Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes

The role of Raf and MAPK (mitogen-activated protein kinase) during the maturation of Xenopus oocytes was investigated. Treatment of oocytes with progesterone resulted in a shift in the electrophoretic mobility of Raf at the onset of germinal vesicle breakdown (GVBD), which was coincident with the activation of MAPK. Expression of a kinase- defective mutant of the human Raf-1 protein (KD-RAF) inhibited progesterone-mediated MAPK activation. MAPK activation was also inhibited by KD-Raf in oocytes expressing signal transducers of the receptor tyrosine kinase (RTK) pathway, including an activated tyrosine kinase (Tpr-Met), a receptor tyrosine kinase (EGFr), and Ha-RasV12. KD- RAF completely inhibited GVBD induced by the RTK pathway. In contrast, KD-RAF did not inhibit GVBD and the progression to Meiosis II in progesterone-treated oocytes. Injection of Mos-specific antisense oligodeoxyribonucleotides inhibited MAPK activation in response to progesterone and Tpr-Met, but failed to inhibit these events in oocytes expressing an oncogenic deletion mutant of Raf-1 (delta N'Raf). Injection of antisense oligodeoxyribonucleotides to Mos also reduced the progesterone- and Tpr-Met-induced electrophoretic mobility shift of Xenopus Raf. These results demonstrate that RTKs and progesterone participate in distinct yet overlapping signaling pathways resulting in the activation of maturation or M-phase promoting factor (MPF). Maturation induced by the RTK pathway requires activation of Raf and MAPK, while progesterone-induced maturation does not. Furthermore, the activation of MAPK in oocytes appears to require the expression of Mos.     

Involvement of protein kinase C in the regulation of oocyte maturation in amphibians (Rana dybowskii)

Ovarian oocytes of Rana dybowskii, isolated early in the hibernation period (late autumn), failed to mature, i.e., germinal vesicle breakdown (GVBD), in response to progesterone during in vitro follicle culture. Oocytes collected during the middle hibernation period matured in response to progesterone, whereas those collected late during the hibernation period (close to the breeding season) underwent spontaneous maturation without added hormone (Kwon et al., '89). The maturational response (GVBD) of oocytes, collected at the three stages of hibernation, to protein kinase C (PKC) activation was investigated and compared to that of progesterone stimulation. A phorbol ester, phorbol 12-myristate 13-acetate (TPA) was used for PKC activation. TPA addition to cultured follicles collected during the early or middle period of hibernation induced oocyte GVBD. The incidence of maturation (% GVBD) induced by TPA varied markedly between animals. TPA (10 microM) induced oocyte maturation in the presence or absence of follicle cells. The time course of the TPA-induced maturation was similar to that of progesterone-stimulated maturation (ED50, 7-9 h). TPA also accelerated the onset of maturation of the follicular oocytes exhibiting spontaneous in vitro maturation. Both TPA- and progesterone-stimulated maturation was blocked by treatment with cycloheximide (1 microgram/2 ml), forskolin (9 microM) (an adenylate cyclase stimulator), and verapamil (0.27 mM) (a calcium transport blocker). Treatment of oocytes with a calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (100 microM) or a PKC inactivator 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) (50 microM) likewise suppressed TPA- or progesterone-induced maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of insulin on spontaneous and progesterone-induced GVBD on Bufo arenarum denuded oocytes

Progesterone is considered as the physiological steroid hormone that triggers meiosis reinitiation in amphibian oocytes. Nevertheless, isolated oocytes can be induced to undergo germinal vesicle breakdown (GVBD) in a saline medium by means of treatment with various hormones or inducing agents such as other steroid hormones, insulin or an insulin-like growth factor. It has been demonstrated that Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. This study was undertaken to evaluate the participation of the purine and phosphoinositide pathway in the insulin-induced maturation of oocytes competent and incompetent to mature spontaneously, as well as to determine whether the activation of the maturation promoting factor (MPF) involved the activation of cdc25 phosphatase in Bufo arenarum denuded oocytes. Our results indicate that insulin was able to induce GBVD in oocytes incompetent to mature spontaneously and to enhance spontaneous and progesterone-induced maturation. In addition, high intracellular levels of purines such as cAMP or guanosine can reversibly inhibit the progesterone and insulin-induced maturation process in Bufo arenarum as well as spontaneous maturation. Assays of the inhibition of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis and its turnover by neomycin and lithium chloride respectively exhibited a different response in insulin- or progesterone-treated oocytes, suggesting that phosphoinositide turnover or hydrolysis of PIP2 is involved in progesterone- but not in insulin-induced maturation. In addition, the inhibitory effect of vanadate suggests that an inactive pre-maturation promoting factor (pre-MPF), activated by dephosphorylation of Thr-14 and Tyr-15 on p34cdc2, is present in Bufo arenarum full-grown oocytes; this step would be common to both spontaneous and hormone-induced maturation. The data presented here strongly suggest that insulin initiates at the cell surface a chain of events leading to GVBD. However, our studies point to the existence of certain differences between the steroid and the peptide hormone pathways, although both involve the decrease in intracellular levels of cAMP, the activation of phosphodiesterase (PDE) and the activation of pre-MPF.     

Boron deficiency disables Xenopus laevis oocyte maturation events

Processes of oocyte maturation that may be affected by boron (B) deficiency were studied to potentially determine a possible biochemical role of B in the Xenopus laevis oocyte. More specifically, the Xenopus oocyte membrane progesterone receptor (OMPR) in B-deficient oocytes was characterized by evaluating progesterone affinity for the OMPR and OMPR responsiveness to progesterone stimulation. The responsiveness of B-deficient oocytes to microinjection of a purified oocyte cytoplasmic fraction (OCF) from B-adequate oocytes was also studied to evaluate which aspects of the maturation process were affected by B deficiency. Results suggested that B deficiency resulted in incomplete oocyte maturation and that maturation could not be induced by the administration of exogenous progesterone. Progesterone successfully induced germinal vesicle breakdown (GVBD) in oocytes from females fed a B-supplemented diet (+B) and females administered a traditional diet of beef liver and lung (B adequate). Addition of exogenous B to the -B oocytes increased the rate of progesterone-induced GVBD slightly. The B-deficient X. laevis oocytes were capable of undergoing GVBD when endogenously stimulated by microinjected purified B-adequate OCF. These results indicated that the inability of the B-deficient oocytes to undergo GVBD was not associated with the cytoplasmic induction process specifically, but possibly in the progesterone receptor or signal transduction pathways. Radio-binding studies found that progesterone binding to the B-deficient OPMR was greatly reduced compared to B-adequate or B-supplemented OMPR. Moreover, washout studies determined that progesterone binding to the OMPR in B-deficient oocytes was more transient than the B adequate or +B oocytes.     

Effect of dehydroleucodine on meiosis reinitiation in Bufo arenarum denuded oocytes

In amphibian oocytes meiosis, the transition from G2 to M phase is regulated by the maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34/cdc2 and cyclin B. In immature oocytes there is an inactive complex (pre-MPF), in which cdc2 is phosphorylated on both Thr-161 and Thr-14/Tyr-15 residues. The dephosphorylation of Thr-14/Tyr-15 is necessary for the start of MPF activation and it is induced by the activation of cdc25 phosphatase. Late, to complete the activation, a small amount of active MPF induces an auto-amplification loop of MPF stimulation (MPF amplification). Dehydroleucodine (DhL) is a sesquiterpenic lactone that inhibits mammalian cell proliferation in G2. We asked whether DhL interferes with MPF activation. For this question, the effect of DhL (up to 30 microM) on the resumption of meiosis was evaluated, and visualized by germinal vesicle break down (GVBD), of Bufo arenarum oocytes induced in vitro by either: (i) removing follicle cells; (ii) progesterone stimulation; (iii) VG-content injection; or (iv) injection of mature cytoplasm. The results show that DhL induced GVBD inhibition, in a dose-dependent manner, in spontaneous and progesterone-induced oocyte maturation. Nevertheless, DhL at the doses assayed had no effect on GVBD induced by mature cytoplasm injection, but exerted an inhibitory effect on GVBD induced by GV content. On the basis of these results, we interpreted that DhL does not inhibit MPF amplification and that the target of DhL is any event in the early stages of the cdc25 activation cascade.     

Involvement of purines and phosphoinositides in spontaneous and progesterone-induced nuclear maturation of Bufo arenarum oocytes

Although progesterone is the established maturation inducer in amphibia, it has been demonstrated that Bufo arenarum oocytes resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called "spontaneous maturation." The present studies were designed to evaluate the participation of purines and phosphoinositides in the spontaneous and progesterone-induced maturation in Bufo arenarum full-grown oocytes. The presented data demonstrate that high intracellular levels of purines such as cAMP or guanosine can inhibit both spontaneous and progesterone-induced maturation in full-grown denuded Bufo arenarum oocytes. Moreover, the fact that the mycophenolic acid was able to induce maturation in denuded oocytes obtained during the nonreproductive period in a manner similar to that of the progesterone and also to increase the percentages of spontaneous maturation suggests that in Bufo arenarum, inosine monophosphate dehydrogenase inhibition is an important step in the resumption of meiosis. Inhibition of the phosphatidylinositol 4,5 bisphosphate hydrolysis by treatment of denuded oocytes with neomycin totally blocks spontaneous and progesterone-induced maturation, suggesting that the products of this hydrolysis (1,2 diacylglycerol and inositol 1,4,5 trisphosphate) may be involved in the maturation process of Bufo. In addition, our results indicate that the activation of protein kinase C is also involved in both types of maturation.     

Microinjected GTP-gamma-S inhibits progesterone-induced maturation of Xenopus oocytes

GTP-gamma-S inhibits progesterone-induced maturation of Xenopus laevis oocytes and induces a rise in their cAMP levels. GTP-gamma-S does not inhibit MPF-induced maturation. Although GTP-gamma-S prevents the progesterone-induced increases in protein synthesis and phosphorylation, it has no effect on the basal rates of either. GTP-gamma-S also prevents the initial DAG drop induced by progesterone. GDP-beta-S effects are ambiguous, but it seems not to affect progesterone-induced maturation. These results suggest that although G-proteins are associated with the pathways affected by progesterone, the effects of progesterone are not mediated by a typical receptor/G-protein/effector interaction.     

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.     

Maturation of Xenopus laevis oocyte by progesterone requires poly(A) tail elongation of mRNA.

Meiotic maturation of Xenopus laevis oocytes by progesterone requires translation of stored maternal mRNAs. We investigated the role of poly(A) tail elongation of mRNAs during this process using cordycepin, which inhibits poly(A) tail elongation of mRNAs. When oocytes were treated with the buffer containing 10 mM cordycepin for 12 h, concentration of 3'-dATP in cytosol of oocytes increased to 0.7 mM, while that of ATP remained constant at around 1.2 mM. Incorporation of [32P]AMP into poly(A) mRNA was inhibited almost completely by this treatment. Progesterone-induced germinal vesicle breakdown (GVBD) was also abolished. Dose dependence of inhibition of progesterone-induced GVBD on cordycepin was similar to that of [32P]AMP incorporation into poly(A) mRNA. However, maturation-promoting factor-induced GVBD was unaffected by treatment of oocytes with cordycepin. Furthermore, the inhibition of GVBD by cordycepin was rescued by removal of cordycepin even in the presence of actinomycin D. Therefore, we concluded that poly(A) tail elongation of mRNA is required for induction of meiotic maturation of X. laevis oocytes. In addition, progesterone induced a 2.7-fold activation of [32P]AMP incorporation into the poly(A) tail of mRNA after a lag period of 3 h whereas GVBD was induced after 6-8 h from the progesterone treatment. Syntheses of most of the proteins were unaffected by treatment of oocytes with progesterone or cordycepin. However, syntheses of several proteins were increased or decreased by progesterone and cordycepin treatment.     

Induction of meiotic maturation in Xenopus oocytes by 12-O-tetradecanoylphorbol 13-acetate

Fully grown Xenopus oocytes are physiologically arrested at the G2/prophase border of the first meiotic division. Addition in vitro of progesterone or insulin causes release of the G2/prophase block and stimulates meiotic cell division of the oocyte, leading to maturation of the oocyte into an unfertilized egg. The possibility that the products of polyphosphoinositide breakdown, diacylglycerol and inositol-1,4,5-trisphosphate (IP3-, are involved in oocyte maturation was investigated. Microinjection of IP3 into oocytes just prior to addition of progesterone or insulin accelerated the rate of germinal vesicle breakdown (GVBD) by up to 25%. Half-maximal acceleration occurred at an intracellular IP3 concentration of 1 microM. Treatment of oocytes with the diacylglycerol analog and tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced GVBD in the absence of hormone. Half-maximal induction of GVBD occurred with 150 nM TPA and was blocked by pretreatment of oocytes with 10 nM cholera toxin. Microinjection of highly purified protein kinase C from rat brain into oocytes did not induce maturation but markedly accelerated the rate of insulin-induced oocyte maturation. However, injection of the enzyme had no effect on progesterone action. In oocytes with a basal intracellular pH below 7.6, TPA increased intracellular pH, but GVBD occurred with TPA in Na-substituted medium. Neomycin, a putative inhibitor of polyphosphoinositide breakdown, reversibly inhibited insulin- but not progesterone-induced maturation. Half-maximal inhibition occurred at 1.6 mM neomycin. These results indicate that protein kinase C is capable of regulating oocyte maturation in Xenopus.     

The role of Ca2+ in progesterone-induced germinal vesicle breakdown of Xenopus laevis oocytes: the synergic effects of microtubule depolymerization and Ca2+

 By monitoring ⁴⁵Ca²⁺ influx and efflux from oocytes a transient increase followed by a transient decrease in the Ca²⁺-content of progesterone-treated oocytes was observed. Chelation of intracellular Ca²⁺ with EGTA or BAPTA-type buffers inhibited progesterone-induced GVBD. Buffers with a mid-range K_d (∼1.5 μm) were most effective in inhibiting GVBD whereas buffers with a K_d above or below this value were less effective. These observations indicate that intracellular Ca²⁺, probably in the form of a localized release, is required for progesterone-induced oocyte maturation. However, Ca²⁺ alone was insufficient to induce GVBD. When the effects of nocodazole and taxol upon this Ca²⁺-requirement were tested, we observed that taxol-induced microtubule polymerization not only delayed progesterone-induced GVBD but also completely inhibited it in combination with BAPTA-AM. Conversely, nocodazole-induced microtubule depolymerization in combination with ionophore A23187 not only accelerated progesterone-induced GVBD, but also induced GVBD in the absence of progesterone. The combined treatment of oocytes with nocodazole and InsP_3, or with cold treatment and ionophore A23187 also induced GVBD in the absence of progesterone. Thus, Ca²⁺ and microtubule depolymerization synergistically promote GVBD. In both nocodazole- and cold-treated oocytes, the GV was displaced to the periphery of the oocyte and underwent GVBD when treated with A23187. However, when the GV was displaced to the cortex by a centrifugal force under conditions that would not cause microtubule depolymerization and the oocyte was treated with A23187, oocytes did not undergo GVBD. Received: 19 January 1996 / Accepted: 21 May 1996     

Inhibition of mos-induced oocyte maturation by protein kinase A

The relationship between the mos protooncogene protein and cAMP- dependent protein kinase (PKA) during the maturation of Xenopus oocytes was investigated. Microinjection of the PKA catalytic subunit (PKAc) into Xenopus oocytes inhibited oocyte maturation induced by the mos product but did not markedly affect the autophosphorylation activity of injected mos protein. By contrast, PKAc did not inhibit maturation promoting factor (MPF) activation or germinal vesicle breakdown (GVBD) that was initiated by injecting crude MPF preparations. In addition, inhibiting endogenous PKA activity by microinjecting the PKA regulatory subunit (PKAr) induced oocyte maturation that was dependent upon the presence of the endogenous mos product. Moreover, PKAr potentiated mos protein-induced MPF activation in the absence of progesterone and protein synthesis. These data are consistent with the hypothesis that progesterone-induced release from G2/M is regulated via PKAc and that PKAc negatively regulates a downstream target that is positively regulated by mos.     

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.     

The effects of the normal and oncogenic forms of the neu tyrosine kinase, and the corresponding forms of an immunoglobulin E receptor/neu tyrosine kinase fusion protein, on Xenopus oocyte maturation.

In this work, we have used Xenopus oocyte maturation as a read-out for examining the ability of the neu tyrosine kinase (p185neu) to participate with the epidermal growth factor (EGF) receptor in a common signal transduction pathway. We find that unlike the case for the EGF receptor, which elicits EGF-dependent maturation of these oocytes as reflected by their germinal vesicle breakdown (GVBD), neither the normal neu tyrosine kinase (p185val664) nor the oncogenic form of neu (p185glu664) are able to effectively trigger this maturation event. However, expression of p185glu664 causes a specific and significant promotion of the progesterone-induced GVBD, reducing the half-time for this maturation even from approximately 9 h to approximately 5 h. Stimulation of the progesterone-induced GVBD did not occur following the expression of a kinase-deficient p185neu protein (in which a lysine residue at position 758 was changed to alanine). Essentially identical results were obtained when the mRNAs coding for fusion proteins comprised of the extracellular domain of the receptor for immunoglobulin E (IgE), and the membrane-spanning and tyrosine kinase domains of normal or oncogenic p185neu (designated IgER/p185val664 and IgER/p185glu664, respectively), were injected into oocytes. Antigen-induced crosslinking of IgER/p185val164 proteins expressed in oocytes caused a reduction in the half-time for the progesterone-stimulated GVBD from approximately 9 h to approximately 7 h. Thus, the aggregation of the membrane-spanning and/or tyrosine kinase domains of p185val664 partially mimics the effects of the oncogenic forms of p185neu. Overall, the results of these studies suggest that the activation of the p185neu tyrosine kinase by a point mutation within its membrane-spanning helix, or an aggregation event, can result in the facilitation of oocyte maturation events that are elicited by other factors (e.g. progesterone). However, the activated p185neu tyrosine kinases are not able to mimic the EGF-stimulated EGF receptor tyrosine kinase in triggering oocyte maturation, which suggests that the EGF receptor and the p185neu tyrosine kinase do not input into identical signal transduction pathways in these cells.     

Changes in protein phosphorylation accompanying maturation of Xenopus laevis oocytes

Protein phosphorylation has been measured after injection of [³²P]phosphate into oocytes of Xenopus laevis undergoing progesterone-induced meiotic maturation. As oocytes mature, there is a burst of nonyolk protein phosphorylation several hours after progesterone exposure and shortly before germinal vesicle breakdown (GVBD). This burst is not due to changes in the specific activity of the phosphate or ATP pool. Enucleated oocytes exposed to progesterone also experience the burst, indicating the cytoplasmic location of phosphoprotein formation. When an oocyte receives an injection of cytoplasm containing the maturation-promoting factor (MPF), a burst of protein phosphorylation occurs immediately, and GVBD occurs shortly thereafter, even in the presence of cycloheximide. Under a variety of conditions promoting or blocking maturation, oocytes which undergo GVBD are the only ones to have experienced the phosphorylation burst. The results suggest that the protein phosphorylation burst is a necessary step in the mechanism by which MPF promotes GVBD.     

Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of Rana pipiens: Studies with phorbol 12-myristate 13-acetate

In ovarian follicles of Rana pipiens, frog pituitary homogenates (FPH) elevate intrafollicular progesterone levels which in turn is thought to induce meiotic resumption in the prophase I arrested oocytes. Calcium plays a role in FPH and steroid-provoked responses in the somatic and gametic components of the follicle, presumably via effects exerted at the plasma membrane of their respective target cells. Many membrane active hormones which utilize Ca²⁺ in their intracellular transduction also provoke membrane phosphoinositide hydrolysis yielding inositol triphosphate (IP₃) and diacyl glycerol (DAG), an activator of the CA²⁺-dependent protein kinase C (PKC). The actions of phorbol 12-myristate 13-acetate (TPA), a potent synthetic activator of PKC, on progesterone production and oocyte maturation was examined in in vitro cultured ovarian follicles. TPA induced germinal vesicle breakdown (GVBD) in intact follicles and in oocytes denuded of somatic components, while the inactive compound phorbol 13-monoacetate was ineffective. Further, TPA induction of GVBD exhibited similarities to progesterone-induced GVBD, being inhibited by treatments which elevate cAMP or inhibit protein synthesis. TPA alone did not elevate intrafollicular or medium progesterone levels, as occurred in FPH-treated follicles. TPA partially inhibited intrafollicular progesterone accumulation induced by FPH or treatments which elevate cAMP levels. These data suggest that activation of PKC plays a role in oocyte maturation independent of follicular progesterone production as occurs in response to FPH. Further, it appears that the somatic cells of the amphibian follicle also possess PKC which when activated, antagonizes cAMP generating pathway in these cells. Results indicate that protein kinase can influence oocyte maturation in Rana follicular oocytes by several mechanisms.     

Is a decrease in cyclic AMP a necessary and sufficient signal for maturation of amphibian oocytes?

Acetylcholine rapidly lowered the intracellular levels of cyclic AMP in stage 5 and 6 Xenopus laevis oocytes. Acetylcholine alone did not induce oocyte maturation, though it did accelerate maturation induced by progesterone. The effect of acetylcholine on oocyte maturation was independent of extracellular calcium concentration. Adenosine increased cyclic AMP and abolished the progesterone-induced decrease in cyclic AMP levels in follicles and in denuded oocytes. This effect of adenosine was blocked by the Ra purinergic receptor antagonist, theophylline. Despite those effects, adenosine alone induced maturation in stage 6 oocytes and accelerated progesterone-induced maturation in both stage 5 and 6 cells. Adenosine also induced a significant increase in the rate of 45Ca efflux from oocytes in the presence and the absence of external calcium. We suggest that the activation of cell surface receptors involved in the release of calcium from cellular stores may induce or accelerate oocyte maturation independently of small changes in intracellular cyclic AMP concentration.     

SNT1/FRS2 mediates germinal vesicle breakdown induced by an activated FGF receptor1 in Xenopus oocytes

The docking protein SNT1/FRS2 (fibroblast growth factor receptor substrate 2) is implicated in the transmission of extracellular signals from the fibroblast growth factor receptor (FGFR), which plays vital roles during embryogenesis. Activating FGFR mutations cause several craniosynostoses and dwarfism syndromes in humans. Here we show that the Xenopus homolog of mammalian FRS-2 (XFRS2) is essential for the induction of oocyte maturation by an XFGFR1 harboring an activating mutation (XFGFR1act). Using a dominant-negative form of kinase suppressor of Ras, we show the Mek activity is required for germinal vesicle breakdown (GVBD) induced by co-expression of XFGFR1act and XFRS2, but this activity is not required for progesterone-induced GVBD. Furthermore, Mek/MAPK activity is critical for the induction and/or maintenance of H1 kinase activity at metaphase of meiosis II in progesterone-treated oocytes. An activated XFGFR1 containing a mutation in the phospholipase Cgamma binding site (XFGFR1actY672F) displayed a reduced ability to induce cell-cycle progression in oocytes, suggesting phospholipase Cgamma may not be necessary but that it augments XFGFR signaling in this system. Oocytes co-expressing XFGFR1act and XFRS2 showed substantial H1 kinase activity, but this activity was blocked when the oocytes were treated with the phosphatidylinositol 3-kinase inhibitor LY294002. Although phosphatidylinositol 3-kinase activity is essential for XFGFR1act/XFRS2-induced oocyte maturation, this activity is not required for maturation induced by progesterone. Finally, ectopic expression of Xspry2, a negative regulator of XFGFR signaling, greatly reduced MAPK activation and GVBD induced by the expression of either XFGFR1act plus XFRS2 or activated Ras (H-RasV12). In contrast, Xspry2 did not prevent GVBD induced by an activated form of Raf1, suggesting that Xspry2 exerts its inhibitory function upstream or parallel to Raf and downstream of Ras.     

Function of the Mos/MAPK pathway during oocyte maturation in the Japanese brown frog Rana japonica

Fully grown immature oocytes acquire the ability to be fertilized with sperm after meiotic maturation, which is finally accomplished by the formation and activation of the maturation-promoting factor (MPF). MPF is the complex of Cdc2 and cyclin B, and its function in promoting metaphase is common among species. The Mos/mitogen-activated protein kinase (MAPK) pathway is also commonly activated during vertebrate oocyte maturation, but its function seems to be different among species. We investigated the function of the Mos/MAPK pathway during oocyte maturation of the frog Rana japonica. Although MAPK was activated in accordance with MPF activation during oocyte maturation, MPF activation and germinal vesicle breakdown (GVBD) was not initiated when the Mos/MAPK pathway was activated in immature oocytes by the injection of c-mos mRNA. Inhibition of Mos synthesis by c-mos antisense RNA and inactivation of MAPK by CL100 phosphatase did not prevent progesterone-induced MPF activation and GVBD. However, continuous MAPK activation and MAPK inhibition through oocyte maturation accelerated and delayed MPF activation, respectively. Furthermore, Mos induced a low level of cyclin B protein synthesis in immature oocytes without the aid of MAPK. These results suggest that the general function of the Mos/MAPK pathway, which is not essential for MPF activation and GVBD in Rana oocytes, is to enhance cyclin B translation by Mos itself and to stabilize cyclin B protein by MAPK during oocyte maturation.     

Calcium,potassium, and sodium exchange by full-grown and maturing Xenopus laevis oocytes

The kinetics of calcium, potassium, and sodium exchange by Xenopus laevis oocytes were monitored with radioactive tracers both before and during progesterone-induced maturation. The rate of ⁴⁵Ca release steadily elevates for several hours during maturation, beginning within 40 min after progesterone exposure. About an hour later, the rate of ⁴⁵Ca uptake also increases. The rate of ⁴⁵Ca release begins to decline 1–2 hr before germinal vesicle breakdown (GVBD); the rate of calcium uptake declines only after GVBD. Similar changes are seen after maturation is induced with other steroids, but not when maturation is blocked by inhibitors. The passive potassium flux initially increases after progesterone treatment to be followed later by a decrease. These observed changes occur coincidently with those of ⁴⁵Ca efflux. The passive sodium flux, on the other hand, steadily increases from the time of progesterone treatment until GVBD.