Intracellular acidification delays hormonal G2/M transition and inhibits G2/M transition triggered by thiophosphorylated MAPK in Xenopus oocytes

Xenopus oocyte maturation is analogous to G2/M transition and characterized by germinal vesicle breakdown (GVBD), spindle formation, activation of MPF and Mos-Xp42(Mpk1) pathways. It is accompanied prior to GVBD by a transient increase in intracellular pH. We determined that a well known acidifying compound, NH(4)Cl, delayed progesterone-induced GVBD in a dose-dependent manner. GVBD(50) was delayed up to 2.3-fold by 10 mM NH(4)Cl. Cyclin B2 phosphorylation, Cdk1 Tyr15 dephosphorylation as well as p39(Mos) accumulation, Xp42(Mpk1) and p90(Rsk) phosphorylation induced by progesterone were also delayed by incubation of oocyte in NH(4)Cl. The delay induced by NH(4)Cl was prevented by injection of MOPS buffer pH 7.7. In contrast to acidifying medium, alkalyzing treatment such as Tris buffer pH 9 injections, accelerated GVBD, MPF and Xp42(Mpk1) activation, indicating that pHi changes control early steps of G2/M dynamics. When injected in an immature recipient oocyte, egg cytoplasm triggers GVBD through MPF auto-amplification, independently of protein synthesis. In these conditions, GVBD and Xp42(Mpk1) activation were delayed by high concentration of NH(4)Cl, which never prevented or delayed MPF activation. Strickingly, NH(4)Cl strongly inhibited thiophosphorylated active MAPK-induced GVBD and MPF activation. Nevertheless, Tris pH 9 did not have any effects on egg cytoplasm- or active MAPK-induced GVBD. Taken together, our results suggest that dynamic of early events driving Xp42(Mpk1) and MPF activation induced by progesterone may be negatively or positively regulated by pH(i) changes. However Xp42(Mpk1) pathway was inhibited by acidification alone. Finally, MPF auto-amplification loop was not sensitive to pH(i) changes.     

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.     

Studies on the Relative Roles of Pituitary and Progesterone in the Induction of Meiotic Maturation in the Amphibian Oocyte

In the amphibian, gonadotropins act on the epithelial cells surrounding the oocyte to produce and/or release progesterone which in turn acts at the oocyte surface to initiate the resumption of meiotic maturation. Since maturation is reported to require continuous exposure to gonadotropins but only brief (5--15 min) exposure to progesterone, it was of interest to reexamine the interrelationships between the two hormonal stimuli as well as the kinetics of progesterone production, metabolism, and biological activity. Germinal vesicle breakdown (GVBD) required continuous exposure to 0.005 pituitaries/ml for 6.0 h to produce 50% GVBD that occurred at 10.1 h. Actinomycin D (10 microgram/ml) completely inhibited pituitary induced GVBD when added during the first 5--6 h; 50% inhibition occurred when added at 7.3 h. Thus, actinomycin D continues to inhibit 1--2 h after the requirement for pituitary stimulation. Pituitary stimulation produced a 4-fold increase in 3H-acetate incorporation into progesterone and acetate conversion to progesterone was relatively constant during successive 2 h pulses throughout the 10 h period leading to GVBD. There was no significant metabolism of 3H-acetate derived progesterone when follicles were treated with pituitary extract, although the same follicles rapidly concentrated and metabolized exogenous 3H- or 14C-progesterone. The length of continuous progesterone exposure required for 50% GVBD varied from 11 h at 0.158 microM to less than 0.08 h at 15.8 microM. The time to 50% GVBD was only delayed by about 10% (1.5 h) when maximal and minimal progesterone levels were compared. A comparison of 3H-progesterone uptake and response (GVBD) as a function of [progesterone]0 indicated that uptake of 2--4 mumol 1(-1) cell water will induce 50% GVBD. These results indicate that a threshold uptake must be reached to initiate resumption of meiosis and that this level must be maintained throughout the period leading to nuclear breakdown. Under physiologic conditions, gonadotropins stimulate progesterone production and this progesterone is protected from, or inaccessible to, steroid metabolizing enzymes.     

Production of progesterone from de novo-synthesized cholesterol in cumulus cells and its physiological role during meiotic resumption of porcine oocytes

To investigate the role of factors secreted by cumulus cells during meiotic resumption of porcine oocytes, 1, 5, 10, or 20 cumulus-oocyte complexes (COCs) were cultured in each well of a culture dish containing 300 microl of maturation medium for 20 h. There was a significant positive correlation between the rate of germinal vesicle breakdown (GVBD) and the number of COCs cultured in each well for 20 h. The level of progesterone in the medium in which COCs had been cultured for 20 h also rose significantly with an increase in the number of COCs cultured in each well. A significantly small proportion of GVBD in oocytes when one COC was cultured in each well for 20 h was improved by the addition of progesterone. This proportion of GVBD was fully comparable to that of COCs cultured in the absence of additional progesterone with 20 COCs. Thus, progesterone secreted by COCs plays a positive role in GVBD induction in porcine oocytes. Furthermore, we also examined the role of sterol biosynthesis on progesterone production by cumulus cells and in oocyte GVBD. The results showed that the addition of ketoconazole, which suppressed the sterol biosynthetic pathway produced by demethylation of lanosterol, decreased the rate of GVBD, as well as progesterone production in COCs cultured for 20 h. However, the suppression of GVBD by ketoconazole was overtaken by the addition of progesterone. These results demonstrate that a high level of progesterone produced by cumulus cells was responsible for an acceleration of GVBD in porcine oocytes.     

Muscarinic Receptor Stimulation Activates a Ca<Superscript>2+</Superscript>-dependent Cl<Superscript>-</Superscript> Conductance in Rat Distal Colon

Recently, it was observed that the acetylcholine analogue carbachol induces a transient stimulation of an apical Cl(-) conductance in basolaterally depolarized rat distal colonic epithelium (Schultheiss et al., 2003). The further characterization of this conductance was the aim of the present study. All experiments were performed at basolaterally depolarized tissues (111.5 mmol.l(-1) KCl buffer at the serosal side); in the absence of a K(+) gradient, a Cl(-) current was driven across the apical membrane (107 mmol.l(-1) K gluconate/4.5 mmol.l(-1) KCl buffer on the mucosal side). Under these conditions, carbachol evoked an atropine-sensitive biphasic change in short-circuit current (I(SC)), consisting of a transient increase followed by a long-lasting decrease, suggesting a stimulation of apical Cl(-) conductance followed by an inhibition. This conductance was inhibited by SITS, but was resistant against glibenclamide, a blocker of CFTR. The carbachol-induced I(SC) was dependent on the presence of mucosal Ca(2+). Ionomycin, a Ca(2+) ionophore, mimicked the effect of carbachol. An antibody against bovine Ca(2+)-activated Cl(-) channel ClCa 1 stained rat colonic epithelial cells both at the cell membrane as well as intracellularly, suggesting that the action of Ca(2+) may be caused by a stimulation of a ClC a-type anion channel. The activation of apical Cl(-) conductance by carbachol was resistant against any blockers of the phospholipase C/IP3/protein kinase C pathway tested (e.g., U-73122, 2-ABP, Li(+), staurosporine), but was inhibited by the NO-synthase blocker L: -NNA. Vice versa, NO-donating compounds such as GEA 3162 or sodium nitroprusside evoked a transient increase of I(SC). Consequently, NO seems to be involved in the transient stimulation of apical Ca(2+)-dependent Cl(-) conductance after muscarinic receptor stimulation.     

Studies on the physiological function of spermine in the process of progesterone induced toad oocyte maturation

Spermidine or spermine but not putrescine inhibited progesterone induced Bufo bufo gargarizans oocyte maturation.The ID50 for spermine inhibition via intra -oocyte microinjection on maturation induced by progesterone was 6.8mM(100nl).Spermine could inhibit MPF induced toad oocyte maturation with a much higher ID50.A 55 kD protein was dephosphorylated during the process of progesterone induced oocyte maturation .Spermine selectively promoted the level of phosphorylation of this protein in both progesterone-stimulated and hormone-untreated oocytes.The extent of its dephosphorylation was fairly Correlated with the percentage of GVBD in the hormone stimulated oocytes.The level of endogenous spermine was reduced by 28% between the perod of 0.40 GVBD50 and 0.60 GVBD50,at which 55 kD protein was dephosphorylated.Spermine inhibited progesterone-stimulated protein synthesis in almost the same dose dependent manner as its inhititory effect on the hormone-induced maturation,The endogenous spermine regulated 55 kD protein dephosphorylation which may trigger the increase of protein dephosphorylation which may trigger the increase of protein synthesis and in turn promote the activation of MPF,It is possible that 55 kD protein may be one of the components of messenger ribonucleoprotein(mRNP) particles.     

Properties of membrane ion conductances evoked by hormonal stimulation of guinea-pig and rabbit isolated hepatocytes

Membrane conductance changes evoked in isolated guinea-pig or rabbit hepatocytes by hormonal stimulation were studied with the whole-cell patch clamp technique. In Cl-containing solutions, noradrenaline (NA), ATP or angiotensin II (AII) evoked an increase of conductance to both K (GK) and Cl (GCl) ions. Activation of GK occurred after a delay of several seconds and was sustained in the presence of hormone. Activation of GCl was transient, lasting several seconds, and arose either at the same time or shortly after the increase in GK. Conductances showed an initial rapid rise and slow oscillatory changes during maintained hormone application. The NA-induced current reversed at -19 mV in Cl solutions, between the equilibrium potentials for chloride (ECl = 0 mV) and potassium ions (EK = -85 mV), and at -75 mV, near EK, in Cl-free solution. In both conditions whole-cell current-voltage curves were linear in the range -100 mV to +40 mV. The conductance increase produced by NA to Cl- ions was about 50 nS, that to K+ ions was 6 nS. The potassium conductance increase was abolished by the polypeptide toxin apamin (50 nM). An increase in membrane current noise was associated with NA-evoked outward K+ current and blocked by apamin. Spectral analysis gave estimates of the elementary K channel conductance of 1.7 pS. Power spectra were fitted by two Lorentzian components, with average half-power frequencies of 2 and 190 Hz. These results are discussed in relation to the single-channel properties and indicate that the open probability of K channels during the NA response is high. In Cl solutions, with apamin to block the K conductance, no increase in current noise was detected during the large Cl conductance evoked by NA. This suggests either that Cl channels are of very low unitary conductance (less than 1 pS) or that Cl transport is due to a membrane carrier. The complex time-course of hormonally evoked conductances is not due to the properties of ion conductances per se but probably to underlying changes of intracellular second-messenger concentration.     

Increased intracellular pH is not necessary for ribosomal protein s6 phosphorylation, increased protein synthesis, or germinal vesicle breakdown in Xenopus oocytes

An increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation during Xenopus oocyte maturation has been reported by several laboratories. In this paper, the question of whether the pHi increase is necessary to induce S6 phosphorylation, an increase in protein synthesis, or germinal vesicle breakdown (GVBD) was assessed using sodium-free medium and the putative Na/H exchange blocker amiloride. Sodium-free medium decreased basal pHi by 0.3 unit and prevented increases in pHi in response to both insulin and progesterone, but S6 phosphorylation occurred normally with both hormones. GVBD occurred normally in sodium-free medium in response to progesterone, but the effect of insulin was reduced by 60%. In sodium-containing medium, amiloride inhibited GVBD and prevented insulin or progesterone-induced increases in pHi but the hormone-induced increase in S6 phosphorylation was unaffected. In the absence of sodium, amiloride inhibited GVBD but did not affect pHi, indicating that amiloride inhibits GVBD by a pHi-independent mechanism. Both progesterone and insulin increased protein synthesis in oocytes by 35%, and amiloride inhibited basal protein synthesis but not the increase with hormone. In the presence of cholera toxin, protein synthesis increases with insulin were inhibited but increased S6 phosphorylation was unaffected. Priming of animals with pregnant mare's serum gonadotropin prior to oocyte isolation reduced the time required for progesterone-induced GVBD, and increased the synchrony of GVBD of the population. Priming also increased oocyte basal pHi and basal protein synthesis as well as the magnitude of the increase in protein synthesis with progesterone but had no effect on S6 phosphorylation. The results indicate that in Xenopus oocytes increased pHi is not necessary for increased S6 phosphorylation, increased protein synthesis, or GVBD in response to insulin or progesterone nor is increased S6 phosphorylation sufficient for GVBD or increased protein synthesis.     

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     

Freeze-fracture electron microscopy of membrane changes in progesterone-induced maturing oocytes and eggs of Xenopus laevis

Using freeze-fracture electron microscopy, compositional changes were analysed in the surface membrane of Xenopus oocytes during maturation after in vitro progesterone treatment, as well as in eggs before and after fertilization. Investigated stages were as follows: (1) defolliculated full-grown oocytes; (2) defolliculated oocytes after 5 min exposure to 5 micrograms/ml progesterone; (3) ditto at germinal vesicle breakdown (GVBD) after 5 h progesterone treatment; (4) unfertilized eggs at oviposition and (5) zygotes 30 min post-fertilization. Comparing the patterns of intramembranous particle (IMP) density and IMP size during these stages the following changes were found: a transient decrease in IMP density was found after 5 min progesterone treatment; a 48% increase during maturation; a further 17% increase after fertilization. In defolliculated oocytes tight-junction-like structures were found, but no gap junctions. These results are discussed with reference to progesterone action, membrane remodelling, protein synthesis and membrane lipid organization.     

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.     

Spontaneous maturation of follicular oocytes in Rana dybowskii in vitro: seasonal influences, progesterone production and involvement of cAMP

Seasonal and hormonal influences regulating oocyte maturation (germinal vesicle breakdown, GVBD) in ovarian follicles of Rana dybowskii were investigated. During the early winter (Dec.-Jan.) GVBD occurred at a low incidence following in vitro culture of intact follicles. Addition of progesterone of frog pituitary homogenate (FPH) to such follicles induced oocyte maturation, whereas IBMX or forskolin inhibited hormone-induced oocyte maturation. The time course of spontaneous in vitro maturation varied markedly with the seasons and between animals. Follicles isolated from the ovaries in early February required 21-24 hours of culture to mature spontaneously, and addition of FPH or progesterone to the culture medium markedly accelerated the time course of GVBD. In contrast, follicles isolated in late February matured very rapidly (less than 6 hours), and FPH or progesterone were ineffective in accelerating the time course of GVBD. IBMX and forskolin separately or in combination stimulated follicular progesterone production, which resembled that seen following FPH stimulation. FPH addition to such follicles shifted the steroid peak to the left (accelerated) and increased the absolute amount of hormone detected in late-maturing follicles (50% GVBD, about 18 hours) but not in rapidly maturing follicles (50% GVBD, 3 hours). In contrast to other amphibians, a high incidence of spontaneous oocyte maturation occurred during in vitro culture. Essentially all animals exhibited spontaneous maturation during the normal breeding season, even those animals collected in the early winter and kept in artificial hibernation at 4 degrees C for extended periods.     

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.     

The effect of insulin on intracellular ph and ribosomal protein S6 phosphorylation in oocytes of Xenopus laevis

Both insulin and progesterone are capable of stimulating germinal vesicle breakdown (GVBD) of large, Stage VI oocytes of Xenopus laevis. Numerous studies have shown an increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation prior to GVBD in oocytes treated with progesterone. In this study the effect of insulin and progesterone on pHi and S6 phosphorylation was compared. Both hormones increased pHi and S6 phosphorylation to similar levels and the time course of pHi change was the same for both hormones. Half-maximal effects of insulin were observed at 7 X 10(-8) M concentrations. In the presence of 1 nM cholera toxin, the ability of progesterone to induce these two responses was inhibited while the action of insulin was unaffected. However, GVBD induced by either hormone was blocked by cholera toxin. In small, Stage IV oocytes that do not undergo GVBD in response to either progesterone or insulin, a partial increase in pHi without S6 phosphorylation occurred in response to progesterone but both events occurred in response to insulin. These results suggest that the inability of Stage IV oocytes to undergo GVBD in response to hormone is not due to a failure to increase pHi or phosphorylate S6. The results in this paper also indicate that these events are regulated differently by insulin and progesterone in Xenopus oocytes.     

Testosterone-induced meiotic maturation of Xenopus laevis oocytes: evidence for an early effect in the synergistic action of insulin

Meiosis reinitiation in oocytes (stage 5-6 of Dumont) isolated free of follicle cells by collagenase treatment from ovarian pieces of Xenopus laevis, was studied in observing the germinal vesicle breakdown (GVBD) provoked by progesterone and testosterone (0.1 nM-1 microM), alone or in association with insulin (30 micrograms/ml). Testosterone, was more active than progesterone to elicit GVBD in vitro, raising the question of the relative roles of both steroids in the physiological maturation process in vivo. The potentiating effect of insulin, already observed on progesterone action, was also demonstrated upon testosterone effect; the results suggested that it occurs during the early phase of hormone-induced meiosis reinitiation.     

Protein Kinase A(PKA)-Restrictcive and PKA-Permissive Phases of Oocyte Maturation

Substantial evidence has indicated that cAMP-dependent protein kinase (protein kinase A orPKA) plays a critical role in maintaining meiotic prophase arrest in vertebrate oocytes.However, PKA activity dynamic and its physiological substrate profile remain poorly defined.We have recently developed a novel PKA substrate construct which we employ to monitor PKAactivity in live oocytes. In the current study, we have employed biochemical and imaginganalyses of single cells to determine PKA activity dynamics during oocyte maturation and toinvestigate the consequence of re-activation of PKA during oocyte maturation. Wedemonstrated here that progesterone caused a rapid and permanent inhibition of PKA during theentire maturation process. However, artificial reactivation of endogenous PKA had differentialconsequences, depending on the timing of PKA reactivation. Reactivation of endogenous PKAat any time prior to GVBD inhibited progesterone-induced GVBD. PKA reactivation at GVBD,or thereafter, did not interfere with meiosis I to meiosis II transition, nor did it interfere withmetaphase II arrest. These results demonstrate for the first time a PKA-restricted phase and aPKA-permissive phase during oocyte maturation.     

Contribution of JNK, Mek, Mos and PI-3K signaling to GVBD in Xenopus oocytes

In Xenopus oocytes, induction of the G2/M transition by progesterone is a complex process that is promoted by a network of signaling molecules whose cumulative effect results in the activation of maturation promoting factor (MPF) and germinal vesicle breakdown (GVBD). We examined the role of Mos, Mek, PI-3 kinase and c-Jun N-terminal kinase (JNK) in progesterone stimulation of GVBD. Expression of an activated form of JNK neither induced nor enhanced progesterone-mediated GVBD in oocytes, suggesting a limited role in cell-cycle progression. We blocked Mek, Mos and PI-3 kinase activities by a variety of means that included expression of dominant-negative kinase suppressor of Ras (DnKSR), expression of a dominant-negative PI-3 kinase (DnPI3K), treatment of oocytes with a Mek inhibitor (U1026) or PI-3 kinase (LY294002) inhibitor, and introduction of Mos antisense morpholinos. Inhibition of any one pathway alone failed to block GVBD induced by either high or low concentrations of progesterone. In contrast, inhibiting Mos or Mek function in addition to abrogating PI-3 kinase activity effectively blocked oocyte maturation. Furthermore, by expressing suboptimal amounts of Mos in conjunction with an activated form of Mek and an activated form of the p110 catalytic subunit of PI-3 kinase, we show cooperation among these signaling molecules toward the induction of GVBD. Moreover, expression of optimal amounts of these three proteins in conjunction with inhibitors of Mos, Mek or PI-3 kinase demonstrated that activated Mek-induced GVBD is independent of Mos or PI-3 kinase activity. In addition, Mos-induced GVBD is dependent upon Mek activity, but does not require PI-3 kinase activity. Finally, Mos appears to be a major contributor to GVBD induced by activated PI-3 kinase, while Mek is a minor contributor to this process.     

Discrimination of the roles of MPF and MAP kinase in morphological changes that occur during oocyte maturation

Maturing amphibian oocytes undergo drastic morphological changes, including germinal vesicle breakdown (GVBD), chromosome condensation, and spindle formation in response to progesterone. Two kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), are involved in these changes, but their precise roles are unknown. Unlike in Xenopus oocytes, discrimination of the functions of MAPK and MPF in Rana oocytes is easy owing to the lack of pre-MPF. We investigated the roles of these kinases by careful observations of chromosomes and microtubules in Rana oocytes. MPF and MAPK activities were manipulated by treatment with progesterone, c-mos mRNA, or cyclin B mRNA in combination with MAPK kinase inhibitors. Activation of one kinase without activation of the other induced only limited events; GVBD was induced by MPF without MAPK, and reorganization of microtubules at GVBD was induced by MAPK without MPF, but other events were not induced. In contrast, coactivation of MPF and MAPK by injection of c-mos and cyclin B mRNA promoted almost all of the morphological changes that occur during maturation without progesterone, indicating that these are controlled by cooperation of MPF and MAPK. The results revealed the functions of MAPK and MPF in each process of sequential morphological changes during oocyte maturation.     

Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes

Oocytes induced to undergo meiotic maturation by progesterone possess a cytoplasmic activity that causes germinal vesicle breakdown (GVBD). The cytoplasmic factor postulated to be responsible for this activity is designated as the maturation promoting factor (MPF). The activity of MPF was assayed by injecting cytoplasm into fully-grown oocytes to induce GVBD. It was found that maturing oocyte cytoplasm possesses MPF activity before GVBD begins. Treatment of progesterone stimulated oocytes with cycloheximide, either applied externally or injected, inhibited the appearance of MPF in the cytoplasm as well as GVBD when the inhibitor treatment was initiated before the cytoplasm exhibited MPF activity. In contrast, the same treatment did not inhibit GVBD when it was applied to oocytes after the cytoplasm possessed MPF activity. Furthermore, cycloheximide treatment of recipient oocytes did not inhibit the induction of GVBD by injected cytoplasm containing MPF. Cytoplasm of oocytes injected with MPF subsequently possessed MPF activity as high as that of the original donor cytoplasm in spite of its extensive dilution. This suggests that amplification of MPF took place in the recipient. Cycloheximide treatment did not inhibit the amplification of MPF. It was concluded that cycloheximide inhibits only the initial phase of induction of MPF activity, but neither its amplification nor its action on the nucleus that causes GVBD. From these results, a hypothesis concerning the cytoplasmic mechanism for the induction of GVBD has been proposed.     

Presence of a calcium-activated chloride current in mouse ventricular myocytes

The properties of several components of outward K(+) currents, including the pharmacological and kinetics profiles as well as the respective molecular correlates, have been identified in mouse cardiac myocytes. Surprisingly little is known with regard to the Ca(2+)-activated ionic currents. We studied the Ca(2+)-activated transient outward currents in mouse ventricular myocytes. We have identified a 4-aminopyridine (4-AP)- and tetraethyl ammonium-resistant transient outward current that is Ca(2+) dependent. The current is carried by Cl(-) and is critically dependent on Ca(2+) influx via voltage-gated Ca(2+) channels and the sarcoplasmic reticulum Ca(2+) store. The current can be blocked by the anion transport blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Single channel recordings reveal small conductance channels (approximately 1 pS in 140 mM Cl(-)) that can be blocked by anion transport blockers. Ensemble-averaged current faithfully mirrors the transient kinetics observed at the whole level. Niflumic acid (in the presence of 4-AP) leads to prolongation of the early repolarization. Thus this current may contribute to early repolarization of action potentials in mouse ventricular myocytes.