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The extraction and purification of boar sperm surface proteins
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Chanida Kupradit and Mariena Ketudat-Cairns*
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School of Biotechnology, Institute of Agricultural Technology, Suranaree University
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of Technology, Nakhon Ratchasima, 30000, Thailand
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* ketudat@sut.ac.th, fax 66 44 22 4154
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Abstract
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Immunological sperm sexing is one of the desirable choices to separate X and
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Y sperm. The basic concept of immunological techniques is based on the different
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proteins on the surface between X and Y sperm. To investigate the sperm surface
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protein, proteomic investigation using 2 dimensional gel (2D-gel) electrophoresis
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technique has been applied. The initial step of protein extraction is very important for
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protein separation by 2D-gel. Thus the suitable strategies for sperm surface proteins
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extraction were considered in this research. The aim of this research was to extract
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proteins from unsorted boar spermatozoa surface for further use in 2D gel analyses.
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The sperm surface proteins were extracted and then purified using Con-A sepharose
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bead. Our research finding demonstrated that after 22 h incubation, 2.625 mg/ml and
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0.186 mg/ml of the total proteins can be obtained using extraction solution with and
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without 0.5% Triton X-100, respectively. The small size proteins (~13-16 kDa) were
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the major products which can be extracted immediately after incubation in extraction
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solution containing Triton X-100. From the purification results, all most all of the
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proteins especially the small size products (~13 and 16 kDa) tightly bound to the Con-
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A bead even in the present of 0.5% Triton X-100. These results indicated that the
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major extracted proteins in Triton X-100 solution were glycosylated proteins. In the
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future, surface proteins from sex sorted sperms (X sperm or Y sperm only) will be
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extracted by this strategy and then the protein pattern on 2D gel will be compared.
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Keywords: Immunological sexing, 2D gel, spermatozoa, surface protein
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1. Introduction
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Predetermination of sex of offspring from agriculturally important animal
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species could have a high significant impact on the efforts of producers to reduce
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production costs (Abeydeera et al., 1998). In pig, combination of sexed-sorting and
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cryopreservation of sperm prior to artificial insemination (AI) or in vitro fertilization
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(IVF) follow by embryo transfer have been applied (Bathgate et al., 2007). However,
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commercialization of sex sorted sperm using flow cytometry technique still have
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problems in term of high economic cost and sperm damage (George E. Seidel, 2003).
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Therefore immunological approaches are highly desirable since this technique is
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cheaper and less invasive for the sperm when compare to the flow cytometry
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technique. The basic concept of immunological techniques is based on the different
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proteins on the surface between X and Y sperm (Seidel and Johnson, 1999). Specific
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protein on X or Y sperm surface can be a good biomarker for monoclonal antibody
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production which specific to only X or Y sperm. Thus the investigation of proteins
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properties from X and Y sperm surface are required.
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According to boar sperm surface properties, it has been demonstrated that the
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ejaculated spermatozoa surface is coated with a number of seminal plasma proteins.
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These surface proteins have various biochemical activities such as haemagglutination,
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heparin-binding or zona pellucida-binding (Ohsako et al., 1997). The majority of
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these protein are the group of 12–16 kDa glycoproteins that bind to the sperm surface
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(Caballero et al., 2008).
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To investigate the sperm surface protein, the science of proteomics is an
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alternative approach used for comparisons of the proteomes which are involved in
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biological process from difference sources (Strzeżek et al., 2005). To separate the
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protein from each sample for protemic investigation, two-dimensional gel (2D-gel)
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electrophoresis has been used (Hamady et al., 2005). The initial step of protein
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extraction is a very importance part in using this technique. Thus the suitable
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strategies for sperm surface protein extraction should be considered.
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The objective of this research was to extract protein from unsorted boar
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spermatozoa surface for further analyze by 2D gel. The groups of 12–16 kDa
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glycoproteins that bind to the sperm surface were used as marker to confirm that the
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protein of sperm surface can be extracted. The extractions of sperm surface protein
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were purified by Con-A sepharose beads (Amersham) to prove that protein derived by
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this investigation were glycosylated surface protein.
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2. Materials and Methods
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2.1 Sperm preparation
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To separate sperm pellets from seminal plasma and dilution medium, 150 ml
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of fresh boar sperm were centrifuged at 4,000 rpm, 25oC for 10 min. Then the sperm
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pellets were re-suspend in 40 ml 1X PBS (phosphate buffer saline) pH 7.4 and
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centrifuged at 4,000 rpm, 25oC for 10 min to wash sperm pellets. These sperm pellets
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were washed 8 times in 1X PBS pH 7.4. These sperm pellets were then solubilized in
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extraction solutions for sperm surface protein extraction.
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2.2 Sperm surface protein extraction
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Washed sperm pellets were directly incubated in 15 ml centrifuged tube
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containing 5 ml extraction solution (20 mM Tris-Cl pH 7.4, 1 mM PMSF, 1X
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protease cocktail inhibitor (Sigma), 0.5% Triton X-100, 0.5 M NaCl) by shaking on-
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ice for 22 h. For the negative control, the equal amount of sperm pellets were
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incubated in the same conditions without 0.5% Triton X-100 and 0.5 M NaCl.
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After 22 h of incubation, numbers of sperm cell were determined by
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heamacytometer. Proteins in the supernatant of each extraction solution, total sperm
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cell suspension and sperm cell pellets were detected by 15% SDS-PAGE. Total
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protein concentrations of each sample were measured by Bradford method (Bradford).
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2.3 Purification of sperm surface protein using Con-A sepharose bead
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Before the purification process, 200 ul of Con-A sepharose 4B beads (GE
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Healthcare) were equilibrated with 5 ml of 20 mM Tris-Cl pH 7.4 containing 0.5 M
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NaCl, 1 mM CaCl2 and 1 mM MnCl2. Then 1 ml of each sperm surface protein
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sample containing 1 mM CaCl2 and 1 mM MnCl2 was incubated with 200 µl bead by
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shaking on ice for 1 h. At the end of binding step, the flow-through fraction was
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collected. The binding steps were repeated 3 times. After flow-through was collected,
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the unbound proteins were eliminated by washing the bead with 5 ml of washing
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solution (20 mM Tris-Cl pH 7.4, 0.5 M NaCl, 100 mM D-glucopyranoside). Washing
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fractions were collected after the washing step. In the step of elution, Con-A beads
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containing bound proteins were incubated on-ice with 0.1-1 ml elution buffer (20 mM
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Tris-Cl pH 7.4, 0.5 M NaCl, 800 mM D-glucopyranoside) for 3 min. Each elution
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fractions were collected during elution step. Finally, the glycosylated proteins were
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eluted with this elution buffer in the total volume of 2 ml. The fractions of flow-
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through, washing and elution were subjected to 15% SDS-PAGE for protein band
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detection.
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3. Results and Discussion
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3.1 Sperm surface protein extraction
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The surface proteins from the unsorted boar sperm (approximately 2 x 109
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cell/ml) were extracted using extraction solution with and without 0.5% Triton X-100.
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After 22 h incubation, 2.625 mg/ml and 0.186 mg/ml of the total proteins were
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obtained using extraction solution with and without 0.5% Triton X-100, respectively.
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The results of protein extraction (Fig 1) showed that the high intensity of small size
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protein bands can be detected in extraction solution containing Triton X-100 but not
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in the control. Similar results have been reported by Ohsako et al. (1997) whom
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extracted miniature swine sperm surface protein using a hypertonic saline solution.
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They observed 13 and 16 kDa proteins in the extraction fraction (detected on 15%
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SDS-PAGE). Both proteins were localized to whole sperm surface. Moreover, they
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reported that the 13 kDa protein had haemagglutination activity while the 16 kDa
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does not.
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In boar, bulk of seminal plasma proteins (>90%) belong to the spermadhesin
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family. They are a group of 12–16 kDa glycoproteins that bind to the sperm surface
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(Caballero et al., 2008). Spermadhesins, AQN (12 kDa), AWN (15 kDa), PSP (14-16
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kDa), and DQH (13 kDa) sperm surface protein are the most abundant seminal protein
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(Calvete et al., 1995; Maňásková and Jonáková, 2008). Heparin binding protein,
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AQN-1 and DQH play a role to stabilize the plasma membrane over the acrosomal
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vesicle and probably participate in formation of sperm oviductal reservoir (Ekhlasi-
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Hundrieser et al., 2005; Maňásková et al., 2007). Non-heparin binding protein,
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heterodimer complex of PSP-I/ PSP-II, mainly localized to the acrosomal area to
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preserved sperm viability, motility, and mitochondrial activity (Caballero et al.,
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2006) . Our research finding suggested that the small protein band (13-16 kDa) should
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be the seminal plasma protein which bound to the sperm surface.
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The comparison of the intense protein bands showed that the extracted protein
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pattern derived from solution containing 0.5% Triton X-100 at 0 and 22 h were not
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different (Fig 1 lane 5 and 6). These results made clear that these surface proteins can
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be extracted immediately after incubated in extraction solution containing Triton X-
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100.
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3.2 Purification of sperm surface protein using Con-A sepharose bead
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Regarding to the purification of surface proteins, Con-A sepharose bead were
Con-A
binds
molecules
containing
α-D-mannopyranosyl,
α-D-
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applied.
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glucopyranosyl and sterically related residues. This affinity ligand can be used for
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applications such as isolation of cell surface glycoproteins from detergent-solubilized
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membranes (Amersham Bioscience).
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To confirm that the sperm surface proteins were solubilized in the extraction
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solution containing 0.5% Triton X-100, sperm surface proteins in each extraction
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solution was purified using Con-A sepharose bead (GE Healthcare). The proteins
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were then eluted with elution solution containing 800 mM D-glucopyranoside. The
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purification results of the extracted proteins using Triton X-100 (Fig 2) showed that
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the small size protein (~ 13 and 16 kDa) tightly bound to the Con-A bead even in the
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present of 0.5 % Triton X-100. These indicated that these small size products were the
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glycosylated protein.
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When compare the pattern of extracted protein bands between Triton X-100
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(Fig 2) and the control without Triton X-100 (Fig 3) that bind to Con-A bead, the
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results showed that the band pattern were different. The protein bands which present
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in extraction solution without Triton X-100 should be some background of
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hydrophilic surface or glycosylated protein. Although some protein can be solubilized
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in the control but with very low concentration when compare to surface proteins
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concentration derived from using Triton X-100. These results made clear that the
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extracted proteins derived from Triton X-100 were the sperm surface proteins.
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However, from the purification results showed that the bound protein can not
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be eluted from Con-A bead even in high concentration of D-glucopyranoside in strong
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elution solution (Fig 2 lane 8 and 15). It is possible that these glycosylated proteins
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tightly bound to Con-A bead and very difficult to elute from the bead. The
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observation of protein band pattern from purification result (Fig 2) showed that almost
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all of the proteins which solubilized in Triton X-100 solution were able to bind to
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Con-A bead. These results demonstrated that the major protein that present in Triton
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X-100 solution were the glycosylated surface proteins.
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4. Conclusions
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Pre-selection sex of offspring in livestock reproduction is important for
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improving reproductive management and reduces reproduction time and cost. Sperm
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sexing by immunological method is one of the desirable choice for sperm sexing. The
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advantages of this technique are low processing cost, less invasive for the sperm and
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no limitation in the yield sperm. To separate the protein from each sample for
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proteomic investigation, 2D-gel has been applied. The suitable strategies of surface
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protein extraction from boar sperm were investigated. The sperm surface protein can
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be extracted by Triton X-100. The small size proteins (~13-16 kDa) were the major
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products which present in the extraction solution containing 0.5% Triton X-100.
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These products should be the seminal plasma proteins that bind to sperm surface.
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These proteins can be extracted immediately after incubated in extraction solution
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containing Triton X-100. The purification of these sperm surface protein using Con-A
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bead indicated that almost all of the proteins which solubilized in Triton X-100
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solution were the glycosylated proteins. Thus all of them were able to bind to Con-A
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sepharose bead.
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For further work, surface proteins from sex sorted sperms (X sperm or Y
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sperm only) will be extracted and then compared the protein pattern on 2D-gel. The
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different proteins from X and Y sperm will be identified as protein marker. The long
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term goal is to try to produce monoclonal antibody to the X and Y specific sperm
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surface proteins for sperm sexing application by immunological technique.
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5. Acknowledgements
SUT farm is thanks for providing the fresh swine sperm. Chanida Kupradit is
supported by CHE-PHD-THA-SUP from the commission on higher education.
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6. References
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Abeydeera, L. R., Johnson, L. A., Welch, G. R., Wang, W. H., Boquest, A. C.,
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Cantley, T. C., Rieke, A., and Day, B. N. (1998). Birth of piglets preselected
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for gender following in vitro fertilization of in vitro matured pig oocytes by X
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and Y chromosome bearing spermatozoa sorted by high speed flow cytometry.
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Theriogenology, 50: 981-988.
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Bathgate, R., Morton, K. M., Eriksson, B. M., Rath, D., Seig, B., Maxwell, W. M. C.,
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and Evans, G. (2007). Non-surgical deep intra-uterine transfer of in vitro
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produced porcine embryos derived from sex-sorted frozen–thawed boar sperm.
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Anim. Reprod. Sci., 99: 82–92.
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Caballero, I., Vázqez, J. M., Garciá, E. M., Roca, J., Martínez, E. A., Calvete, J. J.,
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Sanz, L., Ekwall, H., and Rodríguez-Martínez, H. (2006). Immunolocalization
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and possible functional role of PSP-I/PSP-II heterodimer in highly extended
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boar spermatozoa. J. Andrology, 27(6): 766-773.
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Caballero, I., Vazquez, J. M., Garcıá, E. M., Parrilla, I., Roca, J., Calvete, J. J., Sanz,
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L., and Martínez, E. A. (2008). Major proteins of boar seminal plasma as a
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tool for biotechnological preservation of spermatozoa. Theriogenology, 70(8):
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1352-1355.
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Calvete, J. J., Mann, K., Schäifer, W., Raida, M., Sanz, L., and Töpfer-Petersen, E.
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(1995). Boar spermadhesin PSP-II: location of posttranslational modifications,
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heterodimer formation with PSP-I glycoforms and effect of dimerization on
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the ligand-binding capabilities of the subunits. FEBS Lett., 365: 179-182.
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Ekhlasi-Hundrieser, M., Gohr, K., Wagner, A., Tsolova, M., Petrunkina, A., and
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Töpfer-Petersen, E. (2005). Spermadhesin AQN1 is a candidate receptor
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molecule involved in the formation of the oviductal sperm reservoir in the pig.
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Biol. Reprod., 73: 536-545.
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George E. Seidel, J. (2003). Sexing mammalian sperm-intertwining of commerce,
technology, and biology. Anim. Reprod. Sci., 79(3-4): 145-156.
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Hamady, M., Cheung, T. H. T., Resing, K., Cios, K. J., and Knight, R. (2005). Key
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challenges in proteomics and proteoinformatics. Eng. Med. Biol. Mag., 24(3):
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34-40.
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Maňásková, P., Pěknicová, J., Elzeinová, F., Tichá, M., and Jonáková, V. (2007).
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Origin, localization and binding abilities of boar DQH sperm surface protein
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tested by specific monoclonal antibodies. J. Reprod. Immunol., 74: 103-113.
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Maňásková, P., and Jonáková, V. (2008). Localization of porcine seminal plasma
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(PSP) proteins in the boar reproductive tract and spermatozoa. J. Reprod.
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Immunol., 78: 40-48.
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Ohsako, S., Ikoma, E., Nakanishi, Y., Nagano, R., Matsumoto, M., and
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Nishinakagawa, H. (1997). Isolation of a miniature swine seminal plasma
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haemagglutinin from the sperm surface. J. Reprod. Dev., 43(4): 311-319.
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Seidel, G. E., and Johnson, L. A. (1999). Sexing mammalian sperm-overview.
Theriogenology, 52: 1267-1272.
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Strzeżek, J., Wysocki, P., Kordan, W., Kuklińska, M., Mogielnicka, M., Soliwoda, D.,
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and Fraser, L. (2005). Proteomics of boar seminal plasma – current studies and
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possibility of their application in biotechnology of animal reproduction.
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Reprod. Biol., 5(3): 279-290.
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Figure 1 Boar sperm surface protein analysis using 15% SDS-PAGE. Sperm pellet
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were incubated in extraction solution with (+) or without (-) 0.5% Triton X-
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100 for 0 and 22 h. P: Sperm pellet; T: Total sperm cell suspension; S:
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Sperm surface protein in supernatant; lane 1: Protein molecular marker
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(Fermentas); lane 2: P from 0.5% Triton X-100 + 0.5 M NaCl at 22 h; Lane
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3: T from 0.5% Triton X-100 + 0.5 M NaCl at 0 h; lane 4: T from 0.5%
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Triton X-100 + 0.5 M NaCl at 22 h; lane 5: S from 0.5% Triton X-100 + 0.5
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M NaCl at 0 h; lane 6: S from 0.5% Triton X-100 + 0.5 M NaCl at 22 h; lane
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7: T from Tris-Cl at 0 h; lane 8: T from Tris-Cl at 22 h; lane9: S from Tris-
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Cl at 0 h ; lane 10: S from Tris-Cl at 22 h . The major product of small size
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sperm surface proteins were shown in the box.
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Figure 2 The SDS-PAGE (15%) analysis of boar sperm surface protein purification
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fractions using Con-A sepharose bead. Boar sperm surface proteins were
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extracted by extraction solution containing 0.5% Triton X-100 and 0.5 M
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NaCl for 22 h. lane1: Protein molecular marker (Fermentas); lane 2: soluble
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fraction (s); lane 3: flow-through fraction; lane 4: Con-A bead after binding
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step; lane 5-7 wash fraction number 2, 4, and 6; lane 8: Con-A bead after
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washing step; lane 9-14: elution fraction number 1, 2, 3, 4, 6, 8; lane 15:
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Con-A bead after elution step.
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Figure 3 The SDS-PAGE (15%) analysis of boar sperm surface protein purification
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fractions using Con-A sepharose bead. Boar sperm surface proteins were
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extracted by extraction solution without 0.5% Triton X-100 for 22 h
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(negative control). lane1: Protein molecular marker (Fermentas), lane 2:
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soluble fraction (s); lane 3: flow-through fraction; lane 4: Con-A bead after
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binding step; lane 5-7 wash fraction number 2, 4, and 6; lane 8: Con-A bead
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after washing step; lane 9-14: elution fraction number 1, 2, 3, 4, 6, 8; lane 15:
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Con-A bead after elution step.
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Figure 1
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Figure 2
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Figure 3
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