Tyrosine Kinase Pyk2 In The Rat And Mouse Oocyte Meiosis, Positioning And Function Of Research

Fully-grown mammalian oocytes are arrested at the prophase of the first meiotic division within the ovarian follicles. In vivo, following the stimulation of gonadotropins in each estrus cycle, part of oocytes reinitiate meiosis and ovulated at metaphase II. The oocytes release from the MII arrest complete the meiotic division till fertilization or parthenogenetic activation. The meiotic division is a complex process under the regulation of multiple extra cellular signals. Protein kinases play a key role during meiotic division by regulating the phosphoralation of some proteins. However, studies are focused on the function of Ser/Thr protein kinases, while another important protein kinases, protein tyrosine kinases is scarcely investigated. Proline-rich tyrosine kinase2 (Pyk2), a non-receptor protein tyrosine kinase related to focal adhesion kinase is expressed in many tissues and cell types. Pyk2 stimulated by multiple extra cellular physical and chemical signals, participates in cell signal transduction and have important function in cell adhesion, migration, survival, proliferation, apoptosis and differentiation. Is Pyk2 existed in mammalian oocyte and what role it would play if existed. Nothing is known about this question. To answer it, we have investigated the localization and in particular its function of Pyk2 during rat and mouse oocyte maturation and fertilization by using Western-blot, confocal laser scanning microscopy, drugs treatment, antibody microinjection. This research may throw a light on the molecular mechanisms of mammalian oocyte meiotic maturation and fertilization.We for the first time detected the expression of Pyk2 in rat oocytes by using western-blot analysis and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle or the condensed chromosomes at the germinal vesicle stage or after germinal vesicle breakdown(GVBD). At metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar with that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body emission. These findings suggest the expression and activation of Pyk2 during rat oocyte maturation and also show an important role of Pyk2 for rat oocyte polarity establishment and polar body extrusion by regulating the organization of actin filaments.By using a immunochemistry fluorescence technique Pyk2 was found distributed uniformly in the whole oocyte except for the nucleolus upon it was released from follicle. Pyk2 became concentrated in the nuclear 1h after the release of the oocyte and was associated with the chromosomes just before GVBD. After oocytes underwent GVBD, Pyk2 distributed evenly in the ooplasm and this distribution pattern was maintained till to the first meiotic metaphase II. In contrast, Tyr402 p-Pyk2 was absent in oocyte until 1h after it was released from the follicle. It was distributed throughout the oocyte during the oocyte maturation. In the second meiotic anaphase, which was aroused by the incorporation of sperm, Pyk2 was found in the whole cytoplasm no matter in the in vivo or in vitro fertilization. When the second polar bodies was extruded Pyk2 was tightly associated with the male chromosomes except for its cytoplasm distribution. Pyk2 was also localized in the nucleus of the zygotes. To further study the functions of Pyk2 during mouse oocyte meiosis salicylate, an inhibitor of Pyk2 and Pyk2 antibody were used to treat or microinjected into the oocyte. The GVBD and polar body emission were observed after the treatment and several hours culture. The results showed that salicylate inhibited the oocyte GVBD and polar body emission in a dose dependent manner while the antibody microinjected in the GV oocytes had no function on the GVBD and first polar body emission. These paradoxical results suggest another proteins or kinsase that was required for the mouse oocyte meiosis may also be inhibited by salicylate. In addition, to investigate where Pyk2 was under the regulation of Ca2+ and PKC, A23187 and PMA were used and Pyk2 in MII stage oocyte was found assembled into many granules after the medications. In conclusion, Pyk2 was expressed during the mouse oocyte maturation and fertilization. Tyr402 of Pyk2 was activated 1h after oocyte release from the follicle and kept the activation till to metaphase II. Pyk2 might participate in the condensation of the GV oocyte chromosomes and the decondensation of the sperm according to the results that Pyk2 was co-localized with the oocyte or sperm chromosomes. The inhibition of Pyk2 was not blocked the GVBD and polar body emission and Pyk2 might be regulated by Ca²⁺ and PKC.