Full-grown Oocytes Of Xenopus Laevis Research Articles

Localisation of RNAs into the Germ Plasm of Vitellogenic Xenopus Oocytes

We have studied the localisation of mRNAs in full-grown Xenopus laevis oocytes by injecting fluorescent RNAs, followed by confocal microscopy of the oocyte cortex. Concentrating on RNA encoding the Xenopus Nanos homologue, nanos1 (formerly Xcat2), we find that it consistently localised into aggregated germ plasm ribonucleoprotein (RNP) particles, independently of cytoskeletal integrity. This implies that a diffusion/entrapment-mediated mechanism is active, as previously reported for previtellogenic oocytes. Sometimes this was accompanied by localisation into scattered particles of the “late”, Vg1/VegT pathway; occasionally only late pathway localisation was seen. The Xpat RNA behaved in an identical fashion and for neither RNA was the localisation changed by any culture conditions tested. The identity of the labelled RNP aggregates as definitive germ plasm was confirmed by their inclusion of abundant mitochondria and co-localisation with the germ plasm protein Hermes. Further, the nanos1/Hermes RNP particles are interspersed with those containing the germ plasm protein Xpat. These aggregates may be followed into the germ plasm of unfertilized eggs, but with a notable reduction in its quantity, both in terms of injected molecules and endogenous structures. Our results conflict with previous reports that there is no RNA localisation in large oocytes, and that during mid-oogenesis even germ plasm RNAs localise exclusively by the late pathway. We find that in mid oogenesis nanos1 RNA also localises to germ plasm but also by the late pathway. Late pathway RNAs, Vg1 and VegT, also may localise into germ plasm. Our results support the view that mechanistically the two modes of localisation are extremely similar, and that in an injection experiment RNAs might utilise either pathway, the distinction in fates being very subtle and subject to variation. We discuss these results in relation to their biological significance and the results of others.

Endogenous trypsin receptors in Xenopus oocytes: linkage to internal calcium stores.

The effects of the protease trypsin, externally applied to full-grown oocytes of Xenopus laevis, were studied using electrophysiology and fluorometry. The following results were obtained: trypsin in concentrations of 0.1 microgram/ml to 1 mg/ml liberated Ca2+ from internal stores and evoked large transient currents of up to 5 microA in bath solutions containing 1 mM or no Ca2+. The response desensitized for 50 minutes and recovered at longer times. Transient currents could also be elicited by tryptic impurities in commercially available collagenase used for defolliculation of oocytes. Application of chymotrypsin (0.01 or 1 mg/ml) or of thrombin (3.4 ng/ml or 0.34 mg/ml) neither evoked currents nor desensitized trypsin responses. Incubation with 1 microgram/ml Pertussis toxin for 20 to 25 hours prevented the Ca2+ release from internal stores and the activation of transient currents by trypsin. We propose that endogenous receptors in the oolemma, specific for trypsin, are linked to internal Ca2+ stores via Pertussis toxin-sensitive G proteins. Thus, receptor activation by external trypsin raises internal Ca2+ and thereby opens Ca(2+)-activated Cl channels in the oolemma.

The rapid transient decrease of sn-1,2-diacylglycerol in progesterone-stimulated Xenopus laevis oocytes is the result of an ethanol artifact

Full-grown Xenopus laevis oocytes resume meiosis (meiotic maturation) in response to progesterone stimulation. Three studies have shown that sn-1,2-diacylglycerol (DAG), the intracellular activator of protein kinase C, may be involved in this process ( Wasserman et al., J. Exp. Zool. 255, 63–71, 1990 ; Varnold and Smith, Development 109, 597–604, 1990 ; Stith et al., J. Cell Physiol. 149, 252–259, 1991 ). Two of these studies ( Varnold and Smith, 1990; Stith et al., 1991 ) found a rapid, but transient decrease in the levels of DAG of approximately 25 to 30% within 5 to 30 sec following the addition of progesterone to the oocytes. We have investigated this rapid decline in oocyte DAG. We also found a 20 to 34% decrease in DAG/oocyte within the first 5 to 40 sec following the addition of steroid to the culture medium. However, a similar rapid and transient decrease in oocyte DAG levels was also observed in response to ethanol. Ethanol is used as the vehicle to deliver progesterone to the oocyte culture medium. Therefore, the rapid transient decline in DAG appears to be an artifact of ethanol perturbing the production and/or turnover of DAG within the oocyte and not a physiological response of the oocyte to progesterone.

Purification and characterization of a germ cell-specific form of elongation factor 1α (EF-1α) from Xenopus laevis

Elongation factor 1α (EF-1α) was purified to homogeneity from full-grown oocytes of Xenopus laevis. This protein is encoded by a gene previously shown to be expressed in male and female germ cells, and repressed in somatic cells. The purified protein was identified with EF-1α on criteria of molecular mass, cross-reaction with antibodies raised against Artemia salina EF-1α, affinity for guanine nucleotides, and ability to promote the mRNA-dependent binding of aminoacyl tRNA to 80S ribosomes.

Sn‐1,2‐Diacylglycerol levels increase in progesterone‐stimulated Xenopus laevis oocytes

Full-grown Xenopus laevis oocytes resume meiosis from prophase arrest in response to progesterone stimulation. Recent studies have shown that the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), a very potent activator of protein kinase C, can also induce the resumption of meiosis in amphibian oocytes. We have investigated the possibility that sn-1,2-diacylglycerol (DAG), the intracellular activator of protein kinase C, may be involved in the pathway normally used by progesterone. We have found that full-grown oocytes arrested in meiotic prophase contain 48 +/- 5 pmoles DAG/oocyte. This level increases within the first 5 minutes following the addition of progesterone and reaches a maximum of 75 +/- 10 pmoles following 60 minutes of steroid stimulation. Progesterone induces the up-regulation of a Na+/H+ antiport in the plasma membrane of the oocyte causing an increase in pHi following 60 minutes of steroid stimulation. We have found that the addition of 200 microM DAG (1,2-dioctanoylglycerol, diC8) to the culture medium can cause a partial up-regulation of this Na+/H+ pump in the absence of hormonal stimulation. These results suggest that DAG and protein kinase C may be involved in regulating certain aspects of meiotic maturation in progesterone-stimulated Xenopus oocytes.

Persistence of nonribosome bound 5 S RNA in full-grown oocytes of Xenopus laevis

The 4 and 5 S RNA containing 42 S ribonucleoprotein (RNP) particles characteristic of previtellogenic and white oocytes cannot be detected in full-grown oocytes. When full-grown oocyte RNPs are separated on sucrose gradients 4 and 5 S RNA cannot be detected in the 42 S region. However, not all of the 5 S RNA stored during early oogenesis is incorporated into ribosomes at later stages. A substantial pool (20% of the total) of 5 S RNA remains in a non-ribosome-bound fraction sedimenting at about 7 S in full-grown oocytes.

Carbon dioxide reversibly inhibits meiosis of Xenopus laevis oocyte and the appearance of the maturation promoting factor

Full-grown Xenopus laevis oocytes were incubated in NaHCO 3 buffer equilibrated with carbon dioxide (5 to 100%). Germinal vesicle breakdown never occurred in spite of the appearance of the characteristic white spot at the animal pole. The effect of carbon dioxide was analyzed during progesterone-induced maturation. Carbon dioxide did not inhibit the early steps of maturation whereas it inhibited germinal vesicle breakdown even when applied 4 hr after the initial hormonal trigger. When oocytes were treated transiently in NaHCO 3 buffer equilibrated with carbon dioxide and further incubated in Tris buffer, drastic delay in the kinetic of germinal vesicle breakdown was observed. Inhibition of progesterone-induced maturation by carbon dioxide treatment is coincident with the time of maturation promoting factor appearance (MPF). On the basis of microinjection experiments of MPF into recipient oocytes, it was also shown that MPF expression is not inhibited by carbon dioxide and thus indicates that the late phase of MPF formation and/or MPF amplification is a carbon dioxide-sensitive period.

Adenosine 3′: 5′ Monophosphate Receptor Sites in Xenopus laevis Oocytes

Adenosine 3':5' cyclic monophosphate (cyclic AMP) binds to proteins present in the 100,000 g supernatant fractions of full-grown oocytes of Xenopus laevis. Optimal pH for the binding is 4.0. Two receptor binding sites have been characterized by density gradient centrifugation as peaks with sedimentation constants of 4.6 S and 5.9 S. The apparent dissociation constants for the two cyclic AMP binding sites are 7 nM and 40 nM. The total cyclic AMP binding capacity of oocyte cytosol is 15.8 +/- 2.2 fmoles/oocyte and remains constant during meiotic maturation induced by progesterone.

Synthesis of heterogeneous nuclear RNA in full-grown oocytes of xenopus laevis (Daudin)

At various times following injection of either 3H-GTP or 32PO 4 into full-grown (stage 6) Xenopus laevis oocytes, RNA has been extracted and fractionated on polyacrylamide gels. Based on size, base composition and incorporation data, we have defined the kinetics of synthesis and accumulation of ribosomal RNA (40S, 28S, 18S), heterogeneous RNA of high molecular weight (>40S) and heterogeneous RNA migrating with molecular weights of from 4S to 40S. Nuclear isolations have been performed to determine the cellular distribution of these classes of RNA as a function of time. Evidence is presented which shows that stage 6 oocytes synthesize RNA which by virtue of its size, base composition, rapid turnover and nuclear location is equivalent to the heterogeneous nuclear RNA observed in somatic cells. In addition, the data suggest synthesis of a class of nuclear RNA with a half-life of several hours. A small fraction (5%) of the nuclear RNA is stable, enters the cytoplasm and may represent RNA added to the stockpile of maternal transcripts known to be present in stage 6 oocytes.

Differential accumulation of two size classes of poly(A) associated with messenger RNA during oogenesis in Xenopus laevis

The RNA of full-grown oocytes of Xenopus laevis contains two distinct size classes of poly(A), designated poly(A) S and poly(A) L, which contain 15–30 (mean = 20) and 40–80 (mean = 61) A residues, respectively. Both poly(A) L and poly(A) S are associated with RNA which is heterogeneous in size. The two classes of poly(A) + RNA can be separated by affinity chromatography: Only poly(A) L + RNA binds to oligo(dT)-cellulose under appropriate conditions, but up to 50% of the poly(A) S + RNA can be isolated from the void fraction by binding to poly(U)-Sepharose. Both classes of poly(A) + RNA are active as messenger RNA in an in vitro system and yield identical patterns of in vitro protein products. Previtellogenic oocytes contain almost exclusively poly(A) L, which accumulates up to vitellogenesis but remains almost constant in amount (molecules/oocyte) during vitellogenesis and in the full-grown oocyte. Poly(A) S accumulates (molecules/oocyte) from early vitellogenesis up to the full-grown oocyte. The total number of poly(A) + RNA molecules per oocyte increases throughout oogenesis from 2 × 10 10/previtellogenic oocyte [80–90% poly(A) L] to 20 × 10 10/full-grown oocyte (25–40% poly(A) L). It is argued that poly(A) S is protected from degradation in the oocyte, thus stabilizing the “maternal” poly(A) + mRNA.

Progesterone-induced DNA polymerase activity in full-grown oocytes of Xenopus laevis (1).

DNA polymerase activity increases in full-grown oocytes of Xenopus laevis during in vitro progesterone-induced maturation. This increase is inhibited by cycloheximide. The presence of the oocyte's nucleus (germinal vesicle) seems essential for the induction of this increase: in previously enucleated oocytes, the level of DNA polymerase activity does not change during progesterone treatment. Furthermore, a new form of DNA polymerase is detectable by DEAE chromatography in in vitro matured oocytes.

Nature of progesterone action on amino acid uptake by isolated full-grown oocyte of Xenopus laevis

l-leucine uptake into full-grown oocytes of Xenopus laevis is a saturable process which is Na + dependent and presumably coupled to Na + gradient. Our results indicate that progesterone (10 −6 M) blocks abruptly, around the germinal vesicle breakdown, the saturable transport of l-leucine. p- Chloromercuribenzoate (10 −4 M) induces maturation and after a short lag of time strongly inhibits l-leucine uptake. Cycloheximide prevents progesterone-induced maturation and permeability changes.