Reproduction, Mother and Child Health, Room T3-67, Centre de Recherche du CHU de Québec-Université Laval, CHUL, 2705 Laurier Blvd., Quebec City, QC G1V 4G2, Canada.
Centre for Research in Reproduction, Development and Intergenerational Health, Department of Animal Science, Faculty of Agricultural and Food Sciences, Université Laval, Quebec City, QC G1V 0A6, Canada.
Int J Mol Sci. 2022 Apr 3;23(7):3997. doi: 10.3390/ijms23073997.
Within Leydig cells, steroidogenesis is induced by the pituitary luteinizing hormone (LH). The binding of LH to its receptor increases cAMP production, which then activates the expression of genes involved in testosterone biosynthesis. One of these genes codes for the steroidogenic acute regulatory (STAR) protein. STAR is part of a complex that shuttles cholesterol, the precursor of all steroid hormones, through the mitochondrial membrane where steroidogenesis is initiated. Organochlorine chemicals (OCs) are environmental persistent organic pollutants that are found at high concentrations in Arctic areas. OCs are known to affect male reproductive health by decreasing semen quality in different species, including humans. We previously showed that an environmentally relevant mixture of OCs found in Northern Quebec disrupts steroidogenesis by decreasing STAR protein levels without affecting the transcription of the gene. We hypothesized that OCs might affect STAR protein stability. To test this, MA-10 Leydig cell lines were incubated for 6 h with vehicle or the OCs mixture in the presence or absence of 8Br-cAMP with or without MG132, an inhibitor of protein degradation. We found that MG132 prevented the OC-mediated decrease in STAR protein levels following 8Br-cAMP stimulation. However, progesterone production was still decreased by the OC mixture, even in the presence of MG132. This suggested that proteins involved in steroid hormone production in addition to STAR are also affected by the OC mixture. To identify these proteins, a whole cell approach was used and total proteins from MA-10 Leydig cells exposed to the OC mixture with or without stimulation with 8Br-cAMP were analyzed by 2D SDS-PAGE and LC-MS/MS. Bioinformatics analyses revealed that several proteins involved in numerous biological processes are affected by the OC mixture, including proteins involved in mitochondrial transport, lipid metabolism, and steroidogenesis.
在莱迪希细胞中，类固醇生成是由垂体促黄体生成素 (LH) 诱导的。LH 与受体结合会增加 cAMP 的产生，从而激活参与睾酮生物合成的基因表达。这些基因中的一个编码类固醇急性调节蛋白 (STAR)。STAR 是一种复合物的一部分，该复合物将胆固醇（所有类固醇激素的前体）穿梭通过线粒体膜，从而启动类固醇生成。有机氯化学品 (OCs) 是环境持久性有机污染物，在北极地区的浓度很高。OCs 已知会通过降低不同物种（包括人类）的精液质量来影响男性生殖健康。我们之前表明，在魁北克北部发现的与环境相关的 OCs 混合物通过降低 STAR 蛋白水平而不是影响基因转录来破坏类固醇生成。我们假设 OCs 可能会影响 STAR 蛋白的稳定性。为了验证这一点，MA-10 莱迪希细胞系在存在或不存在 8Br-cAMP 的情况下与载体或 OCs 混合物孵育 6 小时，有或没有 MG132（一种蛋白降解抑制剂）。我们发现，MG132 可防止 OC 介导的 8Br-cAMP 刺激后 STAR 蛋白水平下降。然而，即使存在 MG132，OC 混合物仍会降低孕激素的产生。这表明除了 STAR 之外，参与甾体激素生成的蛋白质也受到 OC 混合物的影响。为了鉴定这些蛋白质，采用了全细胞方法，并用 2D SDS-PAGE 和 LC-MS/MS 分析暴露于 OC 混合物的 MA-10 莱迪希细胞的总蛋白，无论是否用 8Br-cAMP 刺激。生物信息学分析表明，OC 混合物会影响许多生物学过程中的几种蛋白质，包括涉及线粒体转运、脂质代谢和类固醇生成的蛋白质。
