Theriogenology xxx (2015) 1–12

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Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma
ska a, *, Joanna Bukowska b, Anna Hejmej c, Barbara Bilin
ska c, Mariola S1owin d d a Krzysztof Koz1owski , Jan Jankowski , Andrzej Ciereszko a

Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Olsztyn, Poland b In Vitro and Cell Biotechnology Laboratory, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Olsztyn, Poland c Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland d Department of Poultry Science, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 December 2014 Received in revised form 24 March 2015 Accepted 26 March 2015

A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti– Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell–specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: Turkey Seminal plasma Hepatocyte growth factor activator Acrosin Single-domain Kazal-type inhibitor Expression

1. Introduction A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases [1,2]. The amidase * Corresponding author. Tel.: þ48 89 539 31 35; fax: þ48 89 535 74 21.
ska). E-mail address: [email protected] (M. S1owin 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.03.026

activity of turkey seminal plasma is 23- to 28-fold higher than for other birds, and the high level of amidase activity is evident in the vas deferens [1–4]. Turkey seminal plasma proteolytic enzymes are distinctly different from the spermatozoa-associated protease, acrosin; they differ in molecular weight, electrophoretic pattern, and substrate affinity, and they respond differently to an array of

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inhibitors [1]. So far, only one serine proteinase, hepatocyte growth factor activator (HGFA), has been identified in turkey seminal plasma [3,4]. However, the biological role of HGFA in turkey semen remains unknown. Only speculative roles in the maturation of galliform sperm and in antimicrobial defense were suggested [3]. Moreover, the mechanism for the inhibition of activity of HGFA in turkey semen is also unknown. The presence of Kazal-type inhibitors is a characteristic feature of vertebrates’ semen [5–7]. In turkey seminal plasma, two single-domain Kazal-type inhibitors and a multidomain ovoinhibitor were identified [8,9]. In contrast to the ovoinhibitor, which is present both in blood and in the reproductive tract, single-domain Kazal-type inhibitors seem to be specific for the reproductive tract [2]. Gene expression of the ovoinhibitor in the reproductive tract of turkey was recently described [9]. However, the expression of single-domain Kazal-type inhibitors remains to be investigated. An analysis of gene expression of the singledomain Kazal-type inhibitor would allow comparison of expression profiles of the single-domain Kazal-type inhibitor and the multidomain ovoinhibitor. Two forms of the single-domain Kazal-type inhibitor (virgin is the intact form and the modified form has a split peptide bond after enzyme-inhibitor complex dissociation) are present in the ductus deferens [2,8]. The presence of the single-domain Kazal-type inhibitor in the turkey reproductive tract in two forms is an indication of its role in regulating the proteolytic processes in vivo, but the target proteinases in seminal plasma are still unknown. The only available information strongly suggests that Kazal-type inhibitors are involved in control of sperm acrosin activity [10,11]. Recently, using immunoaffinity with antiovoinhibitor immunoglobulin Gs (IgGs) as ligands, a potential target proteinase for the ovoinhibitor, HGFA, was identified [9]. It is unknown at present if HGFA is also controlled by the single-domain Kazal-type inhibitor. In this study, we focused on the identification of the target proteinases for the single-domain Kazal-type inhibitors in turkey seminal plasma. Pure preparations of the Kazal-type inhibitor and the anti–Kazal-type inhibitor monospecific antibodies were used as ligands for proteinase isolation in affinity chromatography. Moreover, gene expression and immunohistochemical localization of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. 2. Materials and methods 2.1. Animals and sampling Semen was routinely collected from individual males at 1-week intervals by abdominal massage [12] from 30 turkeys during the 30 weeks of the reproductive season (period of semen production was from 30–60 weeks of age). The toms were maintained at the Turkey’s Testing Farm of the Department of Poultry Science (University of Warmia and Mazury in Olsztyn). After collection all semen samples were pooled. Semen was centrifuged twice for 10 minutes at 7950 g at 4  C. The supernatant was seminal plasma, which was stored at 26  C.

The liver and the male reproductive tract tissue samples were obtained from six 38-week-old turkeys killed in a local slaughterhouse. For reverse transcription– polymerase chain reaction (RT-PCR), the tissues were immediately frozen in liquid nitrogen, and for immunohistochemical study, the tissues were fixed in Bouin’s fluid. Approval from the Animal Experiments Committee in Olsztyn, Poland, was obtained before the start of any experiments. 2.2. Purification of the single-domain Kazal-type inhibitor from turkey seminal plasma The isolation procedure of the Kazal-type inhibitor was
ska et al. [8]. performed according to the study by S1owin Briefly, virgin and modified forms of the Kazal-type inhibitor were purified by affinity chromatography using methylchymotrypsin-Sepharose 4B. Both forms were separated by ion-exchange chromatography conducted on a column X/K 16/10 of HiLoad Q-Sepharose (GE Healthcare, Uppsala, Sweden). Final purification of the virgin form of the Kazal-type inhibitor was obtained by reverse phase chromatography conducted on a column of BioBasic-8 (Thermo Electron Comparison, Runcorn, England). Reverse phase chromatography produced pure fractions of the virgin form of the Kazal-type inhibitor, which were used for production of polyclonal antibodies against Kazal-type inhibitors and for preparation of affinity chromatography with the Kazaltype inhibitor as a ligand. 2.3. Production and purification of polyclonal antibodies against the single-domain Kazal-type inhibitor Immunization of rabbits and purification of anti–Kazaltype inhibitor IgG were performed according to the study
ska et al. [11]. Rabbit was immunized with 0.5 mg by S1owin of a Kazal-type inhibitor, and the second and third injections with the same Kazal-type inhibitor preparation were given 2 and 4 weeks later. The fractions containing total IgGs were pooled and were designated for isolation of anti–Kazal-type inhibitor monospecific antibodies. 2.4. Isolation of monospecific antibodies against the Kazaltype inhibitor The isolation procedure for anti–Kazal-type inhibitor monospecific antibodies was to couple the Kazal-type inhibitor to the N-hydroxy-succinimide ester (NHS)activated high performance (HP) column and to use this column for affinity purification of anti–Kazal-type inhibitor monospecific IgGs. 2.4.1. Coupling of the single-domain Kazal-type inhibitor to NHS-activated HP column Purified Kazal-type inhibitor (3 mg) was lyophilized, diluted with the standard coupling buffer (0.2 M NaHCO3 and 0.5 M NaCl, pH 8.3), and applied to NHS-activated HP column (GE Healthcare). Any extra active NHS groups were deactivated by sequential washing with 0.5 M ethanolamine and 0.5 M NaCl (pH 8.3) and then 0.1 M acetate and 0.5 M NaCl (pH 4.0). The coupled Kazal-type inhibitor was


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used as a ligand for affinity purification of anti–Kazal-type inhibitor monospecific IgGs and affinity chromatography of turkey seminal plasma proteinases. 2.4.2. Isolation procedure The protein A purified total IgGs (see mentioned earlier) were diluted 1:1 with 100 mM Tris-HCl and 0.5 M NaCl (pH 7.6) and were applied to the Kazal-type inhibitor coupled to the NHS-activated column. The unbound IgGs were washed with 50 mM Tris-HCl and 0.15 M NaCl (pH 7.6). The bound anti–Kazal-type monospecific IgGs were eluted with 0.5 M acetic acid (pH 3.4) and were immediately neutralized by the addition of 1 M Tris-HCl (pH 9.0). The anti–Kazal-type inhibitor monospecific IgGs were used for the Western blot analyses and the immunoaffinity and immunohistochemical studies. 2.5. Affinity chromatography of seminal plasma proteins using the single-domain Kazal-type inhibitor as a ligand Turkey seminal plasma (3 mL) was diluted 1:1 with binding buffer (0.05 M Tris-HCl and 0.15 M NaCl, pH 7.6) and was applied to the NHS-activated column with the Kazal-type inhibitor (2.4.1.). The column was equilibrated with the binding buffer at a flow rate of 0.25 mL/min. The bound proteins were eluted from the column with elution buffer (100 mM glycine-HCl and 0.5 M NaCl, pH 2.0), collected into 1 mL fractions, and immediately neutralized by the addition of 200 mL of 1 M Tris-HCl (pH 9.0). The fractions obtained after four separations were pooled and concentrated to 200 mL with Amicon ultra (cutoff 3 kDa; Millipore, Billerica, MA, USA). 2.6. Affinity chromatography of seminal plasma proteins using anti–Kazal-type inhibitor monospecific antibodies as ligands 2.6.1. Coupling of anti–Kazal-type inhibitor monospecific IgGs to NHS-activated HP column The fractions containing anti–Kazal-type monospecific antibodies (2 mg) were dialyzed overnight against 0.02 M NaHCO3, lyophilized, and diluted with the standard coupling buffer (0.2 M NaHCO3 and 0.5 M NaCl, pH 8.3) and were applied to the NHS-activated HP column (GE Healthcare). 2.6.2. Immunoaffinity with anti–Kazal-type inhibitor monospecific IgGs Immunoaffinity with anti–Kazal-type inhibitor monospecific IgGs was performed according to the study by
ska et al. [9]. S1owin 2.7. Western blot analysis of purified the single-domain Kazal-type inhibitor and seminal plasma with anti–Kazal-type inhibitor monospecific antibodies The pure Kazal-type inhibitor (10 mg of protein) and 40 mg of seminal plasma protein were applied to separating 12.5% acrylamide SDS-PAGE gels under nonreducing conditions according to the method of Laemmli [13] and using

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an SE 250 vertical Mighty Small electrophoresis system (GE Healthcare). For better visualization of the seminal plasma Kazal-type inhibitors, most seminal plasma proteins were precipitated with 0.45 M HClO4 [14]. After 1 hour, the denatured proteins were removed by centrifugation. To remove the perchloric acid, the supernatant was neutralized with 10 mM KOH at 4  C overnight, and the crystals of potassium perchlorate were filtered, and the supernatant was concentrated 10-fold using Amicon ultra (cutoff 3 kDa; Millipore). The concentrated supernatant (20 mg of protein) was applied to SDS-PAGE gels. The
ska Western blot was performed as described by S1owin et al. [11]. The monospecific antibodies against the Kazaltype inhibitor were diluted with Tris-buffered saline (TBS)-T (0.05 M Tris-HCl, 0.15 M NaCl, and 0.1% Tween 20, pH 7.6) at a ratio of 1:250. 2.8. Analytical methods The protein concentration was measured with the method of Lowry et al. [15] and Bradford [16] with Sigma-Aldrich reagents and with bovine serum albumin as the standard.

2.9. Two-dimensional gel electrophoresis 2.9.1. Fraction bound to affinity chromatography Sample preparation, isoelectric focusing, and the second dimension electrophoresis were performed under the same
ska et al. [9]. Approxiconditions as described by S1owin mately 100 mg of protein bound to the Kazal-type inhibitor and 30 mg of protein bound to anti–Kazal-type inhibitor IgGs were used for two-dimensional gel electrophoresis (2DE). Using the immunoaffinity column with anti–Kazaltype monospecific antibodies, we observed that the yield of the column significantly decreased, especially for greater than 20 separations (10 chromatographic separations for 2DE and 10 separations for transfer). Consequently, we had to use a lower protein concentration of protein bound to anti–Kazal-type inhibitor IgGs in our experiment. 2.9.2. Turkey seminal plasma Isoelectric focusing (IEF) was performed with an immobiline DryStrip gel of 24 cm (pH 3–10 NL; GE Healthcare) that was rehydrated overnight at 20  C with a 450-mL protein sample (600 mg of protein) in the rehydration buffer (see mentioned earlier). The IEF was performed at 20  C with step 1 at 500 V for 3.5 hours, step 2 at 1000 V for 1 hour in gradient, step 3 at 8000 V for 3.0 hours in gradient, and step 4 at 8000 V for 5.5 hours (a total focusing time of 60,800 Vh). After equilibration (see mentioned earlier), the second dimension electrophoresis was performed on 12.5% SDS polyacrylamide gels (DALT Gel; a precast polyacrylamide gel; GE Healthcare). The gel was run at 1 W per gel for the first 1 hour and then at 17 W per gel for approximately 4.5 hours until the day front reached the bottom of the gel. The proteins were visible with Coomassie Brilliant Blue G-250 (see mentioned earlier). The visible spots were cut from the gel and were subjected to ingel trypsin digestion and mass spectrometry for identification. Our previous study [17] focused on the identification of

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protein spots of turkey seminal plasma after 2-DE. Therefore, we were able to select correct spots of HGFA for identification.

Screening Tool was used for mapping the HGFA modifications on the 2-D gels [21].

2.10. Detection of phosphospecific proteins in turkey seminal plasma

2.12. Inhibition of HGFA by Kazal-type inhibitor

The protein samples of turkey seminal plasma (100 mg of protein) in the rehydration buffer were loaded onto immobiline DryStrip gel (7 cm, pH 4–7, linear). Then, IEF and the second dimension electrophoresis were performed under
ska et al. [9]. the same conditions as described by S1owin After electrophoresis, the gels were treated with ProQ Diamond phosphoprotein gel stain according to the manufacturer’s recommendations (Molecular Probes, Eugene, OR, USA) and scanned with a Typhoon FLA 9500 from GE Healthcare (excitation at 535 nm). After scanning, the gels were treated with Coomassie Blue, and the visible spots were cut from the gel and were subjected to in-gel trypsin digestion and mass spectrometry for identification. 2.11. Protein identification 2.11.1. Matrix assisted laser desorption/ionization time of flight/ time of flight mass spectrometry protein identification The visible spots were cut from the gel and were subjected to in-gel trypsin digestion using sequencing-grade modified trypsin (Promega, Madison, WI, USA). Desalting was conducted with ZipTip’s mC18 [18] (Millipore). Peptides were eluted with 70% acetonitrile (ACN, Merck, Darmstadt, Germany), dried, and stored at 80  C. Mass spectrometry ana
ska lyses were performed according to the study by S1owin et al. [9] using a matrix-assisted laser desorption/ionization (MALDI) with the autoflex speed time-of-flight (TOF/TOF) mass spectrometer equipped with a smartbeam II laser (355 nm; Bruker Daltonics, Bremen, Germany). 2.11.2. Determination of N-terminal sequence Immediately after 2-DE, spots were electroblotted onto a PVDF membrane using 10 mM 3-(cyclohexylamino)-1propanesulfonic acid-NaOH (pH 11.0) containing 10% methanol. The membrane was stained in 0.1% Coomassie Brilliant Blue R-250 in 40% methanol and 1% acetic acid and destained in 50% methanol. Because of the low intensity of spots 7, 8, and 9 (Supplementary Fig. 3), it was impossible to obtain the Nterminal sequence for an individual spot. On the basis of the same mass spectrum of analyzed spots 7, 8, and 9, we decided to combine all spots for the N-terminal protein sequence. N-terminal protein sequence analysis was performed at BioCentrum Ltd. (Krakow, Poland). The sequentially detached phenylthiohydantoin derivatives of amino acids were identified using the Procise 491 (Applied Biosystems, Foster City, CA, USA) 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). The posttranslational modifications (PTMs), phosphorylation sites, and tyrosine nitration sites were predicted using the NetPhos 2.0 Server http://www.cbs.dtu.dk/services/ NetPhos/ [19] and the GPS-YNO2 Server http://yno2. biocuckoo.org/ [20], respectively. Protein Modification

2.12.1. Isolation of HGFA from turkey seminal plasma Hepatocyte growth factor activator was purified by affinity chromatography conducted on a column HiTrap Benzamidine FF (GE Healthcare) according to the study by Holsberger et al. [3]. Seminal plasma was dialyzed overnight against 25 mM imidazole-HCl, 0.1 M NaCl (pH 6.8), and 2 mL of dialyzed seminal plasma was loaded onto column. Hepatocyte growth factor activator was eluted with 10 mM ammonium formate, 0.1 M NaCl (pH 3), and designated for inhibition assay. The eluted samples were immediately neutralized with 1 M Tris-HCl (pH 9.0). 2.12.2. Inhibition assay The Kazal-type inhibitor activity against HGFA was determined in 50 mM Tris-HCl, pH 8.3 at 25  C. Increasing amounts of the Kazal-type inhibitor from 3.0  106 M to 1.2  104 M were added to a constant HGFA concentration and after 5 minutes of incubation the residual enzyme activity was measured using a turnover of the substrate Nabenzoyl-DL-arginine-p-nitroanilide (Sigma-Aldrich), whose final concentration in the reaction medium was 1 mM. Kinetic measurements were done at 405 nm for 210 seconds. Each reaction was performed in triplicate. 2.13. Ribonucleic acid isolation and real-time PCR To determine KAZAL mRNA expression in the testis, epididymis, and ductus deferens, real-time PCR was per
ska et al. [9]. The formed according to the study by S1owin primer sequences used to identify KAZAL and GAPDH (housekeeping gene) were as follows: 50 TTCTGCTGCTCGTTGTCCTC-30 (sense) and 50 -ATCAGTCCCACAGACTGGGT-30 (antisense) for KAZAL (131 bp), and 50 -GATCCCTTCATCGACCTGAA-30 (sense) and 50 -CATCTGCCCACTTGATGTTG-30 (antisense) for GAPDH (166 bp). The primers were designed based on gene sequences listed at the National Center for Biotechnology Information using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primerblast). The total reaction volume for PCR contained 1 mL of complementary DNA (50 ng), 0.4 mL of (200 nM) forward and reverse primers each, 3 mL of nuclease-free water, 5 mL of KAPA SYBR Fast qPCR master mix, and 0.2 mL of ROX Low reference dye (KAPA Biosystems, Wilmington, MA, USA) in a final volume of 10 mL. Real-time PCR was performed for 40 cycles at 95  C for 20 seconds, at 95  C for 2 seconds, and at 60  C for 20 seconds. To ensure proper amplification of the single-product after each PCR reaction, melting curves were obtained by stepwise increases in temperature. The data obtained by the real-time PCR for the Kazal-type inhibitor were normalized on the basis of GAPDH mRNA content. The data were analyzed using the Miner method [22]. Analysis of the expression of Kazal-type inhibitor mRNA in the turkey reproductive tract and liver was conducted together with the analysis of expression of ovoinhibitor mRNA [9].


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Table 1 Identification of proteins bound to the affinity column with the Kazal-type inhibitor as a ligand. Spot

Protein name

Accession

Theoretical Protein molecular weight

Protein isoelectric point

1–13

Hepatocyte growth factor activator, partial (Meleagris gallopavo) PREDICTED: hepatocyte growth factor activator (M gallopavo) Serum albumin-like (M gallopavo) Immunoglobulin light chain V-J-C region, partial (M gallopavo) PREDICTED: cysteine-rich secretory protein 3-like (M gallopavo)

AAO46038

63,172

7.45

XP003205725 AFA52547 XP003204693

71,871 23,065 29,625

6.66 5.89 6.29

14 15–19a 19a a

Spot no. 19 space was identified as immunoglobulin and cysteine-rich secretory protein.

The analyses were performed at the same time with the same tissue, but with isolated RNA and kits. 2.14. Immunohistochemical detection of the single-domain Kazal-type inhibitor in turkey reproductive tissues and liver Small fragments of testis, epididymis, ductus deferens, and liver were fixed in Bouin’s fluid (saturated picric acid, formaldehyde, and glacial acetic acid at 15:5:1 proportions, respectively) for 24 hours as described recently [9]. The procedures used for immunohistochemistry were similar to
ska et al. [23] and Nynca those described previously by Bilin et al. [24]. Briefly, to optimize immunohistochemical staining, tissue slices were immersed in citrate buffer (10 mM, pH 6.0) and were heated in a microwave oven (8 minutes, 700 W). Nonspecific staining was blocked twice; the first was with 3% hydrogen peroxide (H2O2) in methanol for 15 minutes to inhibit endogenous peroxidase activity, and the second was with 5% normal goat serum for 30 minutes at room temperature to block nonspecific binding sites. The sections were incubated overnight at 4  C in a humidified chamber in the presence of turkey seminal plasma antibodies against the Kazal-type inhibitor (dilution 1:2000). Subsequently, they were incubated with a biotinylated secondary antibody, goat anti-rabbit IgG (1:400; Vector Laboratories, Burlingame, CA, USA) for 60 minutes. After each step in these procedures, the sections were carefully rinsed with TBS (0.05 M Tris-HCl and 0.15 M NaCl, pH 7.6). The staining was developed using avidin biotinylated–horseradish peroxidase complex (1:100, Vectastain Elite ABC Reagent; Vector Laboratories) for 30 minutes. The bound antibodies were made visible by placing tissues in 0.05% 3,30 -diaminobenzidine tetrachloride (Sigma-Aldrich) in TBS containing 0.01% H2O2 and 0.07% imidazole for 6 minutes. The sections were washed and counterstained with Mayer’s hematoxylin, dehydrated, and mounted with DPX

(a mixture of distyrene, a plasticizer, and xylene) mounting media (Sigma-Aldrich). All slides were processed immunohistochemically simultaneously with the same treatment so that staining intensity could be compared among different sections [25]. The cells were immunopositive if a brown reaction product was present and appeared as a signal in reproductive tissue cells, whereas the cells without any specific immunostaining were immunonegative [26]. All immunohistochemical experiments were repeated at least three times. The control sections included omission of the primary antibody and substitution with preimmune goat serum. The sections were examined with a Leica DMR microscope (Leica Microsystems, GmbH Wetzlar, Wetzlar, Germany) with Nomarski interference contrast.

2.15. Statistical analyses The data for gene expression in the male reproductive tract were analyzed by the nonparametric Kruskal-Wallis test (a nonparametric ANOVA) followed by the Dunn’s multiple comparison test using the statistical software program GraphPad5 (GraphPad PRISM v 5.0; GraphPad Software Inc., San Diego, CA, USA). Data are expressed as the mean  standard deviation. 3. Results Western blot analysis revealed that the polyclonal antibodies cross-reacted with the purified preparation of the Kazal-type inhibitor. A single band corresponding to the virgin form of the Kazal-type inhibitor was observed (Supplementary Fig. 1). No signal was observed in the seminal plasma, when 40 mg of protein was loaded onto the gel. However, in the 10 times concentrated supernatant, after the precipitation of most seminal plasma proteins, the

Table 2 Identification of proteins bound to the immunoaffinity column with the anti–Kazal-type monospecific antibodies as ligands. Spot

Protein name

Accession

1 2–4 5a 5a–6 7–9

Kazal-type serine proteinase inhibitor precursor (Meleagris gallopavo) Immunoglobulin light chain V-J-C region, partial (M gallopavo) Proacrosin (M gallopavo) PREDICTED: cysteine-rich secretory protein 3-like (M gallopavo) Hepatocyte growth factor activator, partial (M gallopavo)

CAI46283 AFA52547 CAJ45027 XP003204693 AAO46038

a

Spot no. 5 was identified as proacrosin and cysteine-rich secretory protein.

Theoretical Protein molecular weight

Protein isoelectric point

9045 23,065 38,724 29,625 63,172

7.53 5.89 7.60 6.29 7.45


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Table 3 Identification of the proteoforms of hepatocyte growth factor activator in turkey seminal plasma. Spot Protein accession, description

1

Sequence Mascot Calculated Precursor mass Peptide Peptide sequence coverage (%) score isoelectric point score Observed Theoretical

gij28631143, hepatocyte growth factor 22 activator (Melaegris gallopavo)

276

*gij326919485, PREDICTED: hepatocyte growth factor activator (M gallopavo)

7.45

2481.3561 2480.2046 99 1072.5690 1071.4920 43 2561.2396 2560.0774 70

K.SQFVQPICLPESNTVFPDQFK.C K.CQISGWGHK.H R.SPEIYGTEISENMFCAGYFDSK.S

6.96

2

21

328

3

19

201

4

21

375

5

14

252

6

13

359

7

20

419

2481.2145 2545.0921 1033.5760 1406.6958a 2481.1358 1033.5352 1407.6540a 2566.4276 2481.2634 2545.1333 1033.6051 1407.7358a 2566.4741 2481.3184 1072.5537 1033.6311 1783.7509 1627.6524 1632.7057 1467.7291 1840.7709

2480.2046 2544.0825 1032.5716 1406.6942a 2480.2046 1032.5716 1406.6942a 2565.3631 2480.2046 2544.0825 1032.5716 1406.6942a 2565.3631 2480.2046 1071.4920 1032.5716 1782.7566 1626.6555 1631.7085 1466.7340 1839.7807

117 129 44 93a 123 48 89a 48 103 96 70 90a 40 108 37 38 77 36 60 64 72

1783.7482 1627.6515 1632.7053 2120.0097 1467.7290 1824.7753 1840.7743

1782.7566 1626.6555 1631.7085 2119.0157 1466.7340 1823.7858 1839.7807

41 55 68 56 69 46 81

K.SQFVQPICLPESNTVFPDQFK.C R.SPEIYGTEISENMFCAGYFDSK. R.VNKPGVYTR. R.VTNYVNWINER.Ia K.SQFVQPICLPESNTVFPDQFK.C R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.YILYPQYSVFRPTEHDIALIK.L K.SQFVQPICLPESNTVFPDQFK.C ( R.SPEIYGTEISENMFCAGYFDSK.S R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.YILYPQYSVFRPTEHDIALIK.L K.SQFVQPICLPESNTVFPDQFK.C K.CQISGWGHK.H R.VNKPGVYTR.V R.RTYHCACPEEFTGR.D R.TYHCACPEEFTGR.D K.YCNIVPNHHCYR.G K.AVQLGLGPFSYCR.N R.NPDEDEKPWCYIMK.D Oxidation (M) R.RTYHCACPEEFTGR.D R.TYHCACPEEFTGR.D K.YCNIVPNHHCYR.G K.TTISGHSCLPWNSDLLYR.E K.AVQLGLGPFSYCR.N R.NPDEDEKPWCYIMK.D R.NPDEDEKPWCYIMK.D Oxidation (M)

a Peptide of HGFA with a theoretical molecular weight of 1406.6942 Da was identified using hepatocyte growth factor activator, predicted sequence (accession no XP003206011).

monospecific antibodies recognized the two forms of Kazaltype inhibitors (virgin and modified, Supplementary Fig. 1). The predominant proteins bound to affinity column with the single-domain Kazal-type inhibitor as a ligand were identified as proteoforms of the HGFA (13 spots, Supplementary Fig. 2). Moreover, Ig light chain (five spots), albumin (one spot), and cysteine-rich secretory proteins (one spot) were also identified. The results of the spot identifications are reported in Table 1. Detailed information concerning the identifications is provided in Supplementary Table 1. The identification of HGFA was based on a comparative analysis of two sequences, as shown in Table 3 and Supplementary Tables 1 and 3. In the National Center for Biotechnology Information protein database, two sequences of HGFA were available: HGFA partial (accession no AAO46038 [4]) and HGFA predicted (accession no XP003206011). Although, the predicted sequence was incomplete, it allowed for the identification of one additional peptide of HGFA with a theoretical molecular weight of 1406.6942 Da with the following sequence: R.VTNYVNWINER.I (Tables 3, Supplementary Tables 1 and 3). The Kazal-type inhibitor (one spot) and proacrosin (one spot) were identified in the fraction bound to immunoaffinity column. Moreover, three spots were identified as Ig light chain and two as cysteine-rich secretory proteins (Table 2,

Supplementary Fig. 3). Detailed information concerning the identification is provided in Supplementary Table 2. Spot nos. 7, 8, and 9 (Supplementary Fig. 3) were not identified by MALDI. However, the N-terminal Edman sequencing of the combined spots allowed for the identification of 12 amino acids, IIGGSSSLPGSH, located in the internal part of HGFA (Supplementary Fig. 4). HGFA activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner (Fig. 1). The highest inhibition rate was observed at inhibitor concentrations of 0 to 0.36  104 M (0%–70% inhibition of HGFA). Higher inhibitor concentrations from 0.36 to 0.60  104 M caused 75% to 84% inhibition of HGFA. The further increase in inhibitor concentration to 1.2  104 M increased inhibition by 6%, and 90% inhibition of HGFA was observed (Fig. 1). Up to five proteoforms of HGFA in seminal plasma were identified with six peptides located at positions 371 to 391, 403 to 423, 425 to 432, 458 to 479, 514 to 522, and 523 to 533 (Table 3, Supplementary Figs. 4 and 5). Moreover, two proteoforms of lower molecular weight were identified also as HGFA with another six peptides located at positions 75 to 88, 76 to 88, 170 to 181, 193 to 210, 220 to 232, and 233 to 246 (Table 3, Supplementary Figs. 4 and 5). The ProQ Diamond phosphoprotein gel staining allowed the detection of phosphospecific proteins in turkey


ska et al. / Theriogenology xxx (2015) 1–12 M. Słowin

Fig. 1. Inhibition of HGFA activity by the Kazal-type inhibitor. HGFA, hepatocyte growth factor activator.

seminal plasma. Four phosphospecific spots were identified as HGFA in the turkey seminal plasma (Supplementary Fig. 6, Supplementary Table 3). The Kazal-type inhibitor mRNA transcripts were detected in the turkey reproductive tract and liver. The expression was higher by 188- and 145-fold in the epididymis and the ductus deferens than in the liver, respectively (Fig. 2). The immunohistochemical study revealed positive signals of various intensities for the Kazal-type inhibitor in the turkey testis, epididymis, ductus deferens, and liver (Fig. 3A–D). In the testis, a positive signal for the Kazal-type inhibitor was localized in spermatocytes and round and elongating spermatids (Fig. 3A), and the staining was always localized in the cell cytoplasm. In elongating spermatids, the staining was primarily localized in the cytoplasmic droplets. Sporadically, such droplets were observed also in testicular sperm flagella (Fig. 3A). The intensity of the staining appeared to be correlated with a degree of germ cell differentiation, with staining moderate in spermatocytes and strong to very strong in round and elongating spermatids. By contrast, spermatogonia and mature sperm (without cytoplasmic droplets) were

Fig. 2. Expression of the single domain Kazal-type inhibitor mRNA in turkey reproductive tract tissues and liver. Data represent the mean values  standard error of the mean. Different letters (a,b) indicate statistical significance at P  0.05.

7

immunonegative. Additionally, no positive signal was detected in somatic testicular cells (Sertoli cells, Leydig cells, and myoid cells). In the epithelium of the epididymis, a strong signal for the Kazal-type inhibitor was present throughout the cytoplasm of nonciliated columnar cells (Fig. 3B), and basal cells displayed a weak staining for the Kazal-type inhibitor. In the lumen, positively stained cell cytoplasmic protrusions (i.e., loosened sperm cytoplasmic droplets) were observed (Fig. 3B). No positive signals were observed in ciliated cells or the numerous microvilli that projected from nonciliated cell surfaces into the ductal lumen (bottom insert in Fig. 3B). Additionally, the connective tissues displayed no immunoreaction (Fig. 3B). In the pseudostratified columnar epithelium of the ductus deferens, a strong to very strong staining for the Kazal-type inhibitor was observed primarily within the cell cytoplasm of columnar epithelial cells and basal cells (Fig. 3C). No positive staining was observed in the connective tissues (Fig. 3C). In the liver cells, the positive signal for the Kazaltype inhibitor was of strong intensity and was confined to a few hepatocytes in which the nuclei were strongly stained by hematoxylin (Fig. 3D). In the control sections, in which the primary antibody was omitted and replaced with preimmune serum, no immunopositive signals for the Kazal-type inhibitor were observed (inserts in Fig. 3A–D). 4. Discussion The present study is the first to report on the identification of target serine proteinases for the single-domain Kazal-type inhibitors in turkey seminal plasma. The serine proteinases, HGFA and proacrosin, were identified from the binding fractions to the Kazal-type inhibitors and anti–Kazal-type inhibitor monospecific antibodies. HGFA was present in several phosphorylated proteoforms. The single-domain Kazal-type inhibitor mRNA expression was detected in the turkey reproductive tract and was 150-fold higher than in the liver. The immunohistochemical study revealed the presence of Kazal-type inhibitors in the testicular spermatocytes and spermatids and the epithelia of the epididymis and the ductus deferens. These results are important for better understanding the mechanism of the proteolytic processes within the male reproductive tract. Studies on protease inhibitors in semen have focused mostly on the isolation and characterization of these inhibitors [8,24,27,28]; however, the physiological roles were not determined. So far, the only role assigned to the turkey seminal plasma single-domain Kazal-type inhibitor was the regulation of acrosin activity [10,11]. We found the single-domain Kazal-type inhibitor targeted HGFA, a primary proteinase in turkey seminal plasma. HGFA is a serine proteinase identified in turkey seminal plasma previously by Holsberger et al. [3,4]. In mammals, HGFA is the key enzyme that regulates the activity of hepatocyte growth factor (HGF), which is involved in the regeneration of injured tissues [29–31]. Hepatocyte growth factor has mitogenic, morphogenic, and motogenic effects in a variety of processes, which include embryogenesis, organogenesis, and angiogenesis, as well as tissue remodeling and repair

8


ska et al. / Theriogenology xxx (2015) 1–12 M. Słowin

Fig. 3. Immunohistochemical localization of single domain Kazal-type inhibitor in the turkey testis (A), epididymis (B), ductus deferens (C), and liver (D). Counterstaining with Mayer’s hematoxylin. Nomarski interference contrast. Bars ¼ 10 mm. (A) Positive signals for the Kazal-type inhibitor are localized to spermatocytes (small arrows), round spermatids (arrows), elongating spermatids (double arrowheads), and to sperm possessing cytoplasmic droplets on their flagellas (open arrows). For details see higher magnification (bottom image). No immunopositive reaction is visible in spermatogonia (arrowheads), spermatozoa without cytoplasmic droplet (double open arrows), Leydig cells (LC), Sertoli cells (SC), and myoid cells (cross). (B) Positive signals for the Kazal-type inhibitor are localized to nonciliated columnar cells (arrows), basal cells (small arrows) of the epididymal region, cytoplasmic protrusions in the lumen (asterisks), and connective tissue (crosses). Spermatozoa without cytoplasmic droplet (double open arrows), ciliated cells (double arrowheads), and microvilli projected from nonciliated cell surfaces (arrowheads) are immunonegative. For details see higher magnification (bottom image). (C) Positive signal for the Kazal-type inhibitor is localized to columnar epithelial cells (arrows), basal cells (small arrows). For details see higher magnification (bottom image). Spermatozoa without cytoplasmic droplet (double open arrow) and connective tissue (crosses) are immunonegative. (D) Positive signal for the Kazal-type inhibitor is localized to a few hepatocytes (arrows). No immunopositive signals for the Kazal-type inhibitor are observed in the testis, epididymis, ductus deferens, and liver when the primary antibody is omitted (inserts in A, B, C, D).

[32–38]. For male reproduction, potential roles of HGF in the testis have been postulated for mammals: (1) seminiferous cord formation and development, (2) Leydig cell steroidogenesis and survival, (3) Sertoli cell proliferation and terminal differentiation, and (4) germ cell function and survival [38]. Although the presence of HGF within bird semen has yet to be reported, the presence of HGFA strongly suggested that HGF must be present within the turkey reproductive tract. This needs to be experimentally confirmed, and the roles of the HGFA system in the turkey reproductive system require further study.

The nucleotide sequence of HGFA complementary DNA obtained by Holsberger [4] shows that the HGFA of turkey semen contains 558 amino acids with a calculated molecular mass of 61.38 kDa. The HGFA purified from turkey seminal plasma had a molecular mass of 37 kDa [3]. It was likely that the differences in molecular masses resulted from proteolytic cleavage of HGFA precursors during the activation process, which has been described for human HGFA [39]. The human HGFA precursor of 655 amino acids was cleaved in vitro by thrombin at the bond between Arg407 and Ile408, and the short form of the active HGFA,


ska et al. / Theriogenology xxx (2015) 1–12 M. Słowin

constituting primarily the protease domain, was derived from the COOH-terminal region of a precursor [39]. Our results strongly suggested that the HGFA bound to affinity column represented an active form of HGFA, which was cleaved at position Arg296-Ile297. This suggestion was supported by (1) the N-terminal sequence of HGFA bound to anti–Kazal-type inhibitor IgG started exactly at Ile297 (Supplementary Fig. 4), and (2) the HGFA bound to the affinity column with the Kazal-type inhibitor was identified by MALDI TOF/TOF with six peptides located at the COOHterminal region of the precursor (at positions behind the cleavage site: 371–391, 403–423, 425–432, 458–479, 514– 522, and 523–533; Supplementary Table 1, Supplementary Fig. 4). In summary, activation of the HGFA precursor of turkey semen appeared to be similar to that of human HGFA with cleavage between Arg and Ile. Because we observed neither the precursor form of HGFA nor the Nterminal part of HGFA precursor in the fraction bound to the affinity column, it was assumed that the single-domain Kazal-type inhibitor of turkey seminal plasma was involved in the control of the active form of HGFA. Our results clearly demonstrated that both products of HGFA precursor activation were present in turkey seminal plasma. Using 2-DE electrophoresis and MALDI TOF/TOF, we identified the active form of HGFA in turkey seminal plasma with six peptides at the C-terminal region of the precursor (located behind the cleavage site Arg296-Ile297, as described previously; Supplementary Fig. 4, Supplementary Tables 1 and 3). Moreover, we were able to identify the N-terminal part of the HGFA precursor, which was likely cutoff during activation. This identification was based on the six peptides located at the N-terminal region of HGFA precursor (before the Arg296-Ile297 cleavage site; Supplementary Fig. 4, Table 3). In our study, we were not able to detect the precursor form of HGFA in turkey seminal plasma, and this suggested that HGFA precursor was not present in seminal plasma or was present in concentrations less than detection. Holsberger et al. [3] supported the latter suggestion, and they observed minor bands of 61 kDa after affinity chromatography, which likely represented the precursor form of HGFA. The presence of HGFA mostly in active forms in semen strongly suggested that activation of the precursor occurred earlier within the reproductive tract, presumably in the ductus deferens. The ductus deferens of turkey is a site of intense proteolytic activity [1,2], and it is in this part of the reproductive tract that expression of mRNA for HGFA was found [4]. Moreover, both the distal portions of the efferent duct and the deferent duct were immunoreactive against the active form of HGFA [3]. Kot1owska et al. [2] recorded the appearance of additional serine proteinases of molecular weight in the range of 31 to 40 kDa (which might correspond to active HGFA) in the ductus deferens. In summary, our results and those of Holsberger et al. [3,4]) strongly suggest that the expression, secretion, and activation of HGFA occurred in the ductus deferens, the primary storage location for turkey sperm [40]. The mechanism of HGFA activation in turkey seminal plasma is presently unknown. Thrombin is known to activate human plasma HGFA precursor by cleavage at the bond between Arg407 and Ile408 [39]. However, thrombin

9

has yet to be identified in bird semen. Numerous serine proteinases of molecular weight ranging from 29 to 88 kDa were observed in turkey seminal plasma [2]. It is possible that one of those is responsible for the conversion of the precursor to active HGFA in turkey semen. Future studies should focus on the identification of these serine proteinases and the determination of whether thrombin is present in turkey seminal plasma. Our results clearly demonstrated that HGFA of turkey seminal plasma was represented by numerous proteoforms (the different molecular forms of a protein [41]). Up to nine proteoforms of active HGFA were bound to the Kazal-type inhibitor, which was possible to demonstrate because of protein concentration after affinity chromatography. Our results extend the findings of Holsberger et al. [3] who obtained one band of HGFA after 1-DE. The proteoforms of HGFA were observed at a pI range from 5.5 to 7.0 (Supplementary Figs. 2 and 3). Proteins with many PTMs can be observed as distinct protein proteoform spots on 2-DE gels, as a result of the following modifications: phosphorylation, acetylation, alkylation, cysteine oxidation, or tyrosine nitration [21]. From the analysis of the active HGFA sequence, many potential phosphorylation sites (six serine, three threonine, and five tyrosine) and one tyrosine nitration site were predicted (Supplementary Fig. 4). Using Protein Modification Screening Tool [21], we predicted phosphorylated HGFA and obtained from 1 to 10 phosphorylated HGFA forms (Supplementary Fig. 7), which agreed quite well with our experimental data (13 spots at pI 5.5–7.0 were identified as HGFA). Moreover, we obtained direct evidence for phosphorylation of HGFA because four phosphospecific spots were identified as active HGFA in turkey seminal plasma. In summary, phosphorylation was the primary PTM of HGFA; however, further studies are necessary to identify the specific phosphorylation sites of HGFA in turkey seminal plasma. In our study, we noticed a discrepancy between the identifications of HGFA after affinity chromatography with Kazal-type inhibitors and anti–Kazal-type inhibitor monospecific antibodies. The active form of HGFA was clearly identified by MALDI TOF/TOF in the fraction bound to the affinity column with the Kazal-type inhibitor as a ligand and in turkey seminal plasma, as well. However, the HGFA in the fraction bound to anti–Kazal-type monospecific antibodies was not successfully identified by MALDI; the mass spectrum was completely different than for the identified HGFA. Only the N-terminal sequence allowed for the identification because of the same 12 amino acids as in the identified HGFA. This lack of identification of HGFA in the fraction bound to the IgGs is difficult to explain at present. Perhaps after Kazal-HGFA complexes dissociated, the modified structure of HGFA prevented successful identification by MALDI. Further studies are necessary to identify the differences between these forms of HGFA. The activity of human HGFA is inhibited by naturally occurring serine proteinase inhibitors belonging to different classes, such as the Kunitz domain inhibitors HAI1 (HGFA inhibitor type 1) and HAI-2 (HGFA inhibitor type 2) and the serpin protein C inhibitor (serpin A5) [42–44]. For the first time, we found the single-domain Kazal-type inhibitor was also an HGFA inhibitor. Moreover, in our recent

10


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study, we demonstrated that HGFA was controlled by an ovoinhibitor, which was a multidomain Kazal-type inhibitor [9]. Inhibition of HGFA by Kazal-type inhibitors could be characteristic of poultry semen. Further studies are necessary to test this hypothesis. The single-domain Kazal-type inhibitors present in the male reproductive tract are termed acrosin inhibitors [45]). The postulated physiological functions of the Kazaltype inhibitor of turkey semen are the inactivation of acrosin released from dead or damaged spermatozoa and control of the activation of the proacrosin-acrosin system [10,11]. This postulation is primarily supported by in vitro study [10,11]. We obtained additional evidence that acrosin was controlled by the single-domain Kazal-type inhibitors because acrosin was identified only in the fraction binding to the affinity column with anti–Kazal-type inhibitor IgG as a ligand (Supplementary Fig. 3, Table 2). This confirmed that in vivo Kazal-type inhibitors formed complexes with acrosin, which were bound to the IgGs. Notably, no acrosin was identified in the fraction binding to the affinity column with Kazal-type inhibitors as ligands, which strongly suggested that in turkey seminal plasma acrosin was present only in complexes with Kazaltype inhibitors and not as a free enzyme. It should be underlined that control of acrosin activity seems to be specific for the Kazal-type inhibitor only, because previously lack of acrosin inhibition by ovoinhibitor was demonstrated [9]. In summary, the Kazal-type inhibitors seem to be efficient in neutralizing the acrosin activity of spermatozoa. The Kazal-type inhibitor may also be responsible for controlling the process of proacrosin activation that occurs during acrosome reaction. This suggestion needs to be tested in the further studies. The Kazal-type inhibitor and ovoinhibitor are the main serine proteinase inhibitors in turkey seminal plasma. Activities of both inhibitors were detected in the reproductive tract fluids [2]. However, their participation in the regulation of proteolytic activity of turkey reproductive tract is still to be determined. In this study, we demonstrated for the first time that the single-domain Kazal-type inhibitor was expressed in the turkey reproductive tract, which was similar to the ovoinhibitor [9]. However, different expression profiles were observed for the single-domain Kazal-type inhibitor and the ovoinhibitor. The Kazal-type inhibitor mRNA expression was higher by 99-, 52-, and 14-fold than the ovoinhibitor in the testis, epididymis, and ductus deferens, respectively. By contrast, the ovoinhibitor mRNA expression was higher by 151-fold than the Kazaltype inhibitor in the liver. This result was supported by the presence of Kazal-type inhibitor activity in extracts from the reproductive tract and the absence of activity in the blood [2], which indicated that the single-domain Kazal-type inhibitor was specific for the reproductive tract. On the other hand, the expression of the ovoinhibitor within turkey tissues was broader and indicated its general role in turkey physiology. Immunohistochemistry revealed similar distribution patterns for the single-domain Kazal-type inhibitor and the ovoinhibitor in the turkey reproductive tract [9]. In the testis, the Kazal-type inhibitor was present in spermatocytes and in the round and elongated spermatids, whereas

epithelial cells were the primary secretion sites in the epididymis and ductus deferens. For the secretion sites, the single-domain Kazal-type inhibitors might have several possible roles. First, the germ cell-specific expression of the Kazal-type inhibitors in the testis indicated an important function in spermatogenesis, which was previously suggested for other serine proteinase inhibitors, such as turkey ovoinhibitor and rat eppin [9,46]. Second, secretion by the epithelial cells of epididymis and ductus deferens indicated that the Kazaltype inhibitor was an important factor involved in sperm membrane maturation changes in birds [47,48] and maintaining a microenvironment for sperm, primarily in the ductus deferens. Our previous study strongly suggested that the Kazal-type inhibitor actively participated in the control of proteolytic processes in the ductus deferens because the modified form of the Kazal-type inhibitor (with a split peptide bond in the reactive site after forming the enzyme-inhibitor complex [8]) was observed in ductus deferens. The Kazal-type inhibitor and HGFA collocation in the ductus deferens supported our suggestion that the turkey seminal plasma Kazal-type inhibitor could be responsible for the regulation of HGFA activity [4]. The control of proteolytic processes is important in the maintenance of the microenvironment in which sperm maturation occurs and sperm is stored [40,47–49]. 4.1. Conclusions Activity of HGFA seems to be controlled by the singledomain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma and phosphorylation was the primary PTM of HGFA. In addition to HGFA, acrosin was a target proteinase for the singledomain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and not as a free enzyme. The single-domain Kazaltype inhibitor was specific for the reproductive tract. The germ cell-specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis. The secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in changes in the sperm membrane during maturation and in the maintenance of the microenvironment in which sperm maturation occurs and sperm is stored. Acknowledgments This work was supported by a grant from the National Science Centre (N N311 525840) and funds appropriated to the Institute of Animal Reproduction and Food Research. The authors thank E. Liszewska for her excellent technical assistance and P. Hliwa for the preparation of tissue fragments for the immunohistochemical study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. theriogenology.2015.03.026.


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reproductive tract and immunolocalized in maturing spermatozoa. Mol Reprod Dev 2012;79:832–42. [47] Clulow J, Jones RC. Production, transport, maturation, storage and survival of spermatozoa in the male Japanese quail, Coturnix coturnix. J Reprod Fertil 1982;64:259–66. [48] Esponda P. Spermatozoon maturation in vertebrates with internal fertilization. Microsc Electron Biol Celular 1991;15:1–23. [49] Morris SA, Howarth Jr B, Crim JW, Rodriguez de Cordoba A, Esponda P, Bedford JM. Specificity of sperm-binding Wolffian duct proteins in the rooster and their persistence on spermatozoa in the female host glands. J Exp Zool 1987;242: 189–98.


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12.e1

Supplementary Table 1 Detailed information on the identification of proteins bound to the affinity column with the Kazal-type inhibitor as a ligand. Spot Protein accession, description

Sequence Mascot Calculated Precursor mass Peptide Peptide sequence coverage score pI score Observed Theoretical

1

21%

425

2

19%

235

3

23%

371

4

17%

366

5

15%

345

6

23%

348

7

13%

437

8

15%

412

9

17%

352

10

11%

188

11

15%

100

12

19%

440

13

11%

173

gij28631143, hepatocyte growth factor activator, partial (Melaegris gallopavo) *gij326919485, PREDICTED: hepatocyte growth factor activator (M gallopavo)

7.45 6.96

14

gij326918904, PREDICTED: serum albumin-like (M gallopavo)

50%

394

5.66

15

gij375332007, immunoglobulin light chain V-J-C region, partial (M gallopavo)

27%

246

5.89

16

24%

219

17

35%

227

2481.2140 2545.0944 1033.5872 1407.7077a 2566.4060 2481.2462 1033.5907 1407.7241a 2566.3352 2481.1730 2545.0473 1407.6837a 2566.4011 2481.2302 2545.1060 1033.6009 1407.7249a 2481.3080 2545.1971 1033.6249 1407.7650a 2566.4091 2481.2385 2545.1208 1033.5875 1407.7177a 2481.2550 2545.1359 1033.5970 1407.7281a 2566.4001 2481.2361 2545.1164 1033.5893 1407.7180a 2566.3670 2481.1986 2545.0807 1033.5629 1407.6949a 2481.2476 1033.5913 1407.7226a 1033.5685 1407.6769a 2481.2454 2545.1280 1033.5939 1407.7271a 2481.4191 1033.6528 1407.7726a 1511.8303

2480.2046 2544.0825 1032.5716 1406.6942a 2565.3631 2480.2046 1032.5716 1406.6942a 2565.3631 2480.2046 2544.0825 1406.6942a 2565.3631 2480.2046 2544.0825 1032.5716 1406.6942a 2480.2046 2544.0825 1032.5716 1406.6942a 2565.3631 2480.2046 2544.0825 1032.5716 1406.6942a 2480.2046 2544.0825 1032.5716 1406.6942a 2565.3631 2480.2046 2544.0825 1032.5716 1406.6942a 2565.3631 2480.2046 2544.0825 1032.5716 1406.6942a 2480.2046 1032.5716 1406.6942a 1032.5716 1406.6942a 2480.2046 2544.0825 1032.5716 1406.6942a 2480.2046 1032.5716 1406.6942a 1510.7966

1246.7815 926.5858 2127.3091 2442.1948 2127.0845 1246.7208 926.5686 2127.2012

1245.6717 925.5093 2126.1259 2441.2299 2126.1259 1245.6717 925.5093 2126.1259

124 206 47 94 62 95 53 91a 60 148 120 97a 79 112 66 61 94a 125 126 69 62a 93 93 59 56 85a 140 180 47 91a 76 118 138 48 93a 76 118 138 48 93a 112 65 91 83 54 117 197 76 91a 79 74 86a 61 55 46 53 35 51 127 82 119 35 46 117

K.SQFVQPICLPESNTVFPDQFK.C R.SPEIYGTEISENMFCAGYFDSK.S R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.YILYPQYSVFRPTEHDIALIK.L K.SQFVQPICLPESNTVFPDQFK.C R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.YILYPQYSVFRPTEHDIALIK.L K.SQFVQPICLPESNTVFPDQFK.C R.SPEIYGTEISENMFCAGYFDSK.S R.VTNYVNWINER.Ia K.YILYPQYSVFRPTEHDIALIK.L K.SQFVQPICLPESNTVFPDQFK.C R.SPEIYGTEISENMFCAGYFDSK.S R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.SQFVQPICLPESNTVFPDQFK.C R.SPEIYGTEISENMFCAGYFDSK.S R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.YILYPQYSVFRPTEHDIALIK.L K.SQFVQPICLPESNTVFPDQFK.C R.SPEIYGTEISENMFCAGYFDSK.S R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.SQFVQPICLPESNTVFPDQFK.C R.SPEIYGTEISENMFCAGYFDSK.S R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.YILYPQYSVFRPTEHDIALIK.L K.SQFVQPICLPESNTVFPDQFK.C R.SPEIYGTEISENMFCAGYFDSK.S R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.YILYPQYSVFRPTEHDIALIK.L K.SQFVQPICLPESNTVFPDQFK.C R.SPEIYGTEISENMFCAGYFDSK.S R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.SQFVQPICLPESNTVFPDQFK.C R.VNKPGVYTR.V R.VTNYVNWINER.Ia R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.SQFVQPICLPESNTVFPDQFK.C R.SPEIYGTEISENMFCAGYFDSK.S R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.SQFVQPICLPESNTVFPDQFK.C R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.AVAMITFAQYLQR.C R.VSLLGHFIYSVAR.R R.RHPEFSTQLILR.I R.RPCFTAMGVDTK.Y K.SPGSAPVTVIYR.D K.RPSNIPSR.F K.VAPTITLFPPSKEELDQNK.A K.SPGSAPVTVIYDNTNRPSNIPSR.F K.VAPTITLFPPSKEELDQNK.A K.SPGSAPVTVIYR.D K.RPSNIPSR.F K.VAPTITLFPPSKEELDQNK.A (continued on next page)


ska et al. / Theriogenology xxx (2015) 1–12 M. Słowin

12.e2 Supplementary Table 1 (continued ) Spot Protein accession, description

Sequence Mascot Calculated Precursor mass Peptide Peptide sequence coverage score pI score Observed Theoretical

18

39%

356

19

27%

169

19

gij326916801, PREDICTED: cysteine-rich 14% secretory protein 3-like (M gallopavo)

120

6.29

1246.6945 926.5501 2127.1916 987.5414 1028.5485 987.5310 1028.5513 1960.8519 1281.5802

1245.6717 925.5093 2126.1259 986.5145 1027.5298 986.5145 1027.5298 1959.8645 1280.5642

49 44 94 52 70 54 71 40 43

K.SPGSAPVTVIYR.D K.RPSNIPSR.F K.VAPTITLFPPSKEELDQNK.A R.KGETTPAQR.Q R.VTHDGTAVTK.T R.KGETTPAQR.Q R.VTHDGTAVTK.T R.MEWSPQAAANAQNWANR.C Oxidation (M) K.VGCAVAYCPER.T

a Peptide of HGFA with a theoretical molecular weight of 1406.6942 Da was identified using hepatocyte growth factor activator, predicted sequence (accession no XP003206011).

Supplementary Table 2 Detailed information on the identification of proteins bound to the immunoaffinity column with the anti-Kazal-type monospecific antibodies as ligands. Spot Protein accession, description

Sequence Mascot Calculated Precursor mass Peptide Peptide sequence coverage score pI score Observed Theoretical

53% gij58081895 Kazal-type serine proteinase inhibitor precursor (Meleagris gallopavo) gij375332007, immunoglobulin light 22% chain V-J-C region, partial (M gallopavo)

392

7.53

173

5.89

3 4

22% 22%

130 241

5

gij83423374 9% proacrosin (M gallopavo) gij326916801, PREDICTED: cysteine-rich 23% secretory protein 3-like (M gallopavo)

102

7.6

263

6.29

39%

413

1

2

5

6

1086.5528 2046.9280 2792.3339 1246.7270 926.5593 2127.2367 2127.1772 1246.7087 926.5463 2127.2048 1543.7702

1085.5076 2045.8670 2791.2793 1245.6717 925.5093 2126.1259 2126.1259 1245.6717 925.5093 2126.1259 1542.7889

64 118 160 34 32 79 115 47 46 124 93

K.YSHLPGCPR.D K.TYSNECVLCLSNSEENK.N K.TYSNECVLCLSNSEENKNVQIYK.S K.SPGSAPVTVIYR.D K.RPSNIPSR.F K.VAPTITLFPPSKEELDQNK.A K.VAPTITLFPPSKEELDQNK.A K.SPGSAPVTVIYR.D K.RPSNIPSR.F K.VAPTITLFPPSKEELDQNK.A R.SAAPTQTAEVLQEAK.V

1944.8400 1960.8362 1281.5672 1240.6028 1944.9591 1960.9552 1281.6436 1673.7776 1689.7718 1240.6028

1943.8696 1959.8645 1280.5642 1239.5343 1943.8696 1959.8645 1280.5642 1672.7014 1688.6963 1239.5343

77 111 44 48 125 118 44 92 77 48

R.MEWSPQAAANAQNWANR.C R.MEWSPQAAANAQNWANR.C K.VGCAVAYCPER.T K.NYGCDHSFIK.K R.MEWSPQAAANAQNWANR.C R.MEWSPQAAANAQNWANR.C Oxidation (M) K.VGCAVAYCPER.T K.FHDVYYNCPEMAK.N K.FHDVYYNCPEMAK.N Oxidation (M) K.NYGCDHSFIK.K


ska et al. / Theriogenology xxx (2015) 1–12 M. Słowin

12.e3

Supplementary Table 3 Detailed information on the identification of the phosphospecific proteoforms of hepatocyte growth factor activator in turkey seminal plasma. Spot

Protein accession, description

Sequence coverage

Mascot score

Calculated pI

Precursor mass

Peptide score

Peptide sequence

Observed

Theoretical

1

gij28631143, hepatocyte growth factor activator (Melaegris gallopavo) *gij326919485, PREDICTED: hepatocyte growth factor activator (M gallopavo)

13%

139

7.45 6.69

1033.6402 1407.7816a

1032.5716 1406.6942a

39 49a

R.VNKPGVYTR.V R.VTNYVNWINER.Ia

2

21%

214

3

16%

169

4

17%

229

1072.5521 1033.6226 1407.7577a 1072.5837 1033.6551 1407.7950a 1072.5313 1033.6143 1407.7682a

1071.4920 1032.5716 1406.6942a 1071.4920 1032.5716 1406.6942a 1071.4920 1032.5716 1406.6942a

38 50 87a 32 52 54a 37 75 71a

K.CQISGWGHK.H R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.CQISGWGHK.H R.VNKPGVYTR.V R.VTNYVNWINER.Ia K.CQISGWGHK.H R.VNKPGVYTR.V R.VTNYVNWINER.Ia

a Peptide of HGFA with a theoretical molecular weight of 1406.6942 Da was identified using hepatocyte growth factor activator, predicted sequence (accession no XP003206011).