Differential effects of 6-DMAP, olomoucine and roscovitine on Xenopus oocytes and eggs

Abstract

The effects of the new cyclin-dependent kinase inhibitors, roscovitine and olomoucine, on oocytes and eggs of Xenopus laevis were investigated and compared with those of 6-dimethylamino purine (6-DMAP). The inhibitory properties of 6-DMAP, olomoucine and roscovitine towards p34cdc2-cyclin B isolated from Xenopus eggs revealed K-IC50 values of 300, 40 and 10 μM respectively. The three compounds inhibited progesterone-induced maturation with M-IC50 values of 200, 100 and 20 μM. These values were consistent with the K-IC50 values but the ratio M-IC50/K-IC50 was higher for roscovitine and olomoucine than for 6-DMAP. The disappearance of spindle and condensed chromosomes without pronucleus formation was observed when 1 mM 6-DMAP was applied for 4 h at germinal vesicle breakdown or at metaphase II, whereas no effect was observed using 1 mM olomoucine or 50 μM roscovitine. Changes in the electrophoretic mobility of p34cdc2 and erk2 were observed only in homogenates of matured oocytes or eggs exposed for 4 h to 1 mM 6-DMAP. When the drugs were microinjected into matured oocytes, olomoucine (100 μM) and roscovitine (50 μM) induced pronucleus formation more efficiently than did 6-DMAP (100 μM). Taken together, these results demonstrate that Xenopus oocytes possess a lower permeability to olomoucine and roscovitine and that these new compounds are suitable for in vivo studies after germinal vesicle breakdown provided they are microinjected.

Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract

“…Roscovitine, as an inhibitor of MPF, has been shown to be an effective activation agent in rhesus oocytes (Mitalipov et al 2001). We chose injection of roscovitine rather than culture with roscovitine as an activation treatment in this study because injection was reported to be the more effective treatment in Xenopus (Flament et al 2000), and injection was simply performed in our system by addition of roscovitine to the sperm extract solution. However, this treatment did not increase blastocyst development rates, and resulted in a conceptus that developed abnormally in early gestation and was lost before 114 days.…”

“…Roscovitine, an inhibitor of ATP binding to MPF, has been used via this activity to induce activation of oocytes, either parthenogenetically or after NT (Mitalipov et al 2001). In Xenopus, microinjection of roscovitine was found to be more effective for oocyte activation than was incubation in roscovitine-containing medium (Flament et al 2000). To our knowledge, the effect of injection of roscovitine on oocyte activation in mammalian species has not been evaluated.…”

“…Flow cytometric analysis was used to evaluate the cell-cycle status of donor cells prepared by different donor cell treatments, and its relationship to cell size. Different methods for activation of recombined oocytes, including addition of cycloheximide treatment and roscovitine injection as described by Flament et al (2000) were also evaluated. The viability of embryos produced by NT with roscovitine-treated donor cells was assessed after transfer of these embryos to recipient mares.…”

Nitric Oxide-Donor SNAP Induces Xenopus Eggs Activation

“…Completion of meiosis is attested by the extrusion of the second polar body and pronucleus formation. Following fertilization as well as following parthenogenetic activation, MPF drops within five minutes while MAPK remains active for 30 minutes before decreasing [22], [23]. Inactivation of MPF is mainly due to the degragation of the Cyclin B whereas inactivation of MAPK reflects the degradation of its upstream activator Mos [24].…”

Kicked by Mos and tuned by MPF—the initiation of the MAPK cascade inXenopusoocytes

“…In Xenopus oocytes, Mos had appeared as the best candidate for a MPF-regulated stimulator of the MAPK activation during meiosis, although Mos-independent mechanisms cannot be excluded. Indeed, if the first MPF activation burst at metaphase I is impaired by chemical inhibitors such as roscovitine and olomoucine (Flament et al, 2000) or through the use of Cdc2 negative mutants or Cdc2 inactivating antibodies (Nebreda et al, 1995), no MAPK activation is observed in Xenopus oocytes. Oligonucleotide strategies targeting cyclin B have also been employed to address the role of MPF during meiosis, but failed to prevent the activation peak at meiosis I because the latter relies on cyclins associated to Cdc2 under an inactive stored stock of MPF (called pre-MPF) and is not dependent upon cyclin B synthesis.…”