Research Paper Volume 9, Issue 4 pp 1269—1279

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

Wendan Xu 1, , Qian Zhu 1, , Bei Zhang 1, , Shan Liu 2, , Xiaonan Dai 3, , Chao Gao 1, , Li Gao 1, , Yugui Cui 1, ,

  • 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

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.