Observations on intracellular pH during cleavage of eggs of Xenopus laevis

Direct measurements of intracellular pH was made with recessed-tip pH microelectrodes in fertilized eggs of the frog, Xenopus laevis, from approximately 1 h after fertilization to mid-blastula. The intracellular pH just before first cleavage was 7.65 +/- 0.04 (SD; n = 9). By stage 5 to the middle of stage 6, average intracellular pH was 7.70 +/- 0.06 (SD; n = 16). A statistically significant alkalization of 0.18 +/- 0.03 pH unit (SD; n = 5) was observed beginning in early blastula. A cycle of less than or equal to 0.05 pH unit was occasionally observed during the pre-blastula period, but its significance is unknown. By exposing the early cleavage embryo to saline buffered with sodium propionate, pH 4.7-5.0, it was possible to lower intracellular pH with some degree of control. Apparently, normal cleavage continued to occur when intracellular pH had been forced as much as 0.3 unit below normal. We conclude that this implies no specific involvement of intracellular pH in mitosis and cytokinesis. If intracellular pH was lowered further, cell division ceased at about pH 7.2, and furrow regression began at about pH 7.0. Once furrow regression occurred, subsequent development was usually arrested or abnormal when the embryo was transferred back to normal saline.     

A single-pool model for intracellular calcium oscillations and waves in the Xenopus laevis oocyte.

We construct a minimal model of cytosolic free Ca2+ oscillations based on Ca2+ release via the inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channel (IP3R) of a single intracellular Ca2+ pool. The model relies on experimental evidence that the cytosolic free calcium concentration ([Ca2+]c) modulates the IP3R in a biphasic manner, with Ca2+ release inhibited by low and high [Ca2+]c and facilitated by intermediate [Ca2+]c, and that channel inactivation occurs on a slower time scale than activation. The model produces [Ca2+]c oscillations at constant [IP3] and reproduces a number of crucial experiments. The two-dimensional spatial model with IP3 dynamics, cytosolic diffusion of IP3 (Dp = 300 microns 2 s-1), and cytosolic diffusion of Ca2+ (Dc = 20 microns 2 s-1) produces circular, planar, and spiral waves of Ca2+ with speeds of 7-15 microns.s-1, which annihilate upon collision. Increasing extracellular [Ca2+] influx increases wave speed and baseline [Ca2+]c. A [Ca2+]c-dependent Ca2+ diffusion coefficient does not alter the qualitative behavior of the model. An important model prediction is that channel inactivation must occur on a slower time scale than activation in order for waves to propagate. The model serves to capture the essential macroscopic mechanisms that are involved in the production of intracellular Ca2+ oscillations and traveling waves in the Xenopus laevis oocyte.     

Synthesis and activity of Xenopus laevis oocyte tyrosinase

This study investigates the mechanisms that control pigment synthesis in Xenopus laevis oocytes. Although we find the molecular weight of oocyte tyrosinase to be similar to that of amphibian skin, we were unable to increase its activity by proteases or detergents, as has been reported for skin tyrosinase. On the other hand, by measuring the activity of polysomal-bound enzyme, we were able to correlate increased tyrosinase activity with increased levels of enzyme synthesis. We therefore suggest that in oocytes, the activity of tyrosinase is primarily dependent on its synthesis, whereas in skin, the rate-limiting step is the post-translational activation of the enzyme. We speculate on these differences in relation to the functional role of melanin in skin and oocytes.     

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.     

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.     

A pre-export U1 snRNP in Xenopus laevis oocyte nuclei.

We demonstrate that precursors of U1 snRNA are associated with nuclear proteins prior to export to the cytoplasm. The approximately 15S complexes containing pre-U1 RNA, which we call pre-export U1 snRNPs, were identified in extracts of Xenopus laevis oocyte nuclei that were synthesizing U1 RNAs from injected U1 genes. The U1 snRNP-specific A protein was associated with nuclear pre-U1 RNA since both this protein and the RNA were co-precipitated by antibodies directed against either the m7G-cap of the precursor RNA or the U1-A protein. The interaction of the U1-A protein with pre-U1 RNA required sequences in the loop II region although this region of U1 RNA was not necessary for the association of U1 A protein with mature U1 snRNPs. The U1 A protein helps protect pre-U1 RNA against degradation in the nucleus.     

A comparison of Xenopus laevis oocyte and embryo mRNA

Total RNA, extracted from mature oocytes and tadpoles of Xenopus laevis, was used as a template for in vitro protein synthesis. The oocyte RNA is markedly deficient in abundant mRNA species by comparison to tadpole RNA or other somatic RNAs, in agreement with previous experiments using RNA-cDNA hybridization analysis (S. Perlman and M. Rosbash, 1978, Develop. Biol.63, 197–212). Oocyte pA⁺ RNA is also larger than tadpole pA⁺ RNA or other somatic pA⁺ populations. The larger oocyte pA⁺ RNA and smaller oocyte pA⁺ RNA are equally good templates for in vitro protein synthesis, which implies that much, and perhaps all, of the large oocyte pA⁺ RNA is bona fide mRNA. We suggest that the relatively large size of the oocyte pA⁺ RNA population is due, at least in part, to the relative lack of abundant mRNA species in the population. This suggestion follows from the observation of 0. Meyuhas and R. P. Perry (1979, Cell16, 139–148) that L-cell-abundant mRNAs are preferentially small and rare mRNAs preferentially large. Most of the oocyte pA⁺ sequences are also present in tadpoles and are still adenylated at this stage. Oocyte proteins synthesized in vivo do not appear deficient in abundant proteins, suggesting that a translational control mechanism operates to select certain pA⁺ RNAs at higher frequencies than others.     

Isolation and characterization of a monosialosylgangliopentaosyl ceramide from Xenopus laevis oocyte.

A monosialosylgangliopentaosyl ceramide was isolated from Xenopus laevis oocytes. It represented 5.8% of the total acidic glycosphingolipids. From the results of sugar-composition analysis, enzymatic hydrolysis, permethylation analysis, and negative ion fast atom bombardment mass spectrometry, the structure of the ganglioside was determined to be as follows: [sequence: see text] The predominant species of fatty acids were alpha-hydroxy fatty acids, h22:0, h24:0, and h24:1. The long chain bases of this ganglioside consisted mainly of d18:1 sphingosine and phytosphingosine. Other acidic glycolipids were also characterized. The most abundant component of acidic glycolipids was sulfatide, which represented 85.7% of the total acidic glycolipid mixture. GM3, GM2, GM1a, and GD1a were also detected.     

A DNA binding protein from Xenopus laevis oocyte mitochondria

A DNA binding protein has been isolated, by affinity chromatography on DNA cellulose, from mitochondria and from purified mitDNA-protein complexes from oocytes of Xenopus laevis. This 12,500 daltons protein is polymeric in its native form and binds to DNA with a high efficiency. It exhibits an apparently preferential binding to the single-stranded fiber of the D loop structures.     

Regulation of heart size in Xenopus laevis

The region with the potential to form the heart has traditionally been called the heart field. This region can be approximated by, but is not identical to, the expression domain of the early cardiac gene Nkx2.5. The region expressing Nkx2.5 does not change in size, although there are major shape changes and a subdivision of the region into non-myogenic and myogenic lineages. Using a variety of embryo manipulations, we have sought to determine whether cellular interactions could change the size of the initial Nkx2.5-expressing region and thus change the size of the heart. We have shown that if the heart is isolated from the dorsal half of the embryo, the volume of tissue expressing myocardial differentiation markers increases, indicating that signals restricting the size of the heart come from the dorsal side. Despite the change in myocardial volume, the non-myogenic heart lineages are still present. The ability of dorsal tissues to restrict the size of the heart is further demonstrated by fusing two Xenopus embryos shortly after gastrulation, generating twinned embryos where the heart of one embryo would develop adjacent to different tissues of the second embryo. The final size of the differentiated heart was markedly reduced if it developed in close proximity to the dorso-anterior surface of the head but not if it developed adjacent to the flank or belly. In all cases, the manipulations that restricted the size of the myocardium also restricted the expression of Nkx2.5 and GATA-4, both key regulatory genes in the cardiogenic pathway. These results provide evidence for a model in which signals from dorso-anterior tissues restrict the size of the heart after gastrulation but before neural fold closure.     

Regulation of intracellular pH

The approach that most animal cells employ to regulate intracellular pH (pH(i)) is not too different conceptually from the way a sophisticated system might regulate the temperature of a house. Just as the heat capacity (C) of a house minimizes sudden temperature (T) shifts caused by acute cold and heat loads, the buffering power (beta) of a cell minimizes sudden pH(i) shifts caused by acute acid and alkali loads. However, increasing C (or beta) only minimizes T (or pH(i)) changes; it does not eliminate the changes, return T (or pH(i)) to normal, or shift steady-state T (or pH(i)). Whereas a house may have a furnace to raise T, a cell generally has more than one acid-extruding transporter (which exports acid and/or imports alkali) to raise pH(i). Whereas an air conditioner lowers T, a cell generally has more than one acid-loading transporter to lower pH(i). Just as a house might respond to graded decreases (or increases) in T by producing graded increases in heat (or cold) output, cells respond to graded decreases (or increases) in pH(i) with graded increases (or decreases) in acid-extrusion (or acid-loading) rate. Steady-state T (or pH(i)) can change only in response to a change in chronic cold (or acid) loading or chronic heat (or alkali) loading as produced, for example, by a change in environmental T (or pH) or a change in the kinetics of the furnace (or acid extrudes) or air conditioner (or acid loaders). Finally, just as a temperature-control system might benefit from environmental sensors that provide clues about cold and heat loading, at least some cells seem to have extracellular CO(2) or extracellular HCO(3)(-) sensors that modulate acid-base transport.     

Evidence for intracellular sodium pumps in permeabilized Xenopus laevis oocytes.

Ouabain binding was studied in Xenopus laevis oocytes permeabilized by detergents. The behaviour of markers showed that 10 microM-digitonin selectively disrupts the plasma membrane. In the presence of ATP, oocytes permeabilized at 10 microM-digitonin bound no more ouabain molecules than were required to abolish active 86Rb+ uptake in the intact cells. However, the ouabain binding capacity increased approx. 2-fold when inner membranes were disrupted by SDS or excess digitonin, as judged from the accompanying release of the lysosomal marker beta-hexosaminidase. The results suggest that oocytes have a large internal pool of functional sodium pumps.     

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.     

Immunological relationship between oocyte nuclear proteins of Xenopus laevis and X. borealis

Monoclonal antibodies (mABs) have been raised against oocyte nuclear proteins of Xenopus laevis and X. borealis and have been screened for species specificity. Although about 40% of all germinal vesicle polypeptides differ between the two species as judged by two-dimensional gel analysis, most mABs react with polypeptides of both species. Biochemical analysis of the antigens by immunoblotting revealed that a homologue of each antigen of one species could be detected in the other species, despite frequent differences in molecular structure. Nevertheless, five strictly species-specific mABs have been identified. All five are directed against the same acidic polypeptide B3 of X. borealis, which is a structurally altered homologue of the protein N1, previously described in X. laevis germinal vesicles. In oocyte nuclei of hybrids between X. laevis females and X. borealis males, polypeptide of both species appear to be accumulated equivalently. Exceptions to this rule are most easily explained by differences between individuals and by the loss of certain alleles resulting from the cross.     

The effect of myo-inositol 1,4,5-trisphosphorothioate on Cl- current pattern and intracellular Ca2+ in the Xenopus laevis oocyte.

Microinjection of myo-inositol 1,4,5-trisphosphate into voltage-clamped Xenopus laevis oocytes or the stimulation of the phosphatidylinositol cycle elicits a complex Ca2(+)-dependent Cl- current pattern. Microinjection of myo-inositol 1,3,4,5-tetrakisphosphate causes an immediate release of Ca2+, but elicits a different Cl- current pattern than myo-inositol 1,4,5-trisphosphate. We have studied the effects of myo-inositol 1,4,5-trisphosphorothioate, which can not be converted to myo-inositol 1,3,4,5-tetrakisphosphate. Myo-inositol 1,4,5-trisphosphorothioate caused an immediate release of intracellular Ca2+, as measured by fura-2 imaging. Myo-inositol 1,4,5-trisphosphorothioate generated a Cl- current pattern similar to myo-inositol 1,3,4,5-tetrakisphosphate, not myo-inositol 1,4,5-trisphosphate.     

Transcriptional activity in previtellogenic oocyte germinal vesicles from Xenopus laevis

Receptor interactions of parotid acinar cells with beta-agonists are mediated by cyclic 3',5'-monophosphate (cAMP) and expressed as cAMP-dependent protein kinase (cAPK) activation. In addition to its location in the cytoplasm, we have shown that cAPK is associated with the nuclear non-histone protein (NHP) fraction (0.35 M NaCl extract) of rat parotid acinar cells. Nuclei were prepared from isolated parotid acini with minimal contamination from other cell types or cytoplasmic components. The nuclear cAPK activity was inhibited by the thermostable inhibitor and was stimulated by the addition of exogenous cAMP to the assay, indicating that the enzyme is present in the holoenzyme form. Enzyme activity was not increased in the presence of detergent, suggesting that cAPK is not bound to the nuclear membrane. Photoaffinity-labeling studies with an 8-azido analog of cAMP showed that regulatory subunits of both type I and type II cAPK isozymes are present in parotid cell nuclei. Short-term in vitro stimulation of the acini with 10(-6) M isoproterenol did not alter cAPK activity in the nuclear fraction. These findings indicate that compartmentation of cAPK into nuclear and extranuclear locations in rat parotid acinar cells is similar to that of several other cell types which are responsive to hormonal stimulation.     

Nucleotide excision repair in oocyte nuclear extracts from Xenopus laevis

We have developed efficient DNA repair extracts derived from the unusually large nuclei of Xenopus oocytes. These extracts use nucleotide excision repair (NER) to completely remove bulky adducts from DNA. There is very little or no synthesis on control, undamaged DNA, indicating the extracts do not have significant nonspecific nuclease activity, and repair of cyclobutane pyrimidine dimers (CPDs) occurs in the dark, indicating that NER, and not photolyase, is responsible for CPD repair. The extracts can be inactivated with antibodies specific to repair proteins and then repair activity can be restored by adding purified recombinant protein. Here we describe detailed protocols for preparing Xenopus nuclear repair extracts.