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Germ cells require communication with associated somatic cells for normal gametogenesis, as exemplified by an oocyte that interacts with granulosa cells via paracrine factors as well as gap junctions located at sites of contact between these two cell types. The objective of the present study was to define the mechanisms by which cell-cell contact with the oocyte is controlled and to determine the extent that the oocyte actively participates in this association. Proline-rich tyrosine kinase 2 (PTK2), a focal adhesion kinase, was found to be activated at sites of contact between the oocyte and trans-zonal cell processes from the surrounding granulosa cells. In order to determine the functional significance of oocyte-derived PTK2 signaling in oocyte-follicle communication, an oocyte-specific Ptk2 knockout was produced through a breeding strategy pairing a floxed Ptk2-CAT-eGFP mouse with the Zp3-Cre line. Since Ptk2-null mice never develop to birth, this represents the first opportunity to define the role of PTK2 in oocyte-follicle communication. Ablation of Ptk2 within the developing oocyte resulted in lower fertility with reduced numbers of pups, lower rates of blastocyst formation, and reduced cell numbers per blastocyst. Follicles containing Ptk2-null oocytes exhibited reduced oocyte diameter, reduced numbers of connexin 37 and 43 foci at the oocyte surface, and impaired dye coupling between oocyte and granulosa cells. These findings are consistent with a model in which PTK2 plays a critical role in establishing or maintaining oocyte-granulosa cell contacts that are essential for gap junction-mediated communication between granulosa cells and the oocyte., (© 2014 Wiley Periodicals, Inc.)

Post-ovulatory aging of oocytes results in the progressive loss of fertilization and developmental competence. This degradation of oocyte quality has been the object of numerous investigations, primarily focused on individual signaling pathways which provide limited insight into the status of global signaling events. The purpose of the present investigation was to comprehensively assess broad patterns of signaling pathway activity during in vitro aging as an initial step in defining control points that can be targeted to prevent the reduction in oocyte quality during prolonged culture. An antibody microarray-based phospho-proteome analysis performed on oocytes before and after eight hours of culture revealed significant changes in the abundance or activation state of 43 proteins that function in a wide variety of protein kinase-mediated signaling pathways. Several of the most significantly affected kinases were studied by Western blot and confocal immunofluorescence to corroborate the array results. Prolonged culture resulted in global changes in the abundance and activity of protein kinases that regulate the response to calcium, stress, and cell-cycle control. Examination of intracellular structures revealed a previously unrecognized increase in the abundance of large autophogagic lysosomes, which correlates with changes in protein kinase pathways. These results provide insight into the stresses experienced by oocytes during culture and the diversity of responses that results from them. The observed increase in autophagy-related activity, together with the disruptions in calcium signaling, cell-cycle, and stress-response pathways, have the potential to negatively impact oocyte quality by interfering with the normal sequence of biochemical changes that constitute egg activation following fertilization., (© 2014 Wiley Periodicals, Inc.)

Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte., (Copyright © 2013 Wiley Periodicals, Inc.)

The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre-positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC-family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK-mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC-family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans., (Copyright © 2011 Wiley Periodicals, Inc.)

The control of microtubule and actin-mediated events that direct the physical arrangement and separation of chromosomes during meiosis is critical since failure to maintain chromosome organization can lead to germ cell aneuploidy. Our previous studies demonstrated a role for FYN tyrosine kinase in chromosome and spindle organization and in cortical polarity of the mature mammalian oocyte. In addition to Fyn, mammalian oocytes express the protein tyrosine kinase Fer at high levels relative to other tissues. The objective of the present study was to determine the function of this kinase in the oocyte. Feline encephalitis virus (FES)-related kinase (FER) protein was uniformly distributed in the ooplasm of small oocytes, but became concentrated in the germinal vesicle (GV) during oocyte growth. After germinal vesicle breakdown (GVBD), FER associated with the metaphase-I (MI) and metaphase-II (MII) spindles. Suppression of Fer expression by siRNA knockdown in GV stage oocytes did not prevent activation of cyclin dependent kinase 1 activity or chromosome condensation during in vitro maturation, but did arrest oocytes prior to GVBD or during MI. The resultant phenotype displayed condensed chromosomes trapped in the GV, or condensed chromosomes poorly arranged in a metaphase plate but with an underdeveloped spindle microtubule structure or chromosomes compacted into a tight sphere. The results demonstrate that FER kinase plays a critical role in oocyte meiotic spindle microtubule dynamics and may have an additional function in GVBD., (Copyright © 2010 Wiley-Liss, Inc.)

The objective of the present study was to determine whether Fyn kinase participated in signaling events during sperm-egg interactions, sperm incorporation, and meiosis II. The functional requirement of Fyn kinase activity in these events was tested through the use of the protein kinase inhibitor SKI-606 (Bosutinib) and by analysis of Fyn-null oocytes. Suppression of Fyn kinase signaling prior to fertilization caused disruption of the functional polarity of the oocyte with the result that sperm were able to fuse with the oocyte in the immediate vicinity of the meiotic spindle, a region that normally does not allow sperm fusion. The loss of functional polarity was accompanied by disruption of the microvilli and cortical granule-free zone that normally overlie the meiotic spindle. Changes in the distribution of cortical granules and filamentous actin provided further evidence of disorganization of the oocyte cortex. Rho B, a molecular marker for oocyte polarity, was unaffected by suppression of Fyn activity; however, the polarized association of Par-3 with the cortex overlying the meiotic spindle was completely disrupted. The defects in oocyte polarity in Fyn-null oocytes correlated with a failure of the MII chromosomes to maintain a position close to the oocyte cortex which seemed to underlie the above defects in oocyte polarity. This was associated with a delay in completion of meiosis II. Pronuclei, however, eventually formed and subsequent mitotic cleavages and blastocyst formation occurred normally.