A generalized caspase inhibitor disrupts early mammalian development

Abstract

The role and mechanism of cell death in early mammalian embryos is not well understood. In mouse embryos collected after fertilization and maintained in vitro until blastula formation, two instances of cell death are observed: the polar bodies and one or two cells near the equator, at the junction of the inner cell mass to the prototrophoblast. Inhibitors of caspases do not block the death of the polar bodies. Inhibitors of caspases 3, 7 and 8 do not affect post-cavitation death, but the pan-caspase inhibitor zVAD-FMK, when applied at the 1-2 cell stage, causes an expansion of post-cavitation death and ultimately malformation or death of the embryo. Our results indicate that the early deaths are not caspase-dependent and that there is a role for caspase activity in early embryos, which is not related to cell death. The caspase cascade is a major component of apoptosis in development and in many other situations. Presently most researchers assume that caspases function only in cell death, even though related family members serve, for instance, in the activation pathway for immunologically competent cells. If caspases have other roles in cell function, it is appropriate to ask what roles they play in early development. There have been a number of reports on cell death in pre-implantation embryos. The polar bodies begin to die during earliest cleavage, but cell death in the pre-implantation embryo itself begins at about the 8-cell stage. The cell death in the embryo is apoptotic by the criterion of TUNEL positivity (Matwee, Betts and King, 2000). The expression of caspases 2 and 3 (but not caspase 1) has been shown for blastocysts (Weil, Jacobson, Coles, Davies, Gardner, Raff and Raff, 1996). A few cells routinely die in the blastocyst (Handyside and Hunter, 1986) (Weil, Jacobson, Coles, Davies, Gardner, Raff and Raff, 1996). However, pre-implantation development is not disrupted by individually knocking out Bcl-2, Bax, Bcl-X, Bcl-W, or caspases 1, 2,3,8,9, or 11 (Pampfer and Donnay, 1999).During embryonic development of mammals, zygotic genes are translated as early as the 2-cell stage. The first sign of cell death is seen at the 6-8 cell stage, followed by further deaths at the blastula stage. It is believed that cell death in these stages relates to cavitation and blastocyst formation and the elimination of potential trophectoderm cells from the inner cell mass (Pierce, Lewellyn and Parchment, 1989). Although cavitation occurs synchronously with cell death, at least in embryoid bodies, this death requires apoptosis-inducing factor but is independent of caspase expression and does not manifest all the characteristics of apoptosis (Joza, Susin, Daugas, Stanford, Cho, Li, Sasaki, Elia, Cheng, Ravagnan, Ferri, Zamzami, Wakeham, Hakem, Yoshida, Kong, Mak, Zuniga-Pflucker, Kroemer and Penninger, 2001).To determine if specific caspases have different roles in the different stages of preimplantation mouse embryos, we exposed the developing embryos to known caspase inhibitors, the fluoromethylketone (FMK) deriva...

Life-giving caspases: revealing new roles during mouse embryo preimplantation development

“…As mentioned previously, caspase activities have been related to these processes in different somatic and cancer cells. In embryos, Zakeri et al, showed for the first time that a nonlethal caspase activity is present before implantation (Zakeri et al, 2005). In agreement with those sudies, our results showed that the general caspase inhibitor zVAD significantly affected development, with 2-cell embryos showing an arrest at the 4-to 8-cell stage and thus never reaching the blastocyst stage.…”

“…Although blastomeres are susceptible to programmed cell death in unfavourable culture conditions, the ocurrence of apoptosis under physiological conditions is rare before implantation, suggesting that caspases could have non-lethal functions during preimplantation development (Pampfer et al, 1999). Recenty, Zakeri et al, showed that the culture of mouse embryos with the generalized caspase inibitor zVAD did not only fail to prevent the death of ICM cells in culture but, conversely, disrupted the normal development of embryos to the blastocyst stage (Zakeri et al, 2005).…”

Molar tooth development in caspase-3 deficient mice

“…Such compensation has been described in vitro (Zheng et al, 2000). Multiple caspases and regulators of caspase activation may have evolved to provide mammals with greater plasticity to respond to more diverse stimuli, explaining why knock-outs of only one caspase do not lead to complete developmental arrest (Zheng et al, 1999;Zakeri et al, 2005). Compensatory caspase activation could thus bypass the requirement for caspase-3 in tooth development.…”

Efecto de la Suplementación del Medio de Maduración con Cisteamina y de Dos Medios de Cultivo (KSOMaa y SOF) en la Fecundación in vitro de Ovocitos Bovinos

“…Esto, sumado a la alteración en la dinámica de distribución y función mitocondrial, desencadena la apoptosis en distintas etapas del desarrollo y el bloqueo de embriones en el cuarto ciclo celular (Serrano y Olivera- Angel, 2003 Diversos estudios sugieren que esta apoptosis se debería a la expresión de genes proapoptóticos como el NF8B (Takada et al, 2003) y p53 (Velez-Pardo et al, 2007); sin embargo, estos han sido encontrados en distintas etapas embrionarias y en embriones competentes. Por otro lado, se ha demostrado que el bloqueo de las caspasas resulta nocivo para el embrión, aunque no está claro el mecanismo de acción (Zakeri et al, 2005).…”