Changes In Protein Tyrosine Phosphorylation Research Articles

Effects of preconception exposure to phthalates on mouse sperm capacitation parameters.

Phthalates have been linked to adverse male reproductive health, including poor sperm quality and embryo quality as well as a longer time to pregnancy (months of unprotected intercourse before conception occurs). The present study aimed to evaluate the effect of preconception exposure to two ubiquitous phthalate chemicals, di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP), and their mixture on sperm function, fertilization, and embryo development in mice. Adult male C57BL/6Jmice aged 8-9weeks were exposed to di(2-ethylhexyl) phthalate, di-n-butyl phthalate, or their mixture (di-n-butyl phthalate+di(2-ethylhexyl) phthalate) at 2.5mg/kg/day or vehicle for 40days (equivalent to one spermatogenic cycle) via surgically implanted osmotic pumps. Caudal epididymal spermatozoa were extracted and analyzed for motility using computer-assisted sperm analyses. Sperm phosphorylation of protein kinase A substrates and tyrosine phosphorylation, markers of early and late capacitation events, respectively, were analyzed by Western blots. In vitro fertilization was used to evaluate the sperm fertilizing capacity. While the study did not reveal any significant differences in sperm motility and fertilization potential, abnormal sperm morphology was observed in all phthalate exposures, particularly in the phthalate mixture group. In addition, the study revealed significant differences in sperm concentration between control and exposed groups. Moreover, protein phosphorylation of protein kinase A substrates was decreased in the di(2-ethylhexyl) phthalate and mixture exposure groups, while no significant changes in protein tyrosine phosphorylation were observed in any of the groups. Assessment of the reproductive functionality did not reveal significant effects on in vitro fertilization and early embryo development rates but showed wide variability in the phthalate mixture group. Our findings suggest that preconception phthalate exposure affects sperm numbers and phosphorylation of protein kinase A substrates involved in capacitation. Future research is warranted to examine the associations between phthalate exposure and capacitation in human spermatozoa.

Formation and Dissociation of Sperm Bundles in Monotremes.

Because monotremes are the earliest offshoot of the mammalian lineage, the platypus and short-beaked echidna were studied as model animals to assess the origin and biological significance of adaptations considered unique to therian mammals: epididymal sperm maturation and subsequent capacitation. We show that spermatozoa from both species assemble into bundles of approximately 100 cells during passage through the epididymis and that an epididymal protein-secreted protein, acidic, cysteine-rich (osteonectin; SPARC)-is involved in bundle formation. The bundles persisted during incubation in vitro for at least 1 h under conditions that capacitate therian spermatozoa, and then underwent a time-dependent dissociation to release spermatozoa capable of fertilization. Only after this dissociation could the spermatozoa bind to the perivitelline membrane of a hen's egg, display an altered form of motility reminiscent of hyperactivation, and be induced to undergo an acrosome reaction. It is concluded that the development of sperm bundles in the monotreme epididymis mandates that they require a time-dependent process to be capable of fertilizing an ovum. However, because this functional end point was achieved without overt changes in protein tyrosine phosphorylation (a hallmark of capacitation in therians), it is concluded that the process in monotremes is distinctly different from capacitation in therian mammals.

SILAC-based quantification of changes in protein tyrosine phosphorylation induced by Interleukin-2 (IL-2) and IL-15 in T-lymphocytes.

This data article presents the first large-scale quantitative phosphoproteomics dataset generated to decipher the signaling networks initiated by IL-2 and IL-15 in T-lymphocytes. Data was collected by combining immunoprecipitation of tyrosine phosphorylated proteins and TiO2-based phosphopeptide enrichment with SILAC-based quantitative mass spectrometry. We report all the proteins and phosphotyrosine-containing peptides identified and quantified in IL-2- and IL-15-stimulated T-lymphocytes. The gene ontology analysis of IL-2 and IL-15 effector proteins detected in the present work is also included. The data supplied in this article is related to the research work entitled “Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics” [1]. All mass spectrometry data have been deposited in the ProteomeXchange with the identifier PXD001129.

Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca2+ concentration in boar spermatozoa during cryopreservation

Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 °C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation.

Human mast cell line-1 (HMC-1) cells transfected with FcεRIα are sensitive to IgE/antigen-mediated stimulation demonstrating selectivity towards cytokine production

Mast cells play important roles in allergic and inflammatory diseases. Efforts to better understand human mast cell activation and develop novel inhibitory agents have been hampered by the lack of suitable human mast cell lines. The HMC-1 mast cell line has been extensively used, but lacks native expression of the human high-affinity IgE receptor FcεRI limiting its applications. We have stably transfected HMC-1 cells with the IgE-binding α-subunit of FcεRI to generate HMCα cells that are antigen-responsive. We have used flow cytometry, cell signaling assays, pharmacological pathway inhibitors and cell functional assays to characterize the properties of HMCα cells. IgE/antigen responses were compared with those of the adenosine receptor agonist NECA. Surface expression of FcεRI in HMCα cells was demonstrated and was enhanced by prior sensitization with IgE. Activation of HMCα cells with IgE/antigen did not produce degranulation, but did lead to release of numerous cytokines. Whilst there was no measurable increase of intracellular Ca 2+ or marked general changes in protein tyrosine phosphorylation, IgE/antigen stimulation of HMCα cells enhanced phosphorylation of p38 MAPK and Erk. Inhibitors of these pathways, as well as the src kinase inhibitor PP2, attenuated IgE/antigen-induced cytokine release. In summary, we have generated and characterized HMCα cells and show that they are a useful and relevant human mast cell model to examine FcεRI stabilization, signaling and mediator release. We envisage that HMCα cells will have utility in understanding the importance of mast cells in human allergic disease and in assessing the activity of novel anti-allergic compounds.

Role of glycerol-3-phosphate dehydrogenase 2 in mouse sperm capacitation

A tyrosine phosphoproteome study of hamster spermatozoa indicated that glycerol-3-phosphate dehydrogenase 2 (GPD2), is one of the proteins that enables tyrosine phosphorylation during sperm capacitation. Further, enzymatic activity of GPD2 correlated positively with sperm capacitation [Kota et al., 2009; Proteomics 9:1809-1826]. Therefore, understanding the function of GPD2 would help to unravel the molecular mechanism of sperm capacitation. In this study, involving the use of spermatozoa from Gpd2(+/+) and Gpd2(-/-) mice, it has been demonstrated that in the absence of Gpd2, hyperactivation and acrosome reaction were significantly altered, and a few changes in protein tyrosine phosphorylation were also observed during capacitation. Evidence is provided to demonstrate that GPD2 activity is required for ROS generation in mouse spermatozoa during capacitation, failing which, capacitation is impaired. These results imply that GPD2 is involved in sperm capacitation.

The Trypanosoma brucei Life Cycle Switch TbPTP1 Is Structurally Conserved and Dephosphorylates the Nucleolar Protein NOPP44/46

Trypanosoma brucei adapts to changing environments as it cycles through arrested and proliferating stages in the human and tsetse fly hosts. Changes in protein tyrosine phosphorylation of several proteins, including NOPP44/46, accompany T. brucei development. Moreover, inactivation of T. brucei protein-tyrosine phosphatase 1 (TbPTP1) triggers differentiation of bloodstream stumpy forms into tsetse procyclic forms through unknown downstream effects. Here, we link these events by showing that NOPP44/46 is a major substrate of TbPTP1. TbPTP1 substrate-trapping mutants selectively enrich NOPP44/46 from procyclic stage cell lysates, and TbPTP1 efficiently and selectively dephosphorylates NOPP44/46 in vitro. To provide insights into the mechanism of NOPP44/46 recognition, we determined the crystal structure of TbPTP1. The TbPTP1 structure, the first of a kinetoplastid protein-tyrosine phosphatase (PTP), emphasizes the conservation of the PTP fold, extending to one of the most diverged eukaryotes. The structure reveals surfaces that may mediate substrate specificity and affords a template for the design of selective inhibitors to interfere with T. brucei transmission.

Hyperactivation of Stallion Sperm Is Required for Successful In Vitro Fertilization of Equine Oocytes1

Capacitation is a complex and not well-understood process that encompasses all the molecular changes sperm must undergo to successfully fertilize an oocyte. In vitro fertilization has remained elusive in the horse, as evidenced by low in vitro fertilization (IVF) rates (0%-33%); moreover, only two foals have ever been produced using IVF. Incubation of stallion sperm in modified Whittens supplemented with bovine serum albumin and sodium bicarbonate yielded significant rates of time-dependent protein tyrosine phosphorylation and induced acrosomal exocytosis, consistent with capacitation. The objective of this study was to characterize stallion sperm hyperactivation and to test whether hyperactivation of capacitated sperm supported equine IVF. Treatment of sperm with procaine, an anesthetic shown to induce hyperactivation in other mammalian species, resulted in the decrease of three motility variables indicative of hyperactivation: straight line velocity (P = 0.029), straightness (P = 0.001), and linearity (P = 0.002). We demonstrated that procaine-induced hyperactivation was not regulated by changes in protein tyrosine phosphorylation and that it did not induce acrosomal exocytosis in capacitated sperm compared with calcium ionophore (P > 0.05), similar to findings in the bovine. Most notably, by coupling our capacitating conditions with the induction of hyperactivation using procaine, we have achieved the novel result of substantial and reproducible percentages of fertilized mare oocytes (60.7%) in our IVF experiments. Conversely, sperm incubated in capacitating conditions but not treated with procaine did not fertilize (0%). These results support the hypothesis that capacitation and hyperactivation are required for successful IVF in the equine.

Sperm Mitochondrial Integrity Is Not Required for Hyperactivated Motility, Zona Binding, or Acrosome Reaction in the Rhesus Macaque1

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 microM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque.

α-Tocopherol modifies tyrosine phosphorylation and capacitation-like state of cryopreserved porcine sperm

Sperm cryopreservation is associated with the production of reactive oxygen species (ROS) leading to membrane destabilization, which induces capacitation-like changes, increases protein tyrosine phosphorylation, and decreases their fertilizing ability. α-Tocopherol, a lipid peroxidation inhibitor, preserves the functionality of cryopreserved porcine sperm. Our aim was to evaluate the effect of α-tocopherol on sperm quality parameters as well as capacitation-like changes and modifications in protein tyrosine phosphorylation. Boar sperm frozen with or without 200 μg/mL of α-tocopherol were thawed and maintained at 37 °C for 10 min in BTS. Routine parameters of semen quality were evaluated by optical microscopy and membrane changes were determined by the epifluorescence chlortetracycline technique. Changes in protein tyrosine phosphorylation were examined using a specific anti-phosphotyrosine monoclonal antibody. Motility was higher (18%, P < 0.05) in semen with α-tocopherol. Viability did not differ ( P > 0.05) between treatments. However, there was less ( P < 0.05) capacitation-like changes in semen with α-tocopherol compared to control samples. A MW 32 kDa tyrosine-phosphorylated protein was detected in extracts of cryopreserved sperm; the intensity of immunostaining was lower in semen containing α-tocopherol compared to the control (0.211 ± 0.030 versus 0.441 ± 0.034 arbitrary units). Additionally, this band was not detected in fresh sperm. The addition of α-tocopherol to the extender prior to cryopreservation of boar semen protected sperm membranes against oxidative damage and reduced both tyrosine phosphorylation and the capacitation-like state.

Capacitation-associated protein tyrosine phosphorylation and membrane fluidity changes are impaired in the spermatozoa of asthenozoospermic patients

Sperm protein tyrosine phosphorylation has been associated with capacitation, motility changes, zona binding, and fertilizing ability. We previously demonstrated that gradient-isolated human sperm subpopulations differ in their plasma membrane composition, their ability to phosphorylate proteins in tyrosine residues, and their capacity to undergo hyperactivation. In this study, we have characterized capacitation-associated changes in protein tyrosine phosphorylation and membrane fluidity in spermatozoa of asthenozoospermic and normozoospermic patients consulting for infertility. Semen samples were studied at baseline and after a capacitating incubation with or without the addition of a permeable cAMP analog and a phosphodiesterase inhibitor. Basic sperm and computer-assisted motion parameters, hyperactivation, protein tyrosine phosphorylation (immunofluorescence and Western blot), and membrane fluidity (fluorescent Laurdan probe) were the main study parameters. In comparison with normozoospermic and proven-fertile donor semen, asthenozoospermic samples showed lower motility, velocity, and amplitude of lateral head displacement, both originally and after a 6-h capacitating incubation. Unlike those in normal samples, asthenozoospermic spermatozoa were unable to increase protein tyrosine phosphorylation during capacitation. Such impairment, however, was overcome when they were incubated with a membrane-permeable cAMP analog and a phosphodiesterase inhibitor, indicating a possible membrane defect. Confirming this hypothesis, plasma membranes of asthenozoospermic sperm showed decreased fluidity (increased Laurdan polarization), even after a capacitating incubation. In conclusion, spermatozoa from functional asthenozoospermic samples may owe their poor motility, and their inability to properly capacitate and develop hyperactivation, to an impairment in the tyrosine phosphorylation of critical proteins caused by decreased membrane fluidity. These findings suggest a molecular pathogenetic mechanism for a common seminal pathology associated with male infertility.

Changes in tyrosine phosphorylation associated with true capacitation and capacitation-like state in boar spermatozoa

Capacitation is defined as a series of events that render boar sperm competent to fertilize, either in vivo or in vitro. Moreover, preliminary stages of cryopreservation of spermatozoa involving cooling to 5 degrees C have been shown to induce capacitation-like changes in boar spermatozoa. Capacitation of boar spermatozoa is accompanied by protein phosphorylation, however the relationship between both processes is poorly understood. Capacitation status was assessed by chlortetracycline (CTC) staining. Changes in protein tyrosine phosphorylation were examined in pre-cleared whole cell lysates using a specific anti-phosphotyrosine monoclonal antibody. Our results in boar spermatozoa show a significant positive correlation between p32 tyrosine phosphorylation levels and percentage of capacitated (CTC pattern B) spermatozoa. Moreover, incubation of boar spermatozoa with two unrelated tyrosine kinase inhibitors induces a significant reduction in the percentages of capacitated and acrosome-reacted (AR) boar spermatozoa and a reduction in the p32 tyrosine phosphorylation. In our conditions, cooling boar spermatozoa to 5 degrees C and rewarming to 39 degrees C in a noncapacitating medium results in similar CTC staining patterns to those obtained after incubation of boar sperm for 1 or 4 hr at 39 degrees C in a capacitating medium. However, cooled-rewarmed fails to induce an increase in p32 tyrosine phosphorylation in boar spermatozoa. Moreover, CTC staining patterns of cooled-rewarmed spermatozoa do not change after incubation with a tyrosine kinase inhibitor. In conclusion, our results show a direct relationship between capacitation and tyrosine phosphorylation and suggest that p32 tyrosine phosphorylation levels could be used as a marker of the true capacitation changes observed in boar spermatozoa. Moreover, our results show that true capacitation and capacitation-like changes induced after cooling involve alternative intracellular tyrosine phosphorylation pathways in boar spermatozoa.

Tyrosine phosphorylation and the small GTPase rac cross-talk in regulation of endothelial barrier function

Endothelial barrier function depends on the integrity of intercellular adherens junctions controlled by the association of VE-cadherin/catenin complex with cortical actin filaments. Both tyrosine phosphorylation/dephosphorylation of junctional proteins and actin reorganization mediated by rho-GTPases regulate barrier function but the relationship between these regulatory mechanisms is unclear. Here we studied the effects of factors increasing protein tyrosine phosphorylation, pervanadate (PV) and VEGF, on distribution of VE-cadherin, F-actin polymerization and transendothelial electrical resistance (TER) in human umbilical vein endothelial cells (HUVECs). Changes in protein tyrosine phosphorylation of cytoplasmic and junctional proteins, as well as the activity of rho-GTPase racl, were also measured. We report for the first time that PV and VEGF induced a rapid transient increase in endothelial barrier function accompanied by rac1 activation, a differentiated tyrosine phosphorylation of the VE-cadherin/catenin complex, recruitment of actin filament to cell junctions and ruffle formation. A sustained decrease in endothelial barrier function was observed at later times of PV and VEGF treatment. Expression of dominant negative rac1, N17rac1 abolished the barrier-enhancing effects of PV and VEGF, while the sustained decrease in barrier function was unaffected. These observations bring into focus early short-term effects of protein tyrosine phosphorylation in cells, often overshadowed by more pronounced and long-lasting later effects and may play an important role in the regulation of endothelial barrier function.

A Prototype Antibody Microarray Platform to Monitor Changes in Protein Tyrosine Phosphorylation

Reversible protein phosphorylation is a key regulatory process in all living cells. Deregulation of modification control mechanisms, especially in the case of tyrosine, may lead to malignant transformation and disease. Phosphotyrosine (p-Tyr) accounts for only 0.05% of the total cellular phospho-amino acid content, yet plays an unusually prominent role in eukaryotic signaling, development, and growth. Tracking temporal and positional p-Tyr changes across the cellular proteome, i.e. tyrosine phosphoproteomics, is therefore tremendously valuable. Here, we describe and evaluate a prototype antibody (Ab) microarray platform to monitor changes in protein Tyr phosphorylation. Availability permitting, a virtually unlimited number of Abs, each recognizing a specific cellular protein, may be arrayed on a chip, incubated with total cell or tissue extracts or with biological fluids, and then probed with a fluorescently labeled p-Tyr-specific monoclonal Ab, PY-KD1, specifically generated for this assay as part of the current study. The optimized protocol allowed detection of changes in the Tyr phosphorylation state of selected proteins using submicrogram to low nanogram of total protein extract, amounts that may conceivably be obtained from a thousand to a hundred thousand cells, or less, depending on the cell or tissue type. The assay platform was evaluated by assessing changes in a rationally selected subset of the Tyr phosphoproteome of Bcr-Abl-expressing cells treated with a specific inhibitor, Gleevec, and of epidermal growth factor (EGF)-treated HeLa cells. The results, ratiometric rather than strictly quantitative in nature, conformed with previous identifications of several Bcr-Abl and EGF receptor targets, and associated proteins, as detected by exhaustive mass spectrometric analyses. The Ab microarray method described here offers advantages of low sample and reagent consumption, scalability, detection multiplexing, and potential compatibility with microfluidic devices and automation. The system may hold particular promise for dissecting signaling pathways, molecular classification of tumors, and profiling of novel target-cancer drugs.

Cold exposure induces tyrosine phosphorylation of BIT through NMDA receptors in the rat hypothalamus

The hypothalamus has a central role in maintaining homeostases of physiological conditions including body temperature and energy balance. To examine molecular responses to cold exposure in the hypothalamus, we examined changes in protein tyrosine phosphorylation in the suprachiasmatic nucleus of the hypothalamus after acute cold exposure in rats. It was found that brain immunoglobulin-like molecule with tyrosine-based inhibitory motifs (BIT, also called SHPS-1, SIRPα or p84), a transmembrane glycoprotein with two ITIM motifs , showed enhanced tyrosine phosphorylation after cold exposure. Its tyrosine phosphorylation induced by cold exposure was also found in other hypothalamic nuclei including the paraventricular nucleus, lateral hypothalamic area, ventromedial hypothalamus, and arcuate nucleus. This phosphorylation was blocked by AP-5, an NMDA receptor antagonist, indicating that it was mediated by NMDA receptors. These results suggest that BIT is involved in the mechanism of neuronal responses to cold exposure in the hypothalamus.

Cold exposure induces tyrosine phosphorylation of BIT through NMDA receptors in the rat hypothalamus

The hypothalamus has a central role in maintaining homeostases of physiological conditions including body temperature and energy balance. To examine molecular responses to cold exposure in the hypothalamus, we examined changes in protein tyrosine phosphorylation in the suprachiasmatic nucleus of the hypothalamus after acute cold exposure in rats. It was found that brain immunoglobulin-like molecule with tyrosine-based inhibitory motifs (BIT, also called SHPS-1, SIRPα or p84), a transmembrane glycoprotein with two ITIM motifs, showed enhanced tyrosine phosphorylation after cold exposure. Its tyrosine phosphorylation induced by cold exposure was also found in other hypothalamic nuclei including the paraventricular nucleus, lateral hypothalamic area, ventromedial hypothalamus, and arcuate nucleus. This phosphorylation was blocked by AP-5, an NMDA receptor antagonist, indicating that it was mediated by NMDA receptors. These results suggest that BIT is involved in the mechanism of neuronal responses to cold exposure in the hypothalamus.

Changes in protein tyrosine phosphorylation during mannose and senescence induced cell death in rice

In mammals protein tyrosine phosphorylation plays an important role in the activation of apoptosis. However, tyrosine phosphorylation associated with cell death has not been examined in plants. We monitored changes in tyrosine phosphorylation during cell death in rice (Oryza sativa L.) suspension cultures. Cell death was induced in the cell cultures by mannose treatment or by allowing the cultures to senescence. We have demonstrated that both mannose and senescence induced DNA fragmentation in rice suspension cells. In the presence of mannose, the tyrosine phosphorylation patterns of mannose treated and non-treated cell proteins are basically the same, except the tyrosine phosphorylation intensity is considerably different. In aged suspension-cultured cells, the occurrence of DNA fragmentation was detected. In addition, the tyrosine phosphorylation pattern was changed. These results suggest that protein tyrosine phosphorylation may have a role in distinct signal transduction pathways responding to mannose and senescence. The expression of a gene that encodes mitogen-activated protein kinase (MAPK), OsMAPK2, is up-regulated during mannose treatment, suggesting the possible involvement of rice MAPK in pathways associated with rice cell death induced by >d-mannose.

Hyperglycemia and hypercapnia differently affect post-ischemic changes in protein kinases and protein phosphorylation in the rat cingulate cortex

Hyperglycemia and hypercapnia aggravate intra-ischemic acidosis and subsequent brain damage. However, hyperglycemia causes more extensive post-ischemic damage than hypercapnia, particularly in the cingulate cortex. We investigated the changes in the subcellular distribution of protein kinase Cγ (PKCγ) and the Ca 2+/calmodulin-dependent protein kinase II (CaMKII), as well as changes in protein tyrosine phosphorylation during and following 10 min normoglycemic, hyperglycemic (plasma glucose ∼20 mM) and hypercapnic (paCO 2 ∼300 mm Hg) global cerebral ischemia. During reperfusion period, the translocation to cell membranes of PKCγ, but not CaMKII, was prolonged by intra-ischemic hyperglycemia, while it was only marginally affected by hypercapnia. The tyrosine-phosphorylation of proteins in the synaptosomal membranes, as well as the extracellular signal-regulated kinase (ERK) in the cytosol, markedly increased during reperfusion following hyperglycemic ischemia, but to a lesser degree following hypercapnic ischemia. Our data suggest that PKCγ, tyrosine kinase and ERK systems are involved in the process of ischemic damage in the cingulate cortex, where hyperglycemia may affect these kinases through an additional mechanism other than exaggerated acidosis.

Protein tyrosine phosphatases in Chaetopterus egg activation.

Changes in protein tyrosine phosphorylation are an essential aspect of egg activation after fertilization. Such changes result from the net contributions of both tyrosine kinases and phosphatases (PTP). This study was conducted to determine what role(s) PTP may have in egg activation. We identified four novel PTP in Chaetopterus pergamentaceus oocytes, cpPTPNT6, cpPTPNT7, cpPTPR2B, and cpPTPR2A, that have significant homology to, respectively, human PTPsigma, -rho, -D2 and -BAS. The first two are cytosolic and the latter two are transmembrane. Several PTP inhibitors were tested to see if they would affect Chaetopterus pergamentaceus fertilization. Eggs treated with beta-bromo-4-hydroxyacetophenone (PTP inhibitor 1) exhibited microvillar elongation, which is a sign of cortical changes resulting from activation. Those treated with Na3VO4 underwent full parthenogenetic activation, including polar body formation and pseudocleavage and did so independently of extracellular Ca2+, which is required for the Ca2+ oscillations that initiate development after fertilization. Fluorescence microscopy identified phosphotyrosine-containing proteins in the cortex and around the nucleus of vanadate-activated eggs, whereas in fertilized eggs they were concentrated only in the cortex. Immunoblots of vanadate-activated and fertilized eggs showed tyrosine hyperphosphorylation of approximately 140 kDa protein. These results suggest that PTP most likely maintain the egg in an inactive state by dephosphorylation of proteins independent of the Ca2+ oscillations in the activation process.

Kinetic characterization of the changes in protein tyrosine phosphorylation of membranes, cytosolic Ca2+ concentration and viability in boar sperm populations selected by binding to oviductal epithelial cells

On reaching the oviduct, spermatozoa are retained in the isthmic region of the oviduct until ovulation occurs. The essential steps of capacitation are co-ordinated in this region. In this study, a primary cell culture system of oviductal epithelial cells was established to investigate sperm binding to oviductal epithelium and modulation of sperm function during incubation under capacitating conditions in co-culture with oviductal epithelial cells. Epithelial cells were stripped from the oviducts of sows and cultivated for 5-7 days on Lab-Tek Chamber slides on Matrigel. The preparations on chamber slides and suspensions of control spermatozoa were incubated for 3 h in Tyrode's albumin lactate pyruvate (TALP) medium. At 3, 30, 60, 90 and 180 min the free-swimming spermatozoa were collected by washing, and membrane integrity, tyrosine phosphorylation patterns and [Ca(2+)](i) of bound, unbound and control spermatozoa were assessed with fluorescent probes (propidium iodide, Cy-3 and fluo-3-AM). The cells bound to oviductal epithelial cells showed reduced cytosolic Ca(2+) concentration, reduced and almost absent tyrosine phosphorylation of membrane proteins and higher viability at the time of the first sampling. Increases in Ca(2+) concentration and cell death occurred much more slowly during incubation in cells bound to oviductal epithelial cells compared with free-swimming spermatozoa, and no changes in tyrosine phosphorylation were observed. The preferential binding of viable, low-Ca(2+) cells with suppressed tyrosine phosphorylation and slower functional modulation of boar spermatozoa attached to oviductal epithelial cells might represent a mechanism for selecting functionally competent spermatozoa and prolonging their lifespan by delaying capacitation in the oviductal reservoir.