Research Paper Volume 9, Issue 4 pp 1269—1279

Protective effect of calretinin on testicular Leydig cells via the inhibition of apoptosis

Wendan Xu1, , Qian Zhu1, , Bei Zhang1, , Shan Liu2, , Xiaonan Dai3, , Chao Gao1, , Li Gao1, , Yugui Cui1, ,

  • 1 State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, 210029, China
  • 2 Center of Reproductive Medicine, Bethune International Peace Hospital, Hebei Shijiazhuang, China
  • 3 Nanjing Maternal and Child Care Service Center, Nanjing Medical University, Nanjing, 210005, China

Received: February 8, 2017       Accepted: April 17, 2017       Published: April 24, 2017      

https://doi.org/10.18632/aging.101226
How to Cite

Copyright: © 2017 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

The core mechanism of Late-onset hypogonadism (LOH) is the deficiency of androgen due to the functional and quantitative decline of testicular Leydig cells. Here we explored the protective effect of calretinin, a Ca2+-binding protein, on Leydig cells. We found in MLTC-1 cells transfected with LV-calb2, the cell viability and optical density (OD) were higher (p<0.05), cells in the S phase of the cell cycle were increased (p<0.01) and p-ERK1/2 and p-AKT levels were significantly higher (p<0.01 and p<0.05), while in R2C cells transfected with LV-siRNA-calb2, all of the results mentioned above were adverse (p<0.05). The cell apoptotic index after calretinin over-expressed was significantly lower (p<0.001), while the expression levels of mitochondria-related apoptotic factors such as cleaved caspase-9 and cytochrome C (cyto C) were lower and ratio of Bcl2/Bax was higher (p<0.05). After calretinin down-regulated, the apoptotic index was higher (p<0.05), while the expression levels of mitochondria-related apoptotic factors were higher and the ratio of Bcl2/Bax was lower (p<0.05). Therefore, calretinin increases Leydig cell viability and proliferation, possibly via ERK1/2 and AKT pathways, and suppresses apoptosis possibly via the mitochondria-related apoptotic pathway, which could be beneficial in understanding the pathophysiology of LOH and could lead to the study of new treatments.

Abbreviations

LH: Luteinizing hormone; HPG: hypothalamic-pituitary-gonadal; calb2: calcium retinal protein 2; SCLS: small cell lung cancer cells; WiDr: human colon cancer cell line; FBS: fetal bovine serum; CCK8: Cell Counting Kit-8 assay; Brdu: bromodeoxyuridine; BCA: bicinchoninic acid; EDTA: ethylenediaminetetraacetic acid; APS: ammonium persulfate; SDS: sodium dodecylsulfate; Tris: tris hydroxymethyl aminomethans; LOH: Late-onset Hypogonadism; DMSO: dimethyl sulfoxide; OD: optical density; HRP: horseradish peroxidase; TEMED: tetramethyl ethylenedamine; PVDF: polyvinylidene difluoride; DMEM-F12, Dulbecco's Modified Eagle Media: Nutrient Mixture F-12; APC: Allophycocyanin; BSA: bovine serum albumin; PVDF: polyvinylidene difluoride, RIPA, radioimmunoprecipitation assay; PI: pidium iodide; MOI: multiplicity of infection; ECL: enhanced chemiluminescence.