ERO1α Knockdown Attenuates Palmitic Acid-Inhibited Testosterone Secretion by Endoplasmic Reticulum Stress in Testicular Leydig Cells

Main Article Content

Qi-Zhuang Lv
Ting Qin
Xin-Yun Qin
Xiao-Mei Liang
Ke-Yi Nong
Zi-Feng Gong
Li-Ying Liang
Lei Yang
Yu-lin Zhu

Keywords

: testicular Leydig cells; palmitic acid (PA); endoplasmic reticulum oxidoreduclin-1alpha (ERO1α); endoplasmic reticulum stress (ER stress); testosterone

Abstract

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 (ERO1α), 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 ERO1α 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 ERO1α expression were measured by the Cell Counting Kit 8 (CCK-8) assay, enzyme-linked immunesorbent assay (ELISA), and Western blotting, respectively. Moreover, the expres-sion 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 ofERO1α 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 ERO1α expression, and enhanced the expression of the ER stress marker GRP78 and CHOP, while ERO1α knockdown inhibits ER stress marker expression, promotes testosterone secretion, and enhances cell viability in PA-treated MLTC-1 cells. In addition, ERO1α knock-down 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 ERO1α knockdown ameliorates PA-induced decreases in testosterone via ER stress in testicular Leydig cells. Our results indi-cate the necessity of exploring the potential applications of ERO1α as a target gene for restoring fertility in obese men.

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