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.

Luteinizing hormone modulates intracellular calcium, protein tyrosine phosphorylation and motility during human sperm capacitation

2017 ◽  
Vol 483 (2) ◽  
pp. 834-839 ◽  
Author(s):  
Aideé S. López-Torres ◽  
María E. González-González ◽  
Esperanza Mata-Martínez ◽  
Fernando Larrea ◽  
Claudia L. Treviño ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
Intracellular Calcium ◽  
Tyrosine Phosphorylation ◽  
Human Sperm ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine

scholarly journals Intracellular calcium and protein tyrosine phosphorylation during the release of bovine sperm adhering to the fallopian tube epithelium in vitro

Reproduction ◽  
2005 ◽  
Vol 129 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Roberto Gualtieri ◽  
Raffaele Boni ◽  
Elisabetta Tosti ◽  
Maria Zagami ◽  
Riccardo Talevi
Keyword(s):  
Tyrosine Phosphorylation ◽  
Ionophore A23187 ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Phosphorylated Proteins ◽  
Bovine Sperm ◽  
Surface Remodeling ◽  
Bovine Species

In mammals, sperm adhesion to the epithelial cells lining the oviductal isthmus plays a key role in the maintenance of motility and in the selection of superior quality subpopulations. In the bovine species, heparin and other sulfated glycoconjugates powerfully induce the synchronous release of sperm adhering to tubal epithelium in vitro and may represent the signal which triggers release at ovulation in vivo. Sperm detachment may be due either to surface remodeling or to hyperactivation brought about by capacitation. In this paper, the dynamics of intracellular free Ca2+concentration ([Ca2+]i) and protein tyrosine phosphorylation in sperm during and after heparin-induced release from in vitro cultured oviductal monolayers were assessed to determine whether this event is due to capacitation. Moreover, Ca2+-ionophore A23187, thapsigargin, thimerosal and caffeine were used to determine whether [Ca2+]i increase and/or hyperactivation can induce sperm release. Results showed that: 1. heparin-released sperm have significantly higher [Ca2+]i than adhering sperm; 2. heparin induces a [Ca2+]i elevation in the sperm head followed by detachment from the monolayers; 3. external Ca2+is not required for heparin-induced release; 4. [Ca2+]i increase and/or hyperactivation are unable to release sperm; and 5. heparin-released sperm have an increased level of tyrosine phosphorylated proteins compared with adhering sperm. In conclusion, although heparin is considered a long-lasting capacitation agent, it quickly modulates the capacitation of bovine sperm adhering to the fallopian epithelium, probably leading to surface remodeling and therefore to the release of sperm selected and stored within the oviduct through adhesion.

Cyclic-AMP initiates protein tyrosine phosphorylation independent of cholesterol efflux during ram sperm capacitation

2008 ◽  
Vol 20 (6) ◽  
pp. 649 ◽  
Author(s):  
Carmen Colas ◽  
Peter James ◽  
Liz Howes ◽  
Roy Jones ◽  
José A. Cebrian-Perez ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Maximal Response ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Phosphorylated Proteins ◽  
Pde Inhibitors ◽  
Flagellar Proteins ◽  
Ram Sperm ◽  
Camp Levels

Unlike most other species, ram spermatozoa are difficult to capacitate in vitro. Bicarbonate and Ca2+ are necessary, whereas bovine serum albumin does not appear to be obligatory. In the present investigation we have assessed (1) the ability of the cholesterol-sequestering agent, methyl-β-cyclodextrin (M-β-CD), to initiate protein tyrosine phosphorylation, and (2) the importance of phosphodiesterases (PDEs) in controlling the levels of cAMP. Results show that despite removing significant amounts of membrane cholesterol, as assessed by filipin staining, M-β-CD treatment did not stimulate major increases in protein tyrosine phosphorylation. Addition of a cocktail of PDE inhibitors (theophylline and caffeine), a phosphatase inhibitor (okadaic acid) and dibutyryl-cAMP (db-cAMP), however, stimulated specific tyrosine phosphorylation of several proteins between 30 and 120 kDa. On their own, none of the above reagents were effective but a combination of db-cAMP + PDE inhibitors was sufficient to achieve a maximal response. H-89, a protein kinase-A inhibitor, suppressed tyrosine phosphorylation significantly. Immunofluorescence revealed that the newly-phosphorylated proteins localised mainly in the sperm tail. These findings suggest that in ram spermatozoa cAMP levels are too low to initiate tyrosine phosphorylation of flagellar proteins that are indicative of the capacitation state and that this is caused by unusually high levels of intracellular PDEs.

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