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Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells

  • P.M.M. Leon (a1) (a2), V.F. Campos (a1) (a2), C. Kaefer (a1) (a2), K.R. Begnini (a1) (a2), A.J.A. McBride (a3), O.A. Dellagostin (a3), F.K Seixas (a2), J.C. Deschamps (a1) and T. Collares (a4)...


The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus–oocyte complexes (COC) were harvested and divided into two groups: G1 – morphologically healthy cells; and G2 – less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).


Corresponding author

All correspondence to: Tiago Collares. Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Campus Universitário s/n°, Caixa Postal 354, CEP 96010-900, Pelotas, RS, Brazil. Tel: +55 53 3275 7588. e-mail:


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Altieri, D.C. (2010). Survivin and IAP proteins in cell-death mechanisms. Biochem. J. 430, 199205.
Ambruosi, B., Lacalandra, G.M., Iorga, A.I., De, S.T., Mugnier, S., Matarrese, R., Goudet, G. & Dell'Aquila, M.E. (2009). Cytoplasmic lipid droplets and mitochondrial distribution in equine oocytes: Implications on oocyte maturation, fertilization and developmental competence after ICSI. Theriogenology 71, 1093–104.
Anguita, B., Paramio, M.T., Morato, R., Romaguera, R., Jimenez-Macedo, A.R., Mogas, T. & Izquierdo, D. (2009). Effect of the apoptosis rate observed in oocytes and cumulus cells on embryo development in prepubertal goats. Anim. Reprod. Sci. 116, 95106.
Badr, H., Bongioni, G., Abdoon, A.S., Kandil, O. & Puglisi, R. (2007). Gene expression in the in vitro-produced preimplantation bovine embryos. Zygote 15, 355–67.
Caillaud, M., Dell'Aquila, M.E., De, S.T., Nicassio, M., Lacalandra, G.M., Goudet, G. & Gerard, N. (2008). In vitro equine oocyte maturation in pure follicular fluid plus interleukin-1 and fertilization following ICSI. Anim. Reprod. Sci. 106, 431–9.
Chang, S.H., Cvetanovic, M., Harvey, K.J., Komoriya, A., Packard, B.Z. & Ucker, D.S. (2002). The effector phase of physiological cell death relies exclusively on the posttranslational activation of resident components. Exp. Cell Res. 277, 1530.
Choi, Y.H., Love, L.B., Varner, D.D. & Hinrichs, K. (2006). Blastocyst development in equine oocytes with low meiotic competence after suppression of meiosis with roscovitine prior to in vitro maturation. Zygote 14, 18.
Corn, C.M., Hauser-Kronberger, C., Moser, M., Tews, G. & Ebner, T. (2005). Predictive value of cumulus cell apoptosis with regard to blastocyst development of corresponding gametes. Fertil. Steril. 84, 627–33.
Dell'Aquila, M.E., Albrizio, M., Maritato, F., Minoia, P. & Hinrichs, K. (2003). Meiotic competence of equine oocytes and pronucleus formation after intracytoplasmic sperm injection (ICSI) as related to granulosa cell apoptosis. Biol. Reprod. 68, 2065–72.
Dhali, A., Anchamparuthy, V.M., Butler, S.P., Pearson, R.E., Mullarky, I.K. & Gwazdauskas, F.C. (2007). Gene expression and development of mouse zygotes following droplet vitrification. Theriogenology 68, 1292–8.
Filali, M., Frydman, N., Belot, M.P., Hesters, L., Gaudin, F., Tachdjian, G., Emilie, D., Frydman, R. & Machelon, V. (2009). Oocyte in-vitro maturation: BCL2 mRNA content in cumulus cells reflects oocyte competency. Reprod. Biomed. Online 19, 4309.
Galli, C., Colleoni, S., Duchi, R., Lagutina, I. & Lazzari, G. (2007). Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer. Anim. Reprod. Sci. 98, 3955.
Hinrichs, K. (2010). In vitro production of equine embryos: state of the art. Reprod. Domest. Anim 45, 38.
Hinrichs, K., Choi, Y.H., Walckenaer, B.E., Varner, D.D. & Hartman, D.L. (2007). In vitro-produced equine embryos: production of foals after transfer, assessment by differential staining and effect of medium calcium concentrations during culture. Theriogenology 68, 521–9.
Host, E., Mikkelsen, A.L., Lindenberg, S. & Smidt-Jensen, S. (2000). Apoptosis in human cumulus cells in relation to maturation stage and cleavage of the corresponding oocyte. Acta Obstet. Gynecol. Scand. 79, 936–40.
Jacobson, C.C., Choi, Y.H., Hayden, S.S. & Hinrichs, K. (2010). Recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection. Theriogenology 73, 1116–26.
Jeon, K., Kim, E.Y., Tae, J.C., Lee, C.H., Lee, K.S., Kim, Y.O., Jeong, D.K., Cho, S.K., Kim, J.H., Lee, H.Y., Riu, K.Z., Cho, S.G. & Park, S.P. (2008). Survivin protein expression in bovine follicular oocytes and their in vitro developmental competence. Anim. Reprod. Sci. 108, 319–33.
Kawamura, K., Sato, N., Fukuda, J., Kodama, H., Kumagai, J., Tanikawa, H., Nakamura, A., Honda, Y., Sato, T. & Tanaka, T. (2003). Ghrelin inhibits the development of mouse preimplantation embryos in vitro. Endocrinology 144, 2623–33.
Li, H.J., Liu, D.J., Cang, M., Wang, L.M., Jin, M.Z., Ma, Y.Z. & Shorgan, B. (2009). Early apoptosis is associated with improved developmental potential in bovine oocytes. Anim. Reprod. Sci. 114, 8998.
Matzuk, M.M., Burns, K.H., Viveiros, M.M. & Eppig, J.J. (2002). Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science 296, 2178–80.
McKenzie, L.J., Pangas, S.A., Carson, S.A., Kovanci, E., Cisneros, P., Buster, J.E., Amato, P. & Matzuk, M.M. (2004). Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum. Reprod. 19, 28692874.
Melka, M., Rings, F., Holker, M., Tholen, E., Havlicek, V., Besenfelder, U., Schellander, K. & Tesfaye, D. (2009). Expression of apoptosis regulatory genes and incidence of apoptosis in different morphological quality groups of in vitro-produced bovine pre-implantation embryos. Reprod. Domest. Anim. 45, 915–21.
Mortensen, C.J., Choi, Y.H., Ing, N.H., Kraemer, D.C., Vogelsang, M.M. & Hinrichs, K. (2010). Heat shock protein 70 gene expression in equine blastocysts after exposure of oocytes to high temperatures in vitro or in vivo after exercise of donor mares. Theriogenology 74, 374–83.
Park, S.Y., Kim, E.Y., Cui, X.S., Tae, J.C., Lee, W.D., Kim, N.H., Park, S.P. & Lim, J.H. (2006). Increase in DNA fragmentation and apoptosis-related gene expression in frozen–thawed bovine blastocysts. Zygote. 14, 125–31.
Parolin, M.B. & Reason, I.J. (2001). [Apoptosis as a mechanism of tissue injury in hepatobiliary diseases]. Arq. Gastroenterol. 38, 138–44.
Payton, R.R., Rispoli, L.A. & Edwards, J.L. (2010). General features of certain RNA populations from gametes and cumulus cells. J. Reprod. Dev. 56, 583–92.
Pfaffl, M.W., Horgan, G.W. & Dempfle, L. (2002). Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30, e36.
Pretheeban, T., Gordon, M., Singh, R., Perera, R. & Rajamahendran, R. (2009). Differential mRNA expression in in vivo produced pre-implantation embryos of dairy heifers and mature cows. Mol. Reprod. Dev. 76, 1165–72.
Rizos, D., Clemente, M., Bermejo-Alvarez, P., de La, F.J., Lonergan, P. & Gutierrez-Adan, A. (2008). Consequences of in vitro culture conditions on embryo development and quality. Reprod. Domest. Anim. 43, 4450.
Sabbatini, P., Lin, J., Levine, A.J. & White, E. (1995). Essential role for p53-mediated transcription in E1A-induced apoptosis. Genes Dev. 9, 2184–92.
Smits, K., Goossens, K., Van, S.A., Govaere, J., Hoogewijs, M., Vanhaesebrouck, E., Galli, C., Colleoni, S., Vandesompele, J. & Peelman, L. (2009). Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen–thawed in vitro blastocysts. BMC. Res. Notes 2, 246.
Squires, E.L., Carnevale, E.M., McCue, P.M. & Bruemmer, J.E. (2003). Embryo technologies in the horse. Theriogenology 59, 151–70.
Tremoleda, J.L., Stout, T.A., Lagutina, I., Lazzari, G., Bevers, M.M., Colenbrander, B. & Galli, C. (2003). Effects of in vitro production on horse embryo morphology, cytoskeletal characteristics, and blastocyst capsule formation. Biol. Reprod. 69, 1895–906.
van Montfoort, A.P., Geraedts, J.P., Dumoulin, J.C., Stassen, A.P., Evers, J.L. & Ayoubi, T.A. (2008). Differential gene expression in cumulus cells as a prognostic indicator of embryo viability: a microarray analysis. Mol. Hum. Reprod. 14, 157–68.
Wassarman, P.M. & Letourneau, G.E. (1976). RNA synthesis in fully grown mouse oocytes. Nature 261, 73–4.
Wrenzycki, C., Herrmann, D., Lucas-Hahn, A., Korsawe, K., Lemme, E. & Niemann, H. (2005). Messenger RNA expression patterns in bovine embryos derived from in vitro procedures and their implications for development. Reprod. Fertil. Dev. 17, 2335.
Yang, M.Y. & Rajamahendran, R. (2002). Expression of Bcl-2 and Bax proteins in relation to quality of bovine oocytes and embryos produced in vitro. Anim. Reprod. Sci. 70, 159–69.
Yuan, Y.Q., Van, S.A., Leroy, J.L., Dewulf, J., Van, Z.A., de, K.A. & Peelman, L.J. (2005). Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence. Theriogenology 63, 2147–63.
Zwain, I.H. & Amato, P. (2001). cAMP-induced apoptosis in granulosa cells is associated with up-regulation of P53 and Bax and down-regulation of clusterin. Endocr. Res. 27, 233–49.



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