Abstract
Aim:
To examine the cytotoxic effects of genistein, an isoflavone compound, on early postimplantation embryonic development in vitro.
Methods:
Mouse blastocysts were incubated in medium with or without genistein (25 or 50 μmol/L) or daidzein (50 μmol/L) for 24 h. Cell proliferation and growth was investigated by dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and apoptotic or necrotic cells were visualized by Annexin-V and propidium iodide (PI) staining. Implantation and postimplantation development of embryos were measured by in vitro development analysis.
Results:
TUNEL staining and Annexin-V/PI staining showed that genistein dose-dependently increased apoptosis in mouse blastocysts, while daidzein, another soy isoflavone, had no such effect. The pretreatment of the blastocysts with genistein caused fewer cells than the control group and this effect was primary in the inner cell mass. The genistein-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer genistein-pretreated embryos reached the later stages of embryonic development versus the controls, with many of the former embryos dying at relatively early stages of development. In addition, genistein treatment decreased the development of morulas into blastocysts, and dietary genistein was found to induce cell apoptosis and decrease cell proliferation in an animal assay model of embryogenesis.
Conclusions:
Our results collectively indicate that genistein treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death in vitro, while dietary genistein appears to negatively affect mouse embryonic development in vivo by inducing cell apoptosis and inhibiting cell proliferation. These novel findings provide important new insights into the effect of genistein on mouse blastocysts.
Similar content being viewed by others
Article PDF
References
Ellis RE, Yuan J Y, Horvitz HR . Mechanisms and functions of cell death. Annu Rev Cell Biol 1991; 7: 663–98.
Thompson CB . Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267: 1456–62.
Hardy K . Cell death in the mammalian blastocyst. Mol Hum Reprod 1997; 3: 919–25.
Hardy K, Stark J, Winston RM . Maintenance of the inner cell mass in human blastocysts from fragmented embryos. Biol Reprod 2003; 68: 1165–9.
Byrne AT, Southgate J, Brison DR, Leese HJ . Analysis of apoptosis in the preimplantation bovine embryo using TUNEL. J Reprod Fertil 1999; 117: 97–105.
Dong W, Teraoka H, Yamazaki K, Tsukiyama S, Imani S, Imagawa T, et al. 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the zebrafish embryo: local circulation failure in the dorsal midbrain is associated with increased apoptosis. Toxicol Sci 2002; 69: 191–201.
Little SA, Kim WK, Mirkes PE . Teratogen-induced activation of caspase-6 and caspase-7 in early postimplantation mouse embryos. Cell Biol Toxicol 2003; 19: 215–26.
Hsuuw YD, Chang CK, Chan WH, Yu JS . Curcumin prevents methylglyoxal-induced oxidative stress and apoptosis in mouse embryonic stem cells and blastocysts. J Cell Physiol 2005; 205: 379–86.
Chan WH . Ginkgolides induce apoptosis and decrease cell numbers in mouse blastocysts. Biochem Biophys Res Commun 2005; 338: 1263–7.
Chan WH . Ginkgolide B induces apoptosis and developmental injury in mouse embryonic stem cells and blastocysts. Hum Reprod 2006; 21: 2985–95.
Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, et al. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 1987; 262: 5592–5.
Markovits J, Linassier C, Fosse P, Couprie J, Pierre J, Jacquemin–Sablon A, et al. Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res 1989; 49: 5111–7.
Constantinou A, Kiguchi K, Huberman E . Induction of differentiation and DNA strand breakage in human HL-60 and K-562 leukemia cells by genistein. Cancer Res 1990; 50: 2618–24.
Yanagihara K, Ito A, Toge T, Numoto M . Antiproliferative effects of isoflavones on human cancer cell lines established from the gastrointestinal tract. Cancer Res 1993; 53: 5815–21.
Rauth S, Kichina J, Green A . Inhibition of growth and induction of differentiation of metastatic melanoma cells in vitro by genistein: chemosensitivity is regulated by cellular p53. Br J Cancer 1997; 75: 1559–66.
Brown A, Jolly P, Wei H . Genistein modulates neuroblastoma cell proliferation and differentiation through induction of apoptosis and regulation of tyrosine kinase activity and N-myc expression. Carcinogenesis 1998; 19: 991–7.
Azuma Y, Onishi Y, Sato Y, Kizaki H . Induction of mouse thymocyte apoptosis by inhibitors of tyrosine kinases is associated with dephosphorylation of nuclear proteins. Cell Immunol 1993; 152: 271–8.
Bergamaschi G, Rosti V, Danova M, Ponchio L, Lucotti C, Cazzola M . Inhibitors of tyrosine phosphorylation induce apoptosis in human leukemic cell lines. Leukemia 1993; 7: 2012–8.
McCabe MJ Jr, Orrenius S . Genistein induces apoptosis in immature human thymocytes by inhibiting topoisomerase-II. Biochem Biophys Res Commun 1993; 194: 944–50.
Migita K, Eguchi K, Kawabe Y, Mizokami A, Tsukada T, Nagataki S . Prevention of anti-CD3 monoclonal antibody-induced thymic apoptosis by protein tyrosine kinase inhibitors. J Immunol 1994; 153: 3457–65.
Bronte V, Macino B, Zambon A, Rosato A, Mandruzzato S, Zanovello P, et al. Protein tyrosine kinases and phosphatases control apoptosis induced by extracellular adenosine 5′-triphosphate. Biochem Biophys Res Commun 1996; 218: 344–51.
Johnson KL, Vaillant F, Lawen A . Protein tyrosine kinase inhibitors prevent didemnin B-induced apoptosis in HL-60 cells. FEBS Lett 1996; 383: 1–5.
Heo JS, Han HJ . PKC and MAPKs pathways mediate EGF–induced stimulation of 2–deoxyglucose uptake in mouse embryonic stem cells. Cell Physiol Biochem 2006; 17: 145–58.
Bogoliubova NA, Bogoliubova IO, Parfenov VN, Sekirina GG . Characteristic of structural and functional organization of two-cell mouse embryos exposed to inhibitors of cell proliferation. Tsitologiia 1999; 41: 698–706.
Jacquet P, de Saint-Georges L, Barrio S, Baugnet-Mahieu L . Morphological effects of caffeine, okadaic acid and genistein in one-cell mouse embryos blocked in G2 by X-irradiation. Int J Radiat Biol 1995; 67: 347–58.
Goval JJ, Alexandre H . Effect of genistein on the temporal coordination of cleavage and compaction in mouse preimplantation embryos. Eur J Morphol 2000; 38: 88–96.
Pampfer S, de Hertogh R, Vanderheyden I, Michiels B, Vercheval M . Decreased inner cell mass proportion in blastocysts from diabetic rats. Diabetes 1990; 39: 471–6.
Hardy K, Handyside AH, Winston RM . The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 1989; 107: 597–604.
Gardner RL, Davies TJ . Lack of coupling between onset of giant transformation and genome endoreduplication in the mural trophectoderm of the mouse blastocyst. J Exp Zool 1993; 265: 54–60.
Huang FJ, Wu TC, Tsai M Y . Effect of retinoic acid on implantation and post-implantation development of mouse embryos in vitro. Hum Reprod 2001; 16: 2171–6.
Witschi E . Characterization of developmental stages. Part II. Rat. In: Biology Data Book, 2nd ed. Washington, DC: Federation of American Societies of Experimental Biologies; 1972. p 178–80.
Huang FJ, Shen CC, Chang SY, Wu TC, Hsuuw YD . Retinoic acid decreases the viability of mouse blastocysts in vitro. Hum Reprod 2003; 18: 130–6.
Gu Y, Zhu CF, Iwamoto H, Chen JS . Genistein inhibits invasive potential of human hepatocellular carcinoma by altering cell cycle, apoptosis, and angiogenesis. World J Gastroenterol 2005; 11: 6512–7.
Raffoul JJ, Wang Y, Kucuk O, Forman JD, Sarkar FH, Hillman GG . Genistein inhibits radiation–induced activation of NF-kappaB in prostate cancer cells promoting apoptosis and G2/M cell cycle arrest. BMC Cancer 2006; 6: 107.
Zhou JR, Mukherjee P, Gugger ET, Tanaka T, Blackburn GL, Clinton SK . Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis. Cancer Res 1998; 58: 5231–8.
Uckun FM, Tuel-Ahlgren L, Song CW, Waddick K, Myers DE, Kirihara J, et al. Ionizing radiation stimulates unidentified tyrosine-specific protein kinases in human B-lymphocyte precursors, triggering apoptosis and clonogenic cell death. Proc Natl Acad Sci USA 1992; 89: 9005–9.
Liu Y, Bhalla K, Hill C, Priest DG . Evidence for involvement of tyrosine phosphorylation in taxol-induced apoptosis in a human ovarian tumor cell line. Biochem Pharmacol 1994; 48: 1265–72.
Chan WH, Yu JS . Inhibition of UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermal carcinoma A431 cells by genistein. J Cell Biochem 2000; 78: 73–84.
Cao F, Jin TY, Zhou YF . Inhibitory effect of isoflavones on prostate cancer cells and PTEN gene. Biomed Environ Sci 2006; 19: 35–41.
Pinski J, Wang Q, Quek ML, Cole A, Cooc J, Danenberg K, et al. Genistein-induced neuroendocrine differentiation of prostate cancer cells. Prostate 2006; 66: 1136–43.
Morris C, Thorpe J, Ambrosio L, Santin M . The soybean isoflavone genistein induces differentiation of MG63 human osteosarcoma osteoblasts. J Nutr 2006; 136: 1166–70.
Cross JC, Werb Z, Fisher SJ . Implantation and the placenta: key pieces of the development puzzle. Science 1994; 266: 1508–18.
Mueller SO, Simon S, Chae K, Metzler M, Korach KS . Phyto–estrogens and their human metabolites show distinct agonistic and antagonistic properties on estrogen receptor alpha (ERalpha) and ERbeta in human cells. Toxicol Sci 2004; 80: 14–25.
Murrill WB, Brown NM, Zhang JX, Manzolillo PA, Barnes S, Lamartiniere CA . Prepubertal genistein exposure suppresses mammary cancer and enhances gland differentiation in rats. Carcinogenesis 1996; 17: 1451–7.
Allred CD, Ju YH, Allred KF, Chang J, Helferich WG . Dietary genistein stimulates growth of estrogen-dependent breast cancer tumors similar to that observed with genistein. Carcinogenesis 2001; 22: 1667–73.
Cotroneo MS, Wang J, Fritz WA, Eltoum IE, Lamartiniere CA . Genistein action in the prepubertal mammary gland in a chemo-prevention model. Carcinogenesis 2002; 23: 1467–74.
Jeschke U, Briese V, Richter DU, Bruer G, Plessow D, Waldschlager J, et al. Effects of phytoestrogens genistein and daidzein on production of human chorionic gonadotropin in term trophoblast cells in vitro. Gynecol Endocrinol 2005; 21: 180–4.
Plessow D, Waldschlager J, Richter DU, Jeschke U, Bruer G, Briese V, et al. Effects of phytoestrogens on the trophoblast tumour cell lines Be Wo and Jeg3. Anticancer Res 2003; 23: 1081–6.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Chan, Wh., Lu, Hy. & Shiao, Nh. Effect of genistein on mouse blastocyst development in vitro. Acta Pharmacol Sin 28, 238–245 (2007). https://doi.org/10.1111/j.1745-7254.2007.00498.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1745-7254.2007.00498.x