Regulation of the Aurora B chromosome passenger protein complex during oocyte maturation in Xenopus laevis

The dynamics of the Aurora B protein kinase during Xenopus oocyte meiotic maturation were examined. Resting G2 oocytes express inactive Aurora B that is not associated with other subunits of the chromosome passenger complex (CPC). Activity increases near the time of germinal vesicle breakdown in progesterone-treated oocytes, and this increase is correlated with the synthesis of inner centromere protein (INCENP) and survivin, components of the CPC. Ablation of INCENP synthesis led to the failure of progesterone treatment to activate Aurora B, but biochemical progression through the meiosis I-to-II transition and arrest at metaphase II were not affected. At fertilization, Aurora B was deactivated in concert with the degradation of INCENP, and the levels of Aurora B kinase activity and INCENP oscillated in subsequent embryonic cell cycles. Prevention of the decrease in Aurora B activity at fertilization by expression of ectopic wild-type INCENP, but not kinase-dead Aurora B INCENP, blocked calcium-induced exit from metaphase arrest in egg extracts.     

Interaction of cell cycle kinases, microtubules, and chromatin in ascidian oocytes during meiosis

We used kinase assays and confocal microscopy to study the interaction of cell cycle proteins with microtubule organising centres (MTOC) and chromatin in ascidian oocytes during meiosis. The activity of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) appear not to be correlated in control oocytes. MPF activity peaks during metaphase I and II of the meiotic cell cycle whereas the activity of MAPK peaks at telophase I and is subsequently degraded to remain at low levels for the remainder of meiosis. The protein synthesis inhibitor emetine induces the degradation in MPF activity in unfertilized metaphase-I (M-I) oocytes, while MAPK is unaffected. Emetine does not alter the activities of these cell cycle kinases in fertilized oocytes during meiosis I but MPF activity remains low while MAPK activity is high for an elongated time period and oocytes do not complete meiosis I. Emetine induces maternal MTOC duplication in unfertilized M-I oocytes and prevents sperm aster growth in fertilized oocytes, but it does not alter the M-I meiotic apparatus in unfertilized oocytes. These experiments suggest that neither MPF alone nor emetine-sensitive proteins are responsible for M-I arrest in ascidian oocytes, MAPK may ensure this stability. In addition, we showed that the maternal MTOC is present at M-I but suppressed from duplicating in an emetine-sensitive manner.     

Potential role of alphav and beta1 integrins as oocyte adhesion molecules during fertilization in pigs

Integrin molecules are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction in rodents and humans. The objective of this study was to evaluate whether integrin molecules were present on the surface of pig oocytes, consistent with involvement in sperm-oocyte interaction in this species. Immunocytochemistry and confocal microscopy were used to evaluate the presence of beta1, and alpha1, alpha2, alpha3, alpha4, alpha5, alpha6 and alphav integrin subunits on the plasma membrane of pig oocytes. The beta1 and alphav integrin subunits were present consistently at the surface of pig oocytes; however, the remaining alpha integrin subunits evaluated were not routinely detected. The antibodies to the beta1 and alphav integrin subunits recognized appropriately sized protein bands on western blots of partially purified oocyte plasma membrane. These two antibodies also recognized oocyte plasma membrane protein isolated from a sperm plasma membrane affinity column. Sperm plasma membrane proteins of 137 and 93 kDa appeared to be the ligands for the beta1 integrin subunit as revealed by a western sandwich blot. Antibody to an extracellular domain of the beta1 integrin subunit reduced pig sperm-oocyte binding (P < 0.05), also indicating an assisting role for a beta1 oocyte integrin subunit in sperm-oocyte interaction in pigs. These results are consistent with an alphavbeta1 pig oocyte integrin interacting with a ligand on the sperm plasma membrane during fertilization.     

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.     

Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis

Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the post-vitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.     

Consequence of the presence of two different beta subunit isoforms in a GABA(A) receptor

The major isoforms of GABA(A) receptors are thought to be composed of two alpha, two beta and one gamma subunit(s). GABA(A) receptors containing two beta1 subunits respond differently to the anticonvulsive compound loreclezole and the general anaesthetic etomidate than receptors containing two beta2 subunits. Receptors containing beta2 subunits show a much larger allosteric stimulation by these agents than those containing beta1 subunits. We were interested to know how receptors containing both beta1 and beta2 subunits, in different positions respond to loreclezole and etomidate. To answer this question, subunits were fused at the DNA level to form dimeric and trimeric subunits. Concatenated receptors (alpha1-beta1-alpha1/gamma2-beta1, alpha1-beta2-alpha1/gamma2-beta1, alpha1-beta1-alpha1/gamma2-beta2 and alpha1-beta2-alpha1/gamma2-beta2) were expressed in Xenopus ooctyes and functionally compared in their response to the agonist GABA and to the positive allosteric modulators, loreclezole and etomidate. We have shown that (I) in the presence of both beta1 and beta2 subunits in the same pentamer (mixed receptors) direct gating by etomidate is similar to exclusively beta1 containing receptors; (II) In mixed receptors, stimulation by etomidate assumed characteristics intermediate to exclusively beta1 or beta2 containing receptors, but the values for the concentrations < 10 microM were always much closer to those observed in alpha1-beta1-alpha1/gamma2-beta1 receptors; and (III) mixed receptors show no positional effects.     

Phylogenetic conservation of cytostatic factor related genes in the ascidian Ciona intestinalis

In all vertebrates, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase-I, others at prophase-I. Fertilization induces completion of meiosis and entry into the first mitotic division. Several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (CSF) and the maturation promoting factor (MPF) in metaphase I or II. Recently, we proposed the oocytes of ascidian Ciona intestinalis as a new model to study the meiotic division. Here, taking advantage of the recent publication of the C. intestinalis genome, we presented a phylogenetic analysis of key molecular components of the CSF-related machinery. We showed that the Mos/MAP kinase pathway is perfectly conserved in ascidians. We demonstrated the presence of a CSF-like activity in metaphase-I arrested C. intestinalis oocytes able to block cell division in two-cell embryos. We further investigated the regulation of CSF by demonstrating that both CSF and MPF inactivation, at the exit of metaphase-I, are independent from protein synthesis, indicating the absence of short-lived factors that regulate metaphase stability, as in other invertebrate species. The results obtained suggest that meiotic regulation in C. intestinalis resembles that of vertebrates, such as Xenopus accordingly to the position of this organism in the evolutionary tree.     

Intersubunit interactions in human X,K-ATPases: role of membrane domains M9 and M10 in the assembly process and association efficiency of human, nongastric H,K-ATPase alpha subunits (ATP1al1) with known beta subunits

Na,K- and H,K-ATPase (X,K-ATPase) alpha subunits need association with a beta subunit for their maturation, but the authentic beta subunit of nongastric H,K-ATPase alpha subunits has not been identified. To better define alpha-beta interactions in these ATPases, we coexpressed human, nongastric H,K-ATPase alpha (AL1) and Na,K-ATPase alpha1 (alpha1NK) as well as AL1-alpha1 and alpha1-AL1 chimeras, which contain exchanged M9 and M10 membrane domains, together with each of the known beta subunits in Xenopus oocytes and followed their resistance to cellular and proteolytic degradation and their ER exit. We show that all beta subunits (gastric betaHK, beta1NK, beta2NK, beta3NK, or Bufo bladder beta) can associate efficiently with alpha1NK, but only gastric betaHK, beta2NK, and Bufo bladder beta can form stably expressed AL1-beta complexes that can leave the ER. The trypsin resistance and the forces of subunit interaction, probed by detergent resistance, are lower for AL1-beta complexes than for alpha1NK-beta complexes. Furthermore, chimeric alpha1-AL1 can be stabilized by beta subunits, but alpha1-AL1-gastric betaHK complexes are retained in the ER. On the other hand, chimeric AL1-alpha1 cannot be stabilized by any beta subunit. In conclusion, these results indicate that (1) none of the known beta subunits is the real partner subunit of AL1 but an as yet unidentified, authentic beta should have structural features resembling gastric betaHK, beta2NK, or Bufo bladder beta and (2) beta-mediated maturation of alpha subunits is a multistep process which depends on the membrane insertion properties of alpha subunits as well as on several discrete events of intersubunit interactions.     

Differential regulation of G protein subunit expression in mouse oocytes, eggs, and early embryos

Pertussis toxin ADP-ribosylation and Western blot analysis using G protein-specific antibodies were used to study G protein expression in mouse oocytes, eggs, and early embryos. A pertussis toxin (PT) substrate of about 40 kDa was observed in all stages, but its level was stage dependent. It decreased dramatically between germinal vesicle stage oocytes and unfertilized eggs, remained relatively constant through the early 2-cell stage, and then declined again with each cell division, reaching the lowest level at the 8- to 16-cell stage. Its level, or perhaps that of a different substrate, then increased at the blastocyst stage. Western blot analysis with antisera to the G protein alpha subunit indicated that the decrease between germinal vesicle stage oocytes and unfertilized eggs was less pronounced for the alpha subunit itself than for the PT substrate. Antisera to G protein beta subunit revealed that the difference in the amount of this subunit in germinal vesicle-stage oocytes versus unfertilized eggs was even greater than that of the PT ADP-ribosylation substrate. These results suggest that during oocyte maturation G protein beta gamma levels decline to a greater extent than alpha levels. Additional evidence supporting this hypothesis was obtained by showing that addition of exogenous beta gamma to unfertilized egg preparations increased the amount of PT substrate. These results indicate that G protein subunit expression is differentially regulated during oocyte maturation.     

Stockpiling of DNA polymerases during oogenesis and embryogenesis in the frog, Xenopus laevis

The amounts of the various forms of DNA polymerase (alpha 1, alpha 2, beta, and gamma) have been determined in oocytes, eggs, and embryos of the frog, Xenopus laevis. During oogenesis the relative proportions and absolute levels of all forms changed dramatically. In stage I (early) oocytes, DNA polymerase-gamma, the "mitochondrial" polymerase, was the predominant form. During oocyte growth, DNA polymerase-alpha 1 and -alpha 2 increased by more than 100-fold, DNA polymerase-beta by 15-fold, and DNA polymerase-gamma by only 8-fold. During oocyte maturation and ovulation, the levels of all forms of DNA polymerase roughly doubled. The mature stage VI oocyte contained 5 orders of magnitude more DNA polymerase activity than is found in an individual somatic cell. DNA polymerase-alpha 1 and -alpha 2, the "replicative" polymerases, were the predominant forms in mature oocytes and ovulated unfertilized eggs. During fertilization, the relative proportions and absolute levels of the four forms remained constant. During subsequent stages of embryogenesis, the total amounts of DNA polymerase-alpha 1 and -alpha 2 declined slightly from cleavage through gastrulation, the stages of most rapid chromosomal DNA replication. The rapid increase in cell number during early embryogenesis establishes the same levels of DNA polymerase/cell as are present in adult somatic cells. After neurulation, the absolute levels of DNA polymerase-alpha 1 and -alpha 2 increased in proportion to increases in cell number. The absolute levels of DNA polymerase-beta remained constant, and the levels of DNA polymerase-gamma increased 2-fold throughout embryogenesis.     

Meiosis-associated calcium waves in ascidian oocytes are correlated with the position of the male centrosome

We have used confocal microscopy to measure calcium waves and examine the distribution of tubulin in oocytes of the ascidian Ciona intestinalis during meiosis. We show that the fertilisation calcium wave in these oocytes originates in the vegetal pole. The sperm penetration site and female meiotic apparatus are found at opposite poles of the oocyte at fertilisation, confirming that C. intestinalis sperm enter in the vegetal pole of the oocyte. Following fertilisation, ascidian oocytes are characterised by repetitive calcium waves. Meiosis I-associated waves originate at the vegetal pole of the oocyte, and travel towards the animal pole. In contrast, the calcium waves during meiosis II initiate at the oocyte equator, and cross the oocyte cytoplasm perpendicular to the point of emission of the polar body. Immunolocalisation of tubulin during meiosis II reveals that the male centrosome is also located between animal and vegetal poles prior to initiation of the meiosis II-associated calcium waves, suggesting that the male centrosome influences the origin of these calcium transients. Ascidians are also characterised by an increase in sensitivity to intracellular calcium release after fertilisation. We show that this is not simply an effect of oocyte activation. The data strongly suggest a role for the male centrosome in controlling the mechanism and localisation of post-fertilisation intracellular calcium waves.     

Progesterone-stimulated meiotic cell division in Xenopus oocytes. Induction by regulatory subunit and inhibition by catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase.

Ripe Xenopus oocytes in first meiotic prophase when incubated with progesterone in vitro progress synchronously in 3 to 5 h without interphase to second meiotic metaphase where they remain until fertilization or activation. Using highly purified preparations of regulatory and catalytic subunits of adenosine 3':5'-monophosphate-dependent protein kinase from muscle, this progesterone-stimulated cell division sequence was found to be inhibited by microinjection of the catalytic subunit and induced directly in the absence of progesterone after microinjection of regulatory subunit. Dose-response curves revealed that half-maximal effects of regulatory and catalytic subunits occurred at an internal concentration of approximately 0.1 muM. These results indicate that the catalytic subunit is necessary and sufficient to block progesterone-stimulated meiotic cell division. Other experiments revealed that the catalytic subunit was inhibitory only during the first hour after progesterone exposure, suggesting that initial steps in meiotic cell division are affected. Control experiments demonstrate that the muscle cAMP-dependent protein kinase subunits may interact with the endogenous oocyte protein kinase. The results support a model in which meiotic cell division is regulated by a phosphoprotein subject to control by cAMP-dependent protein kinase.     

蛋白激酶C在小鼠卵母细胞体外成熟和受精中的作用

蛋白激酶是一类重要的丝／苏氨酸蛋白激酶。本实验以小鼠为实验动物,研究了PKC在卵母细胞体外成熟、活化和受精中的可能作用,及两种PKC亚型在卵母细胞中的定位。PKC激活剂PMA可以阻止GV期卵母细胞在体外恢复减数分裂,该作用可被PKC抑制剂CalphostinC抵消,但不能被PLCγ抑制剂U73122或PKCδ专一性抑制剂Rottlerin所克服。Western印迹显示PKCα和βI在卵母细胞发育过程中恒量表达。激光共聚焦显微术研究发现,受精或受到活化刺激后PKCα转位到卵母细胞膜上,同时皮质颗粒排放,说明PKCα可能参与调节卵皮质反应。本实验首次在小鼠中研究了PLCγ与受精的关系,发现不存在PKC对PLCγ的正反馈调节。此外,本研究还对小鼠卵巢中对PKCα和βI进行了蛋白定位研究。     

Activation of protein kinase C induces mitogen-activated protein kinase dephosphorylation and pronucleus formation in rat oocytes

Mammalian oocytes are arrested at metaphase of the second meiotic division (MII) before fertilization. When oocytes are stimulated by spermatozoa, they exit MII stage and complete meiosis. It has been suggested that an immediate increase in intracellular free calcium concentration and inactivation of maturation promoting factor (MPF) are required for oocyte activation. However, the underlying mechanism is still unclear. In the present study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, and their interplay in rat oocyte activation. We found that MAP kinase became dephosphorylated in correlation with pronucleus formation after fertilization. Protein kinase C activators, phorbol 12-myriatate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8), triggered dephosphorylation of MAP kinase and pronucleus formation in a dose-dependent and time-dependent manner. Dephosphorylation of MAP kinase was also correlated with pronucleus formation when oocytes were treated with PKC activators. Effects of PKC activators were abolished by the PKC inhibitors, calphostin C and staurosporine, as well as a protein phosphatase blocker, okadaic acid (OA). These results suggest that PKC activation may cause rat oocyte pronucleus formation via MAP kinase dephosphorylation, which is probably mediated by OA-sensitive protein phosphatases. We also provide evidence supporting the involvement of such a process in fertilization.     

Characterization and expression of a maternal axolotl cyclin B1 during oogenesis and early development

The M phase promoting factor (MPF) is a dimer composed of a catalytic Cdk1 subunit and a Cyclin B regulatory subunit. We have characterized a cDNA containing the entire coding sequence of an axolotl Cyclin B1 protein that is able to promote MPF activity when added to a fraction from prophase I oocytes that contains monomeric Cdk1. The axolotl cyclin B1 gene is expressed as a maternal mRNA in oocytes and early embryos. Its poly(A) tail length increases in metaphase II oocytes and then decreases regularly during the first embryonic cell cycles. Endogenous Cyclin B1 protein is first expressed during oocyte meiotic maturation. Its level oscillates after fertilization and is coordinated to the phosphorylation level of tyrosine 15 residue of Cdk1 (pTyr15), with both maxima preceding each cell division. As expected, when translated into microinjected oocytes, axolotl Cyclin B1 induces the resumption of meiosis. In electrically activated unfertilized eggs (UFE), Cyclin B1 and pTyr15 cyclic accumulations are observed with kinetics different from those of the early embryonic cycles. The axolotl embryo and UFE provide interesting in vivo comparative models for studying events controlling Cyclin B1 regulation during development.     

α-endosulfine (ENSA) regulates exit from prophase I arrest in mouse oocytes

Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M-phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M-phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with Okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with Okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.     

A metaphase pause: Hormone-induced maturation progresses through a long pause at the first meiotic metaphase in oocytes of the starfish, Pisaster ochraceus

In several species of starfish, it has been reported that the meiotic divisions in fertilized oocytes occur precociously compared to those in unfertilized oocytes. The nature of the 'acceleration' of meiosis was studied using Pisaster ochraceus oocytes. The extent of the acceleration of first polar body formation was found to be completely dependent on the time of fertilization (or artificial activation); fertilization at about 100 min after 1–methyladenine application accelerated meiosis I the most, while earlier or later fertilization resulted in a smaller extent of accelerations of meiosis I. Observation of isolated meiotic spindles and fluorescent visualization of meiotic spindles in whole oocytes showed that progression of meiosis I in Pisaster oocytes pauses transiently at metaphase I for more than 40min unless they are activated. The activation shortened the duration of metaphase I, which resulted in the acceleration of first polar body formation. A new term 'metaphase pause' is proposed to define this long duration of metaphase I in starfish oocytes.     

A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits

We show that the five subunits of a gamma-aminobutyric acid type A receptor (GABA(A) receptor) can be concatenated to yield a functional receptor. This concatenated receptor alpha(1)-beta(2)-alpha(1)-gamma(2)-beta(2) has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA(A) receptor subtype selective compounds.     

Casein kinase 1 (α, δ and ?) localize at the spindle poles,but may not be essential for mammalian oocyte meiotic progression

CK1 (casein kinase 1) is a family of serine/threonine protein kinase that is ubiquitously expressed in eukaryotic organism. CK1 members are involved in the regulation of many cellular processes. Particularly, CK1 was reported to phosphorylate Rec8 subunits of cohesin complex and regulate chromosome segregation in meiosis in budding yeast and fission yeast.^1-3 Here we investigated the expression, subcellular localization and potential functions of CK1α, CK1δ and CK1ϵ during mouse oocyte meiotic maturation. We found that CK1α, CK1δ and CK1ϵ all concentrated at the spindle poles and co-localized with γ-tubulin in oocytes at both metaphase I (MI) and metaphase II (MII) stages. However, depletion of CK1 by RNAi or overexpression of wild type or kinase-dead CK1 showed no effects on either spindle organization or chromosome segregation during oocyte meiotic maturation. Thus, CK1 is not the kinase that phosphorylates Rec8 cohesin in mammalian oocytes, and CK1 may not be essential for spindle organization and meiotic progression although they localize at spindle poles.