Maturation Of Xenopus Laevis Oocytes Research Articles

Progesterone-inhibited phosphorylation of an unique Mr 48,000 protein in the plasma membrane of Xenopus laevis oocytes.

The meiotic maturation of Xenopus laevis oocytes is induced in vitro by progesterone which interacts at the cell surface level. A cell-free membrane preparation (P-10,000) incorporated 32P from [gamma-32P]ATP, mostly into two proteins, Mr approximately 56,000 and approximately 48,000 (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Progesterone, added in vitro, specifically inhibited the phosphorylation of the Mr approximately 48,000 protein (named p48). Half-maximal inhibition of p48 phosphorylation occurred with progesterone approximately 8 microM, in good correlation with hormone concentration inducing oocyte maturation. The effect was not due to stimulation of protein phosphatase activity. The potent maturation inducers testosterone and deoxycorticosterone also inhibited p48 phosphorylation, whereas biologically inactive steroids or cholesterol did not. p48 phosphorylation was not affected by cAMP, cGMP, polyamines, calmodulin, and phospholipids + diolein. EGTA had a stimulatory effect which was reversed by added Ca2+. The inhibitory effects of progesterone and Ca2+ were additive, suggesting two distinct sites of action. Phospho-p48 was not detected in yolk platelets, microsomes, and cytosol of oocytes. Contrary to p48 itself, the p48 kinase activity was loosely associated with P-10,000. Progesterone inhibited p48 phosphorylation produced by either cytosol or exogenous pure catalytic subunit of cAMP-dependent protein kinase. Conversely, phosphorylation of casein and histones by protein kinase activity present in P-10,000 was not modified by progesterone. It is then suggested that progesterone regulates p48 phosphorylation by affecting the protein substrate in the membrane, rather than by inhibiting the protein kinase enzyme itself. The data demonstrate a direct effect (not mediated by change of protein synthesis) of steroids on p48 phosphorylation in the plasma membrane, and they suggest that this protein could be implicated in the initial action of progesterone on oocyte maturation.

Cholinergic modulation of progesterone‐induced maturation of Xenopus oocytes in vitro

AbstractMixed and muscarinic cholinergic agonists (acetylcholine, carbamylcholine, methacholine, oxotremorine, and pilocarpine) accelerated in a dose‐dependent manner the progesterone‐induced maturation of Xenopus laevis oocytes. None of these agonists induced oocyte maturation in the absence of progesterone. The accelerating effect of cholinergic agonists was blocked in a dose‐dependent manner by specific muscarinic antagonists (atropine and scopolamine) but not by specific nicotinic antagonists (d‐tubocurarine and hexamethonium). The specific nicotinic agonist, dimethylphenylpiperazine, alone induced maturation in the absence of progesterone. The optimal promoting effect of acetylcholine was observed when oocytes were exposed to acetylcholine for 30 min, 5 min after the addition of progesterone, and was markedly better than when oocytes were exposed to acetylcholine throughout their incubation with progesterone. The effect of acetylcholine was observed in both follicle‐enclosed and in defolliculated oocytes, indicating that follicular cells were not the target of the cholinergic drugs.

The thiol-disulfide balance during maturation of Xenopus laevis oocytes.

We measured several of the components involved in the thiol-disulfide balance of immature and mature oocytes of Xenopus laevis: total soluble native sulfhydryl, reduced and oxidized glutathione, native sulfhydryl content of soluble protein, and disulfide content of soluble protein. None of these values changed during maturation. Both the immature and mature oocyte were characterized by a relatively high disulfide to sulfhydryl ratio. We also measured native sulfhydryl content of germinal vesicle proteins and enucleated oocyte proteins in immature oocytes. Contrary to early cytochemical findings, our measurements suggest that the germinal vesicle has a lower concentration of protein sulfhydryl than does the cytoplasm.

Studies on the mechanism of inhibition of amphibian oocyte adenylate cyclase by progesterone

Progesterone treatment induces the meiotic maturation of Xenopus laevis oocytes. Previous evidence indicates that this hormonal effect may be due to inhibition of oocyte adenylate cyclase. The present work studies several aspects of the mechanism of adenylate cyclase inhibition by this hormone. Forskolin greatly stimulates oocyte adenylate cyclase in the absence of guanine nucleotides and this activity is not sensitive to progesterone inhibition. In addition the forskolin-activated enzyme is not inhibited by a wide range of guanine nucleotide, in the presence or absence of hormone. The time course of cAMP synthesis catalyzed by oocyte adenylate cyclase in the presence of guanyl-5′ l -imidodiphosphate (Gpp(NH)p) shows an initial lag period that does not depend on the concentration of Gpp(NH)p. Progesterone causes a very significant increase in the hysteresis of the reaction, at least doubling the half-time of enzyme activation. The hormonal effect on the lag cannot be reversed by saturating concentrations of Gpp(NH)p. Progesterone also decreases the steady-state rates of the reaction. This effect, however, depends on the concentration of Gpp(NH)p. High concentrations of Gpp(NH)p almost completely reverse the inhibition of the steady-state rates. Progesterone does not inhibit if it is added to the reaction after the initial lag period. Guanosine-5′- O-(2-thiodiphosphate) (GDP-β-S) is an efficient competitive inhibitor of Gpp(NH)p activation of adenylate cyclase. Progesterone inhibition is observed at all concentrations of GDP-β-S and is potentiated at high ratios of GDP-β-S to Gpp(NH)p. These data indicate that progesterone inhibits by interfering with the activation of the N s subunit of the enzyme by guanine nucleotides, rather than through a mechanism involving a separate N i subunit.

The modifications of cortical endoplasmic reticulum during in vitro maturation of Xenopus laevis oocytes and its involvement in cortical granule exocytosis.

In Xenopus laevis eggs, cisternae shells which surround cortical granules (CG) are part of a cortical endoplasmic reticulum (ER) network. In this paper the origin of such ER shells has been studied in full-grown, progesterone-exposed Xenopus oocytes. Furthermore, the possible role of the cortical ER in the activation process has been investigated by pricking maturing oocytes. It has been shown that in full-grown ovarian oocytes ER CG shells are absent and ER cisternae are extensively and randomly distributed throughout the peripheral cytoplasm, where they appear to be continuous with annulate lamellae (AL). Following hormone treatment, the AL completely disaggregate and the ER cisternae gradually migrate to the cortex where they surround the CG constituting the typical cortical network described in uterine eggs. Furthermore, it has been found that 8 h after progesterone treatment (before the first polar body extrusion) the response to pricking (CG exocytosis) occurs only at the animal half; there is no observable response in the vegetal half. At this time ER shells surround CG only in the animal hemisphere. A complete CG exocytosis occurs following the first polar body emission, when the cortical ER is well organized in the whole oocyte cortex. The correlation between the differentiation of the cortical ER and the arousal in the oocyte of the ability to respond to a pricking stimulus is discussed in the light of an involvement of the cortical ER in the propagation of CG exocytosis.

Changes of alanine-sodium co-transport during maturation of xenopus laevis oocytes

Maturation of full grown Xenopus oocytes is associated with a decrease of their capacity to transport amino acids and nucleosides. Nevertheless, the transport continues at a reduced rate until the time of metaphase arrest when the oocytes are ready for shedding. We have found that the L-alanine transport in unfertilized, shed oocytes is a saturable process which is Na +-dependent. The stoichiometrical ratio between sodium and alanine is 2:1. Comparison with the alanine uptake in fulll grown oocytes shows that during maturation V max is strongly reduced while K m and the stoichiometrical ratio of sodium and alanine is unaffected. Lowering the osmolarity of the shedding medium reduces the rate of uptake of alanine below the rates seen after shedding into isotonic media. The transport rates amongst the individual oocytes differ. They do not fit to a Gaussian distribution. In addition, in individual oocytes, there exists no correlation between the variations of the transport rates for amino acids and nucleosides, which are also taken up by the unfertilized, shed oocytes.

Early increase of a 105,000-dalton phosphoprotein during meiotic maturation of Xenopus laevis oocyte

In ovo [32P] phosphoproteins were analyzed during meiotic maturation of Xenopus laevis oocytes. A phosphoprotein of 105,000-dalton was found to increase early (one hour) after progesterone induction of meiosis. The pure heat-stable inhibitor (PKI) of cAMP-dependent protein kinase, which induces maturation, was microinjected into oocytes. Again the early increase in the 105,000-dalton [32P] phosphoprotein occurred. The burst in protein phosphorylation, which takes place at the period of germinal vesicle breakdown, was quantitatively and qualitatively comparable in progesterone and PKI-stimulated oocytes. In order to confirm the inverse relationship between the 105,000 dalton [32P] phosphoprotein increase and cAMP-dependent protein kinase activity, purified C-subunit of the kinase has been microinjected into oocytes. C-subunit which inhibits maturation did not increase significantly the 105,000-dalton [32P] phosphoprotein whereas it increased the total level of in ovo phosphorylation. Enucleation experiments favour the localization of the 105,000-dalton protein in both the oocyte cytoplasm and nucleus. Furthermore the progesterone-induced increase in the phosphorylation of the 105,000-dalton protein was found in the cytoplasmic compartment after oocyte enucleation.

Microinjected progesterone reinitiates meiotic maturation of Xenopus laevis oocytes.

Microinjection of progesterone dissolved in paraffin oil induces the reinitiation of meiotic maturation in the Xenopus oocyte; 50% maturation is obtained when 50 nl of a 50 microM solution is microinjected into the oocyte. The kinetics of the response to microinjected progesterone are similar to the kinetics of response to externally applied hormone. When an aqueous solution of progesterone is microinjected instead of an oil solution, maturation is never observed, a result which confirms previous work. Leakage of the steroid into the external medium was estimated to range from 1.6 pmol/hr when microinjection was performed in oil to 3.6 pmol/hr when it was performed in aqueous solution. Metabolism of the hormone microinjected in oil is weak (less than 20%) as compared to that after aqueous microinjection (greater than 80%). Progesterone microinjected in oil decreases the cAMP content as does externally applied hormone. We therefore conclude that progesterone acts initially on an intracellular site in order to trigger meiotic maturation of the Xenopus oocyte.

Effects of cholera toxin and actinomycin on synthesis of [ 35S]methionine-labeled proteins during progesterone-induced maturation of Xenopus laevis oocytes

The incorporation of [ 35S]methionine into polypeptides during progesterone-induced meiotic maturation of Xenopus laevis oocytes was studied by two-dimensional polyacrylamide gel electrophoresis. Five modifications were consistently observed: two polypeptides of an approximate molecular weight of 150K daltons and p I 5 were new proteins, two represent increased incorporation and one was decreased incorporation. Cholera toxin inhibited the appearance of the modifications induced by progesterone. Actinomycin and enucleation did not significantly alter the modifications. These data indicate that a good correlation exists between the modifications in protein synthesis induced by progesterone and the resumption of meiotic cell division.

Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes.

A single ribosomal protein (Mr, 32,000) becomes phosphorylated during progesterone-induced in vitro maturation of Xenopus laevis oocytes. The protein is identified as 40S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two-dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in unstimulated oocytes. After progesterone treatment, phosphorylation of S6 precedes germinal vesicle breakdown (GVBD) and is maximal at the time when 50% of the oocytes have undergone GVBD. S6, when maximally phosphorylated, exists in derivatives that correspond to the most highly phosphorylated forms observed in other systems, and the increase in S6 phosphorylation occurs at approximately the same time as the increase in the overall protein synthesis rate reported to occur during oocyte maturation. S6 is also maximally phosphorylated in unfertilized eggs following maturation in vivo. Injection of a partially purified preparation of maturation-promoting factor into immature oocytes induces immediate phosphorylation of S6 and rapidly increases the rate of protein synthesis. Moreover, incubation of ribosomes with this factor and radiolabeled ATP results in labeling of S6. These findings suggest that S6 phosphorylation may be important in the control of protein synthesis during maturation and may also play a role in the mechanism of action of maturation-promoting factor.

The pure inhibitor of cAMP-dependent protein kinase initiates Xenopus laevis meiotic maturation : A 4-step scheme for meiotic maturation

The availability of the pure inhibitor of cAMP-dependent protein kinase prompted a re-examination of the inhibitor-induced meiotic maturation of Xenopus laevis oocytes. Injection of the inhibitor (1.5 μM) triggered 100% germinal vesicle breakdown faster than progesterone and slower than the maturation-promoting factor: at 0.15 μM, the inhibitor still triggered 100% meiosis, but with a much slower kinetics. In contrast, injection of 24 μM calmodulin resulted in less than 50% GVBD, and results were variable from female to female. Combined injection of inhibitor and calmodulin failed to show any synergism, which does not favour hypotheses according to which calmodulin acts by activation of cyclic nucleotide phosphodiesterase. The net effect of the inhibitor is to decrease the concentration of the free catalytic sub-unit of cAMP-dependent protein kinase, fully dissociated in the unstimulated oocyte, as shown by the absence of effect of pretreatment with cholera toxin on the inhibitor-induced maturation. After such decrease by about 1 μM, a maturation protein, Mp-P, is dephosphorylated by phosphoprotein phosphatases. Dephospho-Mp triggers the synthesis of MPF in cycloheximide-sensitive steps. Finally, MPF triggers GVBD in steps insensitive to cycloheximide. Evidence for such a 4-step scheme—fall in cAMP levels, then in C sub-unit levels, dephosphorylation of Mp leading to the synthesis of MPF and finally MPF-triggered GVBD—is presented and discussed.

Response : The Role of Zinc and Follicle Cells in Insulin-Initiated Meiotic Maturation of Xenopus laevis Oocytes

<i>Response</i> : The Role of Zinc and Follicle Cells in Insulin-Initiated Meiotic Maturation of <i>Xenopus laevis</i> Oocytes

Increased ornithine decarboxylase activity during meiotic maturation in xenopus laevis oocytes

The activity of ornithine decarboxylase (ornithine carboxylyase E.C. 4.1.1.17) was studied during meiotic maturation induced in vitro by progesterone in follicle cell-free oocytes. Enzyme activity increased 4–6 fold during maturation, preceding germinal vesicle breakdown. The increase in ornithine decarboxylase activity was inhibited by cholera toxin, an agent that blocks meiotic maturation and increases cAMP levels within the cell. It was also prevented by cycloheximide but not by actinomycin D. Treatment of oocytes with D,L-α-difluoromethyl-ornithine, an irreversible inhibitor of ornithine decarboxylase and of putrescine synthesis, effectively abolished enzyme activity without preventing germinal vesicle breakdown. These observations show that the progesterone-induced increase in ornithine decarboxylase activity is not required for completion of meiotic division of the oocyte.

Protein factor(s) from mitotic CHO cells induce meiotic maturation in Xenopus laevis oocytes

Protein factor(s) from mitotic CHO cells induce meiotic maturation in Xenopus laevis oocytes

Metabolism of Poly (A) Sequences of Maternal mRNA during Progesterone-Induced Maturation of Xenopus laevis Oocytes

Changes in the content and the size of poly(A) sequences during progesterone-induced maturation of Xenopus laevis oocytes were determined by [3H]poly(U) hybridization and by labeling with exogenously supplied radioactive adenosine. The poly(A) content increased significantly (by about 20 %) by the time of germinal vesicle breakdown (GVBD), then decreased continuously to one half of the initial content by the end of maturation. Gel electrophoresis revealed that poly(A) sequences were being elongated by 20-30 nucleotides before GVBD except for the smallest class of poly-(A) (ca. 20 nucleotides long), but after GVBD they were extensively degraded which gave rise to the egg poly(A) sequences of quite different sizes. All these changes in poly(A) content and size also occurred in oocytes which either were treated with actinomycin D or enucleated before exposure to progesterone; this signifies that they were not dependent on the nucleus. Labeling experi-ments on maturing oocytes showed that poly(A) sequences were synthesized actively independent of the concomitant synthesis of RNA even when a substantial amount of the poly(A) sequences was being degraded (after GVBD). The size distribution of these labeled poly(A) sequences was the same as that of steady-state poly(A) measured by [3H]poly(U) binding, and they were associated with non-radioactive heterogeneous high molecular weight RNA. Thus, the present findings indicate that the poly(A) sequences associated with cytoplasmic maternal mRNA were being turned over extensively and that a portion of them was completely degraded during oocyte maturation. This poly(A) turnover also was indicated in full-grown oocytes which were not treated with progesterone and which showed no changes in the content and size of poly(A) throughout the experimental period.

Induction of amphibian oocyte maturation by polyvalent cations and alkaline pH in the absence of potassium ions

The maturation of Xenopus laevis oocytes was studied in media free of added potassium salts. Under these conditions maturation could be triggered by 1 m M Mn 2+ and La 3+ and, to a lesser extent, by 2–4 m M Ca 2+ and Mg 2+. Maturation induced by 1.5 m M Mn 2+ was inhibited by K + concentrations above 0.25 m M. Potassium was inhibitory when added up to 2 hr before germinal vesicle breakdown occurred. In potassium-free media, maturation could be induced by incubation of oocytes under mild alkaline media (pH 8.5–9). A high percentage of medium-sized oocytes (stage IV according to Dumont) was induced to mature by progesterone in the absence of potassium. Maturation of oocytes in potassium-free media was normal by the criteria of germinal vesicle breakdown, production of maturation promoting factor, vitelline membrane activation, and inhibition by known maturation inhibitors.

Roles of cyclic amp and calcium in maturation of Xenopus laevis oocytes

Progesterone induces in vitro meiotic maturation in Xenopus oocytes. 1. 1. Early steps of maturation are sensitive to inhibition by phosphodiesterase inhibitors (methyl xanthines) or by cholera toxin, suggesting that elevated levels of intracellular cyclic-AMP block progesterone action. 2. 2. Progesterone causes a significant decrease in the amount of newly synthesized cyclic AMP from microinjected α- 32P-ATP. 3. 3. Ca 2+ ions are involved both in the initiation of progesterone action and in the whole process of the first meiotic division.

Endogenous phosphorylated proteins during maturation of Xenopus laevis oocytes

AbstractDuring the course of maturation of Xenopus laevis oocyte a burst of phosphorylation occurs around germinal vesicle breakdown. At the same time a relative drop in a unique phosphoprotein (protein I; mot wt ∼40,000) is observed. Enucleation of [32P] labeled oocytes has shown the cytoplasmic localization of protein I. Methylxanthines and cholera toxin, which inhibit progesterone‐induced maturation, block the burst of phosphorylation and do not change the amount or the distribution of [32P] phosphoproteins.

Mitotic factors from mammalian cells: a preliminary characterization.

The objective of this study was the preliminary characterization of the factors from mitotic HeLa cells that can induce meiotic maturation in Xenopus laevis oocytes. We found that this factor is a heat-labile, Ca2+-sensitive, nondialyzable protein with a sedimentation value of 4-5S. Furthermore, no new protein synthesis was found to be required for this mitotic factor to induce maturation in the amphibian oocytes. These data suggest that the factors involved in the breakdown of nuclear membrane and the condensation of chromosomes that are associated with three different phenomena, mitosis, meiosis, and premature chromosome condensation, are very similar in different animal species.

Induction of maturation in Xenopus laevis oocytes by a steroid linked to a polymer

Induction of maturation in Xenopus laevis oocytes by a steroid linked to a polymer