The Effect of Insulin-like Growth Factor (IGF-1) on Heat Shock Protein 70 (HSP70) Expression of Yak (Bos grunniens) Cumulus Cells and Its Relation with Apoptosis
Abstract：The aim of this study is to verify whether the expression of HSP70 (heat shock protein, HSP) and apoptosis of yak (Bos grunniens) cumulus cells were regulated by IGF-1 (insulin-like growth factor, IGF-1). The yak cumulus cells were cultured in vitro supplemented with different concentrations of IGF-1 (0, 50, 100 and 200 ng/mL). The relative expression levels of HSP70 mRNA and protein in different groups were detected by Real-time quantitative PCR (qRT-PCR) and Western blot (WB), and location of HSP70 protein was detected by immunofluorescence. Furthermore, relative expression levels of Bax (B-cell lymphoma/leukemia associated x protein, Bax) and Bcl-2 (B-cell lymphoma/Leukemia-2, Bcl-2) mRNA in different groups were also evaluated by qRT-PCR, flow cytometry was used to evaluate the rate of apoptosis. The result showed that: (1) IGF-1 improved the relative levels of HSP70 mRNA and protein; these improvements were highest in the 100 ng/mL IGF-1 group, followed by 200 ng/mL and 50 ng/mL groups, the relative levels of HSP70 mRNA and protein were lowest in control group (0 ng/mL IGF-1); (2) Detection by immunofluorescence showed that HSP70 protein could be observed in yak cumulus cells cytoplasm as well as within nuclei; (3) Relative level of Bax was down-regulated by IGF-1, while Bcl-2 was up-regulated. The Bax mRNA was lowest in groups with 100 ng/mL IGF-1, where the Bcl-2 was the highest. In 200 and 50 ng/mL IGF-1 groups, the relative expression levels of Bax was decreased compared with that of in 100 ng/mL group, while relative expression levels of Bcl-2 was increased. Bax expression in the groups with IGF-1 was lower than the group without IGF-1(control group), while Bcl-2 expression was higher in groups with IGF-1 than without IGF-1; (4) IGF-1 inhibited apoptosis of yak cumulus cells, and the rate of apoptosis in cumulus cells was (6.32±0.84)% in 100 ng/mL IGF-1 group, which was lowest among these groups. In 200 and 50 ng/mL IGF-1 groups, the apoptosis rates were (13.85±2.01)% and (25.42±1.63)%, respectively, both of them were significantly higher than that in 100 ng/mL IGF-1 group (P＜0.05). Apoptosis rate of cumulus cells was (26.14±3.16)% in control group, which was lowest among these groups. The results demonstrated that expressions of HSP70, Bax and Bcl-2 on yak cumulus cells were regulated by IGF-1, and the apoptosis rate of cumulus cells was also reduced; the optimum concentration was 100 ng/mL. The results in this study provide new insights for the molecular mechanism of IGF-1 in inhibiting the apoptosis, and also provide theoretical foundation to explore the functions of IGF-1 during yak oocytes maturation and preimplantation embryo development, which will be helpful to improve the technique of yak embryo cultured in vitro.
Adamson, L., Walum, E. Insulin and IGF-1 mediated inhibition of apoptosis in CHO cells grown in suspension in a protein-free medium (J). Alternatives to laboratory animals, 2007, 35: 349~352.Adashi, E.Y., Resnick, C.E., D'Ercole, A.J., Svoboda, M.E., Van Wyk, J.J. Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function(J). Endocrine reviews, 1985, 6: 400~420.Byrne, A.T., Southgate, J., Brison, D.R., Leese, H.J. Regulation of apoptosis in the bovine blastocyst by insulin and the insulin-like growth factor (IGF) superfamily(J). Molecular reproduction and development, 2002, 62, 489~495.Chen, R.M., Chen, T.L., Chiu, W.T., Chang, C.C. Molecular mechanism of nitric oxide-induced osteoblast apoptosis(J). Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 2005, 23: 462-468.Dolt, K.S., Mishra, M.K., Karar, J., Baig, M.A., Ahmed, Z., Pasha, M.A. cDNA cloning, gene organization and variant specific expression of HIF-1 alpha in high altitude yak (Bos grunniens) (J). Gene, 2007,386: 73~80.Dunn, S.E., Hardman, R.A., Kari, F.W., Barrett, J.C. Insulin-like growth factor 1 (IGF-1) alters drug sensitivity of HBL100 human breast cancer cells by inhibition of apoptosis induced by diverse anticancer drugs(J). Cancer research, 1997, 57: 2687~2693.Gilchrist, R.B., Lane, M., Thompson, J.G. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality(J). Human reproduction update, 2008,14:159~177.Hernandez-Gonzalez, I., Gonzalez-Robayna, I., Shimada, M., Wayne, C.M., Ochsner, S.A., White, L., Richards, J.S. Gene expression profiles of cumulus cell oocyte complexes during ovulation reveal cumulus cells express neuronal and immune-related genes: does this expand their role in the ovulation process? (J). Molecular endocrinology (Baltimore, Md.), 2006,20:1300~1321.Kelly, S., Zhang, Z.J., Zhao, H., Xu, L., Giffard, R.G., Sapolsky, R.M., Yenari, M.A., Steinberg, G.K. Gene transfer of HSP72 protects cornu ammonis 1 region of the hippocampus neurons from global ischemia: influence of Bcl-2(J). Annals of neurology, 2002, 52: 160~167.Kenchappa, P., Yadav, A., Singh, G., Nandana, S., Banerjee, K. Rescue of TNFalpha-inhibited neuronal cells by IGF-1 involves Akt and c-Jun N-terminal kinases(J). Journal of neuroscience research, 2004, 76: 466~474.Leibowitz, B., Yu, J. Mitochondrial signaling in cell death via the Bcl-2 family. Cancer biology & therapy, 2010, 9: 417~422.Lisle, R.S., Anthony, K., Randall, M.A., Diaz, F.J. Oocyte-cumulus cell interactions regulate free intracellular zinc in mouse oocytes(J). Reproduction (Cambridge, England), 2013, 145: 381~390.Lolicato, F., Brouwers, J.F., de Lest, C.H., Wubbolts, R., Aardema, H., Priore, P., Roelen, B.A., Helms, J.B., Gadella, B.M. The cumulus cell layer protects the bovine maturing oocyte against Fatty Acid-induced lipotoxicity(J). Biology of reproduction, 2015, 92: 16.Lourenco, B., Sousa, A.P., Almeida-Santos, T., Ramalho-Santos, J. Relation of cumulus cell status with single oocyte maturity, fertilization capability and patient age(J). Journal of reproduction & infertility, 2014, 15: 15~21.Massey, A.J., Williamson, D.S., Browne, H., Murray, J.B., Dokurno, P., Shaw, T., Macias, A.T., Daniels, Z., Geoffroy, S., Dopson, M., Lavan, P., Matassova, N., Francis, G.L., Graham, C.J., Parsons, R., Wang, Y., Padfield, A., Comer, M., Drysdale, M.J., Wood, M. A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells(J). Cancer chemotherapy and pharmacology, 2010, 66: 535~545.Monget, P., Fabre, S., Mulsant, P., Lecerf, F., Elsen, J.M., Mazerbourg, S., Pisselet, C., Monniaux, D. Regulation of ovarian folliculogenesis by IGF and BMP system in domestic animals(J). Domestic animal endocrinology, 2002, 23: 139~154.Peruzzi, F., Prisco, M., Dews, M., Salomoni, P., Grassilli, E., Romano, G., Calabretta, B., Baserga, R. Multiple signaling pathways of the insulin-like growth factor 1 receptor in protection from apoptosis. Molecular and cellular biology, 1999, 19: 7203~7215.Pfaffl, M.W., Horgan, G.W., Dempfle, L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR(J). Nucleic acids research, 2002, 30: e36.Ravagnan, L., Gurbuxani, S., Susin, S.A., Maisse, C., Daugas, E., Zamzami, N., Mak, T., Jaattela, M., Penninger, J.M., Garrido, C., Kroemer, G. Heat-shock protein 70 antagonizes apoptosis-inducing factor(J). Nature cell biology, 2001, 3: 839~843.Singh, B., Armstrong, D.T. Insulin-like growth factor-1, a component of serum that enables porcine cumulus cells to expand in response to follicle-stimulating hormone in vitro(J). Biology of reproduction, 1997, 56: 1370~1375.Uyar, A., Torrealday, S., Seli, E. Cumulus and granulosa cell markers of oocyte and embryo quality(J). Fertility and sterility, 2013, 99: 979~997.Valleh, M.V., Hyttel, P., Rasmussen, M.A., Strobech, L. Insulin-like growth factor 2: a modulator of anti-apoptosis related genes (HSP70, BCL2-L1) in bovine preimplantation embryos(J). Theriogenology, 2014, 82:942~950.Wang, L.M., Feng, H.L., Ma, Y., Cang, M., Li, H.J., Yan, Z., Zhou, P., Wen, J.X., Bou, S., Liu, D.J. Expression of IGF receptors and its ligands in bovine oocytes and preimplantation embryos(J). Animal reproduction science, 2009,114:99~108.Yaseen, M.A., Wrenzycki, C., Herrmann, D., Carnwath, J.W., Niemann, H. Changes in the relative abundance of mRNA transcripts for insulin-like growth factor (IGF-I and IGF-II) ligands and their receptors (IGF-IR/IGF-IIR) in preimplantation bovine embryos derived from different in vitro systems(J). Reproduction (Cambridge, England), 2001, 122: 601~610.Yerushalmi, G.M., Salmon-Divon, M., Yung, Y., Maman, E., Kedem, A., Ophir, L., Elemento, O., Coticchio, G., Dal Canto, M., Mignini Renzinu, M., Fadini, R., Hourvitz, A. Characterization of the human cumulus cell transcriptome during final follicular maturation and ovulation(J). Molecular human reproduction, 2014, 20: 719~735.Zhang B(张彬), Deng HB(邓红兵), Zhou B(周斌), Tan SP(谭斯品), Jiang BM(蒋碧梅). Heat Shock Protein 70 Protected Against Oxidative Stress-Induced Apoptosis in C2C12Myogenic Cells byUpregulation of Bcl-2. Ch in J A rteriosc ler, 2010, 18: 621~624.Zhao XM(赵学明), Du WH(杜卫华), Hao HS(郝海生), Wang D(王栋), Zhu HB(朱化彬). Effects of Cumulus Cells on Bovine Embryo Produced by In vitro Fertilization. Journal of Agricultural Biotechnology, 2011,19:375-380.Zhu, Q., Xu, Y.M., Wang, L.F., Zhang, Y., Wang, F., Zhao, J., Jia, L.T., Zhang, W.G., Yang, A.G. Heat shock protein 70 silencing enhances apoptosis inducing factor-mediated cell death in hepatocellular carcinoma HepG2 cells(J). Cancer biology & therapy, 2009, 8: 792~98.
