxia group（Normoxia）,hypoxia group（Hypoxia）and hypoxia plus melatonin group(Hypoxia+10-7MT)was detected by flow cytometry.The results showed Hypoxia significantly increases the level of apoptosis of granular cells,and melatonin can reverse this effect to a certain extent.These results indicate that melatonin can reduce the apoptosis of granulosa cells induced by hypoxic conditions.2.Melatonin can reduce the level of ROS in granular cells.Apoptosis rate of porcine granulosa cell induced by hypoxia is closely related to oxidative stress caused by ROS accumulation.In order to explore whether melatonin can eliminate ROS produced by granular cells in hypoxia,the experiment also set up normoxia group,hypoxia group and hypoxia plus melatonin group,respectively.We Used confocal laser microscope and flow cytometer to detect the level of ROS in each group of cells.The results suggested that the hypoxic group significantly increased the level of ROS in granulosa cells,indicating that hypoxic conditions made the granulosa cells in a state of oxidative stress,but the level of ROS,after the addition of melatonin,was significantly lower than hypoxic group.These results indicate that melatonin can eliminate the accumulation of ROS induced by hypoxia in granule cells and relieve the oxidative stress of granule cells.3.Melatonin inhibits hypoxia-induced apoptosis of granulosa cells by scavenging ROS.In order to explore the effect of hypoxia-induced ROS on porcine granulosa cell apoptosis,we added glutathione group as a positive control,and divided the cells into normoxia group（Normoxia）and normoxia plus melatonin group(Normoxia+10-7MT),normoxia plus glutathione group（Normoxia+GSH）,hypoxia group（Hypoxia）,hypoxia plus melatonin group(Hypoxia+10-7MT)and hypoxia plus glutathione group（Hypoxia+GSH）,flow cytometry and Western blot were used to detected the levels of ROS and apoptotic proteins in porcine granulosa cells in each group,respectively.The results showed that ROS level and Cleaved-Caspase 3 in the hypoxic group increased significantly.Subsequently,we extracted the total RNA of granulosa cells in the normoxia group,hypoxia group and hypoxia plus melatonin group,and detected the expression of antioxidant enzyme genes in the granulosa cells of each group by q RT-PCR technology,and found that melatonin can Promote the expression of antioxidant enzyme genes in granular cells under hypoxic environment.In short,melatonin can eliminate ROS and promote the expression of antioxidant enzymes to rescue granular cell apoptosis caused by oxidative stress.4.Explore the mechanism that melatonin rescues granular cell apoptosis.According to reports,follicular granulosa cells are mainly mediated by two apoptosis pathways,namely the death receptor pathway and the mitochondrial pathway.The Cleaved-Caspase 8 and Cleaved-Caspase 9 are the apoptotic active proteins of these two pathways respectively,which can activate the downstream Caspase 3,and eventually lead to cell apoptosis.Therefore,in this experiment,these two apoptotic pathway-related apoptotic proteins Caspase 8 and Caspase 9 were detected,and the results found that the low oxygen environment caused the activation of two proteins,and melatonin could inhibit the activation of Caspase 8 and Caspase 9,and this experiment verified this result with inhibitors of Caspase 8/9,respectively.In addition,We explored that the mechanism of melatonin function by adding melatonin receptor antagonists.Therefore,we added inhibitors of the melatonin membrane receptors MTNR1A,MTNR1B,and nuclear receptors ROR/RZR,and detected the level of the apoptotic protein Cleaved-Caspase 3.The results showed that when MTNR1B was inhibited,the level of the apoptotic protein Cleaved-Caspase 3 increased significantly,and the rate of apoptosis also increased significantly,indicating that melatonin can act by activating its membrane receptor MTNR1B.Subsequently,we further tested the protein concentration and activity of PKA downstream of MTNR1B,and the results showed that hypoxia plus melatonin can lead to a significant increase in the level and activity of PKA.In conclusion,the experiment concluded that melatonin functions through the MTNR1B-PKA pathway. |
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