Galphao/i and Galphas signaling function in parallel with the MSP/Eph receptor to control meiotic diapause in C. elegans

BACKGROUND: A conserved biological feature of sexual reproduction in animals is that oocytes arrest in meiotic prophase and resume meiosis in response to extraovarian signals. In C. elegans, sperm trigger meiotic resumption by means of the major sperm protein (MSP) signal. MSP promotes meiotic resumption by functioning as an ephrin-signaling antagonist and by counteracting inhibitory inputs from the somatic gonadal sheath cells. RESULTS: By using a genome-wide RNAi screen in a female-sterile genetic background, we identified 17 conserved genes that maintain meiotic arrest in the absence of the MSP signal. In vitro binding experiments show that MSP promotes oocyte mitogen-activated protein kinase activation and meiotic maturation in part through direct interaction with the VAB-1 Eph receptor. Four conserved proteins, including a disabled protein (DAB-1), a vav family GEF (VAV-1), a protein kinase C (PKC-1), and a STAM homolog (PQN-19), function with the VAB-1 Eph/MSP receptor in oocytes. We show that antagonistic Galphao/i and Galphas signaling pathways function in the soma to regulate meiotic maturation in parallel to the VAB-1 pathway. Galphas activity is necessary and sufficient to promote meiotic maturation, which it does in part by antagonizing inhibitory sheath/oocyte gap-junctional communication. CONCLUSIONS: Our findings show that oocyte Eph receptor and somatic cell G protein signaling pathways control meiotic diapause in C. elegans, highlighting contrasts and parallels between MSP signaling in C. elegans and luteinizing hormone signaling in mammals.     

Eph and NMDA receptors control Ca2+/calmodulin-dependent protein kinase II activation during C. elegans oocyte meiotic maturation

Fertilization in the female reproductive tract depends on intercellular signaling mechanisms that coordinate sperm presence with oocyte meiotic progression. To achieve this coordination in Caenorhabditis elegans, sperm release an extracellular signal, the major sperm protein (MSP), to induce oocyte meiotic maturation and ovulation. MSP binds to multiple receptors, including the VAB-1 Eph receptor protein-tyrosine kinase on oocyte and ovarian sheath cell surfaces. Canonical VAB-1 ligands called ephrins negatively regulate oocyte maturation and MPK-1 mitogen-activated protein kinase (MAPK) activation. Here, we show that MSP and VAB-1 regulate the signaling properties of two Ca2+ channels that are encoded by the NMR-1 N-methyl D-aspartate type glutamate receptor subunit and ITR-1 inositol 1,4,5-triphosphate receptor. Ephrin/VAB-1 signaling acts upstream of ITR-1 to inhibit meiotic resumption, while NMR-1 prevents signaling by the UNC-43 Ca2+/calmodulin-dependent protein kinase II (CaMKII). MSP binding to VAB-1 stimulates NMR-1-dependent UNC-43 activation, and UNC-43 acts redundantly in oocytes to promote oocyte maturation and MAPK activation. Our results support a model in which VAB-1 switches from a negative regulator into a redundant positive regulator of oocyte maturation upon binding to MSP. NMR-1 mediates this switch by controlling UNC-43 CaMKII activation at the oocyte cortex.     

Elucidation of the signaling pathways that underpin capacitation‐associated surface phosphotyrosine expression in mouse spermatozoa

Recent studies from within our laboratory have demonstrated a causal relationship between capacitation‐associated surface phosphotyrosine expression and the ability of mouse spermatozoa to recognize the oocyte and engage in sperm–zona pellucida interaction. In the studies described herein we have sought to investigate the signaling pathways that underpin the tyrosine phosphorylation of sperm surface protein targets and validate the physiological significance of these pathways in relation to sperm–zona pellucida adhesion. Through selective pharmacological inhibition we have demonstrated that surface phosphotyrosine expression is unlikely to be mediated by the canonical cAMP‐dependent protein kinase A (PKA) signaling cascade that has been most widely studied in relation to sperm capacitation. Rather, it appears to be primarily driven by the extracellular signal‐regulated kinase (ERK) module of the mitogen‐activated protein kinase (MAPK) pathway. Consistent with this notion, the main components of the ERK module (RAS, RAF1, MEK, and ERK1/2) were localized to the periacrosomal region of the head of mature mouse spermatozoa and their phosphorylation status within this region of the cell was positively modulated by capacitation. Furthermore, inhibition of several elements of this pathway suppressed sperm surface phosphotyrosine expression and induced a concomitant reduction sperm–zona pellucida interaction. Collectively, these data highlight a previously unappreciated role of the ERK module in the modification of the sperm surface during capacitation to render these cells functionally competent to engage in the process of fertilization. J. Cell. Physiol. 224:71–83, 2010 © 2010 Wiley‐Liss, Inc.     

A primer on meiotic resumption in starfish oocytes: the proposed signaling pathway triggered by maturation-inducing hormone

This short review updates the maturation-inducing hormonal signaling in starfish oocytes. In this system, the activation of cyclin B-Cdc2 kinase (Cdk1) that leads to meiotic resumption does not require new protein synthesis. The key intracellular mediator after hormonal stimulation by 1-methyladenine is the protein kinase Akt/PKB, which in turn directly downregulates Myt1 and upregulates Cdc25 toward the activation of cyclin B-Cdc2. Mitotic kinases including Aurora, Plk1 and Greatwall are activated downstream of cyclin B-Cdc2. The starfish oocyte thus provides a simple model system for the study of meiotic resumption.     

Phosphorylation of mitogen-activated protein kinase is regulated by protein kinase C, cyclic 3',5'-adenosine monophosphate, and protein phosphatase modulators during meiosis resumption in rat oocytes

Mitogen-activated protein (MAP) kinase, protein kinase C (PKC), cAMP, and okadaic acid (OA)-sensitive protein phosphatases (PPs) have been suggested to be involved in oocyte meiotic resumption. However, whether these protein kinases and phosphatases act by independent pathways or interact with each other in regulating meiosis resumption is unknown. In the present study, we aimed to determine the regulation of meiosis resumption and MAP kinase phosphorylation by PKC, cAMP, and OA-sensitive PPs in rat oocytes using an in vitro oocyte maturation system and Western blot analysis. We found that ERK1 and ERK2 isoforms of MAP kinases existed in a dephosphorylated (inactive) form in germinal vesicle breakdown (GVBD)-incompetent and GVBD-competent germinal vesicle intact (GVI) oocytes as well as GVBD oocytes at equivalent levels. These results indicate that MAP kinases are not responsible for the initiation of normal meiotic resumption in rat oocytes. However, when GVBD-incompetent and GVBD-competent oocytes were incubated in vitro for 5 h, MAP kinases were phosphorylated (activated) in GVBD-competent oocytes, but not in meiotic-incompetent oocytes, suggesting that oocytes acquire the ability to phosphorylate MAP kinase during acquisition of meiotic competence. We also found that both meiosis resumption and MAP kinase phosphorylation were inhibited by PKC activation or cAMP elevation. Moreover, these inhibitory effects were overcome by OA, which inhibited PP1/PP2A activities. These results suggest that both cAMP elevation and PKC activation inhibit meiosis resumption and MAP kinase phosphorylation at a step prior to OA-sensitive protein phosphatases. In addition, inhibitory effects of cAMP elevation on meiotic resumption and MAP kinase phosphorylation were not reversed by calphostin C-induced PKC inactivation, indicating that cAMP inhibits both meiotic resumption and MAP kinase activation in a PKC-independent manner.     

Unique regulation of c-Jun N-terminal kinase by PYK2/CAK-beta in angiotensin II-stimulated vascular smooth muscle cells

Activation of tyrosine kinases is believed to play a central role in angiotensin II (AngII) signaling. Here, we have investigated whether a tyrosine kinase, PYK2, is functionally involved in AngII-induced c-Jun N-terminal kinase (JNK) activation in vascular smooth muscle cells (VSMCs). Adenovirus expressing PYK2 kinase-inactive mutant K457A or a tyrosine phosphorylation site mutant Y402F was transfected in VSMCs. AngII-induced JNK phosphorylation was markedly enhanced by K457A, whereas it was suppressed by Y402F. Protein synthesis induced by AngII was also enhanced by K457A and inhibited by Y402F. In this regard, K457A suppressed PYK2 kinase activation by AngII, whereas it enhanced AngII-induced PYK2 Tyr(402) phosphorylation. By contrast, Y402F inhibited PYK2 Tyr(402) phosphorylation, whereas it markedly enhanced AngII-induced PYK2 kinase activation. Thus, we conclude that PYK2 kinase activity negatively regulates JNK activation and protein synthesis, whereas Tyr(402) phosphorylation positively regulates these events in AngII-stimulated VSMCs, suggesting a unique role of PYK2 in mediating vascular remodeling.     

Targeting of PYK2 to focal adhesions as a cellular mechanism for convergence between integrins and G protein-coupled receptor signaling cascades

The non-receptor tyrosine kinase PYK2 appears to function at a point of convergence of integrins and certain G protein-coupled receptor (GPCR) signaling cascades. In this study, we provide evidence that translocation of PYK2 to focal adhesions is triggered both by cell adhesion to extracellular matrix proteins and by activation of the histamine GPCR. By using different mutants of PYK2 as green fluorescent fusion proteins, we show that the translocation of PYK2 to focal adhesions is not dependent on its catalytic activity but rather is mediated by its carboxyl-terminal domain. Translocation of PYK2 to focal adhesions was attributed to enhanced tyrosine phosphorylation of PYK2 and its association with the focal adhesion proteins paxillin and p130(Cas). Translocation of PYK2 to focal adhesions, as well as its tyrosine phosphorylation in response to histamine treatment, was abolished in the presence of protein kinase C inhibitors or cytochalasin D treatment, whereas activation of protein kinase C by phorbol ester resulted in focal adhesion targeting of PYK2 and its tyrosine phosphorylation in an integrin-clustering dependent manner. Overexpression of a wild-type PYK2 enhanced ERK activation in response to histamine, whereas a kinase-deficient mutant substantially inhibited this response. Furthermore, inhibition of PYK2 translocation to focal adhesions abolished ERK activation in response to histamine treatment. These results suggest that PYK2 apparently links between GPCRs and focal adhesion-dependent ERK activation and can provide the molecular basis underlying PYK2 function at a point of convergence between signaling pathways triggered by extracellular matrix proteins and certain GPCR agonists.     

Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation.

Recently, we have purified a Src-related tyrosine kinase, named Xenopus tyrosine kinase (Xyk), from oocytes of Xenopus laevis and found that the enzyme is activated within 1 min following fertilization [Sato et al. (1996) J. Biol. Chem. 271, 13250-13257]. A concomitant translocation of a part of the activated enzyme from the membrane fraction to the cytosolic fraction was also observed. In the present study, we show that parthenogenetic egg activation by a synthetic RGDS peptide [Y. Iwao and T. Fujimura, T. (1996) Dev. Biol. 177, 558-567], an integrin-interacting peptide, but not by electrical shock or the calcium ionophore A23187 causes the kinase activation, tyrosine phosphorylation, and translocation of Xyk. A synthetic tyrosine kinase-specific inhibitor peptide was employed to analyze the importance of the Xyk activity in egg activation. We found that the peptide inhibits the kinase activity of purified Xyk at IC50 of 8 microM. Further, egg activation induced by sperm or RGDS peptide but not by A23187 was inhibited by microinjection of the peptide. In the peptide-microinjected eggs, penetration of the sperm nucleus into the egg cytoplasm and meiotic resumption in the egg were blocked. Indirect immunofluorescence study demonstrates that Xyk is exclusively localized to the cortex of Xenopus eggs, indicating that Xyk can function in close proximity to the sperm-egg or RGDS peptide-egg interaction site. Taken together, these data suggest that the tyrosine kinase Xyk plays an important role in the early events of Xenopus egg activation in a manner independent or upstream of calcium signaling.     

Regulation of the human sperm tyrosine kinase c-yes. Activation by cyclic adenosine 3',5'-monophosphate and inhibition by Ca(2+)

During the process of capacitation, spermatozoa go through a whole set of signaling cascade events in order to become fully competent at fertilizing the egg. An increase in sperm protein tyrosine phosphorylation has been described during this final maturational event in different animal species as well as in humans. Although the phosphotyrosine content of sperm protein is modulated by cAMP, Ca(2+), BSA, oxygen derivatives, and cholesterol, no protein tyrosine kinase (PTK) nor the phosphotyrosine protein phosphatase (PTPase) directly involved in the control of the phosphotyrosine content of sperm protein has been identified. Therefore, the goal of the present study was to identify the tyrosine kinases putatively responsible for the increases in sperm protein phosphotyrosine content. In the present study, we show that the src-related tyrosine kinase c-yes is present in the head of human spermatozoa in both membranes and Triton X-100-insoluble extracts. Our hypothesis was that c-yes is a tyrosine kinase responsible for at least some of the capacitation-induced increase in protein tyrosine phosphorylation. When spermatozoa were previously incubated in the presence of 3-isobutyl-1-methylxanthine or 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, treatments known to increase the phosphotyrosine content of human sperm proteins, an increase in the kinase activity of immunoprecipitated yes was measured using enolase as a substrate. These results suggest that cAMP activates while Ca(2+) inhibits human sperm c-yes kinase activity.     

Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I.

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2.     

Evidence for a role of capillary pericytes in vascular growth of the developing ovine corpus luteum

The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.     

Fyn kinase activity is required for normal organization and functional polarity of the mouse oocyte cortex

The objective of the present study was to determine whether Fyn kinase participated in signaling events during sperm–egg interactions, sperm incorporation, and meiosis II. The functional requirement of Fyn kinase activity in these events was tested through the use of the protein kinase inhibitor SKI‐606 (Bosutinib) and by analysis of Fyn‐null oocytes. Suppression of Fyn kinase signaling prior to fertilization caused disruption of the functional polarity of the oocyte with the result that sperm were able to fuse with the oocyte in the immediate vicinity of the meiotic spindle, a region that normally does not allow sperm fusion. The loss of functional polarity was accompanied by disruption of the microvilli and cortical granule‐free zone that normally overlie the meiotic spindle. Changes in the distribution of cortical granules and filamentous actin provided further evidence of disorganization of the oocyte cortex. Rho B, a molecular marker for oocyte polarity, was unaffected by suppression of Fyn activity; however, the polarized association of Par‐3 with the cortex overlying the meiotic spindle was completely disrupted. The defects in oocyte polarity in Fyn‐null oocytes correlated with a failure of the MII chromosomes to maintain a position close to the oocyte cortex which seemed to underlie the above defects in oocyte polarity. This was associated with a delay in completion of meiosis II. Pronuclei, however, eventually formed and subsequent mitotic cleavages and blastocyst formation occurred normally. Mol. Reprod. Dev. 76: 819–831, 2009. © 2009 Wiley‐Liss, Inc.     

Sperm‐borne protein,PAWP, initiates zygotic development in Xenopus laevis by eliciting intracellular calcium release

We previously reported postacrosomal sheath WW domain binding protein (PAWP) as a candidate sperm borne, oocyte‐activating factor. PAWP enters the oocyte during fertilization and induces oocyte activation events including meiotic resumption, pronuclear formation, and egg cleavage. However, in order to provide proof that PAWP is a primary initiator of zygotic development it is imperative to show that PAWP initiates intracellular calcium signaling, which is considered essential for oocyte activation. Utilizing Xenopus laevis as our model, we injected recombinant PAWP or Xenopus sperm into metaphase II‐arrested oocytes and observed a significant rise in intracellular calcium levels over controls. Concurring intensities and durations of PAWP and sperm‐induced calcium waves, detected by infrared two‐photon laser‐scanning fluorescence microscopy, were prevented by coinjection of a competitive PPGY‐containing peptide derived from PAWP but not by the point‐mutated form of this peptide. This study also correlates PAWP and sperm‐induced calcium release with meiotic resumption in Xenopus. The similar mode of oocyte activation, and the ability of the competitive peptide in blocking both sperm‐ and PAWP‐induced calcium release, provide evidence for the first time that sperm‐anchored PAWP is a primary initiator of zygotic development. Mol. Reprod. Dev. 77: 249–256, 2010. © 2009 Wiley‐Liss, Inc.     

Xp42(Mpk1) activation is not required for germinal vesicle breakdown but for Raf complete phosphorylation in insulin-stimulated Xenopus oocytes

Fully grown G2-arrested Xenopus oocytes resume meiosis in vitro upon exposure to hormonal stimulation. Progesterone triggers oocyte meiosis resumption through a Ras-independent pathway that involves a p39Mos-dependent activation of the mitogen-activated protein (MAP) kinases. Insulin also triggers meiosis resumption through a tyrosine kinase receptor that activates a Ras-dependent pathway leading to the MAP kinases activation. Antisense phosphorothioate oligonucleotides were used to prevent p39Mos accumulation and Erk-like Xp42(Mpk1) activation during insulin-induced Xenopus oocytes maturation. In contrast to previous works, prevention of p39Mos-induced activation of Xp42(Mpk1) in insulin-treated oocytes did not inhibit but delayed meiotic resumption, like in progesterone-stimulated oocytes. Activations of Xp42(Mpk1), the unique Erk of the oocyte, and of its downstream target p90Rsk, were impaired and phosphorylation of the MAPKK kinase Raf was partially inhibited. Similarly, oocytes treated with the MEK inhibitor U0126, stimulated by insulin exhibited delayed germinal vesicle breakdown, absence of Xp42(Mpk1) activation, and partial phosphorylation of Raf. To summarize, whereas p39Mos-induced activation of MEK/MAPK pathway is dispensable for insulin-induced germinal vesicle breakdown, Xp42(Mpk1) activation induced by insulin is dependent upon p39Mos synthesis. Raf complete phosphorylation appears to require the MEK/MAPK pathway activation both in progesterone and insulin-stimulated oocytes.     

Involvement of MEK-mitogen-activated protein kinase pathway in follicle-stimulating hormone-induced but not spontaneous meiotic resumption of mouse oocytes

Mitogen-activated protein (MAP) kinase has been reported to be activated during oocyte meiotic maturation in a variety of mammalian species. However, the mechanism(s) responsible for MAP kinase activation and the consequence of its premature activation during gonadotropin-induced oocyte meiotic resumption have not been examined. The present experiments were conducted to investigate the possible role of MAP kinase in FSH-induced and spontaneous oocyte meiotic resumption in the mouse. MAP kinase kinase (MAPKK, MEK) inhibitor, PD98059 or U0126, produced a dose-dependent inhibitory effect on both FSH-induced oocyte meiotic resumption and MAP kinase activation in the oocytes. However, the same inhibitor did not block spontaneous meiotic resumption of either denuded or cumulus cell-enclosed mouse oocytes, despite the activity of MAP kinase being totally inhibited. Immunoblotting the oocytes and the cumulus cells with the anti-active MAP kinase antibody showed that MAP kinase activity in the oocytes was detected at 8 h of FSH treatment, prior to germinal vesicle breakdown and increased as maturation progressed in the following culture period. In the cumulus cells, MAP kinase was activated even faster, its activity was detected at 1 h of FSH stimulation and increased gradually until 8 h of FSH treatment, then decreased and diminished after 12 h of FSH action. These data demonstrated that the MEK-MAP kinase pathway is implicated in FSH-induced but not spontaneous oocyte meiotic resumption.     

Activated proline‐rich tyrosine kinase 2 regulates meiotic spindle assembly in the mouse oocyte

Proline‐rich tyrosine kinase 2 (PYK2), a member of the protein tyrosine kinase family, plays an important role in various cellular processes. PYK2 can be phosphorylated on tyrosine 402 by diverse stimuli at the cell surface, and recent studies have shown that this activated form of PYK2 is enriched in oocytes and required for fertilization. However, the subcellular localization and functions of activated PYK2 in oocytes remain elusive. In this study, we demonstrate that the localization of p‐PYK2 undergoes dynamic changes during in vitro maturation of mouse oocytes. The signal of p‐PYK2 is initially dispersed in the cytoplasm, but begins to decorate organized microtubules after the germinal vesicle breakdown and localizes to spindle poles at metaphase. Our data further show that p‐PYK2 colocalizes with γ‐tubulin from the germinal vesicle stage through the end of meiosis in mouse oocytes. Nocodazole treatment and washout experiments confirm that p‐PYK2 associates with the oocyte spindle and spindle poles. Moreover, pharmacological inhibition of PYK2 activity dramatically alters the morphology of the bipolar spindle and prevents oocyte maturation. Together, these data suggest that activated PYK2 may function as a component of the microtubule organizing center to regulate spindle assembly during the meiotic process of mouse oocytes.     

Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-delta

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.     

PDK1 is required for the hormonal signaling pathway leading to meiotic resumption in starfish oocytes

Meiotic resumption is generally under the control of an extracellular maturation-inducing hormone. It is equivalent to the G2-M phase transition in somatic cell mitosis and is regulated by cyclin B-Cdc2 kinase. However, the complete signaling pathway from the hormone to cyclin B-Cdc2 is yet unclear in any organism. A model system to analyze meiotic resumption is the starfish oocyte, in which Akt/protein kinase B (PKB) plays a key mediator in hormonal signaling that leads to cyclin B-Cdc2 activation. Here we show in starfish oocytes that when PDK1 activity is inhibited by a neutralizing antibody, maturation-inducing hormone fails to induce cyclin B-Cdc2 activation at the meiotic G2-M phase transition, even though PDK2 activity becomes detectable. These observations assign a novel role to PDK1 for a hormonal signaling intermediate toward meiotic resumption. They further support that PDK2 is a molecule distinct from PDK1 and Akt, and that PDK2 activity is not sufficient for the full activation of Akt in the absence of PDK1 activity.     

3',5'-cyclic adenosine monophosphate response element binding protein up-regulated cytochrome P450 lanosterol 14alpha-demethylase expression involved in follicle-stimulating hormone-induced mouse oocyte maturation

Cytochrome P450 lanosterol 14alpha-demethylase (CYP51) is a key enzyme in sterols and steroids biosynthesis that can induce meiotic resumption in mouse oocytes. The present study investigated the expression mechanism and function of CYP51 during FSH-induced mouse cumulus oocyte complexes (COCs) meiotic resumption. FSH increased cAMP-dependent protein kinase (PKA) RIIbeta level and induced cAMP response element-binding protein (CREB) phosphorylation and CYP51 expression in cumulus cells before oocyte meiotic resumption. Moreover, CYP51 and epidermal growth factor (EGF)-like factor [amphiregulin (AR)] expression were blocked by (2)-naphthol-AS-Ephosphate (KG-501) (a drug interrupting the formation of CREB functional complex). KG-501 and RS21607 (a specific inhibitor of CYP51 activity) inhibited oocyte meiotic resumption, which can be partially rescued by progesterone. These two inhibitors also inhibited FSH-induced MAPK phosphorylation. EGF could rescue the suppression by KG-501 but not RS21607. Furthermore, type II PKA analog pairs, N(6)-monobutyryl-cAMP plus 8-bromo-cAMP, increased PKA RIIbeta level and mimicked the action of FSH, including CREB phosphorylation, AR and CYP51 expression, MAPK activation, and oocyte maturation. All these data suggest that CYP51 plays a critical role in FSH-induced meiotic resumption of mouse oocytes. CYP51 and AR gene expression in cumulus cells are triggered by FSH via a type II PKA/CREB-dependent signal pathway. Our study also implicates that CYP51 activity in cumulus cells participates in EGF receptor signaling-regulated oocyte meiotic resumption.     

Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals

Mitogen-activated protein kinase (MAPK) is a family of Ser/Thr protein kinases that are widely distributed in eukaryotic cells. Studies in the last decade revealed that MAPK cascade plays pivotal roles in regulating the meiotic cell cycle progression of oocytes. In mammalian species, activation of MAPK in cumulus cells is necessary for gonadotropin-induced meiotic resumption of oocytes, while MAPK activation is not required for spontaneous meiotic resumption. After germinal vesicle breakdown (GVBD), MAPK is involved in the regulation of microtubule organization and meiotic spindle assembly. The activation of this kinase is essential for the maintenance of metaphase II arrest, while its inactivation is a prerequisite for pronuclear formation after fertilization or parthenogenetic activation. MAPK cascade interacts extensively with other protein kinases such as maturation-promoting factor, protein kinase A, protein kinase C, and calmodulin-dependent protein kinase II, as well as with protein phosphatases in oocyte meiotic cell cycle regulation. The cross talk between MAPK cascade and other protein kinases is discussed. The review also addresses unsolved problems and discusses future directions.