Localized activation of Src-family protein kinases in the mouse egg

Recent studies in species that fertilize externally have demonstrated that fertilization triggers localized activation of Src-family protein kinases in the egg cortex. However, the requirement for Src-family kinases in activation of the mammalian egg is different from lower species and the objective of this study was to characterize changes in the distribution and activity of Src-family protein tyrosine kinases (PTKs) during zygotic development in the mouse. Immunofluorescence analysis of mouse oocytes and zygotes with an anti-phosphotyrosine antibody revealed that fertilization stimulated accumulation of P-Tyr-containing proteins in the egg cortex and that their abundance was elevated in the region overlying the MII spindle. In addition, the poles of the MII spindle exhibited elevated P-Tyr levels. As polar body extrusion progressed, P-Tyr-containing proteins were especially concentrated in the region of cortex adjacent to the maternal chromatin and the forming polar body. In contrast, P-Tyr labeling of the spindle poles eventually disappeared as meiosis II progressed to anaphase II. In approximately 24% of cases, the fertilizing sperm nucleus was associated with increased P-Tyr labeling in the overlying cortex and oolemma. To determine whether Src-family protein tyrosine kinases could be responsible for the observed changes in the distribution of P-Tyr containing proteins, an antibody to the activated form of Src-family PTKs was used to localize activated Src, Fyn or Yes. Activated Src-family kinases were found to be strongly associated with the meiotic spindle at all stages of meiosis II; however, no concentration of labeling was evident at the egg cortex. The absence of cortical Src-family PTK activity continued until the blastocyst stage when strong cortical activity became evident. At the pronuclear stage, activated Src-family PTKs became concentrated around the pronuclei in close association with the nuclear envelope. This pattern was unique to the earliest stages of development and disappeared by the eight cell stage. Functional studies using chemical inhibitors and a dominant-negative Fyn construct demonstrated that Src-family PTKs play an essential role in completion of meiosis II following fertilization and progression from the pronuclear stage into mitosis. These data suggest that while Src-family PTKs are not required for fertilization-induced calcium oscillations, they do play a critical role in development of the zygote. Furthermore, activation of these kinases in the mouse egg is limited to distinct regions and occurs at specific times after fertilization.