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 (2016) 540–554

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Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm P. Rodríguez-Villamil a, V. Hoyos-Marulanda a, J.A.M. Martins a,1, A.N. Oliveira a, L.H. Aguiar b, F.B. Moreno c, A.L.M.C.S. Velho a, A.C. Monteiro-Moreira c, R.A. Moreira c, I.M. Vasconcelos d, M. Bertolini b, A.A. Moura a, * a

Animal Physiology Laboratory, Department of Animal Science, Federal University of Ceara, Fortaleza, Brazil Molecular and Developmental Biology Laboratory, University of Fortaleza, Fortaleza, Brazil c School of Pharmacy, University of Fortaleza, Fortaleza, Brazil d Department of Biochemistry and Molecular Biology, Federal University of Ceara, Fortaleza, Brazil b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 May 2015 Received in revised form 17 September 2015 Accepted 18 September 2015

The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n ¼ 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 mg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1  2.7%), 10 mg/mL BSP1 (77.8  3.1%), or 20 mg/mL BSP1 (74  2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8  5.0%) and 10 mg/mL BSP1 (34.1  4.4%), but reduced after 20 mg/mL BSP1 (22.4  2.9%) and 40 mg/mL BSP1 (19.3  4.1%; P < 0.05). In the second experiment, cumulus-oocyte complexes (n ¼ 1213) were incubated with frozen-thawed cauda epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 mg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5  1.3% and 24.7  3.2%, respectively) or without heparin (65.5  1.8% and 27.3  1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2  2.7%–79.0  1.1%) than without heparin (P < 0.05). Also, cleavage was better after Fert-TALP medium incubation with 40 mg/mL BSP1 (79.0  1.1%) than with heparin (68.5  1.3%; P < 0.05). Embryo development was higher (P < 0.05) after treatment with 20 mg/mL BSP1 (35.6  2.5%) and 40 mg/mL (41.1  2%) than after incubations with heparin (24.7  3.2%) or without heparin (27.3  1.6%). Interestingly, BSP1 did not cause reductions in blastocyst rates after fertilization with epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to

Keywords: Seminal plasma BSP1 Embryo Sperm Fertilization

* Corresponding author. Tel.: 558533669697; fax: 558533669701. E-mail address: [email protected] (A.A. Moura). 1 Current address: Academic Unit of Serra Talhada, Federal Rural University of Pernambuco, Serra Talhada, Brazil. 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.09.044

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and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Male gametes are produced in the testis, matured, and stored in the epididymis and eventually released on ejaculation, when they are mixed with secretions of accessory sex glands. At this point, seminal proteins modulate several sperm functional attributes, including capacitation, acrosome reaction, and fertilization [1–3]. Among such molecules, binder of sperm (BSP) is a family of proteins present in the seminal plasma (SP) of several species. They are mainly produced by the seminal vesicles of bulls [4], horses and boars [5], goats [6], buffalos [7], rams [8], and by the epididymides of rats [9] and humans [10]. According to its biochemical characteristics, the group of bovine BSP molecules includes BSP1 (formerly known as BSP-A1/A2 or PDC-109), BSP3 (former BSP-A3), and BSP5 (former BSP30 kDa) [11]. BSP1 is a low molecular weight, glycosylated protein and the most abundant of all BSPs, containing two fibronectin type II domains [12] that allow specific binding to membrane phosphatidylcholine [13]. On ejaculation, BSPs bind to sperm [14–16] and induce efflux of membrane cholesterol and phospholipids [17], a key step to promote capacitation [18,19]. Moreover, in the female reproductive tract, BSPs bound to sperm, continue to promote the efflux of membrane lipids in the presence of high-density lipoproteins or glycosaminoglycans (GAGs), enhancing capacitation rates and, further, the acrosome reaction [20–22]. Due to these attributes, BSP1 has the ability to stabilize the sperm membrane during its passage through the female tract [18] and to mediate sperm’s binding to the epithelium of the oviduct [23]. For in vitro production of embryos, metabolic and physiological needs of sperm, oocytes and embryos themselves must be well established. In this regard, during IVF, it is necessary to simulate the physiological pathways that induce sperm capacitation, an essential step that allows spermatozoa to acquire complete fertilizing capacity [24]. Therefore, previous studies have reported the use of capacitation-inducing factors, such as GAGs [25,26], extracellular calcium [27], bicarbonate [28], BSA [29], and heparin [26]. Similar to BSP proteins, some of these molecules are already present in the SP and/or oviductal fluid, such as BSA, and may act as cholesterol acceptor from the sperm plasma membrane, promoting capacitation [22,29]. Heparin is also used for a similar purpose, and it appears to induce membrane cholesterol loss by interacting with SP BSP proteins [22], although through pathways that are still partially known [22]. In summary, BSPs are not only the major proteins of ruminant SP but also play vital roles in sperm function. Because of this, certain attributes of BSPs have been characterized, especially in the bovine species [12,18,30], but there are no reports about the effect of those proteins on fertilization rates and embryo development. On the basis of these facts, a series of experiments were conducted to evaluate functional aspects of BSP1. First, in the

present work, a purification method was developed to purify BSP1 from bovine SP and seminal vesicle fluid (SVF). As second objective, this study evaluated binding patterns of BSP1 to both epididymal and ejaculated sperm; BSP1 influence on sperm capacitation in vitro; effects of purified BSP1 on cleavage and blastocyst rates after IVF with ejaculated or epididymal sperm. 2. Material and methods The study was conducted at the state of Ceara, in the Northeast of Brazil (05 110 S 39 170 W) and approved by the Department of Animal Science of the Federal University of Ceará, Fortaleza, Brazil, on August 30, 2010. 2.1. Purification of BSP1 from bovine SP and SVF 2.1.1. General approach BSP1 was purified from both SP of Bos taurus bulls and SVF of Bos indicus bulls. Heparin and gelatin affinity chromatographies were performed, and protein profiles from chromatographic fractions were monitored by gel electrophoresis, N-terminal sequencing, mass spectrometry, and Western blots. 2.1.2. Seminal plasma collection and processing Semen was obtained by electroejaculation from four adult, healthy Brown Swiss bulls. Semen samples were mixed with a protease inhibitor cocktail (Sigma–Aldrich, USA) right after collection [31] and centrifuged (700  g, 4  C, 15 minutes) to separate sperm from the supernatant SP. This supernatant was pipetted into clean tubes and centrifuged again (5000  g, 4  C, 60 minutes). After the last centrifugation, samples from all bulls were pooled, aliquoted, and stored at 20  C [32]. An aliquot of this pool was used to determine the soluble protein concentration [33]. 2.1.3. Heparin affinity chromatography of SP proteins Seminal plasma proteins (5 mg) were loaded into a 1 mL HiTrap Heparin HP column attached to an Äkta Prime Plus chromatographic system (GE Healthcare, USA). Column was previously equilibrated with binding buffer (sol. A: 40-mM Tris, 2-mM CaCl2, pH 7.4). Protein was injected into the column at a flow rate of 0.5 mL/min. After 5 minutes, the flow rate was accelerated to 1.0 mL/min, and column was washed with 15 mL for elution of nonabsorbed proteins. Heparin-binding proteins (HBP) were eluted with solution B (1 M NaCl added to Sol. A) with 10 mL at 1.0 mL/min. The HBP fractions were pooled, desalted, and concentrated using the Amicon MWCO 10 kDa filters (Sigma–Aldrich, USA). The HBP fraction from SP was saved for the next purification step. The proportion of HBP in SP was estimated by the percent of total peak area after peak integration using the PrimeView Evaluation software, with the baseline adjusted to 0 mAu at 280 nm (GE Healthcare, USA).

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2.1.4. Gelatin affinity chromatography of SP HBP The HBP fraction of SP was separated by gelatin affinity chromatography using 20 mL of Gelatin Sepharose 4B matrix packed into an XK 16/20 empty column (GE Healthcare, USA). Column was equilibrated with binding buffer (Sol. A: 40 mM Tris, 2 mM CaCl2, pH 7.4) using the Äkta Prime Plus system. Samples were loaded into the column at 0.5 mL/min, and after 5 minutes, the system was paused for 30 minutes to allow the interaction of proteins with the matrix. Then, the flow rate was adjusted to 1.0 mL/min, and column was washed with 40 mL for elution of nonabsorbed proteins. Heparin and/or gelatin-binding proteins (HBPGBP) were eluted with solution B (8 M urea added to Sol. A) with 20 mL at 1.0 mL/min. Heparin and/or gelatin-binding proteins fractions were desalted and buffer exchanged to 50 mM ammonium bicarbonate, using a 5 mL HiTrap Desalting column (GE Healthcare, USA), followed by freezing (80  C) and lyophilization for 24 hours using the VirTis BenchTop Pro ES lyophilizer (SP Industries, Inc., USA) at 40  C and minimum pressure of 60 mTorr. The HBPGBP fraction of SP was stored at 20  C until further analysis. Heparin and/or gelatin-binding proteins peak was estimated as described in the previous chromatographic step. 2.1.5. Confirmation of BSP1 identity purified from SP by mass spectrometry Whole SP and fractions obtained by sequential chromatography (HBP and HBPGBP) were subjected to 2-D electrophoresis [16,32]. Briefly, 400 mg SP, HBP, and HBPGBP proteins were rehydrated with buffer sufficient to make 250 mL. Samples were loaded and incubated with 13-cm immobilized pH gradient strips (pH 3–10) to rehydrate for 20 hours. Isoelectric focusing was carried out in Ettan IPGphor 3 (GE Healthcare, USA) apparatus at 20  C, according to the following strategy: 200 V (200 V h), 500 V (1000 V h), 5000 V (10,000 V h), and 10,000 V (22,000 V h), with a total of 33,200 V h. After focusing, samples were subjected to SDSPAGE (15%), run at 250 V, with 30 mA per gel (Hoefer SE 600; GE Healthcare, USA). All gels were stained in a 2% colloidal solution with Coomassie Blue G-250 (Bio-Rad, USA). Spots of interest were excised from the HBPGBP gel, destained, digested with trypsin, and subjected to tandem mass spectrometry [32,34]. Briefly, gel pieces were washed three times with 400 mL in a solution with ammonium bicarbonate (25 mM) and acetonitrile (50%), pH 8.0, dehydrated after two washes with 200 mL of acetonitrile, and vacuum dried. Gel pieces were then incubated (w20 hours) at 37  C with trypsin (166 ng/spot; Promega, USA). Peptides were extracted from gel pieces by washing three times with 25 mL trifluoroacetic (5%) in ammonium bicarbonate (50 mM) and acetonitrile (50%), for 30 minutes. The extracts were vacuum dried and resuspended in 10-mL injection buffer (95% water, 5% acetonitrile, 0.1% formic acid). For liquid chromatography-tandem mass spectrometry (LC-MS/MS), digested samples were injected using the nanoAcquity UPLC Sample Manager, and the chromatographic separation was performed using a UPLC C18 column (75 mm  10 cm) with a flow of 0.6 mL/min. Mass spectra were acquired in a Synapt G2 HDMS instrument (Waters Co., USA) using a data-dependent acquisition, where the three top peaks were subjected to MS/MS.

Mobile phases A and B consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. The gradient conditions used were as follows: 0 minutes with 3% of B, increasing linearly to 30% B in 20 minutes, then it increased up to 70% B in 40 minutes where it remained until 50 minutes, and in the next minute, it was decreased to 3% of B. Data were processed using Mascot Distiller (Matrix Science Ltd., USA) and subjected to database search (Mascot Server 2.3), using other mammalia as the taxon. Searches were made with the assumption that there was a maximum of one missed trypsin cleavage and that peptides were mono-isotopic and using partially oxidized methionine residues as fixed modifications, and completely carbamidomethylated cysteine residues as variable modifications. Peptide mass tolerance and fragment mass tolerance were set to 0.1 Da for MS/MS ion searching. Candidate peptide IDs were accepted if the m/z values were observed within 0.1 Da (typically <0.05 Da) of the theoretical mass of the candidate ID, as determined when manually reviewing Mascot search results. 2.1.6. Edman’s degradation and N-terminal sequence of SP HBPGBP N-terminal sequencing of SP HBPGBP proteins (purified BSP1) was carried out on a Shimadzu PPSQ-23A Automated Protein Sequencer (Shimadzu, Japan), performing Edman degradation [35]. Briefly, sequences were determined from alquilated HBPGBP proteins (2–5 pmol). Phenylthiohydantoinamino acids were detected at 269 nm after separation on a reversed phase C18 column (4.6  2.5 mm) under isocratic conditions, according to the manufacturer’s instructions. The sequences obtained were submitted to automatic alignment against the PDC-109 (BSP1) annotated in the NCBI databank for Bos taurus, using the Blast tool (blast.ncbi.nlm.nih.gov) [36]. 2.1.7. Seminal vesicle fluid collection and processing Once purification of SP BSP1 was successful (see Section 3), we evaluated whether SVF could also be used to obtain suitable amounts of BSP1. Thus, seminal vesicles were collected from four adult Bos indicus bulls in a local, commercial abattoir and rapidly transported on ice to the laboratory. Then, glands were horizontally opened with a scalpel and gently squeezed to extract their fluids, which were pooled and centrifuged twice as described for SP samples (700  g, 15 minutes; 5000  g, 60 minutes; at 4  C). Protein concentration in SVF was determined [33] and samples stored at 20  C. For collection of seminal vesicles, as well as for collection of ovaries and epididymis (see in the following section), animal management at commercial abattoirs was supervised by a veterinarian and followed all regulations of the Brazilian Department of Agriculture (Brazil, Ministério da Agricultura, Pecuária e Abastecimento 2009). 2.1.8. Heparin and gelatin affinity chromatography of SVF Seminal vesicle fluid proteins (5 mg) were separated by heparin affinity chromatography following the same procedure used for SP samples (described in the previous section). The HBP fraction of SVF was also subjected to gelatin affinity chromatography, and the resulting HBPGBP were stored at 20  C until further analysis.

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2.1.9. Confirmation of BSP1 identity purified from SVF Whole SVF and its HBPGBP fraction obtained by heparin and gelatin affinity chromatography were evaluated by Western blot [31]. In summary, 20 mg SVF and HBPGBP proteins were separated in ECL Precast Gels (GE Healthcare, USA) with an 8 to 16% acrylamide gradient and then transferred (at 208 mA for 90 minutes) to PVDF Hybond-P membrane (GE Healthcare, USA) using a TE 70 transfer unit (GE Healthcare, USA). Membranes were blocked overnight at 4  C with 50 mL of PBS with 0.5% Tween-20 (PBS-T; GE, Healthcare) containing BSA (5% w:v), under mild agitation, followed by a 2-hour incubation with antibodies against bovine BSP1 (1:1000). PVDF membranes were then washed three times in PBS-T and incubated for 2 hours with donkey anti-rabbit IgG (1:5000; Abcam, UK) coupled with alkaline phosphatase, washed again three times in PBS-T, and rinsed twice with Tris–HCl (50 mM). Immunoreaction was visualized by exposing the membranes to BCIP/NBT alkaline phosphatase substrate, pH 9.5 (Thermo Scientific, USA). Reaction was stopped by washing the membranes with ultrapure water. Anti-BSP1 antibodies were purified from lyophilized rabbit antiserum kindly provided by Dr. Puttaswamy Manjunath (School of Medicine, the University of Montreal, Canada), on the basis of a protocol described by [37], with modifications. In brief, the lyophilized antiserum was resuspended in 2-mL PBS (50-mM phosphate buffer, 150-mM NaCl, pH 7.4) and loaded onto a 1-mL HiTrap Protein A HP column (GE Healthcare, USA) that was initially washed with 24-mL PBS, and adsorbed proteins were eluted with 10-mL 0.1-M glycine in PBS (pH 2.5). All chromatographic steps were conducted at a flow rate of 1 mL/min. Fractions containing eluted immunoglobulins, including the anti-BSP antibodies, were collected in tubes with 200-mL 1.5 M Tris (pH 8.8) for rapid adjustment of the pH, and then pooled, aliquoted, and stored at 20  C until use. Whole SVF and HBPGBP fraction were also subjected to 1-D gel electrophoresis for protein identification [31]. Briefly, 20 mg of SVF and HBPGBP proteins were separated using acrylamide precast gels (GE Healthcare, USA), as described in the previous section, and stained with colloidal Coomassie blue. A major low molecular weight band from the whole SVF lane and a band of same molecular weight from the HBPGBP lane were excised from gel, and subjected to tandem mass spectrometry as described in the previous section. 2.1.10. Alignment of peptides sequences Similarity between BSP1 proteins purified from SP and SVF was evaluated by alignment of peptides sequences acquired from mass spectrometry and N-terminal sequence against the sequence of PDC-109 precursor deposited in NCBI database, using the CLC Sequence Viewer v.6.6.2 (CL Bio, USA). 2.2. Binding patterns of purified BSP1 to ejaculated and epididymal sperm The pattern of BSP1 binding to sperm was evaluated by immunocytochemistry, adjusting a protocol that has been previously described [38]. Briefly, frozen-thawed sperm (ejaculated and epididymal) were selected by Percoll and

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incubated in Fert-TALP medium containing heparin (control group) or in Fert-TALP without heparin with different concentrations of BSP1 (0, 10, 20, or 40 mg/mL) for 18 hours (at 38  C in 5% CO2). BSP1 purified from SVF was used in this experiment. Before and after incubation, sperm smears of each treatment were fixed in 2% paraformaldehyde for 10 minutes at 4  C, followed by three washes with 0.3% of Tween-20 in PBS (PBS-T). Then, unspecific binding was avoided by incubating slides at 4  C for 1 hour in PBS-T containing 5% of BSA, followed by the blocking solution (PBS-T plus 2% BSA) and anti-BSP1 antibodies (1:500), at 4  C for 1 hour. Anti-BSP1 antibody was the same used for Western blots, as described previously. After this step, slides were washed three times with PBS-T and incubated with donkey anti-IgG conjugated with FITC (1:300; Santa Cruz, USA) in PBS-T, for 1 hour at room temperature, in a dark room. This was followed by three further washes with PBS-T. Then, slides were covered with 2.5-mg/mL 40 ,6-diamidino-2-phenylindole (DAPI; Life Technology, USA) solution for 10 minutes at room temperature and washed with PBS for 1 minute. All slides were dried in the dark using anti-fade reagent (ProLongGold; Invitrogen Corp., USA), before setting the coverslip. Images were acquired by confocal microscopy LSM 710 and ZEN 2011 software (Zeiss, Germany). Controls were conducted using incubations of sperm cells with the first or the second antibody only. None of these controls generated any detectable fluorescence. 2.3. Effect of purified BSP1 on sperm acrosome reaction Frozen ejaculated and epididymal sperm from three Bos indicus bulls were thawed at 37  C for 30 seconds and separated through Percoll gradients (45%–90%). Sperm were incubated in Fert-TALP medium containing heparin (control group) or in Fert-TALP with no heparin and different concentrations of BSP1 (0, 10, 20, or 40 mg/mL). BSP1 purified from SVF was used in this experiment. After 4 hours of incubation (38  C in 5% CO2), sperm was coincubated in 2 mg/mL of Hoechst (33258) solution for 15 minutes, washed in PBS for 5 minutes, and fixed and stained with 40 mg/mL of FITC for 20 minutes. Then, slides were rinsed in water, air-dried, and covered with a glass coverslip. Sperm capacitation rate was estimated by counting the number of live sperm with reacted acrosome, under differential interference light microscopy. The percentages of live and dead sperm for each treatment were determined by counting 160 to 240 sperms/slide. 2.4. In vitro oocyte maturation and embryo production Bovine ovaries were obtained at a local abattoir and kept at 32  C during transport to the laboratory. Cumulusoocyte complexes (COCs) were aspirated with a disposable syringe from follicles with 3 to 8 mm in diameter and washed in Tissue Culture Medium 199 (Sigma–Aldrich, USA), as reported before [39]. In summary, oocytes were transferred to microdrops containing 100 mL maturation medium (10–20 COCs/drop) and matured for approximately 24 hours at 38.8  C in an atmosphere of saturated humidity and 5% CO2. The maturation medium consisted

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of Tissue Culture Medium 199 supplemented with 10% (v:v) fetal bovine serum, 0.2-mM sodium pyruvate, 35-mg/mL porcine FSH (Folltropin-V; Bioniche, Canada) and penicillin/streptomycin (10 mL/mL; Schering, USA). After IVM, COCs were randomly assigned to the different experimental groups. After each straw of frozen semen (ejaculated or epididymal) was thawed for 1 minute at 37  C, sperm were prepared for fertilization using Percoll discontinuous gradient (45% and 90%), as described before [40]. In summary, the 90% Percoll solution (500 mL) was placed in a 2mL Eppendorf tube, and 500 mL of 45% Percoll was smoothly layered on this. Frozen-thawed semen was layered on top of gradients, and tubes were centrifuged for 15 minutes at 700  g. The pellets were resuspended in the same amount of sperm-TALP medium and centrifuged for 5 minutes at 700  g. The supernatant was removed, and the final concentration of the sperm pellet was adjusted with Fert-TALP to 1  106 sperm/mL for fertilization. To assure the quality of ejaculated sperm used in the IVF studies, a few frozen-thawed straws were chosen at random and evaluated as previously described [40]. In brief, the percentage of progressively motile sperm was determined by using 10 mL of diluted sperm placed on a microscope slide, covered with a coverslip. In this case, a minimum of 200 sperm were counted and evaluated with phase contrast lens (400 ). For sperm morphology analysis, sperm were stained with eosin–nigrosin, and 200 cells were counted under bright field microscope (1000 ). On the basis of these evaluations, it was confirmed that frozenthawed sperm had proper quality for IVF. Fertilization medium consisted of Fert-TALP [40], and COCs were fertilized in 100-mL droplets (8–20 COCs/ droplet) covered with mineral oil. A 10 mL aliquot of 1  106 sperm/mL was used to fertilize the COCs. Synthetic oviductal fluid (SOF) medium supplemented with amino acids, 1.5 mM of D-glucose, and 0.4% of BSA was used for culture medium. After 18 to 20 hours, COCs were pipetted to remove the cumulus cells and excess sperm cells, washed once in SOF and transferred into 50-mL droplets of SOF medium under mineral oil in a controlled atmosphere (5% CO2, 5% O2, and 90% N2) at 38.8  C. Zygotes were cultured in vitro in SOF medium for 7 days at 38.8  C, using a humidified incubator with 5% CO2 and 5% O2. Cleavage and blastocyst rates were evaluated on Days 2 and 7 after fertilization, respectively [41]. Blastocyst rates were based on the total number of oocytes allowed to be fertilized. Oocyte maturation and embryo production were performed under the same conditions for IVF experiments with ejaculated and epididymal sperm, always using BSP1 purified from SVF.

fertilized in Fert-TALP medium without heparin þ 20 mg/mL BSP1; (4) T3: COCs fertilized in Fert-TALP medium without heparin þ 40 mg/mL BSP1. For fertilization, COCs were incubated with ejaculated sperm and the different concentrations of BSP1 (0, 10, 20, 40 mg/mL) for 18 hours. For each IVF routine, conducted weekly, we used the three different bulls across the four treatments. Thus, by the end of the study, this strategy allowed the use of all bulls in all treatments.

2.4.1. Effect of BSP1 addition to fertilization media with ejaculated semen on embryo development Matured COCs (n ¼ 1274) were randomly allocated into treatment groups and fertilized with frozen-thawed ejaculated sperm from three different bulls from a commercial artificial insemination center. Treatments were defined as: (1) Control: COCs fertilized in Fert-TALP medium with heparin (2 mg/mL); (2) T1: COCs fertilized in Fert-TALP medium without heparin þ 10 mg/mL BSP1; (3) T2: COCs

Acrosome reaction rates were compared among all treatments by Fisher’s exact test. For the cleavage and blastocyst rates, data were transformed by square root and then analyzed by ANOVA, considering bulls and BSP1 concentrations as main effects and their interactions. The protected least significant difference test was used for subsequent multiple comparisons when ANOVA revealed statistically significant differences (P < 0.05). All data were analyzed using the SAS software [43].

2.4.2. Effect of BSP1 addition to fertilization media with epididymal sperm on embryo development For this experiment, matured COCs (n ¼ 1213) were randomly allocated into treatment groups and fertilized with frozen-thawed cauda epididymal sperm. Treatments were defined as: (1) Control: COCs fertilized in Fert-TALP medium with heparin (2 mg/mL); (2) Control 1: COCs fertilized in Fert-TALP medium without heparin; (3) T1: COCs fertilized in Fert-TALP medium without heparin þ 10 mg/mL BSP1; (4) T2: COCs fertilized in Fert-TALP medium without heparin þ 20 mg/mL BSP1; (5) T3: COCs fertilized in Fert-TALP medium without heparin þ40 mg/mL BSP1. Epididymal sperm cells were collected from different bulls and, for each IVF routine conducted weekly, sperm straws from a different bull were used, always using the same bull across treatments. For fertilization, oocytes were incubated with the frozen-thawed epididymal sperm cells according to the different treatments and distinct BSP1 concentrations used in the fertilization media (0, 10, 20, 40 mg/mL) for 18 hours. Cauda epididymal sperm used in this experiment were collected and frozen as described before [42]. In brief, testes of crossbred bulls were obtained at a local abattoir and immediately transported to the laboratory at room temperature. Then, after removal of the scrotal skin, spermatozoa were collected by retroperfusion of the vas deferens with Tris buffer, followed by small incisions and gentle pressure on the cauda of epididymides. The recovered spermatozoa were placed in 15-mL tubes containing egg yolk-Tris-glycerol extender. For each animal, sperm from both epididymis were combined. Straws were equilibrated for 4 hours at 5  C and then cooled in liquid nitrogen vapor (80  C and 115  C) for 15 minutes. Finally, straws were plunged into liquid nitrogen for storage. The total sperm numbers in the straws were 20  106. After cryopreservation, straws from each bull were thawed to confirm that frozen-thawed epididymal sperm had proper quality, as described previously for ejaculated sperm samples. 2.5. Statistical analysis

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3. Results Based on the chromatographic profiles, HBP represented 67.3% of SP proteins from Bos taurus bulls (Fig. 1A). Further separation by gelatin affinity chromatography (Fig. 1B) showed that 42.5% of HBPs interacted with gelatin. Heparin/gelatin-binding proteins, in turn, represented 28% of all SP proteins. The 2-D maps of HBP and HBPGBP fractions (Fig. 1C) clearly show the diminishing complexity of SP protein profile during the progression of purification steps with heparin followed by gelatin affinity columns. After these sequential procedures, nine spots were detected in the HBPGBP 2-D gel, and all of them were identified by electrospray ionization quadrupole time-of-flight mass spectrometry as the Chain A, Bull Seminal Plasma Pdc-109 Fibronectin Type II Module, also known as BSP1 (Table 1). Identical strategy using affinity chromatography established that bovine SVF contained 55.1% of HBP (Fig. 2A). Within this group, gelatin-binding proteins represented 43.6% (Fig. 2B), which in turn, made 24% of all proteins of the whole SVF. One-dimensional SDS-PAGE confirmed the diversity of proteins in the SVF, while a lane containing the HBPGBP fraction had only one band (Fig. 3). As determined by electrospray ionization quadrupole time-of-flight mass

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spectrometry, band 1 (excised from the whole SVF lane) contained SP protein PDC-109 (BSP1) mixed with SP protein A3 (BSP3), spermadhesin 1, spermadhesin Z13, epididymal secretory protein E1, metalloproteinase inhibitor 2, and SP protein BSP-30 kDa precursor (BSP5). On the other hand, only BSP1 was identified in band 2 (excised from the HBPGBP fraction lane; Table 2). Western blots detected a major 14-kDa band in the lane containing SVF proteins and a single reaction in the lane with the HBPGBP fraction, also with 14 kDa (Fig. 3). Amino acid sequences based on N-terminal analysis and from tryptic peptides obtained from purified BSP1 proteins of Bos taurus SP and Bos indicus SVF are illustrated in Figure 4. The alignment of the common amino acids shows the high conservation of both proteins. As evaluated by immunocytochemistry, there was no detectable fluorescence on frozen-thawed epididymal sperm before incubation with BSP1 (0 hours; Fig. 5A). On the other hand, intense anti-BSP1 reaction was present on the acrosome, equatorial and post-equatorial segments, and midpiece of frozen-thawed ejaculated sperm before any BSP1 treatment (Fig. 5C). After incubation (18 hours) with purified BSP1, fluorescence was detected on post-equatorial and equatorial segments as well as on the midpiece of both epididymal (Fig. 5B) and ejaculated (Fig. 5D) sperm.

Fig. 1. Chromatographic strategy applied for purification of binder of sperm 1 from Bos taurus seminal plasma. (A) Heparin affinity chromatography of whole seminal plasma proteins. (B) Gelatin affinity chromatography of heparin-binding proteins obtained in the previous (A) chromatographic step. (C) Twodimensional maps of Bos taurus whole seminal plasma, HBP, and HBPGBP fractions after heparin and gelatin affinity chromatography. Numbers in the HBPGBP 2-D gel represent those spots subjected to identification by mass spectrometry and described in Table 1. HBP, heparin-binding proteins; HBPGBP, heparin/gelatin-binding proteins.

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Table 1 Seminal plasma heparin/gelatin-binding proteins (HBPGBP) of Bos taurus bulls identified by two-dimensional electrophoresis and tandem mass spectrometry. Protein and spots

NCBI accession number

MS/MS protein score

Sequence covered (%)

Matched peptides

Chain A, Bull Seminal Plasma Pdc-109 Fibronectin Type II ModuledBSP1 (1) Spot 1 20663779 61 28 DQDEGVSTEPTQDGPAELPEDEECVFPFVYR(31) (1) DQDEGVSTEPTQDGPAELPEDEECVFPFVYR(31) (1) DQDEGVSTEPTQDGPAELPEDEECVFPFVYR(31) (1) DQDEGVSTEPTQDGPAELPEDEECVFPFVYR(31) (14) Spot 2 20663779 454 42 GPAELPEDEECVFPFVYR(31) (22) EECVFPFVYR(31) (42) GSLFPWCSLDADYVGR(57) (48) CSLDADYVGR(57) (49) SLDADYVGR(57) (69) CVFPFIYGGK(78) (92) SWCSLSPNYDK(102) (94) CSLSPNYDK(102) (94) CSLSPNYDKDR(104) (95) SLSPNYDK(127) (95) SLSPNYDKDR(104) (96) LSPNYDKDR(104) (97) SPNYDKDR(104) (98) PNYDKDR(104) (1) Spot 3 20663779 37 28 DQDEGVSTEPTQDGPAELPEDEECVFPFVYR(31) (69) Spot 4 20663779 43 14 CVFPFIYGGK(78) (59) Spot 5 20663779 172 31 KHFDCTVHGSLFPWCSLDADYVGR(57) (60) HFDCTVHGSLFPWCSLDADYVGR(57) (69) CVFPFIYGGK(78) (60) Spot 6 20663779 98 30 HFDCTVHGSLFPWCSLDADYVGR(57) (69) CVFPFIYGGK(78) (59) Spot 7 20663779 2764 55 KHFDCTVHGSLFPWCSLDADYVGR(57) (60) HFDCTVHGSLFPWCSLDADYVGR(57) (69) CVFPFIYGGK(78) (79) KYETCTK(85) (86) IGSMWMSWCSLSPNYDK(102) (86) IGSMWMSWCSLSPNYDKDR(104) (1) Spot 8 20663779 1888 83 DQDEGVSTEPTQDGPAELPEDEECVFPFVYR(31) (59) KHFDCTVHGSLFPWCSLDADYVGR(57) (60) HFDCTVHGSLFPWCSLDADYVGR(57) (69) CVFPFIYGGK(78) (79) KYETCTK(85) (86) IGSMWMSWCSLSPNYDK(102) (86) IGSMWMSWCSLSPNYDKDR(104) (60) Spot 9 20663779 140 30 HFDCTVHGSLFPWCSLDADYVGR(57) (69) CVFPFIYGGK(78)

Ion score

m/z

z

18 16 58 61 41 46 69 62 57 38 32 39 60 33 64 41 68 58 37 43 55 88 29 88 9 79 92 45 25 75 97 85 83 87 42 27 74 97 104 35

1185.8296 1185.8298 1185.8333 1185.8368 1077.4916 673.3102 921.9226 578.2535 498.2367 594.7874 678.7943 542.2382 452.1984 462.2215 597.7872 554.2703 497.7274 454.2108 3555.5130 594.7875 717.5698 913.7282 594.2949 685.5432 594.2967 956.1031 913.7293 594.7679 465.2182 1031.4427 789.3202 1778.3035 956.4316 685.2880 594.7738 465.2193 1047.4247 789.0027 685.5504 593.9848

3 3 3 3 2 2 2 2 2 2 2 2 3 2 2 2 2 2 1 2 4 3 2 4 2 3 3 2 2 2 3 2 3 4 2 2 2 3 4 2

Table includes spots detected in 13-cm gels, within the 3 to 10 pH range. Spot numbers names refer to those shown in Figure 2.

Detected immunoreactions had no apparent differences after incubations with 10, 20, or 40 mg/mL BSP1. Acrosome reaction rates increased (P < 0.05) up to the concentration of 20 mg/mL BSP1 for both ejaculated and epididymal sperm, indicating a dose-dependent effect of BSP1 on capacitation. However, after incubation with heparin only, acrosome reaction rates were 74.5% higher (P < 0.05) for ejaculated than for epididymal sperm (Table 3). For IVF with ejaculated sperm, there were no significant interactions between bulls and BSP1 treatments. As expected, IVF with ejaculated sperm without heparin was associated with lower cleavage and embryo rates than the control group with heparin (data not shown). Cleavage rates after incubations with any concentration of BSP1 were similar to those obtained with heparin (Table 4). Blastocyst rates achieved with 10-mg/mL BSP1 was also equivalent to those observed in the group with only heparin. However, higher concentrations of BSP1 (20 and 40 mg/mL BSP1) significantly decreased the percentage of blastocyst

formation in comparison with the group containing only heparin (P < 0.02). Blastocyst rates after incubation of FertTALP with 40-mg/mL BSP1 was much lower than after treatment with 10-mg/mL BSP1 (P < 0.05; Table 4). With frozen-thawed epididymal sperm, inclusion of heparin in the fertilization medium had no effect on cleavage and blastocyst rates in comparison with the treatment without heparin (Table 4). However, Fert-TALP medium containing all BSP1 concentrations favored better cleavage rates in comparison with medium without heparin (P < 0.05). Also, oocytes fertilized with epididymal sperm in Fert-TALP medium with 40-mg/mL BSP1 yielded higher cleavage rates than in medium supplemented with heparin only (P < 0.05). Fertilization medium treated with all amounts of BSP1 allowed better blastocyst rates than with only heparin (P < 0.05). In comparison with the FertTALP medium containing no heparin, treatments with 20-mg/mL and 40-mg/mL BSP1 also induced higher blastocyst rates (P < 0.0003; Table 4).

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Fig. 2. Chromatographic strategy applied for purification of binder of sperm 1 protein from Bos indicus seminal vesicle fluid (SVF). (A) Heparin affinity chromatography of whole SVF. (B) Gelatin affinity chromatography of heparin-binding proteins obtained in the previous (A) chromatographic step. HBP, heparinbinding proteins; HBPGBP, heparin/gelatin-binding proteins.

4. Discussion The present study describes the effects of BSP1 protein on fertilization of in vitro–matured bovine oocytes. Our strategy included the unique purification of BSP1 from SVF and addition of different amounts of BSP1 or heparin to the fertilization media, using frozen-thawed ejaculated and cauda epididymal sperm. To our knowledge, this is the first description of BSP1 effects on bovine embryo development. BSP proteins comprise approximately 60% of all proteins of the accessory sex gland fluid [37] and SP [30] of Bos taurus bulls and nearly the same amount of Bos indicus SP [1]. BSP1 contains 109 amino acids in its mature form [30] and possesses two fibronectin type II domains arranged in tandem [18,44]. BSP1 is a typical accessory sex gland protein, and there are no reports of its expression in the bovine epididymis [18,37,45]. We presently describe an efficient procedure to purify BSP1 from bovine SP using affinity chromatography steps. In the first one, whole SP proteins were separated by their capacity to bind to heparin, a sulfated GAG. Binding of proteins to heparin occurs when basic amino acids of the protein are in contact with acidic groups on heparin [46]. In the second step, SP proteins eluted from the heparin column were separated based on affinity to gelatin, a derivate from collagen. Gelatin specifically binds to residues of fibronectin type 2 [8], the key domains of BSP1. Thus, sequential chromatographies allowed capture of SP BSP1, as shown by mass spectrometry analysis of spots excised from the HBPGBP 2-D gel. Morever, high purity of BSP1 was confirmed by N-terminal sequencing of the proteins captured in the HBPGBP chromatographic fraction. When we applied an identical approach with SVF, BSP1 was also successfully purified, as confirmed by both mass spectrometry and Western blots. A similar protocol has been used before to purify BSP1 from bovine SP [30], but this is the first report describing its application to obtain BSP1 from SVF. Experimental evidence confirms that BSPs attach to the membrane of ejaculated sperm [14,47,38].

Thus, it is possible that SP obtained by centrifugation of ejaculated semen, even at low g force, contains BSPs released from sperm with or without phospholipids or other sperm components. Also unknown is whether BSPs released from sperm, if present in the SP, behave differently or not from BSP molecules that had never been in contact with spermatozoa. Finally, retrieval of fluid from seminal vesicles allows purification of BSP1 from a medium that had never been in contact with sperm and avoids the risks and costs associated with semen collection from adult bulls. Our BSP1 purification protocol was first established using Bos taurus SP, whereas the SVF came from Bos indicus bulls. On the basis of alignment of amino acid sequences obtained from the mass spectra, the peptides detected by mass spectrometry from both Bos taurus SP and Bos indicus SVF show exactly the same sequence. N-terminal amino acid sequence of BSP1 purified from Bos taurus SP also showed high similarity with the data gathered from tryptic peptides, indicating that BSP1 is a highly conserved protein among subspecies [11]. Once confirmed the amino acid sequence similarity between both BSP1 molecules, we purified sufficient amounts of protein from SVF for the IVF assays. The procedure currently described to purify BSP1 from SP and SVF incorporates some steps that have been used before to purify BSP from SP of ruminants [4,6,8]. However, methods described by these authors include gelatin agarose affinity chromatography as the first step to capture bovine BSPs. In our protocol, however, we established heparin affinity chromatography as the first step and then, heparin-absorbed proteins were further separated by gelatin affinity chromatography. We observed that BSP1 was the only protein present in the final fraction (HBPGBP), suggesting that the other BSPs (BSP3 and BSP5) remained in the nonabsorbed fractions of the first chromatographic step. This is a useful information when BSP1 is the main objective of the survey. Moreover, based on integration of area under the chromatographic peaks, our protocol

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Fig. 3. One-dimensional electrophoretic profile (SDS-PAGE) and Western blotting (WB) of Bos indicus whole seminal vesicle fluid (a) and fractions after heparin and gelatin affinity chromatographydHBPGBP (b). Western blotting was performed using anti–binder of sperm 1 antibodies. Bands 1 and 2 indicated in the left image (SDS-PAGE) were identified by mass spectrometry, as summarized in Table 2. Arrows in the right image (WB) indicate positive immunoreaction of BSP1 antibodies.

allowed a recovery of 8.4-mg BSP1 per mL of raw SP, less than what has been described before [48]. Although our protocol apparently recovered less BSP1 than that reported by other authors, it did not include any procedure that potentially cause damage to proteins, such as protein precipitation or organic solvent elution in reversed phase high-performance liquid chromatography. Interaction patterns of BSP1 with the bovine sperm were evaluated by immunochemistry. As we have shown before [38], epididymal sperm had no contact with BSP proteins, in contrast with ejaculated sperm. After incubation with purified BSP1 for 18 hours, there was a clear interaction of BSP1 with epididymal sperm. In ejaculated sperm, BSP1 binding to the acrosome was lost probably as the result of acrosome reaction after the 18-hour incubation. If BSP1 was bound to the acrosome of epididymal sperm during incubation, such binding was lost after cells went through acrosome reaction. Interestingly, our results

also indicated that the inner acrosome membrane does not interact with BSP1, but reasons for such occurrence are still unknown. Both BSP1 and heparin treatments promoted in vitro capacitation of ejaculated and epididymal sperm, in comparison with no treatment. BSP1 enhanced sperm capacitation in a dose response manner up to 20 mg, but sperm appeared to have reached its maximum responding capacity beyond this point, given the incubation time we chose. These results corroborate with observations described by other authors, showing that BSPs homologous induce capacitation of bovine [49], porcine [21], mouse [19] and human [50] sperm, probably due to their stimulation of cholesterol and phospholipid efflux from the sperm membrane. Moreover, heparin also induced capacitation and acrosome reaction on both epididymal and ejaculated sperm, but such effect was much more efficient in the latter. This result confirm the hypothesis, as other authors

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Table 2 Seminal vesicle fluid heparin and heparin/gelatin-binding proteins (HBPGBP) of Bos indicus bulls identified by one-dimensional electrophoresis and tandem mass spectrometry. Proteins and bands

NCBI accession number

Band 1 (raw seminal vesicle fluid) 20663779 Chain A, Bull Seminal Plasma Pdc-109 Fibronectin Type II Module (BSP1)

MS/MS protein score

Sequence covered (%)

Matched peptides

Ion score

m/z

z

512

83

(79)

KYETCTK(85) CVFPFIYGGK(78) (69) CVFPFIYGGK(78) (69) CVFPFIYGGK(78) (86) IGSMWMSWCSLSPNYDK(102) (86) IGSMWMSWCSLSPNYDK(102) (86) IGSMWMSWCSLSPNYDK(102) (86) IGSMWMSWCSLSPNYDKDR(104) (86) IGSMWMSWCSLSPNYDKDR(104) (86) IGSMWMSWCSLSPNYDKDR(104) (86) IGSMWMSWCSLSPNYDKDR(104) (86) IGSMWMSWCSLSPNYDKDR(104) (86) IGSMWMSWCSLSPNYDKDR(104) (34) KHFDCTVHGSLFPWCSLDADYVGR(57) (34) KHFDCTVHGSLFPWCSLDADYVGR(57) (34) KHFDCTVHGSLFPWCSLDADYVGR(57) (34) KHFDCTVHGSLFPWCSLDADYVGR(57) (34) KHFDCTVHGSLFPWCSLDADYVGR(57) (34) KHFDCTVHGSLFPWCSLDADYVGR(57) (34) KHFDCTVHGSLFPWCSLDADYVGR(57) (34) KHFDCTVHGSLFPWCSLDADYVGR(57) (34) KHFDCTVHGSLFPWCSLDADYVGR(57) (34) KHFDCTVHGSLFPWCSLDADYVGR(57) (34) KHFDCTVHGSLFPWCSLDADYVGR(57) (1) DQDEGVSTEPTQDGPAELPEDEECVFPFVYR(31) (39) EMEDIASGAETK(50) (39) EMEDIASGAETK(50) (123) SWCSLSPNFDEDR(135) (176) SDGSCAWYR(184) (33) EVDSGNDIYGNPIKR(47) (36) EVNVSPCPTQPCK(48) (124) VVVEWELTDDKNQR(137) (167) WCSLTSNYDR(176)

30 30 33 01 37 73 77 98 98 98 87 98 69 95 85 38 79 49 10 87 84 46 80 85 94 70 22 85 67 44 47 54 76

928.4394 1186.5900 1186.5918 1186.5932 2060.8840 2076.8988 2092.8240 2332.0117 2347.9186 2348.0080 2348.0238 2363.9527 2364.0012 2738.1313 2738.1897 2738.1901 2738.1990 2738.2033 2738.2057 2738.2111 2738.2241 2866.2688 2866.2961 2866.3168 3554.5075 1279.5662 1295.5632 1611.6694 1100.4420 1675.8220 1514.6940 1729.8718 1300.5600

2 2 2 2 2 2 2 3 3 3 2 3 2 3 4 4 2 4 3 3 4 2 4 3 3 2 2 2 2 2 2 3 2

(69)

Predicted: seminal plasma protein A3 (BSP3)

297469372

156

17

TIMP-2 protein

4835936

110

12

101

18

76

5

74

9

(36)

EESGVIATYYGPK(58)

74

1412.6898

2

58

11

(79)

ESLEIIEGPPESSNSR(94)

58

1742.8416

2

364

76

(68)

28 33 76 17 98 69 72 70 90

1186.5738 2060.8912 2076.8850 2092.8810 2348.0083 2364.0016 2738.2061 2738.2063 3554.5342

2 2 2 2 3 3 4 3 3

Epididymal secretory 27806881 protein E1 precursor 28849953 Seminal plasma protein BSP-30 kDa precursor (BSP5) Spermadhesin-1 27807111 precursor Spermadhesin Z13 126158907 precursor Band 2 (seminal vesicle fluid HBPGBP) 20663779 Chain A, Bull Seminal Plasma Pdc-109 Fibronectin Type II Module (BSP1)

CVFPFIYGGK(78) IGSMWMSWCSLSPNYDK(102) (86) IGSMWMSWCSLSPNYDK(102) (86) IGSMWMSWCSLSPNYDK(102) (86) IGSMWMSWCSLSPNYDKDR(104) (86) IGSMWMSWCSLSPNYDKDR(104) (35) HFDCTVHGSLFPWCSLDADYVGR(57) (35) HFDCTVHGSLFPWCSLDADYVGR(57) (1) DQDEGVSTEPTQDGPAELPEDEECVFPFVYR(31) (86)

Table includes bands detected in 10-cm gels, within the 8% to 16% acrylamide gradient. Band numbers and protein names refer to those shown in Figure 4. Abbreviation: BSP, binder of sperm.

mentioned, that heparin needs sperm-bound BSP to induce capacitation [22,49,51]. When ejaculated sperm was used in the IVF trials, fertilization rates among bulls were similar regardless of BSP1 concentrations, but sires presented different blastocyst rates. However, it is known that bulls have different amounts and binding patterns of BSP1 that could be correlated with fertility rates and post-thawing sperm survival [38,52]. The reason why we observed such differences could be due to the fact that post-thawing ejaculated sperm already had BSP1 bound to the sperm membrane,

and the exogenous BSP1 added to the fertilization media had a different effect for each sire. This effect of external BSP1 is similar to heparin addition on in vitro embryo production, which increase fertilization and blastocyst rates regardless of the IVF rates of bulls [53,54]. When using ejaculated sperm for IVF, the concentration of 10-mg/mL BSP1 was associated with proper cleavage and embryo development, similar to that obtained with heparin. However, more of BSP1 (20 and 40 mg/mL) added to the Fert-TALP medium reduced blastocyst rates. The present study is the first to report this type of BSP1 damaging effect

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Fig. 4. Alignment of binder of sperm 1 (BSP1) precursor sequence (PDC-109) obtained from NCBI database, N-terminal amino acid sequence of BSP1 purified from Bos taurus seminal plasma (N-terminal: SP BSP1), tryptic peptides of BSP1 from Bos taurus seminal plasma (2D spots: SP BSP1), and Bos indicus seminal vesicle fluid (1D bands: SVF BSP1) acquired by mass spectra. Dotted boxes highlight the peptides present within highly conserved regions (gray background). SP, seminal plasma; SVF, seminal vesicle fluid.

on in vitro embryo development. The precise mechanism explaining such outcome is unknown, but this may be the result of BSP1-induced damage on sperm, COC’s and/or the embryo, given the exposure period during fertilization

(18 hours). In agreement with the present results, studies have reported that excess of BSP proteins and time exposure are harmful to cryopreserved sperm because of membrane destabilization caused by abnormal

Fig. 5. Binding patterns of binder of sperm 1 (BSP1) on the bovine epididymal and ejaculated sperm surfaces before and after incubation with purified BSP1 from Bos indicus seminal vesicle fluid. (A) Epididymal sperm before incubation with BSP1; (B) capacitated epididymal sperm after 18 hours of incubation with BSP1 (20 mg/mL); (C) ejaculated sperm before incubation with BSP1; (D) capacitated ejaculated sperm after 18 hours of incubation with BSP1 (20 mg/mL).

P. Rodríguez-Villamil et al. / Theriogenology 85 (2016) 540–554 Table 3 Effect of different concentrations of BSP1 (0, 10, 20, or 40 mg/mL) or heparin (control) on acrosome reaction rates of ejaculated or epididymal bovine sperm. Sperm

Acrosome reaction (%) BSP1 (mg/mL)

Ejaculated Epididymal

Heparin

0

10

20

40

14.5Aa 16.5Aa

24.5Ab 25.5Ab

25.5Ac 27.5Ac

26.0Ac 27.0Ac

44.5Ad 25.5Bb

BSP1 purified from seminal vesicle fluid was used in this experiment. a,b,c,d Within a row, percentages without a common superscript differed (P < 0.05). A,B Within a column, percentages without a common superscript differed (P < 0.05). Abbreviation: BSP, binder of sperm.

phospholipid and cholesterol efflux [18,20,55]. Also, an earlier investigation reported that the content of BSP 30 kDa (currently known as BSP5) in accessory sex gland fluid has a quadratic association with bull fertility [32], suggesting that too much BSP is detrimental to sperm and/ or embryo development, similar to the context reported in the present study. Although BSP5 is another bovine BSP homologue, both BSP5 and BSP1 proteins possess two fibronectin type II domains arranged in tandem and amino terminal extensions that are O-glycosylated at threonine residues. Such biochemical attributes indicated that both BSP1 and BSP5 interact with sperm by the same manner and modulate ligand-binding activities by an equivalent mechanism [56], sharing functional similarities as well [18]. Compared with the control treatment (FertTALP þ heparin), inclusion of 10-mg/mL BSP1 to the fertilization medium exerted a similar effect on ejaculated

Table 4 Cleavage and blastocyst rates of bovine oocytes fertilized with ejaculated and cauda epididymal sperm in Fert-TALP medium containing different concentrations of BSP1 purified from seminal vesicle fluid. Experiments and treatments Ejaculated sperm Control (heparin) T1 T2 T3 Epididymal sperm Control (heparin) Control 1 (without heparin) T1 T2 T3

Oocytes

% Cleavage

325 316 313 320

74.1 77.8 74.0 65.9

244 242

68.5  1.3bc 65.5  1.8c

24.7  3.2c 27.3  1.6bc

240 242 245

74.2  2.7ab 74.0  1.6ab 79.0  1.1a

33.2  1.1ab 35.6  2.5a 41.1  2.0a

   

2.7ab 3.1a 2.0ab 2.6b

% Blastocyst

40.8 34.1 22.4 19.3

   

5.07a 4.4ab 2.9bc 4.1c

Control: oocytes fertilized in Fert-TALP medium with heparin; Control 1: oocytes fertilized in Fert-TALP medium without heparin; T1: oocytes fertilized in Fert-TALP medium without heparin þ 10 mg/mL BSP1; T2: oocytes fertilized in Fert-TALP medium without heparin þ 20 mg/mL BSP1; T3: oocytes fertilized in Fert-TALP medium without heparin þ 40 mg/mL BSP1. a-c For comparisons within each experiment (ejaculated and epididymal sperm), means within the same column with different superscripts are different (P < 0.05). Abbreviation: BSP, binder of sperm.

551

sperm. We assume that BSP1 had the ability to induce proper sperm capacitation, comparable to heparin, because fertilization and cleavage occurred. The hypothesis is that BSP1 function is analogous to BSA, increasing the acrosome reaction by depletion of cholesterol from the ejaculated sperm surface [57]. Previous studies conducted in vitro highlighted that BSP1 induces the efflux of cholesterol and phospholipid from the plasma membrane, leading to the acrosome reaction [21,55,58,59]. It has also been found that BSP protein acts as an acceptor to remove phospholipids and cholesterol from sperm membranes, leading to capacitation [50]. Given that interaction between BSP and heparin exists, some authors suggest that BSP1 probably acts during capacitation by its ability to bind to heparin on the sperm surface [22,53,55]. Our study did not evaluate whether BSP molecules interacted with heparin precisely in the IVF media, but we suggest that exogenous BSP1 promoted proper sperm capacitation of ejaculated sperm without the presence of heparin because cleavage occurred. In the bovine, as in other ruminants such as rams and goats, BSPs and their homologues are accessory sex gland proteins that coat the sperm membrane on ejaculation [15,16,37,47]. Thus, any study about the effect of exogenous BSP on ejaculated sperm must take into account the fact that these cells already contain BSP bound to their membranes. Given this scenario, we established a series of IVF experiments with cauda epididymal sperm once these cells had never been in contact with BSP proteins. Overall, in comparison with ejaculated sperm, cauda epididymal sperm behaved differently when heparin or BSP1 was added to the fertilization medium. First, there were no differences in cleavage and blastocyst rates between the control groups (Fert-TALP medium with or without heparin). Such results agree with the concept that heparin does not induce acrosome reaction of epididymal sperm unless cells are previously exposed to SP [60]. This is also supported by studies showing that preincubation of epididymal spermatozoa with BSP proteins potentiates heparin-induced capacitation [55] by increasing the number of binding sites for heparin on the sperm surface [61]. Our present results corroborate with these findings given that inclusion of heparin in the IVF media with ejaculated sperm (which already contained BSP bound to them) allowed better cleavage and blastocyst rates (74.1% and 40.8%, respectively) than when only heparin was used with epididymal sperm (68.5% and 24.7%, respectively). With epididymal sperm, all BSP1 concentrations induced more efficient cleavage than the no-heparin control, and 40-mg/mL BSP1 allowed higher cleavage than heparin. Moreover, IVF media with any amount of BSP1 favored more blastocyst rates than with heparin, and media with 20 to 40-mg/mL BSP1 stimulated higher blastocyst rates than without heparin. This strongly suggests that exogenous BSP1 triggered capacitating-related events on epididymal sperm, which allowed further acrosome reaction and normal fertilization [22,57]. As well reported in the bull, BSPs induce sperm capacitation [49]. However, BSP proteins alone do not induce acrosome reaction of bovine epididymal sperm in the absence of heparin, concludes a previous study conducted in vitro [55]. Different

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from that study, our experiment used an IVF system, where BSP1 was added to the fertilization medium. Thus, given that we did not mix heparin with BSP, it is possible that BSP1 induced capacitation of epididymal sperm, and molecules associated with the COCs triggered the acrosome reaction. The mechanism and molecular pathways by which BSP proteins induce capacitation may be different from those triggered by heparin [22]. Other role played by BSP proteins includes the ability to mediate the interaction between spermatozoa and the oviductal epithelium, contributing to formation of the sperm reservoir in the oviduct [51]. BSPs also have the ability to remain bound to bovine sperm membrane not only after ejaculation but also after being mixed with isthmus and ampulla fluids and after acrosome reaction [38]. Such experimental evidence suggests that BSP molecules may reach the site of fertilization still attached to the spermatozoa. Thus, a potential role played by BSPs during fertilization itself, which would go beyond its capacitating effect, cannot be ruled out and needs to be further investigated. We noticed that epididymal sperm without heparin and BSP had similar cleavage rates compared to all treatments (but not T3) and produced blastocysts, although with low efficiency. The explanation for such results relates to the fact that capacitation is a complex process and that not all events associated with it are fully understood. For instance, in addition to heparin and BSPs, other factors can also generate capacitation and even some acrosome reaction, such as BSA, calcium, or magnesium. These substances are usually present in fertilization media and may have contributed to capacitate epididymal sperm, when we carried out our in vitro fertilization assays. Finally, incubation of Fert-TALP media with BSP1 and epididymal sperm did not cause any deleterious effect on embryo development, even when higher concentrations of the protein were used. Reasons for such result are not completely understood yet, but we suggest that it occurred because epididymal sperm did not have any membranebound BSP before they were translated into the fertilization media. Thus, as a hypothesis, most BSP1 added into the IVF media attached to sperm, leaving low quantities of free BSP1 to elicit detrimental effects on oocytes and/or embryos. Also, epididymal sperm had not been exposed to BSPs in the male reproductive tract and were cryopreserved without any contact with BSPs. As cited previously, BSP proteins can be detrimental to cryopreserved sperm because of membrane destabilization. Thus, loss of membrane phospholipids and cholesterol caused by an early contact with BSPs would not occur when epididymal sperm was used, contributing to preservation of membrane integrity during storage. In conclusion, the present study demonstrates a valid method for purification of BSP1 from bull SVF. This purified BSP1 was able to bind to and to capacitate sperm. Also, when added to Fert-TALP media, BSP1 induced in vitro development of bovine embryos. With ejaculated sperm, effects of low BSP1 concentration on cleavage and blastocyst rates were similar to those obtained with heparin. However, more BSP1 was associated with reduced blastocyst rates. With epididymal sperm, blastocyst development with either no heparin or heparin only was minimal,

whereas greater concentrations of BSP1 favored higher blastocyst rates. In contrast to what was detected with ejaculated sperm, BSP1 was not detrimental to the outcome of IVF when oocytes were fertilized with epididymal sperm. It is clear therefore that ejaculated and epididymal sperm have distinct responses to both heparin and BSP1, given the in vitro conditions established in our study. Such responses depend on previous exposure or not of spermatozoa to SP. The physiological pathways and molecular mechanism associated with such responses must be further investigated. Acknowledgments The present research has been supported by the Brazilian Research CouncildCNPq (grants 311272/2013-4, 400818/2013-2 and 562787/2010-0). Scholarships have been awarded to graduate students and post-doctoral fellows by CAPES (Commission for Support of High Education) and FUNCAP (Ceara State Foundation for Research and Scientific Development). The authors also appreciate the “Central Analítica” at the Federal University of Ceara, Brazil, funded by CT-INFRA/MCTI-SISNANO/Pró-Equipamentos CAPES. Competing Interests The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. References [1] Rego JP, Crisp JM, Moura AA, Nouwens AS, Li Y, Venus B, et al. Seminal plasma proteome of electroejaculated Bos indicus bulls. Anim Reprod Sci 2014;148:1–17. [2] Kelly VC, Kuy S, Palmer DJ, Xu Z, Davis SR, Cooper GJ. Characterization of bovine seminal plasma by proteomics. Proteomics 2006;6: 5826–33. [3] Belleannee C, Labas V, Teixeira-Gomes AP, Gatti JL, Dacheux JL, Dacheux F. Identification of luminal and secreted proteins in bull epididymis. J Proteomics 2011;74:59–78. [4] Manjunath P, Sairam MR. Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma. Biochem J 1987;241:685–92. [5] Calvete JJ, Raida M, Gentzel M, Urbanke C, Sanz L, Topfer-Petersen E. Isolation and characterization of heparin- and phosphorylcholinebinding proteins of boar and stallion seminal plasma. Primary structure of porcine pB1. FEBS Lett 1997;407:201–6. [6] Villemure M, Lazure C, Manjunath P. Isolation and characterization of gelatin-binding proteins from goat seminal plasma. Reprod Biol Endocrinol 2003;1:39. [7] Arangasamy A, Singh LP, Ahmed N, Ansari MR, Ram GC. Isolation and characterization of heparin and gelatin binding buffalo seminal plasma proteins and their effect on cauda epididymal spermatozoa. Anim Reprod Sci 2005;90:243–54. [8] Bergeron A, Villemure M, Lazure C, Manjunath P. Isolation and characterization of the major proteins of ram seminal plasma. Mol Reprod Dev 2005;71:461–70. [9] Leblond E, Desnoyers L, Manjunath P. Phosphorylcholine-binding proteins from the seminal fluids of different species share antigenic determinants with the major proteins of bovine seminal plasma. Mol Reprod Dev 1993;34:443–9. [10] Lefievre L, Bedu-Addo K, Conner SJ, Machado-Oliveira GS, Chen Y, Kirkman-Brown JC, et al. Counting sperm does not add up any more: time for a new equation? Reproduction 2007;133:675–84. [11] Manjunath P, Lefebvre J, Jois PS, Fan J, Wright MW. New nomenclature for mammalian BSP genes. Biol Reprod 2009;80: 394–7.

P. Rodríguez-Villamil et al. / Theriogenology 85 (2016) 540–554 [12] Fan J, Lefebvre J, Manjunath P. Bovine seminal plasma proteins and their relatives: a new expanding superfamily in mammals. Gene 2006;375:63–74. [13] Desnoyers L, Manjunath P. Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid. J Biol Chem 1992;267: 10149–55. [14] van Tilburg MF, Salles MG, Silva MM, Moreira RA, Moreno FB, Monteiro-Moreira AC, et al. Semen variables and sperm membrane protein profile of Saanen bucks (Capra hircus) in dry and rainy seasons of the northeastern Brazil (3 degrees S). Int J Biometeorol 2015;59:561–73. [15] Manjunath P, Chandonnet L, Leblond E, Desnoyers L. Major proteins of bovine seminal vesicles bind to spermatozoa. Biol Reprod 1994; 50:27–37. [16] Souza CE, Rego JP, Lobo CH, Oliveira JT, Nogueira FC, Domont GB, et al. Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams. J Proteomics 2012;75:4436–56. [17] Therrien A, Manjunath P, Lafleur M. Chemical and physical requirements for lipid extraction by bovine binder of sperm BSP1. Biochim Biophys Acta 2013;1828:543–51. [18] Manjunath P, Therien I. Role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation. J Reprod Immunol 2002;53:109–19. [19] Plante G, Therien I, Manjunath P. Characterization of recombinant murine binder of sperm protein homolog 1 and its role in capacitation. Biol Reprod 2012;87:1–11. [20] Therien I, Moreau R, Manjunath P. Major proteins of bovine seminal plasma and high-density lipoprotein induce cholesterol efflux from epididymal sperm. Biol Reprod 1998;59:768–76. [21] Lusignan MF, Bergeron A, Crete MH, Lazure C, Manjunath P. Induction of epididymal boar sperm capacitation by pB1 and BSP-A1/A2 proteins, members of the BSP protein family. Biol Reprod 2007; 76:424–32. [22] Parrish JJ. Bovine in vitro fertilization: in vitro oocyte maturation and sperm capacitation with heparin. Theriogenology 2014;81:67– 73. [23] Hung PH, Suarez SS. Alterations to the bull sperm surface proteins that bind sperm to oviductal epithelium. Biol Reprod 2012;87:88. [24] Visconti PE, Kopf GS. Regulation of protein phosphorylation during sperm capacitation. Biol Reprod 1998;59:1–6. [25] Lee CN, Ax RL. Concentrations and composition of glycosaminoglycans in the female bovine reproductive tract. J Dairy Sci 1984;67: 2006–9. [26] Parrish JJ, Susko-Parrish JL, Handrow RR, Sims MM, First NL. Capacitation of bovine spermatozoa by oviduct fluid. Biol Reprod 1989;40:1020–5. [27] Visconti PE, Krapf D, de la Vega-Beltran JL, Acevedo JJ, Darszon A. Ion channels, phosphorylation and mammalian sperm capacitation. Asian J Androl 2011;13:395–405. [28] Harrison RA, Gadella BM. Bicarbonate-induced membrane processing in sperm capacitation. Theriogenology 2005;63:342–51. [29] Xia J, Ren D. The BSA-induced Ca2þ influx during sperm capacitation is CATSPER channel-dependent. Reprod Biol Endocrinol 2009; 7:119. [30] Manjunath P, Sairam MR, Uma J. Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography. Biosci Rep 1987;7:231–8. [31] Martins JAM, Souza CEA, Silva FDA, Cadavid VG, Nogueira FC, Domont GB, et al. Major heparin-binding proteins of the seminal plasma from Morada Nova rams. Small Ruminant Res 2013;113: 115–27. [32] Moura AA, Koc H, Chapman DA, Killian GJ. Identification of proteins in the accessory sex gland fluid associated with fertility indexes of dairy bulls: a proteomic approach. J Androl 2006;27: 201–11. [33] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976;72:248–54. [34] Santos EA, Sousa PC, Martins JA, Moreira RA, Monteiro-Moreira AC, Moreno FB, et al. Protein profile of the seminal plasma of collared peccaries (Pecari tajacu Linnaeus, 1758). Reproduction 2014;147: 753–64. [35] Edman P. A method for the determination of amino acid sequence in peptides. Arch Biochem 1949;22:475. [36] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403–10.

553

[37] Moura AA, Chapman DA, Koc H, Killian GJ. A comprehensive proteomic analysis of the accessory sex gland fluid from mature Holstein bulls. Anim Reprod Sci 2007;98:169–88. [38] Souza CE, Moura AA, Monaco E, Killian GJ. Binding patterns of bovine seminal plasma proteins A1/A2, 30 kDa and osteopontin on ejaculated sperm before and after incubation with isthmic and ampullary oviductal fluid. Anim Reprod Sci 2008;105:72–89. [39] Rodriguez-Villamil P, Ongaratto FL, Fernandez Taranco M, Bo GA. Solid-surface vitrification and in-straw dilution after warming of in vitro-produced bovine embryos. Reprod Domest Anim 2014;49: 79–84. [40] Parrish JJ, Krogenaes A, Susko-Parrish JL. Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development. Theriogenology 1995;44:859–69. [41] Robertson I, Nelson R. Certification and identification of the embryo. In: Stringfellow DA, S S, editors. Manual of the International Embryo Transfer Society: a procedural guide and general information for the use of embryo transfer technology emphasizing sanitary procedures. USA, Savory Hill: International Embryo Transfer Society; 2007. p. 103134. [42] Martins CF, Rumpf R, Pereira DC, Dode MN. Cryopreservation of epididymal bovine spermatozoa from dead animals and its uses in vitro embryo production. Anim Reprod Sci 2007;101: 326–31. [43] SAS. SAS user’s guide. Cary, NC: SAS Institute; 2002. [44] Seidah NG, Manjunath P, Rochemont J, Sairam MR, Chretien M. Complete amino acid sequence of BSP-A3 from bovine seminal plasma. Homology to PDC-109 and to the collagen-binding domain of fibronectin. Biochem J 1987;243:195–203. [45] Moura AA, Souza CE, Stanley BA, Chapman DA, Killian GJ. Proteomics of cauda epididymal fluid from mature Holstein bulls. J Proteomics 2010;73:2006–20. [46] Cardin AD, Weintraub HJ. Molecular modeling of proteinglycosaminoglycan interactions. Arteriosclerosis 1989;9:21–32. [47] van Tilburg MF, Rodrigues MA, Moreira RA, Moreno FB, MonteiroMoreira AC, Candido MJ, et al. Membrane-associated proteins of ejaculated sperm from Morada Nova rams. Theriogenology 2013; 79:1247–61. [48] Calvete JJ, Raida M, Sanz L, Wempe F, Scheit KH, Romero A, et al. Localization and structural characterization of an oligosaccharide O-linked to bovine PDC-109. Quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa. FEBS Lett 1994;350:203–6. [49] Therien I, Soubeyrand S, Manjunath P. Major proteins of bovine seminal plasma modulate sperm capacitation by high-density lipoprotein. Biol Reprod 1997;57:1080–8. [50] Plante G, Thérien I, Lachance C, Leclerc P, Fan J, Manjunath P. Implication of the human Binder of SPerm Homolog I (BSPHI) protein in capacitation. Mol Hum Reprod 2014;20:409–21. [51] Gwathmey TM, Ignotz GG, Suarez SS. PDC-109 (BSP-A1/A2) promotes bull sperm binding to oviductal epithelium in vitro and may be involved in forming the oviductal sperm reservoir. Biol Reprod 2003;69:809–15. [52] D’Amours O, Bordeleau LJ, Frenette G, Blondin P, Leclerc P, Sullivan R. Binder of sperm 1 and epididymal sperm binding protein 1 are associated with different bull sperm subpopulations. Reproduction 2012;143:759–71. [53] Lu KH, Seidel Jr GE. Effects of heparin and sperm concentration on cleavage and blastocyst development rates of bovine oocytes inseminated with flow cytometrically-sorted sperm. Theriogenology 2004;62:819–30. [54] Mendes Jr JO, Burns PD, De La Torre-Sanchez JF, Seidel Jr GE. Effect of heparin on cleavage rates and embryo production with four bovine sperm preparation protocols. Theriogenology 2003; 60:331–40. [55] Therien I, Bleau G, Manjunath P. Phosphatidylcholine-binding proteins of bovine seminal plasma modulate capacitation of spermatozoa by heparin. Biol Reprod 1995;52:1372–9. [56] Calvete JJ, Mann K, Sanz L, Raida M, Topfer-Petersen E. The primary structure of BSP-30K, a major lipid-, gelatin-, and heparinbinding glycoprotein of bovine seminal plasma. FEBS Lett 1996; 399:147–52. [57] Gadella BM, Luna C. Cell biology and functional dynamics of the mammalian sperm surface. Theriogenology 2014;81:74–84. [58] Therien I, Moreau R, Manjunath P. Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm. Biol Reprod 1999;61:590–8.

554

P. Rodríguez-Villamil et al. / Theriogenology 85 (2016) 540–554

[59] Therien I, Bergeron A, Bousquet D, Manjunath P. Isolation and characterization of glycosaminoglycans from bovine follicular fluid and their effect on sperm capacitation. Mol Reprod Dev 2005;71:97–106. [60] Florman HM, First NL. Regulation of acrosomal exocytosis. II. The zona pellucida-induced acrosome reaction of bovine spermatozoa is

controlled by extrinsic positive regulatory elements. Dev Biol 1988; 128:464–73. [61] Moreau R, Therien I, Lazure C, Manjunath P. Type II domains of BSPA1/-A2 proteins: binding properties, lipid efflux, and sperm capacitation potential. Biochem Biophys Res Commun 1998;246:148–54.