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.     

Errata

The pH-dependent control of the rate of protein synthesis in the fertilized sea urchin egg has suggested that pH may have an important role in cell activation at fertilization. We looked for similar changes of intracellular pH during meiotic maturation of the Xenopus oocyte. The basal pH of the oocyte is in the range 7.4–7.8. The higher values were found mostly in oocytes from animals with recent hormonal stimulation, suggesting a correlation with the elevated metabolism of such oocytes. Regardless of basal pH, progesterone-induced maturing oocytes alkalize an average of 0.18 pH unit. Beginning shortly before germinal vesicle breakdown, intracellular pH then decreases to near the original value. The same program is observed when maturation is induced by an injection of “maturation promoting factor.” Maturation is delayed or inhibited if intracellular pH is driven acidic. It is induced in the absence of progesterone by trimethylamine, a weak base which may act via an imposed alkalization. However, maturation still occurs when net alkalization is prevented. These data suggest that the alkalization during maturation is a form of metabolic “insurance” and that there may be both pH-dependent and pH-independent pathways for maturation. There is some evidence suggesting that pH changes are related to movement of other ions.     

Regulation of 40S ribosomal protein S6 phosphorylation in Swiss mouse 3T3 cells

Addition of serum to resting cultures of Swiss mouse 3T3 cells causes an immediate multiple phosphorylation of 40S ribosomal protein S6. After 60 min of stimulation, changing to medium containing no serum led to the net dephosphorylation of S6. During this same period, a second protein, as yet unidentified, became increasingly phosphorylated. Incubation of cells with cycloheximide prior to the addition of serum almost completely blocked the activation of protein synthesis. There was no effect on the serum-induced phosphorylation of S6. If cells were stimulated in the presence of cAMP phosphodiesterase inhibitors theophylline or SQ 20006, both S6 phosphorylation and the activation of protein synthesis were inhibited. Stimulation of cells with serum also led to an immediate drop in total intracellular cAMP levels. This was blocked by prostaglandin E1 (PGE1), which caused a 10 fold increase in total intracellular cyclic AMP. However, PGE1 had no effect on protein synthesis or S6 phosphorylation.     

Comparative studies on sporulation-promotive actions on cyclic AMP, theophylline and caffeine in Saccharomyces cerevisiae.

Cyclic AMP, theophylline and caffeine promoted sporulation when added to a presporulation medium containing glucose. Caffeine promoted sporulation even when added to a presporulation medium containing acetate as the carbon source, but cyclic AMP and theophylline did not. Caffeine did not increase the intracellular cyclic AMP level, while theophylline did significantly when added to a presporulation medium containing glucose. Caffeine inhibited the vegatative DNA synthesis with little effect on RNA and protein synthesis, resulting in the increase in cell volume, dry weight, and RNA and protein contents, but cyclic AMP and theophylline did not show such effects.     

Microsome protein phosphorylation in Acer pseudoplatanus cells as influenced by intracellular alkalinization

Abstract. The authors have previously shown that cell treatments causing intra-cellular alkalinization stimulate the in vivo phosphorylation of a 33-K Dalton polypeptide (33 KP) (Tognoli & Basso, 1987). Here, the authors report that this polypeptide belongs to a protein associated with the microsomal membranes. They show that treatment of cells which induce intracellular alkalinization stimulate 33-KP phosphorylation, whether the phosphorylation is performed in vivo (cells loaded with ³²Pi before treatments) or in vitro (microsomes from control and treated cells, incubated with γ³²P ATP). In both cases, 33 KP is phosphorylated on a serine residue. Microsomes do not show any phosphatase activity towards this phosphorylated protein, indicating involvement of a protein kinase reaction as an effector of changes induced by intracellular alkalinization. The number of phosphorylated sites or molecules of this protein increases as a result of intracellular alkalinization, suggesting that intracellular alkalinization causes topological or conformational modifications to a protein kinase or its substrate protein. The in vitro phosphorylation is not specifically influenced by the pH of the in vitro phosphorylation medium, suggesting that protein phosphorylation is not directly controlled by cytoplasmic pH.     

MSP hormonal control of the oocyte MAP kinase cascade and reactive oxygen species signaling

The MSP domain is a conserved immunoglobulin-like structure that is important for C. elegans reproduction and human motor neuron survival. C. elegans MSPs are the most abundant proteins in sperm, where they function as intracellular cytoskeletal proteins and secreted hormones. Secreted MSPs bind to multiple receptors on oocyte and ovarian sheath cell surfaces to induce oocyte maturation and sheath contraction. MSP binding stimulates oocyte MPK-1 ERK MAP Kinase (MAPK) phosphorylation, but the function and mechanism are not well understood. Here we show that the Shp class protein-tyrosine phosphatase PTP-2 acts in oocytes downstream of sheath/oocyte gap junctions to promote MSP-induced MPK-1 phosphorylation. PTP-2 functions in the oocyte cytoplasm, not at the cell surface to inhibit multiple RasGAPs, resulting in sustained Ras activation. We also provide evidence that MSP promotes production of reactive oxygen species (ROS), which act as second messengers to augment MPK-1 phosphorylation. The Cu/Zn superoxide dismutase SOD-1, an enzyme that catalyzes ROS breakdown in the cytoplasm, inhibits MPK-1 phosphorylation downstream of or in parallel to ptp-2. Our results support the model that MSP triggers PTP-2/Ras activation and ROS production to stimulate MPK-1 activity essential for oocyte maturation. We propose that secreted MSP domains and Cu/Zn superoxide dismutases function antagonistically to control ROS and MAPK signaling.     

Calcium-mediated transduction of the hormonal message in meiosis reinitiation of starfish oocytes: modulation following injection of cholera toxin and cAMP-dependent protein kinase

Injections of the regulatory subunit of type I cAMP-dependent protein kinase, of the heat-stable inhibitor protein of cAMP-dependent protein kinase and of calmodulin have no effect on meiosis reinitiation. Drugs, including theophylline, caffeine and procaine, which have been shown previously to inhibit 1-methyladenine (1-MeAde)-induced Ca²⁺ release, both in living starfish oocytes and from plasma membrane-rich fractions obtained from isolated cortices, inhibit meiosis reinitiation when added before—but not after—the end of the hormone-dependent period (period when presence of the hormone in the medium is required for meiosis to occur). In the same conditions, theophylline suppresses 1-MeAde-induced stimulation of protein phosphorylation. Injection of cholera toxin subunit A increases oocyte sensitivity to 1-MeAde. Catalytic subunit of cAMP-dependent protein kinase (C) inhibits meiosis reinitiation when injected before the end of the hormone-dependent period. Oocytes can be released from inhibition due to C injection by raising 1-Me-Ade concentration. These findings support the view that Ca²⁺ release occurs until transduction of the hormonal message (i.e., its intramembrane transfer) has been completed and show that protein phosphorylation remains under plasma membrane control until that time. They also suggest that transduction of the hormonal message might be modulated by intracellular cAMP and membrane phosphorylation, although cAMP content does not change following 1-MeAde addition.     

Conditions affecting protein synthesis in amphibian oocytes.

The endogenous protein synthesis of Xenopus laevis and Calyptocephalella caudiverbera oocytes was studied by measuring the incorporation into acid-precipitable material of radioactive amino acids placed in the extracellular medium. Large differences of incorporation into protein were observed by using different labeled amino acids. For example, it was found that radioactive aspartic acid or glutamic acid was very poorly incorporated at concentrations under 0.1 mm. These differences are due to differences in uptake constants and in the internal pools of free amino acids which are very large for the acidic amino acids. Both types of oocytes behaved similarly with respect to magnesium ion concentration, temperature optimum and inhibitors of protein synthesis. They differed however in sensitivity to pH since Xenopus laevis oocyte protein synthesis was twofold higher at pH 8.5 than at pH 7 while Calyptocephalella caudiverbera oocytes showed no difference. Isolation of oocyte germinal vesicles allowed a study of the entrance of newly synthesized protein into the cell nuclei.     

Inhibition of Tyrosinase Activity and Protein Synthesis in Melanoma Cells by Calcium lonophore A23187

Calcium ionophore A23187 lowers basal levels of tyrosinase and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. lonophore at a concentration of 10^–6 g/ml causes a 50% reduction in basal levels of tyrosinase and inhibits the MSH stimulated level of enzyme. lonophore A23187 also inhibits the PGEi mediated stimulation of tyrosinase, as well as the rise in enzyme activity observed in cells exposed to either theophylline (1 mM) or dbcAMP (10^–4M). lonophore does not affect basal levels of cyclic AMP nor the elevated levels produced by either MSH or PGEi, suggesting then, that the antagonistic activity of A23187 is localized to a point in the pathway of tyrosinase activation distal to the formation of cAMP. lonophore causes a rapid and marked (> 50%) inhibition of cellular protein synthesis and it is possible that this calcium mobilizing compound may exert its inhibitory effects on tyrosinase activity by causing a general reduction in cellular translation. Since the inhibition of protein synthesis occurs in cells exposed to ionophore in either the presence or absence of calcium in the medium, it seems, likely that the ionophore may exert its effects by causing the release of calcium from intracellular sites.     

Somatic cell-oocyte interactions in mouse oogenesis: stage-specific regulation of mouse oocyte protein phosphorylation by granulosa cells

The relative rate of synthesis of a number of proteins and the protein phosphorylation pattern of growing and fully grown oocytes were influenced by the presence of granulosa cells. In particular, a 74-kDa phosphorylated protein was detected only in granulosa cell-enclosed growing mouse oocytes. When reaggregated with granulosa cells, the growing oocyte displayed the phosphorylated form of the 74-kDa protein but when oocytes were cultured on Sertoli cell monolayers or in granulosa cell-conditioned medium the 74-kDa protein was not phosphorylated. We propose that (1) granulosa cells regulate protein phosphorylation in mouse oocytes; (2) a 74-kDa protein is phosphorylated only in growing oocytes when surrounded by granulosa cells; and (3) granulosa cells, but not Sertoli cells, are competent to send the appropriate "signal" to the growing oocyte.     

Calcium-mediated transduction of the hormonal message in meiosis reinitiation of starfish oocytes : Modulation following injection of cholera to toxin and cAMP-dependent protein kinase

Injections of the regulatory subunit of type I cAMP-dependent protein kinase, of the heat-stable inhibitor protein of cAMP-dependent protein kinase and of calmodulin have no effect on meiosis reinitiation. Drugs, including theophylline, caffeine and procaine, which have been shown previously to inhibit 1-methyladenine (1-MeAde)-induced Ca²⁺ release, both in living starfish oocytes and from plasma membrane-rich fractions obtained from isolated cortices, inhibit meiosis reinitiation when added before—but not after—the end of the hormone-dependent period (period when presence of the hormone in the medium is required for meiosis to occur). In the same conditions, theophylline suppresses 1-MeAde-induced stimulation of protein phosphorylation. Injection of cholera toxin subunit A increases oocyte sensitivity to 1-MeAde. Catalytic subunit of cAMP-dependent protein kinase (C) inhibits meiosis reinitiation when injected before the end of the hormone-dependent period. Oocytes can be released from inhibition due to C injection by raising 1-Me-Ade concentration. These findings support the view that Ca²⁺ release occurs until transduction of the hormonal message (i.e., its intramembrane transfer) has been completed and show that protein phosphorylation remains under plasma membrane control until that time. They also suggest that transduction of the hormonal message might be modulated by intracellular cAMP and membrane phosphorylation, although cAMP content does not change following 1-MeAde addition.     

Inhibition of tyrosinase activity and protein synthesis in melanoma cells by calcium ionophore A23187

Calcium ionophore A23187 lowers basal levels of tyrosinase and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. Ionophore at a concentration of 10(-6) g/ml causes a 50% reduction in basal levels of tyrosinase and inhibits the MSH stimulated level of enzyme. Ionophore A23187 also inhibits the PGE1 mediated stimulation of tyrosinase, as well as the rise in enzyme activity observed in cells exposed to either theophylline (1 mM) or dbcAMP (10(-4)M). Ionophore does not affect basal levels of cyclic AMP nor the elevated levels produced by either MSH or PGE1, suggesting then, that the antagonistic activity of A23187 is localized to a point in the pathway of tyrosinase activation distal to the formation of cAMP. Ionophore causes a rapid and marked (greater than 50%) inhibition of cellular protein synthesis and it is possible that this calcium mobilizing compound may exert its inhibitory effects on tyrosinase activity by causing a general reduction in cellular translation. Since the inhibition of protein synthesis occurs in cells exposed to ionophore in either the presence or absence of calcium in the medium, it seems, likely that the ionophore may exert its effects by causing the release of calcium from intracellular sites.     

A sucrose binding protein homologue from soybean affects sucrose uptake in suspension-cultured transgenic tobacco cells

We have obtained Nicotiana tabacum transgenic cell lines expressing a sucrose binding protein (sbp) homologue gene from soybean (Glycine max L.), designated s-64, either in the sense or antisense orientation. Sense cell lines over-accumulated the S-64 protein, whereas the antisense cell lines had reduced levels of the endogenous homologue protein. Sucrose uptake experiments were conducted by incubating suspension-cultured tobacco cells with radiolabeled sucrose at pH 4.5 or 7.0. Raising the extracellular pH to 7.0 caused an inhibition of radiolabeled carbon uptake efficiency, which was attributed to the pH-sensitivity of cell-wall invertase (EC 3.2.1.26), H⁺/hexose transporter and/or H⁺/sucrose symporter activities. Because SBP-mediated sucrose uptake has been shown to be insensitive to extracellular pH in yeast, we performed the sucrose uptake experiments in sense and antisense cultured cells at pH 7.0. Under this condition, the level of SBP homologue correlated with the efficiency of radiolabeled uptake by the transgenic tobacco cells. Furthermore, manipulation of S-64 levels altered sucrose-cleaving activities in a metabolic compensatory manner. Enhanced accumulation of S-64 caused an increase in intracellular sucrose synthase (cleavage, EC 2.4.1.13) activity with a concomitant decline in cell-wall invertase activity. This result may reflect a metabolic adjustment of the sense cell lines caused by its high efficiency of direct sucrose uptake as disaccharide. In contrast, the level of cell-wall invertase activity was remarkably increased in antisense cells, favoring the invertase-dependent sugar uptake system. Collectively, these results may establish a functional link between radiolabeled influx and S-64 accumulation, suggesting that SBP affects sucrose uptake in suspension-cultured cells.     

Comparative studies on sporulation-promotive actions of cyclic AMP,theophylline and caffeine in Saccharomyces cerevisiae

Cyclic AMP, theophylline and caffeine promoted sporulation when added to a presporulation medium containing glucose. Caffeine promoted sporulation even when added to a presporulation medium containing acetate as the carbon source, but cyclic AMP and theophylline did not. Caffeine did not increase the intracellular cyclic AMP level, while theophylling did significantly when added to a presporulation medium containing glucose Caffeine inhibited the vegetative DNA synthesis with little effect on RNA and protein synthesis, resulting in the increase in cell volume, dry weight, and RNA and protein contents, but cyclic AMP and theophylline did not show such effects.     

Distinct mechanisms for the activation of the RSK kinases/MAP2 kinase/pp90rsk and pp70-S6 kinase signaling systems are indicated by inhibition of protein synthesis.

Previous studies demonstrated that addition of protein synthesis inhibitors to quiescent cells resulted in the stimulation of S6 kinase activity. The present characterization of several growth factor- and oncogene-regulated protein-serine/threonine kinases demonstrated that pp70-S6 protein kinase and not pp90rsk, RSK kinase, or MAP2 kinase activities were rapidly stimulated. Dose-response experiments revealed a close correlation between the extent of protein synthesis inhibition and the level of activation of pp70-S6 kinase activity. Analysis of S6 phosphorylation suggests that activation of pp90rsk S6 phosphotransferase activity, whose Xenopus homologues appear to be responsible for S6 phosphorylation during oocyte maturation, may participate in, but is not essential for, the increase in S6 phosphorylation observed in growth-stimulated somatic animal cells. These studies provide additional evidence for the existence of two distinct, independently regulated protein phosphorylation cascades activated in the early G1 phase of the cell cycle.     

Program of early development in the mammal: changes in the patterns and absolute rates of tubulin and total protein synthesis during oocyte growth in the mouse

Oocytes at several stages of growth have been isolated by enzymatic digestion and/or physical disruption of ovaries excised from juvenile and adult mice. The absolute rates of total protein synthesis and tubulin synthesis in these isolated oocytes were determined by measuring sizes of the endogenous methionine pool and apparent rates of incorporation of [³⁵S]methionine into total protein and tubulin using methods described previously (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978,Proc. Nat. Acad. Sci. USA,75, 4160;R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979,Develop. Biol.,68, 341). The size of the endogenous methionine pool increases approximately 350-fold during oocyte growth, from 0.16 fmole in nongrowing oocytes (12 μm) to 56 fmole in fully grown oocytes (85 μm). Since the volume of mouse oocytes also increases about 350-fold during growth, the concentration of intracellular free methionine remains constant at approximately 170 μM. The absolute rate of protein synthesis increases from 1.1 to 41.8 pg/hr/oocyte for nongrowing and fully grown mouse oocytes, respectively. Since this represents about a 38-fold increase in the absolute rate of protein synthesis, the rate of synthesis per picoliter of cytoplasm actually decreases nearly 10-fold during oocyte growth. These measurements indicate that the growing mouse oocyte itself is capable of synthesizing only about 50% of the protein found in fully grown oocytes. Tubulin is one of the major proteins synthesized by growing mouse oocytes since the absolute rate of tubulin synthesis is, on the average, 1.8% of total protein synthesis. The absolute rate of tubulin synthesis increases from 0.4 to 0.6 pg/hr/oocyte as the oocyte grows from 40 to 85 μm in diameter. However, overall, the percentage of total protein synthesis devoted to the synthesis of tubulin actually declines somewhat during this phase of growth, from 2 to 1.5%. Although equimolar amounts of tubulin subunits are present in microtubules, the ratio of absolute rate of synthesis of the β subunit to that of the α subunit varies from 1.3 to 2.0 throughout oocyte growth. High-resolution two-dimensional gel electrophoretic analyses of [³⁵S]methionine-labeled proteins reveal that many changes take place in the pattern of protein synthesis during oocyte growth.     

Studies of the kinetics and ionic requirements for the phosphorylation of ribosomal protein S6 after fertilization of Arbacia punctulata eggs

Fertilization of the eggs of the sea urchin Arbacia punctulata is followed by the phosphorylation of ribosomal protein S6. The increase in phosphorylation starts at the same time that protein synthesis begins to increase, and leads to the appearance of mono-, di-, and triphosphorylated S6 derivatives. Essentially all the S6 is phosphorylated by first cleavage. This phosphorylation requires the occurrence of both the normal Ca²⁺ transient and the consequent Na⁺H⁺ exchange. Protein synthesis can be partially activated by an increase in intracellular pH brought about by weak bases, but this neither causes S6 phosphorylation, nor the inactivation of the specific S6 phosphatase present in unfertilized Arbacia eggs.     

Activation of the progesterone-signaling pathway by methyl-beta-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa Galphas

Treatment of Xenopus laevis oocytes with cholesterol-depleting methyl-β-cyclodextrin (MeβCD) stimulates phosphorylation of mitogen-activated protein kinase (MAPK) and oocyte maturation, as reported previously [Sadler, S.E., Jacobs, N.D., 2004. Stimulation of Xenopus laevis oocyte maturation by methyl-β-cyclodextrin. Biol. Reprod. 70, 1685-1692.]. Here we report that treatment of oocytes with MeβCD increased levels of immunodetectable 39-kDa mos protein. The protein synthesis inhibitor, cycloheximide, blocked the appearance of Mos, blocked MeβCD-stimulated phosphorylation of MAPK, and inhibited MeβCD-induced oocyte maturation. These observations suggest that MeβCD activates the progesterone-signaling pathway. Chemical inhibition of steroid synthesis and mechanical removal of follicle cells were used to verify that MeβCD acts at the level of the oocyte and does not require production of steroid by surrounding follicle cells. Cortical Gα_s is contained in low-density membrane; and treatment of oocytes with progesterone or MeβCD reduced immunodetectable levels of Gα_s protein in cortices and increased internal levels of 45-kDa Gα_s in cortical-free extracts. Dose-dependent increases in internal Gα_s after treatment of oocytes with progesterone correlated with the steroid-induced maturation response, and the increase in internal Gα_s after hormone treatment was comparable to the decrease in cortical Gα_s. These results are consistent with a model in which release of Gα_s from the plasma membrane is involved in activation of the progesterone-signaling pathway that leads to amphibian oocyte maturation.     

Signaling pathways in ascidian oocyte maturation: the role of cyclic AMP and follicle cells in germinal vesicle breakdown

Many ascidian oocytes undergo 'spontaneous' germinal vesicle breakdown (GVBD) when transferred from the ovary to normal pH 8.2 sea water (SW); however, low pH inhibits GVBD, which can then be stimulated while remaining in the low pH SW. Oocytes of Boltenia villosa blocked from GVBD by pH 4 SW undergo GVBD in response to permeant cyclic AMP (8-bromo-cyclic AMP), phosphodiesterase inhibitors (isobutylmethylxanthine and theophylline) or the adenylyl cyclase activator forskolin. This suggests that cAMP increases during GVBD. Removal of the follicle cells or addition of a protease inhibitor inhibits GVBD in response to raised pH but not to forskolin, theophylline or 8 bromo-cAMP. Isolated follicle cells in low pH SW release protease activity in response to an increase in pH. These studies imply that the follicle cells release protease activity, which either itself stimulates an increase in oocyte cAMP level or reacts with other molecules to stimulate this process. Studies with the mitogen-activated protein (MAP) kinase inhibitors U0126 and CI 1040 suggest that MAP kinase is not involved in GVBD. The Cdc25 inhibitor NSC 95397 inhibits GVBD at 200 n m in a reversible manner.     

The maturation response of stage IV, V, and VI Xenopus oocytes to progesterone stimulation in vitro

Full-grown Xenopus oocytes, Stage VI (1200-1300 microns), undergo meiotic maturation when exposed to progesterone. Smaller stage IV (800 microns) and stage V (1000 microns) oocytes remain in prophase arrest when exposed to this steroid. The larger stage VI oocytes undergo an intracellular alkalization from 7.2 to 7.6, a six- to eightfold increase in the phosphorylation of the 40 S ribosomal protein S-6, and a two- to threefold increase in total protein synthesis when exposed to progesterone. It was found that 800- to 1000-microns oocytes do not undergo these physiological changes when exposed to progesterone. This lack of response could explain the failure of small oocytes to undergo germinal vesicle breakdown (GVBD). However, when stage IV and V oocytes were artificially alkalized to a pHi of 7.6 by the weak bases, trimethylamine, procaine, or methylamine, S-6 phosphorylation was stimulated four- to sixfold and protein synthesis was stimulated two- to threefold, but they still did not undergo GVBD. Stage IV and V oocytes are able to amplify MPF injected into their cytoplasm and undergo GVBD. Thus, 800- to 1000-microns oocytes appear to contain a store of inactive MPF in their cytoplasm. It seems that an additional physiological parameter(s), that is unique to steroid-treated stage VI oocytes, is responsible for activating this MPF which induces GVBD.