The relationships between early ionic events, the pattern of protein synthesis, and oocyte activation in the surf clam, Spisula solidissima

The ionic events linked to activation of surf clam (Spisula solidissima) oocytes include a transient increased Ca2+ influx and an acid release. The aim of the present work was to further elucidate the respective roles of these two ionic events and to clarify the possible role of protein kinase C in the sequence of events leading to oocyte activation. K+-enriched seawater, ammonium chloride, and the phorbol ester 12-O-tetradecanoyl-13-phorbol acetate (TPA), a protein kinase C activator, were tested for their ability to promote germinal vesicle breakdown (GVBD), an acid release, increased 45Ca2+ uptake, and a shift in the pattern of protein synthesis. Oocytes activated by addition of K+ ions release an amount of H+ similar to that induced by fertilization, with the same time course, show an increased, verapamil-sensitive, 45Ca2+ uptake that is proportional to the amount of added K+, and undergo a shift in their pattern of protein synthesis, which requires the presence of external Ca2+. Ammonium chloride, at concentrations causing a higher production of acid than that induced by K+ ions or fertilization, does not trigger GVBD nor any increased 45Ca2+ uptake or any detectable shift in the pattern of protein synthesis. Combined additions of ammonium chloride with subthreshold concentrations of K+ ions allow GVBD to occur, thus revealing a synergistic effect of ammonia and K+ ions. TPA slowly induces GVBD, an Na+-dependent acid release, and a shift in the pattern of protein synthesis, in the absence of increased 45Ca2+ uptake. Our results lead us to propose the following sequence of events for the activation of Spisula oocytes: an increased Ca2+ influx contributes to activate protein kinase C which causes a Na+-dependent acid release leading to a rise of pHi. This rise of pHi, although insufficient by itself, may set the pHi in a permissive range for activation to occur through the action of other protein kinase C-sensitive events leading to the production of meiosis-inducing proteins.     

Evidence for Involvement of Protein Kinase C in Germinal Vesicle Breakdown in Chaetopterus

We have examined the possible involvement of protein kinase C (C-kinase) in the initiation of germinal vesicle breakdown (GVBD) in Chaetopterus oocytes. Two tumor-promoting phorbol esters (phorbol-12, 13-dibezoate and 12-0-tetradecanoylphorbol-13-acetate [TPA]) and a permeant diacylglycerol (1-oleoyl-2-acetylglycerol), potent activators of C-Kinase, triggered GVBD. Two other phorbol esters (phorbol-13-monoacetate and 4α-phorbol-12, 13-didecanoate), which do not activate C-kinase, were inactive. Three C-kinase antagonists (W-7, H-7 and retinol) inhibited both naturally-and TPA-induced GVBD, whereas W–5, a much less inhibitory W–7 analog, had no effect on GVBD. Triggering of GVBD by TPA was independent of extracellular Ca²⁺. Although naturally-induced GVBD was blocked by micromolar concentrations of the calmodulin antagonist, calmidazolium (R24571), and by millimolar concentrations of the permeant cAMP analog, dibutryryl cAMP, TPA-induced GVBD was not affected by these agents. These results support the hypothesis that both C-kinase and calmodulin are involved in the sequence of events leading to GVBD in this species.     

Comparative patterns of protein phosphorylation during activation of surf clam oocytes by different artificial agents

Oocytes from the surf clam Spisula solidissima are arrested at prophase I of meiotic maturation, until fertilization, We analyzed the patterns of phosphorylated proteins under procedures mimicking, to various degrees, the normal sperm-induced activation process. High K⁺-seawater, the phorbol ester TPA, serotonin, or a combination of these were used to analyze their effects on both germinal vesicle breakdown (GVBD) and protein phosphorylation. Oocytes were preloaded with ³⁶S-methionine or ³²P-phosphate, and the pattern of labeled proteins was analyzed by polyacrylamide gel electrophoresis followed by autoradiography. When comparing, in high K⁺-activated oocytes, the pattern of phosphorylated proteins with that of synthesized proteins, it appeared that these two processes were largely unrelated to one another. Activation induced by TPA was slower (60 min for GVBD) than that induced by high K⁺ or serotonin (12–15 min for GVBD), but was similarly sensitive to the protein phosphorylation inhibitor, 6-dimethylaminopurine, and resulted in a qualitatively similar pattern of phosphorylated proteins appearing with slower kinetics, reflecting slower GVBD. When both serotonin and TPA were added to oocytes, the kinetics of GVBD was intermediate (30 min), and so was the appearance of phosphorylated proteins. Finally, the kinetics of development of H1 kinase activities was evaluated in oocytes activated by serotonin, TPA, or both. Similar to the general pattern of phosphorylated proteins, increased histone H1 kinase activities developed to similar degrees but with kinetics reflecting those of GVBD in each case. In conclusion, activations by different artificial agents, utilizing different pathways, resulted in GVBD with different kinetics but similar overall patterns of phosphorylated proteins after a lag typical of the agent used. This suggests that diverse pathways may initially be used to activate oocytes, but that these different pathways eventually merge into a common one, resulting in a highly conserved and regulated sequence of phosphorylation processes. © 1996 Wiley-Liss, Inc.     

Inositol trisphosphate, inositol phospholipid metabolism, and germinal vesicle breakdown in surf clam oocytes

Others have reported that microinjection of inositol 1,4,5-trisphosphate (InsP3) releases stored intracellular Ca2+ and causes fertilization envelope elevation, part of the activation process normally initiated by fertilization in deuterostome eggs. In the protostome, Spisula solidissima, germinal vesicle breakdown (GVBD) is the first visible response of the egg to fertilization. To test the effects of InsP3 on egg activation in this organism, we microinjected the compound into oocytes. Microinjection of 0.4-7.0 x 10(-21) moles of InsP3 (equivalent to 5-80 pM if distributed throughout the cell) elicited GVBD in a dose-dependent manner, demonstrating that increased oocyte InsP3 can mimic part of the activation process in this protostome. Synthesis of InsP3 occurs in vivo when phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is hydrolyzed by phospholipase C. To determine whether stimulus-induced synthesis of InsP3 occurs after fertilization of Spisula oocytes, we labeled oocyte lipids with [32P]orthophosphate and measured the radioactivity in phospholipids after insemination. Fertilization resulted in a rapid, transient loss of radioactivity from PtdInsP2. Because the radioactivity in phosphatidylinositol 4-phosphate and other phospholipids did not change, the loss of radioactivity from PtdInsP2 is most likely due to its hydrolysis, yielding InsP3 and diacylglycerol. The latter compound activates protein kinase C which has also been shown to be involved in regulating Spisula oocyte GVBD. Since both of these compounds appear to be early products of fertilization, they could coordinately activate Ca2+- and protein kinase C-dependent processes involved in Spisula oocyte GVBD. These data indicate that egg activation in this protostome includes pathways similar to those found in deuterostome eggs and in other eukaryotic cells.     

Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.     

Cyclic nucleotide content and protein phosphorylation during maturation of Spisula oocytes

Cyclic nucleotide levels in the oocytes of the surf clam Spisula solidissima were measured during germinal vesicle breakdown (GVBD) induced by fertilization. The level of cAMP and cGMP in untreated oocytes was 8.23 ± 0.95 and 4.89 ± 0.39 pmol/10⁶ oocytes. The ratio of cAMP to cGMP ranged from 1.5 to 2.0. The cAMP level in Spisula oocytes fluctuated after fertilization and before GVBD. The cGMP level showed minimal fluctuation, with a tendency to decrease initially followed by a subsequent rise to the basal level in a nonsynchronous manner. These changes were not statistically significant. There was a general increase in protein phosphorylation during the period after fertilization and before GVBD. The greatest increase occurred with proteins of estimated molecular weights of 52, 18, and 12 kD, analyzed by gel electrophoresis and autoradiography.     

Protein kinase C and meiotic maturation of surf clam oocytes

We report here that phorbol ester, a potent activator of protein kinase C, induces germinal vesicle breakdown in surf clam oocytes. However, phorbol ester-induced activation is slow and is not accompanied by an increased Ca2+ influx. Simultaneous additions of phorbol ester and various amounts of K+ ions, which induce Ca2+ influx of different amplitudes, result in successful activation within the normal time schedule at K+ concentrations inefficient alone in activating the oocytes. In vivo, increased protein phosphorylation triggered by phorbol ester amounts to about one third that seen after fertilization. These results suggest that increased Ca2+ influx and protein kinase C activation act in synergy to cause resumption of meiotic maturation in these oocytes.     

Polymyxin B causes coordinate inhibition of phorbol ester-induced C-kinase activity and proliferation of B lymphocytes

Lymphocytes were found to be rich in phospholipid/Ca2+-dependent (C-kinase) activity. Addition of polymyxin B (PMB) to in vitro assays of endogenous and exogenous phosphorylation resulted in profound inhibition of C-kinase activity. The phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) directly activated C-kinase, leading to increased phosphorylation of the same substrates. TPA also stimulated proliferation of B cells as assessed by 3H-thymidine uptake, and PMB strongly inhibited this effect. This coordinate inhibition of TPA-induced phosphorylation and mitogenesis indicates that PMB is a potentially useful inhibitor of C-kinase activity, and that this enzyme may play an important role in mediating B cell responses.     

Induction of ovulation and oocyte maturation of amphibian (Rana dybowskii) ovarian follicles by protein kinase C activation in vitro.

We previously reported that protein kinase C (PKC) activation induced meiotic maturation (germinal vesicle breakdown, GVBD) of Rana dybowskii follicular oocytes cultured in vitro without hormone treatment. The experiments reported here were carried out to establish whether ovarian follicles ovulated in response to PKC activation during culture. A phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was used for PKC activation. TPA addition (10 microM) to cultured ovarian fragments induced ovulation and maturation of the oocytes similar to that seen following addition of frog pituitary homogenate (FPH, 0.05 pituitary/ml) or progesterone (0.5 microgram/ml). Such changes were not observed when ovarian fragments were treated with inactive phorbol ester. The time course of TPA-induced ovulation was similar to that produced by FPH-stimulated ovulation. Both TPA- and FPH-stimulated ovulation and maturation were blocked by treatment with cycloheximide, forskolin (an adenylate cyclase stimulator), and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7; a PKC inactivator). FPH treatment markedly increased progesterone levels in the medium during ovarian fragment culture whereas TPA treatment failed to elevate progesterone levels. Thus, TPA treatment mimics FPH and progesterone in inducing ovulation and meiotic maturation in cultured amphibian ovarian fragments. The data strongly suggest that PKC plays an important role in regulating ovulation as well as in modulating amphibian oocyte maturation during follicular differentiation.     

Residual inhibition of epidermal growth factor binding by pancreatic secretagogues and phorbol ester in rat pancreas

Cholecystokinin-octapeptide (CCK8) inhibits 125I-labeled epidermal growth factor (EGF) cell-associated radioactivity in pancreatic acini, ostensibly as a result of its ability to mobilize cellular Ca2+. The phorbol ester tetradecanoyl phorbol acetate (TPA), a compound that activates protein kinase C, mimics the inhibitory action of CCK8. In the present study we examined the relationship between occupancy of the cholecystokinin (CCK) receptor, the subsequent inhibition of EGF binding, and the potential role of C-kinase activation in mediating this inhibition. Proglumide and dibutyryl cyclic GMP (dbGMP), two distinct competitive antagonists of CCK8, reversed the inhibitory actions of CCK8. Analysis of steady-state saturation kinetics of 125I-EGF binding indicated that CCK8 decreased the apparent affinity of the EGF receptor, mainly as a result of a marked decrease in the amount of internalized ligand. TPA also inhibited 125I-EGF internalization. Removal of CCK8 and TPA from incubation medium did not abolish their inhibitory actions. Carbachol, but not bombesin, exerted a similar residual inhibitory effect. It is suggested that in addition to acting via Ca2+, certain pancreatic secretagogues may also act through C-kinase to regulate EGF binding.     

MAP and cdc2 Kinase Activities at Germinal Vesicle Breakdown inChaetopterus

In frog oocytes, activation of mitogen-activated protein kinase (MAPK, ERK) leads to activation of cdc2 and germinal vesicle breakdown (GVBD). By contrast, in starfish, MAPK is activated after GVBD. Here we have examined the relative involvements of MAPK and cdc2 in GVBD ofChaetopterusoocytes. MAPK was rapidly tyrosine-phosphorylated and activated (within 1–2 min) in response to exposure of the oocytes either to natural seawater (the normal trigger of GVBD in this organism) or to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which can also elicit GVBD. This response preceded the tyrosine dephosphorylation and activation of cdc2 by several minutes. MAPK phosphorylation and activation were transient, lasting only until GVBD occurred and the spindle migrated to the cortex. The enzyme was not phosphorylated again as a result of egg activation. These results are consistent with the hypothesis that the activation of MAPK has a role in GVBD. However, PD 98059, a potent and selective inhibitor of MEK, the protein kinase that phosphorylates and activates MAPK, blocked the phosphorylation of MAPK but did not block GVBD, the dephosphorylation and activation of cdc2, or spindle formation and migration. Oocytes that underwent GVBD in PD 98059 could be fertilized and cleaved normally. Ionophore A23187, although it caused germinal vesicles to disappear and caused transient phosphorylation of MAPK, did not cause dephosphorylation of cdc2, and therefore this disappearance is artifactual. These results suggest that MAPK activation is neither obligatory nor sufficient for either GVBD or meiotic metaphase arrest inChaetopterusand that activation of MAPK and cdc2 occur on independent, parallel pathways.     

Phorbol ester-induced down-regulation of the 80-kDa myristoylated alanine-rich C-kinase substrate-related protein in Swiss 3T3 fibroblasts. Inhibition by staurosporine.

A polyclonal antiserum raised against an oligopeptide with an amino acid sequence corresponding to a sequence of the myristoylated alanine-rich C-kinase substrate (MARCKS) from mouse macrophages and rat brain recognizes the 80-kDa C-kinase substrate from Swiss 3T3 fibroblasts. Using this antiserum for quantitative determination of the 80-kDa MARCKS-related protein, we found that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a rapid down-regulation of this protein in the fibroblasts. In accordance with earlier reports, TPA causes phosphorylation of the 80-kDa protein which can be inhibited by staurosporine. Staurosporine also suppresses the TPA-induced down-regulation, possibly indicating that the down-regulation of the MARCKS-related protein is dependent on its phosphorylation by protein kinase C.     

The effects of quercetin on meiosis initiation in clam and starfish oocytes

We have treated Spisula and Asterias oocytes with quercetin to determine the effects of this drug on germinal vesicle breakdown (GVBD). Quercetin (100-200 microM) reversibly inhibited GVBD when induced by excess KCl or ionophore A23187. Kinetic studies showed that quercetin blocked an early event in GVBD. Lower concentrations of quercetin (10-20 microM) blocked fertilization. However, quercetin sensitized the oocytes to initiation of GVBD by excess ions which do not normally trigger GVBD. Quercetin (100-200 microM) also blocked 1-methyladenine-induced GVBD in the starfish. In subthreshold concentrations of the hormone or in its absence, lower concentrations (20-40 microM) stimulated GVBD. The results support the hypothesis that quercetin exerts its effects on meiosis initiation through its effects on calcium sequestration.     

Protein phosphorylation during activation of surf clam oocytes.

We have investigated the increase of phosphorylated proteins upon activation of surf clam (Spisula solidissima) oocytes, by measuring the cumulative incorporation of 32P in proteins and by performing an SDS-PAGE and autoradiographic analysis of 32P-labeled proteins, from oocytes initially radiolabeled with 32P-orthophosphate. The phosphorylation inhibitor 6-dimethylaminopurine (6-DMAP) inhibits both germinal vesicle breakdown (GVBD) and the normal increase in phosphorylated proteins observed upon activation by KCl, in a reversible and dose-dependent manner. Using different artificial seawaters (normal, Ca(2+)-free, Na(+)-free), we observed that the increase of phosphorylated proteins, upon K+ stimulation, occurs only when GVBD is allowed to proceed along with an increased Ca2+ influx, in normal or Na(+)-free seawater. Stimulation of oocytes by ammonia, which directly raises intracellular pH (pHi) but does not trigger GVBD, is without effect on the level or pattern of phosphorylated proteins. The link between the Ca2+ influx and the level of phosphorylated proteins was further investigated using conditions altering the duration or the level of Ca2+ influx upon K+ stimulation. In all conditions tested, both GVBD and the level of phosphorylated proteins were similarly affected by alterations of the Ca2+ influx, indicating that these processes are tightly coupled one with another. Upon activation of oocytes, six major proteins of estimated molecular weights of 31, 41, 48, 56, 80 and 86 kDa undergo an increased phosphorylation that is reversibly sensitive to 6-DMAP. Our results suggest that increased protein phosphorylation, sensitive to 6-DMAP, is necessary for GVBD and that it is indirectly linked to the increased Ca2+ influx that stands as an upstream trigger for activation, while an elevated pHi alone has no effect on these processes.     

Protein kinase C mediated regulation of calcium channels in PC-12 pheochromocytoma cells

Depolarization of PC-12 pheochromocytoma cells with K+ produces an immediate increase in catecholamine release. The stimulation of release is blocked by Co2+, removal of extracellular Ca2+ or by dihydropyridine drugs such as nitrendipine. Release is enhanced by other dihydropyridines such as BAY K8644. Release is accompanied by a voltage dependent uptake of 45Ca2+ which is also blocked by Co2+ or nitrendipine and enhanced by BAY K8644. The phorbol ester phorbol 12-myristate-13-acetate (TPA) in the range 10(-9)-10(-6) M produced little effect by itself but augmented the K+ evoked release of catecholamine. An analog of TPA which does not activate protein kinase C was ineffective. In contrast, TPA in the same concentration range blocked influx of 45Ca2+ induced by 70 mM K+ or 70 mM K+/BAY K8644. 45Ca2+ influx produced by A23187 was not blocked by TPA. The results suggest a system by which protein kinase C may regulate the output of transmitters from secretory cells.     

Phosphorylation of ribosomal proteins during meiotic maturation and following activation in starfish oocytes: its relationship with changes of intracellular pH

An increased phosphorylation of ribosomal protein S6 has been shown to be correlated with an increase of intracellular pH (pHi) and with stimulation of protein synthesis in many systems. In this research changes in ribosome phosphorylation following hormone-induced meiotic maturation and fertilization or activation by ionophore A23187 were investigated in starfish oocytes. The hormone was found to stimulate, even in the absence of external Na+, the phosphorylation on serine residues of an Mr 31,000 protein identified as S6, as well as that of an acidic Mr 47,000 protein, presumably S1, on threonine residues. Phosphorylation of ribosomes was an early consequence of hormonal stimulation and did not decrease after completion of meiotic maturation. Fertilization or activation by ionophore of prophase-arrested oocytes also stimulated ribosome phosphorylation. Only S6 was labeled in this case, but to a lesser extent than upon hormone-induced meiotic maturation. Changes in pHi were monitored with ion-specific microelectrodes throughout meiotic maturation and following either fertilization or activation. The pHi did not change before germinal vesicle breakdown (GVBD) following hormone addition, but it increased before first polar body emission. It also increased following fertilization or activation by ionophore or the microinjection of Ca-EGTA. In all cases, alkalinization did not depend on activation of an amiloride-sensitive Na+/H+ exchanger. Microinjection of an alkaline Hepes buffer or external application of ammonia, both of which increased pHi, prevented unfertilized oocytes from arresting after formation of the female pronucleus and induced chromosome cycling. Phosphorylation of S6 was still observed following fertilization or induction of maturation when pHi was decreased by external application of acetate, a treatment which suppressed the emission of polar bodies. Protein synthesis increased in prophase-arrested oocytes after fertilization or activation. It also increased after ammonia addition, although this treatment did not stimulate S6 phosphorylation.     

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)     

Inhibition of Starfish Oocyte Maturation by Tumor-Promoting Phorbol Esters*

Effect of tumor promoters including phorbol esters and teleocidin on 1-methyladenine (1-MeAde)-induced oocyte maturation was studied in the starfish. When isolated immature oocytes were treated with 1-MeAde and 12-O-tetradecanoylphorbol-13-acetate (TPA), 1-MeAde-induced maturation was completely inhibited at more than 2.5 μg/ml. However, if TPA was added after the hormone-dependent period (the minimum period wherein 1-MeAde is required), such maturation-inhibiting effect was no longer observed. Pretreatment with TPA for 5 min showed that its inhibitory action is irreversible. However, when TPA-injected oocytes were treated with 1-MeAde, all oocytes underwent germinal vesicle breakdown (GVBD). GVBD was induced in TPA-treated oocytes upon injection of the cytoplasm of maturing oocytes containing maturation-promoting factor (MPF). These facts show that TPA acts on the oocyte surface to inhibit the production of MPF. Retinoids including retinal, retinol and retinoic acid reversed the inhibitory effect of TPA on 1-MeAde-induced maturation. Experiments with various phorbol esters showed a good correlation between their maturation-inhibiting activity and their known tumor-promoting activity. Further, telecoidin, which is structurally unrelated to phorbol esters, inhibited 1-MeAde action. Since both tumor-promoting phorbol esters and teleocidin are known to activate Ca²⁺ -activated, phospholipid-dependent protein kinase (protein kinase C) and their activation effect is inhibited by retinoids, it appears that the activation of protein kinase C by tumor promoters is involved in blocking of 1-MeAde action.     

Phorbol Ester Inhibition of Current Responses and Simultaneous Protein Phosphorylation in Xenopus Oocyte Injected with Brain mRNA

The role of protein kinase C activation in a coupling of Ca2+-mobilizing receptors/GTP-binding protein/phospholipase C was examined using Xenopus oocytes before and after microinjection of mRNA purified from rat brains. Under voltage-clamp conditions, although the phorbol ester TPA per se never elicited any changes in ionic conductance, chloride current responses of mRNA-injected cells to 5-hydroxytryptamine and acetylcholine (ACh) were suppressed by an 8-min pretreatment of 12-O-tetradecanoyl-4 beta-phorbol-13-acetate (TPA), at nanomolar concentrations. Native ACh response in intact follicular oocytes was also inhibited by the TPA treatment. However, similar current responses triggered by the direct activation of their intracellular signalling pathway with guanosine-5'-O-(3-thio)triphosphate or Ca2+ were not affected by TPA. Biochemical analyses indicated that phosphorylation of 33,000- and 45,000-dalton proteins was markedly enhanced by TPA in vivo, and that stimulation of receptors with agonists as well as TPA treatment increased phosphoproteins in the membrane fraction of mRNA-injected oocytes. These observations suggest that protein kinase C may switch off the signal transduction from receptors to GTP-binding proteins and may participate in the negative feedback modulation of receptor-operated ion channel responses.     

Pharmacology of the serotonin-induced meiosis reinitiation in Spisula solidissima oocytes

Germinal vesicle breakdown (GVBD) is the first visible response of the oocyte of Spisula solidissima to the neurohormone serotonin. Pharmacological characterization of this response was performed by using 24 serotonin-related compounds. Dose-response curves were assessed by quantification of GVBD. Rank orders of potency obtained were among agonists: serotonin greater than 8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide greater than 2-methyl-serotonin greater than 1-(3-trifluoromethylphenyl)piperazine; among antagonists; ritanserin ritanserin greater than ICS205930 greater than mianserin = ketanserin = propranolol greater than metoclopramide = yohimbine greater than spiperone. Various other monoaminergic compounds tested were inefficient, demonstrating the specificity of the oocyte response to serotonin. Transduction mechanisms underlying this response were then investigated. Ca2+ appeared to be involved since serotonin induced an increase in the uptake of 45Ca2+ and since it was inefficient in calcium-free sea water. The absence of synergy between serotonin and KCl suggested that both compounds use a common transduction pathway. Exposure of the oocyte to the protein kinase C activator TPA inhibited serotonin-dependent maturation. Our data thus point to an original, previously uncharacterized pharmacological profile and transduction mechanism by which serotonin induces oocyte meiosis reinitiation in Spisula solidissima.