Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development. -CAT-line [1] with the C57BL/6-TgN(mice which were re-crossed to produce animals. For production of males were crossed with females and the resulting females were used for oocyte or embryo collection. Genotyping of mice was conducted according to a published protocol [1] in addition to the Jackson Laboratory protocol (http://jaxmice.jax.org/strain/003651.html). Oocytes were collected stripped of cumulus cells and zona-pellucida then fertilized in vitro under sperm-limiting conditions as previously described [16]; [7]. Incubation of oocytes with the PYK2 inhibitor PF04594755 (Pfizer Corporation Groton CT) or the SRC-family kinase inhibitor Repaglinide SKI 606 (Bosutinib Wyeth Pearl River NY) was carried Cd300lg out by addition of the inhibitor to medium after a 90 minute recovery from the zona-removal process. Oocytes were incubated with the indicated inhibitor for 30 minutes and washed twice prior to addition of sperm. After a 2hr incubation with sperm oocytes were transferred to a fixative containing 2% formaldehyde and 1% saturated picric acid for 2 hrs then permeabilized with 0.1% triton X100 in PBS. Oocytes were then labeled with 10uM DRAQ5 (Cell Signaling Danvers MA) to label DNA and 25nM phalloidin alexa fluor 488 (Invitrogen Carlsbad CA) to label filamentous actin. The number of bound Repaglinide sperm (those confirmed to be outside of the cortical actin layer) were counted by confocal fluorescence using a 40X objective. Fertilization was established by the presence of sperm heads or expanded pronuclei that were clearly within the cortical actin layer. Oocyte activation was determined by resumption of anaphase as indicated by separation of the meiotic chromosomes. Western blot analysis Samples of 10-20 oocytes were resolved on a 10% SDS-polyacrylamide gel with a 4% stacking gel cast with a micro-scale comb to produce wells 1mm in width. Proteins were electro-transferred to immobilon-P (Millipore Corp. Billeria MA) blocked with 5% BSA in tris-buffered saline containing 0.1% Tween 20 (Fisher scientific Pittsburgh PA) then probed with anti-GAPDH (EMD Millipore Billerica MA) and anti-PYK2 PY579 (Invitrogen Carlsbad CA). Other antibodies used included Repaglinide antibodies to activated FAK (anti-FAK PY861 (Invitrogen Carlsbad CA)) and anti-activated SRC (clone 28) (Biosource International Camarillo CA). Chemiluminescence detection was done using Repaglinide the Femto-Kit (Thermo scientific Rockford IL USA). Statistical analysis of band intensity within different experimental groups was performed by test or by Mann Whitney rank sum analysis using Sigmastat software (Systat Software Inc. Chicago IL). Fluorescence Microscopy Oocytes were fixed and prepared for labeling with Draq 5 to detect DNA and phalloidin alexa fluor 488 (Invitrogen Carlsbad CA) to detect filamentous actin as previously described [16] and imaged with a Nikon TE2000 confocal microscope using either the confocal system or the conventional UV illumination. Total PYK2 PY579 content per cell was measured by slight modification of a published method [2]. Oocytes were grouped in a glass bottom microwell (Delta TBG dish Fisher Scientific St. Louis MO) and imaged in a single frame by fluorescence microscopy at low magnification to keep all oocytes in the same focal plane. The fluorescence intensity of each oocyte was measured with Metafluor software (Meta Imaging Devices Downington PA USA). Ca2+ imaging in live oocytes Zona-free oocytes were incubated in mKSOM with 1μM fura-2 Repaglinide AM/0.02% pluronic F-127 (Invitrogen Carlsbad CA) prior to addition of 10μM PF04594755 or 0.1% DMSO as a control. After a 15 minute fertilization period the fertilization droplets were monitored at 37°C on a.