Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

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

A. Kumaresan A , A. P. Siqueira A , M. S. Hossain B , A. Johannisson B , I. Eriksson C , M. Wallgren A and A. S. Bergqvist A D

A Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, SLU, PO Box 7054, Uppsala SE-750 07, Sweden.

B Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, SLU, PO Box 7011, Uppsala SE-750 07, Sweden.

C Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala Biomedical Centre, BMC, PO Box 582, Uppsala SE-751 23, Sweden.

D Corresponding author. Email: ann-sofi.bergqvist@slu.se

Reproduction, Fertility and Development 24(4) 531-542 http://dx.doi.org/10.1071/RD11074
Submitted: 23 March 2011  Accepted: 20 August 2011   Published: 16 November 2011

Abstract

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.

Additional keywords: flow cytometry, intracellular calcium, in vitro sperm capacitation.


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