Src Family Kinases In Mouse And Rat Oocyte Meiotic Maturation Process In The Positioning And Function

Meiotic maturation of oocyte is a final step of oogenesis and a prerequisite process for the immature oocyte to become fertilization. In mammalian oocyte, meiosis resumes when the envelope of the germinal vesicle breakdown (GVBD), occurs a few hours after the luteinizing hormone (LH) surge in vivo, or oocyte releasing from the follicle in vitro. And then undergo a second arrest at metaphase II (MII), which is maintained until fertilization. Meiotic maturation involves synthesis of a series of specific proteins and changes in the phosphorylation state of kinase and phosphatase activity. Many of the components in the maturation induction pathway are still unknown. Kimura et al., (1996) indicated that protein tyrosine phosphorylation may be implicated in the regulation of mouse oocyte meiosis.The Src family kinase (SFKs) were first identified as transforming proteins encoded by oncogenic retroviruses. They are non-receptor tyrosine kinases and keep in a confirmation of low activity conformation by phosphorylation of C-terminal tyrosine residue. Upon dephosphorylation of C-terminal tyrosine residue or displacement of the Src homology regions 2 (SH2) and 3 (SH3) intramolecular interactions by other proteins, SFKs gains a relaxed conformation that allows autophosphorylation of tyrosine in the catalytic site and stabilizes the high activity state. Earlier Studies indicated that the different subcellular localizations of SFKs could be important for the regulation of specific cellular processes (reviewed by Bjorge et al., 2000). Previous studies showed the function of SFKs in the normal cell cycle control: As to the role of SFKs in oocytes, Spivack et al (1984) and Unger and Steele (1992) demonstrated the accelerated rate of progesterone-induced Xenopus oocyte maturation by microinjection of v-Src protein or by the expression of activated c-Src respectively. Furthermore, studies in echinoderm, ascidian, Xenopus, zebrafish and rat oocytes suggest that activation of an SFK(s) is required for oocyte activation. Numerous studies suggest that SFKs also function to regulate the actin cytoskeleton.We used western blot, confocal microscope immunofluorescence to study the in vitro dynamics of the subcellular distribution of SFK during the mouse oocyte meiotic maturation and evaluated the functions of SFK by PP2, a specific SFKs pharmacological inhibitor. Our results showed that nonphospho-SFK were absent in oocyte upon it was released from follicle. Nonphospho-SFK appeared in cytoplasm 0.5 h after the release of oocyte and were translocated to germinal vesicle before the onset of germinal vesicle breakdown (GVBD). After oocytes underwent GVBD, nonphospho-SFK accumulated in the same area as condensed chromosomes. In metaphase I and telophase I stage oocytes, nonphospho-SFK accumulated in the cortex and the cleavage furrow respectively in addition to distributing throughout the cytoplasm. In metaphase II oocytes, nonphospho-SFK concentrated around the aligned chromosomes. In contrast, phospho-SFK were absent in oocyte until 1 h after it was released from the follicle. Phospho-SFK accumulated in the germinal vesicle and the cortex in addition to their cytoplasm localization immediately prior to GVBD. After GVBD, phosphot-SFK distributed evenly in oocyte. In MII oocyte, phospho-SFK were localized throughout the cytoplasm and under the egg member. When the SFK activity was inhibited, oocyte failed to onset GVBD, MI entry and extrude the first polar body. Our research also shown The interaction between SFKs and F- actin .when the activity of SFKs were inhibited, F-actin were depolymerized, by contraries, The activity of SFKs were severely affected when inhibited F-actin polymerize. We also try to investigated the relationship between SFKs and protein kinase C (PKC) or Ca2+, we found that the activity of SFKs were not be affected when oocytes were treated by PMA or A23187. Furthermore, our study shown that SFKs keep its activity during in vitro fertilization (IVF).Then we used confocal microscope immunofluorescence, specific SFKs pharmacological inhibitor methods to evaluate whether SFKs have the same functions during meiotic maturation in rat oocytes. Our results shown that SFKs were localized under the cell membrane (the same area of cortex) and around the chromosome, and were keeping its activity during all the process of oocyte maturation. So the results in hinted that SFKs may be involved in modulation of meiotic maturation in rat oocytes. Our inhibitor methods data shown that 100μM PP2 had significant effect in oocytes GVBD, it was indicted that the activity of SFKs were necessary for GVBD of rat oocytes. The localization of SFKs was same as the localization of cytoskeleton in rat oocytes, so we suspected that the function of SFKs may be related with F-actin or microtubule. We choose 5μM cytochalasin B (an inhibitor of F-actin polymerize) and 10μg/ml Colchicine (an inhibitor of microtubule organization). Our data shown that the activity of SFKs were not affected when F-actin polymerize or microtubule organization were inhibited. Furthermore, it was not alike in mouse oocytes, when inhibited the activity of SFKs or the polymerization of F-actin during rat oocytes maturation, we did not find any change of the location of SFKs or F-actin. So SFKs may have different functions in rat oocytes than they have in mouse oocytes.In conclusion, our results indicated for the firs time that SFKs were localized in mouse and rat oocytes during meiotic maturation process, and the function of SFKs may be related with cytoskeleton such as F-actin or microtubule, and protein kinase C or Ca2+ had no effect on the activity of SFKs during the process in mouse oocytes.