Pattern Of Protein Tyrosine Phosphorylation Research Articles

A Functional Interplay Between Pyk2 and Src Family Kinases Links Ca2+ Signaling To Protein Tyrosine Phosphorylation In Thrombin-Stimulated Platelets

IntroductionThrombin and other soluble agonists promote platelet activation by binding to G-protein-coupled receptors, which typically recruit phospholipase C and trigger cytosolic Ca2+ increase and protein kinase C stimulation. Platelets also express several tyrosine kinases, including different members of the Src family (mainly Src and Lyn) and of the focal adhesion family (such as FAK and Pyk2), which are known to be activated upon stimulation with thrombin. Both Src family kinases (SFKs) and Pyk2 are required for thrombin-induced aggregation and thromboxane A2 production, and both of them have been shown to be regulated by intracellular Ca2+ (Xiang et al, J Biol Chem 2012; Canobbio et al, Blood 2013). However, the hierarchy of protein tyrosine kinases activation, as well as the molecular mechanism linking agonist-induced increase of cytosolic Ca2+ to protein tyrosine phosphorylation is poorly understood. In this study, we investigated the interplay between intracellular Ca2+, Pyk2 and SFKs in the regulation of protein tyrosine phosphorylation in thrombin-stimulated platelets. ResultsIn the presence of the ADP scavenger apyrase, thrombin caused the time-dependent tyrosine phosphorylation of several proteins in aspirin-treated human, as well as mouse platelets. This effect was severely reduced by the intracellular Ca2+ chelator BAPTA-AM and by the SFKs inhibitor PP2, and was totally suppressed only when BAPTA-AM and PP2 were used in combination, indicating that cytosolic Ca2+ and SFKs activity cooperate in the regulation of the whole process of protein tyrosine phosphorylation elicited by thrombin. Analysis of SFKs activity in human and mouse platelets with an anti-pY416Src antibody, that detects the autophosphorylation site in the catalytic domain, revealed that cytosolic Ca2+ is upstream SFKs activation upon thrombin stimulation. Accordingly, SFKs phosphorylation in the catalytic domain was also directly promoted by the Ca2+ ionophore A23187. Immunoblotting analysis on high resolution gels allowed to distinguish between Src and Lyn, and revealed that PP2 prevented phosphorylation of both kinases, but BAPTA-AM only inhibited Src , but not Lyn phosphorylation caused by thrombin. The Ca2+ ionophore A23187 also triggered the phosphorylation and activation of the Ca2+-dependent focal adhesion kinase Pyk2, and stimulation with thrombin promoted Pyk2 activation by a Ca2+-dependent mechanism. However, thrombin- or A23187-induced activation of Pyk2 was also reduced by the SFKs inhibitor PP2, and both PP2 and BAPTA-AM were required to completely suppress Pyk2 activation. In platelets from Pyk2 knockout mice, stimulation of SFKs by thrombin was strongly reduced, as it was the overall pattern of protein tyrosine phosphorylation. Importantly, also SFKs activation caused by Ca2+ionophore was significantly impaired in Pyk2-deficient platelets. Finally, in the absence of Pyk2, thrombin-induced aggregation was reduced to a level comparable to that of wild type platelets treated with PP2, although inhibition of SFKs by PP2 further reduced aggregation of Pyk2-deficient platelets. ConclusionsThese results indicate that in thrombin-stimulated platelets intracellular Ca2+ increase promotes Pyk2 stimulation, which initiates SFKs activation and establishes a positive loop which reinforces the Pyk2/SFKs axis to allow the subsequent massive tyrosine phosphorylation of multiple platelet substrates. Disclosures:No relevant conflicts of interest to declare.

Boar sperm incubation with reduced glutathione (GSH) differentially modulates protein tyrosine phosphorylation patterns and reorganization of calcium in sperm, in vitro fertilization, and embryo development depending on concentrations

The sperm in the female's reproductive tract undergo changes to fertilize the oocyte (sperm capacitation). These changes are regulated by redox system. However, some assisted reproductive technologies require sperm capacitation under in vitro conditions, though this increases the generation of ROS. Therefore, the aim of this study was to evaluate the effect of GSH as an antioxidant agent during the capacitation of boar sperm [evaluated by calcium compartmentalization, tyrosine phosphorylation (Tyr-P), motility, viability, and acrosomal integrity], in vitro fertilization (evaluated by penetration, monospermy, and efficiency %), and later embryo development (evaluated by cleavage and blastocyst rates, total number of cells per blastocyst and blastocyst diameter). Four experimental groups with different GSH concentrations (0-control, 0.5, 1, and 5 mM) were formed. When 1-GSH was added to the medium, the percentage of capacitated sperm increased after 4 h of incubation; the localization of Tyr-P was modified at 1 h and 4 h of incubation depending on the GSH concentration. Percentages of total and progressive sperm motility also increased at 4 h of incubation, but only in the 5-GSH group compared to control. Viability, acrosomal integrity, and general Tyr-P (Western blot) not differ among the experimental groups. The addition of GSH during gamete interaction increased penetration, monospermy, and efficiency rates in the 1-GSH group compared to the others. However, the effect of GSH was not observed in cleavage and blastocyst rates compared to the control. In conclusion, adding GSH modulates sperm capacitation (by means of calcium compartmentalization and tyrosine phosphorilation pattern) depending on its concentration, and improves IVF output at 1-GSH during gamete interaction.

The role of Aquaporin-4 in T cell activation

Abstract Aquaporins (AQPs) are membrane channels that mediate a variety of biological processes via water and other small molecule movement. Several aquaglyceroporins have been shown to regulate key immune functions. However, the role of AQP4 in immune cells is poorly characterized. We recently reported that AQP4 is expressed by both naïve and memory CD4 and CD8 T cells. Furthermore, AQP4 blockade with a small molecule inhibitor, AER-270, reduced T cell activation and proliferation leading to a significant prolongation of fully MHC-mismatched murine heart allograft survival. The goal of this study was to determine the mechanisms by which AQP4 facilitates T cell activation and TCR signaling. Wild-type (WT) but not AQP4−/− T cells treated with AER-270 were protected from lysis in a hypoosmotic shock assay demonstrating AQP4 requirement for water transport in T cells. Following αCD3/αCD28 stimulation, AQP4−/− T cells demonstrated inferior activation compared to WT T cells. Similar decreased activation was observed after treatment of WT T cells with AER-270. Consistent with reduced T cell activation, AQP4 inhibition diminished Ca2+flux in WT but not AQP4−/− T cells in response to TCR crosslinking. AQP4 inhibition also resulted in altered pattern of protein tyrosine phosphorylation at 2 min following TCR crosslinking. These results indicate that despite T cell expression of several water channels, AQP4 function is critical for optimal T cell activation and functions. Our study identifies a novel role of water channels in T cell biology and suggests AQP4 inhibition as a promising strategy to modulate T cell alloresponses following transplantation.

Boar sperm protein tyrosine phosphorylation in the presence of egg yolk soluble and low density lipoprotein fractions during cooling

Increased protein tyrosine phosphorylation and the appearance of a phosphorylated protein of 32 kD (p32) are reported among the capacitation-like changes in cryopreserved boar sperm. Egg yolk freezing extenders are composed by two fractions: insoluble granules and soluble plasma, which contains the low density lipoproteins (LDL) proposed as responsible for the egg yolk cryoprotective action. The aim of this work was to analyze the effects of complete egg yolk and its insoluble, soluble and LDL fractions on boar sperm quality and protein tyrosine phosphorylation after the first stage of a standard cryopreservation protocol. Semen samples in Androstar® Plus diluent were centrifuged and resuspended in the different egg yolk extenders. Temperature was decreased from 17 °C to 5 °C and sperm quality, protein tyrosine phosphorylation and protein pattern were analyzed. Results showed that complete egg yolk as well as soluble and LDL egg yolk fractions maintained sperm quality after temperature decrease. Cooling without any lipid component or in the presence of the insoluble fraction, significantly reduced sperm motility. About sperm protein tyrosine phosphorylation analysis, the p32 band appeared before treatments or after cooling in Androstar® Plus diluent. Complete egg yolk and its insoluble fraction interfered with sperm tyrosine phosphorylation even after cells were extensively washed. Analysis of extenders revealed a high amount of tyrosine phosphorylated proteins in the insoluble fraction, which may have co-precipitate with sperm in experiments. Samples submitted to temperature decrease from 17 °C to 5 °C in the presence of soluble and LDL egg yolk fractions in Androstar® Plus diluent did not show any change in the p32 band associated with sperm capacitation. However, a tyrosine-phosphorylated protein of 33 kD present in clarified egg yolk was also observed in sperm treated with this extender. Protein transference from plasma and LDL egg yolk extenders was also observed in sperm protein profile. Results suggested that soluble and LDL fractions might have a protective action preventing sperm protein tyrosine phosphorylation during cooling from 17 °C to 5 °C. Further studies are needed to expand the knowledge of the LDL protection mechanism as well as to determine the possible benefits of clarified egg yolk in freezing protocols.

Evaluation of protein phosphorylation in bull sperm during their maturation in the epididymis.

Phosphorylation, or dephosphorylation, is one of the most frequent post-translational modifications regulating protein-protein activity in eukaryotic cells. Whereas mature spermatozoa (as specialized cells) are transcriptionally inactive and do not synthesize new proteins, phosphorylation of sperm proteins is very important for the regulation of the sperm function. Although the post-testicular maturation of spermatozoa is a process common to all mammals, comparative studies showed significant differences in sperm surface proteins and the mechanisms of protein modification during the epididymal maturation. In our study, the evaluation of tyrosine phosphorylation, represented by the fluorescent patterns of used anti-phosphotyrosine antibodies (P-Tyr-01 and 4G10), in spermatozoa isolated from different regions of the epididymis - caput, corpus and cauda - was performed. Although in general both antibodies detected almost the same reaction patterns, we observed some dissimilarity associated with the binding specificity of the antibodies and also the segment-dependent manner of phosphorylated protein localization. These data were filled up by immunohistochemical analysis of testes and epididymides cryosections. Additionally, our phosphoproteomic study focused on evaluation of the changes in the pattern of tyrosine-phosphorylated proteins during the post-testicular maturation of bull spermatozoa (PY20 antibody). To summarize the results, an increasing trend of tyrosine phosphorylation of proteins during the maturation of bull sperm in the epididymis was consistently observed in all the methods/experiments.

Protein modification in the post-mating spermatophore of the signal crayfish Pacifastacus leniusculus: insight into the tyrosine phosphorylation in a non-motile spermatozoon

After mating, spermatophores of signal crayfish are stored on the body of the female for a period before fertilization. This study compared the post-mating protein profile and pattern of protein tyrosine phosphorylation of the signal crayfish spermatophore to that of the freshly ejaculated spermatophore and found substantial differences. Two major bands of tyrosine-phosphorylated proteins of molecular weights 10 and 50kDa were observed in the freshly ejaculated spermatophore of the signal crayfish. While the tyrosine-phosphorylated protein band with molecular weight 10kDa was formed by protein(s) of similar pH, the band with molecular weight of 50kDa consisted of proteins of varying pH. In the post-mating spermatophore, the band with molecular weight of 50kDa was not detected, and an increase in the level of protein tyrosine phosphorylation was observed in the 10kDa band. The microtubular radial arms of the spermatozoon showed a positive reaction to an anti-tyrosine antibody conjugated with gold particles in both the freshly ejaculated and post-mating spermatophores. In conclusion, the male gamete of the signal crayfish undergoes molecular modification during post-mating storage on the body of the female including changes in the level of protein expression and protein tyrosine phosphorylation. Structural similarity of the radial arms in the crayfish immotile spermatozoon with flagellum, which is the main site of protein tyrosine phosphorylation in the mammalian motile spermatozoa, raises questions regarding evolution and function of such organelles across the animal kingdom that must be addressed in the future studies.

Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein.

The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif.

The focal adhesion kinase Pyk2 links Ca2+ signalling to Src family kinase activation and protein tyrosine phosphorylation in thrombin-stimulated platelets.

In blood platelets, stimulation of G protein-coupled receptors (GPCRs) by thrombin triggers the activation of Src family kinases (SFKs), resulting in the tyrosine-phosphorylation of multiple substrates, but the mechanism underlying this process is still poorly understood. In the present study, we show that the time-dependent protein-tyrosine phosphorylation triggered by thrombin in human or murine platelets was totally suppressed only upon concomitant chelation of intracellular Ca(2+) and inhibition of SFKs. Thrombin-induced activation of SFKs was regulated by intracellular Ca(2+) and accordingly the Ca(2+) ionophore A23187 was sufficient to stimulate SFKs. A23187 also triggered the phosphorylation and activation of the Ca(2+)-dependent focal adhesion kinase Pyk2 and Pyk2 activation by thrombin was Ca(2+)-dependent. Stimulation of SFKs by thrombin or A23187 was strongly reduced in platelets from Pyk2 knockout (KO) mice, as was the overall pattern of protein-tyrosine phosphorylation. By immunoprecipitation experiments, we demonstrate that Lyn and Fyn, but not Src, were activated by Pyk2. Inhibition of SFKs by PP2 also reduced the phosphorylation of Pyk2in thrombin or A23187-stimulated platelets. Analysis of KO mice demonstrated that Fyn, but not Lyn, was required for complete Pyk2 phosphorylation by thrombin. Finally, PP2 reduced aggregation of murine platelets to a level comparable to that of Pyk2-deficient platelets, but did not have further effects in the absence of Pyk2. These results indicate that in thrombin-stimulated platelets, stimulation of Pyk2 by intracellular Ca(2+) initiates SFK activation, establishing a positive loop that reinforces the Pyk2/SFK axis and allows the subsequent massive tyrosine phosphorylation of multiple substrates required for platelet aggregation.

Blood plasma collected after adrenocorticotropic hormone administration during the preovulatory period in the sow negatively affects in vitro fertilization by disturbing spermatozoa function

Successful fertilization is essential for reproduction and might be negatively affected by stressful events, which could alter the environment where fertilization occurs. The aim of the study was to determine whether an altered hormonal profile in blood plasma caused by adrenocorticotropic hormone (ACTH) administration could affect in vitro fertilization in the pig model. In experiment 1, gametes were exposed for 24 hours to plasma from ACTH-treated, non-ACTH–treated sows, or medium with BSA. Fertilization, cleavage, and blastocyst rates were lower in the ACTH group compared with the no ACTH or BSA control groups (P < 0.01). In experiment 2, the exposure of matured oocytes for 1 hour before fertilization to the same treatments did not have an impact on their ability to undergo fertilization or on embryo development. In experiment 3, spermatozoa were incubated for 0, 1, 4, and 24 hours under the same conditions. There was no effect of treatment on sperm viability. The percentage of acrosome-reacted spermatozoa remained higher in the ACTH group compared with the non-ACTH–treated group through the incubation period (P < 0.001). Protein tyrosine phosphorylation (PTP) patterns were also affected by treatment (P < 0.001). The presence of an atypical PTP pattern was higher in the ACTH group at all the analyzed time points compared with the BSA and no ACTH groups (P < 0.001). In conclusion, this altered environment may not affect oocyte competence but might affect the sperm fertilizing ability through alterations in the acrosome reaction and correct sequence of PTP patterns.

246 USE OF IONOPHORE A23187 ON BOVINE FROZEN SPERM INCREASES HYPERACTIVATED MOTILITY

Sperm capacitation is critical for oocyte fertilization in mammals. The capacitated state is acquired by the spermatozoon during its passage through the female reproductive tract and can be induced in vitro. At a functional level, sperm capacitation is associated with changes in the motility pattern – the hyperactivated state (HA) – and terminates with the acrosome reaction (AR). These events are characteristically regulated by different Ca2+-signalling pathways. For this reason, Ca2+ ionophores, such as A23187, are commonly used in sperm capacitation studies. The induction of AR and IVF, as well as the assessment of protein phosporylation of tyrosine substrates are useful methods for the evaluation of the capacitation state of spermatozoa. Although the increase of protein tyrosine phosphorylation via PKA is associated with sperm capacitation, in the mouse, A23187 capacitates the spermatozoa, thus bypassing this pathway. The aim of the present work was to test the effect of the ionophore A23187 on acquisition of the capacitated state by evaluation of HA motility and protein phosphorylation pattern in frozen bovine spermatozoa. Motile bovine spermatozoa were isolated by gradient centrifugation (Percoll) from frozen/thawed samples and treated with different concentrations of A23187 (0.05, 0.1, 0.2, and 0.3 µM) in H-TYH medium without BSA. After 10 min of treatment, spermatozoa were washed in medium with BSA (11.2%) and incubated in H-TYH with BSA (6%) for 30 min. For control groups, sperm were incubated in H-TYH medium and DMSO. The motility pattern was visually identified and quantified using a computer-assisted sperm analysis system. The motile/immotile spermatozoa and the HA motility patterns of each group were statistically analysed by applying Fisher's tests. The protein tyrosine phosphorylation pattern was evaluated by a Western immunoblotting assay using heparin as a positive control of sperm capacitation. Our results showed that spermatozoa treated with A23187 had a significant increase in HA motility. The proportions of HA spermatozoa were 10.92, 31.27, and 75.4% for 0.1, 0.2, and 0.3 µM A23187, respectively (P &lt; 0.05). On the other hand, the pattern of PKA-tyrosine phoshorylation characteristic of capacitated spermatozoa was absent after incubation with A23187, similar to the response seen in mouse spermatozoa. The percentage of motile spermatozoa in the control groups (H-TYH medium: 36% and DMSO: 27.95%; P &lt; 0.05) was reduced as compared with that of the basal sperm suspension (65.4%, P &lt; 0.05) after 30 to 40 min of incubation. Motility in the spermatozoa treated with 0.1 and 0.2 µM ionophore was similar – 65.9 and 68.1%, respectively, but it was reduced in the 0.3 group – 57.5% (P &lt; 0.05). Thus, treatment with A23187 increased the percentage of spermatozoa with HA motility, probably suggesting an improvement in fertilizing capacity. Nevertheless, these promising results remain to be confirmed by IVF assay.

In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation.

The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22h of incubation in Ham's F10 medium plus bovine albumin at 37° and 5% CO2 for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22h of incubation, there was a significant (P<0.05) increase in oxygen-free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P<0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmentedDNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity.

Insulin affects sperm capacity in pig through nitric oxide

Insulin (Ins) has recently been demonstrated to have the ability to induce the capacitation process in pig spermatozoa. In various mammalian species, capacitation has been linked to the nitric oxide (NO) signalling; therefore, this study investigated NO production in Ins-treated pig spermatozoa by fluorescence-activated cell sorting. For the same samples, sperm capacitation was evaluated by chlortetracycline staining, protein tyrosine phosphorylation pattern and acrosomal status. A significant increase of the intrasperm NO level and the activation of three capacitation indices were detected in response to Ins treatment. Conversely, sperm preincubation with an NO synthase inhibitor (N-nitro-L-arginine methyl ester) or with the anti-Ins receptor β (IRβ) antibody reversed all of the Ins-related effects. These results suggest that Ins has the capacity to enhance intracellular NO concentrations in pig spermatozoa and indicate a possible NO implication upon Ins promotion of capacitation.

244 PROTEIN TYROSINE PHOSPHORYLATION PATTERN OF SPERM BOUND TO THE PORCINE OVIDUCT DURING THE PERIOVULATION STAGE

The interaction of spermatozoa and oviductal epithelial cells (OEC) is a controlled process that regulates sperm capacitation and the acquisition of fertilizing ability until the time of ovulation. A crucial signalling event involved in capacitation is protein tyrosine phosphorylation. In previous studies, we have demonstrated changes in the pattern of protein tyrosine phosphorylation in boar sperm after the co-culture with OEC. The aim of this study was to characterise the pattern of protein tyrosine phosphorylation in boar sperm bound or unbound to the oviduct of the sow during the periovulation stage. Eight crossbred multiparous sows were inseminated with 3 × 109 sperm. The animals were anesthetized and laparotomies were performed at 36 h after insemination. Ovaries and oviducts were exposed through a midventral incision for collection. Each oviduct was divided into four parts: the ampulla, ampullary-isthmic junction, isthmus, and utero-tubal junction. All segments of the oviduct were flushed to recover spermatozoa, which were subsequently fixed. Tissue obtained from each of the oviduct segments were fixed and embedded in a paraffin block. Sections were mounted on poly-l-lysine-coated slides and deparaffinized. Flushed sperm and oviductal sections were analysed by indirect immunofluorescence using monoclonal antiphosphotyrosine antibodies. Three different sperm subpopulations were determined according to the distribution of protein tyrosine phosphorylation observed: nonphosphorylated spermatozoa (pattern 1), subequatorial segment or subequatorial segment and flagellum phosphorylation (pattern 2), and subequatorial segment and head or flagellum phosphorylation, or both (pattern 3). Data were analysed with SPSS (IBM, Armonk, NY, USA) using one-way ANOVA. After flushing, most sperm were recovered from the utero-tubal junction segment of the oviduct, and sperm exhibited a higher proportion of pattern 2 (81.62%). Unbound sperm showed a high level of protein tyrosine phosphorylation in the subequatorial segment, head and flagellum in the isthmus (32.34%), ampullary-isthmic junction (37.70%), or ampulla region (35.11%; P &lt; 0.05). Very few sperm were attached to OEC, and sperm oviduct binding was mainly found in the isthmus region. The most common tyrosine phosphorylation distribution observed in sperm attached to OEC was pattern 1 (84.21%), although labelling to the subequatorial segment was also observed. Our results showed that only sperm that did not display tyrosine phosphorylation on the sperm acrosome region (head) were found bound to OEC. In conclusion, distinct protein tyrosine phosphorylation patterns were found on sperm bound to OEC. This interaction could be used as a tool for selecting a population of sperm containing low levels of tyrosine phosphorylation.

245 EFFECT OF INCUBATION IN OVIDUCTAL EPITHELIAL CELLS AND CO-CULTURE WITH OOCYTES IN THE PROTEIN TYROSINE PHOSPHORYLATION PATTERN OF BOAR SPERM

The oviductal epithelial cells (OEC) play an important role in the process of sperm capacitation and the acquisition of fertilizing capacity. One of the most important changes that can be observed in spermatozoa is the increase in protein tyrosine phosphorylation. In previous studies, we have demonstrated changes in the pattern of protein tyrosine phosphorylation in boar sperm after the co-culture with OEC. The aim of this study was to characterise the pattern of protein tyrosine phosphorylation in boar sperm after incubation in oviductal cells in the presence of oocytes. Epithelial cells were stripped from the oviducts of cycling sows and cultivated for 7 days on a petri dish. Sperm cells treated by a discontinuous gradient of isotonic Percoll were added to the OEC and incubated for 30 min. After 30 min, the spermatozoa bound and unbound were separated and co-cultured with oocytes for 18 h. Five different samples were analysed by indirect immunofluorescence to determine the localization of proteins phosphorylated in tyrosine residues (Tardif et al., 2001 Biol. Reprod. 65, 784–792): ejaculated control, sperm unbound OEC after 30 min, sperm unbound after 30 min with oocytes, sperm bound after 30 min with oocytes, and sperm bound after 30 min and detached after culture with oocytes. Five patterns were determined according to their surface distribution: nonphosphorylated spermatozoa (pattern 1), head, tail, or both phosphorylated (pattern 2), equatorial segment phosphorylated (pattern 3), equatorial segment and head, tail, or both phosphorylated (pattern 4), and phosphorylation of the tail alone and in combination with other areas of the sperm (pattern 5). Sperm without any treatment showed lower tyrosine phosphorylation levels (87.04%; P &lt; 0.05). Unbound sperm showed a high level of protein tyrosine phosphorylation in the subequatorial segment and head, tail, or both phosphorylated (47.71%; P &lt; 0.05), and this pattern was associated with a high level of capacitation. The same pattern was reduced when unbound sperm were co-cultured with oocytes for 18 h (25.67%; P &lt; 0.05), and was even lower in sperm bound with oocytes. The study of phosphorylation of the tail (pattern 5) showed no significant differences between different samples. We concluded that sperm-bound OEC have lower levels of phosphorylation, indicating a selective function in the sperm-OEC interaction. In addition, phosphorylation of the tails appears to be related more to the hyperactivation of sperm than to the different culture conditions.

Equine spermatozoa stored in the epididymis for up to 96 h at 4 °C can be successfully cryopreserved and maintain their fertilization capacity

After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4°C up to 96h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing–thawing on viability, chromatin condensation, ROS generation, protein tyrosine phosphorylation and heterologous fertilization rate (ICSI and IVF using bovine oocytes) of sperm recovered from the epididymis up to 96h post castration. The average volume (720±159μL) and the concentration (6.5±0.4×109 spermatozoa/mL) of sperm recovered from the epididymis were not affected by storage. Sperm viability after refrigeration at 4°C for up to72h was similar (P<0.01). The effect of sperm dilution in the freezing media showed similar values up to 48h, while viability was preserved up to 72h (P<0.01). Cryopreserved spermatozoa show similar viability between different storage times. Chromatin condensation was not affected by storage time; however, incubation for 30min in freezing medium and freezing–thawing process induced an increase in the chromatin decondensation. ROS generation was not affected by storage up to 96h. Epididymal storage did not affect sperm protein tyrosine phosphorylation patterns; although the pattern of phosphorylation changed to strong staining of the equatorial segment when the sperm where capacitated in sperm–TALP. Finally, successful and similar pronuclear formation (analyzed by ICSI) and in vitro penetration (evaluated with bovine zone free oocyte) was observed using cryopreserved sperm obtained from prolong epididymal storage at 4°C. In conclusion, cryopreservation of epididymal stallion sperm stored for up to 72h in the epididymis at 4°C, maintain both viability and ability to fertilize in vitro.

Effect of Spermine-NONOate on acrosome reaction and associated protein tyrosine phosphorylation in Murrah buffalo (Bubalus bubalis) spermatozoa

The present study was conducted to know the role of Nitric Oxide (NO) on the acrosome reaction (AR) in Murrah buffalo (Bubalus bubalis) spermatozoa. Ejaculated buffalo spermatozoa were washed, suspended in sp-TALP media containing 6mg BSA/mL and cell concentration was adjusted to 50×106cells/mL. The cells were incubated for 6h in the absence or presence of heparin (10μg/mL) to induce capacitation. Fully capacitated spermatozoa were incubated in presence of 100μg/mL Lysophosphatidyl choline (LPC, T1) or 100μM Spermine-NONOate (T2) or 100mM l-NAME (T3) or 100μM Spermine-NONOate+100mM l-NAME (T4) or 1mM db-cAMP+0.1mM IBMX (T5) or 100μM H-89 (T6) or 100μM Spermine-NONOate+100μM H-89 (T7) in combination to induce acrosome reaction. The extent of AR was assessed by dual-staining of spermatozoa with trypan blue/Giemsa stain. AR-associated tyrosine-phosphorylated proteins were detected by SDS-PAGE followed by immunoblotting using monoclonal anti-phosphotyrosine antibody. Significant (P<0.05) number of spermatozoa were acrosome reacted in Spermine-NONOate (T2) treated cells but it was significantly (P<0.05) lower than LPC (T1) induced AR. Addition of Spermine-NONOate+l-NAME (T4) resulted in non significant (P>0.05) decrease in acrosome reaction. On addition of H-89+Spermine-NONOate (T7) to sperm culture medium, resulted in significant (P<0.05) decrease in the percent acrosome reaction. Conversely, addition of db-cAMP+IBMX (T5, cAMP analogue) resulted in the significantly (P<0.05) higher number of acrosome reacted spermatozoa. Pattern of sperm protein tyrosine phosphorylation was also different in NO induced acrosome reaction compared to that of LPC. The present study concluded that nitric oxide is involved in acrosome reaction of buffalo spermatozoa by causing the tyrosine phosphorylation of proteins mainly p17 and p20 and through activation of cAMP/PKA pathway.

Role of FYN Kinase in Spermatogenesis: Defects Characteristic of Fyn-Null Sperm in Mice1

FYN kinase is highly expressed in the testis and has been implicated in testis and sperm function, yet specific roles for this kinase in testis somatic and germ cells have not been defined. The purpose of the present investigation was to identify aspects of spermatogenesis, spermiation, or sperm fertilizing capacity that required FYN for normal reproductive function. Matings between Fyn-null males and wild-type females resulted in normal litter sizes, despite the fact that Fyn-null males exhibited reduced epididymal size and sperm count. Morphological analysis revealed a high frequency of abnormal sperm morphology among Fyn-null sperm, and artificial insemination competition studies demonstrated that Fyn-null sperm possessed reduced fertilizing capacity. Fyn-null sperm exhibited nearly normal motility during capacitation in vitro but reduced ability to undergo the acrosome reaction and fertilize oocytes. The typical pattern of capacitation-induced protein tyrosine phosphorylation was slightly modified in Fyn-null sperm, with reduced abundance of several minor phosphoproteins. These findings are consistent with a model in which FYN kinase plays an important role in proper shaping of the head and acrosome within the testis and possibly an additional role in the sperm acrosome reaction, events required for development of full fertilizing capacity in sperm.

The rhodocetin αβ subunit targets GPIb and inhibits von Willebrand factor induced platelet activation

Rhodocetin, a heterotetrameric snake C-type lectin from Calloselasma rhodostoma is a specific antagonist of α2β1 integrin. Its γδ subunit is responsible for binding to α2β1 integrin. In this study we show that the rhodocetin αβ subunit can bind to platelet glycoprotein GPIb. Binding of the rhodocetin αβ subunit does not depend on divalent cations. When added to washed human platelets the rhodocetin αβ subunit effectively inhibits platelet aggregation induced by von Willebrand factor plus ristocetin. In contrast, it does not affect collagen-induced platelet activation. By itself the rhodocetin αβ subunit does not induce any changes when added to washed platelets or platelet-rich plasma. However, rhodocetin αβ, after biotinylation and cross-linkage with avidin induces small platelet agglutination but not aggregation. These agglutinated platelets change their pattern of protein tyrosine phosphorylation slightly as kinase p72SYK but not p125FAK is phosphorylated.

Protein tyrosine phosphorylation and zona binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa

Many similarities between the changes associated with normal capacitation and cryocapacitation have been demonstrated. The present study was undertaken to determine whether similarities exist in the protein tyrosine phosphorylation pattern and zona binding ability between in vitro capacitated (heparin induced; 20 μg/ml) and frozen-thawed (cryocapacitated) buffalo spermatozoa. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated and frozen in liquid nitrogen. Localization of phosphotyrosine-containing protein was determined using an indirect immunoflourescence assay with anti-phosphotyrosine antibody. For zona binding assay, good quality oocytes collected by aspiration technique from fresh buffalo ovaries were used. The bound spermatozoa were stained with Hoechst 33342 dye and observed under fluorescent microscope. The results revealed sperm head associated protein tyrosine phosphorylation in both in vitro capacitated and frozen-thawed spermatozoa. In the zona binding assay, the mean number of bound spermatozoa was 90.6 ± 1.9 and 104.7 ± 2.2 in fresh semen after incubation in non capacitating media at 0 h and 3 h, respectively. But after incubation in capacitating media with heparin for 3 h, the mean number of spermatozoa attached to zona pellucida was 138.4 ± 2.6. The in vitro capacitated spermatozoa had significantly (P < 0.05) higher binding ability than that of fresh spermatozoa. After freezing and thawing, 2.5 fold reductions in the zona binding ability of cryopreserved spermatozoa was observed compared to in vitro capacitated spermatozoa. The binding ability of in vitro capacitated spermatozoa was significantly (P < 0.01) higher than that of frozen-thawed (cryocapacitated) spermatozoa. The study concluded that both in vitro capacitated and frozen-thawed (cryocapacitated) spermatozoa had similar immune-localization of tyrosine phosphorylated protein pattern, however, differed in the zona binding ability.

Evidence of Abnormal Tyrosine Phosphorylated Proteins in the Urine of Patients With Bladder Cancer: The Road Toward a New Diagnostic Tool?

Since changes in protein phosphorylation are a common feature of cancer cells, we analyzed phosphoproteins in the tissue and urine of patients with bladder cancer and assessed the diagnostic relevance of abnormally phosphorylated proteins as tumor markers. Enrolled in this study were 66 patients and 82 healthy volunteers. From the first 14 patients with bladder cancer we obtained samples of malignant and normal bladder tissue. All patients and volunteers provided a urine sample. Protein extracts of tissue specimens were separated by 2-dimensional gel electrophoresis for comparative analysis of neoplastic and normal tissue. Phosphoproteins were studied by Western blot and characterized by mass spectrometry. Urine samples were analyzed by 1-dimensional gel electrophoresis. Phosphoproteins were measured by affinity dot blotting. Profound changes in the pattern of protein tyrosine phosphorylation were consistently, reproducibly observed in bladder cancer tissues. A total of 24 phosphorylated proteins were differentially expressed in cancer tissue and identified by mass spectrometry. Phosphoproteins were fairly stable in urine samples, leading to accumulation. Urinary tyrosine phosphoproteins showed the most remarkable changes in patients with cancer with an approximately 5-fold increase compared to levels in healthy controls. To our knowledge we investigated for the first time the diagnostic potential of tissue and urinary tyrosine phosphoproteins for bladder carcinoma. Results indicate that phosphorylated proteins may represent a new, valuable class of urinary biomarkers for bladder cancer.