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


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


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).

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.

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.


Download data is not yet available.
Abstract 36 | PDF Downloads 13 XML Downloads 0 HTML Downloads 1


1. Bieniek JM, Kashanian JA, Deibert CM, Grober ED, Lo KC, Brannigan RE, et al. Influence of increasing body mass index on semen and reproductive hormonal parameters in a multi-institutional cohort of subfertile men. Fertil Steril. 2016; 106:1070–5.
2. Chen Z, Wen D, Wang F, Wang CB, Yang L. Curcumin protects against palmitic acid-induced apoptosis via the inhibition of endoplasmic reticu-lum stress in testicular Leydig cells. Reprod Biol Endocrin. 2019; 17:71–80. s12958-019-0517-4
3. Haider SG. Cell biology of Leydig cells in the tes-tis. Int Rev Cytol. 2014;233:181–241. https://doi. org/10.1016/S0074-7696(04)33005-6
4. Park SJ, Kim TS, Park CK, Lee SH, Lee DS. hCG-induced endoplasmic reticulum stress triggers apoptosis and reduces steroidogenic enzyme expression through activating transcription factor 6 in Leydig cells of the testis. J Mol Endocrinol. 2013;50(2):151– 66.
5. Yang L, Lei L, Zhao Q, Gong Y, Guan GP, Huang SX. C-type natriuretic peptide/natriuretic peptide receptor 2 is involved in cell proliferation and testoster-one production in mouse Leydig cells. World J Mens Health. 2019;37(2): 186–98. wjmh.180041
6. Lu ZH, Mu YM, Wang BA, Li XL, Lu JM, Li JY, et al. Saturated free fatty acids, palmitic acid and stearic acid, induce apoptosis by stimulation of ceramide generation in rat testicular Leydig cell. Biochem Biophys Res Co. 2003;303(4):1002–7.
7. Kim JH, Park SJ, Kim TS, Park HJ, Park J, Kim BY, et al. Testicular hyperthermia induces unfolded protein response signaling activation in spermatocyte. Biochem Bioph Res Co. 2013;434(4):861–6. https://
8. Rashid HO, Yadav RK, Kim HR, Chae HJ. ER stress: Autophagy induction, inhibition and selection. Autophagy. 2015;11(11):1956–77. https://doi. org/10.1080/15548627.2015.1091141
9. Sovolyova N, Healy S, Samali A, Logue SE. Stressed to death – Mechanisms of ER stress-induced cell death. Biol Chem. 2014;395(1):1–13.
10. Cnop M, Foufelle F, Velloso LA. Endoplasmic reticulum stress, obesity and diabetes. Trends Mol Med. 2012;18(1):59–68. molmed.2011.07.010
11. Guzel E, Arlier S, Guzeloglu-Kayisli O, Tabak M, Ekiz T, Semerci N, et al. Endoplasmic reticulum stress and homeostasis in reproductive physiology and pathology. Int J Mol Sci. 2017;18(4):792. https://
12. Okumura M, Kadokura H, Hashimoto S, Yutani K, Kanemura S, Hikima T, et al. Inhibition of the functional interplay between endoplasmic reticulum (ER) oxidoreduclin-1alpha (Ero1alpha) and protein-disulfide isomerase (PDI) by the endocrine disruptor bisphenol A. J Biol Chem. 2014;289(39):27004–18.
13. Lv QZ, Guo KK, Zhang GF, Zhang YM. The ORF4 protein of porcine circovirus type 2 (PCV2) antagonizes apoptosis by stabilizing the concentration of ferritin heavy chain (FHC) through physical inter-action. J Gen Virol. 2016;97(7):1636–46. https://doi. org/10.1099/jgv.0.000472
14. Lv QZ, Zhuo YL, Zhang YW, Deng JH, Tan XM, Lin Q. Construction of recombinant lentivirus vectors with porcine Hsp40 gene for over expression and interference. Chin Vet Sci. 2019;49(1):76–84. https://doi.10.16656/j.issn.1673-4696.2019.0220
15. Lv QZ, Guo KK, Xu H, Wang T, Zhang YM. Identification of putative ORF5 protein of porcine circovirus type 2 and functional analysis of GFP-fused ORF5 protein. PLoS One.2015;10(6):e0127859.
16. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta C(T)) method. Methods. 2001;25(4):402–8. meth.2001.1262
17. Zhang CC, Kang KK, Ning PB, Peng YX, Lin Z, Cui HJ, et al. Heat shock protein 70 is associated with CSFV NS5A protein and enhances viral RNA replication. Virology. 2015;482:9–18. http://dx.doi. org/10.1016/j.virol.2015.02.014
18. Aggerholm AS, Thulstrup AM, Toft G, Ramlau-Hansen CH, Bonde JP . Is overweight a risk factor for reduced semen quality and altered serum sex hormone profile? Fertil Steril. 2008;90(3):619–26.
19. Ramlau-Hansen CH, Hansen M, Jensen CR, Olsen J, Bonde JP, Thulstrup AM. Semen quality and reproductive hormones according to birthweight and body mass index in childhood and adult life: Two decades of follow-up. Fertil Steril. 2010;94(2):610– 18.
20. Paasch U, Grunewald S, Kratzsch J, Glander HJ. Obesity and age affect male fertility potential. Fertil Steril. 2010;94(7):2898–901. https://doi. org/10.1016/j.fertnstert.2010.06.047
21. Katib A. Mechanisms linking obesity to male infertility. Cent European J Urol. 2015;68(1):79–85.
22. Kim ED, McCullough A, Kaminetsky J. Oral enclomiphene citrate raises testosterone and pre-serves sperm counts in obese hypogonadal men, unlike topical testosterone: Restoration instead of replacement. BJU Int. 2016;117(4):677–85. https://
23. Roth MY, Amory JK, Page ST. Treatment of male infertility secondary to morbid obesity. Nat Clin Pract Endoc. 2008;4(7):415–19. https://doi. org/10.1038/ncpendmet0844
24. Raman JD, Schlegel PN. Aromatase inhibitors for male infertility. J Urol. 2002;167(2):624–9. https://
25. Kajiwara T, Tanaka T, Kukita K, Kutomi G, Tamura Y. Hypoxia augments MHC class I antigen presentation via facilitation of ERO1-alpha-mediated oxidative folding in murine tumor cells. Eur J Immunol. 2016;46(12):2842–51. https://doi. org/10.1002/eji.201646525
26. Lee S, Lee E, Ko E, Ham M, Lee HM, Kim ES, et al. Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-alpha and MMP-9. Cancer Lett. 2018;437:25–34. https://
27. Lai L, Liu Y, Liu YY, Zhang N, Wu D. Role of endoplasmic reticulum oxidase 1α in H9C2 cardiomyocytes following hypoxia/reoxygenation injury. Mol Med Rep. 2020;22:1420–8. mmr.2020.11217