Potential upstream regulators and downstream targets of AMP-activated kinase signaling during oocyte maturation in a marine worm

Unlike in mice, where the onset of oocyte maturation (germinal vesicle breakdown, GVBD) is blocked by cAMP and triggered by AMP-activated kinase (AMPK), oocytes of the marine nemertean worm Cerebratulus undergo GVBD in response to cAMP elevations and AMPK deactivation. Since the pathways underlying AMPK's effects on mammalian or nemertean GVBD have not been fully defined, follicle-free nemertean oocytes were treated with pharmacological modulators and subsequently analyzed via immunoblotting methods using phospho-specific antibodies to potential regulators and targets of AMPK. Based on such phosphorylation patterns, immature oocytes possessed an active LKB1-like kinase that phosphorylated AMPK's T172 site to activate AMPK, whereas during oocyte maturation, AMPK and LKB1-like activities declined. In addition, given that MAPK can deactivate AMPK in somatic cells, oocytes were treated with inhibitors of ERK1/2 MAPK activation. However, these assays indicated that T172 dephosphorylation during maturation-associated AMPK deactivation did not require MAPK and that an observed inhibition of GVBD elicited by the MAPK kinase blocker U0126 was actually due to ectopic AMPK activation rather than MAPK inactivation. Similarly, based on tests using an inhibitor of maturation-promoting factor (MPF), T172 dephosphorylation occurred upstream to, and independently of, MPF activation. Alternatively, active MPF and MAPK were necessary for fully phosphorylating a presumably inhibitory S485/491 site on AMPK. Furthermore, in assessing signals possibly linking AMPK deactivation to MPF activation, evidence was obtained for maturing oocytes upregulating target-of-rapamycin activity and downregulating the cyclin-dependent kinase inhibitor Kip1. Collectively, these findings are discussed relative to multiple pathways potentially mediating AMPK signaling during GVBD.     

Endocytosis in the mouse oocyte and its contribution to cAMP signaling during meiotic arrest

Mammalian oocytes are arrested at prophase I of meiosis until a preovulatory surge of LH stimulates them to resume meiosis. Prior to the LH surge, high levels of cAMP within the oocyte maintain meiotic arrest; this cAMP is generated in the oocyte through the activity of the constitutively active, G(s)-coupled receptor, G-protein-coupled receptor 3 (GPR3) or GPR12. Activated GPRs are typically targeted for desensitization through receptor-mediated endocytosis, but a continuously high level of cAMP is needed for meiotic arrest. The aim of this study was to examine whether receptor-mediated endocytosis occurs in the mouse oocyte and whether this could affect the maintenance of meiotic arrest. We found that constitutive endocytosis occurs in the mouse oocyte. Inhibitors of receptor-mediated endocytosis, monodansylcadaverine and dynasore, inhibited the formation of early endosomes and completely inhibited spontaneous meiotic resumption. A red fluorescent protein-tagged GPR3 localized in the plasma membrane and within early endosomes in the oocyte, demonstrating that GPR3 is endocytosed. However, overexpression of G-protein receptor kinase 2 and β-arrestin-2 had only a modest effect on stimulating meiotic resumption, suggesting that these proteins do not play a major role in GPR3 endocytosis. Inhibition of endocytosis elevated cAMP levels within oocytes, suggesting that there is an accumulation of GPR3 at the plasma membrane. These results show that endocytosis occurs in the oocyte, leading to a decrease in cAMP production, and suggest that there is a balance between cAMP production and degradation in the arrested oocyte that maintains cAMP levels at an appropriate level during the maintenance of meiotic arrest.     

Contribution of high p34cdc2 kinase activity to premature chromosome condensation of injected somatic cell nuclei in rat oocytes

The present study was undertaken to clarify the relationship between the p34cdc2 kinase activity of in vitro-aged or enucleated rat oocytes and the premature chromosome condensation (PCC) of microinjected cumulus cell nuclei. Wistar rat oocytes were placed in vitro up to 120 min after the animal was killed. The p34cdc2 kinase activity of the oocytes decreased in a time-dependent manner. The incidence of PCC was higher when nuclear injection into intact oocytes was completed in 15-45 min rather than 46-120 min. When rat oocytes were enucleated for subsequent nuclear injection, the p34cdc2 kinase activity transiently increased soon after enucleation but drastically decreased after 30 min. Removal of the cytoplasm instead of the meta-phase-plate did not affect the p34cdc2 kinase activity even after 60 min. PCC occurred in intact and cytoplasm-removed oocytes but not in enucleated oocytes. In contrast, oocytes from BDF1 mice exhibited a p34cdc2 kinase level twice that of rat oocytes and supported PCC despite enucleation. The p34cdc2 kinase level of intact rat oocytes was reduced to the equivalent level of aged (120 min) or enucleated (+60 min) oocytes by a 45 min treatment with roscovitine, an inhibitor of p34cdc2 kinase. None of the roscovitine-treated oocytes supported PCC while half of the control oocytes did. When rat oocytes were treated with MG132, a proteasome inhibitor, delayed inactivation of the p34cdc2 kinase was observed in the MG132-treated oocytes. A significantly higher proportion of the MG132-treated oocytes supported PCC when compared with the control oocytes. Moreover, a higher proportion of MG132-treated and enucleated oocytes carried two pseudo-pronuclei after cumulus cell injection and developed to the two-cell stage when compared with the enucleated oocytes at the telophase-II stage. These results suggest that the decreased level of p34cdc2 kinase activity in aged or enucleated rat oocytes is responsible for their inability to support PCC of microinjected donor cell nuclei and that inhibition of p34cdc2 kinase inactivation by chemicals such as MG132 is in part effective for rat oocytes to promote PCC and further development.     

Temporal detection of caspase-3 and -7 in bovine in vitro produced embryos of different developmental capacity

Embryo quality is most frequently evaluated at the blastocyst stage, although quality parameters further back along the developmental axis, such as early developmental kinetics or oocyte quality, can be equally valuable. Despite the fact that previous studies in bovine have linked oocyte diameter and early developmental kinetics with blastocyst formation and viability, their relation with the incidence of apoptosis during embryo development remains relatively unexplored. Therefore, we related non-invasive parameters of oocyte and embryo quality, such as embryo kinetics, embryo morphology, and oocyte diameter, to the incidence of apoptosis throughout embryo development using fluorescent detection of active caspase-3 and -7. First, bovine in vitro embryos were selected according to developmental kinetics and morphology at four set times during culture and subjected to fluorescent detection of active caspase-3 and -7. Caspase activity was significantly higher in slow developing embryos in comparison with fast cleavers (P < 0.05), but was not related to embryo morphology. Second, bovine oocytes were divided into three groups on the basis of oocyte diameter and the resulting embryos were used for staining at the same four set times. Caspase activity was significantly higher in embryos derived from growing oocytes compared with those of fully grown oocytes at 45, 80, and 117 hours post-insemination (hpi; P < 0.05), but not at 168 hpi.     

Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1

In this study, we performed proteomic analysis of porcine oocytes during in vitro maturation. Comparison of oocytes at the initial and final stages of meiotic division characterized candidate proteins that were differentially synthesized during in vitro maturation. While the biosynthesis of many of these proteins was significantly decreased, we found four proteins with increased biosynthetic rate, which are supposed to play an essential role in meiosis. Among them, the ubiquitin C-terminal hydrolase-L1 (UCH-L1) was identified by mass spectrometry. To study the regulatory role of UCH-L1 in the process of meiosis in pig model, we used a specific inhibitor of this enzyme, marked C30, belonging to the class of isatin O-acyl oximes. When germinal vesicle (GV) stage cumulus-enclosed oocytes were treated with C30, GV breakdown was inhibited after 28 h of culture, and most of the oocytes were arrested at the first meiosis after 44 h. The block of metaphase I-anaphase transition was not completely reversible. In addition, the inhibition of UCH-L1 resulted in elevated histone H1 kinase activity, corresponding to cyclin-dependent kinase(CDK1)-cyclin B1 complex, and a low level of monoubiquitin. These results supported the hypothesis that UCH-L1 might play a role in metaphase I-anaphase transition by regulating ubiquitin-dependent proteasome mechanisms. In summary, a proteomic approach coupled with protein verification study revealed an essential role of UCH-L1 in the completion of the first meiosis and its transition to anaphase.     

Cytoplasmic polyadenylation controls cdc25B mRNA translation in rat oocytes resuming meiosis

Resumption of meiosis in oocytes represents the entry into M-phase of the cell cycle and is regulated by the maturation-promoting factor (MPF). Activation of MPF is catalyzed by the dual specificity phosphatase, cdc25. In mammals, cdc25 is represented by a multigene family consisting of three isoforms: A, B and C. A recent report that female mice lacking cdc25B exhibit impaired fertility suggests a role for this isoform in regulating the G2- to M-transition in mammalian oocytes. Supporting the above-mentioned observation, we demonstrate herein that microinjection of neutralizing antibodies against cdc25B interfered with the ability of rat oocytes to undergo germinal vesicle breakdown (GVB). We also show accumulation of cdc25B in GVB oocytes and a transient reduction in its amount at metaphase I of meiosis. The accumulation of cdc25B was associated with its mRNA cytoplasmatic polyadenylation and was prevented by the protein synthesis inhibitor cyclohexamide as well as by the polyadenylation inhibitor cordycepin. Immunofluorescence staining revealed translocation of cdc25B to the metaphase II spindle apparatus. Taken together, our findings provide evidence that cdc25B is involved in resumption of meiosis in rat oocytes. We further demonstrate for the first time, a periodic accumulation of cdc25B throughout meiosis that is translationally regulated and involves cdc25B mRNA polyadenylation.     

Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation

Mammalian oocytes grow and undergo meiosis within ovarian follicles. Oocytes are arrested at the first meiotic prophase, held in meiotic arrest by the surrounding follicle cells until a surge of LH from the pituitary stimulates the immature oocyte to resume meiosis. Meiotic arrest depends on a high level of cAMP within the oocyte. This cAMP is generated by the oocyte, through the stimulation of the G(s) G-protein by the G-protein-coupled receptor, GPR3. Stimulation of meiotic maturation by LH occurs via its action on the surrounding somatic cells rather than on the oocyte itself. LH induces the expression of epidermal growth factor-like proteins in the mural granulosa cells that act on the cumulus cells to trigger oocyte maturation. The signaling pathway between the cumulus cells and the oocyte, however, remains unknown. This review focuses on recent studies highlighting the importance of the oocyte in producing cAMP to maintain arrest, and discusses possible targets at the level of the oocyte on which LH could act to stimulate meiotic resumption.     

Control of division arrest and entry into meiosis by extracellular alkalisation in Saccharomyces cerevisiae

Limitation of nutrients allows yeast cells to arrest proliferation at G1 phase of the cell cycle and to enter the so-called stationary phase. We show here another pathway for cytostasis, which is associated with extracellular accumulation of bicarbonate and the resulting alkalisation of medium during the proliferation of cells respiring acetate. Alkalisation of medium by addition of bicarbonate or alkaline buffers ceased proliferation at G1 phase of logarithmically growing cells and caused a severe drop in G1-cyclin (CLN1 and CLN2) mRNAs. The arrested cells were heat-shock resistant, suggesting that the cells entered the stationary phase. Cells confluently grown on acetate re-entered into the cell cycle after acidification of the culture medium. These results indicate that external alkalisation is a primary cause of the cytostasis. The alkali-induced G1 arrest was shown to be cyclic AMP (cAMP)-independent using mutant cells which lack a functional Ras/cAMP signaling pathway. Alkalisation of medium also stimulated meiosis and sporulation in rich acetate medium, confirming our previous proposal that environmental alkalisation but not nitrogen limitation is a key condition for entry into meiosis and sporulation. © 1998 John Wiley & Sons, Ltd.     

Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep

The aim of this study was to test the Brilliant Cresyl Blue (BCB) stain to select prepubertal sheep oocytes for in vitro blastocyst production. Oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity and mRNA relative expression (RE) of genes related to metabolism (ATPase Na(+)/K(+) transporting α 1 (ATP1A1) and cytochrome c oxidase subunit 1 (COX1)) and constitutive function of the cell (cytoplasmic polyadenylation-element-binding protein (CPEB) and S100A10) were assessed. Immature oocytes were exposed to different BCB concentrations (13, 26, 39 and 52 μM) and classified according to their cytoplasm colouration as grown BCB+ (blue cytoplasm) and growing BCB- (colourless cytoplasm). Staining oocytes with 13 μM BCB during 60 min allows selection of (BCB+) the largest (123.66 μm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71 ± 6.19 s.e.m.) compared with non-stained BCB- oocytes (106.82 μm, 9% and 45.91 ± 3.35 s.e.m. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCB+ than in BCB- oocytes after in vitro maturation (3369 and 1565 AU respectively). MPF activity was assessed by CDC2 kinase activity assay showing significantly higher activity at metaphase II stage in BCB+ than in BCB- oocytes (1.479 ± 0.09 and 1.184 ± 0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A 10, did not show significant effect in mRNA RE between BCB selected oocytes. In conclusion, BCB stains larger and more competent oocytes to develop to the blastocyst stage with more active mitochondria and MPF activity and higher blastocyst cell number.     

Delay of nuclear maturation and reduction in developmental competence of pig oocytes after mineral oil overlay of in vitro maturation media

Steroid hormones, such as progesterone, oestrogen, androgen and meiosis activating sterols, are secreted from cumulus cells that are stimulated by gonadotrophins during maturation of oocytes in vitro. These steroid hormones may be absorbed by mineral oil or paraffin oil; however, in vitro maturation of pig oocytes is commonly performed using medium covered by oil. In this study, high concentrations of progesterone, oestradiol and testosterone were detected in the culture medium after pig cumulus-oocyte complexes (COCs) were cultured with FSH and LH for 44 h in medium without an oil overlay. However, high concentrations of these steroid hormones were not detected in medium when COCs were cultured with the mineral oil overlay. When high concentrations of these steroid hormones were secreted by COCs, germinal vesicle breakdown (GVBD) and the activation of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase in oocytes occurred earlier in comparison with oocytes cultured in medium covered with mineral oil. Moreover, a decrease in p34(cdc2) kinase activity during meiotic progression beyond metaphase I was observed in oocytes cultured in conditions under which high concentrations of steroid hormones were secreted by COCs. In addition, the rate of development to the blastocyst stage after IVF was higher in oocytes matured in medium without an oil overlay. These adverse effects of oil may be explained by absorption by the oil of cumulus-secreted steroids or by the release of toxic compounds into the medium.     

牛α-乳白蛋白-油酸复合物(BAMLET)对SGC-7901细胞的促凋亡作用及凋亡机制的研究

通过研究α-乳白蛋白-油酸复合物(BAMLET)对SGC-7901细胞的促凋亡作用及其对cAMP/PKA信号途径的影响来探讨BAMLET促进细胞凋亡作用的机制。方法:倒置显微镜下观测细胞形态学变化;MTT法检测细胞增殖抑制率;流式细胞术检测细胞的凋亡;酶联免疫试剂盒检测人胃癌细胞中cAMP和PKA的浓度变化。实验结果表明,α-乳白蛋白-油酸复合物处理细胞24、48、72h后,细胞增殖明显受到抑制,且抑制率随时间延长而增加;流式细胞仪检测药物作用细胞24、48、72h后细胞发生不同程度的凋亡且凋亡率逐渐升高。cAMP及PKA实验结果表明,cAMP的浓度在药物作用细胞20min时达到最大值且明显高于对照组、PKA的活性明显高于对照组且差异性显著。上述结果表明BAMLET能够激活cAMP/PKA信号通路,发挥其促进细胞凋亡的作用。     

Catalytic isoforms Tpk1 and Tpk2 of Candida albicans PKA have non‐redundant roles in stress response and glycogen storage

Candida albicans cAMP‐dependent protein kinase (PKA) is coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1). In this organism the cAMP/PKA signalling pathway mediates basic cellular processes, such as the yeast‐to‐hyphae transition and cell cycle regulation. In the present study, we investigated the role of C. albicans PKA in response to saline, heat and oxidative stresses as well as in glycogen storage. To fine‐tune the analysis, we performed the studies on several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in a different BCY1 genetic background. We observed that tpk1Δ/tpk1Δ strains developed a lower tolerance to saline exposure, heat shock and oxidative stress, while wild‐type and tpk2Δ/tpk2Δ mutants were resistant to these stresses, indicating that both isoforms play different roles in the stress response pathway. We also found that regardless of the TPK background, heterozygous and homozygous BCY1 mutants were highly sensitive to heat treatment. Surprisingly, we observed that those strains devoid of one or both TPK1 alleles were defective in glycogen storage, while strains lacking Tpk2 accumulated higher levels of the polysaccharide, indicating that Tpk1 and Tpk2 have opposite roles in carbohydrate metabolism. Copyright © 2009 John Wiley & Sons, Ltd.     

Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes

Mad2 is a pivotal component of the spindle assembly checkpoint (SAC) which inhibits anaphase promoting complex/cyclo-some (APC/C) activity by sequestering Cdc20 thereby regulating the destruction of securin and cyclin B. During mitosis, spindle depolymerisation induces a robust Mad2-dependent arrest due to inhibition of securin and cyclin B destruction. In contrast to mitosis, the molecular details underpinning the meiosis I arrest experienced by mouse oocytes exposed to spindle depolymerisation remain incompletely characterised. Notably, the role of Mad2 and the fate of the anaphase-marker, securin, are unexplored. As shown previously, we find that spindle depolymerisation by nocodazole inhibits first polar body extrusion (PBE) and stabilises cyclin B and cyclin-dependent kinase 1 activity in mouse oocytes. Here we show that stabilisation of cyclin B in nocodazole can be sustained for several hours and is associated with stabilisation of securin. These effects are SAC-mediated as, in oocytes depleted of the majority of Mad2 by morpholino antisense, securin and cyclin B are destabilised and 15% of oocytes undergo PBE. This reflects premature APC/C activation as a mutant form of cyclin B lacking its APC/C degradation signal is stable in Mad2-depleted oocytes. Moreover, homologues do not disjoin during the prolonged meiosis I arrest (> 18 h) induced by nocodaozole indicating that a non-cleavage mechanism is insufficient on its own for resolution of arm cohesion in mammalian oocytes. In conclusion, when all kinetochores lack attachment and tension, mouse oocytes mount a robust Mad2-dependent meiosis I arrest which inhibits the destruction of securin and cyclin B.     

Cytoplasmic dynein participates in meiotic checkpoint inactivation in mouse oocytes by transporting cytoplasmic mitotic arrest-deficient (Mad) proteins from kinetochores to spindle poles

The present study was designed to investigate the localization and function of cytoplasmic dynein (dynein) during mouse oocyte meiosis and its relationship with two major spindle checkpoint proteins, mitotic arrest-deficient (Mad) 1 and Mad2. Oocytes at various stages during the first meiosis were fixed and immunostained for dynein, Mad1, Mad2, kinetochores, microtubules, and chromosomes. Some oocytes were treated with nocodazole before examination. Anti-dynein antibody was injected into the oocytes at germinal vesicle (GV) stage before the examination of its effects on meiotic progression or Mad1 and Mad2 localization. Results showed that dynein was present in the oocytes at various stages from GV to metaphase II and the locations of Mad1 and Mad2 were associated with dynein's movement. Both Mad1 and Mad2 had two existing states: one existed in the cytoplasm (cytoplasmic Mad1 or cytoplasmic Mad2), which did not bind to kinetochores, while the other bound to kinetochores (kinetochore Mad1 or kinetochore Mad2). The equilibrium between the two states varied during meiosis and/or in response to the changes of the connection between microtubules and kinetochores. Cytoplasmic Mad1 and Mad2 recruited to chromosomes when the connection between microtubules and chromosomes was destroyed. Inhibition of dynein interferes with cytoplasmic Mad1 and Mad2 transportation from chromosomes to spindle poles, thus inhibits checkpoint silence and delays anaphase onset. These results indicate that dynein may play a role in spindle checkpoint inactivation.     

Fertilization differently affects the levels of cyclin B1 and M-phase promoting factor activity in maturing and metaphase II mouse oocytes

Fertilization affects levels of cyclin B1 and M-phase promoting factor (MPF) activity in maturing and metaphase II mouse oocytes in two distinct ways. In metaphase II oocytes, it leads to a Ca(2)(+)-dependent, continuous degradation of cyclin B1 and inactivation of cyclin dependent kinase (CDC2A)-cyclin B1 complex (MPF). In this paper, we show that neither mono- nor polyspermic fertilization of prometaphase I and metaphase I oocytes triggered degradation of cyclin B1. However, polyspermic fertilization of prometaphase I oocytes led to a transient decrease in MPF activity that lasted for 2 h. The inactivation of MPF in polyspermic prometaphase I oocytes did not depend on the fertilization-induced increase in the cytoplasmic concentration of free Ca(2)(+) ions, but was caused, at least in part, by dephosphorylation of CDC2A at threonine 161 (Thr161). We found that polyspermic fertilization did not affect glutathione levels in prometaphase I oocytes, and concluded that the decrease in MPF activity and dephosphorylation of CDC2A at Thr161 in polyspermic prometaphase I oocytes were not caused by a change in the redox status of the cell induced by an introduction of excessive amount of sperm protamines. Instead, we propose that inactivation of MPF activity in polyspermic maturing oocytes is caused by a change in nucleo-cytoplasmic ratio that leads to a 'titration' of kinases and phosphatases responsible for keeping MPF in an active state. This idea is supported by the finding that oocytes fused with thymocytes rather than spermatozoa also showed a transient decrease in MPF activity.     

Independent activation of MAP kinase and MPF during the initiation of meiotic maturation in pig oocytes

Mitogen-activated protein (MAP) kinase is universally activated during oocyte maturation in all vertebrates studied to date. Its role in the resumption of meiosis and in the activation of maturation-promoting factor (MPF) remains unclear, especially in domestic species such as the pig. This study aimed to clarify the temporal and causal relationships between MAP kinase and MPF during meiotic maturation, particularly during the resumption of meiosis. Pig oocytes were matured synchronously in culture by treatment with cycloheximide. Kinase activities were analysed using a sensitive in vitro double-kinase assay and the specific MAP kinase pathway inhibitor U0126. MAP kinase and MPF were activated simultaneously at the time of germinal vesicle breakdown (GVBD; 6 h after removal of cycloheximide); they reached significant activity at 7 h (P < 0.05). The activities increased in parallel during GVBD (6-10 h) and peaked when the oocytes entered metaphase I (MI; 10 h). Whereas MAP kinase remained stable at peak activity thereafter, MPF activity significantly declined during the MI-MII transition (16-20 h) but increased to a second peak at MII (22 h). MAP kinase activity in denuded and cumulus-cell enclosed oocytes was completely inhibited by 20 and 80 mmicro mol U0126 l(-1), respectively. Oocytes without detectable MAP kinase activity underwent normal GVBD in terms of nuclear morphology and timing, although later meiotic stages were abnormal. The kinetics of MPF activity during GVBD were unaffected by U0126. This study has demonstrated that MAP kinase is activated simultaneously with MPF at GVBD, but that its activation is not essential for the activation of MPF nor for the resumption of the first meiosis in pig oocytes.