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

2012 ◽  
Vol 79 (10) ◽  
pp. 727-739 ◽  
Author(s):  
Hiroshi Harayama ◽  
Taichi Noda ◽  
Shou Ishikawa ◽  
Osamu Shidara
Keyword(s):  
Cyclic Amp ◽  
Tyrosine Phosphorylation ◽  
Extracellular Calcium ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Dependent Protein ◽  
Boar Spermatozoa

93 CALCIUM REMOVAL INCREASES THE PROTECTIVE EFFECTS OF β-CYCLODEXTRIN PLUS CHOLESTEROL ON PORCINE SPERM DURING COLD SHOCK

2006 ◽  
Vol 18 (2) ◽  
pp. 155 ◽  
Author(s):  
H. Galantino-Homer ◽  
W. Zeng ◽  
S. Megee ◽  
M. Modelski ◽  
I. Dobrinski
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cold Shock ◽  
Cholesterol Content ◽  
Extracellular Calcium ◽  
Shock Treatment ◽  
Membrane Cholesterol ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Viability ◽  
Sperm Capacitation ◽  
Protein Tyrosine

Porcine sperm are extremely sensitive to the damaging effects of cold shock and cryopreservation. Cholesterol-binding molecules, such as 2-hydroxypropyl-�-cyclodextrin (HBCD), improve post-thaw and post-cooling porcine sperm viability when added to an egg yolk-based extender, but also enhance sperm capacitation in other species. Depending upon the environmental cholesterol content, HBCD can act either as a cholesterol shuttle or sink to increase or decrease, respectively, sperm plasma membrane cholesterol content. Increasing the sperm cholesterol to phospholipid ratio reduces cold shock sensitivity whereas decreasing the ratio initiates the process of sperm capacitation. An increase in protein tyrosine phosphorylation correlates with sperm capacitation and has been shown to be dependent upon the presence of extracellular calcium. Sperm intracellular calcium also increases during cold shock. The objective of this study was to determine the combined effects of extracellular calcium and membrane cholesterol manipulation on porcine sperm viability and protein tyrosine phosphorylation following cold shock (10�C for 10 min). Viability was assessed using CFDA/propidium iodide staining. Protein tyrosine phosphorylation, previously shown to correlate with porcine sperm capacitation, was evaluated via antiphosphotyrosine (clone 4G10) immunoblots. We report here that following cold shock, porcine sperm incubated in defined medium containing both 0.8 mM HBCD and 0.5 mM cholesterol 3-sulfate (ChS) incubated in the absence of added extracellular calcium and the presence of 6 mM EGTA have significantly improved viability (90.5 � 6.3%, n = 3) when compared with cold-shocked sperm incubated in either the same medium with calcium (46.1 � 3.8%), without HBCD or ChS (26.5 � 7.4% with calcium; 46.5 � 13.1% without calcium), or with HBCD alone (17.0 � 7.4% with calcium, 36.8 � 7.5% without calcium). As we have found previously, treatment with 0.8 mM HBCD plus 0.5 mM ChS completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment. Although protein tyrosine phosphorylation correlates with porcine sperm capacitation, the ability of cold shock treatment to induce the same phosphorylation pattern indicates that other processes or pathways may contribute to its appearance. Removing extracellular calcium consistently decreased, but did not completely eliminate, the protein tyrosine phosphorylation induced by cold shock. These results indicate that cold shock-induced protein tyrosine phosphorylation is not dependent upon, but can be modulated by, extracellular calcium. The combined effects of calcium, HBCD and ChS on viability suggest that porcine sperm viability following cold shock is best maintained by removing extracellular calcium and increasing membrane cholesterol content via the cholesterol shuttle activity of HBCD. This work was supported by grants from PA Dept. Ag. (ME 443291) and the NIH (5-K08-HD041430).

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

2012 ◽  
Vol 24 (4) ◽  
pp. 531 ◽  
Author(s):  
A. Kumaresan ◽  
A. P. Siqueira ◽  
M. S. Hossain ◽  
A. Johannisson ◽  
I. Eriksson ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Tyrosine Phosphorylation ◽  
Mammalian Species ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Quantitative Evidence ◽  
Phosphorylated Proteins ◽  
Straight Line ◽  
Cryopreserved Sperm ◽  
Boar Spermatozoa

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.

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

2019 ◽  
Vol 86 (7) ◽  
pp. 751-761 ◽  
Author(s):  
Beatriz Macías‐García ◽  
Luis J. García‐Marín ◽  
María J. Bragado ◽  
Lauro González‐Fernández
Keyword(s):  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphorylation ◽  
Calcium Sensing Receptor ◽  
Protein Tyrosine ◽  
Calcium Sensing ◽  
Boar Spermatozoa

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

2015 ◽  
Vol 469 (2) ◽  
pp. 199-210 ◽  
Author(s):  
Ilaria Canobbio ◽  
Lina Cipolla ◽  
Gianni F. Guidetti ◽  
Daria Manganaro ◽  
Caterina Visconte ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Blood Platelets ◽  
Src Family Kinases ◽  
G Protein Coupled Receptors ◽  
Protein Tyrosine Phosphorylation ◽  
Kinase Activation ◽  
Protein Tyrosine ◽  
Dependent Protein ◽  
G Protein Coupled

We address the mechanism for Src family kinases activation downstream of G-protein-coupled receptors (GPCRs) in thrombin-stimulated blood platelets and we describe a novel interplay between Pyk2 and the Src kinases Fyn and Lyn in the regulation of Ca2+-dependent protein-tyrosine phosphorylation.

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