Several signaling pathways are involved in the control of cattle oocyte maturation

The main limit of in vitro production of domestic mammal embryos comes from the low capacity of in vitro matured oocytes to develop after fertilization. As soon as they are separated from follicular environment, oocytes spontaneously resume meiosis without completion of their terminal differentiation. Roscovitine (ROS), an inhibitor of M-phase promoting factor (MPF) kinase activity reversibly blocks the meiotic resumption in vitro. However, in cattle maturing oocytes several cellular events such as protein synthesis and phosphorylation, chromatin condensation and nuclear envelope folding escape ROS inhibition suggesting the alternative pathways in oocyte maturation. We compared the level of synthesis and phosphorylation of several protein kinases during bovine cumulus oocyte complex (COC) maturation in vitro in the presence or not of epidermal growth factor (EGF) and ROS. We showed that during the EGF-stimulated maturation, ROS neither affected the decrease of EGF receptor (EGFR) nor did inhibit totally its phosphorylation in cumulus cells and also did not totally eliminate tyrosine phosphorylation in oocytes. However, ROS did inhibit the Phosphoinositide 3-kinase (PI3) activity when oocytes mature without EGF. Accumulation of Akt/PKB (protein kinase B), JNK1/2 (jun N-terminal kinases) and Aurora-A in oocytes during maturation was not affected by ROS. However, the phosphorylation of Akt but not JNKs was diminished in ROS-treated oocytes. Thus, PI3 kinase/Akt, JNK1/2 and Aurora-A are likely to be involved in the regulation of bovine oocyte maturation and some of these pathways seem to be independent to MPF activity and meiotic resumption. This complex regulation may explain the partial meiotic arrest of ROS-treated oocytes and the accelerated maturation observed after such treatment.     

Impaired maturation of the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 deficient for the cytochrome c biogenesis protein CcmC

Here we show that during the meiotic maturation of Xenopus oocytes, histone H3 becomes phosphorylated on serine-10 at about the time of maturation promoting factor activation and meiosis I entry. However, overexpression of cAMP-dependent protein kinase that blocks entry into M phase, also leads to massive serine-10 phosphorylation of histone H3 in intact Xenopus oocytes but does not cause chromosome condensation. We also show that the phosphorylation of histone H3 during oocyte maturation requires the activation of the mitogen-activated protein kinase/p90Rsk pathway. Our results indicate that in G2-arrested oocytes, which are about to enter M phase, histone H3 phosphorylation is not sufficient for chromosome condensation.     

Regulation of Src kinase activity during Xenopus oocyte maturation

Expression of constitutively active Src protein tyrosine kinase in Xenopus oocytes has been shown to accelerate oocyte maturation suggesting that Src may be involved in meiotic progression. However, meiotic regulation of endogenous Src kinase in oocytes has not been investigated in detail. To address this problem, we measured the activity, expression level, and phosphorylation state of the endogenous Xenopus Src (xSrc) and overexpressed xSrc mutants in the process of progesterone-induced oocyte maturation. We found that the enzyme is first transiently activated in the plasma membrane-containing fraction of oocytes within 3 min of progesterone administration. This event represents one of the earliest responses of oocytes to the hormone and should be related to triggering some early signaling pathways of maturation. Thereafter, xSrc activity increases again at the time of germinal vesicle breakdown (GVBD) and remains elevated till the completion of maturation. This elevation of xSrc activity is associated with a 2-fold increase of xSrc protein content in the absence of change in its specific activity and xSrc mRNA content. No significant changes in the phosphorylation state of C-terminal regulatory phosphotyrosine can be registered either in endogenous xSrc or in overexpressed kinase-negative and wild-type xSrc proteins during maturation. Altogether, these results indicate that upregulation of xSrc in the meiotic metaphase occurs at the translation level. We also demonstrate here that the expression of constitutively active xSrc in Xenopus oocytes is accompanied by the activation of mitogen-activated protein kinase (MAPK). Our data suggest that the Src kinase acts through the MAPK pathway to accelerate oocyte maturation.     

Xp38gamma/SAPK3 promotes meiotic G(2)/M transition in Xenopus oocytes and activates Cdc25C

We have studied the role of p38 mitogen-activated protein kinases (MAPKs) in the meiotic maturation of Xenopus oocytes. Overexpression of a constitutively active mutant of the p38 activator MKK6 accelerates progesterone-induced maturation. Immunoprecipit ation experiments indicate that p38gamma/SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38gamma (Xp38gamma) and show that co-expression of active MKK6 with Xp38gamma induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38gamma requires neither protein synthesis nor activation of the p42 MAPK-p90Rsk pathway, but it is blocked by cAMP-dependent protein kinase. A role for the endogenous Xp38gamma in progesterone-induced maturation is supported by the inhibitory effect of kinase-dead mutants of MKK6 and Xp38gamma. Furthermore, MKK6 can rescue the inhibition of oocyte maturation by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38gamma can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38gamma. Our results indicate that phosphorylation of XCdc25C by Xp38gamma/SAPK3 is important for the meiotic G(2)/M progression of Xenopus oocytes.     

Possible involvement of phosphatidylinositol 3-kinase, but not protein kinase B or glycogen synthase kinase 3beta, in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica

It is known that amphibian oocytes undergo maturation through the formation and activation of maturation-promoting factor (MPF) in response to stimulation by the maturation-inducing hormone progesterone; however, the signal transduction pathway that links the hormonal stimulation on the oocyte surface to the activation of MPF in the oocyte cytoplasm remains a mystery. The aim of this study was to investigate whether the signal transduction mediated by phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3beta (GSK3beta) is involved in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica. Inhibitors of PI3K, wortmannin and LY294002, inhibited progesterone-stimulated germinal vesicle breakdown (GVBD) only when the oocytes were treated at the initial phase of maturation, suggesting that PI3K is involved in the progesterone-induced maturation of Rana oocytes. However, we also obtained results suggesting that PKB and GSK3beta are not involved in Rana oocyte maturation. A constitutively active PKB expressed in the oocytes failed to induce GVBD in the absence of progesterone despite its high level of kinase activity. A Myc-tagged PKB expressed in the oocytes (used to monitor endogenous PKB activity) was not activated in the process of progesterone-induced oocyte maturation. Overexpression of GSK3beta, which is reported to retard the progress of Xenopus oocyte maturation, had no effect on Rana oocyte maturation. On the basis of these results, we propose that PI3K is involved in the initiation of Rana oocyte maturation, but that neither PKB nor GSK3beta is a component of the PI3K signal transduction pathway.     

S6 phosphorylation is regulated at multiple levels in Xenopus laevis oocytes

It is known that the 40s ribosomal protein S6 undergoes a dramatic increase in its level of phosphorylation during Xenopus oocyte meiotic maturation in response to progesterone stimulation. During prophase arrest, the majority of S6 has 0 moles phosphate per mole protein; this increases to 4-5 moles phosphate per mole protein by the time of germinal vesicle breakdown (GVBD). Our in vitro and in vivo studies indicate that the accumulation of phosphate on S6 is the net result of a 4-5-fold increase in S6 kinase activity and a 30-50% decrease in the rate of dephosphorylation and/or turnover of phosphate groups on S6 in maturing oocytes. In addition, the level of phosphorylation of S6 on 80s monosomes injected into non-hormone-stimulated oocytes was unexpectedly high. This indicates that the S6 kinase/phosphatase ratio in prophase arrested oocytes is higher than anticipated from previous studies. This observation implies that the majority of the oocyte ribosomes may be sequestered from any S6 kinase during meiotic prophase. Furthermore, these observations suggest that a portion of the increased accumulation of phosphate on S6 may be the result of increased accessibility of the ribosomes to S6 kinase during oocyte meiotic maturation.     

Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.     

FSH Modulates PKAI and GPR3 Activities in Mouse Oocyte of COC in a Gap Junctional Communication (GJC)-Dependent Manner to Initiate Meiotic Resumption

Many studies have shown that cyclic adenosine-5′-monophosphate (cAMP)-dependent protein kinase A (PKA) and G-protein-coupled receptor 3 (GPR3) are crucial for controlling meiotic arrest in oocytes. However, it is unclear how gonadotropins modulate these factors to regulate oocyte maturation, especially by gap junctional communication (GJC). Using an in vitro meiosis-arrested mouse cumulus-oocyte complex (COC) culture model, we showed that there is a close relationship between follicle-stimulating hormone (FSH) and the PKA type I (PKAI) and GPR3. The effect of FSH on oocyte maturation was biphasic, initially inhibitory and then stimulatory. During FSH-induced maturation, rapid cAMP surges were observed in both cumulus cells and oocyte. Most GJC between cumulus cells and oocyte ceased immediately after FSH stimulation and recommenced after the cAMP surge. FSH-induced maturation was blocked by PKAI activator 8-AHA-cAMP. Levels of PKAI regulatory subunits and GPR3 decreased and increased, respectively, after FSH stimulation. In the presence of the GJC inhibitor carbenoxolone (CBX), FSH failed to induce the meiotic resumption and the changes in PKAI, GPR3 and cAMP surge in oocyte were no longer detected. Furthermore, GPR3 was upregulated by high cAMP levels, but not by PKAI activation. When applied after FSH stimulation, the specific phosphodiesterase 3A (PDE3A) inhibitor cilostamide immediately blocked meiotic induction, regardless of when it was administered. PKAI activation inhibited mitogen-activated protein kinase (MAPK) phosphorylation in the oocytes of COCs, which participated in the initiation of FSH-induced meiotic maturation in vitro. Just before FSH-induced meiotic maturation, cAMP, PKAI, and GPR3 returned to basal levels, and PDE3A activity and MAPK phosphorylation increased markedly. These experiments show that FSH induces a transient increase in cAMP levels and regulates GJC to control PKAI and GPR3 activities, thereby creating an inhibitory phase. After PDE3A and MAPK activities increase, meiosis resumes.     

Wee1B is an oocyte-specific kinase involved in the control of meiotic arrest in the mouse

In most species, the meiotic cell cycle is arrested at the transition between prophase and metaphase through unclear somatic signals. Activation of the Cdc2-kinase component of maturation promoting factor (MPF) triggers germinal vesicle breakdown after the luteinizing hormone (LH) surge and reentry into the meiotic cell cycle. Although high levels of cAMP and activation of protein kinase A (PKA) play a critical role in maintaining an inactive Cdc2, the steps downstream of PKA in the oocyte remain unknown. Using a small-pool expression-screening strategy, we have isolated several putative PKA substrates from a mouse oocyte cDNA library. One of these clones encodes a Wee1-like kinase that prevents progesterone-induced oocyte maturation when expressed in Xenopus oocytes. Unlike the widely expressed Wee1 and Myt1, mWee1B mRNA and its protein are expressed only in oocytes, and mRNA downregulation by RNAi injection in vitro or transgenic overexpression of RNAi in vivo causes a leaky meiotic arrest. Ser15 residue of mWee1B is the major PKA phosphorylation site in vitro, and the inhibitory effects of the kinase are enhanced when this residue is phosphorylated. Thus, mWee1B is a key MPF inhibitory kinase in mouse oocytes, functions downstream of PKA, and is required for maintaining meiotic arrest.     

Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts.

Meiotic maturation of Xenopus and sea star oocytes involves the activation of a number of protein-serine/threonine kinase activities, including a myelin basic protein (MBP) kinase. A 44-kDa MBP kinase (p44mpk) purified from mature sea star oocytes is shown here to be phosphorylated at tyrosine. Antiserum to purified sea star p44mpk was used to identify antigenically related proteins in Xenopus oocytes. Two tyrosine-phosphorylated 42-kDa proteins (p42) were detected with this antiserum in Xenopus eggs. Xenopus p42 chromatographs with MBP kinase activity on a Mono Q ion-exchange column. Tyrosine phosphorylation of Xenopus p42 approximately parallels MBP kinase activity during meiotic maturation. These results suggest that related MBP kinases are activated during meiotic maturation of Xenopus and sea star oocytes. Previous studies have suggested that Xenopus p42 is related to the mitogen-activated protein (MAP) kinases of culture mammalian cells. We have cloned a MAP kinase relative from a Xenopus ovary cDNA library and demonstrate that this clone encodes the Xenopus p42 that is tyrosine phosphorylated during oocyte maturation. Comparison of the sequences of Xenopus p42 and a rat MAP kinase (ERK1) and peptide sequences from sea star p44mpk indicates that these proteins are close relatives. The family members appear to be tyrosine phosphorylated, and activated, in different contexts, with the murine MAP kinase active during the transition from quiescence to the G1 stage of the mitotic cell cycle and the sea star and Xenopus kinases being active during M phase of the meiotic cell cycle.     

Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos.

In somatic cells, the Raf-1 serine/threonine protein kinase is activated by several polypeptide growth factors. We investigated the role of Raf-1 in progesterone-induced meiotic maturation of Xenopus laevis oocytes. Raf-1 enzymatic activity and phosphorylation (reflected by a mobility shift on sodium dodecyl sulfate gels) were increased in oocytes following progesterone stimulation. The increase in Raf-1 activity was concurrent with an elevation in the activity of mitogen-activated protein (MAP) kinase. When RNA encoding an oncogenic form of Raf-1 (v-Raf) was injected into immature oocytes, MAP kinase mobility shift, germinal vesicle breakdown, and histone H1 phosphorylation increased markedly. When RNA encoding a dominant-negative version of Raf-1 was injected, progesterone-induced oocyte maturation was blocked. When RNA encoding Xenopus mos (mosxe) was injected into oocytes, Raf-1 and MAP kinase mobility shifts were observed after several hours. Also, when antisense mosxe oligonucleotides were injected into oocytes, progesterone-induced Raf-1 and MAP kinase mobility shifts were blocked. Finally, when antisense mosxe oligonucleotides were coinjected with v-Raf RNA into oocytes, histone H1 kinase activation, germinal vesicle breakdown, and MAP kinase mobility shift occurred. These findings suggest that Raf-1 activity is required for progesterone-induced oocyte maturation and that Raf-1 is downstream of mosxe activity.     

Phosphatidylinositol 3-kinase and Akt participate in the FSH-induced meiotic maturation of mouse oocytes

Phosphatidylinositol 3-kinase (PI3K) is known to play critical roles in signal transduction processes related to a variety of cellular activities. In the present study, we investigated the role of PI3K during meiotic maturation in mouse oocytes using a specific inhibitor, LY294002. In follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest of cumulus oocyte complexes (COCs), LY294002 suppressed germinal vesicle breakdown (GVBD), first polar body (PB1) emission, and cumulus expansion. To examine the effect of LY294002, denuded oocytes (DOs) were cultured in medium containing follicular fluid meiosis-activating sterol (FF-MAS) since absence of gonadotropin receptors in oocytes has been reported and FSH did not stimulate meiotic maturation of DOs in the presence of hypoxanthine. In FF-MAS-induced maturation of DOs, LY294002 suppressed PB1emission, but not GVBD. In spontaneous gonadotropin-independent oocyte maturation, LY294002 had no effect on COCs and DOs. Akt/protein kinase B, a serine-threonine kinase, is a key downstream effector of the PI3K pathway. Therefore, we also examined the distribution of Akt during FSH-induced meiotic maturation. The distribution of Ser(473) phosphorylated Akt was similar to the localization of microtubules, while Thr(308) phosphorylated Akt was present in the pericentriolar materials (PCM) in metaphase I (MI) and II (MII) oocytes. LY294002 decreased the amount of Thr(308) phosphorylated Akt to very low to undetectable levels in MI and MII oocytes. Ser(473) phosphorylated Akt showed aberrant distribution and very low to undetectable levels of expression in LY294002-treated MI and MII oocytes, respectively. These results suggest that PI3K and Akt participate in mouse meiotic maturation.     

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.     

Cdc2-cyclin B triggers H3 kinase activation of Aurora-A in Xenopus oocytes

Xenopus oocytes are arrested in meiotic prophase I and resume meiotic divisions in response to progesterone. Progesterone triggers activation of M-phase promoting factor (MPF) or Cdc2-cyclin B complex and neosynthesis of Mos kinase, responsible for MAPK activation. Both Cdc2 and MAPK activities are required for the success of meiotic maturation. However, the signaling pathway induced by progesterone and leading to MPF activation is poorly understood, and most of the targets of both Cdc2 and MAPK in the oocyte remain to be determined. Aurora-A is a Ser/Thr kinase involved in separation of centrosomes and in spindle assembly during mitosis. It has been proposed that in Xenopus oocytes Aurora-A could be an early component of the progesterone-transduction pathway, acting through the regulation of Mos synthesis upstream Cdc2 activation. We addressed here the question of Aurora-A regulation during meiotic maturation by using new in vitro and in vivo experimental approaches. We demonstrate that Cdc2 kinase activity is necessary and sufficient to trigger both Aurora-A phosphorylation and kinase activation in Xenopus oocyte. In contrast, these events are independent of the Mos/MAPK pathway. Aurora-A is phosphorylated in vivo at least on three residues that regulate differentially its kinase activity. Therefore, Aurora-A is under the control of Cdc2 in the Xenopus oocyte and could be involved in meiotic spindle establishment.     

Potential role of protein tyrosine phosphatase nonreceptor type 13 in the control of oocyte meiotic maturation

Protein tyrosine phosphatase nonreceptor type 13 (PTPN13) is a tyrosine phosphatase with multiple interacting domains that has been implicated previously in the regulation of apoptosis. We provide evidence that PTPN13 plays an important role in the control of the meiotic cell cycle. A cDNA coding for PTPN13 was isolated during the screening for the substrate of protein kinase A expressed in mammalian oocytes. PTPN13 is expressed in both mouse and Xenopus oocytes and is a substrate for protein kinase A in vitro and in vivo. Expression of a truncated constitutively-active PTPN13 in Xenopus oocytes synergizes with progesterone in the induction of germinal vesicle breakdown, the translation of Mos, the phosphorylation of Erk and the dephosphorylation of Cdc2. The phosphatase activity of PTPN13 is required for this synergism. Oocyte injection with specific small interference RNA downregulates the expression of mRNA for PTPN13 and blocks oocyte maturation induced by progesterone, a blockade that can be overcome by Cdc25 overexpression. These findings indicate that PTPN13 is involved in the regulation of the meiotic cell cycle.     

Progesterone-inhibited phosphorylation of an unique Mr 48,000 protein in the plasma membrane of Xenopus laevis oocytes

The meiotic maturation of Xenopus laevis oocytes is induced in vitro by progesterone which interacts at the cell surface level. A cell-free membrane preparation (P-10,000) incorporated 32P from [gamma-32P]ATP, mostly into two proteins, Mr approximately 56,000 and approximately 48,000 (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Progesterone, added in vitro, specifically inhibited the phosphorylation of the Mr approximately 48,000 protein (named p48). Half-maximal inhibition of p48 phosphorylation occurred with progesterone approximately 8 microM, in good correlation with hormone concentration inducing oocyte maturation. The effect was not due to stimulation of protein phosphatase activity. The potent maturation inducers testosterone and deoxycorticosterone also inhibited p48 phosphorylation, whereas biologically inactive steroids or cholesterol did not. p48 phosphorylation was not affected by cAMP, cGMP, polyamines, calmodulin, and phospholipids + diolein. EGTA had a stimulatory effect which was reversed by added Ca2+. The inhibitory effects of progesterone and Ca2+ were additive, suggesting two distinct sites of action. Phospho-p48 was not detected in yolk platelets, microsomes, and cytosol of oocytes. Contrary to p48 itself, the p48 kinase activity was loosely associated with P-10,000. Progesterone inhibited p48 phosphorylation produced by either cytosol or exogenous pure catalytic subunit of cAMP-dependent protein kinase. Conversely, phosphorylation of casein and histones by protein kinase activity present in P-10,000 was not modified by progesterone. It is then suggested that progesterone regulates p48 phosphorylation by affecting the protein substrate in the membrane, rather than by inhibiting the protein kinase enzyme itself. The data demonstrate a direct effect (not mediated by change of protein synthesis) of steroids on p48 phosphorylation in the plasma membrane, and they suggest that this protein could be implicated in the initial action of progesterone on oocyte maturation.     

tpr-met oncogene product induces maturation-producing factor activation in Xenopus oocytes.

tpr-met, a tyrosine kinase oncogene, is the activated form of the met proto-oncogene that encodes the receptor for hepatocyte growth factor/scatter factor. The tpr-met product (p65tpr-met) was tested for its ability to induce meiotic maturation in Xenopus oocytes. While src and abl tyrosine kinase oncogene products have previously been shown to be inactive in this assay, p65tpr-met efficiently induced maturation-promoting factor (MPF) activation and germinal vesicle breakdown (GVBD) together with the associated increase in ribosomal S6 subunit phosphorylation. tpr-met-mediated MPF activation and GVBD was dependent on the endogenous c-mosxe, while the increase in S6 protein phosphorylation was not significantly affected by the loss of mos function. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine inhibits tpr-met-mediated GVBD at concentrations that prevent insulin- but not progesterone-induced oocyte maturation. Moreover, maturation triggered by tpr-met is also inhibited by cyclic AMP-dependent protein kinase. This is the first demonstration that a tyrosine kinase oncogene product, p65tpr-met, can induce meiotic maturation in Xenopus oocytes and activate MPF through a mos-dependent pathway, possibly the insulin or insulinlike growth factor 1 pathway.     

Calmodulin triggers the resumption of meiosis in amphibian oocytes

The calcium-binding protein, calmodulin, has been purified from Xenopus laevis oocytes. This 18,500-dalton protein, pl 4.3, has two high-affinity calcium-binding sites per mole protein having a dissociation constant of 2.8 x 10(-6) M. Full-grown Xenopus oocytes, arrested in late G2 of the meiotic cell cycle, resumed meiosis when microinjected with 60-80 ng (3-4 pmol) of calmodulin in the form of a calcium-calmodulin complex. The timing of the meiotic events in these recipient oocytes was the same as that normally induced by progesterone. Xenopus ovarian calmodulin stimulated bovine brain phosphodiesterase (PDE) 3- to 10-fold in a calcium-dependent manner, but it had no apparent effect on ovarian PDE activity. A calcium-calmodulin-dependent protein kinase has been isolated from Xenopus oocytes using a calmodulin-Sepharose 4B affinity column. The possible role for this kinase in regulating the G2-M transition in oocytes has been discussed.     

Phosphorylation of conserved serine residues does not regulate the ability of mosxe protein kinase to induce oocyte maturation or function as cytostatic factor

Expression of the mosxe protein kinase is required for the normal meiotic maturation of Xenopus oocytes and overexpression induces maturation in the absence of other stimuli. In addition, mosxe functions as a component of cytostatic factor (CSF), an activity responsible for arrest of the mature egg at metaphase II. After microinjection of Xenopus oocytes with in vitro synthesized RNA encoding either wild-type mosxe or kinase-inactive mosxe(R90), both proteins are phosphorylated exclusively on serine residues and exhibit essentially identical chymotryptic maps. Since the phosphorylated kinase-inactive mosxe(R90) protein was recovered from resting oocytes that have not yet begun to translate endogenous mosxe, this indicates that the major phosphopeptides of mosxe(R90) are phosphorylated by a preexisting protein kinase present in resting oocytes, and are not the result of autophosphorylation. The results presented here also indicate that the mosxe protein does not undergo significant phosphorylation at unique sites during oocyte maturation. If the biological activity of mosxe were regulated by phosphorylation, a site of regulatory phosphorylation would most likely be conserved among mos proteins of different species. Site-directed mutagenesis was used to construct 13 individual serine----alanine mutations at conserved residues (3, 16, 18, 25, 26, 57, 71, 76, 102, 105, 127, 211, and 258). These 13 mutants were analyzed for their abilities to induce oocyte maturation and to function as CSF. Results obtained with the mosxe(A105) mutant revealed that serine-105 is required for both maturation induction and CSF activity, even though serine-105 does not represent a major site of phosphorylation. All of the remaining serine----alanine mosxe mutants induced oocyte maturation and exhibited CSF activity comparable with the wild type. These results demonstrate that none of the conserved serines examined in this study function as regulatory phosphorylation sites for these biological activities. Peptide mapping of the remaining mosxe mutants identified serine-3 as a major phosphorylation site in vivo, which is contained within the chymotryptic peptide MPSPIPVERF.     

Phosphatidylinositol 3-kinase activity is important for progesterone-induced Xenopus oocyte maturation.

In somatic cells, phosphatidylinositol 3-kinase (PI3 kinase) is a critical intermediary in growth factor-induced mitogenesis. We have examined the role of this enzyme in meiotic maturation of Xenopus laevis oocytes. PI3 kinase activity was present in immunoprecipitates of the p85 subunit of PI3 kinase from immature oocytes and markedly increased following progesterone stimulation. Injection of bacterially expressed protein corresponding to the C-terminal SH2 domain of p85 (SH2-C) inhibited progesterone-induced PI3 kinase activation and meiotic maturation. Injection of protein corresponding to the N-terminal SH2 domain or the SH3 domain of p85 did not inhibit PI3 kinase activation or maturation. SH2-C did not inhibit oocyte maturation induced by c-mos RNA injection. In addition, radiolabelled SH2-C was used to probe oocyte lysates, revealing that a novel 200-kDa protein bound to SH2-C. This protein may be an important mediator of progesterone-induced lipid metabolism in oocytes.