Src-Family Protein Tyrosine Kinases Are Required for Meiotic Maturation in the Mouse.

Abstract

Oocyte maturation involves both cytoplasmic maturation processes as well as meiotic divisions that prepare the oocyte for fertilization and progressive development. Three members of the Src-family of protein tyrosine kinases (PTKs) are expressed in mammalian oocytes: Fyn, Yes and Src. These PTKs have been implicated in both the first and second meiotic maturation processes. The objective of the current studies was to examine the specific biochemical and cellular events requiring Src-family PTKs during oocyte maturation. We have used a combination of chemical inhibitors and siRNA knock-down to impair Src PTKs in order to examine Src PTK involvement in meiotic maturation. Oocytes were collected from mature CF1 mice with or without 300 μM dbcAMP to maintain GV arrest. Maturation media were supplemented with 0.2 IU rhFSH and 10 ng/ml EGF. To test the requirement for Src PTKs, oocytes were matured in the presence of Src-specific inhibitors PP2 (10, 20 or 50 μM) or SKI606 (1, 5 or 10 μM). Both inhibitors had no effect on GVBD, but inhibited meiotic progression from MI to MII. This inhibition was dose dependant. At lower doses, some oocytes progressed to metaphase-II but displayed abnormalities in spindle and chromatin organization; the percentage of abnormal oocytes was also dose dependant. Continuous 17-h exposure of COC to 10 μM SKI606 arrested all oocytes at metaphase of MI. Wash-out experiments in which oocytes were held for 1, 2.5 or 5 h with or without cAMP and/or SKI606 and were returned to control medium demonstrated that the effects of SKI606 were reversible. As an alternative approach, we have used siRNA for a specific knock-down of Fyn PTK in GV stage oocytes, since our previous studies implicated Fyn kinase in the completion of meiosis following fertilization. The siRNA injected GV oocytes were maintained in cAMP media for 5 h to allow time for the siRNA actions prior to cAMP removal thus allowing meiotic resumption. Following 17 h of maturation, 61% of the Fyn knock-down eggs arrested at the MI stage (39% matured to MII, n=96), compared to scrambled control siRNA injected and non-injected control eggs which matured to MII (86% n=112 and 93% n=27, respectively). These studies demonstrate that Src PTK activity is required during the early stages of meiotic maturation and further demonstrates the involvement of Src PTKs in meiotic spindle morphogenesis and cell cycle progression at the first metaphase anaphase transition. Fyn PTK specifically appears critical for healthy meiotic maturation and early developmental progression of the mammalian egg.