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At fertilization, an increase of cytosolic calcium ions ([Ca.sup.2+]) triggers various activation responses in animal eggs (1,2). In vertebrates, these responses include exit from metaphase arrest in meiosis II (MII [...]

In vertebrates, unfertilized eggs are arrested at meiotic metaphase II (meta-II) by cytostatic factor (CSF), with Cdc2 activity maintained at a constant, high level. CSF is thought to suppress cyclin B degradation through the inhibition of the anaphase-promoting complex/cyclosome (APC/C)-Cdc20 while cyclin B synthesis continues in unfertilized eggs. Thus, it is a mystery how Cdc2 activity is kept constant during CSF arrest. Here, we show that the APC/C-Cdc20 can mediate cyclin B degradation in CSF-arrested Xenopus eggs and extracts, in such a way that when Cdc2 activity is elevated beyond a critical level, APC/C-Cdc20-dependent cyclin B degradation is activated and Cdc2 activity consequently declines to the critical level. This feedback control of Cdc2 activity is shown to be required for keeping Cdc2 activity constant during meta-II arrest. We have also shown that Mos/MAPK pathway is essential for preventing the cyclin B degradation from inactivating Cdc2 below the critical level required to sustain meta-II arrest. Our results indicate that under CSF arrest, Mos/MAPK activity suppresses cyclin B degradation, preventing Cdc2 activity from falling below normal meta-II levels, whereas activation of APC/C-Cdc20-mediated cyclin B degradation at elevated levels of Cdc2 activity prevents Cdc2 activity from reaching excessively high levels. Keywords: Cytostatic factor; Metaphase II arrest; Xenopus egg; Cyclin B degradation; The anaphase-promoting complex/cyclosome; Cdc20; Mos; MAP kinase

In the early embryonic cell cycle, exit from M phase is immediately followed by entry into S phase without an intervening gap phase. To understand the regulatory mechanisms for the cell cycle transition from M to S phase, we examined dependence on Cdc2 inactivation of cell-cycle events occurring during the M-S transition period, using Xenopus egg extracts in which the extent of Cdc2 inactivation at M phase exit was quantitatively controlled. The result demonstrated that MCM binding to and the initiation of DNA replication of nuclear chromatin occurred depending on the decrease of Cdc2 activity to critical levels. Similarly, we found that Cdc2 inhibitory phosphorylation and cyclin B degradation were turned on and off, respectively, depending on the decrease in Cdc2 activity. However, their sensitivity to Cdc2 activity was different, with the turning-on of Cdc2 inhibitory phosphorylation occurring at higher Cdc2 activity levels than the turning-off of cyclin B degradation. This means that, when cyclin B degradation ceases at M phase exit, Cdc2 inhibitory phosphorylation is necessarily activated. In the presence of constitutive synthesis of cyclin B, this condition favors the occurrence of the Cdc2 inactivation period after M phase exit, thereby ensuring progression through S phase. Thus, M phase exit and S phase entry are coordinately regulated by the Cdc2 activity level in the early embryonic cell cycle. Key Words: Cdc2; early embryonic cell cycles; Cdc2 inhibitory phosphorylation; S phase entry; Xenopus.

A required nonproteolytic proteasome function for the dissociation of Cdc2 and cyclin B at the end of M phase has been found to exist in Xenopus egg extracts. The 20S proteasome could not dissociate the two subunits, but the 26S proteasome could.

In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus blastula, chromatin tethering to the NE depends on nuclear filamentous actin that develops in a blastula-specific manner. To investigate whether chromatin tethering operates in the blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2–3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus blastula but was elevated after the blastula stage. When the LBR level was precociously elevated in the blastula by injecting LBR mRNA, it induced alterations in nuclear lamina architecture and nuclear morphology and caused DNA damage and abnormal mitotic spindles, depending on the N2–3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus blastula, as it is detrimental to embryonic development. [ABSTRACT FROM AUTHOR]

The ability to reprogram in vivo a somatic cell after differentiation is quite limited. One of the most impressive examples of such a process is transdifferentiation of pigmented epithelial cells (PECs) to lens cells during lens regeneration in newts. However, very little is known of the molecular events that allow newt cells to transdifferentiate. Histone B4 is an oocyte-type linker histone that replaces the somatic-type linker histone H1 during reprogramming mediated by somatic cell nuclear transfer (SCNT). We found that B4 is expressed and required during transdifferentiation of PECs. Knocking down of B4 decreased proliferation and increased apoptosis, which resulted in considerable smaller lens. Furthermore, B4 knock-down altered gene expression of key genes of lens differentiation and nearly abolished expression of-crystallin. These data are the first to show expression of oocyte-type linker histone in somatic cells and its requirement in newt lens transdifferentiation and suggest that transdifferentiation in newts might share common strategies with reprogramming after SCNT. [ABSTRACT FROM AUTHOR]

To investigate the regulatory mechanisms of the cell cycle transition from M phase to M phase in meiotic cycles, a Xenopus oocyte extract that performs the M-M transition has been developed. Using the meiotic extract, we found that a low level of Cdc2 activity remained at the exit of meiosis I (MI), due to incomplete degradation of cyclin B. The inactivation of the residual Cdc2 activity induced both entry into S phase and tyrosine phosphorylation on Cdc2 after MI. Quantitative analysis demonstrated that a considerable amount of Wee1 was present at the MI exit and Cdc2 inhibitory phosphorylation during this period was suppressed by the dominance of Cdc2 over Wee1. Consistently, the addition of more than a critical amount of Wee1 to the extract induced Cdc2 inhibitory phosphorylation, changing the M-M transition into an M-S-M transition. Thus, the Cdc2 activity remaining at MI exit is required for suppressing entry into S phase during the meiotic M-M transition period. [ABSTRACT FROM AUTHOR]