Latrunculin A depolarizes starfish oocytes.

Depolymerization of the actin cytoskeleton may liberate Ca2+ from InsP3-sensitive stores in some cell types, including starfish oocytes, while inhibiting Ca2+ influx in others. However, no information is available on the modulation of membrane potential (V(m)) by actin. The present study was aimed to ascertain whether the widely employed actin depolymerizing drug, latrunculin A (Lat A), affects V(m) in mature oocytes of the starfish Astropecten aranciacus. Lat A induced a membrane depolarization which was mimicked by cytochalasin D, another popular actin disruptor, and prevented by jasplakinolide, a stabilizer of the actin network. Lat A-elicited depolarization consisted in a positive shift in V(m) which reached the threshold of activation of voltage-gated Ca2+ channels (VGCC), thus triggering an action potential. Lat A-promoted depolarization lacked the action potential in Ca2+-free sea water, while it was abolished upon removal of external Na+. Moreover, membrane depolarization was prevented by pre-injection of BAPTA and heparin, but not ryanodine. These data indicate that Lat A induces a membrane depolarization by releasing Ca2+ from InsP3Rs. The Ca2+ signal in turn activates a Ca2+-dependent Na+ entry, which causes the positive shift in V(m) and stimulates the VGCC.     

Induced nondisjunction in drosophila oocytes.

Nondisjunction of the sex chromosomes and compound-second chromosomes was induced in Drosophila melanogaster by irradiation of immature oocytes and cold treatment of mature oocytes. These two treatments cause nondisjunction by different mechanisms. This results in a differential for stage sensitivity and the production of relatively different frequencies of the various exceptional progeny classes.     

Incidence of nondisjunction in mouse oocytes.

The chromosomes of more than 3000 ovulated mouse oocytes from strains C3H/Han, NMRI/Han, and (101 X C3H)F1 have been analyzed after spontaneous and hormonally induced ovulation. No significant difference in the incidence of nondisjunction was observed among the three strains with or without hormonal pretreatment. The incidence of nondisjunction was estimated to be 0.47% in NMRI/Han, 0.62% in C3H/Han, and 0.81% in (101 X C3H/F1. The incidence of chromosomal imbalance after the first meiotic division was slightly higher after adding the events following presegregation. Diploidy-spontaneous and hormonally induced-reached a significant leve in NMRI/Han. This may be interpreted as a consequence of hormonal interference with a genetically defined malfunction of gene product(s) during the late phase of oogenesis.     

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.     

Receptor-mediated vitellogenin binding to chicken oocytes.

The specific binding of vitellogenin to chicken oocyte membranes was characterized. This major hen serum phospholipoglycoprotein and one of its lower-molecular-weight components, phosvitin, bound to oocyte membranes with KD values of approx. 6 x 10-7 M. The optimum pH for binding was 6.0, the same as the pH of yolk contents. Phosvitin and vitellogenin compete with each other for binding; other proteins tested do not compete to the same degree. Phosvitin, which contains 10% phosphate by weight, appears to be the polypeptide recognized by the receptor. RNA failed to compete with either vitellogenin or phosvitin for binding, suggesting that the binding specificity may require more than polymeric phosphate. The binding was tissue-specific in that phosvitin and vitellogenin bound to oocyte surfaces (at both pH 6.0 and 7.5), but not to chicken erythrocytes (at either pH).     

Angiotensin II receptors in Xenopus oocytes.

Electrical recordings were used to study the sensitivity of native Xenopus oocytes to the octapeptide angiotensin II (AII). AII elicited oscillatory currents associated with an increase in membrane conductance to Cl-. Responsiveness to AII varied greatly between oocytes taken from different frogs, and to a lesser extent between oocytes from the same ovary. Oocytes from frogs showing high sensitivity had response thresholds between 0.5-1.0 nM AII, and at a holding potential of -60 mV, responded to 1 microM AII with currents greater than 3 microA. In contrast, oocytes from some frogs gave no response, even to 10 microM AII. A total of 618 oocytes from 79 frogs were tested for sensitivity to AII, and oocytes from 85% of frogs gave detectable electrical responses. Oscillatory Cl- currents elicited by AII were largely independent of extracellular Ca2+, were abolished by chelation of intracellular Ca2+ using EGTA and were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3). In addition to oscillatory Cl- currents, AII also evoked an influx of extracellular Ca2+, giving rise to a transient inward Cl- current on membrane hyperpolarizing steps. These experiments all suggested that AII responses were elicited through activation of an intracellular messenger pathway triggered by hydrolysis of inositolphospholipids, mobilization of intracellular Ca2+ by inositol polyphosphates, and activation of Ca(2+)-gated Cl- channels. The effect of manual or enzymic defolliculation on AII responses was studied in nine separate experiments recording from 70 defolliculated oocytes. Efficacy of defolliculation procedures was assayed using scanning electron microscopy, which confirmed removal of 90 to greater than 98% of follicular cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Export of proteins from oocytes of Xenopus laevis.

When human lymphoblastoid mRNA was microinjected into X. laevis oocytes, titers of interferon rapidly reached a maximum inside the oocyte while accumulation of interferon continued in the incubation medium for at least 45 hr. If interferon protein was injected into oocytes it was rapidly inactivated. Significantly, newly synthesized interferon but not injected interferon was found to be membrane-associated. Further experiments involving the co-injection of mRNAs coding for secretory proteins (guinea pig milk proteins and human interferon) and nonsecretory proteins (rabbit globin) revealed that only the secretory proteins were exported from the oocyte. Moreover, different proteins were exported at different rates. A distinct subclass of newly synthesized oocyte proteins of unknown function also accumulated in the incubation medium. Since the information encoded in the messenger RNAs of secretory proteins is sufficient to specify synthesis, compartmentation and secretion of these proteins, the oocyte may provide a complete system for the analysis of the secretory process.     

Endogenous L-glutamate transport in oocytes of Xenopus laevis.

The existence of an endogenous Na(+)-glutamate cotransporter in the oocytes of Xenopus laevis is demonstrated. The transporter does not accept D-glutamate as substrate. The dependence on substrate displays two saturating components with low (K1/2 = 9 mM) and high (K1/2 = 0.35 microM) affinities for L-glutamate. The dependence on external Na+ exhibits a saturating component with a K1/2 value of about 5 mM and a component that has not saturated up to 110 mM Na+. In voltage-clamped oocytes, it is possible to demonstrate that Na(+)-dependent L-glutamate transport is directly coupled to countertransport of Rb+. The analysis of the voltage dependence of the Na+,K(+)-dependent L-glutamate uptake suggests that positive charges are moved inwardly during the transport cycle.     

Protein phosphorylation during activation of surf clam oocytes.

We have investigated the increase of phosphorylated proteins upon activation of surf clam (Spisula solidissima) oocytes, by measuring the cumulative incorporation of 32P in proteins and by performing an SDS-PAGE and autoradiographic analysis of 32P-labeled proteins, from oocytes initially radiolabeled with 32P-orthophosphate. The phosphorylation inhibitor 6-dimethylaminopurine (6-DMAP) inhibits both germinal vesicle breakdown (GVBD) and the normal increase in phosphorylated proteins observed upon activation by KCl, in a reversible and dose-dependent manner. Using different artificial seawaters (normal, Ca(2+)-free, Na(+)-free), we observed that the increase of phosphorylated proteins, upon K+ stimulation, occurs only when GVBD is allowed to proceed along with an increased Ca2+ influx, in normal or Na(+)-free seawater. Stimulation of oocytes by ammonia, which directly raises intracellular pH (pHi) but does not trigger GVBD, is without effect on the level or pattern of phosphorylated proteins. The link between the Ca2+ influx and the level of phosphorylated proteins was further investigated using conditions altering the duration or the level of Ca2+ influx upon K+ stimulation. In all conditions tested, both GVBD and the level of phosphorylated proteins were similarly affected by alterations of the Ca2+ influx, indicating that these processes are tightly coupled one with another. Upon activation of oocytes, six major proteins of estimated molecular weights of 31, 41, 48, 56, 80 and 86 kDa undergo an increased phosphorylation that is reversibly sensitive to 6-DMAP. Our results suggest that increased protein phosphorylation, sensitive to 6-DMAP, is necessary for GVBD and that it is indirectly linked to the increased Ca2+ influx that stands as an upstream trigger for activation, while an elevated pHi alone has no effect on these processes.     

Centrifugation of developing oocytes leading to embryonic defects.

Drosophila melangaster females were centrifuged for 6 hr at 670g. Eggs collected from centrifuged flies frequently developed into abnormal embryos and larvae which were unable to hatch. A typical abnormality was the fusion of segments, identified by the cuticle hairs which mark larval segmental boundaries. Malformation of segments occurred most frequently in the middle region of the larva (segments 6–9 of the 12 larval segments). The induction of abnormalities is stage specific. They occurred in embryos developing from eggs laid up to 33 hr after centrifugation; eggs laid later than 33 hr (stage 7, King, 1970) showed no abnormalities.     

Messenger RNA competition in living Xenopus oocytes.

When calf lens crystallin mRNA and rabbit globin mRNA are competing for factors limiting protein synthesis in living Xenopus oocytes, no mRNA species is preferentially selected for translation. Differences in the intrinsic translational efficiency of the mRNA species exist, but the relative efficiencies are the same at high and low mRNA concentrations. mRNAs already being translated, in particular endogenous oocyte mRNAs, are less sensitive to competitive inhibition by injected mRNAs. As injected mRNAs gradually become incorporated into the protein-synthesizing machinery of the oocyte, they acquire the same status as the oocyte's own active mRNAs. Exogenous mRNAs this become endogenous mRNAs. These results, together with previous estmates of the translational efficiency of injected heterologous mRNA species, are compatible with the assumption that a large proportion of the endogenous mRNAs is not competing for the translational apparatus of the oocyte and, therefore, probably is present in the temporarily inactivated form.