Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14)

Bruna Alves Felix Kamila de Sousa Otávio Jorge André Matias Martins Fágner Cavalcante Patrocínio dos Santos Ana Luiza Malhado Cazaux de Souza Velho Fábio Roger Vasconcelos Maria Júlia Barbosa Bezerra Arlindo A. Moura About the authors

ABSTRACT

The objective of the present study was to develop a methodology to obtain the enriched fraction of ram seminal vesicle protein 14 (RSVP14). The study was developed using Morada Nova rams, from which semen samples were collected weekly. Seminal plasma proteins were precipitated with cold ethanol, and then 6.15 mg/mL of total proteins were subjected to liquid gelatin affinity chromatography using a Gelatin-Sepharose matrix coupled to an automated chromatographic system. Proteins were eluted into four fractions (A, B, C, and D), in which A and B contained non-gelatin-binding proteins, and C and D fractions contained gelatin-binding proteins. Gels were analyzed by Quantity One software, in which five protein bands were detected in fraction D, with molecular weights between 12 and 30 kDa. The gelatin-binding proteins (fraction D) were loaded into a HiTrap™ Heparin HP affinity column. Two chromatographic fractions were separated (D1 and D2), in which D1 contained non-heparin-binding proteins, and D2 contained heparin-binding proteins. Proteins from the last two peaks were subjected to 12.5% SDS-PAGE and Western Blot. Two bands with molecular weight of 14 and 24 kDa, contained in fraction D1, were excised from gel and subjected to tandem mass spectrometry, identifying the proteins RSVP14 and RSVP24. Thus, the chromatographic methods of the present study are efficient to capture the enriched fraction of RSVP14.

Keywords:
Ovis aries ; proteomics; spermatozoa

1. Introduction

The seminal plasma is a complex physiological secretion originated from the testis, epididiymides, and accessory sex glands of the male reproductive tract, playing important roles in sperm capacitation (Plante et al., 2012Plante, G.; Thérien, I. and Manjunath, P. 2012. Characterization of recombinant murine binder of sperm protein homolog 1 and its role in capacitation. Biology of Reproduction 87:1-11. https://doi.org/10.1095/biolreprod.111.096644
https://doi.org/10.1095/biolreprod.111.0...
) and other important events of male reproductive physiology, such as sperm motility and protection, acrosome reaction, fertilization, and initial embryonic development (Moura et al., 2018Moura, A A.; Memili, E.; Portela, A. M. R.; Viana, A. G.; Velho, A. L. C.; Bezerra M. J. B. and Vasconselos, F. R. 2018. Seminal plasma proteins and metabolites: effects on sperm function and potential as fertility markers. Animal Reproduction 15:691-702. https://doi.org/10.21451/1984-3143-AR2018-0029
https://doi.org/10.21451/1984-3143-AR201...
). The most abundant proteins in ruminant seminal plasma are members of two families of proteins, binder of sperm proteins (BSP) and spermadhesins. Proteins from BSP family bind to phospholipids and induce the efflux of cholesterol from the spermatic membrane to promote sperm capacitation (Plante et al., 2012Plante, G.; Thérien, I. and Manjunath, P. 2012. Characterization of recombinant murine binder of sperm protein homolog 1 and its role in capacitation. Biology of Reproduction 87:1-11. https://doi.org/10.1095/biolreprod.111.096644
https://doi.org/10.1095/biolreprod.111.0...
).

Bergeron et al. (2005)Bergeron, A.; Villemure, M.; Lazure, C. and Manjunath, P. 2005. Isolation and characterization of the major proteins of ram seminal plasma. Molecular Reproduction and Development 71:461-470. https://doi.org/10.1002/mrd.20310
https://doi.org/10.1002/mrd.20310...
indicated that BSP homologues identified in the seminal plasma of rams are defined as ram seminal vesicle proteins (RSVP). According to van Tilburg et al. (2013)van Tilburg, M. F.; Rodrigues, M. A. M.; Moreira, R. A.; Moreno, F. B.; Monteiro-Moreira, A. C. O.; Cândido, M. J. D. and Moura, A. A. 2013. Membrane-associated proteins of ejaculated sperm from Morada Nova rams. Theriogenology 79:1247-1261. https://doi.org/10.1016/j.theriogenology.2013.03.013
https://doi.org/10.1016/j.theriogenology...
, the main proteins present in ram seminal plasma are RSVP14 proteins (representing approximately 30% of the intensity of all spots identified in 2-D SDS PAGE). Amino acid sequences and disulfide bond assignments confirm the structural similarity between RSVP and BSP. Also, RSVP are specifically secreted by ram seminal vesicles (Fernández-Juan et al., 2006Fernández-Juan, M.; Gallego, M.; Barrios, B.; Osada, J.; Cebrián-Pérez, J. A. and Muiño-Blanco, T. 2006. Immunohistochemical localization of sperm-preserving proteins in the ram reproductive tract. Journal of Andrology 27:588-595. https://doi.org/10.2164/jandrol.05187
https://doi.org/10.2164/jandrol.05187...
) and bind to the plasma membrane of sperm (Souza et al., 2012Souza, C. E. A.; Rego, J. P. A.; Lobo, C. H.; Oliveira, J. T. A.; Nogueira, F. C. S.; Domont, G. B.; Fioramonte, M.; Gozzo, F. C.; Moreno, F. B.; Monteiro-Moreira, A. C. O.; Figueiredo, J. R. and Moura, A. A. 2012. Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams. Journal of Proteomics 75:4436-4456. https://doi.org/10.1016/j.jprot.2012.05.039
https://doi.org/10.1016/j.jprot.2012.05....
), inducing capactitation (Leahy et al., 2019Leahy, T.; Rickard, J. P.; Bernecic, N. C.; Druart, X. and de Graaf, S. P. 2019. Ram seminal plasma and its functional proteomic assessment. Reproduction 157:R243-R256. https://doi.org/10.1530/REP-18-0627
https://doi.org/10.1530/REP-18-0627...
).

Ovine BSP also act to protect sperm from damages caused by cryopreservation and detergent treatments (Luna et al., 2015Luna, C.; Colás, C.; Casao, A.; Serrano, E.; Domingo, J.; Pérez-Pé, R.; Cebrián-Pérez, J. A. and Muiño-Blanco, T. 2015. Ram seminal plasma proteins contribute to sperm capacitation and modulate sperm-zona pellucida interaction. Theriogenology 83:670-678. https://doi.org/10.1016/j.theriogenology.2014.10.030
https://doi.org/10.1016/j.theriogenology...
; Barrios et al., 2005Barrios, B.; Fernández-Juan, M.; Muiño-Blanco, T. and Cebrián-Pérez, J. A. 2005. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold-shock. Journal of Andrology 26:539-549. https://doi.org/10.2164/jandrol.04172
https://doi.org/10.2164/jandrol.04172...
; Pini et al., 2018Pini, T.; Farmer, K.; Druart, X.; Teixeira-Gomes, A. P.; Tsikis, G.; Labas, V.; Leahy, T. and Graafa, S. P. 2018. Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage. Cryobiology 82:78-87. https://doi.org/10.1016/j.cryobiol.2018.04.005
https://doi.org/10.1016/j.cryobiol.2018....
). The interaction of these proteins with the sperm membrane occurs due to the presence of fibronectin type II domains in RSVP of 14 kDa (Barrios et al., 2005Barrios, B.; Fernández-Juan, M.; Muiño-Blanco, T. and Cebrián-Pérez, J. A. 2005. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold-shock. Journal of Andrology 26:539-549. https://doi.org/10.2164/jandrol.04172
https://doi.org/10.2164/jandrol.04172...
), which is characteristic of BSP proteins. Besides, RSVP14 is part of the protein structure surrounding the sperm membrane, stabilizing its phospholipids (Barrios et al., 2005Barrios, B.; Fernández-Juan, M.; Muiño-Blanco, T. and Cebrián-Pérez, J. A. 2005. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold-shock. Journal of Andrology 26:539-549. https://doi.org/10.2164/jandrol.04172
https://doi.org/10.2164/jandrol.04172...
).

Therefore, given the importance of RSVP for sperm function and male fertility, the present study was conducted to develop a methodology to obtain an enriched fraction of RSVP14.

2. Material and Methods

The present study was accepted by the institutional animal ethics committee (case no. 1120270318).

Five 1-2-year old Morada Nova rams were used in the current study, and semen was collected for seven months weekly by means of electroejaculation, as reported before (Souza et al., 2010Souza, C. E. A.; Araújo, A. A.; Oliveira, J. T. A.; Lima Souza, A. C.; Neiva, J. N. M. and Moura, A. A. 2010. Reproductive development of Santa Inês rams during the first year of life: body and testis growth, testosterone concentrations, sperm parameters, age at puberty and seminal plasma proteins. Reproduction in Domestic Animals 45:644-653. https://doi.org/10.1111/j.1439-0531.2008.01322.x
https://doi.org/10.1111/j.1439-0531.2008...
). Semen samples were mixed with a protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA) shortly after collection (Martins et al., 2013Martins, J. A. M.; Souza, C. E. A.; Silva, F. D. A.; Cadavid, V. G.; Nogueira, F. C.; Domont, G. B.; Oliveira, J. T. A. and Moura, A. A. 2013. Major heparin-binding proteins of the seminal plasma from Morada Nova rams. Small Ruminant Research 113:115-127. https://doi.org/10.1016/j.smallrumres.2013.01.005
https://doi.org/10.1016/j.smallrumres.20...
) and centrifuged (700 × g, 4 °C 15 min) to separate the sperm cells from the supernatant. The supernatant was pipetted into clean tubes and centrifuged again (5000 × g, 4 °C 60 min) to remove cell debris (Rodríguez-Villamil et al., 2016Rodríguez-Villamil, P.; Hoyos-Marulanda, V.; Martins, J. A. M.; Oliveira, A. N.; Aguiar, L. H.; Moreno, F. B.; Velho, A. L. M. C. S.; Monteiro-Moreira A. C.; Moreira R. A.; Vasconcelos, I. M.; Bertolini, M. and Moura, A. A. 2016. Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm. Theriogenology 85:540-554. https://doi.org/10.1016/j.theriogenology.2015.09.044
https://doi.org/10.1016/j.theriogenology...
). Then, the samples from all animals were grouped in pools and stored at −80 °C until further analysis.

Ram seminal plasma proteins were precipitated according to Bergeron et al. (2005)Bergeron, A.; Villemure, M.; Lazure, C. and Manjunath, P. 2005. Isolation and characterization of the major proteins of ram seminal plasma. Molecular Reproduction and Development 71:461-470. https://doi.org/10.1002/mrd.20310
https://doi.org/10.1002/mrd.20310...
. In summary, nine volumes of pure ethanol stored at low temperature were added to 15 mL of seminal plasma in agitation for 90 min at 4 °C and then centrifuged at 10,000 × g for 10 min. After three subsequent washes with ethanol, the precipitates were solubilized in 50 mM ammonium bicarbonate and lyophilized. About 370 mg of dry seminal plasma powder were recovered after this protocol. Lyophilized seminal proteins were resuspended in 30 mL of binding buffer (Solution A: 40 mM Tris, 2 mM CaCl2, pH 7.4), purchased from GE Healthcare (Piscataway, NJ, USA) and packed into 15 microtubes of 2000 μL and stored at −80 °C. An aliquot of this pool was used to determine the soluble protein concentration (Bradford, 1976Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248-254. https://doi.org/10.1016/0003-2697(76)90527-3
https://doi.org/10.1016/0003-2697(76)905...
).

The soluble protein concentration of seminal plasma pool was 12.3 mg/mL, and 6.15 mg/mL were applied for chromatographic runs (the retention capacity of the gelatin column was 4.5-8.0 mg). Fractions containing 6.15 mg/mL of precipitated seminal plasma proteins were subjected to gelatin affinity chromatography using a 25-mL-Gelatin Sepharose 4B matrix packed in an empty XK 16/20 column (GE Healthcare; Piscataway, NJ, USA). The column was equilibrated with binding buffer (Solution A: 40 mM Tris, 2 mM CaCl2, pH 7.4), coupled to the Äkta Prime Plus chromatography system (GE Healthcare, Piscataway, NJ, USA). A low initial flow of 1 mL/min binding buffer was applied to allow the gelatin-binding proteins to interact with the column. After 5 min, the flow rate was increased to 2 mL/min for 40 min, and gelatin binding proteins were eluted using 8 M of urea added to the binding buffer. Peaks containing non-gelatin-binding and gelatin-binding proteins were determined after this chromatography run. Fractions with gelatin-binding components were concentrated using 10-kDa filters (VivaSpin MWCO 10 kDa, GE Healthcare, Piscataway, NJ, USA) and the material retained by the filters was quantified according to Bradford's method (Bradford, 1976Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248-254. https://doi.org/10.1016/0003-2697(76)90527-3
https://doi.org/10.1016/0003-2697(76)905...
). Fractions without affinity to gelatin were treated in a similar manner. To estimate the percentage of proteins bound to gelatin, peak integration was performed using PrimeView Evaluation Software (GE Healthcare, Piscataway, NJ, USA) (Rodríguez-Villamil et al., 2016Rodríguez-Villamil, P.; Hoyos-Marulanda, V.; Martins, J. A. M.; Oliveira, A. N.; Aguiar, L. H.; Moreno, F. B.; Velho, A. L. M. C. S.; Monteiro-Moreira A. C.; Moreira R. A.; Vasconcelos, I. M.; Bertolini, M. and Moura, A. A. 2016. Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm. Theriogenology 85:540-554. https://doi.org/10.1016/j.theriogenology.2015.09.044
https://doi.org/10.1016/j.theriogenology...
). Gelatin-binding proteins obtained at this first chromatographic run were stored at −20 °C for the next step.

Concentration of soluble gelatin-binding proteins was determined by Bradford's method, and 1.72 mg/mL of gelatin-binding proteins was applied in the next chromatographic run. Briefly, proteins were diluted in 100 mL of binding buffer (Sol. A: 40 mM Tris, 2 mM CaCl2, pH 7.4), respecting the maximum retention capacity of the heparin column (3 mg). The fraction of proteins with affinity to gelatin was separated by heparin affinity chromatography using a HiTrap Heparin HP column of 1 mL (GE Healthcare, Piscataway, NJ, USA), coupled to the Äkta Prime Plus chromatography system (GE Healthcare, Piscataway, NJ, USA). The column was equilibrated with binding buffer. An initial flow of 0.5 mL/min of binding buffer was applied to allow heparin-binding proteins to interact with the column. After 5 min, the flow was increased to 1 mL/min and for 30 min and the retained proteins were eluted using 1 M of sodium chloride (NaCl) added to the binding buffer. After this second chromatographic run, two peaks were formed, corresponding to non-heparin-binding and heparin-binding proteins. These two peaks were separated and concentrated using 10-kDa filters. The concentration of soluble proteins retained in the filter was also determined (Bradford, 1976Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248-254. https://doi.org/10.1016/0003-2697(76)90527-3
https://doi.org/10.1016/0003-2697(76)905...
). To estimate the percentage of non-heparin-binding and heparin-binding proteins after the chromatographic run, peak integration was performed and analyzed using PrimeView Evaluation Software, with the baseline adjusted to 0 mAu at 280 nm. The concentrations of heparin-binding and non-heparin binding proteins were approximately 1.2 and 1.0 mg/mL, respectively. The two fractions were stored at −20 °C for further analysis.

For 1-D SDS-PAGE, a volume containing 20 μg of proteins from each fraction obtained from affinity chromatographic runs were mixed with 20 μL of sample buffer [0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% (v/v) glycerol, 0.2 M Dithiothreitol (DTT), 0.02% bromophenol blue; GE Healthcare, Piscataway, NJ, USA], boiled for 90 s, and pipetted to the wells of a stacking gel (4% of acrylamide), placed on a 12.5% gradient polyacrylamide gel. An equal amount of seminal plasma proteins was also applied as a positive control. In one well of the stacking gel, 10 μL of Amersham ECL Full-Range Rainbow Molecular Weight (GE Healthcare, Piscataway, NJ, USA) were loaded to allow molecular weight estimation of the protein bands. The 1-D SDS-PAGE was run in a SE600 Ruby apparatus (GE Healthcare, Piscataway, NJ, USA), at 500 V, 25 mA/gel, 90 W. The gel was stained with Coomassie Brilliant Blue (CBB-R250) for 12 h and destained after several washes in a solution containing methanol (40%) and acetic acid (10%) in double-distilled water. After destaining, the gel was scanned at 300 dpi (Image Scanner, GE Healthcare, Piscataway, NJ, USA) and saved as a TIFF file. The image was analyzed using Quantity One software, v.4.6.3 (Bio-Rad, Rockville, MD, USA). The molecular weight of each band was determined according to the molecular marker using a point-to-point regression model available in the Quantity One software.

Proteins separated by 1-D SDS-PAGE were destained and trypsin-digested as previously described in detail (Martins et al., 2013Martins, J. A. M.; Souza, C. E. A.; Silva, F. D. A.; Cadavid, V. G.; Nogueira, F. C.; Domont, G. B.; Oliveira, J. T. A. and Moura, A. A. 2013. Major heparin-binding proteins of the seminal plasma from Morada Nova rams. Small Ruminant Research 113:115-127. https://doi.org/10.1016/j.smallrumres.2013.01.005
https://doi.org/10.1016/j.smallrumres.20...
; Moura et al., 2007Moura, A. A.; Chapman, D. A.; Koc, H. and Killian, G. J. 2007. A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls. Animal Reproduction Science 98:169-188. https://doi.org/10.1016/j.anireprosci.2006.03.012
https://doi.org/10.1016/j.anireprosci.20...
). In summary, the selected bands were cut into approximately 1 mm3 pieces, transferred to clean tubes and washed three times with solution containing acetonitrile (50%) and ammonium bicarbonate (50%; 25 mM at pH 8.0) and washed again twice with 200 μL acetonitrile for 5 min and dried under vacuum. The bands were incubated with trypsin for 20 h at 37 °C. The peptides were then extracted by triple washing with 5% trifluoroacetic acid in 50% acetronitrile and ammonium bicarbonate (50 mM) for 30 min. Supernatants were concentrated in microtubes and vacuum-dried (Eppendorf, Hauppauge, NY, USA). The peptides were separated on a C18 BEH300 column (100 μm × 100 mm) using the nanoAcquity ™ system (Waters Corp, Milford, CT, EUA) and eluted at 600 μL/min with acetronitrile gradient (5–85%) containing 0.1% of formic acid. The liquid chromatography system was connected to a nanospray mass ionization source (Synapt hdms system, Waters Corp, Milford, CT, EUA). The mass spectrometer was operated in positive mode using 90 °C capillary and 3.5 kV voltage. Instrument calibration was performed using double protonated [Glu1] -fibrinopeptide B fragments (m/z 785.84), and the Lock-mass used during acquisition was the intact ion. The LC-MS/MS procedure was performed according to the data-dependent acquisition (DDA) method by selecting double or triple charge precursor ions MS/MS. The ions were fragmented by induced collision dissociation using argon as the collision gas and collision energy ramp that varied according to the charge state of the selected precursor ion. Data collection was performed at a range of m/z 300–2100 for MS sampling (1 scan/s), identifying ions with m/z ranging from 50–2500 for MS/MS. Data were collected using MassLynx 4.1 software, processed using Protein Lynx Global Server 2.4 server (Waters Corp, Milford, CT, EUA), and converted to peak list text file (pkl) for database searching.

The ionic spectra obtained for each pkl peptide were searched using the MASCOT search tool (Matrix Science, London, UK, v.2.6) in the NCBInr database using the MS/MS ion search mode. For the search, we considered loss values of at most one tryptic cleavage, monoisotopic peptides with a load of +1, +2, and +3, with variable methionine residue oxidation and fixed variation of carbamidomethylated cysteine residues. Identification was considered unambiguous when the protein score was significant (P<0.05), and due to the close coincidence of the theoretical and experimental protein molecular weight and isoelectric point.

Twenty micrograms of seminal plasma and 20 μg of non-gelatin-binding and gelating-binding were subjected to molecular separation by 12.5% SDS-PAGE and then transferred (at 45 mA for 90 min) to a PVDF Hybond-P membrane (GE Lifesciences, Piscataway, NJ, USA) using a TE 70 transfer unit (GE Healthcare, Piscataway, NJ, USA). The membranes were blocked overnight and incubated with primary antibody as described previously by Souza et al. (2012)Souza, C. E. A.; Rego, J. P. A.; Lobo, C. H.; Oliveira, J. T. A.; Nogueira, F. C. S.; Domont, G. B.; Fioramonte, M.; Gozzo, F. C.; Moreno, F. B.; Monteiro-Moreira, A. C. O.; Figueiredo, J. R. and Moura, A. A. 2012. Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams. Journal of Proteomics 75:4436-4456. https://doi.org/10.1016/j.jprot.2012.05.039
https://doi.org/10.1016/j.jprot.2012.05....
, at 4 °C with 30 mL of PBS with 0.5% Tween-20 (PBS-T, GE Healthcare, Piscataway, NJ, USA) containing skimmed milk (5% w/p), under agitation, followed by 1 h incubation with antibody against the sperm protein ligand (1:6000 for anti-BSP) based on the protocol described by Moura et al. (2007)Moura, A. A.; Chapman, D. A.; Koc, H. and Killian, G. J. 2007. A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls. Animal Reproduction Science 98:169-188. https://doi.org/10.1016/j.anireprosci.2006.03.012
https://doi.org/10.1016/j.anireprosci.20...
, with modifications. The PVDF membranes were then washed three times in 1X Phosphate-Buffered Saline and Tween Detergent (PBS-T, GE Healthcare, Piscataway, NJ, USA), and incubated for 1 h with anti-rabbit IgG (1:5000, Abcam, UK) along with 30 mL PBS with 0.5% Tween-20 (PBS-T, washed again three times in PBS-T and washed once with Tris-HCl 50 mM). Immunoreaction was visualized by exposing the membranes to BCIP/NBT alkaline phosphatase substrate, pH 9.5 (Thermo Scientific, Waltham, MA, EUA). The reaction was stopped by washing the membranes with ultrapure water.

3. Results

Based on chromatographic profiles, gelatin-binding proteins represented approximately 30% of ram seminal plasma total protein. Chromatograms of gelatin affinity chromatography presented four peaks well separated and eluted in 13, 20, 45, and 53 min, respectively (Figure 1). Fractions A and B were eluted during the column washing with binding buffer, for 30 min, with absorbances of 100 and 40 mAu, respectively. The last two peaks, grouped in fractions C and D, were eluted in the presence of 8 M urea for 60 min, with an absorbance of 18 and 60 mAu, respectively (Figure 1). After separation by heparin affinity chromatography, the non-heparin-binding proteins represented the peak of interest with 30.64% of proteins obtained in the first chromatographic step. Chromatograms of heparin affinity chromatographies presented two peaks eluted in 3 and 20 min, respectively (Figure 2).

Figure 1
Gelatin affinity chromatography with ovine seminal plasma proteins.
Figure 2
Heparin affinity chromatography of gelatin binding proteins obtained in the previous chromatographic step.

Gelatin-binding proteins and whole seminal plasma proteins separated by 1-D SDS PAGE were analyzed and compared using Quantity One software. Bands representing fractions A, B, C, and D indicated the presence of 28 proteins with molecular weights between 14 and 102 kDa (Figure 3). Fraction D (Figure 3) represented the peak of interest, with 28.44% of proteins bound to gelatin. Fractions A and B (Figure 3) included non-gelatin-binding proteins with 12-73 kDa. An immunodetection test was performed with anti-BSP antibodies (Figure 4), and the four peaks of the gelatin column reacted to that antibody. The chromatographic conditions applied to obtain fraction D were efficient to separate the proteins into two peaks, grouped in D1 (non-heparin-binding proteins) and D2 (heparin-binding proteins) (Figure 5).

Figure 3
SDS-PAGE of whole seminal plasma proteins (SP) and gelatin-binding proteins without (A and B) and with (C and D) affinity to gelatin.
Figure 4
Western blotting performed using anti-binder of sperm antibodies.
Figure 5
SDS-PAGE of whole seminal plasma proteins (SP) and heparin-binding proteins without (D1) and with (D2) affinity to heparin.

One-dimensional electrophoresis indicated that the enriched fraction shown in lane D1 had only two bands with molecular weights of 14 and 24 kDa. As determined by mass spectrometry, band 1 contained binder of sperm 1 precursor (Ovis aries), and band 2 contained binder of sperm 5 precursor (Ovis aries; Table 1). Western blots using anti-BSP antibodies confirmed the presence of BSP in heparin-binding (D1) and non-heparin-binding (D2) fractions (Figure 6).

Table 1
Binder of sperm proteins (BSP) precursors enriched fractions from Ovis aries seminal plasma identified by one-dimensional SDS-PAGE and tandem mass spetrometry
Figure 6
Western blotting performed using anti-binder of sperm antibodies with proteins bound and non-bound to heparin.

4. Discussion

In the present study, we described a new method to obtain a RSVP14 enriched fraction from ram seminal plasma, consisting of two sequential steps of affinity chromatography. Binder of sperm proteins have affinity to gelatin, heparin, and other glycosaminoglicans as the result of hydrophobic and ionic interactions (Plante et al., 2016Plante, G.; Prud'homme, B.; Fan, J.; Lafleur, M. and Manjunath, P. 2016. Evolution and function of mammalian binder of sperm proteins. Cell and Tissue Research 363:105-127. https://doi.org/10.1007/s00441-015-2289-2
https://doi.org/10.1007/s00441-015-2289-...
; Gasset et al., 1997Gasset, M.; Saiz, J. L.; Laynez, J.; Sanz, L.; Gentzel, M.; Töpfer-Petersen, E. and Calvete, J. J. 1997. Conformational features and thermal stability of bovine seminal plasma protein PDC-109 oligomers and phosphorylcholine-bound complexes. European Journal of Biochemistry 250:735-744. https://doi.org/10.1111/j.1432-1033.1997.00735.x
https://doi.org/10.1111/j.1432-1033.1997...
). Also, BSP proteins have conserved structures composed of a N-terminal domain variable and two fibronectin type II domains arranged in tandem (Plante et al., 2016Plante, G.; Prud'homme, B.; Fan, J.; Lafleur, M. and Manjunath, P. 2016. Evolution and function of mammalian binder of sperm proteins. Cell and Tissue Research 363:105-127. https://doi.org/10.1007/s00441-015-2289-2
https://doi.org/10.1007/s00441-015-2289-...
). Our method, thus, used BSP chemical attributes to obtain an RSVP14 enriched fraction of complex seminal plasma samples from rams.

In the present study, the group of proteins with 14-15 and 22-24 kDa were the most predominant in fraction D (Figure 3). According to Bergeron et al. (2005)Bergeron, A.; Villemure, M.; Lazure, C. and Manjunath, P. 2005. Isolation and characterization of the major proteins of ram seminal plasma. Molecular Reproduction and Development 71:461-470. https://doi.org/10.1002/mrd.20310
https://doi.org/10.1002/mrd.20310...
, such proteins are defined as RSVP-14, RSVP-15, RSVP-22, and RSVP-24 kDa. We found results (Figure 3) similar to those described by Villemure et al. (2003)Villemure, M.; Lazure, C. and Manjunath, P. 2003. Isolation and characterization of gelatin-binding proteins from goat seminal plasma. Reproductive Biology and Endocrinology 1:39. https://doi.org/10.1186/1477-7827-1-39
https://doi.org/10.1186/1477-7827-1-39...
, who suggested that proteins present in fractions A and B could be BSP homologues along with other proteins of the same molecular weight.

Bovine BSP1 is glycosylated, being visually differentiated with two protein isoforms by immunoblot analysis. The BSP proteins in bulls, stallions, wild boars, and humans tend to form clusters in solution (Calvete et al., 1995Calvete, J. J.; Reinert, M.; Sanz, L. and Töpfer-Petersen, E. 1995. Effect of glycosylation on the heparin-binding capability of boar and stallion seminal plasma proteins. Journal of Chromatography A 711:167-173. https://doi.org/10.1016/0021-9673(95)00011-B
https://doi.org/10.1016/0021-9673(95)000...
; Kumar et al., 2008Kumar, V.; Hassan, M. I.; Kashav, T.; Singh, T. P. and Yadav, S. 2008. Heparin-binding proteins of human seminal plasma: purification and characterization. Molecular Reproduction and Development 75:1767-1774. https://doi.org/10.1002/mrd.20910
https://doi.org/10.1002/mrd.20910...
; Manjunath et al., 1988Manjunath, P.; Baillargeon, L.; Marcel, Y. L.; Seidah, N. G.; Chrétien, M. and Chapdelaine, A. 1988. Diversity of novel proteins in gonadal fluids. p.259-273. In: Molecular biology of brain and endocrine peptidergic systems. Chrétien M. and McKerns, K. W., eds. Springer, Boston. https://doi.org/10.1007/978-1-4684-8801-2_17
https://doi.org/10.1007/978-1-4684-8801-...
; Plante et al., 2016Plante, G.; Prud'homme, B.; Fan, J.; Lafleur, M. and Manjunath, P. 2016. Evolution and function of mammalian binder of sperm proteins. Cell and Tissue Research 363:105-127. https://doi.org/10.1007/s00441-015-2289-2
https://doi.org/10.1007/s00441-015-2289-...
). In the study of Villemure et al. (2003)Villemure, M.; Lazure, C. and Manjunath, P. 2003. Isolation and characterization of gelatin-binding proteins from goat seminal plasma. Reproductive Biology and Endocrinology 1:39. https://doi.org/10.1186/1477-7827-1-39
https://doi.org/10.1186/1477-7827-1-39...
, the comparison of the N-terminal sequence of proteins from RSVP family indicated a high degree of structural relationships between RSVP. This suggests the existence of different glycoforms of RSVP14 in ovine seminal plasma, which proves that RSVP proteins exist in different molecular forms and glycoforms. Martins et al. (2013)Martins, J. A. M.; Souza, C. E. A.; Silva, F. D. A.; Cadavid, V. G.; Nogueira, F. C.; Domont, G. B.; Oliveira, J. T. A. and Moura, A. A. 2013. Major heparin-binding proteins of the seminal plasma from Morada Nova rams. Small Ruminant Research 113:115-127. https://doi.org/10.1016/j.smallrumres.2013.01.005
https://doi.org/10.1016/j.smallrumres.20...
reported that RSVP of 14 and 22 kDa had no affinity to heparin, results that are in agreement with the Western blots conducted in our study.

The BSP homologues of other species such as goats (Villemure et al., 2003Villemure, M.; Lazure, C. and Manjunath, P. 2003. Isolation and characterization of gelatin-binding proteins from goat seminal plasma. Reproductive Biology and Endocrinology 1:39. https://doi.org/10.1186/1477-7827-1-39
https://doi.org/10.1186/1477-7827-1-39...
) and bisons (Boisvert et al., 2004Boisvert, M.; Bergeron, A.; Lazure, C. and Manjunath, P. 2004. Isolation and characterization of gelatin-binding bison seminal vesicle secretory proteins. Biology of Reproduction 70:656-661. https://doi.org/10.1095/biolreprod.103.023069
https://doi.org/10.1095/biolreprod.103.0...
) do not bind to heparin either. Amino acid substitutions can modify the affinity of molecules to heparin (Ward, 2010Ward, W. S. 2010. Function of sperm chromatin structural elements in fertilization and development. Molecular Human Reproduction 16:30-36. https://doi.org/10.1093/molehr/gap080
https://doi.org/10.1093/molehr/gap080...
), so it is possible that differences in amino acids and sequences of fibronectin II domains in RSVP prevent them from binding to heparin (Martins et al., 2013Martins, J. A. M.; Souza, C. E. A.; Silva, F. D. A.; Cadavid, V. G.; Nogueira, F. C.; Domont, G. B.; Oliveira, J. T. A. and Moura, A. A. 2013. Major heparin-binding proteins of the seminal plasma from Morada Nova rams. Small Ruminant Research 113:115-127. https://doi.org/10.1016/j.smallrumres.2013.01.005
https://doi.org/10.1016/j.smallrumres.20...
).

The RSVP contained in D1 fraction (Figure 5) did not bind to heparin, similarly to the result described by Villemure et al. (2003)Villemure, M.; Lazure, C. and Manjunath, P. 2003. Isolation and characterization of gelatin-binding proteins from goat seminal plasma. Reproductive Biology and Endocrinology 1:39. https://doi.org/10.1186/1477-7827-1-39
https://doi.org/10.1186/1477-7827-1-39...
in goats. Binder of sperm proteins play roles in sperm capacitation, sperm interaction with the oviduct epithelium, and fertilization. Also, BSP interact with components of semen extenders, suggesting that BSP can be used for the development of new protocols for sperm cryopreservation and artificial insemination (Moura et al., 2018Moura, A A.; Memili, E.; Portela, A. M. R.; Viana, A. G.; Velho, A. L. C.; Bezerra M. J. B. and Vasconselos, F. R. 2018. Seminal plasma proteins and metabolites: effects on sperm function and potential as fertility markers. Animal Reproduction 15:691-702. https://doi.org/10.21451/1984-3143-AR2018-0029
https://doi.org/10.21451/1984-3143-AR201...
).

Artificial insemination in sheep is challenging but a feasible technique. Early studies have observed a wide variety of sublethal freezing effects of cryopreservation on sperm (Peris et al., 2007Peris, S. I.; Bilodeau, J. F.; Dufour, M. and Bailey, J. L. 2007. Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm. Molecular Reproduction and Development 74:878-892. https://doi.org/10.1002/mrd.20686
https://doi.org/10.1002/mrd.20686...
). Thus, in the study developed by Ari and Daskin (2010)Ari, U. C. and Daskin, A. 2010. Freezing of washed angora goat semen with extenders added bull or ram seminal plasma. Kafkas Universitesi Veteriner Fakültesi Dergisi 16:233-237., the addition of ram seminal plasma protected and significantly improved the stabilization of acrosome membrane of goat sperm post-thaw. In the study by Barrios et al. (2005)Barrios, B.; Fernández-Juan, M.; Muiño-Blanco, T. and Cebrián-Pérez, J. A. 2005. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold-shock. Journal of Andrology 26:539-549. https://doi.org/10.2164/jandrol.04172
https://doi.org/10.2164/jandrol.04172...
, seminal plasma proteins of approximately 14 and 20 kDa were responsible for protection of ram sperm membrane. More recently, studies conducted in Australia indicated that seminal plasma proteins, including BSP, can prevent damages to ram sperm caused by cryopreservation (Barrios et al., 2005Barrios, B.; Fernández-Juan, M.; Muiño-Blanco, T. and Cebrián-Pérez, J. A. 2005. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold-shock. Journal of Andrology 26:539-549. https://doi.org/10.2164/jandrol.04172
https://doi.org/10.2164/jandrol.04172...
; Luna et al., 2015Luna, C.; Colás, C.; Casao, A.; Serrano, E.; Domingo, J.; Pérez-Pé, R.; Cebrián-Pérez, J. A. and Muiño-Blanco, T. 2015. Ram seminal plasma proteins contribute to sperm capacitation and modulate sperm-zona pellucida interaction. Theriogenology 83:670-678. https://doi.org/10.1016/j.theriogenology.2014.10.030
https://doi.org/10.1016/j.theriogenology...
; Pini et al., 2018Pini, T.; Farmer, K.; Druart, X.; Teixeira-Gomes, A. P.; Tsikis, G.; Labas, V.; Leahy, T. and Graafa, S. P. 2018. Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage. Cryobiology 82:78-87. https://doi.org/10.1016/j.cryobiol.2018.04.005
https://doi.org/10.1016/j.cryobiol.2018....
). Thus, there seems to be sufficient pieces of evidence to support the concept that certain seminal plasma proteins are beneficial to sperm and protect these cells during cryopreservation. This knowledge can be exploited to improve methods of commercial sperm freezing (Pini et al., 2018Pini, T.; Farmer, K.; Druart, X.; Teixeira-Gomes, A. P.; Tsikis, G.; Labas, V.; Leahy, T. and Graafa, S. P. 2018. Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage. Cryobiology 82:78-87. https://doi.org/10.1016/j.cryobiol.2018.04.005
https://doi.org/10.1016/j.cryobiol.2018....
).

5. Conclusions

Affinity chromatographic methods used in the present study were efficient to obtain an enriched fraction of RSVP14 from ram seminal plasma. This is a significant step to obtain RSVP from whole seminal fluid, but the use of additional chromatographic approaches, such as ionic exchange or gel filtration, are still required to obtain highly purified RSVP. Enriched RSVP14 fractions or purified RSVP can be used in studies designed to understand the precise roles of the major seminal plasma proteins of rams. Purified seminal proteins can also be used in extenders to improve the results of commercial freezing and the outcome of artificial insemination in ovine species.

Acknowledgments

The present research was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Scholarships were granted to graduate students and post-doctoral fellows by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP). The authors also appreciate Dr. Puttaswamy Manjunath (Department of Medicine, University of Montréal, Canada) for providing antibodies against BSP proteins and Dr. Celso Nagano (Laboratório de Espectrometria de Massa do Nordeste, Universidade Federal do Ceará, Brasil).

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Publication Dates

  • Publication in this collection
    30 Oct 2020
  • Date of issue
    2020

History

  • Received
    22 Apr 2019
  • Accepted
    11 Aug 2020
Sociedade Brasileira de Zootecnia Universidade Federal de Viçosa / Departamento de Zootecnia, 36570-900 Viçosa MG Brazil, Tel.: +55 31 3612-4602, +55 31 3612-4612 - Viçosa - MG - Brazil
E-mail: rbz@sbz.org.br