Plasma membrane destination of the classical Xenopus laevis progesterone receptor accelerates progesterone-induced oocyte maturation

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a non-genomic mechanism initiated at the cell membrane. Recently, two Xenopus oocyte progesterone receptors have been cloned; one is the classical progesterone receptor (xPR-1) involved in genomic actions and the other a putative seven-transmembrane-G-protein-couple receptor. Both receptors are postulated to be mediating the steroid-induced maturation process in the frog oocyte. In this study, we tested the hypothesis that the classical progesterone receptor, associated to the oocyte plasma membrane, is participating in the reinitiation of the cell cycle. Addition of a myristoilation and palmytoilation signal at the amino terminus of xPR-1 (mp xPR-1), increased the amount of receptor associated to the oocyte plasma membrane and most importantly, significantly potentiated progesterone-induced oocyte maturation sensitivity. These findings suggest that the classical xPR-1, located at the plasma membrane, is mediating through a non-genomic mechanism, the reinitiation of the meiotic cell cycle in the X. laevis oocyte.     

Aven is dynamically regulated during Xenopus oocyte maturation and is required for oocyte survival

We have analyzed the expression and function of the cell death and cell cycle regulator Aven in Xenopus. Analysis of Xenopus Aven expression in oocytes and embryos revealed a band close to the predicted molecular weight of the protein (36 kDa) in addition to two bands of higher molecular weight (46 and 49 kDa), one of which was determined to be due to phosphorylation of the protein. The protein is primarily detected in the cytoplasm of oocytes and is tightly regulated during meiotic and mitotic cell cycles. Progesterone stimulation of oocytes resulted in a rapid loss of Aven expression with the protein levels recovering before germinal vesicle breakdown (GVBD). This loss of Aven is required for the G2–M1 cell cycle transition. Aven morpholino knockdown experiments revealed that early depletion of the protein increases progesterone sensitivity and facilitates GVBD, but prolonged depletion of Aven results in caspase-3 activation and oocyte death by apoptosis. Phosphorylated Aven (46 kDa) was found to bind Bcl-x_L in oocytes, but this interaction was lost in apoptotic oocytes. Thus, Aven alters progesterone sensitivity in oocytes and is critical for oocyte survival.     

Chronic boron or copper deficiency induces limb teratogenesis in Xenopus

Sets of adult male and female Xenopus laevis were administered a boron-deficient (−B) diet under low-boron culture conditions, a boron-supplemented (+B) diet under ambient boron culture conditions, a copper-deficient (−Cu) diet under low-copper culture conditions, or a copper-supplemented (+Cu) diet under ambient copper culture conditions, for 120 d. Adults from each group were subsequently bred, and the progeny were cultured and bred. Results from these studies indicated that although pronounced effects on adult reproduction and early embryo-larval development were noted in the −B F₁ generation, no effects on limb development were observed. No significant effects on reproduction, early embryogenesis, or limb development were noted in the +B group, irrespective of generation. Highly specific forelimb and hindlimb defects, including axial flexures resulting in crossed limbs and reduction deficits, were observed in −B F₂ larvae, but not in the +B F₂ larvae. As was noted in the boron-deficiency studies, significant effects on reproduction and early embryo development were observed in the −Cu F₁ generation, but not in the +Cu F₁ generation. Unlike the effects associated with boron deficiency, maldevelopment of the hindlimbs (32 responders, n=40) was found in the F₁ generation.     

Endogenous lactate transport in Xenopus laevis oocyte: dependence on cytoskeleton and regulation by protein kinases

Carbon flux in Xenopus laevis oocyte is glycogenic and an endogenous monocarboxylate transporter is responsible for intracellular lactate uptake. The aim of the present study was to determine if direct activation of protein kinases C and A modulates the activity of lactate transporter, as well as to investigate the possible role of cytoskeleton in these regulatory phenomena. The modulation was studied in isolated Xenopus oocytes of stage V–VI by measuring ¹⁴C-lactate uptake, both in the absence and in the presence of cytoskeletal-perturbing toxins. We found that the basal lactate transporter activity depends on the integrity of the cytoskeleton since it is partially inhibited by cytoskeleton disorganisation. Both PKA and PKC activation caused a significant decrease in transport activity and this decrease could be blocked by specific protein kinase inhibitors. The evidenced effects were not additive. Transport inhibition was annulled by agents that destabilize actin filaments or microtubules. We conclude that both protein kinases A and C, whose effects are mediated by cytoskeleton, negatively regulate the endogenous lactate transporter of Xenopus oocyte, suggesting that these kinases may have a role in the control of cytosolic pyruvate/lactate pool in the oocyte. All the experiments in this study comply with the current laws of Italy.     

G(alpha)s levels regulate Xenopus laevis oocyte maturation

Progesterone, produced by follicular cells, induces Xenopus laevis oocyte maturation through a very early event that inhibits the activity of the adenylyl cyclase effector system. The participation of a G-protein has been implicated, based on the fact that the inhibitory effect of the steroid is GTP-dependent, and it has been proposed that progesterone acts interfering with G(alpha)s function at the plasma membrane. Here we investigate whether the change in oocyte G(alpha)s levels affects the maturation process induced by progesterone. Overexpression of X. laevis wild type (wt) G(alpha)s and the constitutive activated G(alpha)s(QL) mutant, both blocked progesterone-induced maturation, G(alpha)s(QL) being much more effective than the wt protein. On the other hand, depletion of G(alpha)s, by the use of antisense oligonucleotides, caused spontaneous maturation measured as MAPK activation, indicating clearly that the presence of G(alpha)s is necessary to keep oocytes arrested. Overexpression of three different G-protein coupled receptors (GPCR), the beta2-adrenergic receptor and the m4 and m5 muscarinic receptors, all caused inhibition of MAPK activation induced by progesterone. These receptors, upon their activation with the respective ligands, might be inducing the release of G(beta)gamma from their respective G(alpha), which together with endogenous G(alpha)s-GTP, activate adenylyl cyclase. Our results indicate that G(alpha)s plays an important role in the maturation process and support previous findings of G(beta)gamma participation, suggesting the presence of a mechanism where a constitutively activated G(alpha)s subunit, together with the G(beta)gamma heterodimer, both maintain high levels of intracellular cAMP levels, blocking the G2/M transition.     

Impact of boron deficiency on Xenopus laevis: a summary of biological effects and potential biochemical roles

The toxicity of boron has been understood for many years. However, limited data currently exist concerning the nutritional essentiality of B in chordates. Results from an ongoing research program evaluating the nutritional essentiality of B in the South African clawed frog, Xenopus laevis, found that X. laevis fed a low-B diet in a low-B culture media produced a substantially higher number of necrotic eggs and fertilized embryos than frogs fed a boron-sufficient diet. Markedly decreased embryo cell counts at mid-blastula transition and an increased frequency of abnormal gastrulation were also noted in embryos from adult frogs fed the B-deficient diet. By 96 h of development, none of the larvae collected from the B-deficient adults and maintained in low-boron culture media developed normally. Reproductive effects associated with B deficiency in female Xenopus included ovary atrophy, oocyte necrosis, and incomplete oocyte maturation. In males, a decrease in testis weight and sperm count was noted. These studies suggest that these adverse effects resulting from B deficiency could be found during gametogenesis, gamete maturation embryonic development, and larval maturation. The studies also confirmed that B deficiency was capable of interrupting the X. laevis life cycle. Additional studies evaluating the role of B in the thyroid axis and the oocyte plasma membrane progesterone receptor provide the first line of direct evidence for a biochemical role of boron in X. laevis. Combined together, this research program provides firm evidence that B is nutritionally essential in X. laevis.     

Aurora-A kinase Ser349 phosphorylation is required during Xenopus laevis oocyte maturation

Xenopus laevis Aurora-A is phosphorylated in vivo onto three amino acids: Ser53, Thr295 and Ser349. The activation of the kinase depends on its autophosphorylation on Thr295 within the T-loop. The phosphorylation of Ser53 by still unknown kinase(s) prevents its degradation. The present work focused on the regulation of Aurora-A function via Ser349 phosphorylation. Mutagenesis of Ser349 to alanine (S349A) had few impact in vitro on the capability of the kinase to autophosphorylate as well as on its activity. These data in addition to in gel kinase assays and site-specific proteolytic digestion experiments prove that Ser349 is clearly neither a primary autophosphorylation site, nor an autophosphorylation site depending on the priming phosphorylation of Thr295. Using specific antibodies, we also show that the phosphorylation of Aurora-A Ser349 is a physiological event during Xenopus oocyte maturation triggered by progesterone. A peak of phosphorylation paralleled the decrease of Aurora activity observed between meiosis I and II. In response to progesterone, X. laevis stage VI oocytes microinjected with the Aurora-A S349A mutant proceeded normally to germinal vesicle breakdown (GVBD), but degenerated rapidly soon after. Since phosphorylation of Ser349 is responsible for a decrease in kinase activity, our results suggest that a down-regulation of Aurora-A activity involving Ser349 phosphorylation is required in the process of maturation.     

Modulation of O-GlcNAc glycosylation during Xenopus oocyte maturation

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a post-translational modification, which is believed antagonises phosphorylation. We have studied the O-GlcNAc level during Xenopus oocyte meiotic resumption, taking advantage of the high synchrony of this model which is dependent upon a burst of phosphorylation. Stimulation of immature stage VI oocytes using progesterone was followed by a 4.51 +/- 0.32 fold increase in the GlcNAc content, concomitantly to an increase in phosphorylation, notably on two cytoplasmic proteins of 66 and 97 kDa. The increase of O-GlcNAc for the 97 kDa protein, which we identified as beta-catenin was partly related to its accumulation during maturation, as was demonstrated by the use of the protein synthesis inhibitor--cycloheximide. Microinjection of free GlcNAc, which inhibits O-glycosylated proteins-lectins interactions, delayed the progesterone-induced maturation without affecting the O-GlcNAc content. Our results suggest that O-GlcNAc glycosylation could regulate protein-protein interactions required for the cell cycle kinetic.     

Spontaneous oocyte maturation in Rana pipiens: Estrogen and follicle wall involvement

Immature (germinal vesicle stage) Rana pipiens oocytes typically remain arrested in prophase I of meiosis even after extended periods of in-vitro culture, if not stimulated with hormones. We have, however, sporadically observed “spontaneous” occurrences of oocyte maturation in vitro without the addition of hormones. This study documents some of our observations on this phenomenon and presents experimental results concerning the effects and possible involvement of estrogen and follicle wall components in regulating spontaneous oocyte maturation. Estrogen was found to inhibit spontaneous oocyte maturation (GVBD) in a dose-dependent fashion. Follicles in which spontaneous maturation was inhibited by estrogen retained their responsiveness (GVBD) to both frog pituitary homogenate (FPH) and progesterone stimulation. Inhibitory effects of estrogen on spontaneous maturation, however, were not reversed following incubation of washed follicles in plain culture medium without added hormones. Possible involvement of progesterone synthesis in spontaneous oocyte maturation was ascertained by simultaneously monitoring endogenous progesterone synthesis and the occurrence of spontaneous GVBD over the course of the maturation process. In spontaneous maturing follicle there was a gradual increase in basal levels of progesterone synthesis that preceded GVBD. Significantly, addition of estrogen abolished both the spontaneous progesterone production and spontaneous oocyte maturation. When FPH was added to follicles exhibiting spontaneous oocyte maturation, progesterone production was augmented and the time course of oocyte maturation was greatly accelerated. Involvement of ovarian components in the maturation process was investigated by selective removal of various follicle layers by microdissection. Removal of follicle epithelium and theca layer (defolliculation) markedly decreased spontaneous and FPH-induced maturation, whereas removal of the entire follicle wall (denudation) completely blocked it. Our results suggest that both spontaneous and FPH-induced maturation involve an estrogen sensitive process in the follicle wall. Thus, somatic follicle cells appear to serve as a common mediator for both types of maturation, which are linked by some intrafollicular mechanism involving steroidogenesis. Hence, estrogen may play an important role as an endogenous intrafollicular regulator of oocyte meiotic maturation.     

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

Mixed 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.     

Possible mechanisms in the rearrangement of non-yolk cytoplasmic materials during maturation of theXenopus laevis oocyte

Using the monoclonal antibody (MoAb) Xa5B6 as probe, the authors examined the mechanisms of cytoplasmic rearrangement occurring during maturation of theXenopus oocyte. The antigen molecules recognized by the MoAb are arranged in radial striations of the oocyte cytoplasm. The radial striations were disorganized in vitro by progesterone treatment, and the antigen molecules were uniformly distributed, predominantly in the animal hemisphere. Even when the germinal vesicle was mechanically removed or when germinal vesicle breakdown was suppressed in a K⁺-free medium, progesterone induced a disorganization of the radial striations. This progesterone-induced disorganization was inhibited by the protein synthesis inhibitor cycloheximide. When full-sized oocytes were treated with cytochalasin B, the radial striations were also disorganized, but the antigen molecules did not disperse into the large mass. Colchicine treatment had little effect. Antigen molecules were no longer arranged in radial striations and were completely dispersed when the oocyte was simultaneously treated with both drugs. These results indicate that the two compartments in the oocyte cytoplasm, the yolk-free cytoplasm and yolk column, are organized by different types of cytoskeletal system. It is also suggested that the maturation-promoting factor (MPF) activated during progesterone-induced maturation disrupts these cytoskeletal systems and disorganizes the radial striations. Correspondence to: A.S. Suzuki     

Studies on the physiological function of spermine in the process of progesterone induced toad oocyte maturation

Spermidine or spermine but not putrescine inhibited progesterone induced Bufo bufo gargarizans oocyte maturation.The ID50 for spermine inhibition via intra -oocyte microinjection on maturation induced by progesterone was 6.8mM(100nl).Spermine could inhibit MPF induced toad oocyte maturation with a much higher ID50.A 55 kD protein was dephosphorylated during the process of progesterone induced oocyte maturation .Spermine selectively promoted the level of phosphorylation of this protein in both progesterone-stimulated and hormone-untreated oocytes.The extent of its dephosphorylation was fairly Correlated with the percentage of GVBD in the hormone stimulated oocytes.The level of endogenous spermine was reduced by 28% between the perod of 0.40 GVBD50 and 0.60 GVBD50,at which 55 kD protein was dephosphorylated.Spermine inhibited progesterone-stimulated protein synthesis in almost the same dose dependent manner as its inhititory effect on the hormone-induced maturation,The endogenous spermine regulated 55 kD protein dephosphorylation which may trigger the increase of protein dephosphorylation which may trigger the increase of protein synthesis and in turn promote the activation of MPF,It is possible that 55 kD protein may be one of the components of messenger ribonucleoprotein(mRNP) particles.     

Regulation of Src kinase activity during Xenopus oocyte maturation

Expression of constitutively active Src protein tyrosine kinase in Xenopus oocytes has been shown to accelerate oocyte maturation suggesting that Src may be involved in meiotic progression. However, meiotic regulation of endogenous Src kinase in oocytes has not been investigated in detail. To address this problem, we measured the activity, expression level, and phosphorylation state of the endogenous Xenopus Src (xSrc) and overexpressed xSrc mutants in the process of progesterone-induced oocyte maturation. We found that the enzyme is first transiently activated in the plasma membrane-containing fraction of oocytes within 3 min of progesterone administration. This event represents one of the earliest responses of oocytes to the hormone and should be related to triggering some early signaling pathways of maturation. Thereafter, xSrc activity increases again at the time of germinal vesicle breakdown (GVBD) and remains elevated till the completion of maturation. This elevation of xSrc activity is associated with a 2-fold increase of xSrc protein content in the absence of change in its specific activity and xSrc mRNA content. No significant changes in the phosphorylation state of C-terminal regulatory phosphotyrosine can be registered either in endogenous xSrc or in overexpressed kinase-negative and wild-type xSrc proteins during maturation. Altogether, these results indicate that upregulation of xSrc in the meiotic metaphase occurs at the translation level. We also demonstrate here that the expression of constitutively active xSrc in Xenopus oocytes is accompanied by the activation of mitogen-activated protein kinase (MAPK). Our data suggest that the Src kinase acts through the MAPK pathway to accelerate oocyte maturation.     

Minor-class splicing occurs in the nucleus of the Xenopus oocyte

A small fraction of premessenger RNA introns in certain eukaryotes is excised by the minor spliceosome, which contains low-abundance small nuclear ribonucleoproteins (snRNPs). Recently, it was suggested that minor-class snRNPs are localized to and function in the cytoplasm of vertebrate cells. To test whether U12-type splicing occurs in the cytoplasm of Xenopus oocytes, we performed microinjections of the well-characterized P120 minor-class splicing substrate into the nucleus or into the cytoplasm. Our results demonstrate that accurate splicing of this U12-dependent intron occurs exclusively in the nuclear compartment of the oocyte, where U12 and U6atac snRNPs are primarily localized. We further demonstrate that splicing of both a major-class and a minor-class intron is inhibited after nuclear envelope breakdown during meiosis.     

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.     

Casein kinase G may be the target of spermine during progesterone-induced oocyte maturation

Casein kinase G (CKG) with more than 2500-fold enrichraent was purified from Bufo bufo gargarizans ovaries. The catalytic activity of the enzyme was found to be associated with its 42 kD subunit, and its 26 kD subunit was found to be the major tsrget for the enzyme autophos phorylation. Each fuU-grown oocyte contained 1.9 units of CKG corresponding to an intracellular concentration of 93 nM. After injecting an amount of 0,38 units of the enzyme into the oocyte, approximately 50% of the progesterone-induoed maturation was inhibited. The inhibitory effect was enhanced in oocytes pretreated with spermine, which was consistent with the results that the enzyme was activated in vitro in the presence of spermine, The MPF-induced oocyte maturation was delayed and even prohibited in the kinase-microinjected oocytes. A 55 kD oocyte protein was identified as an suhstrate of CKG both in vivo and in vitro, and the enhancement of the 55 kD protein phosphory[ation was associated with kinase inhibition on maturation and on protein synthesis in kinase-microinjected oocytes. As the endogenous spermine level decreased in the course of progesteroneinduced oocyte maturation. 55 kD protein was dephosphorylated, Heparin, a specific inhibitor of CKG, potentiated the progesterone-induced oocyte maturation. Altogether the experimental reSults indicated Strongly that CKG may be the physiological target of spermine.     

A predominant basic alpha-tubulin isoform present in prophase Xenopus oocyte decreases during meiotic maturation.

Xenopus oocytes are blocked in prophase of the first meiotic division. During the G2/M transition drastic changes occur both in the cytoskeletal organization and in the capacity of tubulin to polymerize. Posttranslational modification of tubulin isoforms might be one of the factors that control the dynamic properties of microtubules. We have therefore analysed, by two-dimensional polyacrylamide gel electrophoresis, the isotubulins purified from Xenopus oocytes, and we show that tubulin is resolved into at least four alpha-isoforms and four beta-isoforms. We have identified a basic alpha (alpha b)-tubulin isoform which is specific to prophase arrested oocyte and that progressively disappears during meiotic maturation; its decrease is initiated when the nuclear envelope breaks down and is controlled by the nucleus. Using 35S methionine labelled oocytes we demonstrate that the disappearance of the alpha b isotubulin results from both an arrest of its biosynthesis after maturation, and from posttranslational modification which induces a shift of this alpha-isoform to a more acidic pI. Moreover, in vitro experiments using 35S prelabelled tubulin purified from prophase oocytes show that metaphase extracts containing MPF activity are able to induce the acidification of the alpha b-isoform, suggesting that the observed posttranslational modification might be regulated by p34cdc2. However, the nature of this modification remains to be elucidated.     

Calcium signaling differentiation during Xenopus oocyte maturation

Ca(2+) is the universal signal for egg activation at fertilization in all sexually reproducing species. The Ca(2+) signal at fertilization is necessary for egg activation and exhibits specialized spatial and temporal dynamics. Eggs acquire the ability to produce the fertilization-specific Ca(2+) signal during oocyte maturation. However, the mechanisms regulating Ca(2+) signaling differentiation during oocyte maturation remain largely unknown. At fertilization, Xenopus eggs produce a cytoplasmic Ca(2+) (Ca(2+)(cyt)) rise that lasts for several minutes, and is required for egg activation. Here, we show that during oocyte maturation Ca(2+) transport effectors are tightly modulated. The plasma membrane Ca(2+) ATPase (PMCA) is completely internalized during maturation, and is therefore unable to extrude Ca(2+) out of the cell. Furthermore, IP(3)-dependent Ca(2+) release is required for the sustained Ca(2+)(cyt) rise in eggs, showing that Ca(2+) that is pumped into the ER leaks back out through IP(3) receptors. This apparent futile cycle allows eggs to maintain elevated cytoplasmic Ca(2+) despite the limited available Ca(2+) in intracellular stores. Therefore, Ca(2+) signaling differentiates in a highly orchestrated fashion during Xenopus oocyte maturation endowing the egg with the capacity to produce a sustained Ca(2+)(cyt) transient at fertilization, which defines the egg's competence to activate and initiate embryonic development.     

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.