The determination of the relationship between endoplasmic reticulumassociated protein degradation (ERAD) with steroidogenesis in mouse testis and leydig cell lines

In recent studies with ERAD and steroidogenesis, it has shown that ERAD is regulated by androgens. It has reported that SVIP is ERAD inhibitor. There was no study conducted on interactions of ERAD proteins with proteins involved in steroidogenesis. We investigated expressions of SVIP, p97/VCP, StAR, CYP17A1 and 3β-HSD in mouse testicular tissue and Leydig cells to determine roles of ERAD proteins in steroidogenesis by immunofluorescence, immunohistochemistry, co-immunoprecipitation, Western blot, qRT-PCR and ELISA. We found StAR, CYP17A1 and 3β-HSD were colocalized with SVIP and p97/VCP in Leydig cells. A decrease in CYP17A1, 3β-HSD and StAR expressions was observed as a result of suppression of SVIP siRNAs and p97/VCP siRNAs expressions in MA10, TM3 and HLC in Leydig cells. While significant increase in SVIP expression was observed after transfection with SVIP siRNA and treatment of cells with hCG, there was no significant change in CYP17A1, 3β-HSD and StAR protein expressions after transfection with p97/VCP siRNA and treatment of cells with hCG in human Leydig cells. While significant decrease in gene expressions of SVIP and p97/VCP was observed in human Leydig cells transfected with control siRNA, SVIP siRNA and p97/VCP siRNA, no significant changes were observed in CYP17A1, 3β-HSD and StAR. StAR, CYP17A1 and 3β-HSD in HLC cells that were precipitated with p97/VCP, and it has determined that these proteins interact with p97/VCP. When testosterone levels were compared in cell media of transfected with control siRNA, SVIP siRNA and p97/VCP siRNA in human Leydig cells, significant decrease in testosterone levels was observed in SVIP siRNA and p97/VCP siRNA transfected groups. Testosteron levels were significantly increased after transfection with SVIP siRNA and treatment with hCG compared to transfected group with SVIP siRNA. We can say that; SVIP and p97/VCP, affect steroidogenesis in Leydig cells and SVIP and p97/VCP play role in testosterone synthesis.