Gonadotropins and the timing of progesterone-induced meiotic maturation of Xenopus laevis oocytes

Isolated oocytes from 30 unstimulated Xenopus laevis females required from 2.50 +/- 0.13 to 14.59 +/- 0.77 hr after progesterone exposure for the first 50% of each group to complete meiotic maturation. Injecting 8 females with an amount of hCG not causing ovulation (25 micrograms, 96 IU) lowered oocyte maturation times by 45-83%. An enzyme-linked immunosorbent assay (ELISA) of the blood of 18 unstimulated animals found a constituent which bound to anti-hCG in amounts (equivalent to 0-1.03 micrograms/ml hCG) that had a direct relationship to the rates of GVBD in oocytes. Preincubation of manually isolated follicles in 0.25-1.25 micrograms/ml hCG shortens oocyte maturation times by 18-50% in a direct, nonlinear fashion and this priming effect is reversed when hCG is withdrawn. The action of gonadotropins in facilitating germinal vesicle breakdown (GVBD) mimics the previously reported priming effect produced by preincubation of oocytes in subthreshold levels of progesterone. Evidence suggests that individual variation in the time course of progesterone-induced meiotic maturation of amphibian oocytes is the result of priming differences caused by the action on follicle cells of fluctuating blood levels of an LH-like hormone.     

Antipain microinjection prevents progesterone to inhibit adenyl cyclase in Xenopus oocytes

Microinjection of antipain, an inhibitor of thiol and Ca2+-dependent proteases, in immature Xenopus oocytes inhibited meiotic maturation induced by progesterone, but not by transfer of cytoplasm taken from maturing oocytes. Oocytes could be released from antipain inhibition by increasing progesterone concentration. alpha-32P-ATP was microinjected to study adenylcyclase in ovo. As already reported, neosynthesis of cAMP was decreased following progesterone application. This decrease was not observed, or it was considerably reduced, in oocytes previously injected with antipain. In amphibian, full-grown ovarian oocytes are arrested at first meiotic prophase, and have a large nucleus known as the germinal vesicle. Progesterone induces the production of a cytoplasmic maturation-promoting factor (MPF), which itself triggers germinal vesicle breakdown (GVBD), and subsequent events of meiotic maturation (Masui and Markert, 1971; Gerhart et al., 1984). A considerable body of evidences support the view that release from prophase block is due to inactivation of a cAMP-dependent protein kinase (reviewed by Maller, 1983). On the other hand, progesterone has been shown to induce a transient decrease in cAMP level (Speaker and Butcher, 1977; Schorderet-Slatkine et al., 1982; Cicirelli et al., 1985), and this initial drop of cAMP, along with a number of studies indicating a decrease in adenylate cyclase activity (Mulner et al., 1979; Baltus et al., 1981; Sadler and Maller, 1981; Finidori-Lepicard et al., 1981; Jordana et al., 1981), provided key support to the theory that an early drop in cAMP led to the dephosphorylation of a hypothetical protein which initiates maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transforming ras proteins accelerate hormone-induced maturation and stimulate cyclic AMP phosphodiesterase in Xenopus oocytes.

Transforming Harvey (Ha) ras oncogene products accelerated the time course of Xenopus oocyte maturation induced by insulin, insulinlike growth factor 1, or progesterone. The transforming constructs, [Val-12]Ha p21 and [Val-12, Thr-59]Ha p21, displayed equal potency and efficacy in their abilities to accelerate the growth peptide-induced response. Normal Ha p21 was only 60% as powerful and one-fifth as potent as the mutants containing valine in the 12 position. In contrast, two nontransforming constructs, [Val-12, Ala-35, Leu-36, Thr-59]Ha p21 and [Val-12, Thr-59]Ha(term-174) p21, had no effect on the time course of hormone-induced maturation. Effects of the transforming ras proteins on hormone-induced maturation correlated with their abilities to stimulate in vivo phosphodiesterase activity measured after microinjection of 200 microM cyclic [3H] AMP. When p21 injection followed 90 min of insulin treatment, there was no increase in phosphodiesterase activity over that measured after hormone treatment or p21 injection alone, but additive effects of p21 and insulin on enzyme activity were observed during the first 90 min of insulin treatment. Even though normal Ha p21 and transforming [Val-12, Thr-59]Ha p21 stimulated oocyte phosphodiesterase to equal levels when coinjected with substrate at the initiation of the in vivo assay, the transforming protein elicited a more sustained stimulation of enzyme activity. These results suggest that stimulation of a cyclic AMP phosphodiesterase activity associated with insulin-induced maturation is involved in the growth-promoting actions of ras oncogene products in Xenopus oocytes.     

G protein beta gamma 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.     

Freeze-fracture electron microscopy of membrane changes in progesterone-induced maturing oocytes and eggs of Xenopus laevis

Using freeze-fracture electron microscopy, compositional changes were analysed in the surface membrane of Xenopus oocytes during maturation after in vitro progesterone treatment, as well as in eggs before and after fertilization. Investigated stages were as follows: (1) defolliculated full-grown oocytes; (2) defolliculated oocytes after 5 min exposure to 5 micrograms/ml progesterone; (3) ditto at germinal vesicle breakdown (GVBD) after 5 h progesterone treatment; (4) unfertilized eggs at oviposition and (5) zygotes 30 min post-fertilization. Comparing the patterns of intramembranous particle (IMP) density and IMP size during these stages the following changes were found: a transient decrease in IMP density was found after 5 min progesterone treatment; a 48% increase during maturation; a further 17% increase after fertilization. In defolliculated oocytes tight-junction-like structures were found, but no gap junctions. These results are discussed with reference to progesterone action, membrane remodelling, protein synthesis and membrane lipid organization.     

LH-stimulation of the adenylyl cyclase system in follicles and oocytes of Xenopus laevis

The presence of an adenylyl cyclase sensitive to LH in Xenopus laevis is studied. The assay for adenylyl cyclase in membranes and homogenates from Xenopus laevis follicles and oocytes is characterized and the aim is centered on the appearance of LH-response through oogenesis. The results show a stimulation by LH in whole follicle and oocytes surrounded by the follicle cells. The oocytes become responsive to LH from stage III onwards, suggesting an action of the gonadotropin on the monolayer of follicular cells in early stages of follicle development.     

Xenopus oocytes and the biochemistry of cell division

The control of cell proliferation involves both regulatory events initiated at the plasma membrane that control reentry into the cell cycle and intracellular biochemical changes that direct the process of cell division itself. Both of these aspects of cell growth control can be studied in Xenopus oocytes undergoing meiotic maturation in response to mitogenic stimulation. All mitogenic signaling pathways so far identified lead to the phosphorylation of ribosomal protein S6 on serine residues, and the biochemistry of this event has been investigated. Insulin and other mitogens activate ribosomal protein S6 kinase II, which has been cloned and sequences in oocytes and other cells. This enzyme is activated by phosphorylation on serine and threonine residues by an insulin-stimulated protein kinase known as MAP-2 kinase. MAP kinase itself is also activated by direct phosphorylation on threonine and tyrosine residues in vivo. These results reconstitute one step of the insulin signaling pathway evident shortly after insulin receptor binding at the membrane. Several hours after mitogenic stimulation, a cell cycle cytoplasmic control element is activated that is sufficient to cause entry into M phase. This control element, known as maturation-promoting factor or MPF, has been purified to near homogeneity and shown to consist of a complex between p34cdc2 protein kinase and cyclin B2. In addition to apparent phosphorylation of cyclin, regulation of MPF activity involves synthesis of the cyclin subunit and its periodic degradation at the metaphase----anaphase transition. The p34cdc2 kinase subunit is regulated by phosphorylation/dephosphorylation on threonine and tyrosine residues, being inactive when phosphorylated and active when dephosphorylated. Analysis of phosphorylation sides in histone H1 for p34cdc2 has revealed a consensus sequence of (K/R)S/TP(X)K/R, where the elements in parentheses are present in some but not all sites. Sites with such a consensus are specifically phosphorylated in mitosis and by MPF in the protooncogene pp60c-src. These results provide a link between cell cycle control and cell growth control and suggest that changes in cell adhesion and the cytoskeleton in mitosis may be regulated indirectly by MPF via protooncogene activation. S6 kinase II is also activated upon expression of MPF in cells, indicating that MPF is upstream of S6 kinase on the mitogenic signaling pathway. Further study both of the signaling events that lead to MPF activation and of the substrates for phosphorylation by MPF should lead to a comprehensive understanding of the biochemistry of cell division.     

Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I.

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2.     

Expression of rat mRNA coding for hormone-stimulated adenylate cyclase in Xenopus oocytes

Beta-Adrenergic agonist-stimulated hyperpolarization, whole-cell cAMP accumulation, and activity of isoproterenol-stimulated membrane-bound adenylate cyclase (EC 4.6.1.1) in Xenopus laevis ovarian oocytes are entirely dependent on the presence of nascent follicle cells. A method was developed to remove rapidly and completely all extra-oocyte cell types to yield defolliculated oocytes that exhibited normal viability and resting membrane potentials yet lacked beta-adrenergic receptor (beta AR)-stimulated responses. Purified follicle membranes contained beta AR-stimulated adenylate cyclase activity, whereas oocyte cell membranes did not. Purified oocyte membrane preparations from X. laevis oocytes previously microinjected with C6-2B rat astrocytoma mRNA, and subsequently defolliculated, exhibited novel beta AR and forskolin-stimulated adenylate cyclase activity. These experiments demonstrate that oocytes expressed rat C6-2B mRNA coding for the beta-adrenergic receptor and the components necessary for forskolin-stimulated adenylate cyclase activity.     

Analysis of the p21 ras system during development of meiotic competence in Xenopus laevis oocytes.

The ability of Xenopus oocytes to undergo insulin- or insulin-like growth factor 1-induced meiotic maturation develops during oogenesis, with cells 1.0 mm in diameter or larger responding in a size-dependent manner. Since insulin-induced oocyte maturation was shown previously to be p21 ras-dependent, experiments were performed to test whether a deficiency in the p21 ras system might account for meiotic incompetence in small oocytes (less than or equal to 0.9 mm diameter). Both small and large oocytes were found to contain comparable levels of membrane-associated p21, as determined by protein immunoblotting. Treatment of both small and large oocytes with 2 microM insulin for 2 hr increased endogenous levels of membrane-associated p21 by approximately 70%. Stimulation of microinjected p21-membrane association by insulin was observed to be both time- and concentration-dependent in large oocytes with an EC50 of 50 nM. In addition, comparable levels of GTPase activating protein were measured in extracts prepared from oocytes ranging from 0.8 to 1.3 mm in diameter. Therefore, the p21 system is apparently not limiting during oogenesis, and expression of some other cellular component must account for development of meiotic competence in Xenopus oocytes.     

Polylysine activates membrane-bound adenylyl cyclase from Xenopus laevis oocytes through the Gs transducing protein.

1. The activity of the adenylyl cyclase found in the membranes of Xenopus laevis can be affected by polylysine and other polycations. 2. The activity of the catalytic subunit measured with forskolin is inhibited by polylysine and polyarginine at concentrations above 10 microM and by spermine above 3 mM. 3. The adenylyl cyclase activity stimulated by GTP-gamma-S or F- through the stimulatory G protein (Gs) can be increased by polylysine, polyornithine and spermine but not by polyarginine. 4. Polylysine stimulation of Gs dependent activity is due to the increase in the apparent affinity for GTP-gamma-S and to a lowering of the requirement for Mg2+ concentration.     

Protein kinase C and progesterone-induced maturation in Xenopus oocytes

Though progesterone-induced maturation has been studied extensively in Xenopus oocytes, the mechanism whereby the prophase block arrest is released is not well understood. The current hypothesis suggests that a reduction in cAMP and subsequent inactivation of cAMP-dependent protein kinase is responsible for reentry into the cell cycle. However, several lines of evidence indicate that maturation can be induced without a concomitant reduction in cAMP. We show that the mass of diacylglycerol in whole oocytes and plasma membranes decreases 29% and 10% respectively, within the first 15 sec after the addition of progesterone. Diacylglycerol in plasma membranes further decreased 59% by 5 min. We also show that the protein kinase C inhibitors sphingosine and staurosporine can induce oocyte maturation. In addition, the synthetic diglyceride, DiC8, and microinjected PKC can inhibit or delay progesterone-induced maturation. These results together suggest that a transient decrease in protein kinase C activity may regulate entry into the cell cycle. The mechanism whereby DAG is decreased in response to progesterone is unclear. Initial studies show that progesterone leads to a decrease in IP3 suggesting that progesterone may act by reducing the hydrolysis of PIP2. On the other hand, progesterone caused a decrease in the amount of [3H]arachidonate labelling in DAG during the same time suggesting that progesterone may stimulate lipase activity. The relationship between postulated changes in the PKC pathway and those hypothesized for the PKA pathway are discussed.     

Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.     

Rabies mRNA translation in Xenopus laevis oocytes.

Two rabies virus-specific mRNA species were identified by analysis of their encoded proteins after translation of the partially purified species in Xenopus laevis oocytes. One of these coded for the virion surface glycoprotein (G protein), and the other coded for the major structural protein of the virion nucleocapsid (N protein). The G-mRNA sedimented in a sucrose density gradient at about 18S, and the N-mRNA had a sedimentation coefficient of approximately 16S. Their respective translation products were identified in a radioimmunoassay with specific monoclonal antibody probes that recognized only G or N proteins. Immunoprecipitates formed between the radiolabeled viral antigens synthesized in programmed oocytes and their respective monoclonal antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The glycoprotein antigen translated from G-mRNA in oocytes migrated in the gel ahead of the virion G protein with a migration rate that was similar to that of nonglycosylated intracellular glycoproteins from virus-infected cells. The results suggested that the branched-chain carbohydrate of G protein was not required for recognition by the particular monoclonal antibody used. The nucleocapsid antigen translated from N-mRNA in oocytes migrated to the same position in the gel as marker virion N protein. Both the electrophoretic mobility of virus-specific antigens in sodium dodecyl sulfate-polyacrylamide gel and the antibody concentration dependence for immunoprecipitations were criteria for identifying the individual viral proteins encoded by the two rabies mRNA's.     

Amino acid uptake in Xenopus laevis oocytes.

The isolated oocytes from Xenopus laevis are able to take up radioactive amino acids from the exogenous medium. Most amino acids tested are taken up to reach concentrations higher than the extracellular medium. The initial uptake velocities vary with the external amino acid concentration in a Michaelis-Menten fashion. Aspartic acid requires concentrations an order of magnitude higher than the five other amino acids tested to reach half the maximal uptake velocity. The uptake mechanism seems to be specific for groups of analogous amino acids, as can be determined by competition studies. The amino acid groups for which there is some evidence of uptake specificity would be aromatic, aliphatic, acidic and basic. Amino acid pools of oocytes show that these cells can concentrate amino acids from Xenopus blood, as well as from artificial media.     

Sodium-dependent and sodium-independent phosphate uptake by full-grown, prophase-arrested oocytes of Xenopus laevis before and after progesterone-induced maturation

The uptake of inorganic phosphate by full-grown, prophase-arrested oocytes occurs by two essentially independent transport systems, which accomplish the delivery of extracellular phosphate into two different cellular compartments. One transport system is sodium-dependent, the other is sodium-independent. Both systems can be inhibited by sulfhydryl reagents and the sodium-dependent system becomes inhibited during progesterone-induced oocyte maturation.