Isolation and characterization of an ovoinhibitor, a multidomain Kazal-like inhibitor from Turkey (Meleagris gallopavo) seminal plasma.

BIOLOGY OF REPRODUCTION (2014) 91(5):108, 1–15 Published online before print 24 September 2014. DOI 10.1095/biolreprod.114.118836

Isolation and Characterization of an Ovoinhibitor, a Multidomain Kazal-Like Inhibitor from Turkey (Meleagris gallopavo) Seminal Plasma1 Mariola Słowin´ska,2,3 Ewa Liszewska,3 Joanna Nynca,3 Joanna Bukowska,4 Anna Hejmej,5 Barbara Bilin´ska,5 Jarosław Szubstarski,6 Krzysztof Kozłowski,7 Jan Jankowski,7 and Andrzej Ciereszko3 3

Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Olsztyn, Poland 4 In Vitro and Cell Biotechnology Laboratory, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Olsztyn, Poland 5 Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland 6 The Veterinary Diagnostic Laboratory SLW BioLab, Ostro´da, Poland 7 Department of Poultry Science, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland ABSTRACT

antibacterial activity, domestic animal reproduction, expression, male reproductive tract, ovoinhibitor, seminal plasma, turkey

INTRODUCTION Kazal inhibitors are canonical serine proteinase inhibitors that interact with their cognate enzymes through their reactive site. The reactive site of Kazal inhibitors is variable, but the domain is structurally conserved. The reactive site residue, P1, generally corresponds to the specificity of the cognate enzyme; therefore, inhibitors with P1 Arg or Lys tend to inhibit trypsin, and those with P1 Tyr, Phe, Leu, and Met inhibit chymotrypsin [1]. Moreover, Kazal inhibitors are classified as single domain if they have only one reactive site/domain and are classified as multidomain if several active sites/domains are present on the same inhibitor molecule [1]. In the male reproductive tracts of vertebrates, only single-domain Kazal inhibitors have been identified [2–4]. They are present in high concentrations in seminal plasma and can be attached to the spermatozoa surface [5]. The primary postulated physiological function of semen Kazal inhibitors is the protection of the reproductive tissues, seminal plasma proteins, or viable spermatozoa from the proteolytic action of proteases, including the acrosin liberated from the acrosomes of dead and damaged spermatozoa [1]. One of the well-known multi-domain inhibitors is the avian egg ovoinhibitor that, depending on the avian species, possesses six or seven Kazal-type domains [1, 6, 7]. It has been identified in the blood and all egg compartments, including the egg white, egg yolk, vitelline membranes, and eggshell [8, 9]. The synthesis and secretion of the ovoinhibitor occurs in the oviduct [8]. The ovoinhibitor is a specific inhibitor of the serine proteinases such as trypsin, chymotrypsin, subtilisin, porcine elastase, proteinases K and F, and fungal proteinase [1, 8]. This type of inhibitor also plays a significant role in antibacterial egg defense against Bacillus spp., preventing contamination of table eggs and protecting the embryo [9]. However, the presence of the ovoinhibitor has been confirmed only in the avian female reproductive tract and eggs and not in the male reproductive tract. Turkey seminal plasma contains three serine proteinase inhibitors [10]. Two of them are of low molecular weight and have been identified as virgin and modified single-domain Kazal-type inhibitors [11] (Fig. 1B). These inhibitors form a tight reversible complex with turkey acrosin, which strongly suggests that the acrosin is a target enzyme for these inhibitors

1 Supported by a grant from the National Science Centre (N N311 525840) and funds appropriated to the Institute of Animal Reproduction and Food Research. Presented in part at the XXIV International Poultry Symposium PB WPSA, September, 12–14, 2012, Kołobrzeg, Poland, and the 3rd Winter Workshop of the Society for Biology of Reproduction, January 31–February 1, 2013, Zakopane, Poland. 2 Correspondence: Mariola Słowin´ska, Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10–748 Olsztyn, Poland. E-mail: [email protected]

Received: 4 March 2014. First decision: 31 March 2014. Accepted: 19 September 2014. Ó 2014 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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Turkey seminal plasma contains three serine proteinase inhibitors. Two of them, with low molecular masses (6 kDa), were identified as single-domain Kazal-type inhibitors responsible for regulating acrosin activity. Our experimental objective was to isolate and characterize the inhibitor with the high molecular weight from turkey seminal plasma. The inhibitor was purified using hydrophobic interaction and affinity chromatography. Pure preparations of the inhibitor were used for identification by mass spectrometry, for determination of physicochemical properties (molecular weight, pI, and content and composition of the carbohydrate component), for kinetic studies, and for antibacterial tests. Gene expression and immunohistochemical detection of the inhibitor were analyzed in the testis, epididymis, and ductus deferens. The inhibitor with a high molecular weight from turkey seminal plasma was identified as an ovoinhibitor, which was found in avian semen for the first time. The turkey seminal plasma ovoinhibitor was a six-tandem homologous Kazal-type domain serine proteinase inhibitor that targeted multiple proteases, including subtilisin, trypsin, and elastase, but not acrosin. Our results suggested that hepatocyte growth factor activator was a potential target proteinase for the ovoinhibitor in turkey seminal plasma. The presence of the ovoinhibitor within the turkey reproductive tract suggested that its role was to maintain a microenvironment for sperm in the epididymis and ductus deferens. The turkey seminal plasma ovoinhibitor appeared to play a significant role in an antibacterial semen defense against Bacillus subtilis and Staphylococcus aureus.

´ SKA ET AL. SŁOWIN

for 10 min at 7950 3 g. We considered the supernatant as seminal plasma, which was stored at 268C. The liver and the male reproductive tract tissue samples were obtained from six 38-wk-old turkeys killed in a local slaughterhouse. For quantitative RTPCR, the tissues were immediately frozen in liquid nitrogen, and for immunohistochemical study, the tissues were fixed in Bouin fluid. Approval from the Animal Experiments Committee in Olsztyn, Poland, was obtained before the start of the experiments.

under physiological conditions [12]. Moreover, turkey seminal plasma single-domain Kazal inhibitors are involved in the control of proacrosin activation in turkey spermatozoa [13]. The inhibitor with high molecular weight from turkey seminal plasma has not been identified, and information concerning this inhibitor is very limited. It is capable of inhibiting trypsin and chymotrypsin [14], which suggests that it has broader specificity than the previously identified singledomain Kazal inhibitors. In contrast to the other seminal plasma Kazal inhibitors, the high-molecular-weight inhibitor is present in the blood [10]. In this study, we focused on the isolation and characterization of the high-molecular-weight inhibitor from turkey seminal plasma. Pure preparations of this inhibitor were obtained and used for identification by mass spectrometry, for determination of physicochemical properties (molecular weight, pI, and content and composition of the carbohydrate component), for kinetic studies, and for antibacterial tests. The potential target protease for the ovoinhibitor was identified in seminal plasma, and the reproductive tract of turkey toms was analyzed for inhibitor gene expression.

Purification of Inhibitor of High Molecular Weight from Turkey Seminal Plasma The seminal plasma (5 ml) was brought to 40% saturation with the addition of saturated ammonium sulfate. The sample was then stored for 20 min at 48C and centrifuged at 10 000 3 g for 10 min. After centrifugation, the supernatant was discarded, and the precipitate was dissolved in 1 M (NH4)2SO4 in 50 mM Tris-HCl, pH 7.6. The solution was applied to the RESOURCE PHE, a hydrophobic interaction column (6.4 mm i.d. 3 30 mm) prepacked with SOURCE 15PHE (GE Healthcare) that had been equilibrated with 1 M (NH4)2 SO4 in 50 mM Tris-HCl, pH 7.6. Unbound materials were eluted with 10 column volumes of the equilibrating buffer. The inhibitor was bound under these conditions, and the bound proteins were eluted using a linearly decreasing concentration of (NH4)2SO4 (from 1 to 0 M) at a flow rate of 0.5 ml/min with ¨ KTA purifier system (GE Healthcare) and were detected at 280 nm. The an A presence of inhibitor activity in the collected fractions was determined with spectrophotometric (by trypsin activity inhibition with measurement at 405 nm; see below in Analytical Methods) and electrophoretic methods. Fractions of the inhibitor, obtained after seven separations, were pooled, dialyzed for 48 h against 20 mM Tris-HCl buffer, pH 7.6, at 48C, and subjected to Hi Trap NHS (N-hydroxy-succinimide ester)-activated HP (High Performance) coupled with subtilisin (Sigma-Aldrich). The Hi Trap NHS-activated HP column (0.7 3 2.5 cm), prepacked with NHS-activated Sepharose High Performance and coupled with subtilisin, was prepared according to the manufacturer’s instructions (GE Healthcare). The column was first equilibrated using 50 mM Tris, 150 mM NaCl, pH 7.4, and then incubated with the bound fraction to the Resource PHE. The column was washed in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, and elution was performed using 100 mM glycine and 0.5 M NaCl, pH 2.0. The eluted samples were immediately neutralized with 1 M Tris-HCl, pH 9.0.

MATERIALS AND METHODS Animals and Sampling Blood, eggs, and semen were obtained from turkeys of the BUT Big-6 line. The blood was taken by venipuncture of the brachial vein and centrifuged for 10 min at 1605 3 g, and the blood plasma was stored at 268C. The egg whites from 1-day-old eggs were subjected to separation of the ovoinhibitor-rich fraction with the method described by Davis et al. [15]. First, egg white (250 ml) was blended and dialyzed against continuously changing deionized water for 2 days at 48C. The mucinous precipitate was centrifuged and discarded. Next, the supernatant was brought to 40% saturation with the addition of saturated ammonium sulfate. After centrifugation the supernatant was discarded, and the precipitate was dissolved in 50 ml of water and reprecipitated by the addition of saturated ammonium sulfate to 40% saturation. The precipitate was dissolved in 15 ml of water and stored at 268C. Semen was routinely collected at 1-wk intervals by abdominal massage [16] from 27 individual male turkeys during the 30 wk of the reproductive season (period of semen production was from 30 to 60 wk of age). Toms were maintained at the Turkey Testing Farm of the Department of Poultry Science (University of Warmia and Mazury in Olsztyn). Semen was centrifuged twice

Analytical Methods The protein concentration was measured with the Lowry et al. [17] method and the method of Bradford [18] with Sigma-Aldrich reagents and bovine serum albumin as the standard. Total antitrypsin activity (ATA) was evaluated spectrophotometrically by inhibition of bovine trypsin amidase activity

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FIG. 1. Purification of the ovoinhibitor from turkey seminal plasma. Lanes: SP, seminal plasma; HIC, fraction that binds to Resource PHE; and Affsub, fraction eluted from Hi Trap NHS-activated HP coupled with subtilisin. Protein samples were analyzed by SDS-PAGE under nonreducing conditions after Coomassie blue staining (A), by native PAGE combined with reverse zymography (B), by SDS-PAGE gelatin under nonreducing conditions combined with zymography (C), and by Western blot using anti-chicken ovoinhibitor IgG (D). For better detection of the ovoinhibitor in seminal plasma and HIC fraction, the strips were cut off and stained for a prolonged time. Ovo, ovoinhibitor; Kazal, virgin; and Kazal*, modified single-domain Kazal-type inhibitors.

OVOINHIBITOR IN TURKEY SEMINAL PLASMA

FIG. 2. Mass spectrometry coverage of the ovoinhibitor sequence. Peptides identified by mass spectrometry are shaded in gray and the signal peptide is underlined. Peptide identified after N-deglycosylation is shaded in yellow. Predicted post-translational modifications: N-glycosylation sites are shown in bold, phosphorylation sites are denoted with stars, and S-palmitoylation sites are double underlined. Six reactive sites of the ovoinhibitor are inside black boxes. Posttranslational modification N, O-glycosylation sites were predicted with NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) [27] and NetOGlyc 3.1 Server (http://www.cbs.dtu.dk/services/NetOGlyc/) [28], phosphorylation sites with NetPhos 2.0 Server (http://www.cbs.dtu.dk/ services/NetPhos/) [29], and S-palmitoylation sites with CSS-Palm 3.0 (http://csspalm.biocuckoo.org) [30].

Electrophoretic Methods After each purification step, the eluted protein fractions were analyzed by SDS-PAGE with a SE 250 vertical Mighty Small electrophoresis system (GE Healthcare) under nonreducing conditions on a separating 12.5% acrylamide gel according to the method of Laemmli [22]. The samples were not boiled. The gels were stained for proteins with 0.025% Coomassie brilliant blue R-250 in 40% methanol and 7% acetic acid. Antiproteinase activity after PAGE was identified using bovine trypsin or chymotrypsin (Sigma-Aldrich) by reverse zymography according to Uriel and Berges [23], as described by Kotłowska et al. [10]. Aliquots (20 lg protein) of seminal plasma, 15 lg protein of hydrophobic interaction chromatography (HIC) fraction, and 10 lg of pure ovoinhibitor were loaded into each well (Fig. 1B). For the comparative studies of blood, egg white, and seminal plasma inhibitors, 20 lg of protein was loaded into the wells. Following electrophoresis, the gels were incubated at 378C for 15 min with bovine trypsin or chymotrypsin in 0.1 M phosphate buffer (pH 7.4) and then transferred into a solution containing a chromogenic substrate (acetyl-DLphenylalanine-b-naphthyl ester) for trypsin/chymotrypsin. The zones that possessed inhibitory activity against the chosen enzymes appeared as unstained bands on a colored background [23]. The stained gels were stored in 2% acetic acid. Electrophoretic detection of gelatinase activity under nonreducing conditions was performed according to the method of Siegel and Polakoski [24], as described previously [10]. Aliquots (20 lg protein) of seminal plasma, 15 lg protein of HIC fraction, and 10 lg of pure ovoinhibitor were loaded into each well (Fig. 1C). Dithiothreitol (DTT) was omitted from the appropriate buffers, and the samples were not boiled. SDS-PAGE was performed with a SE 250 vertical Mighty Small electrophoresis system using self-cast 12.5% separating polyacrylamide gel according to the method of Laemmli [22]. A run was conducted at 10 mA/gel for 0.5 h and then at 20 mA/gel until the dye reached the bottom. After electrophoresis, the gels were washed at room temperature with 2.5% Triton X-100 for 40 min and then incubated in development solution (50 mM Tris-HCl, 200 mM NaCl, 0.02% Triton X-100 with 5 mM CaCl2, pH 7.5) for 20 h at 378C. After incubation, the gels were stained with 0.025% Coomassie brilliant blue for 48 h. The stained gels were stored in 2% acetic acid. Areas of proteolysis appeared as clear zones against a blue background. Molecular mass estimations were made using prestained SDS-PAGE standards (201.2–6.3 kDa; Bio-Rad, Hoefer). The molecular weights of the proteinase bands were estimated with the use of the Kodak 1D program (Eastman Kodak Company).

Identification of the Ovoinhibitor Determination of N-terminal sequence of the ovoinhibitor. Samples of the ovoinhibitor were boiled, subjected to SDS-PAGE electrophoresis, and

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electroblotted onto a polyvinylidene fluoride membrane using 10 mM 3(cyclohexylamino)-1-propanesulfonoic acid-NaOH (pH 11.0) containing 10% methanol. The membrane was stained with 0.1% Coomassie brilliant blue R250 in 40% methanol and 1% acetic acid and was destained in 50% methanol. The N-terminal protein sequence analysis was performed at BioCentrum Ltd. The sequentially detached phenylthiohydantoin derivatives of amino acids were identified with the Procise 491 (Applied Biosystems) automatic sequence analysis system, according to the standard protocol of the manufacturer. Sequence comparisons were performed using the database SWISSProt (http:// www.ncbi.nlm.nih.gov/blast). In-gel digestion and identification by liquid chromatography tandem mass spectrometry. The SDS-PAGE band corresponding to 45–50 kDa was cut from the gel after Coomassie blue staining and further rinsed with water and acetonitrile (ACN). After washing, the gel slice was transferred into a 1.5-ml reaction tube and equilibrated twice with 50 mM NH4HCO3 for 10 min. The gel slice was incubated for 30 min in 50 mM NH4HCO3 with 45 mM dithioerythritol at 658C, which was followed by a 30-min incubation step in 50 mM NH4HCO3 with 100 mM iodoacetamide to reduce and block the cysteine residues. After being washed twice for 15 min in 50 mM NH4HCO3, the gel slice was minced and subjected to overnight digestion at 378C with 1 lg porcine trypsin (Promega). The supernatant was collected and preserved. The peptides were further extracted with 50 ll of 50 mM NH4HCO3 and 60 ll of 80% ACN. The ACN supernatant and the NH4HCO3 fractions were combined and concentrated with a SpeedVac concentrator (Bachover, Vacuum Concentrator). The peptide sample was reconstituted with 10 ll of 0.1% formic acid and centrifuged for 15 min at 12 000 3 g at room temperature. The liquid chromatography tandem mass spectrometry (LC-MS/MS) separation was performed on a multidimensional liquid chromatography (LC) system (Ettan MDLC; GE Healthcare) coupled to a linear IT mass spectrometer (LTQ; Thermo Electron). A sample was injected onto a C18 trap column (C18 ˚ , 300 lm 3 5 mm column size; LC PepMap100, 5 lm particle size, 100 A Packings Dionex) and subsequently separated by reversed-phase chromatography with a nano-LC column (C18 PepMap100, 3 lm bead size, 75 lm i.d., 15 cm length; LC Packings Dionex) at a flow rate of 280 nl/min. Electrospray ionization was performed with distal coated SilicaTips (FS-360-50-15-D-20; New Objective) at a needle voltage of 1.4 kV. The MS and MS/MS analyses were performed using cycles of one MS scan (mass range 400–1600 m/z) and three subsequent dependent MS/MS scans (collision energy 35%). MS/MS data were searched with a Mascot version 2.1.03 (Matrix Science) against the NCBInr 20120510 database and the following parameters: 1) enzyme: trypsin; 2) fixed modification: carbamidomethyl; 3) variable modifications: oxidation (M); 4) peptide mass tolerance: 2 Da; 5) MS/MS mass tolerance: 0.8 Da; 6) peptide charges: 1 þ, 2 þ, and 3 þ; 7) instrument: electrospray ionization trap; and 8) missed cleavage: one allowed. The search results were filtered with a significant threshold of P , 0.05 and a MASCOT ion score cutoff of 30. Identifications were accepted when at least two peptides per protein fulfilled these thresholds. The putative signal peptide was determined with SignalP 4.1 Server (http:// www.cbs.dtu.dk/services/SignalP/ [25]). The theoretical molecular mass and isoelectric point of the ovoinhibitor were calculated from the protein sequence with the ProtParamToll program (http://us.expasy.org/tools/protparam.html) [26]. The posttranslational modification N- and O-glycosylation sites were predicted with NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/ NetNGlyc/) [27] and NetOGlyc 3.1 Server (http://www.cbs.dtu.dk/services/ NetOGlyc/) [28], the phosphorylation sites with NetPhos 2.0 Server (http://

according to the method of Geiger and Fritz [19] with modifications previously described by Ciereszko et al. [20, 21]. The measurements were made at 405 nm. N-a-benzoyl-DL-arginine-p-nitroanilide was used as the substrate. To measure Kazal inhibitor activity, the ovoinhibitor was precipitated with 0.45 M HClO4. The remaining activity, corresponding to Kazal activity, was measured in the supernatant. The ovoinhibitor activity was calculated as the difference between total ATA and Kazal inhibitor activity.


www.cbs.dtu.dk/services/NetPhos/) [29], and the S-palmitoylation sites with CSS-Palm 3.0 (http://csspalm.biocuckoo.org) [30]. A multiple alignment was performed with CLUSTALW software (http://www.genome.jp/tools/clustalw/), which was followed by BoxShade software (http://www.ch.embnet.org/ software/BOX_form.html) on the ovoinhibitor sequences to analyze the conserved regions.

Determination of Molecular Weight and Isoelectric Point The molecular mass was determined with matrix-assisted laser desorption/ ionization (MALDI) with the autoflex speed time-of-flight (TOF) mass spectrometer (Bruker Daltonics). The isoelectric point was determined with isoelectric focusing (IEF). The IEF of the ovoinhibitor was conducted under nondenaturing conditions in ready IEF gels with a pH range of 5–8 with cathode and anode IEF buffers (Bio-Rad) for 2.5 h in total, starting with a constant voltage of 100 V for 1 h, then increasing to 250 V for the next 1 h and then to 500 V for 30 min. For pI calibration, the IEF marker kit (Bio-Rad) was used. The isoelectric point of the ovoinhibitor was estimated with the use of the Kodak 1D program.

Interaction with Lectins and Enzymatic Deglycosylation The procedure described in the protocol of the digoxigenin (DIG) Glycan Differentiation kit (Roche Diagnostics) was used for the characterization of the glycoprotein moiety of the ovoinhibitor. Interactions of the ovoinhibitor were tested with the following DIG-labeled lectins: Galanthus nivalis agglutinin, Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin, peanut agglutinin (PNA), and Datura stramonium agglutinin. Carboxypeptidase T, transferrin, fetuin, and asialofetuin were used as control proteins (Roche). Ovoinhibitors and control proteins were transferred onto nitrocellulose after gel electrophoretic separation in 12.5% SDS-PAGE [22, 31]. Visualization of the lectins bound to the carbohydrate moieties of the ovoinhibitor was based on the

FIG. 4. Deglycosylation of the ovoinhibitor with PNGase F ( þN), Oglycosidase ( þO), neuraminidase ( þneur) and endoglycosidase H ( þenH). Protein samples were denatured and analyzed by SDS-PAGE on a separating 12.5% acrylamide gel under nonreducing conditions.

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FIG. 3. Multiple sequence alignment of ovoinhibitors from several bird species: saker (F. cherrug), peregrine falcon (F. peregrinus), flycatcher (F. albicollis), pigeon (C. livia), turkey (M. gallopavo), chicken (G. gallus) and duck (A. platyrhynchos). A multiple alignment was performed with CLUSTALW software (http://www.genome.jp/tools/clustalw/), followed by BoxShade software (http://www.ch.embnet.org/software/BOX_form.html) on the ovoinhibitor sequences to analyze conserved regions.


FIG. 5. Effect of the ovoinhibitor on serine proteinase activity. The molar ratios of ovoinhibitor (I):enzyme (E) are indicated on the x-axis.

Mass Spectrometry Analysis of the Ovoinhibitor and Deglycosylated Ovoinhibitor Matrix assisted laser desorption/ionization time of flight/time of flight mass spectrometry sample preparation. The SDS-PAGE bands corresponding to the ovoinhibitor and deglycosylated ovoinhibitor were cut from the gel and subjected to reduction, alkylation, and in-gel trypsin digestion as described above (In-gel digestion and identification by liquid chromatography tandem mass spectrometry). The tryptic peptides were concentrated and desalted in

FIG. 6. Antimicrobial activity of the turkey seminal plasma ovoinhibitor against B. subtilis and S. aureus. Data represent means 6 SEM. Different letters indicate statistical significance at P 0.05.

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ZipTips lC18 [32] (Millipore). The peptides were eluted with 70% ACN, dried, and stored at 808C until MS analysis. The matrix was prepared by dissolving 5 mg of a-cyano-4-hydroxycinnamic acid (Bruker Daltonics) in 1 ml of 50% ACN and 0.1% trifluoroacetic acid (TFA). The dried in-gel digests were reconstituted with 1 ll of 10% ACN and 0.1% TFA. The samples were spotted following the dried-droplet method, where 1 ll reconstituted in-gel digest sample was spotted initially on an MTP 384 target plate ground steel (Bruker Daltonics), followed by 1 ll of matrix, and mixed on the spot by gentle up-and-down motion of the pipette tip. Additionally, a peptide calibration standard (Bruker Daltonics) was spotted with the matrix for the calibration of MS. Matrix assisted laser desorption/ionization time of flight/time of flight mass spectrometry protein identification. Mass spectra were acquired in the range of 700–3500 m/z with a MALDI-TOF autoflex speed TOF/TOF mass spectrometer equipped with a Smartbeam II laser (355 nm; Bruker Daltonics). The operating conditions were as follows: ion source 1 ¼ 19.00 kV, ion source 2 ¼ 16.75 kV, lens voltage ¼ 7.50 kV, reflector voltage ¼ 21.00 kV, optimized pulsed ion extraction time ¼ 120 ns, matrix suppression ¼ 500 Da, and positive reflectron mode. The strongest precursors were selected for MS/MS analysis. Peak lists were generated from MS spectra with flex Analysis version 3.3 (Bruker Daltonics). The spectra were then subjected to searches with a Mascot version 2.4 (Matrix Science). The database search criteria were as follows: enzyme: trypsin; taxonomy: turkey (Meleagris gallopavo 16 228 entries); fixed modification: carbamidomethylation (C); variable modifications: oxidation (M); peptide mass tolerance 6 0.5 Da; fragment mass tolerance 0.5 Da; and one missed cleavage allowed. The search results were filtered with a significant threshold of P , 0.05 and a MASCOT ion score cutoff of 30.

staining reaction using 0.06% 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium chloride phosphate. Enzymatic deglycosylation of denatured ovoinhibitors was performed using N-glycosidase F (PNGase F) from Flavobacterium meningosepticum, Oglycosidase from Streptococcus pneumoniae, and neuraminidase (Sialidase) from Arthrobacter ureafaciens (Roche). Briefly, 1 lg of ovoinhibitors was denatured through boiling in the presence of 0.1% SDS in 20 mM sodium phosphate buffer (pH 7.0) for 4 min. Then, Triton X-100 was added to the final concentration of 1%, followed by 2 U of PNGase F, 2 mU of O-glycosidase, and 2 mU of neuraminidase. The reaction was conducted at 378C for 18 h. The final reaction products were analyzed by SDS-PAGE on a separating 12.5% acrylamide gel. The control sample of ovoinhibitor was incubated without enzymes. The molecular masses of deglycosylated inhibitors were estimated with the use of the Kodak 1D program. To check for the presence of the N-linked oligosaccharides of the high mannose type, deglycosylation with endoglycosidase H from Streptomyces plicatus was performed. The ovoinhibitor was denatured by boiling in the presence of 0.02% SDS in 20 mM sodium phosphate buffer (pH 5.5) for 4 min. Then, Triton X-100 was then added to the final concentration of 0.2%, followed by 5 mU of endoglycosidase H.


FIG. 9. Native PAGE of blood (B), egg white (EW), and seminal plasma (SP) serine proteinase inhibitors. Inhibitors were detected by reverse zymography after incubation with trypsin (T) or chymotrypsin (CH). Ovo, ovoinhibitor; Kazal, virgin; and Kazal*, modified single-domain Kazaltype inhibitors.

FIG. 7. Two-dimensional gel electrophoresis image of proteins bound to immunoaffinity column with anti-ovoinhibitor IgGs. Proteins were identified as follows: ovoinhibitor (spot nos. 1–23), HGFA (spot nos. 24– 31), immunoglobulin light chain V-J-C region, partial (spot nos. 32–36), and serum albumin-like (spot nos. 37–40).

Antimicrobial Properties Antibacterial tests were performed according to the method described by Bourin et al. [9]. Bacillus subtilis ATCC 6633, Bacillus thuringiensis ATCC 10792 and ATCC 33679, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 25923 were purchased from Microbiologics. Bacteria were grown on tryptic soy agar overnight at 308C, and then a few colonies were picked with a sterile loop and diluted in modified Dulbecco PBS without calcium chloride and magnesium chloride (buffer A; Sigma-Aldrich). The bacteria (3 3 105 colony-forming units/150 ll) were incubated with various concentrations of the ovoinhibitor (3.5–7.0 3 106 M) diluted in buffer A for 3 h at room temperature. Twenty microliters of the bacteria-ovoinhibitor mix was then sampled and serially diluted. The different dilutions were plated onto tryptic soy agar and incubated overnight at 308C. The colonies were counted after overnight growth.

Production and Purification of Polyclonal Antibodies Against the Ovoinhibitor and Western Blot Analysis Recombinant chicken ovoinhibitor (Cusabio), which shared 71% sequence identity with the turkey ovoinhibitor (calculated by BLAST; http://blast.ncbi. nlm.nih.gov), was used for the production of antibodies and was suspended in 20 mM Tris-HCl, 8 M urea, pH 8.0 to reach a protein concentration of 1 mg/ml. Next, 0.3 ml of the ovoinhibitor suspension was mixed with 0.3 ml of PBS, emulsified with 0.6 ml of Freund complete adjuvant (FCA), and injected intradermally into a rabbit. A second injection with the same ovoinhibitor preparation was given 2 wk later. The control rabbit was injected with a mixture of an equal volume of 20 mM Tris-HCl, 8 M urea, pH 8.0, with PBS and FCA. Two weeks after the second injection, the rabbits were bled from the marginal ear vein, and the presence of antibodies was determined by Western blot analysis. The rabbits were then anesthetized before bleeding, and the collected blood was allowed to clot at 48C overnight. Centrifugation at 3500 3 g for 10 min was performed to obtain the blood serum. The polyclonal anti-ovoinhibitor immunoglobulin G (IgG) was purified from rabbit serum using 1 ml Hi Trap Protein A HP (GE Healthcare) affinity column [13]. Fractions containing IgG were pooled and tested for further analyses, including Western blot, immunohistochemistry, and affinity chromatography.

FIG. 8. Cross-reactivity between polyclonal antibodies against the ovoinhibitor (Ovo) and purified preparations of Ovo and blood (B), egg white (EW), and seminal plasma (SP) proteins after SDS-PAGE. For better detection of Ovo in SP, the strip with SP protein was cut off and stained for a prolonged time.

Inhibition Assay Trypsin from bovine pancreas, subtilisin A type VIII, elastase from porcine pancreas type IV, a-chymotrypsin from bovine pancreas type II, N a-BenzoylD,L-arginine 4-nitroanilide hydrochloride (BAPNA), N-p-Tosyl-Gly-Pro-Arg p-nitroanilide acetate salt, N-Succinyl-Ala-Ala-Ala-p-nitroanilide, and NSuccinyl-Ala-Ala-Pro-Phe p-nitroanilide were all purchased from SigmaAldrich. Acrosin I from turkey spermatozoa was isolated according to

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Słowin´ska et al. [12]. The ovoinhibitor activity of turkey seminal plasma was assayed in 0.1 M Tris-HCl, 0.005% Triton X-100, pH 8.3. The reaction mixture consisted of 0.3 mM substrate: BAPNA (trypsin, acrosin), N-p-Tosyl-Gly-ProArg p-nitroanilide (subtilisin), N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (chymotrypsin), and N-Succinyl-Ala-Ala-Ala-p-nitroanilide (elastase). The enzyme concentrations used were 0.1 3 106 M for trypsin, subtilisin, and acrosin; 0.025 3 106 M for elastase; and 2 3 109 M for chymotrypsin. Proteases were independently incubated with an increasing concentration of purified ovoinhibitor in the buffer for 20 min at 378C. The remaining activity of proteases was evaluated after the addition of their respective substrates by measuring the absorbance at 410 nm. Each reaction was performed in triplicate.


Coupling of Anti-Ovoinhibitor IgG to NHS-Activated HP Column and Isolation of the Ovoinhibitor and/or Ovoinhibitor-Proteinase Complex from Turkey Seminal Plasma Fractions containing anti-ovoinhibitor polyclonal antibodies were dialyzed overnight against 0.02 M NaHCO3, lyophilized, diluted with the standard coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3), and applied to the NHS-activated HP column (GE Healthcare). Any extra active NHS groups were deactivated by sequential washing with buffer A (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and buffer B (0.1 M acetate, 0.5 M NaCl, pH 4.0). Coupled anti-ovoinhibitor polyclonal antibodies were used as a ligand for affinity purification of the ovoinhibitor and the ovoinhibitor-proteinase complex from turkey seminal plasma. Turkey seminal plasma (1 ml) was diluted 1:1 with binding buffer (0.05 M Tris-HCl, 0.15 M NaCl, pH 7.6) and applied to the NHS-activated column with the anti-ovoinhibitor polyclonal antibody. The column was equilibrated with the binding buffer at a flow rate of 0.25 ml/min. Bound proteins were eluted from the column with elution buffer (0.5 M acetic acid, pH 3.4), collected into 1-ml fractions, and neutralized with the addition of 200 ll of 1 M Tris-HCl, pH 9.0. Fractions obtained after seven separations were pooled and concentrated to 200 ll with Amicon ultra (cutoff 3 kDa; Millipore). Preparation of NHS-activated HP column without ligand (anti-ovoinhibitor IgG) for detection of nonspecific binding proteins is described in the Supplemental Materials and Methods (Nonspecific binding on the bead of Hi Trap NHS-activated HP; Supplemental Data are available online at www. biolreprod.org).

Immunohistochemical Detection of the Ovoinhibitor in Turkey Reproductive Tissues and Liver Fragments of the testis, epididymis, ductus deferens, and liver were fixed in Bouin fluid (saturated picric acid, formaldehyde, glacial acetic acid at 15:5:1 proportions) for 24 h, dehydrated in an increasing gradient of ethanol, and embedded in paraffin. All sections (5 lm) were mounted on slides coated with 3-aminopropyltriethoxysilane (Sigma-Aldrich) deparaffinized in xylene, rehydrated gradually through a series of ethanol dilutions, and washed in water. The procedures used for immunohistochemistry were similar to those described previously by Bilin´ska et al. [34] and Nynca et al. [35]. To achieve antigen retrieval the slices were immersed in citrate buffer (10 mM, pH 6.0) and heated in a microwave oven (8 min, 750 W). Endogenous peroxidase activity was blocked by incubation in a solution of hydrogen peroxide (0.3%, v/v) in Trisbuffered saline (TBS; 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.6), whereas nonspecific binding was prevented with normal goat serum (5%, v/v). The sections were incubated overnight at 48C in a humidified chamber in the presence of antibodies against the ovoinhibitor (dilution 1:1000). Subsequently, they were incubated with biotinylated secondary antibodies, anti-rabbit IgG (1:400; Vector Laboratories), for 60 min. After each step in these procedures, the sections were carefully rinsed with TBS; the antibodies were also diluted in TBS buffer. The staining was developed using avidin-biotinylated horseradish peroxidase complex (ABC/HRP; 1:100; VECTASTAIN Elite ABC Reagent, Vector Laboratories) for 30 min followed by 0.05% 3,3 0 -diaminobenzidine tetrachloride (DAB; Sigma-Aldrich) in TBS containing 0.01% H2O2 and 0.07% imidazole for 6 min. Thereafter, the sections were washed, counterstained with Mayer hematoxylin, dehydrated, and mounted using DPX mounting media (Sigma-Aldrich). All slides were processed immunohistochemically simultaneously with the same treatment so that staining intensity could be compared among different sections [36]. The cells were considered immunopositive if a brown reaction product was present and appeared as a signal in reproductive tissue cells; the cells without any specific immunostaining were considered immunonegative [37]. Negative controls included sections incubated with preimmune goat serum instead of the primary antibody. All of the immunohistochemical experiments were repeated three times. The sections were examined with a Leica DMR microscope (Leica Microsystems GmBH Wetzlar) with Nomarski interference contrast.

Two-Dimensional Gel Electrophoresis After concentration, the proteins were precipitated with the 2-D Clean-up Kit (GE Healthcare). The pellet was resuspended in 125 ll of rehydration buffer consisting of 7 M urea, 2 M thiourea, 2% w/v CHAPS, 18 mM DTT, 2% buffer, and a trace of bromophenol blue and loaded onto immobiline DryStrip gel (7 cm; pH 4–7; GE Healthcare) with passive rehydration (10 h). Isoelectric focusing was performed at 208C on an Ettan IPGphor apparatus (GE Healthcare), with the current limited to 50 lA/strip and the following voltage program: 300 V for 1 h, 1000 V for 0.5 h in gradient, 5000 V for 1.5 h in gradient, and 5000 V for 0.5 h (a total focusing time of 8125 Vh). Prior to SDS-PAGE, the IPG strip was equilibrated in 6 M urea, 75 mM Tris-HCl (pH 8.8), 2% SDS, 30% glycerol, and 1% DTT (w/v) for 15 min, and for 15 min in the same solution but with 2.5% iodoacetamide (w/v) instead of DTT and a trace of bromophenol blue. SDS-PAGE was performed with self-cast 12.5% separating polyacrylamide gel according to the method of Laemmli [22], but without stacking gels, with a SE 250 vertical Mighty Small electrophoresis system (GE Healthcare). A run was conducted at 10 mA/gel for 0.5 h and then at 20 mA/gel until the dye reached the bottom. After electrophoresis, the gel was processed using the Coomassie staining method. The gel was fixed in fixing solution (40% ethanol and 10% acetic acid) for 40 min. After removing the fixing solution, the gel was stained using 0.08% Coomassie brilliant blue G-250. Water was used for removing residual stain. Visible spots were cut from the gel and subjected to ingel trypsin digestion and mass spectrometry identification (for details see Mass Spectrometry Analysis of the Ovoinhibitor and Deglycosylated Ovoinhibitor).

RNA Isolation and Real-Time PCR Tissues frozen in liquid nitrogen were mechanically disrupted and then treated with Fenozol buffer and homogenized using Lysing Matrix D (BLIRT S.A.) in a FastPrep-24 instrument (MP Biomedicals). Total RNA was extracted with the Total RNA Mini kit (A&A Biotechnology) according to the manufacturer’s instructions. RNA concentration and quality were determined spectrophotometrically with a ND-1000 spectrophotometer (NanoDrop Technologies) and with agarose gel electrophoresis. Total RNA (2 lg) was reverse transcribed to cDNA

Statistical Analyses To test the effects of ovoinhibitor concentration on bacteria growth and gene expression in the male reproductive tract, the Kruskal-Wallis test (a nonparametric ANOVA) was used and was followed with the Dunn multiple comparison test in the statistical software program GraphPad5 (GraphPad PRISM v 5.0, GraphPad Software Inc.). Data are shown as means 6 SD. The

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with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s protocol. The reverse transcription (RT) reaction was preceded by DNase I digestion (Invitrogen Life Technologies, Inc.) and performed in a total volume of 40 ll containing 1 ll of RT Buffer, 1 ll of dNTP mix, 2.5 lM of RT random primers, 1 U/ll of RNase inhibitor, and 2.5 U/ll of MultiScribe Reverse Transcriptase. The RT reaction was conducted at 258C for 10 min, at 378C for 120 min, and then at 858C for 5 min. Real-time PCR was performed on the ABI Viia 7 sequence detection system with KAPA SYBR Fast qPCR master mix (KAPA Biosystems). Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the ovoinhibitor were designed using Primer-BLAST (http://www.ncbi.nlm.nih. gov/tools/primer-blast) based on information listed at the National Center for Biotechnology Information (NCBI). The primers were 5 0 -ATTGCCAAGGGATCCAGCAA-3 0 (sense) and 5 0 -TGCTCTCCAGGTATGCGTTG-3 0 (antisense) for the ovoinhibitor (120 bp), and 5 0 -GATCCCTTCATCGACCTGAA3 0 (sense) and 5 0 -CATCTGCCCACTTGATGTTG-3 0 (antisense) for GAPDH (166 bp). The reaction mixture for PCR contained 1 ll of cDNA (50 ng), 0.4 ll of (200 nM) forward and reverse primers each, 1 ll of nuclease-free water, 5 ll of KAPA SYBR Fast qPCR master mix, and 0.2 ll of ROX Low reference dye in a final volume of 10 ll. The thermal cycling conditions during the real-time PCR were performed with the following profile: 958C for 20 sec (initial denaturing), followed by 40 repetitive cycles of 958C for 2 sec (denaturing) and 608C for 20 sec (annealing). Proper amplification of the genes of interest was verified through melting curve analyses. The data obtained by the real-time PCR for the ovoinhibitor were normalized on the basis of GAPDH mRNA content, and the data were analyzed using the Miner method [33]. Based on our earlier study [10], we knew that the ovoinhibitor was present in blood and was likely produced in the liver. Therefore, for RT-PCR, we decided to use liver as a positive control. However, statistical analyses were performed only with data from the reproductive tract.

Western blot analysis was used to check the cross-reactivity between antibody anti-ovoinhibitors and turkey blood, egg white, seminal plasma, HIC fraction, and pure ovoinhibitor. The samples (40 lg of protein) of blood, egg white, and seminal plasma, 30 lg protein of HIC fraction, and 10 lg of pure ovoinhibitor were applied on 12.5% SDS-PAGE gels. The Western blot was performed as described by Słowin´ska et al. [13]. Polyclonal antibodies against the ovoinhibitor were diluted with TBS-T (0.05 M Tris-HCl, 0.15 M NaCl, 0.1% Tween 20, pH 7.6) at a ratio of 1:2000. For better detection of the ovoinhibitor in seminal plasma and HIC fraction, the strips were cut off and stained for a prolonged time (approximately 4–5 min).

´ SKA ET AL. SŁOWIN TABLE 1. Isolation of the ovoinhibitor from turkey seminal plasma. Purification step Seminal plasma Hydrophobic interaction chromatography Affinity chromatography

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Fold

Yield (%)

54.76 3.2 0.05

3174 231 152

58.0 72.2 3040.0

1 1.2 52.4

100 7.3 4.8

data for total ATA, Kazal, and ovoinhibitor activity during the reproductive period were analyzed with repeated-measures one-way ANOVA. Correlations between seminal plasma protein concentration and inhibitor activity measured in seminal plasma obtained from turkeys 30–60 wk old were calculated using the Spearman correlation test.

the ovoinhibitor contained from 4.4% to 5.7% carbohydrate moieties. After incubation with O-glycosidase, neuraminidase, and endoglycosidase H, no differences in the molecular weights of the ovoinhibitors were observed compared with control samples (Fig. 4). Additional protein bands were observed in the ovoinhibitor samples after deglycosylation, but those bands were introduced with the preparation of enzymes used for the deglycosylation. By comparing the MS result obtained with the ovoinhibitor and the deglycosylated ovoinhibitor, we identified one additional peptide 411RCVFCNAYLESNRT424. Asparagine, at position 424, was predicted to be N-glycosylated with NetNGlyc (Table 2 and Fig. 2).

RESULTS Isolation of Inhibitor of High Molecular Weight from Seminal Plasma

Antiproteinase Activity of the Purified Ovoinhibitor The turkey seminal plasma ovoinhibitor displayed the strongest activity against subtilisin. A 1:2 molar ratio of ovoinhibitor to subtilisin produced 97.7% inhibition (Fig. 5). Strong ovoinhibitor activities were also observed against trypsin and elastase. A 1.5:1 and 2:1 molar ratio of ovoinhibitor to trypsin and to elastase caused 99.9% and 99.5% inhibition, respectively. A weaker activity was observed against chymotrypsin, and a 20:1 molar ratio of ovoinhibitor to chymotrypsin produced 90% inhibition. No activity was detected against sperm acrosin.

Identification of the Ovoinhibitor from Turkey Seminal Plasma

Antimicrobial Assay The purified ovoinhibitor from the turkey seminal plasma was able to inhibit B. subtilis and S. aureus (Fig. 6). Incubation of B. subtilis and S. aureus with 7 3 106 M of the ovoinhibitor decreased colony formation units by 32.2% and 31.8%, respectively.

N-terminal Edman sequencing of the ovoinhibitor allowed for the identification of the 12 amino acids from the internal part of the inhibitor: DGNVMVACPRIL. The ovoinhibitor was also identified with mass spectrometry with 14 unique peptides (Table 2) that corresponded to 40% sequence coverage of the ovoinhibitor-like protein (Fig. 2). The multiple sequence alignment of the ovoinhibitor from several bird species, including the saker (Falco cherrug), peregrine falcon (Falco peregrinus), flycatcher (Ficedula albicollis), pigeon (Columba livia), turkey (M. gallopavo), chicken (Gallus gallus), and duck (Anas platyrhynchos), is shown in Figure 3.

Identification of Protein Bound to Immunoaffinity with AntiOvoinhibitor IgG The proteins bound to the anti-ovoinhibitor polyclonal antibodies were identified as follows: ovoinhibitor (23 spots), hepatocyte growth factor activator (HGFA; 8 spots), immunoglobulin light chain (5 spots), and albumin (4 spots; Fig. 7). The results of the spot identification are reported in Table 3. Detailed information concerning the identifications is provided in Supplemental Table S1. Nonspecific binding proteins to the NHS-activated HP column without ligands (anti-ovoinhibitor IgG) were identified as follows: serum albumin-like, cysteine-rich secretory protein 3-like, and immunoglobulin light chain (Supplemental Fig. S1). The results of the spot identification are reported in Supplemental Table S2.

Determination of Molecular Weight and Isoelectric Point The molecular masses of two forms of the ovoinhibitor measured by mass spectrometry were 53.52 and 51.50 kDa. With IEF, the isoelectric points of the two forms of the ovoinhibitor were estimated to be 5.3 and 5.2. Interaction with Lectins and Enzymatic Deglycosylation A positive reaction of the two bands of the ovoinhibitor was observed with SNA. According to the manufacturer’s instructions (2006 DIG Glycan Differentiation kit; Roche Diagnostics), a positive reaction with SNA indicated the presence of sialic acid linked (2–6) to galactose. Electrophoretic analysis of the ovoinhibitor after deglycosylation with PNGase F revealed decreases in the molecular masses from 47.5 to 45.6 and from 45.4 to 43.0 (Fig. 4). Thus,

Western Blot Analysis of the Purified Ovoinhibitor and Blood, Egg White, and Seminal Plasma Proteins using Polyclonal Antibody Anti-Ovoinhibitor The Western blot analysis revealed that polyclonal antibodies cross-reacted with the purified preparation of the ovoinhi8

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With the two-step isolation procedure we obtained a 52.4fold purification of the inhibitor with a yield of 4.8% (Table 1). Using HIC as the first step in purification, we separated the inhibitor from Kazal inhibitors and most seminal plasma proteins (Fig. 1). At this stage of isolation, the ovoinhibitor was not visible using protein staining (Fig. 1A) and was identified only by staining against trypsin activity and Western blot (Fig. 1, B and D). Moreover, the fractions that contained the isolated inhibitor activity were contaminated with proteolytic enzymes (Fig. 1C). The final purification of the ovoinhibitor was achieved by affinity chromatography with Hi Trap NHSactivated HP coupled with subtilisin (Fig. 1). The electrophoretic profile of purified inhibitor revealed a broad band of activity after PAGE and two main bands as well as smaller bands of lower intensity after SDS-PAGE and Western blot (Fig. 1, A and D).

OVOINHIBITOR IN TURKEY SEMINAL PLASMA TABLE 2. Characteristics of the ovoinhibitor peptides identified by mass spectrometry.

Protein accession, description gi|326928512, ovoinhibitor-like (Meleagris gallopavo)

Sequence coverage

Mascot score

Calculated pI

40%

520

6.22

3%

Precursor mass Observed

Theoretical

Peptide score

815.9456 1015.0196 1108.9398 1119.0608 1121.0758 1147.5986 1465.6296 1616.7198 1877.2308 2048.6086 2778.2656 3047.9212

815.4324 1014.4917 1108.5513 1117.5009 1120.5434 1147.5081 1465.6065 1615.7923 1876.8374 2046.9065 2777.2207 3046.3042

35 36 33 53 52 47 65 39 53 52 60 50

3084.8182 1532.7121*

3083.3172 1531.6548

81 36

Peptide sequence R.TLVACPR.I K.HITIDCTR.Y K.YPSTVSKDGR.T R.DGNVMVACPR.I K.LEIGSVDCTK.Y K.DGTSWVACPR.N R.CTEEVPELDCSK.Y R.QVVRDGNVMVACPR.I þoxidation K.EHSTPLDCTQYLGNSR.N R.EHSANVEKEYDGECRPK.H R.NLKPVCGTDGSTYSNECGICLYNR.E K.EHCQGIQQVSPICTMEYIPHCGSDGK.T þoxidation R.ILSPVCGTDGFTYDNECGICAHNAEQR.T R.CVFCNAYLESNR.T

* Peptide identified after deglycosylation by MALDI.

Immunohistochemical Detection of the Ovoinhibitor in Turkey Reproductive Tissues and Liver The immunohistochemical study revealed positive signals, but of various intensities, for the ovoinhibitor in the turkey testis, epididymis, ductus deferens, and liver (Fig. 11). In the testis, a positive signal for the ovoinhibitor was localized to germ cells, except for the spermatogonia, with the signal being of weak intensity in spermatocytes and of strong and to moderate intensity in round and elongating spermatids and sperm, respectively (Fig. 11A). The staining was always localized to the cell cytoplasm. In elongating spermatids and testicular sperm, the staining was mainly localized to the cytoplasmic droplet. Spermatozoa without cytoplasmic droplets were immunonegative. Less frequently, a positive signal was also detected in the Sertoli cell cytoplasm (Fig. 11A). No positive immunoreaction was noticed in Leydig and myoid cells. Additionally, the epithelium around blood vessels displayed no positive signal (not shown). In the epithelial cells of the more proximal part of the epididymis, a strong signal for the ovoinhibitor was confined to the apical cytoplasm (Fig. 11B, left panel). There was no positive signal for the ovoinhibitor in ciliated cells, basal cells, or the numerous microvilli projected from nonciliated cell surfaces into the ductal lumen (Fig. 11B, left panel). Interestingly, the nonciliated cells and basal cells displayed a moderate to strong staining for the ovoinhibitor in the main epididymis (Fig. 11B, right panel). There was no positive signal in epididymal sperm because the cytoplasmic droplets were still not present on the flagella. Some epithelial cells projected considerable amounts of the positively stained cytoplasm into the lumen, as did loose cytoplasmic droplets (Fig. 11B, right panel). There was no positive staining in the connective tissue (Fig. 11B). In the pseudostratified columnar epithelium of the ductus deferens, a strong staining for the ovoinhibitor was observed

Antiproteinase Profiles of Blood, Egg White, and Seminal Plasma of Turkey The broad bands of inhibitors with low migration rates, corresponding to the migration rate of the seminal plasma ovoinhibitor, were detected in the blood and egg whites of turkeys with the use of trypsin and chymotrypsin for the detection of antiproteinase activity (Fig. 9). However, clear differences in the migration rate of the ovoinhibitor were visible among these body fluids. The fastest, moderate, and slowest migration rates of the ovoinhibitor were recorded for seminal plasma, blood, and egg-white inhibitors, respectively. In addition to the ovoinhibitor, inhibitors of faster migration rates were also observed. For the seminal plasma, two inhibitors were detected with the use of trypsin (previously identified as Kazal inhibitors [11]). For egg white, two inhibitors were detected with the use of trypsin and chymotrypsin, and blood was characterized by the presence of one additional moderate-migration-rate inhibitor, which inhibited chymotrypsin but not trypsin. Expression of Ovoinhibitor mRNA in the Male Reproductive Tract The ovoinhibitor mRNA transcript was detected in the turkey reproductive tract and liver. The expression was higher by 4 and 12 times in the epididymis and the ductus deferens, respectively, than in the testis (Fig. 10). The level of ovoinhibitor mRNA transcripts in the liver was 175-, 42-, and 15-fold higher than in the testis, epididymis, and ductus deferens, respectively.

TABLE 3. Identification of the proteins bound to the immunoaffinity column with anti-ovoinhibitor IgG. Spot 1–23 24–31 32–36 37–40

Protein name

Accession

Protein molecular mass (Da)

Protein pI

Predicted: ovoinhibitor-like Hepatocyte growth factor activator Immunoglobulin light chain V-J-C region, partial Serum albumin-like

XP003210422 AAO46038 AFA52547 XP003205725

49905 63172 23065 71871

6.22 7.45 5.89 6.66

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bitor. In turkey blood, egg white, and seminal plasma, these antibodies allowed for the identification of a broad band of proteins with similar migration rates as the purified ovoinhibitor (Fig. 8).


primarily within the epithelial cell cytoplasm (Fig. 11C). Basal cells were weakly stained. Sperm were immunonegative. There was no positive staining in the connective tissue (Fig. 11C). In the liver cells, the positive signal for the ovoinhibitor was of moderate intensity and localized to various hepatocytes (Fig. 11D). The epithelium of capillaries was immunonegative (Fig. 11D). In the control sections, in which the primary antibody was omitted and replaced with preimmune serum, no immunopositive signals for the ovoinhibitor were observed (upper inserts in Fig. 11, A–D). Activities of the Ovoinhibitor and Kazal Family Inhibitors of Turkey Seminal Plasma During the Reproductive Period The reproductive period influenced the total ATA and the ovoinhibitor and Kazal inhibitor activities (Fig. 12). The increase of total ATA at the end of the reproductive period corresponded to an increase in the ovoinhibitor activity. The total ATA and ovoinhibitor activity were positively correlated with protein concentration (r ¼ 0.33 and r ¼ 0.36, respectively; P , 0.0001).

FIG. 10. Expression of the ovoinhibitor mRNA in turkey reproductive tract tissues. Data represent means 6 SEM. Different letters indicate statistical significance at P 0.05.

Downloaded from www.biolreprod.org. FIG. 11. Immunohistochemical localization of the ovoinhibitor in the turkey testis (A), epididymis (B), ductus deferens (C), and liver (D). Immunostaining was performed using antibody against the ovoinhibitor, followed by anti-rabbit IgG and ABC/HRP visualized by DAB. Counterstaining with Mayer hematoxylin with Nomarski interference contrast. Bars ¼ 10 lm. A) Strong and moderate signals for the ovoinhibitor are localized to round (arrows) and elongating spermatids (double arrowheads) and sperm possessing cytoplasmic droplets on the flagella (open arrows). Weak signals are localized to spermatocytes (small arrows) and Sertoli cells (SC). No immunopositive reaction is visible in Leydig cells (LC) and myoid cells (cross). At higher magnification (bottom [boxed image from main panel]), note a positive signal localized to round spermatids (arrows), cytoplasmic droplets of elongating spermatids (double arrowheads) and sperm flagella (open arrows), and a loosened droplet (asterisk). Spermatozoa without a cytoplasmic droplet are immunonegative (double open arrows). B) In the proximal epididymal region (left panel and at a higher magnification, bottom left [boxed image from main panel]), a strong signal for the ovoinhibitor is localized to the apical cytoplasm of ciliated epithelial cells (double arrowheads), whereas nonciliated cells (arrows), microvilli projected from nonciliated cells (arrowhead), basal cells (small arrows), and connective tissue are immunonegative (cross). Spermatozoa without a cytoplasmic droplet are immunonegative (double open arrows). In the main epididymis (right panel and at a higher magnification, bottom right [boxed image from main panel]), strong and moderate signals are localized to nonciliated cells (arrows) and basal cells (small arrows). Ciliated cells (double arrowheads), spermatozoa without a cytoplasmic droplet (double open arrows), and connective tissue (cross) are immunonegative. Positively stained cell cytoplasmic protrusions (loosened sperm cytoplasmic droplets) are visible in the lumen (asterisks). C) Strong staining for the ovoinhibitor is localized to columnar epithelial cells (arrows), whereas in basal cells (small arrow) a weak signal is observed. Note the immunonegative spermatozoa (double open arrows) and connective tissue (crosses). At a higher magnification (bottom [boxed image from main panel]), a cytoplasmic pattern of the staining is visible in columnar epithelial cells (arrow). Spermatozoa without a cytoplasmic droplet are immunonegative (double open arrows). D) A moderate signal for the ovoinhibitor is localized to hepatocytes (arrows). At a higher magnification (bottom [boxed image from main panel]), note the immunonegative epithelium of capillaries (arrowheads). No immunopositive staining for the ovoinhibitor is observed in the testis, epididymis, ductus deferens, or liver when the primary antibody is omitted (upper insets in A, B, C, and D).

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FIG. 12. Total ATA, Kazal inhibitor activity, and ovoinhibitor (Ovo) activity of turkey seminal plasma during the reproductive period. Effects of the reproductive period on inhibitor activity were significant at **P 0.01 and ***P 0.001. Data were from six individual toms.

DISCUSSION The present study is the first to report the isolation and identification of the ovoinhibitor from turkey seminal plasma. The ovoinhibitor was identified by mass spectrometry, and the physicochemical characteristics such as molecular mass, isoelectric point, and glycoprotein structure were determined. The ovoinhibitor displayed strong activities against subtilisin, trypsin, and elastase, and was able to inhibit B. subtilis and S. aureus. Ovoinhibitor mRNA expression was detected in the turkey reproductive tract. The immunohistochemical study revealed the presence of the ovoinhibitor in the testicular spermatids and in the epithelium of the epididymis and ductus deferens. Hepatocyte growth factor activator was identified in the binding fraction to anti-ovoinhibitor IgG. The polyclonal anti-ovoinhibitor antibodies cross-reacted with blood, egg white, and seminal plasma ovoinhibitors. Higher ovoinhibitor activity was found at the end of the reproductive period for turkey toms. In this study, we developed an original method for the purification of the ovoinhibitor from turkey seminal plasma. It was isolated through sequential application of HIC and affinity chromatography with immobilized subtilisin. Affinity chromatography with immobilized serine proteinases (trypsin and chymotrypsin) was previously used for the purification of ovoinhibitors from chicken egg white and yolk [9, 38, 39]. Rhodes et al. [40] even obtained 0.6–0.7 g of chicken ovoinhibitor from 1 L of egg white. The turkey seminal plasma ovoinhibitor displayed strong activities against subtilisin, so we applied it as a ligand for ovoinhibitor isolation. However, we observed a low yield from the isolation procedure for the turkey ovoinhibitor from seminal plasma. Moreover, during the purification process, proteolytic degradation of the ovoinhibitor was observed. After Western blot analysis, polyclonal antibodies recognized two main bands of the ovoinhibitor as well as bands of lower molecular weight (Figs. 1 and 8). In summary, the ovoinhibitor from the turkey seminal plasma was quite difficult to isolate and affinity chromatography with immobilized serine proteinase, like subtilisin, appears to be a critical step for successful purification. The analysis of the sequence indicated that the ovoinhibitor from turkey seminal plasma was similar to other avian ovoinhibitors (Fig. 3). It should be noted that mass spectrometry analysis with identified peptides allowed for the confirmation of most of the sequence, which was quite interesting because its sequence remains a predicted sequence in databases. The turkey seminal plasma ovoinhibitor was a 11

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45.2-kDa (calculated mass), 410-amino-acid-residue single polypeptide, consisting of six tandem homologous Kazal-type domains. For comparison, ovoinhibitors from the Japanese quail and chicken and the pigeon possess six and seven Kazaltype domains, respectively [1, 6, 7]. Eight domains were identified in the zebra finch ovoinhibitor (accession no. XP_002194461). The alignment of the entire sequence of the turkey (M. gallopavo) ovoinhibitor with the sequences of the avian ovoinhibitors indicated that the ovoinhibitor was conserved among avian species (Fig. 3). The conserved region constituted six reactive sites and a cysteine residue responsible for the formation of disulfide bridges in the ovoinhibitor domain. After conducting comparative analysis of the entire sequence of the turkey ovoinhibitor, the predicted and partial sequences of reptile (turtle and alligator) ovoinhibitors were found in the NCBI database (accession nos. XP_006110272.1, XP_005300113.1, XP_007065083.1, XP_006032271.1, and XP_006268670.1). The amino acid at position P1 in the reactive site of each domain primarily determined the inhibitory specificity [1]. The P1 residues of the six domains of the turkey seminal plasma ovoinhibitor were, in order, Arg, Arg, Arg, Phe, Met, and Met (Fig. 2), which showed that three trypsin-type reactive sites were followed by three chymotrypsin-, subtilisin-, and elastase-type reactive sites. The inhibition of these proteinases was confirmed in the present study by kinetics analysis and the zymogram method using either bovine trypsin or chymotrypsin. The molecular masses of the turkey ovoinhibitor measured by MALDI mass spectrometry were 53.52 and 51.50 kDa, which were 11.6%–15.5% higher than the calculated molecular mass (45.2 kDa). The differences could be explained by the presence of carbohydrate components in the turkey ovoinhibitor. However, a low carbohydrate content that ranged from 1.6% to 10% was determined for Japanese quail and chicken ovoinhibitors, respectively [41, 42], and in the present study, we estimated only from 4% to 5.4% for carbohydrate moieties. Therefore, the carbohydrate components alone could not explain the differences between measured and calculated molecular masses. The analysis of the amino acid sequence of the turkey ovoinhibitor revealed that in addition to glycosylation sites there were also multiple potential phosphorylation and S-palmitoylation sites (Fig. 2). Further studies are necessary to determine if these sites of potential posttranslational modifications can explain the differences between calculated and measured molecular weights for the turkey ovoinhibitor. Our study indicated that the turkey seminal plasma ovoinhibitor was a glycoprotein with the presence of N-linked carbohydrate chains to asparagine at position 422. However, it is unknown if the ovoinhibitor was also glycosylated in the second predicted N-glycosylation site, asparagine at position 141. The application of O-glycosidase, neuraminidase, and endoglycosidase H for deglycosylation was attempted but no changes in electrophoretic migration rates were observed. These results are in agreement with the amino acid sequence analysis in which two potential N-glycosylation sites and the lack of O-glycosylation sites were predicted (Fig. 2). For comparison, three N-glycosylation sites were predicted for chicken and zebra finch ovoinhibitors, and four sites were predicted for the pigeon ovoinhibitor. Similarly to the turkey ovoinhibitor, no O-glycosylation sites were predicted in other avian ovoinhibitors. Therefore, it can be suggested that avian ovoinhibitors were N-glycosylated but not O-glycosylated. The binding of SNA lectin to the carbohydrate moieties of the turkey seminal plasma ovoinhibitor indicated the presence of N-terminally linked sialic acid to galactose or N-acetylgalac-


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appeared to be removed within a cytoplasmatic droplet. It is worth noting that during sperm maturation the cytoplasmic droplet migrates down the flagellum, from its proximal to distal part, to be detached from the sperm. A similar stage-specific expression of a protease inhibitor was also demonstrated for rat Eppin (epididymal protease inhibitor) by Bian et al. [44]. These authors suggested that Eppin might be important during spermatogenesis, particularly for spermatid elongation. Perhaps the stage-specific production of the turkey ovoinhibitor reflected its strict translational regulation in the germ cell, particularly in spermiogenesis. The stage-specific secretion of inhibitors in turkey and rat spermatids indicated the important function of serine proteinase inhibitors in spermatogenesis. In contrast to the testis, in the epididymis and ductus deferens the ovoinhibitor was primarily secreted by epithelial cells. The secretory and absorptive functions of the epithelium are important for the maintenance of the microenvironment in which sperm maturation occurs and in which sperm is stored [50, 51]. It was demonstrated that proteins secreted in the Wolffian duct (vas deferens) were an important factor involved in the sperm membrane maturation changes in birds [52, 53]. The ovoinhibitor secreted by the epididymis and ductus deferens might play an important function in maintaining a microenvironment for sperm, primarily with its antimicrobial properties or with a wide range of antiproteinase activity (see below). In the performance of the physiological function, the ovoinhibitor might be supported by Kazal family inhibitors present in the turkey reproductive tract [10]. Ovoinhibitors from avian eggs were able to inhibit some serine proteinases, such as trypsin, chymotrypsin, subtilisin, porcine elastase, proteinases K and F, and fungal proteinase from Aspergillus oryzae [8, 9, 41, 54]. The data from the kinetic analysis indicated that the ovoinhibitor from turkey seminal plasma efficiently inhibited subtilisin, trypsin, and elastase but was less effective against chymotrypsin. The inhibition of these proteinases was also deduced from the amino acid at position P1 in the reactive site of the six domains (see above). Previous reports demonstrated that Kazal-type inhibitors with inhibitory activities towards subtilisin might possess bacteriostatic activity against the soil-dwelling bacteria B. subtilis and B. thuringiensis [9, 55–57]. It was suggested that the inhibitors could influence microorganism growth by inhibiting the subtilisin that they secrete; weak subtilisin inhibition correlated with low bacteriostatic activity on B. subtilis [57]. We observed that the turkey seminal plasma ovoinhibitor displayed strong activities against subtilisin and therefore might be an important antimicrobial agent of turkey semen. In our study, we demonstrated for the first time the antimicrobial activity of the turkey seminal plasma ovoinhibitor. The ovoinhibitor reduced the growth of B. subtilis and S. aureus. Antimicrobial activity against B. subtilis can likely be explained by the strong inhibition of subtilisin. However, the antimicrobial activity against S. aureus cannot be explained by such a mechanism because subtilisin is not produced by S. aureus. Perhaps some serine proteases secreted by S. aureus may be inhibited by the ovoinhibitor. Such proteinases have been identified in S. aureus [58, 59]. Our results strongly suggested that the seminal plasma ovoinhibitor played a significant role in antibacterial semen defense against B. subtilis and S. aureus, protecting the semen during semen collection and hen insemination. Elastase is responsible for catalyzing the breaking down of elastin. Increased degradation and fragmentation of elastic fibers may play a significant role in the disease process [60]. Avian egg white ovoinhibitors [1, 8, 54, 61] and the

tosamine. Sialic acid was also identified in domain I of the chicken ovoinhibitor [43]. Overall, our data demonstrated that the turkey seminal plasma ovoinhibitor was a N-glycosylated glycoprotein, and its carbohydrate component seemed to be similar to that of other avian ovoinhibitors. The differences in carbohydrate content and the presence of a few glycosylation sites in the avian ovoinhibitor structure were responsible for the heterogeneity of the ovoinhibitors; for example, five bands were identified for the chicken ovoinhibitor [8]. It is possible that the difference in the carbohydrate components was responsible for the heterogeneity of the turkey seminal plasma ovoinhibitor, because the two bands identified after SDSPAGE suggested that there were various glycosylated forms of the ovoinhibitor. Using anti-polyclonal antibodies against the ovoinhibitor, we confirmed its presence in the blood, egg white, and seminal plasma of turkey. A positive reaction suggested that the seminal plasma ovoinhibitor was immunologically identical with blood and egg white inhibitors and therefore presumably homologous with them. Immunological similarities of serum and egg white ovoinhibitors were also described for the chicken [38] and serum and egg white ovoinhibitors shared an identical sequence for the first 30 residues [1]. The presence of the ovoinhibitor in the egg white and semen suggested that its synthesis was not linked only to the production of egg white but that it might have a function related to male reproduction. Ovoinhibitors from avian eggs differed in carbohydrate content [8]. These differences can affect the isoelectric point and the molecular weight of a protein. In our study, we observed slight differences in migration rates of the ovoinhibitors after PAGE electrophoresis from blood, egg white, and seminal plasma. Therefore, it can be suggested that the ovoinhibitors from different tissues differed in glycosylation patterns, but this remains to be experimentally confirmed. Gene expressions of serine proteinase inhibitors are found in the male reproductive tract of mammals. Inhibitors such as Eppin, SPINK2, and HUSI-II were specifically expressed in the testis, epididymis, and seminal vesicle [2, 3, 44–46]. In this study, we have demonstrated for the first time that turkey ovoinhibitor mRNA was expressed in the reproductive tract, which provides the evidence for its synthesis within the turkey reproductive tract. A higher level of ovoinhibitor mRNA expression in the epididymis and ductus deferens was in agreement with the immunohistochemical data; a high level of secretion of the ovoinhibitor was observed in the epididymis and ductus deferens (see below). The high expression and secretion of the ovoinhibitor in the ductus deferens correlated with high proteolytic activity in the ductus deferens [10], where the amidase activity was 7–10-fold higher than in the epididymis and testis [47] and where the expression of HGFA was evident [48, 49]. Avian spermatozoa developed fertilizing ability in the testis, and sperm maturation appeared in the epididymis and ductus deferens [50]. Moreover, the ductus deferens is the main storage place for bird sperm [50, 51]. Perhaps the ovoinhibitor might be a part of the system that was responsible for the maintenance of the microenvironment for sperm maturation and storage in the male ductus deferens. With immunohistochemistry, we revealed the presence of the ovoinhibitor in the turkey testis, epididymis, ductus deferens, and liver. The expression of the ovoinhibitor in the testis and epididymis and ductus deferens differed among the tissues studied. Our results suggested that the germ cellspecific expression was characteristic for the ovoinhibitor in the testis. Strong and moderate intensity was observed in the cytoplasm of round and elongating spermatids and sperm (Fig. 11A). When the sperm was mature, most of the ovoinhibitor


ACKNOWLEDGMENT The authors thank Dr. Georg J. Arnold and Thomas Fro¨hlich for their involvement in the LC-MS/MS analysis and P. Hliwa for the preparation of tissue fragments for immunohistochemical study. The authors would like to thank the two anonymous reviewers for their valuable comments and suggestions.

REFERENCES 1. Laskowski M Jr, Kato I. Protein inhibitors of proteinases. Annu Rev Biochem 1980; 49:593–626. 2. Ma L, Yu H, Ni Z, Hu S, Ma W, Chu C, Liu Q, Zhang Y. Spink13, an epididymis-specific gene of the Kazal-type serine protease inhibitor (SPINK) family, is essential for the acrosomal integrity and male fertility. J Biol Chem 2013; 288:10154–10165. 3. Moritz A, Lilja H, Fink E. Molecular cloning and sequence analysis of the cDNA encoding the human acrosin-trypsin inhibitor (HUSI-II). FEBS Lett 1991; 278:127–130. 4. Perry AC, Jones R, Hall L. Sequence analysis of monkey acrosin-trypsin inhibitor transcripts and their abundant expression in the epididymis. Biochim Biophys Acta 1993; 1172:159–160. 5. Jonakova V, Calvete JJ, Mann K, Schafer W, Schmid ER, Topfer-Petersen

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formed complexes with HGFA, and under the reduction conditions of two-dimensional gel electrophoresis, the ovoinhibitor/HGFA complexes dissociated to a free enzyme and the inhibitor. Thus, the separate spots were identified as ovoinhibitor and HGFA. The presence of HGFA in the fraction bound to immunoaffinity column might indicate that ovoinhibitor/ HGFA complexes were present in turkey seminal plasma. HGFA is a serine proteinase identified in turkey seminal plasma by Holsberger at al. [48, 49] and is secreted in the efferent and deferent duct epithelial cells of the turkey reproductive tract [48]. The gene for HGFA is expressed in the deferent duct cells [49]. HGFA is the key enzyme that regulated the activity of the hepatocyte growth factor involved in the regeneration of the injured tissues [65]. However, the regulation of HGFA activity and its role in turkey reproductive physiology is unknown. Increased values of biochemical parameters of turkey seminal plasma during the reproductive season, including antiproteinase activity, were previously reported [66, 67]. However, the participation of particular inhibitors in total ATA was not determined. In our study, we confirmed that total ATA increased during the reproductive season. Moreover, we found different profiles for Kazal and ovoinhibitor activities. Up to the 50th wk of age, an increase in total ATA was mainly caused by Kazal inhibitors, whereas at the end of the season, an increase in total ATA corresponded mainly to increased ovoinhibitor activity. Moreover, ovoinhibitor activity was positively correlated with protein concentration. Increased seminal plasma protein concentration is a characteristic feature of yellow semen syndrome (semen with reduced fertility and hatchability [68–70]). Further study should be conducted to test the relationship between the ovoinhibitor, sperm aging, and YSS to evaluate if the ovoinhibitor can be a marker of semen quality. In conclusion, turkey semen contains the ovoinhibitor—a multidomain Kazal-type serine proteinase inhibitor targeting multiple proteases, including subtilisin, trypsin, and elastase. The presence of the ovoinhibitor within the turkey reproductive tract suggested a role in maintaining a microenvironment for sperm in the epididymis and ductus deferens. Our results suggested that HGFA was a potential target proteinase for the ovoinhibitor in turkey seminal plasma. Moreover, the turkey seminal plasma ovoinhibitor played a significant role in antibacterial semen defense against B. subtilis and S. aureus.

ovoinhibitor from turkey seminal plasma displayed strong activities against elastase (this study). Moreover, it was demonstrated that chicken ovoinhibitor prevented the adsorption of elastase on elastin; the complex of porcine elastase and chicken ovoinhibitor was not adsorbed on elastin whereas elastase was adsorbed [62]. Elastinolytic enzymes might be a target proteinase for the turkey seminal plasma ovoinhibitor. This might have importance in the protection of the connective tissue of the turkey reproductive tract by the prevention of elastinolytic activity of elastase in vivo. The turkey ovoinhibitor did not inhibit sperm acrosin, a trypsin-like enzyme located in sperm acrosome [63]. The absence of inhibition of acrosin was difficult to explain because of the presence of Arg in the P1 residues of the first three domains; inhibitors with Arg or Lys at P1 tend to inhibit trypsin and trypsin-like enzymes [1]. It should be noted that proteolytic degradation of the ovoinhibitor was observed during the purification process and could have been partially responsible for the lack of effect on acrosin activity. However, we performed the antiproteinase assay with the same ovoinhibitor preparation for all tested proteinases and only acrosin activity was not inhibited. It should be emphasized that the mechanism for turkey proacrosin activation is unknown and activation could be possible either through autoactivation or by the action of another trypsin-like proteinase, which could be potentially inhibited by the ovoinhibitor. Further studies directed to reveal the mechanism of proacrosin activation are necessary to evaluate potential involvement of the ovoinhibitor in this process. High inhibition efficiency of the ovoinhibitor on subtilisin and elastase suggested that the physiological function of the turkey ovoinhibitor was connected with the protection of semen and the reproductive tract against inflammatory episodes. However, turkey sperm acrosin activity was likely controlled by single-domain Kazal inhibitors identified previously in seminal plasma [11, 12]. The high association constant (7.6 3 107 M1) determined for the turkey Kazal inhibitor/ acrosin interaction indicated that a Kazal inhibitor could effectively inhibit acrosin in vivo. In summary, the antiproteinase system of turkey seminal plasma consisted of one multidomain ovoinhibitor, which could control subtilisin and elastase activity and single-domain Kazal inhibitors responsible for controlling acrosin activity. The differentiated inhibition properties of turkey seminal plasma serine inhibitors presumably helped to maintain a balance between the protease and antiprotease activity of turkey semen. Turkey seminal plasma contains serine proteinases, which could potentially be inhibited by the ovoinhibitor based on its P1 specificity. The high amidase activity of the seminal plasma was inhibited by benzamidine (a serine proteinase inhibitor) as reported by Thurston et al. [47]. With gelatin zymography, serine proteinases of molecular mass ranging from 29 to 88 kDa were identified in turkey seminal plasma. Further studies are necessary to test which of these serine proteinases could be a target for the ovoinhibitor in vivo. In our study, the immunoaffinity column with antiovoinhibitor IgG as a ligand was used to purify the ovoinhibitor and/or ovoinhibitor/proteinase complexes from turkey seminal plasma. In addition to the ovoinhibitor and HGFA, the fraction bound to the immunoaffinity column also contained immunoglobulin light chain and serum albumin-like. It is possible that these proteins represented the nonspecific binding proteins directly on the bead itself [64] (Supplemental Fig. S1 and Supplemental Table S2). The ovoinhibitor and one serine proteinase-HGFA were identified in the binding fraction to anti-ovoinhibitor IgG. It is possible that the ovoinhibitor


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Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma.

Reticuloendotheliosis in a wild turkey (Meleagris gallopavo) from coastal Georgia.

Isolation and characterization of luteinizing hormone and follicle-stimulating hormone from pituitary glands of the turkey (Meleagris gallopavo).

Isolation and characterization of a herpesvirus from wild turkeys (Meleagris gallopavo osceola) in Florida.

Comparative gait analysis of two strains of turkey, Meleagris gallopavo.

Genetic analysis of production and feed efficiency traits in an Orlopp turkey line (Meleagris gallopavo).

The potential for archiving and reconstituting valuable strains of turkey (Meleagris gallopavo) using primordial germ cells.

Morphology of the epididymal region and ductus deferens of the turkey (Meleagris gallopavo).

Response of the hepatic transcriptome to aflatoxin B1 in domestic turkey (Meleagris gallopavo).

Dilated Cardiomyopathy in a Rio Grande Wild Turkey (Meleagris gallopavo intermedia) in Southern Utah, USA, 2013.

Histopathology of hereditary muscular dystrophy of the domestic turkey (Meleagris gallopavo).

Vasoactive intestinal peptide is a hypothalamic prolactin-releasing neuropeptide in the turkey (Meleagris gallopavo).

Cloning, Sequencing, and Expression of Selenoprotein Transcripts in the Turkey (Meleagris gallopavo).

Ultrastructure of epithelial cells in the epididymal region of the turkey (Meleagris gallopavo).

Purification and characterization of a sperm motility-dynein ATPase inhibitor from boar seminal plasma.

Plasma calcium levels in the laying turkey (Meleagris gallapovo).

Changes in the plasma concentrations of prolactin, luteinising hormone, progesterone and D-(beta)-hydroxybutyrate in turkey hens (Meleagris gallopavo), during treatment of broodiness under commercial conditions.

Mitochondrial DNA-Based Analyses of Relatedness Among Turkeys, Meleagris gallopavo.

Modulation of the spleen transcriptome in domestic turkey (Meleagris gallopavo) in response to aflatoxin B1 and probiotics.

Characterization of dissimilar steroid productions by granulosa, theca interna and theca externa cells during follicular maturation in the turkey (Meleagris gallopavo).

Serological and microbial survey of Mycoplasma gallisepticum in wild turkeys (Meleagris gallopavo) from six western states.

Enhanced vasoactive intestinal peptide-induced prolactin secretion from anterior pituitary cells of incubating turkeys (Meleagris gallopavo).

Preparation and properties of luteinizing hormone (LH) subunits from the turkey (Meleagris gallopavo) and their recombination with subunits of ovine LH.

An acrosin inhibitor in ram spermatozoa that does not originate from the seminal plasma.