Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo)

Abstract

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation.

Introduction

A successful fertilization depends on several factors, including the ability of spermatozoa to undergo the acrosome reaction [1]. The acrosome reaction results in the release of acrosomal proteolytic enzymes that facilitate the sperm penetration of egg investments [1], [2], [3]. One of these enzymes is acrosin, a trypsin-like protease. Acrosin is synthesized and stored in the sperm acrosome as proacrosin, an inactive zymogen form [4]. During the acrosome reaction proacrosin is converted into mature acrosin. Acrosin activity is controlled by Kazal inhibitors [5], [6]. These inhibitors are present in high concentration in turkey seminal plasma [7]. However, it is unknown if Kazal inhibitors from avian semen can also control proacrosin activation.

The presence of the proacrosin/acrosin system was demonstrated in turkey spermatozoa [6]. The proacrosin/acrosin system of turkey spermatozoa consists of two forms of acrosin, I and II, which are characterized by similar physicochemical characteristics [6], [8]. Acrosin I and II were found to be glycoproteins with molecular weights of 30 and 41 kDa when estimated by SDS-PAGE and 30.8 kDa for both acrosins when measured by mass spectrometry [6], [8]. Similarities between turkey acrosins were also confirmed immunologically by Western blot analysis using anti-acrosin antibodies against acrosin I. The presence of the two forms of acrosins in spermatozoa agrees with the concept of functional redundancy of proteolytic enzymes in the reproductive system [9].

The liquid storage of semen is of practical interest in the management of male turkeys. However, even under optimal storage conditions, sperm functionality and viability decline and therefore diluted semen cannot be stored longer than 6 h after semen collection [10], [11]. Lost fertilizing ability and motility were found to be accompanied by significant losses in the phospholipids of the plasma membrane [12], [13], lipid peroxidation [14], [15], [16] and greater susceptibility to damage of cold-stored sperm membranes [17]. Kotłowska et al. [18] suggested that during the liquid storage, disturbances to the acrosome occur due to increased sperm acrosin-like activity, measured as amidase activity. This suggestion was confirmed by Lemoine et al. [19] who demonstrated that the sperm's ability to undergo the acrosome reaction was affected after liquid storage. However, there is a lack of detailed information concerning changes in the proacrosin/acrosin system during liquid storage.

Significant research efforts have been conducted to develop suitable protocols for avian sperm cryopreservation. However, similarly to liquid storage, the poultry industry has not been able to take advantage of cryopreservation [20], [21]. Avian spermatozoa are highly sensitive to damage caused by cryopreservation [22]. Damage occurring during freezing-thawing procedures mainly affect cellular membranes (plasmatic and mitochondrial) and the nuclei. Such damage affects fertility as well as fertility duration, which is a very important factor in birds due to the long-term semen storage in the uterovaginal glands of the female [21], [23]. Recently it was shown that similarly to liquid storage, the ability of chicken sperm to undergo the acrosome reaction in vitro was significantly reduced during the freezing process [24]. It is possible that reduced ability of sperm to undergo the acrosome reaction is connected with changes in the proacrosin/acrosin system. However, changes in the proacrosin/acrosin system during cryopreservation has not yet been studied.

The objective of the present work was to evaluate the effect of liquid storage and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa.