胰岛素样生长因子-1(IGF-1)对牦牛卵丘细胞热休克蛋白70 (HSP70)表达的影响及其与细胞凋亡的关联性分析

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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.

Key words： Insulin-like growth factor (IGF-1) Yak Cumulus cells Heat shock protein 70 (HSP70) Apoptosis

Received: 16 February 2015 Published: 09 July 2015

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	PAN Yang-Yang
	CUI Yan
	FAN Jiang-Feng
	LI Lu-Ru
	YU Si-Jiu

Cite this article:

PAN Yang-Yang,CUI Yan,FAN Jiang-Feng, et al. 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[J]. , 2015, 23(9): 1208-1216.

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http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2015/V23/I9/1208

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.