Critical effect of pigWee1B on the regulation of meiotic resumption in porcine immature oocytes.
Porcine immature oocytes require protein synthesis for meiotic resumption, thus the importance of Cdc2 inhibitory phosphorylation in their meiotic arrest remains controversial. We examined the involvement of Cdc2 phosphorylation in the meiotic arrest of porcine oocytes with a special focus on Wee1B, an oocyte-specific Wee1 family member recently reported in mouse oocytes. We cloned a Wee1B homologue of pig by RT-PCR followed by 5'- and 3'-RACE. Overexpression of pigWee1B in porcine immature oocytes by the injection of pigWee1B mRNA almost completely blocked the germinal vesicle breakdown (GVBD) under the low cAMP concentration, which could not block their spontaneous meiotic resumption by itself. The MPF activation and cyclin B synthesis were inhibited in these oocytes. Conversely, downregulation of pigWee1B expression by the injection of specific antisense mRNA induced GVBD in the oocytes, the spontaneous meiotic resumption of which was blocked by the high concentration of cAMP (dbcAMP). In these oocytes, the MPF activity was elevated and cyclin B was accumulated. Downregulation of pigMyt1, another Wee1 family member, could not induce the GVBD under the same condition. The inhibition of tyrosine phosphatase by vanadate blocked the GVBD even in the pigWee1B-downregulated oocytes. These results suggest that the inhibitory phosphorylation of CDC2, which is catalyzed by pigWee1B, but not pigMyt1, is involved in the meiotic arrest of porcine oocytes, and that the inactivation of Wee1B in combination with the phosphatase activation induces the conversion of pre-MPF to the active MPF and starts the cyclin B synthesis, follwed by a further increase of MPF and meiotic resumption.
Shimaoka
Takuma
T
Laboratory of Applied Genetics, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Nishimura
Takanori
T
Kano
Kiyoshi
K
Naito
Kunihiko
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D019926:Cyclin B / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D016200:Maturation-Promoting Factor / Q000378:metabolism*; D008540:Meiosis / Q000187:drug effects / Q000502:physiology*; D000090204:Mesothelin; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D016372:RNA, Antisense / Q000378:metabolism; D016415:Sequence Alignment; D013552:Swine; D014638:Vanadates / Q000494:pharmacology
D019926:Cyclin B; D004268:DNA-Binding Proteins; C000715528:Msln protein, mouse; D016372:RNA, Antisense; D014638:Vanadates; D011505:Protein-Tyrosine Kinases; D016203:CDC2 Protein Kinase; D016200:Maturation-Promoting Factor; D017027:Protein Tyrosine Phosphatases; D000090204:Mesothelin