Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos

Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.     

Temporal regulation of embryonic M-phases

Temporal regulation of M-phases of the cell cycle requires precise molecular mechanisms that differ among different cells. This variable regulation is particularly clear during embryonic divisions. The first embryonic mitosis in the mouse lasts twice as long as the second one. In other species studied so far (C. elegans, Sphaerechinus granularis, Xenopus laevis), the first mitosis is also longer than the second, yet the prolongation is less pronounced than in the mouse. We have found recently that the mechanisms prolonging the first embryonic M-phase differ in the mouse and in Xenopus embryos. In the mouse, the metaphase of the first mitosis is specifically prolonged by the unknown mechanism acting similarly to the CSF present in oocytes arrested in the second meiotic division. In Xenopus, higher levels of cyclins B participate in the M-phase prolongation, however, without any cell cycle arrest. In Xenopus embryo cell-free extracts, the inactivation of the major M-phase factor, MPF, depends directly on dissociation of cyclin B from CDK1 subunit and not on cyclin B degradation as was thought before. In search for other mitotic proteins behaving in a similar way as cyclins B we made two complementary proteomic screens dedicated to identifying proteins ubiquitinated and degraded by the proteasome upon the first embryonic mitosis in Xenopus laevis. The first screen yielded 175 proteins. To validate our strategy we are verifying now which of them are really ubiquitinated. In the second one, we identified 9 novel proteins potentially degraded via the proteasome. Among them, TCTP (Translationally Controlled Tumor Protein), a 23-kDa protein, was shown to be partially degraded during mitosis (as well as during meiotic exit). We characterized the expression and the role of this protein in Xenopus, mouse and human somatic cells, Xenopus and mouse oocytes and embryos. TCTP is a mitotic spindle protein positively regulating cellular proliferation. Analysis of other candidates is in progress.     

Program of early development in the mammal: changes in patterns and absolute rates of tubulin and total protein synthesis during oogenesis and early embryogenesis in the mouse.

The absolute rates of total protein synthesis and tubulin synthesis during oogenesis and early embryogenesis in the mouse have been determined by measuring specific activities of the endogenous methionine pool and rates of incorporation of [³⁵S]methionine into total protein and tubulin. The absolute rate of protein synthesis decreases from 43 to 33 pg/hr/oocyte during meiotic maturation, while the size of the endogenous methionine pool remains essentially unchanged at 65 fmole/oocyte (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978, Proc. Nat. Acad. Sci. USA,75, 4160). The one-cell mouse embryo synthesizes protein at a rate of 45 pg/hr/embryo, so that fertilization is accompanied by about a 40% increase in the absolute rate of total protein synthesis. The eight-cell compacted embryo synthesizes protein at the rate of 51 pg/hr/embryo. The size of the endogenous methionine pool increases dramatically during early embryogenesis, from 74 fmole in the unfertilized ovum to 137 and 222 fmole in the one-cell embryo and eight-cell compacted embryo, respectively. Tubulin is one of the major proteins synthesized by the mouse oocyte and embryo since the absolute rate of tubulin synthesis is, on the average, 1.3% that of total protein synthesis. The absolute rate of tubulin synthesis decreases from 0.61 to 0.36 pg/hr/oocyte during meiotic maturation and then increases to 0.60 pg/hr/embryo in the one-cell embryo and to 0.66 pg/hr/embryo in the eight-cell compacted embryo. During meiotic maturation and early embryogenesis the direction and magnitude of changes in the rate of tubulin synthesis closely parallel those of total protein synthesis. Although equimolar amounts of tubulin subunits are present in microtubules, the ratio of the absolute rate of synthesis of the β subunit to that of the α subunit is about 2.0 throughout meiotic maturation and early embryogenesis.High-resolution two-dimensional gel electrophoretic analysis of [³⁵S]methionine-labeled proteins reveals that many of the newly synthesized proteins that first appear during meiotic maturation of the oocyte continue to be synthesized in the one-cell embryo. Nearly all of the proteins synthesized in the one-cell embryo are also synthesized in the unfertilized ovum, although some changes in the pattern of protein synthesis are associated with fertilization. Therefore, the developmental program for early embryogenesis in the mouse appears to be activated during meiotic maturation of the oocyte. These results are compared with those obtained using oocytes and embryos from nonmammalian animal species.     

Cell-cycle control during meiotic maturation

The meiotic cell cycle, which is comprised of two consecutive M-phases, is crucial for the production of haploid germ cells. Although both mitotic and meiotic M-phases share cyclin-B-Cdc2/CDK1 as a key controller, there are meiosis-specific modulations in the regulation of cyclin-B-Cdc2. Recent insights indicate that a common pattern in these modulations can be found by considering the particular activities of mitogen-activated protein kinase (MAPK) during meiosis. The G(2)-phase arrest of meiosis I is released via specific, MAPK-independent signalling that leads to cyclin-B-Cdc2 activation; thereafter, however, the meiotic process is under the control of interplay between MAPK and cyclin-B-Cdc2.     

Intracellular pH regulation in the early embryo

Intracellular pH (pHi) regulation is a homeostatic function of all cells. Additionally, the plasma membrane-based transporters controlling pHi are involved in growth factor activation, cell proliferation and salt transport – all processes active in early embryos. pHi regulation in the early embryos of many species exhibits unique features: in mouse preimplantation embryos, mechanisms for correcting excess acid apparently are inactive, while excess base is removed by the mechanism common in differentiated cells. Additionally, unlike differentiated cells, mouse preimplantation embryos are highly permeable to H⁺ until the blastocyst stage, where the epithelial cells surrounding the embryo are impermeable. In several non-mammalian species, of which the best-studied is sea urchin, cytoplasmic alkalinization at fertilization is necessary for development of the embryo, and elevated pHi must be maintained during early development. Thus, pHi regulatory mechanisms appear to be important for early embryo development in many species.     

Program of early development in the mammal: Changes in absolute rates of synthesis of ribosomal proteins during oogenesis and early embryogenesis in the mouse

The absolute rates of synthesis of specific ribosomal proteins have been determined during growth and meiotic maturation of mouse oocytes, as well as during early embryogenesis in the mouse. These measurements were made possible by the development of a high-resolution twodimensional gel electrophoresis procedure capable of resolving basic proteins with isoelectric points between 9.1 and 10.2. Mouse ribosomal proteins were separated on such gels and observed rates of incorporation of [³⁵S]methionine into each of 12 representative ribosomal proteins were converted into absolute rates of synthesis (femtograms or moles synthesized/hour/oocyte or embryo) by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and embryos (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978,Proc. Nat. Acad. Sci. USA,75, 4160;R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979,Develop. Biol.,68, 341–359). Ribosomal proteins were synthesized at all stages of oogenesis and early embryogenesis examined and, while equimolar amounts of ribosomal proteins were found in ribosomes, they were always synthesized in nonequimolar amounts during development. Rates of synthesis of individual ribosomal proteins differed from each other by more than an order of magnitude in some cases. Synthesis of ribosomal proteins accounted for 1.5, 1.5, and 1.1% of total protein synthesis during growth of the oocyte, in the fully grown oocyte, and in the unfertilized egg, respectively. During meiotic maturation of mouse oocytes the absolute rate of synthesis of ribosomal proteins decreased about 40%, from 620 to 370 fg/hr/cell, as compared to a 23% decrease in the rate of total protein synthesis during the same period. On the other hand, during early embryogenesis the absolute rates of synthesis of each of the 12 ribosomal proteins examined increased substantially as compared with those of the unfertilized egg, such that at the eight-cell stage of embryogenesis synthesis of ribosomal proteins (4.17 pg/hr/embryo) accounted for about 8.1% of the total protein synthesis in the embryo. Consequently, while the absolute rate of total protein synthesis increased about 1.5-fold during development from an unfertilized mouse egg to an eight-cell compacted embryo, the absolute rate of ribosomal protein synthesis increased more than 11-fold during the same period. These results seem to reflect the differences reported for the patterns of ribosomal RNA synthesis during early development of mammalian, as compared to nonmammalian, animal species. The results are compared with those obtained using oocytes and embryos fromXenopus laevis.     

KIF20A Regulates Porcine Oocyte Maturation and Early Embryo Development

KIF20A (Kinesin-like family member 20A), also called mitotic kinesin-like proteins 2 (MKLP2), is a mammalian mitotic kinesin-like motor protein of the Kinesin superfamily proteins (KIFs), which was originally involved in Golgi apparatus dynamics and thought to essential for cell cycle regulation during successful cytokinesis. In the present study, we investigated whether KIF20A has roles on porcine oocyte meiotic maturation and subsequent early embryo development. By immunofluorescence staining, KIF20A was found to exhibit a dynamic localization pattern during meiosis. KIF20A was restricted to centromeres after germinal vesicle breakdown (GVBD), transferred to the midbody at telophase I (TI), and again associated with centromeres at metaphase II (MII). Inhibition of endogenous KIF20A via a specific inhibitor, Paprotrain, resulted in failure of polar body extrusion. Further cell cycle analysis showed that the percentage of oocytes that arrested at early metaphase I (MI) stage increased after KIF20A activity inhibition; however, the proportion of oocytes at anaphase/telophase I (ATI) and MII stages decreased significantly. Our results also showed that KIF20A inhibition did not affect spindle morphology. In addition, KIF20A was localized at the nucleus of early embryos, and KIF20A inhibition resulted in failure of early parthenogenetic embryo development. These results demonstrated that KIF20A is critical for porcine oocyte meiotic maturation and subsequent early embryo development.     

Effects of PKC activation on the meiotic maturation, fertilization and early embryonic development of mouse oocytes

Protein kinase C (PKC) is a family of Ser/Thr protein kinase widely distributed in eukaryotes. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. However, the mechanism of PKC's actions and the PKC isoforms responsible for these actions are poorly understood. In this study, we reveal in mouse eggs and early embryos: (1) the effects of PKC on the meiotic and mitotic cell cycle progression during oocyte maturation, egg activation and embryonic cleavages; (2) the functional importance of classical PKC subclasses in these processes; and (3) the subcellular localization of the PKC alpha isoform during development from GV stage oocytes to the blastocyst stage embryos. The results indicate that the PKC activator phorbol 12-myristate 13-acetate (PMA) inhibits the meiotic resumption of cumulus-free mouse oocytes by a mechanism dependent not only on classical PKC activity but also on other PKC isoforms. PKC activation after germinal vesicle breakdown leads to the inhibition of mitogen-activated protein kinase phosphorylation and the arrest of cell cycle at MI stage. The second polar body emission and the cleavages of early embryos are blocked after prolonged PKC activation. The subcellular localization of PKC alpha isoform in mouse oocytes and embryos is developmental-stage associated. All these results suggest that PKC has multiple functional roles in the cell cycle progression of mouse oocytes and embryos.     

RNA synthesis in immature mouse oocyte development

Oocyte development in several nonmammalian species is characterized by the synthesis of large quantities of ribonucleic acids during lampbrush stages of meiosis. These are stored in the oocyte and used during later oocyte maturation and early embryogenesis. This autoradiographic study examined the incorporation and persistence of ribonucleic acid in mouse oocytes during comparable stages of development. At each age examined, fetal through juvenile, the radiolabeled RNA precursors were incorporated into mouse oocytes during the growth stages. The RNAase-digestible label appeared first over nucleoli and meiotic chromosomes, becoming cytoplasmic after 24 hours, and remaining cytoplasmic through all remaining stages. Once incorporated the label persisted during subsequent oocyte growth and maturation through preimplantation embryo stages with apparently undiminished levels. It is suggested that this persistently labeled RNA represents maternal RNA stored for use during early embryonic development.     

A rat oocyte differentiation antigen (OA-1) detected by a monoclonal antibody

Cell surface antigenic changes associated with differentiation of the rat oocyte and early embryo have been demonstrated with a monoclonal antibody (anti-OA-1). Antigen is first detectable coincident with initiation of oocyte growth, is a constant feature of all growing oocytes and displays a redistribution during meiotic maturation. Following fertilization, antigen is detectable on the surface of the embryo through the four-cell stage. This first monospecific marker for the rat oocyte and embryo should prove useful in probing structure/function relationships in oocyte growth, meiotic maturation fertilization, and/or early embryonic development.     

Developmentally acquired PKA localisation in mouse oocytes and embryos

Localisation of Protein Kinase A (PKA) by A-Kinase Anchoring Proteins (AKAPs) is known to coordinate localised signalling complexes that target cAMP-mediated signalling to specific cellular sub-domains. The cAMP PKA signalling pathway is implicated in both meiotic arrest and meiotic resumption, thus spatio-temporal changes in PKA localisation during development may determine the oocytes response to changes in cAMP. In this study we aim to establish whether changes in PKA localisation occur during oocyte and early embryo development.Using fluorescently-labelled PKA constructs we show that in meiotically incompetent oocytes PKA is distributed throughout the cytoplasm and shows no punctuate localisation. As meiotic competence is acquired, PKA associates with mitochondria. Immature germinal vesicle (GV) stage oocytes show an aggregation of PKA around the GV and PKA remains co-localised with mitochondria throughout oocyte maturation. After fertilisation, the punctuate, mitochondrial distribution was lost, such that by the 2-cell stage there was no evidence of PKA localisation. RT-PCR and Western blotting revealed two candidate AKAPs that are known to be targeted to mitochondria, AKAP1 and D-AKAP2. In summary these data show a dynamic regulation of PKA localisation during oocyte and early embryo development.     

Current concepts of cell-cycle regulation and its relationship to oocyte maturation, fertilization and embryo development

Genetic and biochemical approaches have contributed to an explosion of literature on cell-cycle control. Regulation of the cell-cycle is controlled by a series of kinases and phosphatases. Key control points are during the G(1)-S and G(2)-M transitions. During both transitions, cyclins interact with a specific kinase to allow a cell to pass through that phase. The meiotic maturation of oocytes, fertilization and embryo development are all events influenced by cell-cycle regulation. Understanding cell-cycle control should provide new ways for gamete and embryo biologists to approach culture and development problems.     

Calcium and meiotic maturation of the mammalian oocyte

The role of calcium in the regulation of both the meiotic and mitotic cell cycles has been the subject of considerable investigation in the nonmammalian field. In contrast, the mechanisms for signalling meiotic maturation in the mammalian oocyte are not as well documented nor as clearly defined. In the mammalian oocyte, calcium is associated with both spontaneous and hormone-induced meiotic maturation. A transient release of endogenously stored calcium precedes germinal vesicle breakdown and can override cyclic AMP maintained meiotic arrest; it thus may signal the resumption of meiosis. Additionally, extracellular calcium is apparently required for meiotic progression past metaphase I. The time sequence for meiotic resumption and progression is very varied between species. The timing of cell cycle protein synthesis during meiosis suggests that cyclins may be expressed in oocytes of some species much earlier in their development than in others. A generic model is proposed for the mechanism for triggering meiotic resumption in the mammalian oocyte. In this model, the critical components of meiotic resumption involve the temporal relationship of cyclin synthesis and the subsequent activation of the MPF complex by the calcium signal generated, which accounts for differences among species. © 1995 Wiley-Liss, Inc.     

哺乳动物卵母细胞及早期胚胎蛋白质组学研究进展

雌性生殖细胞发育是动物繁殖的基石,哺乳动物卵母细胞和早期胚胎在其生长发育过程中有许多独特的现象和规律,涉及一系列蛋白质合成/降解和磷酸化等状态的动态改变。对卵母细胞分裂、成熟调控机理以及植入前胚胎发育规律的研究是发育生物学领域的一项重要课题。蛋白质组学是以细胞或组织内全部的蛋白质为研究对象,系统鉴定、定量蛋白质并研究这些蛋白质功能的科学。随着蛋白质分离、鉴定技术的快速发展,蛋白质组学为卵母细胞发生、分化、成熟以及质量控制等相关研究提供了新的方法和内容,如在蛋白质定量、修饰、定位和相互作用等方面提供其他组学技术不可获得的重要信息。这些信息将有助于揭示哺乳动物卵母细胞成熟和早期胚胎发育的分子机制,对于进一步完善卵母细胞的体外成熟培养体系,提高胚胎体外生产、体细胞克隆和转基因动物生产效率具有重要意义。     

Expression and activity of p40MO15, the catalytic subunit of cdk-activating kinase, during Xenopus oogenesis and embryogenesis.

Threonine 161 phosphorylation of p34cdc2 and its equivalent threonine 160 in p33cdk2 by cdk-activating kinase (CAK) is essential for the activation of these cyclin-dependent kinases. We have studied the expression and associated kinase activity of p40MO15, the catalytic subunit of CAK, during Xenopus oogenesis, meiotic maturation, and early development to understand in more detail how cdk kinases are regulated during these events. We find that p40MO15 is a stable protein with a half-life > 16 h that is accumulated during oogenesis. p40MO15 protein and its associated CAK activity are localized predominantly to the germinal vesicle; however, a small but significant proportion is found in the cytoplasm. The amount of p40MO15 detected in stage VI oocytes remains unchanged through meiotic maturation, fertilization, and early embryogenesis. Significantly, p40MO15 was found to be constitutively active during oogenesis, meiotic maturation, and the rapid mitotic cycles of early development. This suggests that regulation of p34cdc2 and p33cdk2 activity during cell cycle progression does not involve changes in the level or activity of p40MO15/CAK.     

Phosphorylated ERK5/BMK1 transiently accumulates within division spindles in mouse oocytes and preimplantation embryos

MAP kinases of the ERK family play important roles in oocyte maturation, fertilization, and early embryo development. The role of the signaling pathway involving ERK5 MAP kinase during meiotic and mitotic M-phase of the cell cycle is not well known. Here, we studied the localization of the phosphorylated, and thus potentially activated, form of ERK5 in mouse maturing oocytes and mitotically dividing early embryos. We show that phosphorylation/dephosphorylation, i.e. likely activation/inactivation of ERK5, correlates with M-phase progression. Phosphorylated form of ERK5 accumulates in division spindle of both meiotic and mitotic cells, and precisely co-localizes with spindle microtubules at metaphase. This localization changes drastically in the anaphase, when phospho-ERK5 completely disappears from microtubules and transits to the cytoplasmic granular, vesicle-like structures. In telophase oocytes it becomes incorporated into the midbody. Dynamic changes in the localization of phospho-ERK5 suggests that it may play an important role both in meiotic and mitotic division.     

Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures.     

Beclin-1 knockdown shows abscission failure but not autophagy defect during oocyte meiotic maturation

Cytokinesis is the final step in cell division that results in the separation of a parent cell into daughter cells. Unlike somatic cells that undergo symmetric division, meiotic division is highly asymmetric, allowing the preservation of maternal resources for embryo development. Beclin-1/BECN1, the mammalian homolog of yeast Atg6, is a key molecule of autophagy. As part of a class III phosphatidylinositol 3-kinase (PI3K-III) complex, BECN1 initiates autophagosome formation by coordinating membrane trafficking. However, emerging evidence suggests that BECN1 regulates chromosome segregation and cytokinesis during mitosis. Thus, we investigated the function of BECN1 during oocyte meiotic maturation. BECN1 was widely distributed during meiotic maturation forming small vesicles. Interestingly, BECN1 is also detected at the midbody ring during cytokinesis. Depletion of BECN1 impaired the cytokinetic abscission, perturbing the recruitment of ZFYVE26 at the midbody. Similar phenotypes were observed when PI3K-III activity was inhibited. However, inhibition of autophagy by depleting Atg14L did not disturb meiotic maturation. Therefore, our results not only demonstrate that BECN1 as a PI3K-III component is essential for cytokinesis, but also suggest that BECN1 is not associated with autophagy pathway in mouse oocytes.