Changes of PKA and PDK1 in the principal piece of boar spermatozoa treated with a cell-permeable cAMP analog to induce flagellar hyperactivation

A cAMP-induced protein tyrosine phosphorylation and flagellar hyperactivation are controlled via complicated signaling cascades in mammalian spermatozoa. For instance, these events seem to be regulated positively by the PKA-mediated signaling and negatively by the PI3K/PDK1-mediated signaling. In this article, we have shown molecular changes of PKA and PDK1 in cAMP analog (cBiMPS)-treated boar spermatozoa in order to disclose possible roles of these kinases in protein tyrosine phosphorylation and hyperactivation. Ejaculated spermatozoa were incubated with cBiMPS, and then they were used for biochemical analyses of sperm kinases by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The first 30-min incubation with cBiMPS highly activated PKA of the principal piece to the accompaniment of autophosphorylation on Thr-197 of catalytic subunits. However, protein tyrosine phosphorylation and hyperactivation were fully induced in the sperm samples after the 180-min incubation. A potentially active form of PDK1 (54/55-kDa phospho-PDK1) was detected in the principal piece of the spermatozoa during the 90-min incubation. Another potentially active form (59-kDa phospho-PDK1) gradually increased during the same incubation period. However, the PDK1 suddenly became inactive by the dephosphorylation after the 180-min incubation, namely coincidently with full induction of protein tyrosine phosphorylation and hyperactivation. Additionally, existence of PI3K-dependently suppressing mechanisms for protein tyrosine phosphorylation was confirmed in the principal piece by pharmacological experiments with LY294002 and biochemical analyses with anti-PI3K p85 antibodies. These findings suggest that dephosphorylation of PDK1 may be a molecular switch for enhancement of protein tyrosine phosphorylation and flagellar hyperactivation in boar spermatozoa.     

Relationship between cyclic AMP‐dependent protein tyrosine phosphorylation and extracellular calcium during hyperactivation of boar spermatozoa

In mammalian spermatozoa, the state of protein tyrosine phosphorylation is modulated by protein tyrosine kinases and protein tyrosine phosphatases that are controlled via cyclic AMP (cAMP)‐protein kinase A (PKA) signaling cascades. The aims of this study were to examine the involvement of cAMP‐induced protein tyrosine phosphorylation in response to extracellular calcium and to characterize effects of pharmacological modulation of the cAMP‐induced protein phosphorylation state and calmodulin activity during hyperactivation in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell‐permeable cAMP analog) and CaCl₂ at 38.5°C to induce hyperactivation, and then used for Western blotting and indirect immunofluorescence of phosphorylated proteins and for the assessment of motility. Both cBiMPS and CaCl₂ were necessary for hyperactivation. The increase in hyperactivated spermatozoa exhibited a dependence on the state of cBiMPS‐induced protein tyrosine phosphorylation in the connecting and principal pieces. The addition of calyculin A (an inhibitor for protein phosphatases 1/2A (PP1/PP2A), 50–100 nM) coincidently promoted hyperactivation and cAMP‐induced protein tyrosine phosphorylation in the presence of cBiMPS and CaCl₂. Moreover, the addition of W‐7 (a calmodulin antagonist, 2–4 µM) enhanced the percentages of hyperactivated spermatozoa after incubation with cBiMPS and CaCl₂, independently of protein tyrosine phosphorylation. These findings indicate that cAMP‐induced protein tyrosine phosphorylation in the connecting and principal pieces is involved in hyperactivation in response to extracellular calcium, and that calmodulin may suppress hyperactivation via the signaling cascades that are independent of cAMP‐induced protein tyrosine phosphorylation. Mol. Reprod. Dev. 79: 727–739, 2012. © 2012 Wiley Periodicals, Inc.     

A unique mechanism for cyclic adenosine 3',5'-monophosphate-induced increase of 32-kDa tyrosine-phosphorylated protein in boar spermatozoa

A cAMP-induced increase of tyrosine-phosphorylated proteins is involved in the expression of fertilizing ability in mammalian spermatozoa. We (Harayama, 2003: J Androl 24:831-842) reported that incubation of boar spermatozoa with a cell-permeable cAMP analog (cBiMPS) increased a 32-kDa tyrosine-phosphorylated protein (TyrP32). The purpose of this study is to characterize the signaling cascades that regulate the cAMP-induced increase of TyrP32. We examined effects of tyrosine kinase inhibitor (lavendustin A), tyrosine phosphatase inhibitor (Na3VO4), cell-permeable calcium chelator (BAPTA-AM), and cholesterol acceptor (methyl-beta-cyclodextrin: MBC) on the increase of TyrP32 and the change and loss of acrosomes in boar spermatozoa. The spermatozoa were used for detection of tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence and for examination of acrosomal integrity by Giemsa staining. At least eight tyrosine-phosphorylated proteins including TyrP32 exhibited the cAMP-dependent increase during incubation with cBiMPS. In many proteins of them, this increase was reduced by lavendustin A but was enhanced by Na3VO4. In contrast, the cAMP-induced increase of TyrP32 was abolished by Na3VO4 but was hardly affected by lavendustin A. Giemsa staining showed that the increase of spermatozoa with weakly Giemsa-stained acrosomes (severely damaged acrosomes) or without acrosomes was correlative to the cAMP-induced increase of TyrP32. Moreover, the lack of calcium chloride in the incubation medium or pretreatment of spermatozoa with BAPTA-AM blocked the change and loss of acrosomes and the increase of TyrP32, suggesting these events are dependent on the extracellular and intracellular calcium. On the other hand, incubation of spermatozoa with MBC in the absence of cBiMPS could mimic the change and loss of acrosomes and increase of TyrP32 without increase of other tyrosine-phosphorylated proteins. Based on these results, we conclude that the cAMP-induced increase of TyrP32 is regulated by a unique mechanism that may be linked to the calcium-dependent change and loss of acrosomes.     

The calcium‐sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa

Regulation of protein tyrosine phosphorylation is required for sperm capacitation and oocyte fertilization. The objective of the present work was to study the role of the calcium‐sensing receptor (CaSR) on protein tyrosine phosphorylation in boar spermatozoa under capacitating conditions. To do this, boar spermatozoa were incubated in Tyrode's complete medium for 4 hr and the specific inhibitor of the CaSR, NPS2143, was used. Also, to study the possible mechanism(s) by which this receptor exerts its function, spermatozoa were incubated in the presence of specific inhibitors of the 3‐phosphoinositide dependent protein kinase 1 (PDK1) and protein kinase A (PKA). Treatment with NPS2143, GSK2334470, an inhibitor of PDK1 and H‐89, an inhibitor of PKA separately induced an increase in tyrosine phosphorylation of 18 and 32 kDa proteins, a decrease in the serine/threonine phosphorylation of the PKA substrates together with a drop in sperm motility and viability. The present work proposes a new signalling pathway of the CaSR, mediated by PDK1 and PKA in boar spermatozoa under capacitating conditions. Our results show that the inhibition of the CaSR induces the inhibition of PDK1 that blocks PKA activity resulting in a rise in tyrosine phosphorylation of p18 and p32 proteins. This novel signalling pathway has not been described before and could be crucial to understand boar sperm capacitation within the female reproductive tract.     

Regulation of tyrosine kinase activity during capacitation in goat sperm

Protein tyrosine phosphorylation is a key event accompanying sperm capacitation. Although this signaling cascade generates an array of tyrosine-phosphorylated polypeptides, their molecular characterization is still limited. It is necessary to differentiate the localization of the tyrosine-phosphorylated proteins in spermatozoa to understand the link between the different phosphorylated proteins and the corresponding regulated sperm function. cAMP plays a pivotal role in the regulation of tyrosine phosphorylation. The intracellular cAMP levels were raised in goat spermatozoa by the addition of the phosphodiesterase inhibitor, IBMX in conjugation with caffeine. Tyrosine phosphorylation was significantly up-regulated following treatment with these two reagents. Treatment of caudal spermatozoa with IBMX and caffeine, time dependent up-regulated phosphorylation of the protein of molecular weights 50 and 200 kDa was observed. Increased phosphorylation was observed with a combination of IBMX and caffeine treatment. Tyrosine phosphorylation in caput spermatozoa was not affected significantly under these conditions. The expression level of tyrosine kinase in sperm was examined with specific inhibitors and with anti-phosphotyrosine antibody. The indirect immunofluorescence staining was carried out on ethanol permeabilized sperm using anti-phosphotyrosine antibody. Western blot analysis was done using two separate PKA antibodies: anti-PKA catalytic and anti-PKA RIα. Almost no difference was found in the intracellular presence of the PKA RIα and RIIα subunits in caput and caudal epididymal spermatozoa. However, the catalytic subunit seemed to be present in higher amount in caudal spermatozoa. The results show that caprine sperm displays an enhancement of phosphorylation in the tyrosine residues of specific proteins under in vitro capacitation conditions.     

Effect of reactive oxygen species on capacitation and associated protein tyrosine phosphorylation in buffalo (Bubalus bubalis) spermatozoa

In the present study, the effect of two particular reactive oxygen species (ROS), superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)) on buffalo (Bubalus bubalis) sperm capacitation and associated protein tyrosine phosphorylation was studied. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50 x 10(6)/mL and incubated at 38.5 degrees C for 6h with or without heparin (10(g/mL; a positive control), or xanthine (X; 0.5mM)-xanthine oxidase (XO; 0.05 U/mL)-catalase (C; 2100 U/mL) system that generates O(2)(-) or NADPH (5mM) that stimulates the endogenous O(2)(-) production or H(2)O(2) (50 microM). The specific effect of O(2)(-), H(2)O(2) and NADPH on buffalo sperm capacitation and protein tyrosine phosphorylation was assessed by the addition of superoxide dismutase (SOD), catalase and diphenylene iodonium (DPI), respectively, to the incubation medium. Each of X+XO+C system, NADPH and H(2)O(2) induced a significantly higher percentage (P<0.05) of capacitation in buffalo spermatozoa compared to control. However, DPI inhibited this NADPH-induced capacitation and protein tyrosine phosphorylation and suggested for existence of an oxidase in buffalo spermatozoa. Using immunoblotting technique, at least seven tyrosine-phosphorylated proteins (20, 32, 38, 45, 49, 78 and 95 kDa) were detected in capacitated buffalo spermatozoa. Out of these, the tyrosine phosphorylation of p95 was induced extensively by both O(2)(-) as well as exogenous source of H(2)O(2) and using specific activators and inhibitors of signaling pathways, it was found this induction was regulated through a cAMP-dependent PKA pathway. Further, immunofluorescent localization study revealed that these ROS-induced tyrosine-phosphorylated proteins are mostly distributed in the midpiece and principal piece regions of the flagellum of capacitated spermatozoa and suggested for increased molecular activity in flagellum during capacitation. Thus, the study revealed that both O(2)(-) and H(2)O(2) promote capacitation and associated protein tyrosine phosphorylation in buffalo spermatozoa and unlike human and bovine, a different subset of sperm proteins were tyrosine-phosphorylated during heparin- and ROS-induced capacitation and regulation of these ROS-induced processes were mediated through a cAMP/PKA signaling pathway.     

Pentoxifylline induced signalling events during capacitation of hamster spermatozoa: significance of protein tyrosine phosphorylation.

To fertilize the oocyte, mammalian spermatozoa must undergo capacitation and acrosome reaction. These events are believed to be associated with various biochemical changes primarily mediated by cAMP, Ca2+ and protein kinases. But the precise signaling mechanisms governing sperm function are not clear. To study this, we used pentoxifylline (PF), a sperm motility stimulant and a cAMP-phosphodiesterase inhibitor, during capacitation and acrosome reaction of hamster spermatozoa. PF induced an early onset of sperm capacitation and its action involved modulation of sperm cell signaling molecules viz, cAMP, [Ca2+]i and protein kinases. The PF-induced capacitation was associated with an early and increased total protein phosphorylation coupled with changes in the levels of reactive oxygen species. Protein kinase (PK)-A inhibitor (H-89) completely inhibited phosphorylation of a 29 kDa protein while PK-C inhibitor (staurosporine) did not inhibit phosphorylation. Interestingly, PF induced protein tyrosine phosphorylation of a set of proteins (Mr 45-80 K) and a greater proportion of PF-treated spermatozoa exhibited protein tyrosine phosphorylation, compared to untreated controls (82 + 9% vs 34 +/- 10%; p < 0.001); tyrosine-phosphorylated proteins were localized specifically to the mid-piece of the sperm. The profile of protein tyrosine phosphorylation was inhibitable by higher concentrations (> 0.5 mM) of tyrosine kinase inhibitor, tyrphostin A47. However, at lower (0.1-0.25 mM) concentrations, the compound interestingly induced early sperm capacitation and protein tyrosine phosphorylation, like PF. These results show that protein tyrosine phosphorylation in the mid-piece segment (mitochondrial sheath) appears to be an early and essential event during PF-induced capacitation and a regulated level of tyrosine phosphorylation of sperm proteins is critical for capacitation of hamster spermatozoa.     

Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP‐dependent hyperactivation in the spermatozoa of Japanese Black bulls

The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen‐stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell‐permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine‐phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen‐stored spermatozoa were classified as having a high‐grade acrosome condition before incubation, sperm tyrosine‐phosphorylated proteins, including the 33‐kDa tyrosine‐phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41‐ and 33‐kDa sperm tyrosine‐phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine‐phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen‐stored sperm were classified as having a low‐grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine‐phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine‐phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP‐dependent hyperactivation for the spermatozoa of individual Japanese Black bulls. Mol. Reprod. Dev. 77:910–921, 2010. © 2010 Wiley‐Liss, Inc.     

A cyclic adenosine 3',5'-monophosphate-dependent protein kinase C activation is involved in the hyperactivation of boar spermatozoa

An intracellular cAMP-PKA signaling plays a pivotal role in the expression of fertilizing ability in mammalian spermatozoa. The aim of this study is to disclose biological function of serine/threonine protein kinases that are activated by the action of the cAMP-PKA signaling in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) at 38.5 degrees C up to 180 min, and then they were used for biochemical analyses of PKCs by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The incubation of spermatozoa with cBiMPS gradually activated PKCs in the connecting piece. The activation of sperm PKCs was accompanied with changes of their electrophoretic mobility by the PKA-mediated serine/threonine phosphorylation. In coincidence with the PKC activation, the cBiMPS-incubated spermatozoa were capable of exhibiting hyperactivation of flagellar movement. Moreover, the cBiMPS-induced hyperactivation was dramatically suppressed by the addition of either of specific PKC inhibitors (Ro-32-0432 and bisindolylmaleimide I) to the sperm suspensions. On the other hand, experiments using a calcium-deficient medium showed that the cBiMPS-induced hyperactivation of flagellar movement and activation of PKCs required the extracellular calcium. Based on the obtained data, we have concluded that a cAMP-PKA signaling can induce activation of calcium-sensitive PKCs that is leading to the hyperactivation of flagellar movement in boar spermatozoa. Moreover, the cAMP may have a unique role as the up-regulator of PKCs during the expression of fertilizing ability in boar spermatozoa.     

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 6 mg BSA/mL and cell concentration was adjusted to 50×10(6) cells/mL. The cells were incubated for 6h in the absence or presence of heparin (10 μg/mL) to induce capacitation. Fully capacitated spermatozoa were incubated in presence of 100 μg/mL Lysophosphatidyl choline (LPC, T1) or 100 μM Spermine-NONOate (T2) or 100 mM L-NAME (T3) or 100 μM Spermine-NONOate+100 mM L-NAME (T4) or 1 mM db-cAMP + 0.1 mM IBMX (T5) or 100μM H-89 (T6) or 100 μM Spermine-NONOate+100 μM 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.     

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta.

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.     

Different signaling pathways in bovine sperm regulate capacitation and hyperactivation

Hyperactivated sperm motility is characterized by high-amplitude and asymmetrical flagellar beating that assists sperm in penetrating the oocyte zona pellucida. Other functional changes in sperm, such as activation of motility and capacitation, involve cross talk between the cAMP/PKA and tyrosine kinase/phosphatase signaling pathways. Our objective was to determine the role of the cAMP/protein kinase A (PKA) signaling pathway in hyperactivation. Western blot analyses of detergent extracts of whole sperm and flagella were performed using antiphosphotyrosine antibody. Bull sperm capacitated by 10 microg/ml heparin and/or 1 mM dibutyryl-cAMP plus 100 microM 3-isobutyl-1-methylxanthine exhibited increased protein tyrosine phosphorylation without becoming hyperactivated. Procaine (5 mM) or caffeine (10 mM) immediately induced hyperactivation in nearly 100% of motile sperm but did not increase protein tyrosine phosphorylation. After 4 h of incubation with caffeine, sperm expressed capacitation-associated protein tyrosine phosphorylation but hyperactivation was significantly reduced. Sperm initially hyperactivated by procaine or caffeine remained hyperactivated for at least 4 h in the presence of Rp-cAMPS (cAMP antagonist) or PKA inhibitors H-89 or H-8. Pretreatment with inhibitors also failed to block induction of hyperactivation; however, the inhibitors did block protein tyrosine phosphorylation when sperm were incubated with capacitating agents, thereby verifying inhibition of the cAMP/PKA pathway. While induction of hyperactivation did not depend on cAMP/PKA, it did require extracellular Ca(2+). These findings indicate that hyperactivation is mediated by a Ca(2+) signaling pathway that is separate or divergent from the pathway associated with acquisition of acrosomal responsiveness and does not involve protein tyrosine phosphorylation downstream of the actions of procaine or caffeine.     

Regulation of the human sperm tyrosine kinase c-yes. Activation by cyclic adenosine 3',5'-monophosphate and inhibition by Ca(2+)

During the process of capacitation, spermatozoa go through a whole set of signaling cascade events in order to become fully competent at fertilizing the egg. An increase in sperm protein tyrosine phosphorylation has been described during this final maturational event in different animal species as well as in humans. Although the phosphotyrosine content of sperm protein is modulated by cAMP, Ca(2+), BSA, oxygen derivatives, and cholesterol, no protein tyrosine kinase (PTK) nor the phosphotyrosine protein phosphatase (PTPase) directly involved in the control of the phosphotyrosine content of sperm protein has been identified. Therefore, the goal of the present study was to identify the tyrosine kinases putatively responsible for the increases in sperm protein phosphotyrosine content. In the present study, we show that the src-related tyrosine kinase c-yes is present in the head of human spermatozoa in both membranes and Triton X-100-insoluble extracts. Our hypothesis was that c-yes is a tyrosine kinase responsible for at least some of the capacitation-induced increase in protein tyrosine phosphorylation. When spermatozoa were previously incubated in the presence of 3-isobutyl-1-methylxanthine or 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, treatments known to increase the phosphotyrosine content of human sperm proteins, an increase in the kinase activity of immunoprecipitated yes was measured using enolase as a substrate. These results suggest that cAMP activates while Ca(2+) inhibits human sperm c-yes kinase activity.     

Purification of a 72-kDa protein-tyrosine kinase from rat liver and its identification as Syk: involvement of Syk in signaling events of hepatocytes

Syk protein-tyrosine kinase (PTK) has been implicated in a variety of hematopoietic cell responses including immunoreceptor signaling. However, so far, there has been no evidence of the expression of Syk or Syk-related PTK in non-hematopoietic tissues. In this study, we have purified from blood cell-depleted rat liver a 72-kDa cytoplasmic PTK which shows cross-reactivity with anti-Syk antibody. Partial amino acid sequence analysis revealed that this 72-kDa PTK is identical to Syk. Immunohistochemical and RT-PCR analyses demonstrated that Syk is expressed in human hepatocytes and two rat liver-derived cell lines, JTC-27 and RLC-16. Furthermore, Syk is significantly tyrosine-phosphorylated in response to angiotensin II in JTC-27 cells, and angiotensin II-induced MAP kinase activation is blocked by the treatment of cells with a Syk-selective inhibitor, piceatannol. These results suggest that Syk plays an important role in signaling events of hepatocytes, such as signaling steps leading to MAP kinase activation by G-protein-coupled receptors. This is the first report of the expression of Syk in non-hematopoietic tissue.     

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.     

Intracellular signaling cascades induced by relaxin in the stimulation of capacitation and acrosome reaction in fresh and frozen-thawed bovine spermatozoa

Relaxin is one of the 6-kDa peptide hormones, which acts as a pleiotropic endocrine and paracrine factor. Our previous studies revealed that sperm capacitating medium containing relaxin induced capacitation and acrosome reaction (AR) in fresh and frozen-thawed porcine or bovine spermatozoa. However, the intracellular signaling cascades involved with capacitation or AR induced by relaxin was unknown. Therefore, the present study was designed to investigate the intracellular signaling cascades involved with capacitation and AR induced by relaxin in fresh and frozen-thawed bovine spermatozoa. Spermatozoa were incubated in sperm Tyrode's albumin lactate pyruvate (Sp-TALP) medium supplemented with (40 ng ml(-1)) or without relaxin, and subjected to evaluation of chlortetracycline staining pattern, cholesterol efflux, Ca(2+)-influx, intracellular cyclic adenosine monophosphate (cAMP) and protein tyrosine phosphorylation. Capacitation and AR were increased (P<0.05) in both fresh and frozen-thawed spermatozoa incubated with relaxin. Cholesterol effluxes were greater in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa incubated with relaxin than the spermatozoa incubated without relaxin. Ca(2+)-influxes were also significantly stimulated by relaxin in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa. The Sp-TALP medium containing relaxin influenced the generation of intracellular cAMP in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa, and exhibited higher exposure of protein tyrosine phosphorylation in both sperm types than the medium devoid of relaxin. Therefore, the results postulate that relaxin exerts the intracellular signaling cascades involved with capacitation and AR through accelerating the cholesterol efflux, Ca(2+)-influx, intracellular cAMP and protein tyrosine phosphorylation in fresh and frozen-thawed bovine spermatozoa.     

The effect of oviductal fluid on protein tyrosine phosphorylation in cryopreserved boar spermatozoa differs with the freezing method

Sperm capacitation takes place in the oviduct and protein tyrosine phosphorylation of sperm proteins is a crucial step in capacitation and acquisition of fertilizing potential. Cryopreserved spermatozoa show altered expression of protein tyrosine phosphorylation in the oviduct. The present study compared two freezing methods (conventional-conventional freezing (CF) and simplified-simplified freezing (SF) methods) for their effect on the ability of boar spermatozoa to undergo protein tyrosine phosphorylation in response to oviductal fluid (ODF). Cryopreserved boar-spermatozoa were incubated with pre- and post-ovulatory ODF for 6 h at 38 °C under 5% CO₂. Aliquots of sperm samples were taken at hourly intervals and analyzed for kinematics and protein tyrosine phosphorylation. Global protein tyrosine phosphorylation in spermatozoa was measured using flow cytometry and different patterns of phosphorylation were assessed using confocal microscopy. Immediately after thawing, no significant difference was observed in post-thaw sperm motility, velocity and global tyrosine phosphorylation between the two methods of freezing although the freezing method significantly (P < 0.05) influenced the effect of oviductal fluid on these parameters during incubation. While spermatozoa frozen by the CF method showed a significantly higher (P < 0.001) proportion of phosphorylation in response to preovulatory ODF during incubation, spermatozoa frozen by the SF method did not elicit such significant response as there was no significant difference in the proportion of tyrosine phosphorylated spermatozoa between treatments at any given time during incubation. If the CF method was used, the proportion of spermatozoa displaying either tail or full sperm phosphorylation increased in response to both preovulatory (EODF) and postovulatory oviductal fluid. However, if the SF method was used, a significant increase in these patterns was noticed only in the EODF treated group. The present study demonstrates that preovulatory isthmic ODF induce tyrosine phosphorylation in a higher proportion of boar spermatozoa compared to the post-ovulatory fluid and that the method of freezing significantly influences the response of post-thaw spermatozoa to porcine ODF.     

Capacitation-like alterations in cooled boar spermatozoa: assessment by the chlortetracycline staining assay and immunodetection of tyrosine-phosphorylated sperm proteins

This study was undertaken in order to characterize alterations occurring in cooled boar spermatozoa by a chlortetracycline (CTC) staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Spermatozoa were collected from 10 mature boars, washed and then resuspended in a Tris-citric acid-glucose (TCG) solution. The sperm suspensions were slowly cooled to 4 degrees C over 5 h and held for 2 days. Aliquots of the sperm suspensions were recovered before and after the cooling treatment and then used for the CTC staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Before the cooling treatment, almost all of the spermatozoa stained with CTC were characterized by uniform fluorescence over the whole head (an F pattern: uncapacitated spermatozoa). After the cooling treatment, however, significant higher percentages of spermatozoa exhibited a B pattern with a dark band of diminished fluorescence in the post acrosomal region and a relatively bright fluorescence in the acrosomal region (the pattern of capacitated spermatozoa). Coincidently, a 32 kDa tyrosine-phosphorylated protein appeared in the spermatozoa. However, these alterations occurring in the cooled spermatozoa were attenuated by the supplementation to the sperm suspensions with seminal plasma (20% (v/v)). Additionally, the same alterations were observed in the spermatozoa incubated in a capacitation-supporting medium (a modified Krebs-Ringer bicarbonate; mKRB) for 5 h. These results suggest that cooling could induce capacitation-like alterations in boar spermatozoa that were associated with the tyrosine phosphorylation of the 32 kDa sperm protein.     

Reactive oxygen species and protein kinases modulate the level of phospho-MEK-like proteins during human sperm capacitation

Capacitation is an essential process by which spermatozoa acquire fertilizing ability. Reactive oxygen species (ROS), protein kinase A (PKA), protein kinase C (PKC), protein tyrosine kinases (PTKs), and the extracellular signal-regulated protein kinase (ERK or mitogen-activated protein kinase [MAPK]) pathway regulate sperm capacitation. Our aim was to evaluate the phosphorylation of MEK (MAPK kinase or MAP2K) or MEK-like proteins in human sperm capacitation and its modulation by ROS and kinases. Immunoblotting using an anti-phospho-MEK antibody indicated that the phosphorylation of three protein bands (55, 94, and 115 kDa) increased in spermatozoa treated with fetal cord serum ultrafiltrate (FCSu), BSA, or isobutylmethylxanthine plus dibutyryl cAMP as capacitating agents. These phospho-MEK-like proteins are localized along the sperm flagellum. The MEK-inhibitors PD98059 and U126 prevented this phosphorylation, suggesting that these proteins are MEK-like proteins. The ROS scavengers prevented, and the addition of H(2)O(2) or spermine-NONOate (nitric oxide donor) triggered, the increase of phospho-MEK-like proteins. The capacitation-related increases in phospho-MEK-like proteins induced by FCSu, H(2)O(2), and spermine-NONOate were similarly modulated by PKA, PKC, and PTK, suggesting ROS as mediators in this phenomenon. These results indicate that phospho-MEK-like proteins are modulated by ROS and kinases and probably represent an intermediary step between the early events and the late tyrosine phosphorylation associated with capacitation.     

Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa

Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.