Mechanism of Action of an Environmentally Relevant Organochlorine Mixture in Repressing Steroid Hormone Biosynthesis in Leydig Cells

Abstract

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.

Differential Response of Transcription Factors to Activated Kinases in Steroidogenic and Non-Steroidogenic Cells

“…Penicillin and streptomycin sulphate were added to the cell culture media to a final concentration of 50 mg/L, and all cell lines were kept at 37 °C, 5% CO 2 in a humidified incubator. All cell lines were validated by morphology and Leydig cell lines by quantifying steroidogenic output (progesterone for MA-10) as previously described [ 25 , 27 , 39 , 73 , 74 , 75 , 76 , 77 ]. MA-10 (60,000 cells per well) and CV-1 (25,000 cells per well) were transiently transfected using polyethylenimine hydrochloride (PEI) (Sigma-Aldrich Canada, Oakville, ON, Canada) as previously described [ 39 , 78 ] or the calcium phosphate co-precipitation method as described in [ 23 , 24 ].…”

A 35-bp Conserved Region Is Crucial for Insl3 Promoter Activity in Mouse MA-10 Leydig Cells

“…Penicillin and streptomycin sulphate were added to the cell culture media to a final concentration of 50 mg/L, and cells were kept at 37 °C, 5% CO 2 in a humidified incubator. MA-10 Leydig cells were validated by morphology and by quantifying steroidogenic output, as previously described [ 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 ]. MA-10 cells were transiently transfected using polyethylenimine hydrochloride (PEI) (Sigma-Aldrich Canada, Oakville, ON, Canada), as previously described [ 61 , 63 , 64 ], or the calcium phosphate co-precipitation method, as described in [ 22 , 35 , 48 , 65 ].…”