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.     

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.     

A Gbetagamma stimulated adenylyl cyclase is involved in Xenopus laevis oocyte maturation

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a nongenomic mechanism that implicates the inhibition of the effector system adenylyl cyclase (AC). Recently, it has been shown that the G protein betagamma heterodimer is involved in oocyte maturation arrest. Since AC is the proposed target for Gbetagamma action, we considered of importance to identify and characterize the Gbetagamma regulated AC isoform(s) that are expressed in the Xenopus oocyte. Through biochemical studies, we found that stage VI plasma membrane oocyte AC activity showed attributes of an AC2 isoform. Furthermore, exogenous Gbetagamma was capable to activate oocyte AC only in the presence of the activated form of Galphas (Galphas-GTPgammaS), which is in agreement with the Ggammabeta conditional activation reported for the mammalian AC2 and AC4 isotypes. In order to study the functional role of AC in oocyte maturation we cloned from a Xenopus oocyte cDNA library a gene encoding an AC with high identity to AC7 (xAC7). Based on this sequence, we constructed a minigene encoding the AC-Gbetagamma interacting region (xAC7pep) to block, within the oocyte, this interaction. We found that microinjection of the xAC7pep potentiated progesterone-induced maturation, as did the AC2 minigene. From these results we can conclude that a Gbetagamma-activated AC is playing an important role in Xenopus oocyte meiotic arrest in a Galphas-GTP dependent manner.     

Stimulation of Xenopus laevis oocyte maturation by methyl-beta-cyclodextrin

The cholesterol-depleting drug methyl-beta-cyclodextrin (Me-beta-CD) was tested for its effects on amphibian oocyte maturation, cholesterol depletion, and low-density membrane recovery. Progesterone-induced oocyte maturation was accelerated by pretreatment of cells with 5-50 mM Me-beta-CD in a dose-dependent manner. Treatment of oocytes with 50 mM Me-beta-CD alone was sufficient to induce germinal vesicle breakdown, stimulate formation of meiotic spindles, and stimulate phosphorylation of mitogen-activated protein kinase over time courses longer than those observed after progesterone treatment. After short-term (30 min) labeling of oocytes with [(3)H]cholesterol, 30-90 min of treatment with 5-50 mM Me-beta-CD removed 50%-70% of cell- associated label, and cholesterol depletion was not observed with alpha-cyclodextrin. After long-term (20-23 h) labeling of oocytes with [(3)H]cholesterol, Me-beta-CD treatment resulted in dose- dependent cholesterol depletion in the 5-50 mM range, and 50 mM Me-beta-CD removed approximately 50% of cell-associated label after 9 h. Treatment of oocytes with 5-50 mM Me-beta-CD also decreased recovery of low-density membrane by detergent-free sucrose gradient centrifugation. These results implicate cholesterol and low-density membrane domains in the signaling mechanisms leading to germinal vesicle breakdown in amphibian oocytes.     

Boron deficiency disables Xenopus laevis oocyte maturation events

Processes of oocyte maturation that may be affected by boron (B) deficiency were studied to potentially determine a possible biochemical role of B in the Xenopus laevis oocyte. More specifically, the Xenopus oocyte membrane progesterone receptor (OMPR) in B-deficient oocytes was characterized by evaluating progesterone affinity for the OMPR and OMPR responsiveness to progesterone stimulation. The responsiveness of B-deficient oocytes to microinjection of a purified oocyte cytoplasmic fraction (OCF) from B-adequate oocytes was also studied to evaluate which aspects of the maturation process were affected by B deficiency. Results suggested that B deficiency resulted in incomplete oocyte maturation and that maturation could not be induced by the administration of exogenous progesterone. Progesterone successfully induced germinal vesicle breakdown (GVBD) in oocytes from females fed a B-supplemented diet (+B) and females administered a traditional diet of beef liver and lung (B adequate). Addition of exogenous B to the -B oocytes increased the rate of progesterone-induced GVBD slightly. The B-deficient X. laevis oocytes were capable of undergoing GVBD when endogenously stimulated by microinjected purified B-adequate OCF. These results indicated that the inability of the B-deficient oocytes to undergo GVBD was not associated with the cytoplasmic induction process specifically, but possibly in the progesterone receptor or signal transduction pathways. Radio-binding studies found that progesterone binding to the B-deficient OPMR was greatly reduced compared to B-adequate or B-supplemented OMPR. Moreover, washout studies determined that progesterone binding to the OMPR in B-deficient oocytes was more transient than the B adequate or +B oocytes.     

Influence of cytochalasin B on oocyte maturation in Xenopus laevis

Cytochalasin B (CB) exerts an inhibiting effect on the formation, migration and anchoring in the cortex of the meiotic spindle in maturing Xenopus laevis oocytes. Regional sensitivity to CB (CB-sensitive zones) has been found in the oocytes which varies with reference to the stage of oocyte maturation at which CB is applied. Light and electron microscopy has shown that in these CB-sensitive zones the yolk and pigment granules, unlike the cortical ones, are displaced into the cytoplasm centripetally under the influence of CB.     

The development of competence for meiotic maturation during oogenesis in Xenopus laevis

Meiotic maturation of large, 1.2-1.4 mm in diameter, stage VI oocytes of Xenopus laevis can be induced to mature in vitro by exposure to progesterone or by microinjection of maturation-promoting factor (MPF). Small, 0.95 mm in diameter, stage IV oocytes do not respond to progesterone but do undergo germinal vesicle breakdown (GVBD) in response to microinjection of MPF. The possibility that small oocytes are nonresponsive to progesterone due to a specific defect in an event known to occur with large oocytes is investigated. Both large and small oocytes possess a plasma membrane steroid receptor (Mr = 110,000) as measured by photoaffinity labeling with [3H]R5020, but the density of receptors in small oocytes is only 20% of that in large oocytes. Adenylate cyclase activity stimulated by guanyl-5'-yl-imidodiphosphate is equally inhibited by steroid (50%) in plasma membranes from both large and small oocytes with an apparent IC50 of 2 X 10(-7) M progesterone. Microinjection of the heat-stable inhibitor protein of cAMP-dependent protein kinase induces GVBD in large but not in small oocytes. These results indicate that the nonresponsiveness of small, stage IV oocytes to progesterone is due to a deficiency in an event(s) subsequent to cAMP fluctuations but prior to MPF action.     

The subcellular localization of cyclin B2 is required for bipolar spindle formation during Xenopus oocyte maturation

Cyclins B1 and B2 are subtypes of cyclin B, a regulatory subunit of a maturation/M-phase promoting factor, and they are also highly conserved in many vertebrate species. Cyclin B1 is essential for mitosis, whereas cyclin B2 is regarded as dispensable. However, the overexpression of the cyclin B2 N-terminus containing the cytoplasmic retention signal, but not cyclin B1, inhibits bipolar spindle formation in Xenopus oocytes and embryos. Here we show that endogenous cyclin B2 was localized in and around the germinal vesicle. The perinuclear localization of cyclin B2 was perturbed by the overexpression of its N-terminus containing the cytoplasmic retention signal, which resulted in a spindle defect. This spindle defect was rescued by the overexpression of bipolar kinesin Eg5, which is located at the perinuclear region in the proximity of endogenous cyclin B2. These results demonstrate that the proper localization of cyclin B2 is essential for bipolar spindle formation in Xenopus oocytes.     

New B-type cyclin synthesis is required between meiosis I and II during Xenopus oocyte maturation

Progression through meiosis requires two waves of maturation promoting factor (MPF) activity corresponding to meiosis I and meiosis II. Frog oocytes contain a pool of inactive "pre-MPF" consisting of cyclin-dependent kinase 1 bound to B-type cyclins, of which we now find three previously unsuspected members, cyclins B3, B4 and B5. Protein synthesis is required to activate pre-MPF, and we show here that this does not require new B-type cyclin synthesis, probably because of a large maternal stockpile of cyclins B2 and B5. This stockpile is degraded after meiosis I and consequently, the activation of MPF for meiosis II requires new cyclin synthesis, principally of cyclins B1 and B4, whose translation is strongly activated after meiosis I. If this wave of new cyclin synthesis is ablated by antisense oligonucleotides, the oocytes degenerate and fail to form a second meiotic spindle. The effects on meiotic progression are even more severe when all new protein synthesis is blocked by cycloheximide added after meiosis I, but can be rescued by injection of indestructible B-type cyclins. B-type cyclins and MPF activity are required to maintain c-mos and MAP kinase activity during meiosis II, and to establish the metaphase arrest at the end of meiotic maturation. We discuss the interdependence of c-mos and MPF, and reveal an important role for translational control of cyclin synthesis between the two meiotic divisions.     

Eya1 protein distribution during embryonic development of Xenopus laevis

Eya1 and other Eya proteins are important regulators of progenitor proliferation, cell differentiation and morphogenesis in all three germ layers. At present, most of our knowledge of Eya1 distribution is based on in situ hybridization for Eya1 mRNA. However, to begin to dissect the mechanisms underlying Eya1 functions, we need a better understanding of the spatiotemporal distribution of Eya1 proteins during embryonic development, their subcellular localization and their levels of expression in various tissues. Here we report the localization of Eya1 protein throughout embryonic development from neural plate stages to tadpole stages of Xenopus laevis using a specific antibody for Xenopus Eya1. Our study confirms the expression of Eya1 protein in cranial placodes, placodally derived sensory primordia (olfactory epithelium, otic vesicle, lateral line primordia) and cranial ganglia, as well as in somites, secondary heart field and pharyngeal endoderm. In addition, we report here a novel expression of Eya1 proteins in scattered epidermal cells in Xenopus. Our findings also reveal that, while being predominantly expressed in nuclei in most expression domains, Eya1 protein is also localized to the cytoplasm, in particular in the early preplacodal ectoderm, some placode-derived ganglia and a subset of epidermal cells. While some cytoplasmic roles of Eya1 have been previously described in other contexts, the functions of cytoplasmic Eya1 in the preplacodal ectoderm, cranial ganglia and epidermal cells remain to be investigated.     

Regulation of the Aurora B chromosome passenger protein complex during oocyte maturation in Xenopus laevis

The dynamics of the Aurora B protein kinase during Xenopus oocyte meiotic maturation were examined. Resting G2 oocytes express inactive Aurora B that is not associated with other subunits of the chromosome passenger complex (CPC). Activity increases near the time of germinal vesicle breakdown in progesterone-treated oocytes, and this increase is correlated with the synthesis of inner centromere protein (INCENP) and survivin, components of the CPC. Ablation of INCENP synthesis led to the failure of progesterone treatment to activate Aurora B, but biochemical progression through the meiosis I-to-II transition and arrest at metaphase II were not affected. At fertilization, Aurora B was deactivated in concert with the degradation of INCENP, and the levels of Aurora B kinase activity and INCENP oscillated in subsequent embryonic cell cycles. Prevention of the decrease in Aurora B activity at fertilization by expression of ectopic wild-type INCENP, but not kinase-dead Aurora B INCENP, blocked calcium-induced exit from metaphase arrest in egg extracts.     

Characterization of the O-GlcNAc protein modification in Xenopus laevis oocyte during oogenesis and progesterone-stimulated maturation

Little information exists about single N-acetylglucosamine modifications on proteins in growth and developmental model systems. To explore these phenomena, Xenopus laevis oocytes from stages I-VI of oogenesis were isolated and proteins analyzed on SDS-PAGE. The proteins were probed with antibodies specific for O-GlcNAc. Levels of the O-GlcNAc protein modification were highest in stages I and II, while decreasing in stages III-VI. The reduction in amount of O-GlcNAc-modified proteins was correlated to increases in apparent O-GlcNAcase (streptozotocin-inhibitable neutral hexosaminidase), activity involved in removing protein monoglycosylations. The O-GlcNAc modification was also characterized during progesterone-stimulated oocyte maturation. Although O-GlcNAcase activity appeared relatively constant between quiescent and matured stage VI oocytes, a small decrease in the levels of both total and specific O-GlcNAc-modified proteins was observed. Investigating the function of O-GlcNAc during maturation, oocytes were incubated with compounds known to modulate the levels of the O-GlcNAc protein modification and then stimulated to mature. Oocytes treated with compounds known to increase O-glycosylation consistently matured slower than non-treated controls, while oocytes treated with compounds that decrease O-glycosylation matured slightly faster than controls. The O-GlcNAc modification may play important roles in both the developmental and cell division processes of X. laevis oocytes.