Department of Anatomy & Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., mail stop 3038, Kansas City, KS 66160, USA.
J Assist Reprod Genet. 2010 Apr;27(4):169-82. doi: 10.1007/s10815-010-9391-x. Epub 2010 Feb 20.
To ask whether distinct kinase signaling pathways mediate cytoplasmic or nuclear maturation of mouse oocytes and if in vitro maturation influences the distribution and timing of these phosphorylation events.
Mouse cumulus oocyte complexes (COCs) were matured under conditions known to influence oocyte quality (basal or supplemented media) and assayed with epitope specific antibodies that would distinguish between Cdk1 or tyrosine kinase targets at 0, 2, 4, 8, and 16 hrs. Semi-quantitative image analysis was used to assess the topographical patterns of protein phosphorylation during in vitro maturation. In vitro fertilization and embryo culture were used to examine the effects of culture conditions on developmental potential.
Protein tyrosine phosphorylation increased during meiotic progression from methaphase-I to metaphase-II. Levels were significantly higher in the oocyte cortex. Levels of cortical staining are enhanced in oocytes matured in supplemented media that displayed higher developmental competence. In contrast, bulk substrates for Cdk1 kinase localize to the meiotic spindle while cytoplasmic levels of kinase activity increase throughout meiotic progression; culture media had no measurable effect. Ablation of the tyrosine kinase Fyn significantly reduced cortical levels of tyrosine phosphorylation.
The findings indicate that distinct signaling pathways mediate nuclear and cytoplasmic maturation during in vitro maturation in a fashion consistent with a role for tyrosine kinases in cortical maturation and oocyte quality.
探讨不同的激酶信号通路是否介导了小鼠卵母细胞的胞质或核成熟，以及体外成熟是否会影响这些磷酸化事件的分布和时间。
采用已知影响卵母细胞质量的条件（基础或补充培养基）对小鼠卵丘-卵母细胞复合物（COCs）进行体外成熟，并使用表位特异性抗体进行检测，这些抗体可区分 Cdk1 或酪氨酸激酶靶标在 0、2、4、8 和 16 小时的分布情况。采用半定量图像分析来评估体外成熟过程中蛋白质磷酸化的拓扑模式。体外受精和胚胎培养用于研究培养条件对发育潜能的影响。
在从第一次减数分裂中期到第二次减数分裂中期的过程中，蛋白质酪氨酸磷酸化增加。卵母细胞皮质中的水平明显更高。在补充培养基中成熟的卵母细胞中，皮质染色水平增强，显示出更高的发育能力。相比之下，Cdk1 激酶的大量底物定位于减数分裂纺锤体，而细胞质中激酶活性水平在整个减数分裂过程中增加；培养基没有可测量的影响。酪氨酸激酶 Fyn 的消融显著降低了皮质中的酪氨酸磷酸化水平。
这些发现表明，不同的信号通路在体外成熟过程中通过与酪氨酸激酶在皮质成熟和卵母细胞质量中的作用一致的方式来介导核和胞质成熟。
