Raf-1 kinase, a potential regulator of intracellular pH in Xenopus oocytes.

Xenopus laevis oocytes undergo an increase in intracellular pH (pHi) from 7.2 to 7.7 due to the up-regulation of Na+/H+ antiporters in their plasma membrane during oocyte meiotic maturation. Up-regulation of Na+/H+ exchangers (NHE) found in other cell systems appears to be controlled, in some cases, by direct phosphorylation of the exchanger. A number of active protein kinases can be found in maturing Xenopus oocytes. These include, c-mos kinase, Raf-1 kinase, mitogen-activated kinase kinase (MAPKK), MAPK, ribosomal S6 kinase (RSK), and histone H-1 kinase. Our previous study indicated that c-mos kinase, was involved in regulating the increase in oocyte pHi. In the current study, we show that when mRNA coding for a constitutively active form of Raf-1 kinase (delta N-Raf-1) was microinjected into oocytes, the protein product induced an increase in oocyte pHi. On the contrary, the injection of mRNA coding for wild-type Raf-1 (WT-Raf-1) or a kinase-deficient form of Raf-1 (KD-Raf-1) had no effect on the recipient oocyte pHi. 8-Br-cAMP and forskolin blocked the increase in pHi during oocyte meiotic maturation, but had no effect on the Raf-1-induced increase in oocyte pHi. Studies using antisense c-mos oligos demonstrated that Raf-1 was not working via a feedback loop to endogenous c-mos mRNA within the recipient oocytes. Experiments using the selective MAPKK inhibitor, PD 98059, indicated that the Raf-1 effect on oocyte pHi was not mediated by the downstream kinase, MAPKK. Therefore, Raf-1 appears to act independently of c-mos kinase in a pathway, not involving MAPKK, leading to the up-regulation of the Na+/H+ antiporters in Xenopus oocytes.

Analysis of R59022 actions in Xenopus laevis oocytes.

R59022, a diacylglycerol (DAG) kinase inhibitor, stimulated meiotic maturation of Xenopus laevis oocytes when applied extracellularly. The time course of R59022-induced oocyte maturation was proportional to the concentration of R59022 in the low micromolor range, and the 30 microM-induced response was a fast or faster than progesterone-induced maturation. Dose-response analysis yielded an apparent EC50 for R59022-induced oocyte maturation of approximately 15 microM. An increase in total oocyte DAG levels was observed following treatment with 10 microM R59022. Treatment of oocytes with R59022 also resulted in a significant increase in intracellular pH similar to the increase observed with progesterone. When various phosphodiesterase (PDE) inhibitors were tested for their effects on R59022-induced oocyte maturation, papaverine (a potent nonselective inhibitor of PDE) and CI-930 (a selective PDE III inhibitor) were observed to significantly inhibit the R59022-stimulated response. The sensitivity of R59022-induced oocyte maturation to inhibition by papaverine was intermediate between the sensitivities of the IGF-1- or progesterone-induced responses. Treatment of oocytes with R59022 did not significantly affect the level of oocyte PDE activity measured in vivo, suggesting that elevated levels of DAG may parallel observed increases in PDE but do not directly lead to a stimulation of PDE. The t ime course for stimulation of ribosomal S6 kinase activity by R59022 followed the pattern for stimulation of ribosomal S6 kinase activity by R59022 followed the pattern for stimulation by progesterone rather than IGF-1. Treatment of isolated membranes with R59022 resulted in inhibition of membrane-associated adenyl cyclas e activity that was not mimicked by DAG analogs. Thus, in addition to elevating oocyte levels of DAG, R59022 also has steroid-like actions.

Protooncogene product, c-mos kinase, is involved in upregulating Na+/H+ antiporter in Xenopus oocytes.

Progesterone-stimulated Xenopus laevis oocytes undergo an increase in their intracellular pH from 7.3 to 7.7 because of the activation of Na+/H+ antiporters in their plasma membrane. Activation of Na+/H+ exchangers (NHE) in other cell systems appears to be regulated by phosphorylation of the NHE protein. In the current study we demonstrated that cytoplasm taken from steroid-stimulated oocytes rapidly induced an increase in intracellular pH when microinjected into full-grown stage VI recipient oocytes. The protein within the cytoplasm that appears to be responsible for this activity is c-mos kinase. Microinjected pure mosxe kinase protein rapidly activated the Na+/H+ exchangers in full-grown recipient oocytes. Furthermore, injected mosxe protein rapidly activated the Na+/H+ exchangers in smaller progesterone-insensitive stage IV oocytes. Therefore, it appears that the protooncogene product, p39 c-mos kinase, which is normally synthesized in full-grown stage VI oocytes in response to progesterone stimulation, is involved in the upregulation of the Na+/H+ antiporters during oocyte meiotic maturation.

Forskolin and methylxanthines block the increase in intracellular pH during meiosis in Xenopus laevis oocytes.

Xenopus oocytes, arrested in late prophase of the meiotic cell cycle, undergo nuclear membrane breakdown (Germinal Vesicle Breakdown, GVBD) in response to progesterone stimulation. During this prophase/M-phase transition the oocytes undergo an increase in their intracellular pH (pHi) from 7.3 to 7.7 in response to the steroid. This increase in pHi appears to be due to the activation of Na+/H+ antiporters in the oocyte plasma membrane. Several studies have shown that the pathway leading to GVBD is blocked by forskolin or methylxanthines which elevate cAMP levels within the oocyte. We have found that these same compounds also blocked the up-regulation of the Na+/H+ exchangers during oocyte meiotic maturation.

Oocyte storage affects the kinetics of insulin-induced GVBD in Xenopus laevis oocytes.

Xenopus laevis oocytes underwent meiotic maturation in response to progesterone or insulin stimulation. However, the kinetics of the oocytes' response to the steroid was much faster than the response induced by insulin even under saturating hormone concentrations. This time differential was seen in oocytes that were stimulated with either hormone shortly after they were isolated from the animal. On the contrary, if oocytes were first cultured in MBS medium for 12-14 hours and then treated with either hormone, the oocytes responded to insulin and progesterone with similar kinetics. This result was not due to the ionic strength, potassium concentration or pH of the medium. The effect of storage in MBS was ligand specific for insulin. The extra 12-14 hours in MBS medium had no effect on the oocytes' response to progesterone.

The rapid transient decrease of sn-1,2-diacylglycerol in progesterone-stimulated Xenopus laevis oocytes is the result of an ethanol artifact.

Full-grown Xenopus laevis oocytes resume meiosis (meiotic maturation) in response to progesterone stimulation. Three studies have shown that sn-1,2-diacylglycerol (DAG), the intracellular activator of protein kinase C, may be involved in this process (Wasserman et al., J. Exp. Zool. 255, 63-71, 1990; Varnold and Smith, Development 109, 597-604, 1990; Stith et al., J. Cell Physiol. 149, 252-259, 1991). Two of these studies (Varnold and Smith, 1990; Stith et al., 1991) found a rapid, but transient decrease in the levels of DAG of approximately 25 to 30% within 5 to 30 sec following the addition of progesterone to the oocytes. We have investigated this rapid decline in oocyte DAG. We also found a 20 to 34% decrease in DAG/oocyte within the first 5 to 40 sec following the addition of steroid to the culture medium. However, a similar rapid and transient decrease in oocyte DAG levels was also observed in response to ethanol. Ethanol is used as the vehicle to deliver progesterone to the oocyte culture medium. Therefore, the rapid transient decline in DAG appears to be an artifact of ethanol perturbing the production and/or turnover of DAG within the oocyte and not a physiological response of the oocyte to progesterone.

sn-1,2-diacylglycerol levels increase in progesterone-stimulated Xenopus laevis oocytes.

Full-grown Xenopus laevis oocytes resume meiosis from prophase arrest in response to progesterone stimulation. Recent studies have shown that the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), a very potent activator of protein kinase C, can also induce the resumption of meiosis in amphibian oocytes. We have investigated the possibility that sn-1,2-diacylglycerol (DAG), the intracellular activator of protein kinase C, may be involved in the pathway normally used by progesterone. We have found that full-grown oocytes arrested in meiotic prophase contain 48 +/- 5 pmoles DAG/oocyte. This level increases within the first 5 minutes following the addition of progesterone and reaches a maximum of 75 +/- 10 pmoles following 60 minutes of steroid stimulation. Progesterone induces the up-regulation of a Na+/H+ antiport in the plasma membrane of the oocyte causing an increase in pHi following 60 minutes of steroid stimulation. We have found that the addition of 200 microM DAG (1,2-dioctanoylglycerol, diC8) to the culture medium can cause a partial up-regulation of this Na+/H+ pump in the absence of hormonal stimulation. These results suggest that DAG and protein kinase C may be involved in regulating certain aspects of meiotic maturation in progesterone-stimulated Xenopus oocytes.

S6 phosphorylation is regulated at multiple levels in Xenopus laevis oocytes.

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

Effect of ouabain on the meiotic maturation of stage IV-V Xenopus laevis oocytes.

Full-grown stage VI Xenopus laevis oocytes (1,200 to 1,300 micron) respond to progesterone stimulation by undergoing a series of physiological and morphological changes that are referred to as meiotic maturation. Oocytes in earlier stages of oogenesis (I through V) do not undergo these changes and remain in prophase arrest when exposed to this steroid. We have found that oocytes ranging from 850 micron (stage IV) to 1,000 micron (stage V) are capable of responding to progesterone under the appropriate conditions. Oocytes greater than or equal to 850 micron in diameter underwent germinal vesicle breakdown (GVBD) after 10-12 hr of exposure to progesterone when ouabain was added to the medium at a concentration greater than 2.5 X 10(-6) M. Under this culture condition, progesterone was now able to induce a 0.3- to 0.4-unit increase in the intracellular pH of stage IV-V oocytes, a 4- to 5-fold increase in 40s ribosomal protein S-6 phosphorylation, and a 2.3-fold increase in their rate of protein synthesis. All of these physiological changes are characteristic of full-grown stage VI oocytes undergoing meiotic maturation. In addition, we have found that oocytes greater than or equal to 750 micron are capable of amplifying maturation promoting factor (MPF) in their cytoplasm leading to GVBD. Therefore, stage IV-V Xenopus oocytes have the potential for undergoing meiotic maturation, but they are blocked at a point in prophase that appears to be alleviated by the combination of progesterone and ouabain.

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.

The regulation of ribosomal protein S-6 phosphorylation in Xenopus oocytes: a potential role for intracellular pH.

The intracellular pH of full-grown Xenopus oocytes increases from 7.2 to 7.6 in response to progesterone stimulation. Phosphorylation of the 40 S ribosomal protein S-6 increases six- to eightfold in these stimulated cells and this phosphorylation coincides with the intracellular alkalization. This is followed by a two- to threefold increase in the protein synthetic rate. Progesterone-treated cells cultured in choline chloride substituted Na-free medium fail to alkalize and S-6 is not phosphorylated. Weak bases, such as trimethylamine, methylamine, and procaine, artificially alkalize the cell cytoplasm and stimulate S-6 phosphorylation in medium containing or lacking sodium. The methylxanthine theophylline, suppresses S-6 phosphorylation and inhibits protein synthesis. This inhibition does not appear to involve cAMP. Rather, theophylline acidifies the oocyte cytoplasm. Thus, S-6 phosphorylation appears to be regulated by the intracellular pH of the cell. In addition, the level of protein synthesis in the oocyte seems to be correlated with the level of S-6 phosphorylation.

Intracellular pH plays a role in regulating protein synthesis in Xenopus oocytes.

Full-grown Xenopus oocytes undergo meiotic maturation in response to progesterone stimulation. Using [14C]dimethyloxazolidine dione (DMO), we have measured a cytoplasmic alkalization in these oocytes starting at pH 7.14 +/- 0.17 during the germinal vesicle (GV) stage, and increasing to 7.56 +/- 0.14 at the time of germinal vesicle breakdown (GVBD). During this period, the rate of protein synthesis increases 2-fold from 18.9 +/- 3.1 to 37.7 +/- 8.8 ng/hr/oocyte. Artificial alkalization of GV stage oocytes to pHi 7.68 +/- 0.16, by exposure to the weak bases trimethylamine, methylamine, procaine, or imidazole, led to a 1.8-fold increase in the synthetic rate. Intracellular acidification from 7.5 back to 7.0 had no apparent effect on the elevated rate of protein synthesis following GVBD. Therefore, a cytoplasmic alkalization in the range of 7.5 to 7.6 seems to be one of the events that is necessary for initiating the increase in protein synthesis in maturing Xenopus oocytes; however, it does not appear that an elevated pHi is necessary to maintain the increased synthetic rate following GVBD.

Calmodulin triggers the resumption of meiosis in amphibian oocytes.

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

Progesterone induces a rapid increase in [Ca2+]in of Xenopus laevis oocytes.

Progesterone causes a rapid increase in the intracellular free calcium level in fully grown amphibian oocytes. When albino Xenopus laevis oocytes were microinjected with the Ca-specific photoprotein aequorin, the calcium-induced luminescence from this protein increased at 40-60 sec after the addition of progesterone and returned to the control level within 5-6 min. No further change in the aequorin glow could be detected through the remainder of the maturation period. This transient increase in cytoplasmic free calcium may be involved in controlling the resumption of oocyte maturation.

The cyclic behavior of a cytoplasmic factor controlling nuclear membrane breakdown.

The activity of a cytoplasmic factor (MPF), capable of inducing nuclear membrane breakdown (germinal vesicle breakdown) when injected into amphibian oocytes, has been studied during the course of early cleavage in amphibian embryos. Mature egg cytoplasm was found to contain high levels of this activity, but this was quickly lost after fertilization or artificial activation. MPF activity later reappeared in the egg cytoplasm and started to cycle with time. The peak of embryonic MPF activity during each cycle coincided with the time the embryonic nuclei were entering the G2-M transition, i.e., mitosis. However, in colchicine-arrested embryos, this activity remained at an elevated level and no longer oscillated. The timing of the appearance and disappearance of this activity appeared to be under the control of the cytoplasm because such behavior was still observed in enucleated eggs. Continued protein synthesis in the embryo was required for the reappearance, but not for the disappearance, of this activity. MPF, previously thought to be restricted to oocyte maturation, may play a more general role in controlling nuclear membrane breakdown during mitosis as well as meiosis.

A cytoplasmic factor promoting oocyte maturation: its extraction and preliminary characterization.

Cytoplasm of maturing amphibian oocytes possesses a factor that induces germinal vesicle breakdown. This factor was extracted from Rana pipiens eggs and assayed by microinjection in Xenopus laevis oocytes. The activity of this factor is Mg-dependent, Ca-sensitive, and associated with heat-labile protein. Centrifugation on a sucrose density gradient revealed that the factor exists in three different molecular sizes.