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.     

Meiotic initiation by the mos protein in Xenopus.

When fully grown Xenopus oocytes are stimulated by progesterone, a period of protein synthesis is necessary for maturation. Synthesis of the mos proto-oncogene product, pp39mos, is necessary for the activation of M-phase promoting factor (MPF) in meiosis I. On the basis that mos is translated de novo on hormonal stimulation of Xenopus oocytes and that injecting mos RNA into oocytes induces their maturation, we have proposed that the mos protein is a candidate initiator of oocyte maturation, needed to trigger the conversion of precursor MPF into its active form. To determine whether mos is the only protein required for initiating maturation, we have produced a soluble, active recombinant mos protein and injected it into Xenopus oocytes. We report here that in the absence of protein synthesis that mos protein efficiently induces germinal vesicle breakdown and the activation of MPF. The oocytes, however, do not proceed into meiosis II. Thus, the mos protein fulfills the requirements of an initiator protein, but the synthesis of one or more additional proteins may be necessary to complete oocyte maturation.     

四氯化碳对雌性小鼠卵母细胞成熟和体外受精等影响

探讨了四氯化碳对小鼠生殖作用的影响.采用小鼠卵母细胞体外培养、体外受精的方法研究了四氯化碳对小鼠卵母细胞的成熟和体外受精的影响.结果表明:四氯化碳对小鼠脏器、超排卵的卵母细胞数量和卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率并可降低体外受精率.四氯化碳可以破坏卵母细胞减数分裂进行,降低卵母细胞的受精能力,具有潜在的生殖毒性.     

Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation

Vertebrate eggs awaiting fertilization are arrested at metaphase of meiosis II by a biochemical activity termed cytostatic factor (CSF). This activity inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that triggers anaphase onset and mitotic/meiotic exit by targeting securin and M-phase cyclins for destruction. On fertilization a transient rise in free intracellular calcium causes release from CSF arrest and thus APC/C activation. Although it has previously been shown that calcium induces the release of APC/C from CSF inhibition through calmodulin-dependent protein kinase II (CaMKII), the relevant substrates of this kinase have not been identified. Recently, we characterized XErp1 (Emi2), an inhibitor of the APC/C and key component of CSF activity in Xenopus egg extract. Here we show that calcium-activated CaMKII triggers exit from meiosis II by sensitizing the APC/C inhibitor XErp1 for polo-like kinase 1 (Plx1)-dependent degradation. Phosphorylation of XErp1 by CaMKII leads to the recruitment of Plx1 that in turn triggers the destruction of XErp1 by phosphorylating a site known to serve as a phosphorylation-dependent degradation signal. These results provide a molecular explanation for how the fertilization-induced calcium increase triggers exit from meiosis II.     

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.     

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.     

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.     

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.     

Nitric oxide produced by cumulus cells stimulates maturation of mouse oocytes

In recent years, embryo engineering concerning animal cloning, animal transgene and bioreactor technique has been developed rapidly. Since the operation of each of these techniques needs matured mammalian oocytes, the demand for highly qualified oocytes is expanding. On the other hand, various factors existing in follicle might be involved in oocyte meiotic maturation, and the process involved is extremely complex and inadequately defined. As a result, the mechanism of oocyte maturation regula…     

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.     

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.     

体外受精过程中多精受精发生机制

对哺乳动物体外受精（IVF）过程中多精受精的发生原因进行了阐述，指出多精受精的发生是影响各种哺乳动物体外受精效率的重要原因，其中在猪和人的IVF中表现得尤为突出。大量研究表明，除了物种的特异性以外，不完全的卵母细胞胞质成熟、异常透明带、高精子浓度、受精培养基中不适当的添加物以及在受精过程中的其他一些异常因素都与多精受精密切相关。     

体外受精过程中多精受精发生机制

对哺乳动物体外受精(IVF)过程中多精受精的发生原因进行了阐述,指出多精受精的发生是影响各种哺乳动物体外受精效率的重要原因,其中在猪和人的IVF中表现得尤为突出。大量研究表明,除了物种的特异性以外,不完全的卵母细胞胞质成熟、异常透明带、高精子浓度、受精培养基中不适当的添加物以及在受精过程中的其他一些异常因素都与多精受精密切相关。     

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.     

Release of mature starfish oocytes from interphase arrest by microinjection of human centrosomes

Mature oocytes (unfertilized eggs) are arrested at definite cell-cycle stages which vary from species to species. In frogs and mammals, the oocytes are arrested at the second metaphase of meiosis whereas in echinoderms they are blocked later, at the pronucleus stage. What causes the maturing oocytes to stop at some point in the cell cycle is not entirely clear. In frogs, the metaphase arrest seems to be maintained by a cytostatic factor. In echinoderms, which stop at interphase, no such a factor has so far been found. The fertilization process, beyond the introduction of paternal chromosomes, releases the oocyte from cell-cycle arrest and provides a functional centrosome to replace the endogenous centrosome which is apparently lost during oogenesis in most species. Several lines of evidence suggest that release from cell-cycle arrest is mediated by a Ca2+ burst which is associated with fertilization, and it is known that the functional centrosome provided by the sperm is necessary for mitotic spindle formation and cleavages. We report here that microinjection of purified human centrosomes into mature starfish oocytes is sufficient to release them from arrest at interphase and to support many cleavages leading to the occasional formation of normal embryos. In this species centrosome induced re-entry into the cell cycle does not require a transient calcium burst nor does it require intact microtubules.     

Nitric oxide produced by cumulus cells stimulates maturation of mouse oocytes

The effect of sodium nitroprusside (SNP), a donor of nitric oxide, on meiotic maturation of mouse oocytes was studied by injecting N^w-nitro-L-arginine methyl ester (L-NAME) intra-peritoneal (ip), a nitric oxide synthase inhibitor, or culturing oocytes in the medium supplemented with L-NAME or hypoxanthine (HX) to arrest the spontaneous oocyte maturation in vitro. The results showed that the inhibitory effect of L-NAME by injecting 10 mg/kg ip on extrusion of the first polar body only could be reversed by injecting 2.5 mg/kg SNP with L-NAME simultaneously (P < 0.05). Half an hour later ten mice died when given 10 mg/kg SNP ip. The treatment of some concentrations of SNP (10^–7, 10^–6, 10^–5mol/L) significantly stimulated meiotic maturation to metaphase Ⅱ stages in cumulus enclosed oocytes in the presence of HX. However, other concentrations of SNP (10^–8, 10^–4, 10^–3 mol/L) had no effect on HX-arrested oocyte meiotic maturation. The optimal concentration of SNP on CEOs had no effect on DOs. The dose of 10^–3 mol/LL-NAME demonstrated a significant suppression in formation of PB₁, but not in GVBD. This inhibition was reversed by the addition of SNP. These results indicated that the physiological levels of NO produced by cumulus cells could stimulate meiotic maturation of mouse oocytes both in vivo and in vitro.     

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.     

精子和酒精对小鼠卵母细胞激活的比较

对不同卵龄的小鼠卵母细胞被精子和酒精激活后的激活率进行了比较。结果显示,卵母细胞对常规的体外受精和酒精的人工激活的激活率存在卵龄的差异。注射hCG后15～24h的卵母细胞容易被酒精的人工刺激所激活,20h卵龄的卵母细胞激活率最高,平均为81．6％,且速即卵裂率也最高,平均为48％,卵龄更大的卵母细胞激活率降低,而13h的卵母细胞难以被酒精激活。另一方面,13～15h的卵母细胞容易被精子激活而受精,卵龄较大的卵母细胞在体外难以被精子激活受精。这表明,精子和酒精对卵母细胞的激活机制有所不同。