Progesterone-induced Xenopus Oocyte Maturation Research Articles

Effects of Thioglycolic Acid on Progesterone-Induced Maturation of Xenopus Oocytes

In order to examine the effects of thioglycolic acid (TGA) on reproduction, Xenopus oocytes were treated with different concentrations of TGA. During culture, frequencies of germinal vesicle breakdown (GVBD) and MI–MII transition were determined. Samples collected at indicated times were subjected to immunoblotting. Data indicated that TGA accelerated the frequncy of GVBD, but inhibited polar body extrusion and formation of MII-arrested eggs in a concentration-dependent manner. At 4 h after progesterone addition, phosphorylation of extracellular signal-regulated kinase (ERK) and p90 ribosomal S6 kinase, two members of the mitogen-activated protein kinase (MAPK) pathway, was upregulated in TGA-treated oocytes. The regulatory subunit of M-phase promoting factor (MPF)-cyclin B was also upregulated by TGA, while phospho-Cdc2 was downregulated. At 8 h, Cdc2 dephosphorylation and cyclin B1 were downregulated by TGA treatment. However, TGA exerted no effect on Mos, an MAPKKK (MAPK kinase kinase). In conclusion, TGA has the potential to inhibit in vitro maturation of Xenopus oocyte with increased GVBD frequency accompanied by alterations in protein expression and phosphorylation involved in MPF and MAPK pathways. Since egg formation is essential to maintain appropriate reproductive capacity, our findings may have certain toxicological implications.

Survivin mRNA is down-regulated during early Xenopus laevis embryogenesis.

One of the hallmarks of early development is the rapid proliferation of cells immediately after fertilization. Many of the rules that govern cell division in normal somatic cells, such as contact inhibition and apoptosis, seem temporarily suspended in the early embryo. A similar suspension of mechanisms normally regulating cell division occurs in the development of cancer. Survivin, an inhibitor of apoptosis and a positive regulator of progression through the cell cycle, localizes to the mitotic spindle and interacts with several proapoptotic caspases. Survivin protein expression has been studied during the development of the salivary gland in mouse. However, the regulation of survivin during the critical transitions defining oocyte maturation and the early restriction of developmental potential are not easily examined in the mouse. We therefore studied survivin mRNA expression during oogenesis and early embryogenesis in Xenopus laevis. We found that survivin mRNA is present in the earliest stages of Xenopus oocytes and that it accumulates during oogenesis. Progesterone-induced maturation of Xenopus oocytes leads to polyadenylation of the survivin transcript. Survivin mRNA is also present in early Xenopus embryos. After the onset of zygotic transcription, however, the amount of survivin mRNA declines rapidly to undetectable levels. This decrease in survivin mRNA correlates temporally with both the slowing of the cell cycle and the onset of endogenous embryonic apoptosis. With the exception of the ovary, survivin mRNA was undetectable in all adult Xenopus tissues examined.

Overexpression of Hp95 induces G1 phase arrest in confluent HeLa cells

Xp95, a protein recently identified in Xenopus laevis, is potentially involved in progesterone-induced Xenopus oocyte maturation. In this study, we cloned a human homologue of Xp95, designated Hp95, and examined the effect of its overexpression on the growth properties of human malignant HeLa cells which have lost the contact inhibition of cell proliferation. We observed that although HeLa cells did not undergo G1 phase arrest at any stage after confluence, they were able to downregulate their G1 phase CDK activities in response to confluence. When Hp95 was overexpressed in HeLa cells by transfection with a constitutive or an inducible expression vector containing a full-length Hp95 transgene, HeLa cells became able to undergo G1 phase arrest and form a monolayer culture after confluence. However, the G1 phase CDK activities in these Hp95 overexpressing cells were not inhibited further as compared to control cells after confluence. These results indicate that the defects in HeLa cells that cause the loss of contact inhibition of cell proliferation are in components downstream of the G1 phase CDKs and that overexpression of Hp95 counteracts some of these defects.

G Proteins Impede Maturation

Steroid hormones can influence cellular events by two very different mechanisms: interaction with ligand-activated transcription factors to regulate gene expression or interaction with cell-surface proteins to regulate nongenomic signaling pathways. Lutz et al. studied progesterone-induced maturation of Xenopus oocytes, which have high and low affinity-binding sites for progesterone at the plasma membrane. Injection of a variety of RNAs and chemicals to influence G protein subunit activity suggested that constitutively active free Gβγ subunits act as inhibitors of oocyte maturation. Treatments that depleted free Gβγ enhanced progesterone-stimulated oocyte maturation and promoted tyrosine phosphorylation of extracellular signal-regulated kinase 1 (ERK1) and treatments that increased free Gβγ levels inhibited progesterone-stimulated maturation and decreased ERK1 phosphorylation. The authors propose that progesterone acts via an unknown mechanism (possibly by antagonizing a constitutively active G protein-linked receptor) to sequester these inhibitory Gβγ subunits and promote maturation. Lutz, L.B., Kim, B., Jahani, D., and Hammes, S.R. (2000) G protein βγ subunits inhibit nongenomic progesterone-induced signaling and maturation of Xenopus laevis oocytes. J. Biol. Chem. 275 : 41512-41520. [Abstract] [Full Text]

G Protein βγ Subunits Inhibit Nongenomic Progesterone-induced Signaling and Maturation in Xenopus laevis Oocytes: EVIDENCE FOR A RELEASE OF INHIBITION MECHANISM FOR CELL CYCLE PROGRESSION

Progesterone-induced maturation of Xenopus oocytes is a well known example of nongenomic signaling by steroids; however, little is known about the early signaling events involved in this process. Previous work has suggested that G proteins and G protein-coupled receptors may be involved in progesterone-mediated oocyte maturation as well as in other nongenomic steroid-induced signaling events. To investigate the role of G proteins in nongenomic signaling by progesterone, the effects of modulating Galpha and Gbetagamma levels in Xenopus oocytes on progesterone-induced signaling and maturation were examined. Our results demonstrate that Gbetagamma subunits, rather than Galpha, are the principal mediators of progesterone action in this system. We show that overexpression of Gbetagamma inhibits both progesterone-induced maturation and activation of the MAPK pathway, whereas sequestration of endogenous Gbetagamma subunits enhances progesterone-mediated signaling and maturation. These data are consistent with a model whereby endogenous free Xenopus Gbetagamma subunits constitutively inhibit oocyte maturation. Progesterone may induce maturation by antagonizing this inhibition and therefore allowing cell cycle progression to occur. These studies offer new insight into the early signaling events mediated by progesterone and may be useful in characterizing and identifying the membrane progesterone receptor in oocytes.

The classical progesterone receptor mediates Xenopus oocyte maturation through a nongenomic mechanism.

Xenopus laevis oocytes are physiologically arrested at G(2) of meiosis I. Resumption of meiosis, or oocyte maturation, is triggered by progesterone. Progesterone-induced Xenopus oocyte maturation is mediated via an extranuclear receptor and is independent of gene transcription. The identity of this extranuclear oocyte progesterone receptor (PR), however, has remained a longstanding problem. We have isolated the amphibian homologue of human PR from a Xenopus oocyte cDNA library. The cloned Xenopus progesterone receptor (xPR) functioned in heterologous cells as a progesterone-regulated transcription activator. However, endogenous xPR was excluded from the oocyte nucleus and instead appeared to be a cytosolic protein not associated with any membrane structures. Injection of xPR mRNA into Xenopus oocytes accelerated the progesterone-induced oocyte maturation and reduced the required concentrations of progesterone. In enucleated oocytes, xPR accelerated the progesterone-induced mitogen-activated protein kinase activation. These data suggest that xPR is the long sought after Xenopus oocyte receptor responsible for progesterone-induced oocyte maturation.

Identification and Cloning of Xp95, a Putative Signal Transduction Protein in Xenopus Oocytes

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.

Ras-interacting domain of RGL blocks Ras-dependent signal transduction in Xenopus oocytes

RalGDS family members (RalGDS and RGL) interact with the GTP-bound form of Ras through its effector loop. The C-terminal region (amino acids 602–768) of RGL is responsible for binding to Ras. In this paper we characterized a Ras-interacting domain of RGL using deletion mutants of RGL(602–768). RGL(602–768), RGL(632–768), and RGL(602–734) bound to the GTP-bound form of Ras and inhibited the GAP activity of NF-1. RGL(646–768) showed a low binding activity to Ras and inhibited GAP activity of NF-1 weakly. None of RGL(659–768), RGL(685–768), RGL(602–709), and RGL(602–686) bound to Ras or inhibited GAP activity of NF-1. These results indicate that amino acids 632–734 of RGL constitute a nearly minimal domain that contains the binding element for Ras. RGL(632–734) inhibited v-Ras- but not progesterone-induced Xenopus oocyte maturation. Furthermore, RGL(632–734) inhibited v-Ras- but not v-Raf-dependent extracellular signal-regulated kinase activation in Xenopus oocytes. These results clearly demonstrate that the Ras-interacting domain of RGL is important for Ras-dependent signal transduction in vivo.

Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.

The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone.

Phosphatidylinositol 3-kinase activity is important for progesterone-induced Xenopus oocyte maturation.

In somatic cells, phosphatidylinositol 3-kinase (PI3 kinase) is a critical intermediary in growth factor-induced mitogenesis. We have examined the role of this enzyme in meiotic maturation of Xenopus laevis oocytes. PI3 kinase activity was present in immunoprecipitates of the p85 subunit of PI3 kinase from immature oocytes and markedly increased following progesterone stimulation. Injection of bacterially expressed protein corresponding to the C-terminal SH2 domain of p85 (SH2-C) inhibited progesterone-induced PI3 kinase activation and meiotic maturation. Injection of protein corresponding to the N-terminal SH2 domain or the SH3 domain of p85 did not inhibit PI3 kinase activation or maturation. SH2-C did not inhibit oocyte maturation induced by c-mos RNA injection. In addition, radiolabelled SH2-C was used to probe oocyte lysates, revealing that a novel 200-kDa protein bound to SH2-C. This protein may be an important mediator of progesterone-induced lipid metabolism in oocytes.

Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos.

In somatic cells, the Raf-1 serine/threonine protein kinase is activated by several polypeptide growth factors. We investigated the role of Raf-1 in progesterone-induced meiotic maturation of Xenopus laevis oocytes. Raf-1 enzymatic activity and phosphorylation (reflected by a mobility shift on sodium dodecyl sulfate gels) were increased in oocytes following progesterone stimulation. The increase in Raf-1 activity was concurrent with an elevation in the activity of mitogen-activated protein (MAP) kinase. When RNA encoding an oncogenic form of Raf-1 (v-Raf) was injected into immature oocytes, MAP kinase mobility shift, germinal vesicle breakdown, and histone H1 phosphorylation increased markedly. When RNA encoding a dominant-negative version of Raf-1 was injected, progesterone-induced oocyte maturation was blocked. When RNA encoding Xenopus mos (mosxe) was injected into oocytes, Raf-1 and MAP kinase mobility shifts were observed after several hours. Also, when antisense mosxe oligonucleotides were injected into oocytes, progesterone-induced Raf-1 and MAP kinase mobility shifts were blocked. Finally, when antisense mosxe oligonucleotides were coinjected with v-Raf RNA into oocytes, histone H1 kinase activation, germinal vesicle breakdown, and MAP kinase mobility shift occurred. These findings suggest that Raf-1 activity is required for progesterone-induced oocyte maturation and that Raf-1 is downstream of mosxe activity.

Microtubule organization during maturation of Xenopus oocytes: Assembly and rotation of the meiotic spindles

Assembly of the meiotic spindles during progesterone-induced maturation of Xenopus oocytes was examined by confocal fluorescence microscopy using anti-tubulin antibodies and by time-lapse confocal microscopy of living oocytes microinjected with fluorescent tubulin. Assembly of a transient microtubule array from a disk-shaped MTOC was observed soon after germinal vesicle breakdown. This MTOC-TMA complex rapidly migrated toward the animal pole, in association with the condensing meiotic chromosomes. Four common stages were observed during the assembly of both M1 and M2 spindles: (1) formation of a compact aggregate of microtubules and chromosomes; (2) reorganization of this aggregate resulting in formation of a short bipolar spindle; (3) an anaphase-B-like elongation of the prometaphase spindle, transversely oriented with respect to the oocyte A-V axis; and (4) rotation of the spindle into alignment with the oocyte axis. The rate of spindle elongation observed in M1 (0.7 μm min −1) was slower than that observed in M2 (1.8 μm min −1). Examination of spindles by immunofluorescence with antitubulin revealed numerous interdigitating microtubules, suggesting that prometaphase elongation of meiotic spindles in Xenopus oocytes results from active sliding of antiparallel microtubules. A substantial number of maturing oocytes formed monopolar microtubule asters during M1, nucleated by hollow spherical MTOCs. These monasters were subsequently observed to develop into bipolar M1 spindles and proceed through meiosis. The results presented define a complex pathway for assembly and rotation of the meiotic spindles during maturation of Xenopus oocytes.

Regulation of alpha 1-beta 3-NA(+)-K(+)-ATPase isozyme during meiotic maturation of Xenopus laevis oocytes.

During progesterone-induced maturation of Xenopus oocytes, the transport and ouabain binding capacity of Na(+)-K(+)-ATPase at the plasma membrane is completely downregulated. To elucidate the mechanism and the physiological significance of this process, we have followed the fate of oocyte alpha-beta 3-Na(+)-K(+)-ATPase complexes during meiotic maturation and early embryonic development. An immunocytochemical follow-up of the catalytic alpha-subunit, ouabain binding studies, cell surface iodination, and oocyte cell fractionation combined with immunochemical subunit detection provides evidence that following progesterone treatment Na(+)-K(+)-ATPase molecules are retrieved from the oocyte plasma membrane. The enzyme complexes are recovered in an active form in an intracellular compartment in both in vitro and in vivo matured eggs. Exogenous Xenopus alpha 1- and beta 1-complexes expressed in the oocyte from injected cRNAs are regulated by progesterone similar to endogenous Na(+)-K(+)-ATPase complexes. Finally, active Na(+)-K+ pumps internalized during oocyte maturation appear to be redistributed to plasma membrane fractions during blastula formation in Xenopus embryos. In conclusion, our data suggest that endocytosis of alpha 1- and beta 3-complexes during meiotic maturation of Xenopus oocytes is responsible for downregulation of Na(+)-K(+)-ATPase activity and results in an intracellular pool of functional enzymes, which might be reexpressed during early development in response to physiological needs.

A Follicular Fluid Factor InhibitingXenopusOocyte Maturation

A factor was isolated from bovine follicular fluid (FF) that blocked progesterone-induced maturation of Xenopus oocytes. The factor was purified by ultrafiltration through PM-10 membrane, gel filtration on Sephadex G-25 column, gel permeation chromatography on Superose 12 column and reversed-phase HPLC on Partisil ODS-3 column. The active factor is a peptide with an estimated Mr of 8000 and it effectively blocked progesterone-induced maturation of Xenopus oocytes at a concentration of 100 micrograms/ml. On the other hand, it did not influence the spontaneous maturation of isolated cumulus-enclosed or denuded mouse oocytes. The present findings suggest that the FF factor may be an inhibitor or antagonist of progesterone action.

Microinjected GTP-γ-S inhibits progesterone-induced maturation of Xenopus oocytes

GTP-γ-S inhibits progesterone-induced maturation of Xenopus laevis oocytes and induces a rise in their cAMP levels. GTP-γ-S does not inhibit MPF-induced maturation. Although GTP-γ-S prevents the progesterone-induced increases in protein synthesis and phosphorylation, it has no effect on the basal rates of either. GTP-γ-S also prevents the initial DAG drop induced by progesterone. GDP-β-S effects are ambiguous, but it seems not to affect progesterone-induced maturation. These results suggest that although G-proteins are associated with the pathways affected by progesterone, the effects of progesterone are not mediated by a typical receptor/G-protein/effector interaction.

Regulatory changes of membrane transport and ouabain binding during progesterone-induced maturation of Xenopus oocytes.

During progesterone-stimulated maturation of defolliculated full-grown Xenopus oocytes, the activities of the transport systems for L-alanine, thymidine, chloride, phosphate, and alkali ions decrease. Differences of the extent and time course of these changes suggest that they are controlled by at least partially independent mechanisms. A closer investigation of the Na-K ATPase has shown that in unstimulated oocytes, ouabain produces maximal inhibition when 8-12 X 10(9) molecules are bound per cell. This number is bound during the first phase of a diphasic uptake process. Since this phase can be suppressed by increasing the concentration of external K+ to 45 mmol/liter or more, it is concluded that it refers to binding to the Na-K pump in the plasma membrane. Ouabain bound prior to progesterone-induced germinal vesicle breakdown (GVBD) remains bound after the breakdown, although the Na-K pump loses the capacity to bind ouabain after GVBD in oocytes that had not been exposed to ouabain preceding GVBD. In the presence of Mg++ membranes isolated before regulatory inhibition of pumping and ouabain binding show a Na+-dependent incorporation of 32P from gamma-[32P]-ATP that can be reversed by the addition of K+. The phosphorylation site migrates on LiDS-polyacrylamide gel electropherograms at about 98,000 daltons and can be identified as a Commassie blue-stainable band.

Acetylcholine promotes progesterone-induced maturation of Xenopus oocytes.

Progesterone-induced maturation of follicle-enclosed and denuded Xenopus laevis oocytes was significantly shortened by a concomitant exposure to acetylcholine. The promotion of maturation by acetylcholine was blocked by the specific muscarinic antagonist atropine. The action of acetylcholine was dose dependent, and the neurotransmitter was effective at very low concentrations. Progesterone progressively reduced the electrophysiological responses of X. laevis oocytes to acetylcholine, which completely disappeared close to the time of germinal vesicle breakdown. Progesterone alone did not elicit any electrophysiological responses. The in vitro effect of acetylcholine on oocyte maturation might reflect a physiological influence of the cholinergic system on an in vivo maturation process.

A stage-specific inhibitory effect of benzamil on Xenopus oocyte maturation located at the cell surface

Benzamil, a drug reported to block passive Na + transport, also inhibits progesterone-induced maturation of Xenopus oocytes when given early in the maturation period. The localization of tritiated benzamil at the oocyte surface was verified by autoradiography using a method that prevents diffusion of substances. These data indicate that the primary site of action of benzamil is at the cell surface.