Quetglas M D, Adona P R, de Bem T H C, Pires P R L, Leal C L V
Departamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, São Paulo, Brazil.
Reprod Domest Anim. 2010 Dec;45(6):1074-81. doi: 10.1111/j.1439-0531.2009.01498.x.
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.
本研究旨在评估细胞周期蛋白依赖性激酶（CDK）抑制对牛卵母细胞减数分裂控制相关因子的影响：成熟促进因子（MPF）（p34^（cdc2）和细胞周期蛋白B1）以及丝裂原活化蛋白激酶（MAPK）。将卵母细胞在体外以10 μM的CDK抑制剂丁内酯I（BLI）维持在生发泡（GV）期24小时（抑制组）。在此期间后，将部分卵母细胞转移至体外成熟（IVM）培养24小时（抑制并成熟组）。对照卵母细胞在卵泡抽吸后立即评估（未成熟组）或在体外成熟24小时后评估（成熟组）。实时PCR分析表明，无论是否用BLI抑制CDK，在未成熟和抑制的卵母细胞中均检测到p34^（cdc2）和MAPK的转录本，且成熟后其水平降低。在所有卵母细胞组中，细胞周期蛋白B1的检测水平相似。通过蛋白质印迹法在所有卵母细胞组中检测到p34^（cdc2）和MAPK蛋白的水平相似，且仅在成熟后检测到细胞周期蛋白B1蛋白。免疫荧光检测显示，p34^（cdc2）定位于未成熟卵母细胞的细胞质和GV中，成熟后则遍布整个细胞质。在所有卵母细胞组的细胞质中均检测到细胞周期蛋白B1和MAPK。对于用或未用BLI处理的卵母细胞，在成熟的大部分时间里，成熟促进因子和MAPK活性相似。总之，CDK抑制不影响MPF和MAPK的表达（mRNA和蛋白质水平）及定位，并且对成熟过程中的激酶活性几乎没有影响。
