Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes

The c-mos proto-oncogene is expressed as a maternal mRNA in oocytes and early embryos of Xenopus laevis, but its translation product pp39mos is detectable only during progesterone-induced oocyte maturation. Microinjection of mos-specific antisense oligonucleotides into oocytes not only prevents expression of pp39mos, but also blocks germinal vesicle breakdown, indicating that it functions during reinitiation of meiotic division.     

A direct link of the Mos-MAPK pathway to Erp1/Emi2 in meiotic arrest of Xenopus laevis eggs

In vertebrates, unfertilized eggs (or mature oocytes) are arrested at metaphase of meiosis II by a cytoplasmic activity called cytostatic factor (CSF). The classical Mos-MAPK pathway has long been implicated in CSF arrest of vertebrate eggs, but exactly how it exerts CSF activity remains unclear. Recently, Erp1 (also called Emi2), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C) required for degradation of the mitotic regulator cyclin B (ref. 5), has also been shown to be a component of CSF in both Xenopus and mice. Erp1 is destroyed on fertilization or egg activation, like Mos. However, despite these similarities the Mos-MAPK (mitogen-activated protein kinase) pathway and Erp1 are thought to act rather independently in CSF arrest. Here, we show that p90rsk, the kinase immediately downstream from Mos-MAPK, directly targets Erp1 for CSF arrest in Xenopus oocytes. Erp1 is synthesized immediately after meiosis I, and the Mos-MAPK pathway or p90rsk is essential for CSF arrest by Erp1. p90rsk can directly phosphorylate Erp1 on Ser 335/Thr 336 both in vivo and in vitro, and upregulates both Erp1 stability and activity. Erp1 is also present in early embryos, but has little CSF activity owing, at least in part, to the absence of p90rsk activity. These results clarify the direct link of the classical Mos-MAPK pathway to Erp1 in meiotic arrest of vertebrate oocytes.     

Protein kinase C mediates neural induction in Xenopus laevis

Inductive cell interactions are essential in early embryonic development, but virtually nothing is known about the molecular mechanisms involved. Recently factors resembling fibroblast growth factor and transforming growth factor-beta were shown to be involved in mesoderm induction in Xenopus laevis, suggesting that membrane receptor-mediated signal transduction is important in induction processes. Here we report direct measurements of protein kinase C (PKC) activity in uninduced ectoderm, and in neuroectoderm shortly after induction by the involuting mesoderm, in Xenopus laevis embryos. Membrane-bound PKC activity increased three to fourfold in the induced neuroectoderm while the cytosolic PKC activity was decreasing, indicating that PKC activity was translocated during neural induction. A similar time- and dose-dependent translocation of activity was seen after incubation with the PKC activator 12-O-tetradecanoyl phorbol-13-acetate, which also induced neural tissue in competent ectoderm, suggesting that PKC is involved in the response to the endogenous inducing signal during neural induction.     

Cdc6 synthesis regulates replication competence in Xenopus oocytes

The early division cycles of an embryo rely on the oocyte's ability to replicate DNA. During meiosis, oocytes temporarily lose this ability. After a single round of pre-meiotic S-phase, oocytes enter meiosis and rapidly arrest at prophase of meiosis I (G2). Upon hormonal stimulation, arrested oocytes resume meiosis, re-establish DNA replication competence in meiosis I shortly after germinal vesicle breakdown (GVBD), but repress replication until fertilization. How oocytes lose and regain replication competence during meiosis are important questions underlying the production of functional gametes. Here we show that the inability of immature Xenopus oocytes to replicate is linked to the absence of the Cdc6 protein and the cytoplasmic localization of other initiation proteins. Injection of Cdc6 protein into immature oocytes does not induce DNA replication. However, injection of Cdc6 into oocytes undergoing GVBD is sufficient to induce DNA replication in the absence of protein synthesis. Our results show that GVBD and Cdc6 synthesis are the only events that limit the establishment of the oocyte's replication competence during meiosis.     

Polymorphism and absence of Leu-enkephalin sequences in proenkephalin genes in Xenopus laevis

The structures of the genes coding for the opioid peptide precursors proopiomelanocortin, proenkephalin (proenkephalin A) and prodynorphin (proenkephalin B), are known for some mammalian species. To gain insight into the evolutionary history of these precursors, we have examined the proenkephalin gene in the South African clawed toad, Xenopus laevis, which diverged from the principal line of vertebrate evolution some 350 Myr ago. The human proenkephalin gene consists of four exons, of which the main exon (exon IV) contains all known biologically active peptides--six Met-enkephalin sequences and one Leu-enkephalin sequence. We report here the primary structures of the putative main exons of two proenkephalin genes in X. laevis, each of which codes for seven Met-enkephalin sequences but no Leu-enkephalin, indicating that Met-enkephalin preceded Leu-enkephalin in the evolution of the proenkephalin gene. The organization of the main exons of the toad genes is remarkably similar to that of the human gene and conserved regions provide evidence for functionally significant structures. We also detect a polymorphism in one of the toad proenkephalin genes, mapping 1.5 kilobases (kb) 5' of the main exon; it is caused by an insertion/deletion of a 1-kb repetitive sequence which has the characteristics of a transposable element.     

Inositol 1,4,5-trisphosphate mimics muscarinic response in Xenopus oocytes

The enhanced metabolism of phosphoinositides, which is associated with a wide variety of stimuli and physiological responses, has been studied intensively. Berridge and his collaborators demonstrated that the first measurable reaction following cell membrane receptor activation is a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and that the product of this reaction, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), could cause a release of non-mitochondrial calcium. These findings have been verified in other systems. Although the relationship between the hydrolysis of PtdIns(4,5)P2 and the mobilization of intracellular calcium was clearly demonstrated, the direct link between Ins(1,4,5)P3 production and the physiological response was only implied. We have investigated the possibility that the intracellular release of Ins(1,4,5)P3 mediates the muscarinic-cholinergic response is Xenopus oocytes, and we show here that intracellularly injected Ins(1,4,5)P3 mimics the muscarinic depolarizing chloride current in Xenopus oocytes. This is the first demonstration of a direct link between phosphoinositides metabolism and a neuro-transmitter-induced physiological response.     

乙酰胆碱对非洲爪蟾卵母细胞ATP-激活电流的调制作用

研究乙酰胆碱对爪蟾卵母细胞上ATP激活电流的影响及其机理.用双电极电压钳技术记录爪蟾卵母细胞的细胞外液加入乙酰胆碱对ATP激活电流（IATP）的影响.结果显示,乙酰胆碱对大多数细胞IATP具有增强作用,而对少数细胞IATP具有抑制作用,且均呈现浓度依赖性;乙酰胆碱对IATP的调制作用是通过毒蕈碱样乙酰胆碱受体（M受体）激活实现的.     

Specific degradation of keratin in Xenopus laevis egg extracts undergoing apoptosis

Cytochrome c activates apoptosis specific protease XCPP32 when being added to Xenopus laevis egg extracts, and induces apoptosis in this cell-free system. During apoptosis, cyto-skeleton proteins in egg extracts are degraded. Western blot assay indicates that 42-ku acidic keratin in egg extracts has been degraded by XCPP32. The degradation of 42-ku keratin may be crucial in apoptosis.