Effect of cyclin-dependent kinase (CDK) inhibition on expression, localization and activity of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) in bovine oocytes.
This study aimed to assess the effects of cyclin-dependent kinase (CDK) inhibition on factors involved in the control of meiosis in bovine oocytes: maturation promoting factor (MPF) (p34(cdc2) and cyclin B1) and mitogen activated protein kinase (MAPK). Oocytes were maintained at germinal vesicle (GV) stage in vitro with 10 μM of the CDK inhibitor butyrolactone I (BLI) for 24 h (inhibited). After this period, some of the oocytes were transferred to in vitro maturation (IVM) culture for 24 h (inhibited and matured). Control oocytes were assessed immediately after follicle aspiration (immature) or after in vitro maturation for 24 h (matured). Real-time PCR analyses showed that transcripts for p34(cdc2) and MAPK were detected in immature and inhibited oocytes and decreased after maturation, irrespective of CDK inhibition with BLI. Cyclin B1 was detected at similar levels in all oocyte groups. The p34(cdc2) and MAPK proteins were detected by Western blotting at similar levels in all oocyte groups, and cyclin B1 protein was detected only after maturation. Immunofluorescence detection showed that p34(cdc2) was localized in the cytoplasm and GV of immature oocytes, and then throughout the cytoplasm after maturation. Cyclin B1 and MAPK were detected in the cytoplasm in all oocyte groups. Maturation promoting factor and MAPK activities were similar throughout most of maturation for oocytes treated with or without BLI. In conclusion, CDK inhibition did not affect the expression (mRNA and protein levels) and localization of MPF and MAPK, and had nearly no effect on kinase activities during maturation.
Quetglas
M D
MD
Departamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, São Paulo, Brazil.
Adona
P R
PR
de Bem
T H C
TH
Pires
P R L
PR
Leal
C L V
CL
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002417:Cattle / Q000502:physiology*; D056744:Cyclin B1 / Q000235:genetics / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D005786:Gene Expression Regulation / Q000187:drug effects*; D016200:Maturation-Promoting Factor / Q000235:genetics / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D056744:Cyclin B1; D012333:RNA, Messenger; C082868:butyrolactone I; D018844:Cyclin-Dependent Kinases; D016200:Maturation-Promoting Factor; D020929:Mitogen-Activated Protein Kinase Kinases; D015107:4-Butyrolactone