Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate

Abstract

Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10 mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca²⁺]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P < 0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca²⁺]i between P1 and SRF-P1 in fresh as well as in frozen–thawed semen. A higher (P < 0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32 kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.

Introduction

Cryopreservation leads to death of a large number of spermatozoa due to insults associated with the process, including cold shock, osmotic stress and intracellular ice crystal formation during freezing and thawing [35]. In practice, half of the spermatozoa are rendered immotile by freezing and thawing, and the surviving population also shows a shortened lifespan and altered fertilizing potential compared to freshly ejaculated spermatozoa [1], [36]. In view of these facts, the use of frozen–thawed boar semen has not achieved widespread acceptability for commercial breeding by artificial insemination [22] and the status of boar semen cryopreservation is still considered poor-to-fair [18]. Several reasons have been attributed to the low fertility of frozen–thawed boar spermatozoa, including premature capacitation-like changes (cryocapacitation) during the process of cryopreservation [1], [35] that leads to a shorter lifespan and consequently reduced sperm fertilizing ability [2], [6].

During cryopreservation, boar spermatozoa undergo alterations similar to those occurring during capacitation, leading to membrane destabilization, changes in motility pattern, increased protein tyrosine phosphorylation (PTP) and decreased fertilizing ability [1]. Since ejaculated sperm cells are highly compartmentalized, transcriptionally inactive and unable to synthesis new proteins, protein phosphorylation is an important means of modifying their function [19], [32]. Although similarities in the PTP pattern of boar spermatozoa between cryocapacitation and true capacitation have been reported [11], [30], the mechanism is still obscure. The phosphorylation status of tyrosine proteins can be used as a marker for cryocapacitation of boar spermatozoa [37]. Moreover, PTP has been shown to be related to the tolerance of spermatozoa to survive frozen storage [12] in bovine. It is generally believed that the increase in tyrosine phosphorylated proteins results from activation of protein tyrosine kinases and inactivation of protein tyrosine phosphatases [33]. Seminal plasma contains acid phosphatases that are known to dephosphorylate the sperm proteins, and one of the roles of seminal plasma is to maintain spermatozoa in a decapacitated state [7]. However; most cryopreservation protocols remove seminal plasma before processing spermatozoa for cryopreservation, potentially leading to alterations in the ratio of kinases to phosphatases, leading to disturbances in the phosphorylation–dephosphorylation status of sperm proteins.

Various approaches have been used to improve the quality of frozen–thawed boar spermatozoa, including different freezing protocols [9], [28], incorporation of various additives [20], [24] and the use of different portions of the ejaculate [22]. To identify the most suitable fraction of the ejaculate that will best survive the freezing and thawing process, previous studies have further fractioned the sperm-rich fraction (SRF) into the sperm peak portion (first 10 mL) and the rest of the SRF [22], [26]. In first 10 mL of the SRF, the cauda epididymal fluid content is high, whereas the rest of the SRF continues to have high numbers of spermatozoa, mixed with a richer protein secretion from the vesicular glands. Spermatozoa present in the sperm peak portion (P1) better sustain cooling and freezing compared to those present in the rest of the ejaculate [20]. Since the ejaculate portion had a significant effect on sperm kinetics, membrane integrity and capacitation-like changes [20], we hypothesized that spermatozoa in different portions differ in their ability to undergo PTP during the cryopreservation process, particularly with reference to the biochemical differences between the ejaculate portions. Thus, the aim of the present study was to determine whether there were any differences between spermatozoa in either portion in terms of sperm kinematics, intracellular calcium concentrations and patterns of PTP at different stages during the cryopreservation process and after thawing.