Testosterone and Progesterone Rapidly Attenuate Plasma Membrane Gβγ-Mediated Signaling in Xenopus laevis Oocytes by Signaling through Classical Steroid Receptors

2007 ◽  
Vol 21 (1) ◽  
pp. 186-196 ◽  
Author(s):  
Kristen Evaul ◽  
Michelle Jamnongjit ◽  
Bala Bhagavath ◽  
Stephen R. Hammes
Keyword(s):  
Xenopus Laevis ◽  
G Protein ◽  
G Proteins ◽  
Oocyte Maturation ◽  
Live Cells ◽  
Inhibition Mechanism ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Potassium Influx

Abstract Many transcription-independent (nongenomic) steroid effects are regulated by G proteins. A well-established, biologically relevant example of steroid/G protein interplay is steroid-triggered oocyte maturation, or meiotic resumption, in Xenopus laevis. Oocyte maturation is proposed to occur through a release of inhibition mechanism whereby constitutive signaling by Gβγ and other G proteins maintains oocytes in meiotic arrest. Steroids (androgens in vivo, and androgens and progesterone in vitro) overcome this inhibition to promote meiotic resumption. To test this model, we used G protein-regulated inward rectifying potassium channels (GIRKs) as markers of Gβγ activity. Overexpression of GIRKs 1 and 2 in Xenopus oocytes resulted in constitutive potassium influx, corroborating the presence of basal Gβγ signaling in resting oocytes. Testosterone and progesterone rapidly reduced potassium influx, validating that steroids attenuate Gβγ activity. Interestingly, reduction of classical androgen receptor (AR) expression by RNA interference abrogated testosterone’s effects on GIRK activity at low, but not high, steroid concentrations. Accordingly, androgens bound to the Xenopus progesterone receptor (PR) at high concentrations, suggesting that, in addition to the AR, the PR might mediate G protein signaling when androgens levels are elevated. In contrast, progesterone bound with high affinity to both the Xenopus PR and AR, indicating that progesterone might signal and promote maturation through both receptors, regardless of its concentration. In sum, these studies introduce a novel method for detecting nongenomic steroid effects on G proteins in live cells in real time, and demonstrate that cross talk may occur between steroids and their receptors during Xenopus oocyte maturation.

scholarly journals Nuclear transport of U1 snRNP in somatic cells: differences in signal requirement compared with Xenopus laevis oocytes.

1994 ◽  
Vol 125 (5) ◽  
pp. 971-980 ◽  
Author(s):  
U Fischer ◽  
J Heinrich ◽  
K van Zee ◽  
E Fanning ◽  
R Lührmann
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Import ◽  
Nuclear Transport ◽  
Somatic Cells ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Nuclear Targeting ◽  
Transport Pathway ◽  
U1 Snrnp

The signal requirement for the nuclear import of U1 RNA in somatic cells from different species was investigated by microinjection of both digoxygenin-labeled wild type and mutant U1 RNA molecules and in vitro reconstituted U1 snRNPs. U1 RNA was shown to be targeted to the nucleus by a temperature-dependent process that requires the prior assembly of RNPs from the common proteins and the microinjected RNA. Competition in the cell between immunoaffinity-purified U1 snRNPs and digoxygenin-labeled U1 snRNPs reconstituted in vitro showed that the transport is saturable and should therefore be a mediated process. The transport of a karyophilic protein under the same conditions was not affected, indicating the existence of a U snRNP-specific transport pathway in somatic cells, as already seen in the Xenopus laevis oocyte system. Surprisingly, the signal requirement for nuclear transport of U1 snRNP was found to differ between oocytes and somatic cells from mouse, monkey and Xenopus, in that the m3GGpppG-cap is no longer an essential signaling component in somatic cells. However, as shown by investigation of the transport kinetics of m3GpppG- and ApppG-capped U1 snRNPs, the m3GpppG-cap accelerates the rate of U1 snRNP import significantly indicating that it has retained a signaling role for nuclear targeting of U1 snRNP in somatic cells. Moreover, our data strongly suggest that cell specific rather than species specific differences account for the differential m3G-cap requirement in nuclear import of U1 snRNPs.

Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes

1988 ◽  
Vol 8 (10) ◽  
pp. 4257-4269
Author(s):  
A Shimamura ◽  
D Tremethick ◽  
A Worcel
Keyword(s):  
Xenopus Laevis ◽  
Transcriptional Activation ◽  
High Speed ◽  
Histone H1 ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
5S Rna ◽  
Rna Transcription

We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.

scholarly journals The Xenopus laevis Isoform of G Protein-Coupled Receptor 3 (GPR3) Is a Constitutively Active Cell Surface Receptor that Participates in Maintaining Meiotic Arrest in X. laevis Oocytes

2008 ◽  
Vol 22 (8) ◽  
pp. 1853-1865 ◽  
Author(s):  
James Deng ◽  
Stephanie Lang ◽  
Christopher Wylie ◽  
Stephen R. Hammes
Keyword(s):  
Cell Surface ◽  
Xenopus Laevis ◽  
G Protein ◽  
Oocyte Maturation ◽  
Cell Surface Receptor ◽  
Xenopus Laevis Oocytes ◽  
Intracellular Camp ◽  
G Protein Coupled Receptor ◽  
Meiotic Arrest ◽  
G Protein Coupled

Abstract Oocytes are held in meiotic arrest in prophase I until ovulation, when gonadotropins trigger a subpopulation of oocytes to resume meiosis in a process termed “maturation.” Meiotic arrest is maintained through a mechanism whereby constitutive cAMP production exceeds phosphodiesterase-mediated degradation, leading to elevated intracellular cAMP. Studies have implicated a constitutively activated Gαs-coupled receptor, G protein-coupled receptor 3 (GPR3), as one of the molecules responsible for maintaining meiotic arrest in mouse oocytes. Here we characterized the signaling and functional properties of GPR3 using the more amenable model system of Xenopus laevis oocytes. We cloned the X. laevis isoform of GPR3 (XGPR3) from oocytes and showed that overexpressed XGPR3 elevated intraoocyte cAMP, in large part via Gβγ signaling. Overexpressed XGPR3 suppressed steroid-triggered kinase activation and maturation of isolated oocytes, as well as gonadotropin-induced maturation of follicle-enclosed oocytes. In contrast, depletion of XGPR3 using antisense oligodeoxynucleotides reduced intracellular cAMP levels and enhanced steroid- and gonadotropin-mediated oocyte maturation. Interestingly, collagenase treatment of Xenopus oocytes cleaved and inactivated cell surface XGPR3, which enhanced steroid-triggered oocyte maturation and activation of MAPK. In addition, human chorionic gonadotropin-treatment of follicle-enclosed oocytes triggered metalloproteinase-mediated cleavage of XGPR3 at the oocyte cell surface. Together, these results suggest that GPR3 moderates the oocyte response to maturation-promoting signals, and that gonadotropin-mediated activation of metalloproteinases may play a partial role in sensitizing oocytes for maturation by inactivating constitutive GPR3 signaling.

scholarly journals Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination

1991 ◽  
Vol 11 (6) ◽  
pp. 3278-3287 ◽  
Author(s):  
E Maryon ◽  
D Carroll
Keyword(s):  
Homologous Recombination ◽  
Xenopus Laevis ◽  
Kinetic Properties ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Two Dimensional Gel Electrophoresis ◽  
Recombination Mechanism ◽  
Single Strand Annealing ◽  
Recombination Pathway

Homologous recombination between DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient if injected molecules have overlapping homologous ends. Earlier work demonstrated that ends of linear molecules are degraded by a 5'----3' exonuclease activity, yielding 3' tails that participate in recombination. Here, we have characterized intermediates further advanced along the recombination pathway. The intermediates were identified by their unique electrophoretic and kinetic properties. Two-dimensional gel electrophoresis and hybridization with oligonucleotide probes showed that the intermediates had heteroduplex junctions within their homologous overlaps in which strands ending 3' were full length and those ending 5' were shortened. Additional characterization suggested that these intermediates had formed by the annealing of complementary 3' tails. Annealed junctions made in vitro were rapidly processed to products, indicating that they are on the normal recombination pathway. These results support a nonconservative, single-strand annealing mode of recombination. This recombination mechanism appears to be shared by many organisms, including bacteria, fungi, plants, and mammals.

scholarly journals Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes.

1988 ◽  
Vol 8 (10) ◽  
pp. 4257-4269 ◽  
Author(s):  
A Shimamura ◽  
D Tremethick ◽  
A Worcel
Keyword(s):  
Xenopus Laevis ◽  
Transcriptional Activation ◽  
High Speed ◽  
Histone H1 ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
5S Rna ◽  
Rna Transcription

We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.

scholarly journals 3'-end-dependent formation of U6 small nuclear ribonucleoprotein particles in Xenopus laevis oocyte nuclei.

1992 ◽  
Vol 12 (7) ◽  
pp. 3032-3040 ◽  
Author(s):  
M P Terns ◽  
E Lund ◽  
J E Dahlberg
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Antigen ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
La Protein ◽  
Shift Analysis

We have identified and characterized a U6 small nuclear (sn) ribonucleoprotein particle (RNP) present in the nuclei of Xenopus laevis oocytes. The structure of this U6 snRNP was investigated by native gel shift analysis and a combination of RNA-protein UV cross-linking, RNase T1 fingerprinting, and immunoprecipitation assays. These analyses demonstrate that certain forms of U6 snRNA associate with the 50-kDa nuclear antigen La both in vivo and in vitro. The La protein binds the stretch of uridylates at the 3' hydroxyl end of newly synthesized U6 snRNA. La does not bind to mature U6 snRNAs that have 2',3'-cyclic phosphate (greater than p) groups at their 3' ends (E. Lund and J. E. Dahlberg, Science 255:327-330, 1992) or to U6 snRNAs in anti-Sm-precipitable U4/U6 snRNPs. We propose that 3'-end modification, including posttranscriptional UMP addition, modulates the binding of La protein to U6 snRNA which, in turn, may affect the function of this RNA.

scholarly journals Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins.

1987 ◽  
Vol 7 (3) ◽  
pp. 1285-1288 ◽  
Author(s):  
A K Deshpande ◽  
H F Kung
Keyword(s):  
Monoclonal Antibody ◽  
Xenopus Laevis ◽  
Oocyte Maturation ◽  
Neutralizing Antibody ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Ras Proteins ◽  
Ras Protein ◽  
P21 Ras

Microinjection of transforming p21 ras protein induces maturation of Xenopus laevis oocytes, and the induction is blocked by coinjection of monoclonal antibody (Y13-259) against p21 ras proteins. Similar to other inducing agents, the effect of p21 ras protein is mediated via the appearance of maturation or meiosis-promoting factor activity. In addition, the neutralizing antibody markedly reduces oocyte maturation after insulin induction, whereas it fails to inhibit progesterone induction. Our results suggest that insulin induces maturation of oocytes via a different pathway than that of steroidal agents. The induction by insulin is ras dependent, and the action of ras may be directed at the steps before meiosis-promoting factor autocatalytic activation. These results suggest a role of p21 ras protein in the events associated with amphibian oocyte maturation.

scholarly journals 3'-end-dependent formation of U6 small nuclear ribonucleoprotein particles in Xenopus laevis oocyte nuclei

1992 ◽  
Vol 12 (7) ◽  
pp. 3032-3040
Author(s):  
M P Terns ◽  
E Lund ◽  
J E Dahlberg
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Antigen ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
La Protein ◽  
Shift Analysis

We have identified and characterized a U6 small nuclear (sn) ribonucleoprotein particle (RNP) present in the nuclei of Xenopus laevis oocytes. The structure of this U6 snRNP was investigated by native gel shift analysis and a combination of RNA-protein UV cross-linking, RNase T1 fingerprinting, and immunoprecipitation assays. These analyses demonstrate that certain forms of U6 snRNA associate with the 50-kDa nuclear antigen La both in vivo and in vitro. The La protein binds the stretch of uridylates at the 3' hydroxyl end of newly synthesized U6 snRNA. La does not bind to mature U6 snRNAs that have 2',3'-cyclic phosphate (greater than p) groups at their 3' ends (E. Lund and J. E. Dahlberg, Science 255:327-330, 1992) or to U6 snRNAs in anti-Sm-precipitable U4/U6 snRNPs. We propose that 3'-end modification, including posttranscriptional UMP addition, modulates the binding of La protein to U6 snRNA which, in turn, may affect the function of this RNA.

Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins

1987 ◽  
Vol 7 (3) ◽  
pp. 1285-1288
Author(s):  
A K Deshpande ◽  
H F Kung
Keyword(s):  
Monoclonal Antibody ◽  
Xenopus Laevis ◽  
Oocyte Maturation ◽  
Neutralizing Antibody ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Ras Proteins ◽  
Ras Protein ◽  
P21 Ras

Microinjection of transforming p21 ras protein induces maturation of Xenopus laevis oocytes, and the induction is blocked by coinjection of monoclonal antibody (Y13-259) against p21 ras proteins. Similar to other inducing agents, the effect of p21 ras protein is mediated via the appearance of maturation or meiosis-promoting factor activity. In addition, the neutralizing antibody markedly reduces oocyte maturation after insulin induction, whereas it fails to inhibit progesterone induction. Our results suggest that insulin induces maturation of oocytes via a different pathway than that of steroidal agents. The induction by insulin is ras dependent, and the action of ras may be directed at the steps before meiosis-promoting factor autocatalytic activation. These results suggest a role of p21 ras protein in the events associated with amphibian oocyte maturation.

scholarly journals Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination.

1991 ◽  
Vol 11 (6) ◽  
pp. 3278-3287 ◽  
Author(s):  
E Maryon ◽  
D Carroll
Keyword(s):  
Homologous Recombination ◽  
Xenopus Laevis ◽  
Kinetic Properties ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Two Dimensional Gel Electrophoresis ◽  
Recombination Mechanism ◽  
Single Strand Annealing ◽  
Recombination Pathway

Homologous recombination between DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient if injected molecules have overlapping homologous ends. Earlier work demonstrated that ends of linear molecules are degraded by a 5'----3' exonuclease activity, yielding 3' tails that participate in recombination. Here, we have characterized intermediates further advanced along the recombination pathway. The intermediates were identified by their unique electrophoretic and kinetic properties. Two-dimensional gel electrophoresis and hybridization with oligonucleotide probes showed that the intermediates had heteroduplex junctions within their homologous overlaps in which strands ending 3' were full length and those ending 5' were shortened. Additional characterization suggested that these intermediates had formed by the annealing of complementary 3' tails. Annealed junctions made in vitro were rapidly processed to products, indicating that they are on the normal recombination pathway. These results support a nonconservative, single-strand annealing mode of recombination. This recombination mechanism appears to be shared by many organisms, including bacteria, fungi, plants, and mammals.

Sign in / Sign up

Export Citation Format

Share Document