Expression of adenylate cyclase-coupled osseous parathyroid hormone and parathyroid hormone-like peptide receptors in Xenopus oocytes.

Functional parathyroid hormone (PTH) and PTH-like peptide receptors were expressed in Xenopus laevis oocytes after injection of poly(A)+ RNA isolated from the rat osteogenic sarcoma cell line, UMR 106. Increases in cAMP were seen in individual oocytes in response to added bovine (b) PTH-(1-34) (10(-6) M), human (h) PLP-(1-34) (hPLP-(1-34), 10(-6) M), isoproterenol (10(-4) M), and forskolin (10(-4) M). Although both intracellular and extracellular cAMP levels were stimulated approximately 1.5-2-fold by these agonists, intracellular concentrations of cAMP were substantially higher than extracellular concentrations. Peak increases with bPTH-(1-34) occurred after a 30-min incubation with the hormone 48 h after oocyte injection. bPTH-(1-34) caused a concentration-dependent augmentation of cAMP in injected oocytes, and the in vitro antagonist hPLP-(3-34) produced dose-dependent inhibition of both bPTH-(1-34)- and hPLP-(1-34)-stimulated cAMP accumulation. Specific binding of PTH to oocyte membranes was also demonstrated 48 h after oocyte injection with UMR 106 cell mRNA. Following size fractionation of isolated UMR 106 poly(A)+ RNA by sucrose density gradients, mRNA directing the expression of both PTH- and PLP-stimulated cAMP in oocytes appeared in the 3.5-4.9-kilobase fraction. These results demonstrate that adenylate cyclase-coupled osseous PTH and PLP receptors can be expressed after injection of naturally occurring mRNA into Xenopus oocytes, that PTH- and PLP-stimulated increases in cAMP concentrations can be detected in individual oocytes injected with bone cell-derived mRNA, that PTH and PLP appear to cross-react at a common receptor after injection of UMR 106 cell mRNA into oocytes, and that size selection of mRNA encoding the PTH and PLP receptors can be achieved by density gradient centrifugation. These studies, therefore, indicate the potential usefulness of the Xenopus oocyte system in expression cloning of PTH and PLP receptor cDNAs and illustrate the feasibility of employing this system to examine the biology of PTH and PLP receptors.     

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.     

Changes in protein phosphorylation accompanying maturation of Xenopus laevis oocytes

Protein phosphorylation has been measured after injection of [³²P]phosphate into oocytes of Xenopus laevis undergoing progesterone-induced meiotic maturation. As oocytes mature, there is a burst of nonyolk protein phosphorylation several hours after progesterone exposure and shortly before germinal vesicle breakdown (GVBD). This burst is not due to changes in the specific activity of the phosphate or ATP pool. Enucleated oocytes exposed to progesterone also experience the burst, indicating the cytoplasmic location of phosphoprotein formation. When an oocyte receives an injection of cytoplasm containing the maturation-promoting factor (MPF), a burst of protein phosphorylation occurs immediately, and GVBD occurs shortly thereafter, even in the presence of cycloheximide. Under a variety of conditions promoting or blocking maturation, oocytes which undergo GVBD are the only ones to have experienced the phosphorylation burst. The results suggest that the protein phosphorylation burst is a necessary step in the mechanism by which MPF promotes GVBD.     

Expression of rat jejunal cystine carrier in Xenopus oocytes

Functional interactive cystine-lysine carriers have been expressed in Xenopus oocytes following the injection of RNA extracted from rat intestinal mucosa. Lysine-inhibitable cystine uptake was able to be measured 16 h after oocyte injection with RNA. The longer the oocytes were maintained after injection, the more cystine transport capability was induced. Uninjected or water-injected oocytes showed virtually no lysine-inhibitable cystine uptake, and no system developed after the oocytes had been isolated and maintained in vitro. The cystine uptake expressed after RNA injection was selectively inhibited by dibasic amino acids and phenylalanine but not by other amino acids or alpha-methyl-D-glucoside. Expression of the interactive cystine-lysine system was induced only by RNA isolated from intestinal tissue and not by RNA from rat liver. The Km for cystine uptake in RNA-injected oocytes was 0.01 mM and appears identical to the single system found in the RNA source tissue.     

Pre-MPF is absent in immature oocytes of fishes and amphibians except Xenopus

Maturation-promoting factor (MPF), a final trigger for initiating oocyte maturation, is activated in the oocyte cytoplasm, in response to maturation-inducing hormone (MIH) secreted from follicle cells surrounding the oocyte. MPF consists of cdc2 and cyclin B. We investigated the state of cdc2 and cyclin B in immature and mature oocytes of fishes (carp, catfish and lamprey) and amphibians ( Xenopus, frog [ Rana ] and toad [ Bufo ]) using monoclonal antibodies raised against mouse cdc2, which also recognize fish and amphibian cdc2, and monoclonal antibodies against goldfish cyclin B1 and polyclonal antibodies against Xenopus cyclins B1 and B2. Anti-cdc2 and anti-cyclin B immunoblotting of oocyte extracts fractionated by gel filtration chromatography showed that immature oocytes from all of these species with the exception of Xenopus contained only monomeric cdc2. Cyclin B-bound inactive cdc2 (pre-MPF) was present only in immature Xenopus oocytes. Cdc2-cyclin B complex was, however, found in mature oocytes from all the species examined. After the oocyte is induced to mature by MIH, cdc2 should therefore bind to cyclin B in all of these species, except Xenopus. These results suggest that the complex formation of cdc2 and cyclin B in response to MIH stimulation at the oocyte surface is a critical step for initiating oocyte maturation in fishes and amphibians, with the exception of Xenopus , in which pre-MPF already exists in immature oocytes and only its chemical modification is required for MPF activation.     

A three-step scheme for gray crescent formation in the rotated axolotl oocyte

It has been shown that various inhibitors of protein synthesis can elicit the precocious appearance of a gray crescent (GC) in in vitro maturing, nonactivated Ambystoma mexicanum oocytes. However, evidence has now been obtained that these treatments fail to induce GC formation when the oocytes are enucleated before initiation of maturation. The ability to form a GC is reestablished in enucleated oocytes by the injection of nucleoplasm from a normal oocyte, either before or after the injection of the inhibitor. In the latter case, the GC appears very rapidly, even though protein synthesis is at about 1/10th that of the control enucleated oocyte, after treatment with diphtheria toxin (final concentration 10(-8) M) as an inhibitor. One or several nuclear factors, in conjunction with inhibition of protein synthesis, are therefore essential for early symmetrization. The corrective nuclear factor is already present in the germinal vesicle of young oocytes, at the very beginning of vitellogenesis. It is not species specific, since enucleated axolotl oocytes can be symmetrized with Pleurodeles or even Xenopus oocyte nucleoplasm. Moreover, it has been shown that the nuclear-cytoplasmic interaction is possible only when cytoplasmic maturation has been proceeding for at least 10 hr after exposure to progesterone (at 18 degrees C). A three-step process as a prerequisite of GC formation in the oocyte is proposed: Cytoplasmic maturation must proceed till a reactive state is attained, allowing interactions with nuclear factors; Nuclear factor(s) interact(s) with matured cytoplasm; Inhibition of protein synthesis triggers GC formation. Sequence of steps 2 and 3 can be experimentally inverted but must always be preceded by step 1. Since a sharp reduction in amino acid incorporation has also been found in normally fertilized eggs just prior to GC formation, it is suggested that the scheme described above could be also applicable to normal symmetrization in this model system.     

Assay for characterization of human follicular oocyte maturation inhibitor using Xenopus oocytes

Human follicular fluid from healthy mature Graafian follicles and from pathologic ovarian cyst fluid was found to be inhibitory to progesterone-induced meiotic maturation of oocytes from the South African clawed toad, Xenopus laevis. Human follicular fluid but not human serum, collected from the same individuals, demonstrated a linear dose-response inhibition on the maturation of oocytes in the Xenopus assay system. These findings indicate that the human follicular and cyst fluids contain oocyte maturation inhibitor (OMI). This human OMI was inactivated when subjected to a boiling water bath for 2 min. The OMI action was shown to be reversible in its inhibitory action. The fact that OMI can act directly on the oocyte was demonstrated by its inhibitory action on maturation in defolliculated oocytes. The findings demonstrate that the inhibitory action of human OMI is not species-specific. Xenopus oocytes provide a simple, readily available, year-round bioassay material for testing follicular oocyte maturation inhibitor.     

Stability and movement of mRNAs and their encoded proteins in Xenopus oocytes

The stability and movement of several polyadenylated (poly A+) and nonpolyadenylated (poly A-) mRNAs in Xenopus oocytes have been examined. At least 50% of the poly A+ mRNA molecules (9S rabbit globin mRNA, chicken ovalbumin, and lysozyme) were stable in oocytes over a 48-h period, irrespective of the amount injected. About 50% of injected poly A- reovirus mRNAs was degraded within the first 24 h of injection, irrespective of the amount injected, although no further degradation was observed over an additional 24 h. The movement of all poly A+ mRNAs injected at either the animal or vegetal pole of the oocyte was very slow. Little movement of RNA from the animal half to the vegetal half was observed even 48 h after injection. In contrast, similar amounts of mRNA were present in both halves 48 h after vegetal pole injection. Similar results were obtained after injection of poly A- reovirus mRNAs. The movement of the proteins encoded by the poly A+ mRNAs was studied in the 6-h period after injection when little mRNA movement had occurred. 85% of the globin synthesized accumulated in the animal half irrespective of injection site. The movement of the sequestered secretory proteins ovalbumin and lysozyme in the same oocytes as globin was much slower; very little lysozyme appeared in the half of the oocyte opposite the site of injection.     

Induced formation of asters and cleavage furrows in oocytes of Xenopus laevis during in vitro maturation

We have previously reported that injection of purified basal bodies or sperm into unfertilized eggs of Xenopus laevis induced the formation of asters and irregular cleavage furrows. Fully grown oocytes were found to be unable to form asters or cleavage furrows. In this paper we show that the oocyte acquires the ability to form asters upon basal body injection at the time of germinal vesicle breakdown during in vitro maturation. Our evidence indicates that aster formation requires progesterone-stimulated changes in the oocyte and mixing of cytoplasm and germinal vesicle plasm. The ability of the oocyte to form cleavage furrows arises six to eight hours after germinal vesicle breakdown. We infer that some maturational change in the cell cortex occurs to enable the egg surface to furrow. Experiments on the relationship of aster formation to furrow initiation indicates that asters stimulate furrow formation. However, some furrowing could be induced without aster formation in mature oocytes and unfertilized eggs by an activation stimulus, showing that asters are not essential for cleavage initiation. The significance of these observations are discussed in the light of our current understanding of meiotic maturation, cell cleavage and aster growth.     

Trimer formation determines the rate of influenza virus haemagglutinin transport in the early stages of secretion in Xenopus oocytes

We have previously shown that influenza haemagglutinin (HA) acquires Endo H resistance en route to the cell surface after microinjection of its mRNA into Xenopus oocytes (Ceriotti, A. and A. Colman. 1989. J. Cell Biol. 109:1439-1444.) In this paper we use the injection of varying amounts of mRNA (0.05-5 ng/oocyte) to effect a 30-fold change in HA protein synthesis within the oocyte. Using the Endo H assay as an indicator of protein movement from the ER to the medial Golgi we find that this movement is reduced, sometimes dramatically, when intracellular HA levels fall. This reduction in movement is closely correlated with a decreased rate of trimer formation as assessed both by trypsin resistance and sedimentation analysis, leading us to conclude that trimer formation is not only, as has been shown before essential for ER-Golgi complex movement, but is the major rate limiting step in this movement. Interestingly at least 50% of unassembled HA monomers that accumulate after low HA synthesis can be rescued into trimers over 24 h later, after a second injection of concentrated HA mRNA. In contrast when we repeated this experiment with another membrane protein, the human low density lipoprotein, or with murine secretory immunoglobulin we found that the rate of movement was insensitive to the protein concentration. This latter result seemed surprising since earlier work had shown that unassembled IgG heavy chains (like monomeric HA) remain in the oocyte ER; however in these present experiments we have been unable to detect any unassembled heavy chains even at the lowest expression levels, indicating that tetramerization of Ig is much faster than trimerization of HA.     

Regulation of Xenopus oocyte meiosis arrest by G protein betagamma subunits

BACKGROUND: Progesterone induces the resumption of meiosis (maturation) in Xenopus oocytes through a nongenomic mechanism involving inhibition of an oocyte adenylyl cyclase and reduction of intracellular cAMP. However, progesterone action in Xenopus oocytes is not blocked by pertussis toxin, and this finding indicates that the inhibition of the oocyte adenylyl cyclase is not mediated by the alpha subunits of classical G(i)-type G proteins. RESULTS: To investigate the possibility that G protein betagamma subunits, rather than alpha subunits, play a key role in regulating oocyte maturation, we have employed two structurally distinct G protein betagamma scavengers (G(t)alpha and betaARK-C(CAAX)) to sequester free Gbetagamma dimers. We demonstrated that the injection of mRNA encoding either of these Gbetagamma scavengers induced oocyte maturation. The Gbetagamma scavengers bound an endogenous, membrane-associated Gbeta subunit, indistinguishable from Xenopus Gbeta1 derived from mRNA injection. The injection of Xenopus Gbeta1 mRNA, together with bovine Ggamma2 mRNA, elevated oocyte cAMP levels and inhibited progesterone-induced oocyte maturation. CONCLUSION: An endogenous G protein betagamma dimer, likely including Xenopus Gbeta1, is responsible for maintaining oocyte meiosis arrest. Resumption of meiosis is induced by Gbetagamma scavengers in vitro or, naturally, by progesterone via a mechanism that suppresses the release of Gbetagamma.     

Possible involvement of Nemo-like kinase 1 in Xenopus oocyte maturation as a kinase responsible for Pumilio1, Pumilio2, and CPEB phosphorylation

Members of the mitogen-activated protein kinase (MAPK) family play important roles in Xenopus oocyte maturation. Nemo-like kinase (NLK), an atypical MAPK, is known to function in multiple developmental processes in vertebrates and invertebrates, but its involvement in gametogenesis and gamete maturation is unknown. In this study, we biochemically examined NLK1 during Xenopus oocyte maturation. NLK1 is expressed in immature oocytes, and its protein level remains constant during maturation. NLK1 is inactive in immature oocytes but is activated during maturation, depending on Mos protein synthesis but not on p42 MAPK activation. Overexpression of NLK1 by injection of 5 ng of mRNA accelerates progesterone-induced oocyte maturation by enhancing Cyclin B1 protein synthesis through the translational activation of its mRNA, in accordance with precocious phosphorylation of Pumilio1 (Pum1), Pumilio2 (Pum2), and cytoplasmic polyadenylation element-binding protein (CPEB), key regulators of the translational control of mRNAs stored in oocytes. A higher level of NLK1 expression by injection of 50 ng of mRNA induces Pum1/Pum2/CPEB phosphorylation, CPEB degradation, Cyclin B1 protein synthesis, and oocyte maturation in the absence of progesterone. NLK1 phosphorylates Pum1, Pum2, and CPEB in vitro. These findings provide the first evidence for the involvement of NLK1 in Xenopus oocyte maturation. We suggest that NLK1 acts as a kinase downstream of Mos and catalyzes phosphorylation of Pum1, Pum2, and CPEB to regulate the translation of mRNAs, including Cyclin B1 mRNA, stored in oocytes.     

A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation, leading us to question what the Mos target might be if Mos is still required. We now demonstrate that Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF. We have found that Myt1 is likely to be the Mos target in this process, as Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. We propose that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes.     

Reconstitution of the Golgi apparatus after microinjection of rat liver Golgi fragments into Xenopus oocytes

We have studied the reconstitution of the Golgi apparatus in vivo using an heterologous membrane transplant system. Endogenous glycopeptides of rat hepatic Golgi fragments were radiolabeled in vitro with [3H]sialic acid using detergent-free conditions. The Golgi fragments consisting of dispersed vesicles and tubules with intraluminal lipoprotein-like particles were then microinjected into Xenopus oocytes and their fate studied by light (LM) and electron microscope (EM) radioautography. 3 h after microinjection, radiolabel was observed by LM radioautography over yolk platelet-free cytoplasmic regions near the injection site. EM radioautography revealed label over Golgi stacked saccules containing the hepatic marker of intraluminal lipoprotein-like particles. At 14 h after injection, LM radioautographs revealed label in the superficial cortex of the oocytes between the yolk platelets and at the oocyte surface. EM radioautography identified the labeled structures as the stacked saccules of the Golgi apparatus, the oocyte cortical granules, and the plasmalemma, indicating that a proportion of microinjected material was transferred to the surface via the secretion pathway of the oocyte. The efficiency of transport was low, however, as biochemical studies failed to show extensive secretion of radiolabel into the extracellular medium by 14 h with approximately half the microinjected radiolabeled constituents degraded. Vinblastine (50 microM) administered to oocytes led to the formation of tubulin paracrystals. Although microinjected Golgi fragments were able to effect the formation of stacked saccules in vinblastine-treated oocytes, negligible transfer of heterologous material to the oocyte surface could be detected by radioautography. The data demonstrate that dispersed fragments of the rat liver Golgi complex (i.e., unstacked vesicles and tubules) reconstitute into stacked saccules when microinjected into Xenopus cytoplasm. After the formation of stacked saccules, reconstituted Golgi fragments transport constituents into a portion of the exocytic pathway of the host cell by a microtubule-regulated process.     

A characterization of cytostatic factor activity from Xenopus eggs and c-mos-transformed cells

In Xenopus oocytes, the mos proto-oncogene product is required during meiosis I for the activation of maturation promoting factor (MPF) and the subsequent breakdown of the germinal vesicle (GVBD). In addition, the mos product has been shown to be a candidate "initiator" of meiotic maturation and is an active component of cytostatic factor (CSF), an activity responsible for metaphase II arrest. Here we demonstrate that pp39mos is required throughout oocyte maturation. We found that in progesterone stimulated oocytes, depletion of mos RNA immediately before GVBD terminally decreased MPF. Likewise, oocytes depleted of mos RNA and induced to mature with crude MPF proceeded through GVBD but lacked the MPF activity required to arrest mature oocytes at metaphase II. Thus, during maturation the mos product is required, directly or indirectly, to sustain MPF activity. On the other hand, mouse NIH/3T3 cells transformed by the constitutive expression of pp39mosxc possessed CSF activity but lacked constitutive levels of MPF or its associated histone H1 kinase activity. Moreover, cytosols prepared from transformed NIH/3T3 cells or Xenopus eggs had similar levels of CSF activity, but pp39mos levels were greater than 40-fold higher in the transformed cell extract. These analyses show that maintenance of CSF during interphase does not result in the maintenance of MPF.     

Oogenesis in Xenopus laevis (Daudin). II. The induction and subcellular localization of tyrosinase activity in developing oocytes

Tyrosinase activity is increased at specific stages of development in Xenopus laevis oocytes in mature females by an injection of 1000 units of human chorionic gonadtropin (HCG). Enzyme activity is stimulated slightly in stage II oocytes, greatly (5- to 6-fold) in stage III and early stage IV oocytes, slightly in late stage IV, and not at all in stage V oocytes. Tyrosinase activity has been localized cytochemically in oocytes by the DOPA-reaction. The DOPA-reaction product is found in the distal cisterna of the Golgi complex and in an anastomosing network of smooth-surfaced tubules associated with the Golgi complex. No reaction product is found in the clustered elements of smooth endoplasmic reticulum which gives rise to the premelanosomes. Substantial melanization of the premelanosomes does not occur until the DOPA-positive Golgi complexes move into proximity with the premelanosomes at the oocyte periphery. Biochemical assay of the isolated melanin granules shows that premelanosomes isolated from stage III and IV oocytes contain significant tyrosinase activity. This activity appears to decrease in the later stages of melanization. It is concluded that the metabolic activities leading to the formation of oocyte pigmentation are stimulated by gonadotropins and the degree of response to the stimulation is quantitatively regulated according to parameters typical of the specific stage of oocyte development.     

Regulation of Src kinase activity during Xenopus oocyte maturation

Expression of constitutively active Src protein tyrosine kinase in Xenopus oocytes has been shown to accelerate oocyte maturation suggesting that Src may be involved in meiotic progression. However, meiotic regulation of endogenous Src kinase in oocytes has not been investigated in detail. To address this problem, we measured the activity, expression level, and phosphorylation state of the endogenous Xenopus Src (xSrc) and overexpressed xSrc mutants in the process of progesterone-induced oocyte maturation. We found that the enzyme is first transiently activated in the plasma membrane-containing fraction of oocytes within 3 min of progesterone administration. This event represents one of the earliest responses of oocytes to the hormone and should be related to triggering some early signaling pathways of maturation. Thereafter, xSrc activity increases again at the time of germinal vesicle breakdown (GVBD) and remains elevated till the completion of maturation. This elevation of xSrc activity is associated with a 2-fold increase of xSrc protein content in the absence of change in its specific activity and xSrc mRNA content. No significant changes in the phosphorylation state of C-terminal regulatory phosphotyrosine can be registered either in endogenous xSrc or in overexpressed kinase-negative and wild-type xSrc proteins during maturation. Altogether, these results indicate that upregulation of xSrc in the meiotic metaphase occurs at the translation level. We also demonstrate here that the expression of constitutively active xSrc in Xenopus oocytes is accompanied by the activation of mitogen-activated protein kinase (MAPK). Our data suggest that the Src kinase acts through the MAPK pathway to accelerate oocyte maturation.