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

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.

Proteomic characteristics of human sperm cryopreservation

“…The researchers concluded that these changes in the capacitation‐like state were detrimental to fertility and sperm were rendered unstable and had a short lifespan. Yet other studies revealed a different picture , in which [Ca 2+ ] i decreased significantly in freeze‐thawed sperm. We found lower levels of [Ca 2+ ] i in live freeze‐thawed sperm (7‐AAD‐negative) compared with fresh at three different time points.…”

Effects of storage time on the motility, mortality and calcium levels of Atlantic salmon Salmo salar spermatozoa

“…Such conditions could account for the ability of the milt dilution to suffer low mortality and maintain intracellular calcium levels for longer periods of time (Ubilla & Valdebenito, 2011). In mammalian spermatozoa, activated cells produce a large number of metabolites that can alter their properties (Kumaresan et al, 2012). Salmo salar spermatozoa seem to maintain their function over time.…”

Oviductal fluid modulates the dynamics of tyrosine phosphorylation in cryopreserved boar spermatozoa during capacitation

“…Yet, capacitation‐like changes (cryocapacitation) occur in spermatozoa subjected to cooling and/or cryopreservation, and these changes are similar to true capacitation (Bailey et al, 2000; Green and Watson, 2001). A substantial proportion of boar spermatozoa may undergo protein tyrosine phosphorylation during the cryopreservation process (Kumaresan et al, 2011, 2012a,b). Recently, Harayama et al (2010) also reported a relationship between the level of tyrosine phosphorylation in frozen‐thawed semen and tolerance to frozen storage in bulls.…”