Actin in Xenopus oocytes

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.     

Hyperpolarization-activated chloride currents in Xenopus oocytes

During hyperpolarizing pulses, defolliculated Xenopus oocytes have time- and voltage-dependent inward chloride currents. The currents vary greatly in amplitude from batch to batch; activate slowly and, in general, do not decay; have a selectivity sequence of I- > NO3- > Br- > Cl- > propionate > acetate; are insensitive to Ca2+ and pH; are blocked by Ba2+ and some chloride channel blockers; and have a gating valence of approximately 1.3 charges. In contrast to hyperpolarization- activated chloride currents induced after expression of phospholemman (Palmer, C. J., B. T. Scott, and L. R. Jones. 1991. Journal of Biological Chemistry. 266:11126; Moorman, J. R., C. J. Palmer, J. E. John, J. E. Durieux, and L. R. Jones. 1992. 267:14551), these endogenous currents are smaller; have a different pharmacologic profile; have a lower threshold for activation and lower voltage- sensitivity of activation; have different activation kinetics; and are insensitive to pH. Nonetheless, the endogenous and expressed current share striking similarities. Recordings of macroscopic oocyte currents may be inadequate to determine whether phospholemman is itself an ion channel and not a channel-modulating molecule.     

Mechanism of chromatin assembly in Xenopus oocytes

We have analyzed the chromatin assembly reaction catalyzed by the Xenopus oocyte extract (S-150). A 50 S complex is formed upon mixing the 17 S pUC DNA and the S-150. Mature histones are not detected in this complex, which contains relaxed DNA and protein, and generates subnucleosomal 7 S particles upon digestion with micrococcal nuclease. The relaxed nucleoprotein is gradually supercoiled into nucleosomal chromatin in the S-150, via a pathway that requires ATP and is blocked by novobiocin, and this process is accompanied by the appearance of mature histones H3 and H4. Isolated complexes also supercoil in vitro, which implies the complex is a kit that contains histone precursors, as well as topoisomerases and other enzymes required for assembly. We discuss the biological implications of these findings.     

Cytoskeleton in Xenopus oocytes and eggs

The Xenopus egg is a huge cell divided into compartments with distinct characteristics. The organization of the cytoskeleton reflects both the size of the egg and its regional differences. We review the information concerning the deployment and function of cytoskeletal elements during the changes in cellular organization accompanying oogenesis, oocyte maturation, and following fertilization.     

Synthesis of carp proinsulin in Xenopus oocytes

Total poly(A)-containing RNA from Brockmann boides of carp (Cyprinus carpio) directs the synthesis of authentic carp proinsulin in Xenopus oocytes. Neither preproinsulin nor further processing of the proinsulin to insulin can be detected in the oocytes.     

Induction of membrane excitability in Xenopus oocytes

Immature oocytes from the African toad Xenopus laevis are not known to be excitable cells, which means that they do not generate an action potential in response to small depolarizations. However, a regenerative response is produced if successive depolarizing currents of large magnitude are applied to the oocyte membrane. This response is characterized by the occurrence of a positive transmembrane potential that can last for several minutes. The opening of voltage-dependent channels, highly selective for sodium ions, underlies the depolarization thus obtained. These channels exhibit unconventional electrophysiological and pharmacological properties, which set them apart from other types of voltage-dependent sodium channels found in excitable tissues. The opening of the oocyte sodium channels is a complex process, which includes an induction phase. During this phase, the channels change from an electrical state of inexcitability into an excitable voltage-dependent state. The induction mechanism is modulated by the temperature of the bathing medium, by the activation of enzymes (namely a phospholipase C and a protein kinase C) and by the release of calcium ions from intracellular phosphatidyl-inositol trisphosphate stores. The results summarized in this review point out the possible role that these sodium channels may play in the physiology of the oocyte.     

Cap-independent translation initiation in Xenopus oocytes.

Eukaryotic cellular mRNAs contain a cap at their 5'-ends, but some viral and cellular mRNAs bypass the cap-dependent mechanism of translation initiation in favor of internal entry of ribosomes at specific RNA sequences. Cap-dependent initiation requires intact initiation factor eIF4G (formerly eIF-4gamma, eIF-4Fgamma or p220), whereas internal initiation can proceed with eIF4G cleaved by picornaviral 2A or L proteases. Injection of recombinant coxsackievirus B4 protease 2A into Xenopus oocytes led to complete cleavage of endogenous eIF4G, but protein synthesis decreased by only 35%. Co-injection of edeine reduced synthesis by >90%, indicating that eIF4G-independent synthesis involved ongoing initiation. The spectrum of endogenous proteins synthesized was very similar in the presence or absence of intact eIF4G. Translation of exogenous rabbit globin mRNA, by contrast, was drastically inhibited by eIF4G cleavage. The N-terminal cleavage product of eIF4G (cpN), which binds eIF4E, was completely degraded within 6-12 h, while the C-terminal cleavage product (cpC), which binds to eIF3 and eIF4A, was more stable over the same period. Thus, translation initiation of most endogenous mRNAs inXenopusoocytes requires no eIF4G, or perhaps only cpC, suggesting a cap-independent mechanism.     

Gonadotropin-releasing hormone receptor expression in Xenopus oocytes

The rodent GnRH receptor was characterized in Xenopus oocytes injected with RNA isolated from rat pituitary and from a gonadotrope cell line, alpha T3, derived from a transgenic mouse. Three to 4 days after 150-200 ng RNA injection, 93% of the oocytes, which were recorded by voltage clamp, responded to 10(-7) M GnRH. The mean inward currents obtained after RNA injection were 620 +/- 88 nA (n = 22) with pituitary RNA and 1415 +/- 598 (n = 4) with alpha T3 RNA. The threshold GnRH concentration able to evoke the dose dependent current after pituitary RNA injection was 3 x 10(-9) M GnRH. The GnRH receptor response of the oocyte was antagonized by [D-Phe2,6,Pro3] GnRH and [N-Ac-D-Na](2)1, D-alpha D-Me, pCl-Phe2, D-Arg6, D-Ala10-NH2]GnRH and could be elicited by D-Ser(But)6,Pro9-N-ethylamide GnRH (buserelin). The reversal potential of the GnRH generated current as determined by voltage-ramp was -22.5 +/- 1.0 mV (n = 7) and -25.6 +/- 3.3 mV (n = 3) in pituitary and cell line RNA-injected oocytes respectively, consistent with the chloride reversal potential. The GnRH receptor response was virtually eliminated by intracellular EGTA injection but was unaffected by ligand application in calcium-free perfusate. The GnRH-evoked response is mimicked by intracellular injection of inositol 1,4,5-trisphosphate. To determine the size of the GnRH receptor mRNA, alpha T3 RNA was size fractionated through a sucrose gradient. The maximal GnRH response was induced by a fraction larger than the 28S ribosomal peak. Thus we find that oocytes injected with RNA from an appropriate source develop an electrophysiological response to GnRH which is dependent on intracellular calcium mobilization, is independent of extracellular calcium, and may be mediated by inositol 1,4,5-trisphosphate.     

Effect of lipid hydroperoxide on Xenopus oocytes and on neurotransmitter receptors synthesized in Xenopus oocytes injected with exogenous mRNA

The effect of 13-L-hydroperoxylinoleic acid (LOOH) on both Xenopus oocytes and neurotransmitter receptors synthesized in the oocytes was studied by electrophysiological and ion flux measurement. Addition of LOOH to the incubation mixture of the oocytes raised the membrane potential and decreased the membrane resistance of the oocytes. These effects of LOOH on the oocytes were reversed within a few hours by incubation with frog Ringer solution. Addition of LOOH also caused an increase of Li+ and 45Ca2+ uptake into the oocytes. However, production of alkoxy radicals by the addition of FeCl2 to the incubation mixture containing LOOH did not accelerate the damage to the oocytes by LOOH. So essential toxicity is caused possibly by an increase in the membrane permeability resulting from disturbance of the lipid bilayer arrangement, not from production of active alkoxy radicals during decomposition of LOOH. Nicotinic acetylcholine and gamma-aminobutyric acid receptors were synthesized in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and rat brain. LOOH noncompetitively inhibited the function of these receptors and also increased the rate of desensitization of the receptors.     

Tetrodotoxin-sensitive sodium current in native Xenopus oocytes

Depolarization of oocytes of Xenopus laevis usually elicits mainly passive currents, and a calcium-dependent chloride current. However, oocytes obtained from some donors show, in addition, a transient inward current on depolarization to potentials beyond ca. -40 mV. This current is abolished by tetrodotoxin at submicromolar concentrations, and is prolonged by veratrine; thus, it probably arises through sodium channels of a type similar to those found in nerve and muscle cells. However, the kinetics of the sodium currents varied between oocytes from different donors; this result suggests that genes encoding different sodium channels may be expressed in oocytes from different donors. The presence of these native channels may complicate experiments to study the expression of exogenous sodium channels encoded by foreign messenger RNAs injected into the oocyte.     

Transient potassium current in native Xenopus oocytes

Depolarization of follicle-enclosed oocytes of Xenopus laevis obtained from some donors elicits, in addition to other responses, a fast transient outward current. After holding the membrane potential at -100 mV this response begins to be activated by depolarizations to around -30 mV, and increases progressively as the voltage is raised further. A striking characteristic is that the current recovers only slowly (several seconds) from inactivation following a depolarizing pulse. Because of its outward direction and insensitivity to removal of extracellular chloride or addition of tetrodotoxin, the current probably arises largely through a flux of potassium ions. The current was abolished after treatment of oocytes with collagenase to remove enveloping cells, and although it was blocked by barium and zinc ions, tetraethylammonium was relatively ineffective. In addition, the potassium current was unaffected by 5 mM manganese, suggesting that it does not arise as a consequence of an influx of calcium into the oocyte.     

Polyadenylic acid-containing RNA in Xenopus laevis oocytes

The quantity of poly(A)-containing RNA is measured in Xenopus laevis oocytes as a function of developmental stage. The amount of poly(A)-containing RNA per oocyte, 0.7 to 1.0% of the total RNA, remains relatively constant from early vitellogenesis until ovulation. It is largely present in the cytoplasm of the oocyte in the form of a ribonucleoprotein complex. The poly(A) sequence is approximately 100 bases in length and is attached to molecules of heterogeneous sedimentation coefficients.     

Microinjected GTP-gamma-S inhibits progesterone-induced maturation of Xenopus oocytes

GTP-gamma-S inhibits progesterone-induced maturation of Xenopus laevis oocytes and induces a rise in their cAMP levels. GTP-gamma-S does not inhibit MPF-induced maturation. Although GTP-gamma-S prevents the progesterone-induced increases in protein synthesis and phosphorylation, it has no effect on the basal rates of either. GTP-gamma-S also prevents the initial DAG drop induced by progesterone. GDP-beta-S effects are ambiguous, but it seems not to affect progesterone-induced maturation. These results suggest that although G-proteins are associated with the pathways affected by progesterone, the effects of progesterone are not mediated by a typical receptor/G-protein/effector interaction.     

Expression of epithelial Na channels in Xenopus oocytes

Epithelial Na channel activity was expressed in oocytes from Xenopus laevis after injection of mRNA from A6 cells, derived from Xenopus kidney. Poly A(+) RNA was extracted from confluent cell monolayers grown on either plastic or permeable supports. 1-50 ng RNA was injected into stage 5-6 oocytes. Na channel activity was assayed as amiloride- sensitive current (INa) under voltage-clamp conditions 1-3 d after injection. INa was not detectable in noninjected or water-injected oocytes. This amiloride-sensitive pathway induced by the mRNA had a number of characteristics in common with that in epithelial cells, including (a) high selectivity for Na over K, (b) high sensitivity to amiloride with an apparent K1 of approximately 100 nM, (c) saturation with respect to external Na with an apparent Km of approximately 10 mM, and (d) a time-dependent activation of current with hyperpolarization of the oocyte membrane. Expression of channel activity was temperature dependent, being slow at 19 degrees C but much more rapid at 25 degrees C. Fractionation of mRNA on a sucrose density gradient revealed that the species of RNA inducing channel activity had a sedimentation coefficient of approximately 17 S. Treatment of filter-grown cells with 300 nM aldosterone for 24 h increased Na transport in the A6 cells by up to fivefold but did not increase the ability of mRNA isolated from those cells to induce channel activity in oocytes. The apparent abundance of mRNA coding for channel activity was 10-fold less in cells grown on plastic than in those grown on filters, but was increased two- to threefold by aldosterone.