Androgenesis Regulated By Calretinin In Testicular Leydig Cells And Mechanisms

Background and Objectives:Spermatogenesis and androgenensis are the main roles of the testis,the former completed in seminiferous tubules and the latter in Leydig cells.The synthesis and secretion of testosterone are controlled by the hypothalamus-pituitary-gonad (HPG) axis.LH combining with its receptor (LHR), activates the enzyme cyclic adenosine, converting the ATP to cAMP, and then activates the protein kinase A (PKA) dependent on cAMP;activates phospholipase C (PLC), and then phosphatidyl inositol can be divided into two acyl glycerin (DAG) and three phosphoinositide(IP3),activating PKC.Both can promote the expression of steroid hormone enzymes such as STAR, leading to promote the synthesis of testosterone.IP3 activates the sarcoplasmic reticulum calcium pool and the cytoplasm calcium ions increased,together with DAG,activates PKC;Ca2+ as a second messenger, is also invoLVed in the synthesis of sex hormones.In addition to the endocrine regulation, androgen synthesis is also influenced by paracrine and autocrine, such as insulin-like growth factor, prolactin, thyroid hormone, interleukin 1 (IL-1), promoting cell division factor, tumor necrosis factor (TNF alpha), etc.So androgen synthesis of leydig cells is adjusted by the complex network of endocrine, autocrine and paracrine factors.Calcium retinal protein (calretinin) is a kind of calcium binding protein, mainly expressing in the nervous system, also existing in the ovary, testis.The main effect of calretinin is to prevent calcium overload,as the buffer of intracellular calcium ion concentration.Also it can be used as a Ca2+ receptor.Preliminary studies showed that calretinin was expressed in Leydig cells in rats and Leydig cells expresseed highest calretinin at the adult time when highest androgen was systhesed;testosterone was increased after calretinin was over-expressed in leydig cells.In conclusion, we assume that the calretinin as the local factor, can affect the intracellular calcium ion concentration,promote Leydig cells androgen synthesis by PKC pathway, at the same time, calretinin may also promot Leydig cell proliferation and inhibit apoptosis to indirectly promote the testicular androgen synthesis.To test our hypothesis, we use leydig cells as the model to study the influence of calretinin on androgen synthesis of Leydig cells,and the mechanism of action.Materials and methods:(l)Verify the LV-calb2 and LV-siRNA-calb2 lentivirus, respectively transfecting mouse Leydig cell line MLTC-1 and rat Leydig cells R2C,Then the validity of transfection virus expression was provesd by immunofluorescence technique, Western blot method;(2)MLTC-1, R2C cells were transfected by the LV-calb2 and LV-siRNA-calb2 lentivirus respectively. After in vitro 96 h, apoptosis was detected by flow cytometry technology.(3)MLTC-1, R2C cells were transfected by the LV-calb2 and LV-siRNA-calb2 lentivirus respectively. The change of the expression of the proliferation and apoptosis related factors AKT, p-AKT, Bcl2, Bax, cytochromeC, cleaved-caspase3/9, caspase-3/9,cleaved-PARP, PARP was observed by Western blot.(4)R2C cells were transfected by LV-siRNA-calb2 lentivirus. After in vitro 96 h, serum steroid hormone levels were determinated via ria method.(5)R2C cells were transfected by LV-siRNA-calb2 lentivirus.The change of the expression of protein invoLVed in PKC signaling pathways,such as PLC,p-PKCu (PKD),PKD,p-MARCKS,MARCKS,CREB,STAR was observed by Western blot.(6)MLTC-1,R2C cells were transfected by the LV-calb2 and LV-siRNA-calb2 lentivirus respectively The change of cytoplasmic Ca2+was tested by the confocal microscopy with the calciumfluorescent probe X-Rhod-1;(7)Statistical methods. The SPSS 17.0 software was used for statistical analysis. All data were presented as meanistandard deviation (Means ± SD). The single-factor sample was compared using independent sample t test. P<0.05 was represented significant difference.Results:(1)MLTC-1 was infected by LV-calb2,after 96 hours,the expression of calretinin was up-regulated(p<0.001) whilethe vector group didn’t express calretinin.R2C cells were infected by LV-siRNA-calb2, after 96 hours,the expression of calretinin was down-regulated (p<0.05)and the interference efficiency was up to 60%, showing that the building of LV-calb2 and LV-siRNA-calb2 lentiviral vectors was efficient(2) After the over-expression of calretinin, apoptosis rate was reduced in MLTC-1 (p< 0.001) and after the intervention of calretinin,apoptosis rate in R2C was inceased (p< 0.05).(3)After over expression of calretinin in MLTC-1 cells,the p-AKT, Bcl2/ Baxexpression was increased (p< 0.05) while,CytochromeC, cleaved caspase3/9, caspase-3/9, cleaved-PARP, PARP expression was decreased(p< 0.05),expression of AKT was not changed(p>0.05).;After intervention of calretinin in R2C expression of p-AKT, Bcl2/Baxwas reduced (p< 0.05) while CytochromeC(p< 0.05), cleaved caspase3/9, caspase-3/9, cleaved-PARP, PARP expression was increased expression of AKT was not changed.(4)After gene interference of calretinin, secretion of progesteron in R2C cells was decreased (p< 0.05).(5)After the intervention of calretinin,theexpression of PKC pathway signal molecules:PLC, p-PKD, p-MARCKS, MARCKS, CREB,STAR,was reduced (p< 0.05) and expression of PKD was not changed(p>0.05).(6) After the over-expression of calretinin intracellular calcium level(p<0.001) was increasesed while interference calretinin intracellular calcium levels wasreducted(p<0.05).Conclusion:(1) Calretinin promptes the steriodogenesis in leydig cells.(2) Calretinin may act as a local factor to regulate steriodogenesis in leydig cells by the PLC-Ca2+-PKC signaling pathways.(3) Calretininpromotes the activity of Leydig cells, increases cells in S phase meaning increase of cell proliferation activity, inhibits the apoptosis of leydig cells and its mechanism may be through strengthening PI3K-AKT and ERK1/2 signal to increase cell proliferation activityand suppress the mitochondrial apoptotic pathway in order to inhibits the cell apoptosis.(4) This research proves calretinin participates in the regulation of testicular androgen synthesis and leydig cell proliferation and apoptosis, and also discusses its mechanism of action, theoretically significant to further clarify intracellular signal mechanism of the testosterone synthesis in Leydig cells, and clinically significant to study and remedy male gonadal dysfunction diseases such as LOH.