Protein kinase B/Akt is essential for the insulin- but not progesterone-stimulated resumption of meiosis in Xenopus oocytes.
In the present study, we have characterized the Xenopus Akt expressed in oocytes from the African clawed frog Xenopus laevis and tested whether its activity is required for the insulin- and progesterone-stimulated resumption of meiosis. A cDNA encoding the Xenopus Akt was isolated and sequenced, and its expression in the Xenopus oocyte was confirmed by reverse transcription PCR and Northern blotting. Using phosphospecific antibodies and enzyme assays, a large and rapid activation of the Xenopus Akt was observed upon insulin stimulation of the oocytes. In contrast, progesterone caused a modest activation of this kinase with a slower time course. To test whether the activation of Akt was required in the stimulation of the resumption of meiosis, we have utilized two independent approaches: a functional dominant negative Akt mutant and an inhibitory monoclonal antibody. Both the mutant Akt, as well as the inhibitory monoclonal antibody, completely blocked the insulin-stimulated resumption of meiosis. In contrast, both treatments only partially inhibited (by approx. 30%) the progesterone-stimulated resumption of meiosis when submaximal doses of this hormone were utilized. These data demonstrate a crucial role for Akt in the insulin-stimulated cell cycle progression of Xenopus oocytes, whereas Akt may have an ancillary function in progesterone signalling.
Andersen
Carsten B
CB
Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA 94305, U.S.A.
Sakaue
Hiroshi
H
Nedachi
Taku
T
Kovacina
Kristina S
KS
Clayberger
Carol
C
Conti
Marco
M
Roth
Richard A
RA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D019556:COS Cells; D004305:Dose-Response Relationship, Drug; D005260:Female; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D008540:Meiosis / Q000187:drug effects / Q000502:physiology*; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D010802:Phylogeny; D011374:Progesterone / Q000378:metabolism / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology; D014982:Xenopus laevis / Q000502:physiology*
D000911:Antibodies, Monoclonal; D007328:Insulin; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011374:Progesterone; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt