| Melatonin (MEL) is a natural antioxidant which is of strong effect in body. It not only can remove free radicals, but also can enhance the effect of antioxidant enzymes to reduce the damage of ROS to the body. At present the research about the antioxidant function of melatonin are mostly focused on mammalian oocytes, but the study for the granulosa cells which also play an important role in the process of follicular is less, especially the related reports have not yet seen in birds. The female maternal line of TianFu goose was taken as experimental material to study the levels of melatonin in goose egg yolk and the expression level of antioxidant enzymes (SOD1, GPxl, CAT) in granular cells of different size follicles, discussing the antioxidant effect of melatonin in the process of follicular development. The goose granulosa cells were as a model at the same time, we preliminary discussed the antioxidant effect of melatonin in goose granulosa cells, and H2O2 was used to simulate oxidative stress induce granulosa cell be damaged, finally the granulosa cells were pretreated with melatonin before the effect of H2O2, further study the melatonin can protect granulosa cell from the damage caused by H2O2, confirming the antioxidant function of melatonin in granulosa cells. The main results were as follows:(1)The level of melatonin in goose egg yolk of different size follicles was measured. It was found that the content of melatonin showed a increasing trend in the process of follicular maturation, the highest (8.865 pg/ml) was F1, and the follicles were higher than plasma (5.811 pg/ml). These results suggested that melatonin play an important role in the process of follicular development.(2)The gene expression level of antioxidant enzymes SOD1, GPxl and CAT in granular cells of different size follicles were detected. There were not obvious development trend for gene expression of the three antioxidant enzymes. The expressional peak of SOD1 and GPxl occurred in F2 (P< 0.05), separately up to 23.5 and 10.4. However as for CAT, there were higher expression in 4-6 mm,8-10 mm, especially in F4 (P< 0.05).(3) Goose granulosa cells which used as models were incubated with exogenous MEL (0, 0.1,1,10 ng/ml). After incubation, the cell viability was improved (P<0.05), the contents of ROS in cells were less, the expression level of MEL receptor (P<0.05), antioxidant enzymes SOD, GPx, CAT (P<0.05) and anti-apoptotic gene BCL-2 (P>0.05) were up, the pro-apoptotic gene CASPASE3 (P<0.05) was inhibited. These results initially indicated MEL played an antioxidant effect in goose granulosa cells so that inhibiting cell apoptosis.(4) The granulosa cells were incubated with H2O2 (0,10,100,1000 umol/L) to study the effect of oxidative stress on granulosa cells. We found that H2O2 reduced the activity of granulosa cells (P<0.05), inhibited gene (P<0.05) and protein (P>0.05) expression of SOD, promoted gene and protein expression of GPx, CAT, and MEL receptor (P<0.05) The gene expression of BCL-2 was inhibited and CASPASE3 was increased by H2O2 (P<0.05). Above findings showed H2O2 could induce granulosa cell apoptosis.(5) The granulosa cells were incubated with 0.1 ng/ml and 1 ng/ml MEL for 24h, and then treated with 1000 umol/L H2O2 for 24h. We found that MEL could reduce ROS quantity and cell apoptotic rate caused by H2O2, the gene expressions of CAT, MEL1C and CASPASE3 which increased by H2O2 were lower under the effect of MEL (P<0.05), and BCL-2 was upregulated (P<0.05). These results further indicated MEL play an important antioxidant effect in the growth procession of granulosa cells which can protect granulosa cell from the damage caused by H2O2. |
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