Roles of protein kinase C in oocyte meiotic maturation and fertilization

Protein kinase C (PKC) is a superfamily of Ser/Thr protein kinases that is distributed widely in eukaryotes. It plays key regulatory roles at multiple steps of oocyte meiotic maturation and fertilization. During the process of meiotic maturation, the activation of PKC in cumulus cells stimulates meiotic maturation, whereas the activation of PKC in oocytes results in the inhibition of germinal vesicle breakdown. PKC activity increases following the meiotic maturation, and decreases at the transition of metaphase/anaphase in meiosis I, so as to facilitate the release of the first polar body and the entry of meiosis II. In fertilization of mammalian oocytes, PKC may act as one of the downstream targets of Ca2+ to stimulate the cortical granule exocytosis, release the oocytes from MII arrest and to induce pronucleus formation. PKC is also involved in the regulation of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Several PKC isoforms have been identified in mammalian oocytes, and there is evidence showing that classical PKCs may be the principal mediator of oocyte cortical reaction.     

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.     

Competence to replicate in the unfertilized egg is conferred by Cdc6 during meiotic maturation

Meiotic maturation, the final step of oogenesis, is a crucial stage of development in which an immature oocyte becomes a fertilizable egg. In Xenopus, the ability to replicate DNA is acquired during maturation at breakdown of the nuclear envelope by translation of a DNA synthesis inducer that is not present in the oocyte. Here we identify Cdc6, which is essential for recruiting the minichromosome maintenance (MCM) helicase to the pre-replication complex, as this inducer of DNA synthesis. We show that maternal cdc6 mRNA but not protein is stored in the oocyte. Cdc6 protein is synthesized during maturation, but this process can be blocked by degrading the maternal cdc6 mRNA by oligonucleotide antisense injections or by translation inhibition. Rescue experiments using recombinant Cdc6 protein show that Cdc6 is the only missing replication factor whose translation is necessary and sufficient to confer DNA replication competence to the egg before fertilization. The licence to replicate is given by Cdc6 at the end of meiosis I, but the cytostatic factor (CSF) pathway, which maintains large amounts of active Cdc2/Cyclin B2, prevents the entry into S phase until fertilization.     

Function and interaction of maturation-promoting factor and mitogen-activated protein kinase during meiotic maturation and fertilization of oocyte

Mitogen-activated protein kinase (MAP kinase) cascade and maturation-promoting factor (MPF) play very important roles during meiotic maturation and fertilization of oocyte. Interaction between MAP kinase and MPF influences meiotic maturation and fertilization of oocyte throughout the animal kingdom, including stimulation of germinal vesicle breakdown (GVBD), suppression of DNA replication, control of meiotic chromosome segregation, maintenance of metaphase II arrest, and resumption and completion of second meiosis. This review focuses on the function and interaction of MAP kinase and MPF during meiotic maturation and fertilization of oocyte.     

A positive-feedback-based bistable 'memory module' that governs a cell fate decision

The maturation of Xenopus oocytes can be thought of as a process of cell fate induction, with the immature oocyte representing the default fate and the mature oocyte representing the induced fate. Crucial mediators of Xenopus oocyte maturation, including the p42 mitogen-activated protein kinase (MAPK) and the cell-division cycle protein kinase Cdc2, are known to be organized into positive feedback loops. In principle, such positive feedback loops could produce an actively maintained 'memory' of a transient inductive stimulus and could explain the irreversibility of maturation. Here we show that the p42 MAPK and Cdc2 system normally generates an irreversible biochemical response from a transient stimulus, but the response becomes transient when positive feedback is blocked. Our results explain how a group of intrinsically reversible signal transducers can generate an irreversible response at a systems level, and show how a cell fate can be maintained by a self-sustaining pattern of protein kinase activation.     

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.     

Specific proteolysis of the c-mos proto-oncogene product by calpain on fertilization of Xenopus eggs

The Xenopus c-mos proto-oncogene product, pp39mos, accumulates in the unfertilized egg during maturation, is hyperphosphorylated and exhibits protein kinase activity. On fertilization, or soon after the completion of meiosis, the accumulated pp39mos undergoes selective proteolysis. Using an in vitro protease assay system, we show here that this specific proteolysis is caused by the calcium-dependent cysteine protease, calpain.     

富勒烯及其衍生物对小鼠卵母细胞成熟和激活的作用

为探索富勒烯及其水溶性衍生物对哺乳动物卵母细胞减数分裂的作用,使用富勒烯膦酸衍生物(2P)、富勒烯-PVP和富勒醇作用于体外培养的小鼠GV期卵母细胞和超排卵母细胞,通过观察第一极体排出率和原核形成率以判断卵母细胞的成熟和激活,并探讨光照对这一作用的影响.结果表明,在光照和非光照下,富勒烯的PVP水溶液、富勒醇对卵母细胞的成熟没有明显影响,而2P在光照下对卵母细胞的成熟和孤雌激活具有明显抑制作用,其作用有浓度依赖性.     

Roles of MAP kinase signaling pathway in oocyte meiosis

Mitogen-activated protein kinase (MAPK) is a family of Ser/Thr protein kinases expressed widely in eukaryotic cells. MAPK is activated by a cascade of protein kinase phosphorylation and plays pivotal roles in regulating meiosis process in oocytes. As an important physical substrate of MAPK, p90^rsk mediates numerous MAPK functions. MAPK was activated at G2/M transition during meiosis. Its activity reached the peak at MⅠ stage and maintained at this level until the time before the pronuclear formation after fertilization. There is complex interplay between MAPK and MPF in the meiosis regulation. Furthermore, other intracellular signal transducers, such as cAMP, protein kinase C and protein phosphotase, ect., also regulated the activity of MAPK at different stages during meiosis in oocytes. In the present article, the roles of MAPK signaling pathway in oocyte meiosis are reviewed and discussed.     

Activation at M-phase of a protein kinase encoded by a starfish homologue of the cell cycle control gene cdc2+

In both starfish and amphibian oocytes, the activity of a major protein kinase which is independent of Ca2+ and cyclic nucleotides increases dramatically at meiotic and mitotic nuclear divisions. The in vivo substrates of this kinase are unknown, but phosphorylation of H1 histone can be used as an in vitro assay. We have purified this kinase from starfish oocytes. The major band in the most highly purified preparation contained a polypeptide of relative molecular mass (Mr) 34,000 (34K). This is the same size as the protein kinase encoded by cdc2+, which regulates entry into mitosis in fission yeast and is a component of MPF purified from Xenopus. Here, we show that antibodies against p34 recognize the starfish 34K protein and propose that entry into meiotic and mitotic nuclear divisions involves activation of the protein kinase encoded by a homologue of cdc2+. Given the wide occurrence of cdc2+ homologues from budding yeast to Xenopus and human cells, this activation may act as a common mechanism controlling entry into mitosis in eukaryotic cells.     

Independent inactivation of MPF and cytostatic factor (Mos) upon fertilization of Xenopus eggs.

In vertebrates, mature eggs are arrested at the second meiotic metaphase by the cytostatic factor (CSF), now known to be the c-mos proto-oncogene product (Mos). Fertilization or egg activation triggers a transient increase in the cytoplasmic free calcium and releases the meiotic arrest by inactivating maturation/mitosis-promoting factor (MPF). CSF or Mos, which is also inactivated by the calcium transient, seems to stabilize MPF in mature eggs and CSF-injected embryos. Thus, it was assumed that CSF inactivation is the primary cause of MPF inactivation on meiotic release. We have directly compared the degradation kinetics of CSF (Mos) and MPF during meiotic release, using the same batch of Xenopus eggs. We report here that, at the molecular level, cyclin subunits of MPF are degraded before Mos is degraded and, at the physiological level, that MPF activity is inactivated before CSF activity during activation of Xenopus eggs. These results, in conjunction with circumstantial evidence, support the novel view that a calcium transient on fertilization induces a CSF-independent pathway for MPF inactivation, whereas CSF inactivation during meiotic release serves only to allow the fertilized egg to enter mitosis.     

Go protein as signal transducer in the pertussis toxin-sensitive phosphatidylinositol pathway

Receptors stimulating phospholipase C do so through heterotrimeric GTP-binding proteins to produce two second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol. In spite of the detailed understanding of phospholipase C structure and phosphatidyl inositol signalling, the identity of the GTP-binding protein involved is so far unknown. To address this issue, we have used the Xenopus oocyte in which muscarinic receptors couple to phospholipase C through a pertussis toxin-sensitive GTP-binding protein. In this cell, InsP3 mobilizes intracellular Ca2+ to evoke a Cl- current. The magnitude of this Cl- current is proportional to the amount of InsP3 in the cell, and therefore can be used as an assay for InsP3 production. We report here that the activated alpha-subunit of the GTP-binding protein GO, when directly injected into oocytes, evokes a Cl- current by mobilizing Ca2+ from intracellular InsP3-sensitive stores. We also show that holo-GO, when injected into oocytes, can specifically enhance the muscarinic receptor-stimulated Cl- current. These data indicate that GO can serve as the signal transducer of the receptor-regulated phospholipase C in Xenopus oocytes.     

The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs

The c-mos proto-oncogene product, pp39mos, is present in unfertilized Xenopus eggs, and disappears on fertilization. Microinjection of synthetic mos RNA into two-cell embryos induces cleavage arrest at metaphase. By contrast, egg cytosol extracts, when immunodepleted of endogenous pp39mos, lose their cleavage-arresting activity in injected embryos. These results demonstrate that Mos protein is the cytostatic factor CSF, long known as an endogenous meiotic inhibitor in vertebrate eggs.     

Reconstruction of human embryos derived from somatic cells

Reconstruction of human nuclear transfer embryos is a necessary step of therapeutic cloning. In this study we injected somatic cell nuclei into M Ⅱ oocytes and activated reconstructed oocytes with calcium ionophore A23187 (CaA) and 6-dimethylaminopurine (6-DMAP). After oocyteactivation and 2PN formation, we removed the female PN.By using this method, we avoided the application of DNA fluorescent stain and ultraviolet light for oocyte enucleation,and over elimination of ooplasm was also mitigated. Some reconstructed embryos developed into the blastocyst stage in vitro.     

The Xenopus localized messenger RNA An3 may encode an ATP-dependent RNA helicase

The maternal messenger RNA An3 was originally identified localized to the animal hemisphere of Xenopus laevis oocytes, eggs and early embryos. Xenopus embryos depend on mRNA and protein present in the egg before fertilization (maternal molecules) to provide the information needed for early development. Localization of maternal mRNA gives cells derived from different regions of the egg distinctive capacities for protein synthesis. We show here that An3 mRNA encodes a protein with 74% identity to a protein encoded by the testes-specific mRNA PL10 found in mouse, which is proposed to have RNA helicase activity. Because the gene encoding An3 mRNA is reactivated after gastrulation and remains active throughout embryogenesis, we have examined its distribution in embryonic and adult tissues. Unlike PL10 mRNA, which is primarily restricted to the testes, An3 mRNA is broadly distributed in later development.     

椭圆背角无齿蚌卵子发生的研究

作者通过石蜡切片技术与苏木精－伊红染色的组织学方法及超薄切片技术和透射电镜观察，较系统地研究了椭圆背角无齿蚌卵细胞的发生、发育与成熟过程的细胞学特征：原始生殖细胞在滤泡壁上分裂增殖，成为卵原细胞、卵原细胞发育、膨大成为卵母细胞而排入滤泡腔。在滤泡腔中卵母细胞不断地进行卵黄的合成与积累，体积继续增大。最后成熟的卵母细胞，在未完成减数分裂的情况下直接排入外鳃叶腔，与随水流入的精子相遇而受精，在观察的基础上，作者比较分析了双壳纲淡水贝类生殖过程中所具有的一般特征，着重讨论了卵子发生过程中的双核仁现象与卵黄发生，成熟卵母细胞减数分裂的阻断与重新起始。     

Steroid-induced meiotic division in Xenopus laevis oocytes: surface and calcium.

Progesterone reinitiates meiotic maturation in Xenopus oocytes. Evidence is reported which indicates that the steroid acts at the level of the cell surface and suggests that an induced change of Ca2+ distribution triggers in turn a cascade of cytoplasmic events including protein synthesis and germinal vesicle (nucleus) breakdown. These novel features of steroid hormone action in amphibian oocytes are discussed in relation to presently accepted views of the mechanism of action of steroid hormones in somatic cells.     

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.     

Phosphorylation of Erp1 by p90rsk is required for cytostatic factor arrest in Xenopus laevis eggs

Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF), which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins-regulatory proteins of meiosis and mitosis-for degradation. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest through activation of p90 ribosomal S6 kinase (p90rsk), is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.