Effect Of Sulfur Dioxide On Primary Mice Testis Leydig Cells Testosterone Synthesis And Autophagy

[Objecktive]Sulfur dioxide(SO2)is abundant in the environment.In this study,for investigating the effects of SO2 on the testosterone biosynthesis,we measured the testosterone content in the serum and cell culture medium,moreover,we measure the expression of genes involved in testosterone biosynthesis.To further study the exact mechanism,we added the ERK inhibitors(U0126)prior to treatment with SO2 derivatives,then investigate the testosterone biosynthesis and expression of genes involved in testosterone biosynthesis.To investigate the effects of SO2 on primary mice Leydig cells autophagy,we measure the expression of genes and protein involved in autophagy.[Method]In this study,we build an experimental model with male Kunming mice and primary mice Leydig cells.In vivo,twenty-four of the above male mice were randomly divided into control group and SO2 groups.One group of mice exposure to clean air is controls.Other groups were exposed in mixed gas containing 0.5 ppm sulfur dioxide.After exposure to SO2 for 60 days,the mice were injected with 20%urethane solution for euthanized.We collected serum and isolate testis carefully for further study.In vitro,the primary Leydig cells were cultured,the concentration of SO2 derivatives(Na2SO3 and NaHSO3 combination with 3:1 mole ratio)was confirmed with MMT proliferation assay.After exposure to 0.5 ppm SO2 and different concentrations of SO2 derivatives,the testosterone content in the serum and cell culture medium were measured by ELISA kit.The expression of genes involved in testosterone biosynthesis and autophagy was measured by RT-PCR.To further study the exact mechanism,we investigated the ERK1/2 genes and p-ERK1/2 protein expressions in primary leydig cells.In addition,we added the ERK inhibitors(U0126)prior to treatment with SO2 derivatives,then investigate the testosterone biosynthesis and expression of genes involved in testosterone biosynthesis.We used the Western Bloting to investigate the star and protein involved in autophagy.[Result]Compare with control,SO2 and SO2 derivatives can significantly increase the testosterone concentration in the mice serum and supernatant of culturing primary mice(P<0.01,P<0.001).When we added the concentration of SO2 derivatives,we found that the testosterone production in supernatant started to decreased.Genes involved in testosterone biosynthesis in mice testis and primary testis leydig cells(Star,Cypllal,Hsd3bl,Cypl7al and Hsdl7b3)were measured by qRT-PCR.In vivo,genes expression of Star,Cypllal,Cypl7al and Hsd17b3 was up-regulation(P<0.05,P<0.01,P<0.001),while Hsd3bl invariant exposure to sulfur dioxide in testis of mice.In vitro,after treating with various concentrations of SO2 derivatives for 24h and 48h,Star,Cyp11a1,Cyp17al and Hsd3bl were up-regulation,moreover,treating for the 48h,Hsd17b3 gene was up-regulation in primary leydig cells(P<0.05,P<0.01).Through the immunofluorescence,we found that SO2 derivatives can up-regulation the expression of P-ERK1/2 protein(P<0.01).When treating cells with ERK inhibitor in advance,the effect of SO2 derivatives on testosterone and genes involved in testosterone biosynthesis is recovered in primary mice Leydig cells.In addition,compare with control,the group of treating with SO2 derivatives can decrease the expression of genes involved in autophagy(P<0.05).When we added the concentration of SO2 derivatives,we find the expression of genes involved in autophagy was increased(P<0.05,P<0.01,P<0.001).We measured the expression of Star and the protein involved in autophagy,we found SO2 derivatives can increase the expression of Star protein(P<0.01),while decrease the expression of protein involved in autophagy(P<0.05,P<0.01).[Conclution]SO2 and SO2 derivatives contribute to testosterone biosynthesis maybe related to the ERK pathway.Moreover SO2 derivatives can affect the mice Leydig cells autophagy.