ERO1α KNOCKDOWN ATTENUATES PALMITIC ACID-MEDIATED INHIBITION OF TESTOSTERONE SECRETION BY INHIBITING ENDOPLASMIC RETICULUM STRESS IN TESTICULAR LEYDIG CELLS

Background and objective Palmitic acid (PA), the most common saturated free fatty acid (FFA) in food, is related to obesity-related male infertility. The possible mechanism is PA-mediated inhibition of testosterone secretion. Endoplasmic reticulum (ER) oxidoreductin-1alpha (ERO1 alpha), an oxidase that is localized in the ER, plays an essential role in maintaining ER homeostasis and is related to hormone secretion. However, the role and underlying mechanisms of ERO1 alpha in PA-mediated inhibition of testosterone secretion have not been reported. Material and methods Murine Leydig tumor cell line 1 (MLTC-1) cells were treated with different doses of PA. Cell viability, testosterone secretion, and ERO1 alpha expression were measured by the Cell Counting Kit 8 (CCK-8) assay, enzyme-linked immunesorbent assay (ELISA), and Western blotting, respectively. Moreover, the expression of ER stress marker proteins (glucose-regulated protein 78 [GRP78] and CCAAT/enhancer-binding protein homologous protein [CHOP]) was also measured after treatment. Subsequently, the expression of ERO1 alpha was knocked down, and cell viability, testosterone secretion, and ER stress were measured after treatment with the PA or the ER stress agonist thapsigargin (TG, an ER stress inducer). Also, testosterone secretion was measured by ELISA when ER stress was inhibited by 4-phenylbutyric acid (4-PBA, an ER stress inhibitor). Results PA treatment reduced cell viability, induced ERO1 alpha expression, and enhanced the expression of the ER stress marker GRP78 and CHOP, while ERO1 alpha knockdown inhibits ER stress marker expression, promotes testosterone secretion, and enhances cell viability in PA-treated MLTC-1 cells. In addition, ERO1 alpha knockdown rescued the TG-induced the decrease in testosterone secretion and cell viability. Conclusions These findings suggest that PA inhibits testosterone secretion via ER stress and that ERO1 alpha knockdown ameliorates PA-induced decreases in testosterone via ER stress in testicular Leydig cells. Our results indicate the necessity of exploring the potential applications of ERO1 alpha as a target gene for restoring fertility in obese men.