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Laboratorio di fisiologia comparata, Dipartimento di Scienze e Tecnologie biologiche ed ambientali,2University of Lecce, 73100 Lecce, Italy
Marine Aquaculture and Fisheries Research Centre (M.A.R.C.),3 73100 Lecce, Italy
Dipartimento di Biologia,4 Difesa e Biotecnologie Agro-Forestali, University of Basilicata, 85100 Potenza, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, fertilization, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sea bass (Dicentrarchus labrax) is one of the most cultured fish species in the Mediterranean area and, for this reason, it is important to collect data to improve its brood stock management. Previous studies have demonstrated that cryopreserved sea bass spermatozoa had similar fertilization rate and class motility (according to a report by Suquet et al. [11]: class 0 for immotile sperm, class 1 for 0%–20% motile cells, class 2 for 20%–40% motile cells, class 3 for 40%–60% motile cells, class 4 for 60%–80% motile cells, and class 5 for 80%–100% motile cells) compared with fresh sperm, but showed a decline in motility duration [12, 13] and changes in metabolism [14]. In addition, eggs inseminated with frozen-thawed sea bass sperm showed a lower hatching rate compared with those fertilized with fresh sperm (personal observation).
These changes could be attributable to the effect of the cryopreservation protocol on cellular proteins. Protein screening has become an excellent approach with which to evaluate changes in expression due to different stresses. Using this method it has been demonstrated that the reduction in motility observed in boar and human spermatozoa following cryopreservation was associated with a decrease in heat shock protein 90 during cooling [8, 9]. Similarly, the loss of P25b (a protein associated with the plasma membrane covering the acrosome) may be responsible, at least in part, for the decrease in fertility following the freezing-thawing procedure of bull semen [10]. Few data are available on fish protein pattern by two-dimensional polyacrylamide gel electrophoresis (2-DE) [15–18], and no information addresses the effect of the freezing-thawing procedure on sperm protein expression.
In the present study we used the 2-DE and matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to verify whether the cryopreservation procedure, applied to sea bass milt, affected the expression of proteins involved in the control of sperm functions. The present work shows that the use of the cryopreservation procedure causes the degradation of sperm proteins, and could be responsible (at least partially) for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals, reagent grade, were obtained from Merck (Darmstadt, Germany), Sigma (St. Louis, MO), and Fluka-Chemika (Buchs, Switzerland).
Gamete Collection
The study was carried out on a sexually mature brood stock of reared Dicentrarchus labrax males (3 yr old, length 25–30 cm, weight 200–250 g). The experiments were carried out during the reproductive period, which in this location, runs from January to February. The brood stock was kept in an indoor tank at a density of 0.6 kg/m3. The brood stock tank was replenished with seawater at a rate of 1 L/sec, while compressed air was provided through air stones. Sea bass were given pellets daily, and fish food was provided once a week. The water temperature ranged between 13.5 and 15.5°C, and the salinity was 34.2
. When handled, animals were anesthetized using 0.1 ml/L 2-phenoxy ethanol. Semen was collected by a gentle pressure applied to the testes and sperm ducts in order to obtain a drop of milt on the previously cleaned area of the gonophore. Urine and potentially urine-polluted semen (checked by color changes and viscosity variations) were carefully discarded. Sperm were collected in 2-ml syringes and stored at 4°C on ice for a maximum of 15 min. After collection, sperm were diluted at a ratio of 1:3. Aliquots of sperm to be used fresh were diluted using sperm motility-inhibiting saline solution (3.5 mg/ml NaCl, 0.11 mg/ml KCl, 1.23 mg/ml MgCl2, 0.39 mg/ml CaCl2, 1.68 mg/ ml NaHCO3, 0.08 mg/ml glucose, and 10 mg/ml BSA pH 7.7) while sperm aliquots to be cryopreserved were diluted using Mounib medium. These diluted samples were used to extract proteins for two-dimensional analysis. For egg collection, females were separated at 13°C in individual tanks (1 m3). Eggs were collected by stripping 72 h after a single injection of 10 µg/kg of D-Trp6 LHRHa. As described by Fauvel et al. [19], the quality of ova was estimated from their morphological features (i.e., perfect rotundity, development of a perivitelline space, vitellus translucency) under a dissecting microscope.
Cryopreservation of Semen
The freezing protocol developed by Fauvel et al. [12] was applied for semen cryopreservation. Semen samples were diluted in Mounib medium at 4°C (10.01 mg/ml KHCO3, 1.99 mg/ml reduced glutathione, 42.78 mg/ ml sucrose, 10 mg/ml BSA, and 10% dimethyl sulfoxide pH 7.8; osmolality 310) at a ratio of 1:3 (semen:Mounib medium) and immediately placed into 0.25-ml straws. The dilution was not allowed to equilibrate but was directly subjected to a freezing protocol [20]. Straws were placed horizontally on a tray 6.5 cm above the liquid nitrogen surface. After a freezing period of 15 min, straws were plunged into nitrogen and then stored. For thawing, straws were immersed for 5 sec in a water bath (35°C).
Sperm Motility Assessment
Because of a high sperm concentration in milt, the assessment of sperm motility requires a high dilution rate [21]. Fresh and frozen-thawed sperm were both diluted 1:150. Frozen-thawed sperm were first diluted 1:3 in Mounib medium (before cryopreservation) and 1:50 in the sperm motility-inhibiting saline solution after thawing. Fresh sperm were directly diluted 1:150 using sperm motility-inhibiting saline solution, and sperm motility was assessed immediately after gamete collection. Sperm motility was assessed as follows: 6 µl of diluted sample was placed on microscope slides and activated by adding 54 µl of seawater (1:10). Motility was evaluated within the first 10 sec after activation using a microscope (Eclipse E600; Nikon) connected to a video monitor (TM-A14OPN; JVC). Two independent observers in three replicates for each sample counted the percentage of initially motile sperm. The total duration of motility was timed by stopwatch when 95% of the sperm ceased moving. Only forward-moving sperm were judged to be motile; those simply vibrating or turning on their axes were considered to be immotile [22]. Motility was classified as the percentage of motile sperm.
Fertilization Assays
Sea bass sperm is highly concentrated and for this reason, dilution is also normally used for sea bass semen [23, 24].
The fertilizing ability of fresh and frozen-thawed semen was evaluated using the following experimental protocol. Fresh and frozen-thawed sperm were both diluted 1:10. Frozen-thawed sperm were first diluted 1:3 in Mounib medium (before cryopreservation) and after, 1:3.3 using sperm motility-inhibiting saline solution (after thawing). Fresh sperm were directly diluted 1:10 using sperm motility-inhibiting saline solution. Triplicate 5-ml batches of ova were inseminated with 150 µl of diluted sperm. Frozen sperm samples were thawed and diluted individually just before insemination in order to avoid a possible decrease in fertility due to the post-thaw delay. The sperm to egg ratio used was 1:200 x 103. Gametes were activated by the addition of 2.5 ml of seawater (38 g/L, 13°C). Sperm was allowed to fertilize eggs for 3 min. Inseminated eggs were then transferred into 100-ml vials of seawater in which early development took place. After 3 h, the vial contents were poured onto a net, and both floating and sinking eggs were placed onto a counting plate. The resulting fertilization rate was assessed under a dissecting microscope on 200 randomly chosen eggs. Eggs were assumed to be fertilized when they exhibited the four-cell stage, which occurred 3 h after fertilization at 13°C [24].
Preparation of Protein Sample
Fresh and cryopreserved semen samples (previously diluted 1:3) were centrifuged at 800 x g for 10 min at 4°C. The pellet (containing the spermatozoa) was washed two times in the sperm motility-inhibiting saline solution for sea bass sperm without BSA at 800 x g for 10 min at 4°C; and finally resuspended in the lysis buffer containing 8 M urea, 2% Chaps, and 18.6 mM dithiothreitol. The first centrifugation step allowed the separation of spermatozoa from seminal plasma, the other two washing steps avoided possible contamination of spermatozoa by seminal plasma. After incubation for 1 h at room temperature the samples were centrifuged at 12 000 x g for 5 min at 4°C. The supernatants were then recovered, divided in aliquots of 10 µg/ml of protein, and stored at –70°C until the electrophoresis analyses were carried out. The protein concentration was measured using the Quick Start Bradford Protein Assay (Bio-Rad) using BSA as the standard.
Isoelectric Focusing
Isoelectric focusing (IEF) was performed on immobilized pH gradients (IPG; pH 3–10, 13 cm) with IPGphor (Amersham Biosciences). A total of 60 µg of protein was used for analytical runs, and 800 µg of proteins was used for preparative runs to a total volume of 250 µl of rehydrating buffer (8 M urea, 2% Chaps, 18.6 mM dithiothreitol, and 1% IPG buffer pH 3–10 [Amersham Biosciences]). Strips were rehydrated for 12 h. Focusing was performed with 50 µA per strip for 1 h at 500 V, 1 h at 1000 V, and 2 h at 8000 V at 20°C. After IEF the strips were equilibrated in the first step with 6 M urea, 30% glycerol, 2% SDS, 50 mM Tris pH 6.8, and 2% dithiothreitol for 15 min; and the second step with 2.5% iodoacetamide instead of dithiothreitol for another 15 min. As a tracking dye, a few grains of bromophenol blue were added.
SDS-PAGE
Separation of the second dimension was performed in 12.5% SDS/ polyacrylamide gels (14 x 16 cm) using the Hofer SE 600 Ruby System (Amersham Biosciences). The running conditions were 15 mA/gel for 15 min and 30 mA/gel for 5 h. Once the bromophenol blue had reached the anode, the gels were fixed and stained by a standard silver staining protocol (Amersham Biosciences). Coomassie R-250 staining protocol (Roti-Blue; Roth, Karlsruhe, Germany) was used to visualize protein spots in preparative gels.
Acquisition and Analysis of Two-Dimensional Gels
The stained two-dimensional gels were scanned on ImageMaster Gel Scanner (Amersham Biosciences). The image analysis was performed using the Imagemaster 2D Elite software version 3.1 (Amersham Biosciences). Protein spots were detected using automated routines from the software combined with manual editing to remove artifacts. One gel from a fresh sperm sample was selected as the basis for the construction of a reference gel against which the remaining gels were matched using standard routines from within the software. Each spot within the reference gel was assigned a spot number that was used in the subsequent description to refer to individual spots.
Tryptic in Gel Digestion of 2-DE-Resolved Proteins
Spots of interest were excised from Coomassie stained two-dimensional gels. The gel pieces were placed in 0.5-ml Eppendorf tubes and destained overnight with a solution of 5 mM ammonium bicarbonate/50% acetonitrile. After removing the supernatant, the gel pieces were dehydrated by acetonitrile, swelled by rehydratation in 5 mM ammonium bicarbonate, and shrunk again by addition of acetonitrile [25]. After removing the liquid phase, gel pieces were dried in speedvac, proteins were digested overnight at 37°C with trypsin (Promega, Madison, WI; modified trypsin), and the resulting peptide mixtures were analyzed by MALDI-TOF mass spectrometry.
MALDI-TOF Mass Spectrometry Analysis
For MALDI-TOF mass spectrometry analysis, 2 µl of each peptide mixture was mixed with 2 µl of 
-cyano-4-hydroxy-cinnamic acid solution in 50% v/v acetonitrile/0.5% v/v trifluoroacetic acid. Subsequently, 0.4 µl of this matrix-peptides mixture was applied to a target disk and allowed to air-dry. Spectra were acquired using an Ettan MALDI-TOF Pro mass spectrometer (Amersham Biosciences). Spectra were calibrated using two internal standard peptides: (ile7)AngIII (M+H 897.531, monoisotopic) and hACTH 18–39 (M+H 2465.191, monoisotopic). Mass fingerprinting database searching was carried out using the on-line software packages PeptIdent (http://www.expasy.ch), Mascot (http://www.matrixscience.com), and MS-Fit (http://prospector.ucsf.edu). The comparison of the search in different databases is a valuable check of the protein identification by informatics analysis [26].
The search of protein databases was performed as suggested by the available documentation found on the database Web sites (see above) and by M.R. Wilkins and K.L. Williams [27]. Briefly, "missed cleavage sites" take into account partial cleavages that could occur during protein digestion. We chose one missed cleavage. Because the unknown proteins have been reduced and alkylated by iodoacetamide, we specified this modification to perform the protein identification. Finally, proteins separated by gel electrophoresis often show an oxidation of methionines. It is possible to specify this modification, and the program will modify the theoretical masses of all methionine-containing peptides accordingly, before matching with user-specified peptides. We used this option to identify the protein.
The criteria used to select spots for the analysis were 1) more than a two-fold difference in protein quantities (normalized spot volume) between fresh and frozen-thawed sperm, and 2) a high-normalized volume (to simplify the detection of the spot in the preparative gel and to obtain a sufficient amount of each protein).
Statistical Analysis
The amount of protein present in a spot was taken as the area of the spot multiplied by the density and referred to as the volume. Following removal of background the spot volumes were normalized to the total protein detected for each gel by dividing the individual spot volume by the sum of all spot volumes and multiplying by 100. The normalized spot volume is referred to as abundance. Comparison between proteins of fresh and cryopreserved sperm was assessed using the Mann-Whitney test, and relationships were considered statistically significant when P < 0.05.
The Animal Care and Treatment Committee of the University of Lecce approved the animal studies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sperm samples with similar fertilization rates (70%– 90%) and percentage of motility (80%–100%) before and after cryopreservation were used to extract proteins for two-dimensional analysis. These samples showed lower motility duration after the cryopreservation procedure (Table 1). The two-dimensional experiments were repeated six times (in six different sperm samples from different individual fish, before and after the freezing-thawing procedure) with similar qualitative results. The number of spots detected on the two-dimensional gels performed on fresh sperm samples resulted in 163 spots in 5 gels and 168 in 1 gel, with molecular masses ranging between 190 and 10 kDa and isoelectric points (pI) between 3.5 and 8.0 (Fig. 1). Only 163 spots were detected in all gels, and these were used for comparative analysis. Figure 1 shows the results of a typical experiment performed on sperm samples before (Fig. 1A) and after (Fig. 1B) cryopreservation. The protein profiles appeared similar, but some differences became evident first on visual inspection, and subsequently by using image analysis software. In fact, in the cryopreserved sperm samples, among the 163 spots considered, 13 (named 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 19, 20, and 21) were significantly (P < 0.05) less expressed, and 8 were entirely absent (6, 12, 13, 14, 15, 16, 17, and 18); these are listed in Table 2. All of these 21 spots are highlighted in Figure 1. Of these spots, five (5, 6, 8, 13, and 20) were analyzed with MALDI-TOF.
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Identification of Protein Spots by MALDI-TOF
In accordance with the above-mentioned criteria, five spots were analyzed with MALDI-TOF. Three were selected from among the spots that significantly decreased after cryopreservation (5, 8, and 20) and two (6 and 13) were taken from among those that were absent in the gel obtained with frozen-thawed sperm (Fig. 1 and Table 2). Spots were excised from the gels and processed for mass spectrometry analysis as detailed in Materials and Methods. For protein identification, peptide fragment masses were used to search different databases for protein homologies using three search programs (PeptIdent, Mascot, and MS-Fit). Protein homologies were obtained for three spots. These proteins were identified from protein sequences already described in other teleost species and amphibians. In particular, two were from Brachidanio rerio (spots 5 and 20) and one was from Xenopus laevis (spot 13). Table 3 summarizes the data of the bio-informatics analysis for these proteins. In particular, spot 5 showed homology with a novel protein of zebrafish (similar to SKB1 of human and mouse), spot 13 showed homology with G1/S-specific cyclin E2, and spot 20 showed homology with the hypothetical protein DKFZp566A1524.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Five of the protein spots shown in Table 2 were analyzed by MALDI-TOF for protein identification. Three out of five sea bass proteins processed for peptide mapping were found to have homologies as suggested by Pappin et al. [30] (at least four peptides matched, and coverage was 
20%) with existing sequences in the databases we used (Table 3).
For spot 5, the search engine PeptIdent found a homology with a protein of Brachidanio rerio (similar to SKB1 of human and mouse). This is a highly conserved cytoplasmic protein with methyltransferase activity that interacts with the members of the Janus family tyrosine kinases (JAK) [31].
Genome activation is one of the first critical events in the life of a new organism. Both the timing of genome activation and the array of genes activated must be controlled correctly, and these events depend on changes in chromatin structure and availability of transcription factors [32].
The presence of members of the JAK/STAT proteins has recently been demonstrated in human sperm. Their possible contribution to the pool of transcription factors during sperm-oocyte fusion has been hypothesized as well as in the signal transmission to the oocyte nucleus [33]. Our results suggest that the observed reduction in SBK1 proteins in cryopreserved sperm could be responsible for abnormal early embryo development, which in turn, could determine the lower hatching rate observed (personal observation).
The spot protein 13 matched in Mascot and MS-Fit with a G1/S-specific cyclin E2 protein, which is essential in the control of the cell cycle at the G1/S (start) transition [34]. Cyclin E is involved in the activation of cyclin-dependent kinase 2 (cdk2). Recently, it has been demonstrated that cdk2 phosphorylates the protein phosphatase, PP1gamma2, a key enzyme in the development and regulation of sperm motility [35]. The observed reduction in sea bass sperm motility duration in frozen-thawed spermatozoa could be a consequence of the cyclin E degradation.
The protein spot 20 matched, in MS-Fit, with the hypothetical protein DKFZp566A1524 of unknown function.
The present work shows that the use of the cryopreservation procedure causes the degradation of 21 sperm proteins, and among these, 2 could be at least partially responsible for the observed decrease in sperm motility duration and the lower hatching rate of eggs fertilized with cryopreserved sperm. In addition, these observations suggest that two-dimensional electrophoresis coupled with MALDI-TOF analysis could be used as a tool to improve cryopreservation procedures.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 13 September 2004.
First decision: 4 November 2004.
Accepted: 12 January 2005.
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REFERENCES |
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