ESTIMACIÓN DE COSTOS

The metaphase state is characterised by the activity of two kinases. These are MPF (M phase/maturation-promoting factor) and mitogen-activated protein (MAP) kinase, although evidence for the role of MAP kinase at mitotic metaphase is hmited (see below). MPF is composed of a regulatory cyclin B subunit (Evans et al., 1983; Lohka et al., 1988) and cychn-dependent kinase (CDKl) (Gautier et al., 1988; Labbe et al., 1989; Nurse and Thuriaux, 1980). The activation of cyclin Bl-CDK is dependent upon cychn binding and a series of phosphorylation and dephosphorylation reactions (Morgan, 1997). Cyclin B l- CDK inactivation is triggered by the proteolytic destruction of ubiquitin-tagged cyclin B via the APC (Imiger gr aZ., 1995; King et al., 1995; Sudakin et al., 1995). It has been shown that cyclin B destruction is dependent on the N-terminal 90 amino acids (Murray and Kirschner, 1989), which contain the destruction box sequence (see above) that confers the ability to become ubiquitinated (Glotzer et a l, 1991).

Progression of the meiotic cell cycle is controlled by the activity of MPF (Masui and Markert, 1971; Murray and Kirschner, 1989; Newport and Kirschner, 1984). Primary oocytes have low levels of MPF activity. Progesterone secretion (stimulated by LH) induces the activation of MPF, leading to meiosis I (MI). MPF activity peaks at metaphase of MI, falls briefly between the two meiotic divisions, rises again as the MU spindle is

assembled, and then remains high during the ME arrest (Gerhart et al., 1984). The rapid transition from MI to MU and the MU arrest are maintained by an activity named cytostatic factor (CSF). CSF stabilises cyclin (by blocking the abihty of active MPF to induce cychn degradation) and maintains MPF levels (Masui, 1974) and involves the Mos/MAP kinase pathway (Colledge e ta l, 1994; Hashimoto e ta l, 1994; Verlhac e ta l, 1996). Experiments have shown that MAP kinase activity is capable of maintaining the meiotic metaphase state, characterised by condensed chromosomes and the absence of a nuclear envelope (Moos et a l, 1996b; Verlhac e ta l, 1994).

Fertihsation leads rapidly to inactivation of MPF and interphase of the first mitotic ceU cycle. MPF activity is no longer stabilised by the MAP kinase pathway (Palmer and Nebreda, 2000). The activity of MPF declines just before second polar body formation and that of Mos/MAP kinase decreases just before formation of the pronuclei (Verlhac et a l,

1994). Inactivation of CSF was originally believed to be due to degradation of the c-mos product by the Ca^^-dependent cysteine protease calpain (Watanabe et a l, 1989) but is now thought to be caused by degradation of the c-mos product by the 26S proteasome (Ishida et a l, 1993). Although CSF stabilises MPF at the ME arrest, MPF inactivation and CSF inactivation at egg activation are independent events, at least in Xenopus eggs (Watanabe et a l, 1991).

After fertilisation, each mitosis is initiated by the activation of MPF and terminated by its inactivation. MPF activity thus rises each time the embryo enters mitosis and then falls as it enters the next interphase (Gerhart et a l, 1984). However, the role of the MAP kinase cascade in mitotic cell cycles is unclear. Some studies have shown that MAP kinase activity is elevated during mitosis, at least in mammalian ceU lines (Shapiro et a l, 1998) and early sea urchin embryos (Chiri et a l, 1998; Philipova and Whitaker, 1998). However, MAP kinase activity is thought to be low in mouse oocytes during mitosis (Verlhac et a l, 1994).

It has been demonstrated that cyclin B turns over during the metaphase II arrest in mouse oocytes. A constant protein level is maintained for several hours through the continuous balanced synthesis and degradation of cyclin (Kubiak et a l, 1993). The equihbrium between these two processes is thought to be dependent in part upon CSF. The degradation of cychn B during metaphase arrest, unlike stimulated degradation and release from metaphase arrest at egg activation (see below), is not dependent on a Ca^^ increase. The presence of the intact metaphase spindle was originally found to be required for cychn destruction both during the metaphase arrest and at exit from M-phase in mouse oocytes (Kubiak et a l, 1993). An intact metaphase spindle has since been shown to be necessary for cychn destruction in many other ceh types. An exception is the cycling extracts of fertilised Xenopus egg, in which MPF cycles independently of an intact spindle (Gerhart et a l, 1984), although at very high densities of sperm nuclei, microtubule depolymerisation arrests extracts in mitosis (Minshull et a l, 1994). In cells other than the Xenopus oocyte,

exposure to the microtubule-depolymerising drug nocodazole is associated with a reduced rate of cyclin B degradation and it has been proposed that cyclin B and the proteins that degrade it must physically interact on the spindle to allow effective degradation and entry into interphase (Kubiak et al., 1993). This is supported by the finding that, after oocyte bisection, MPF activity is predominantly associated with the spindle (Kubiak et a l, 1993). Furthermore, oocytes fertilised or parthenogenetically activated in the presence of nocodazole, fail to progress to interphase, despite responding with normal Ca^^ transient patterns (Winston et a l, 1995). The reason for this spindle “checkpoint” is that the chromosomes must be distributed accurately to the daughter cells (fertilised oocyte and second polar body) upon egg activation and errors will arise if chromosomes are improperly attached to the meiotic spindle. Arresting fertilised oocytes at metaphase of the second meiotic division by disrupting the spindle can be an important tool used to investigate the effect of the cell cycle on processes associated with sperm-egg fusion.