Endogenous phosphorylated proteins during maturation of Xenopus laevis oocytes

During 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.     

Induction of maturation in small Xenopus laevis oocytes

The competence of Xenopus laevis oocytes in various stages of growth to respond to progesterone treatment was investigated. Full-grown (stage 6) oocytes undergo nuclear membrane dissolution and resume meiosis in response to progesterone exposure, while smaller oocytes (stages 3-5; less than 1100 micron in diameter) do not. The defect which prevents 750- to 1050-micron oocytes from responding to progesterone can be overcome by microinjecting cytoplasm withdrawn from a stage 6 oocyte. Germinal vesicle breakdown in these small oocytes occurs on a timetable similar to that of stage 6 oocytes exposed to progesterone and is accompanied by a twofold increase in protein synthesis as well as the activation of MPF. The results argue that a cytoplasmic factor(s) which probably first appears at late stage 5 is required for progesterone responsiveness. The identity and role of the factor(s) in the development of maturation competence and the regulation of maternal mRNA translation are discussed.     

Phospholipase-induced maturation of Xenopus laevis oocytes: Mitogenic activity of generated metabolites

Signal transduction induced by generation of second messengers from membrane phopholipids is considered a major regulatory mechanism in control of cell proliferation. We report here that in the Xenopus laevis oocytes model, microinjection of the three most relevant types of phospholipases acting on membrane phospholipids (A2, C, and D) are capable of inducing oocyte maturation with similar efficiencies. This effect is mediated by the generation of known second messengers such as lyso-phospholipids, arachidonic acid, diacylglycerol, and phosphatidic acid. Specific inhibitors of protein kinase C made it possible to identify alternative independent signalling pathways for induction of oocyte maturation. Our results indicate that while phospholipase C seems to be dependent on protein kinase C (PKC), phospholipase A2, and phospholipase D are completely independent of protein kinase C function. Thus, the oocyte system is a powerful tool for the analysis of the potential mitogenic activity of lipid metabolites. It is also an excellent tool for unravelling the different routes involved in the regulation of cell growth.     

Calmodulin synthesis and accumulation during oogenesis and maturation of Xenopus laevis oocytes

The calmodulin levels in stage 6 Xenopus oocytes averaged 89 +/- 24 (SD) ng/oocyte and had largely accumulated by stage 3 of oogenesis. From stage 3 to early stage 6, calmodulin levels did not increase further. However, in large stage 6 oocytes (greater than 1.25 mm diam) calmodulin levels again rose to a level as high as 121 ng/oocyte. Calmodulin levels did not change during the maturation of stage 6 oocytes and the results of measurements on animal and vegetal oocyte halves from control and mature oocytes showed no evidence of a redistribution of calmodulin during maturation. Measurements of calmodulin synthesis in stages 1 and 2 oocytes, stage 4 oocytes, and stage 6 oocytes indicated that calmodulin was being synthesized continuously during oogenesis and that the rate of synthesis increased during oogenesis. In stage 1 and 2 oocytes (combined), the synthesis rate was 3.5 pg/hr/oocyte; in stage 4 oocytes it was 48 pg/hr/oocyte, and in large stage 6 oocytes the rate had increased to 160 pg/hr/oocyte. These changes in the rates of synthesis were discussed as they relate to the pattern of calmodulin accumulation during oogenesis.     

Freeze-fracture analysis of structural reorganization during meiotic maturation in oocytes of Xenopus laevis

Summary During meiotic maturation, the cortex of oocytes of Xenopus laevis undergoes structural reorganization, visualized in this study by freeze-fracture electron microscopy. In the full-grown but immature oocyte, annulate lamellae are dispersed throughout the subcortex of the egg, 5 to 20 m from the plasma membrane. The annulate lamellae consist of well-organized stacks of membrane with visible pores. Stimulation of meiotic maturation by progesterone leads to disruption of the annulate lamellae and formation of an elaborate cortical endoplasmic reticulum which surrounds the cortical granules and intertwines throughout the cortex of the mature egg. Pore-like structures similar to those previously observed in the subcortical annulate lamellae are observed in the mature cortical endoplasmic reticulum. The cortical endoplasmic reticulum is often in close apposition with the plasma membrane and with membranes of cortical granules, but no junctions are visualized. This study provides further evidence that the cortical endoplasmic reticulum develops during progesterone-stimulated meiotic maturation in vitro, and that the annulate lamellae are precursors to the cortical endoplasmic reticulum.     

Ribosomal preparations may induce maturation in Xenopus laevis oocytes

Xenopus laevis oocytes undergo maturation when they are injected with large quantities of crude ribosomes from various origins: X laevis full-grown or matured oocytes, Xenopus ovaries and embryos, Xenopus liver or mouse liver. All have the same efficiency, whatever their origin: they include 50-90% maturation in the injected oocytes at about the same speed as progesterone treatment. The ribosomal preparations are inactive wen injected into recipient oocytes pretreated with cholera toxin or cycloheximide. After dissociation with the high salt extract, but not with the subunits. Hypotheses concernning the mode action of this ribosomal extract are disussed.     

The effect of trifluoperazine on maturation of Xenopus laevis oocytes

Full grown Xenopus oocytes were incubated with trifluoperazine (TFP) or injected with TFP. Incubation of oocytes in TFP resulted in normal-appearing meiotic maturation, as judged by the presence of the white spot and the absence of the germinal vesicle. Cortical granule breakdown in TFP-incubated oocytes was not normal. Abnormal cortical granule breakdown was also observed when progesterone-maturated oocytes were activated in the presence of TFP. Oocytes microinjected with TFP and incubated with progesterone appeared to mature in a normal manner, as judged by the absence of the germinal vesicle; these underwent cortical granule breakdown following activation, but frequently lacked the white spot. Oocytes microinjected with TFP did not mature in the absence of progesterone. We conclude that incubation, although not microinjection, of oocytes with TFP induces essentially normal resumption of meiotic maturation.     

Changes in protein phosphorylation accompanying maturation of Xenopus laevis oocytes

Protein phosphorylation has been measured after injection of [³²P]phosphate into oocytes of Xenopus laevis undergoing progesterone-induced meiotic maturation. As oocytes mature, there is a burst of nonyolk protein phosphorylation several hours after progesterone exposure and shortly before germinal vesicle breakdown (GVBD). This burst is not due to changes in the specific activity of the phosphate or ATP pool. Enucleated oocytes exposed to progesterone also experience the burst, indicating the cytoplasmic location of phosphoprotein formation. When an oocyte receives an injection of cytoplasm containing the maturation-promoting factor (MPF), a burst of protein phosphorylation occurs immediately, and GVBD occurs shortly thereafter, even in the presence of cycloheximide. Under a variety of conditions promoting or blocking maturation, oocytes which undergo GVBD are the only ones to have experienced the phosphorylation burst. The results suggest that the protein phosphorylation burst is a necessary step in the mechanism by which MPF promotes GVBD.     

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.     

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.     

Induced formation of asters and cleavage furrows in oocytes of Xenopus laevis during in vitro maturation

We have previously reported that injection of purified basal bodies or sperm into unfertilized eggs of Xenopus laevis induced the formation of asters and irregular cleavage furrows. Fully grown oocytes were found to be unable to form asters or cleavage furrows. In this paper we show that the oocyte acquires the ability to form asters upon basal body injection at the time of germinal vesicle breakdown during in vitro maturation. Our evidence indicates that aster formation requires progesterone-stimulated changes in the oocyte and mixing of cytoplasm and germinal vesicle plasm. The ability of the oocyte to form cleavage furrows arises six to eight hours after germinal vesicle breakdown. We infer that some maturational change in the cell cortex occurs to enable the egg surface to furrow. Experiments on the relationship of aster formation to furrow initiation indicates that asters stimulate furrow formation. However, some furrowing could be induced without aster formation in mature oocytes and unfertilized eggs by an activation stimulus, showing that asters are not essential for cleavage initiation. The significance of these observations are discussed in the light of our current understanding of meiotic maturation, cell cleavage and aster growth.     

Digitoxigenin, a digitalis steroid, induces meiotic maturation of Xenopus laevis oocytes

Digitoxigenin, a C23 digitalis steroid induces meiotic maturation of Xenopus oocyte. The dose of digitoxigenin which induces half maximal response is 3.3 +/- 2.10(-5)M. In contrast the conjugated digitalis steroid, digitoxin (digitoxigenine + 3 digitoxoses) never triggered maturation at any of the doses tested. These experiments which show that only free digitoxigenin mimics progesterone action, suggest that both digitoxigenin and progesterone possess a common initial site of action which is not localized at the level of the outer leaflet of the oocyte plasma membrane.     

Protein kinase activities during maturation in xenopus laevis oocytes

Protein kinase activities have been compared in ovarian oocytes and in ovulated eggs of $\text{Xenopus laeyis}$.In ovaries and ovarian oocytes, we have detected, in addition to an already known (1) cyclic AMP stimulated phosphoprotein kinase, a second very active phosphoprotein kinase which is cAMP-independent.Besides these two activities, a third protein kinase activity becomes detectable after maturation and ovulation: it is a cAMP and cGMP-dependent histone kinase.     

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.     

Cortical membrane-trafficking during the meiotic resumption of Xenopus laevis oocytes

Summary The giant mucous cells in the skin of the terrestrial banana slug Ariolimax columbianus secret intact granules containing mucins. Electron microscopy, after ultrarapid freezing and freeze-substitution in osmium, shows that the secreted granules are bounded by two distinct membranes, presumably derived from the Golgi apparatus and the plasmalemma. Relatively stable, intact granules can be obtained in great quantity in our in vitro system. Rapid lysis of the granules was induced by adenosine triphosphate. At much higher concentrations, adenosine diphosphate and 5-adenylimido-diphosphate also caused lysis. Other nucleotides and related compounds, as well as 1,4,5-inositol triphosphate and molluscan neurotransmitters and neuropeptides, had no effect on the granules. The stability of secreted granules varied with the ionic composition of the isosmotic medium in which they were suspended. When the predominant cation in the medium was potassium, and calcium was also present, granules lysed if exposed to shear stress (stirring of the suspension). This did not occur if sodium was the major cation present. None of the other ions in the suspension media had detectable effects on the stability of the granules.     

Cyclic AMP levels during the maturation of Xenopus oocytes

While several studies have suggested that the induction of oocyte maturation results from a transient decrease in cAMP levels, attempts to demonstrate such a change have led to inconsistent results with respect to whether or not a decrease occurs as well as timing of the decrease. In this report the results of experiments designed to demonstrate small changes in cAMP content in Xenopus laevis oocytes are presented and a statistically significant 20% decrease in cAMP content from between 2 and 50 min postprogesterone addition is found. The cAMP content subsequently rises to 12% higher than control levels and then becomes indistinguishable from control values for the remainder of the maturation period.