| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Swine Reproduction-Development Program, 4-10 Ag/For Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada.
| Correspondence to: Dr Michael Dyck, 4-10 Ag/For Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada (e-mail: michael.dyck{at}ualberta.ca). |
| Received for publication January 21, 2009; accepted for publication August 18, 2009. |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Key words: Male fertility, artificial insemination, semen, pig, 2-D gel electrophoresis, tandem mass spectrometry
Seminal plasma is composed of secretions from the male accessory sex glands and epididymis, which contains many organic and inorganic components that have effects on sperm quality (Foxcroft et al, 2008). The proteins secreted into seminal plasma may play an important role during sperm capacitation and fertilization (Rodríguez-Martínez et al, 1998) and may also serve to protect sperm from damage or to maintain their longevity. In the boar, the ejaculate consists of a large volume of semen with a relatively dilute sperm concentration. Boar semen is ejaculated in specific sequential fractions; namely, the first sperm-rich fraction, followed by the relatively sperm-free fraction prior to the second sperm-rich fraction, and then the gel fraction at the end of the ejaculate collection. Pig semen destined for use in AI is often composed of the pooled first sperm-rich fraction, whereas the remainder of the ejaculate is usually discarded. During natural mating, the sperm are usually only exposed to proteins in the initial sperm-rich fraction, and it has been shown that the exposure of sperm to the later fractions of the ejaculate has a detrimental effect on sperm quality and relative fertility (as reviewed by Rodríguez-Martínez et al, 2008). For example, previous studies from our laboratory showed differences in oocyte penetration rates in vitro when sperm were preincubated with different fractions of seminal plasma from the same ejaculate (Zhu et al, 2000). One of the goals of this study was to evaluate the specific protein content of these different fractions to better understand their individual contributions to fertility potential and the roles of these proteins in fertility.
Using two-dimensional (2-D) gel electrophoresis, Killian et al (1993) identified 2 seminal plasma proteins with high fertility in bulls (26 and 55 kDa) and 2 proteins that were correlated with low fertility (16 and 16 kDa). The 55-kDa fertility-associated protein has been identified as osteopontin (Cancel et al, 1999), and the 26-kDa fertility-associated protein has been identified as lipocalin-type prostaglandin D synthase (Gerena et al, 1998). In stallions, Brandon et al (1999) reported that a 72-kDa seminal plasma protein, also identified as horse osteopontin, was positively correlated with fertility. Osteopontin has recently been localized on ejaculated bull sperm and may play a role in fertilization and also as a block to polyspermy (Erikson et al, 2007). In the pig, addition of osteopontin during in vitro fertilization also reduced polyspermy rates (Hao et al, 2006) and improved embryo development after fertilization (Hao et al, 2008).
Specific proteins from the spermadhesin family, such as porcine seminal protein (PSP), AWN, and AQN, coat the sperm surface during ejaculation, producing structural changes to the sperm plasma membrane that affect sperm performance during the fertilization process (Manásková et al, 2003). The major seminal plasma glycoprotein (PSP-I), isolated and identified from boar seminal plasma by Rutherfurd et al (1992), may prevent premature capacitation and the acrosome reaction (Kwok et al, 1993; Töpfer-Petersen et al, 1998). This protein may also have immunoregulatory activity (Kwok et al, 1993; Yang et al, 1998; Assreuy et al, 2002, 2003). AWN-1 is another spermadhesin that affects zona pellucida–binding activity (Sanz et al, 1992; Rodríguez-Martínez et al, 1998) and may also have a role in the capacitation process (Calvete et al, 1997; Töpfer-Petersen et al, 1998). AWN-1 is synthesized by the rete testis, prostate, seminal vesicles (Sinowatz et al, 1995), and female reproductive tract (Ekhlasi-Hundrieser et al, 2002). Given the functions of these spermadhesins, they may potentially be candidate markers of boar fertility.
The main objective of the present study was to evaluate specific seminal plasma proteins—PSP-I, AWN-I, and osteopontin—in different ejaculate fractions that could be effective predictors of relative boar fertility using a population of boars that 1) would be considered acceptable for use in AI programs on the basis of ejaculate/sperm characteristics measured in most commercial AI centers, and 2) have established differences in in vivo fertility when only 1.5 billion sperm per AI dose are used for insemination (Ruiz-Sánchez et al, 2006). The seminal plasma proteome in the pooled sperm-rich fraction used for AI was also compared across boars of low and high fertility to further investigate potential markers of fertility.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Semen Collection for Seminal Plasma Evaluation
Ejaculates were collected into a series of sterile, prewarmed 15-mL Falcon
tubes (VWR Canlab, Mississauga, Canada) as described by Xu et al
(1996,
1998)
(Figure 1), and 3 fractions
were defined on the basis of sperm concentration
(Ruiz-Sánchez et al,
2006). Briefly, tubes containing the first Sperm-Rich fraction
were identified both visually and using a calibrated colorimeter (model 254;
Sherwood Scientific Ltd, Cambridge, United Kingdom). The Sperm-Peak fraction
was considered to be the tube containing the highest sperm concentration
within the Sperm-Rich fraction (usually the first 10-mL fraction collected).
The last tubes of the first Sperm-Rich fraction were measured to identify the
final tube with a concentration of 100 x 106 or more
spermatozoa per milliliter; this and the previous tubes were included as part
of the first Sperm-Rich fraction. Finally, tubes containing less than 100
x 106 spermatozoa per milliliter, between the first
Sperm-Rich fraction and the second Sperm-Rich fraction, were considered to be
part of the Sperm-Free fraction. All of the tubes from the first Sperm-Rich
fraction were then combined in a prewarmed thermos and filtered through gauze
to eliminate any gel component, and this combined first Sperm-Rich fraction
was then used for routine semen evaluation (twice a week during the evaluation
period) and for breeding by AI (every 3 weeks out of 4).
|
On 4 occasions during the evaluation period, 3-mL aliquots of each identified fraction (Sperm-Peak, the combined Sperm-Rich, and the Sperm-Free) were used for seminal plasma protein evaluation. After collection, semen samples were centrifuged at 1020 x g for 30 minutes at 4°C, and the seminal plasma supernatant recovered was frozen with liquid nitrogen and stored at –20°C until analysis.
Seminal Plasma Protein Evaluation In Vitro
Seminal plasma samples were thawed and immediately centrifuged at 10 000
x g for 15 minutes to remove any cellular debris. Total protein
concentration was then quantified using the Pierce BCA Protein Assay (Pierce
Biotechnology, Rockford, Illinois) according to the manufacturer's
directions.
PSP-I Analysis by Western Blot
Based on the preliminary studies of Zhu et al
(2000), an aliquot of each
sample was deglycosylated to remove the N-linked oligosaccharides from the
PSP-I glycoprotein, allowing quantification of a single PSP-I band by Western
blot analysis. Aliquots of 100 µg of total protein seminal plasma samples
were diluted to a final concentration of 1 mg/mL with phosphate buffer (20 mM
sodium phosphate, pH 7.5; 50 mM EDTA; and 0.02% [wt/vol] sodium azide)
supplemented with 0.5% (wt/vol) sodium dodecyl sulfate (SDS) and 5% (vol/vol)
β-mercaptoethanol and boiled for 2 minutes to denature the proteins.
Then, 2.5% (vol/vol) Nonidet P-40 and 2 units of Peptide-N-Glycosidase (Roche,
Penzberg, Germany) were added and incubated for 16–18 hours at 37°C.
After protein digestion, the deglycosylated proteins were precipitated with 3
volumes of cold acetone for 45 minutes and centrifuged at 3000 x
g for 20 minutes. The pellet was washed twice with cold acetone and
stored at –20°C until further analysis.
The PSP-I antibody was kindly donated by Dr Kwok (Department of Obstetrics and Gynecology, Albert Einstein Medical Center, Philadelphia, Pennsylvania). PSP-I was evaluated in both glycosylated and deglycosylated (D-PSP-I) samples from the 3 fractions and 4 time periods (collections) from each of the 9 boars. Both deglycosylated and glycosylated samples were separated on 15% SDS-polyacrylamide gels. A total of 5 µg of total protein from each sample and a control (pooled) sample was loaded onto each gel. After electrophoresis, proteins were transferred to Hybond ECL nitrocellulose membrane (GE Healthcare, Quebec, Canada) using a constant current of 75 mA for 15 hours at 4°C. Membranes were blocked with tris-buffered saline (TBS) supplemented with 0.1% (vol/vol) Tween 20 (TBS-T) and 5% (wt/vol) nonfat milk for 1 hour at room temperature. Membranes were then washed 3 times (5 minutes each) with TBS-T and incubated for 1 hour with primary antiserum raised against PSP-I (1:20 000 dilution), followed by three 5-minute washes. The membranes were then incubated with anti-rabbit immunoglobulin G (IgG) and peroxidase-linked donkey antibody (GE Healthcare) for 30 minutes at room temperature (1:16 000 dilution) and washed 3 times with TBS-T for 5 minutes. Immunoactivity was detected by ECL chemiluminescence (GE Healthcare) according to the manufacturer's instructions. The membrane was exposed to Hyperfilm-ECL (GE Healthcare) for 10 seconds and developed. The films were scanned using an imaging densitometer (Bio-Rad Laboratories, Hercules, California), and immunoreactive PSP-I bands were quantified using densitometry analysis software (Molecular Analyst v2.01; Bio-Rad Laboratories). To standardize the gels, an internal control (pooled) sample was run in each blot. The relative abundance of PSP-I (arbitrary units per microgram of total protein) was calculated using the volume (optical density by surface area) of the sample PSP-I band divided by the volume of the internal control band and then divided by the amount of protein loaded (5 µg). The PSP-I concentration per milliliter of seminal plasma (PSP-I/mL) was estimated as PSP-I abundance x the total protein concentration. Finally, the total amount of PSP-I per AI dose (PSP-I/AI dose) was calculated as PSP-I/mL x estimate volume of seminal plasma included in each AI dose of semen. AWN-1 and osteopontin protein concentrations were calculated in the same manner.
AWN-I Analysis by Western Blotting
Because of limitations in the amount of AWN-1 antibody available (kindly
provided by Dr F. Sinowatz, Department of Veterinary Anatomy, University of
Munich, Munich, Germany; and Dr E. Töpfer-Petersen, Institut für
Reproduktionsmedizin, Tierärzttliche Hochschule Hannover, Hannover,
Germany), AWN-1 analysis was limited to seminal plasma samples of the 2
highest-fertility (R-2 and Y-2) and the 2 lowest-fertility (G-1 and R-1)
boars, based on their fertility index assessed in vivo
(Table 1); for each boar, 3
different seminal plasma fractions from the 4 ejaculates collected were
analyzed. The Western blot protocol was as used for PSP-I with the following
differences: The dilutions used for the first (AWN-1) and second (rabbit
anti-chicken IgG, IgY peroxidase antibody; A9046; Sigma Chemical, St Louis,
Missouri) antibodies were 1:1000 and 1:10 000, respectively.
|
Osteopontin Analysis by Western Blotting
Equivalent samples from the same 4 boars analyzed for AWN-1 were also
evaluated for osteopontin (OPN), again using the PSP-I Western blotting
protocol with the following variations based on Johnson et al
(1999). Proteins (25 µg)
were loaded onto each well and separated on a 15% polyacrylamide gel. A sample
of sow's milk was also used as an OPN-positive control because osteopontin is
known to be enriched in milk. The membranes were initially blocked overnight
at 4°C with TBS-T 5% nonfat milk, incubated overnight with a cocktail of
rabbit polyclonal antibodies against recombinant human OPN (LF-123 and LF-124;
1:1000) (Fisher et al, 1995),
and then incubated for 2 hours at room temperature with a
peroxidase-conjugated donkey anti-rabbit IgG secondary antibody (1:10 000).
The membrane was exposed to a Hyperfilm ECL for 15 seconds. Specificity of the
first antibody was previously tested by Cancel et al
(1997) to identify OPN in bull
semen and by Johnson et al
(1999) to identify OPN in the
uterus.
2-D Gel Electrophoresis of Seminal Plasma Proteins
Proteomics analysis was restricted to the Sperm-Rich fraction of the same
samples used for AWN-1 and OPN analysis because this was representative of the
seminal plasma proteins in the dose used for artificial insemination. The
Sperm-Rich fraction was extracted using the same acetone precipitation method
described earlier, and 100 µg of protein was solubilized in rehydration
buffer (7 M urea; 2 M thiourea; 4% [wt/vol] CHAPS; 0.5% [vol/vol] pharmalytes,
pH 3–10; and 20 mM dithiothreitol [DTT]) for 1 hour at room temperature
before loading the sample onto 7-cm linear pH 3–10 Immobiline (GE
Healthcare) strips for overnight rehydration. The 1-D separation program on an
Ettan IPGPhor isoelectric focusing apparatus (GE Healthcare) was 10 minutes at
500 V, 10 minutes at 1000 V, 1.5 hours at 4000 V, and 1 hour at 5000 V for a
total of 11 250 Volt-hours. After focusing, strips were equilibrated in 50 mM
tris-HCl, 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) SDS, and bromophenol
blue for 15 minutes with 1% (wt/vol) DTT, and then alkylated using 2.5%
(wt/vol) iodoacetamide for 15 minutes. The gel strips were then loaded onto
simultaneously cast 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels
to separate proteins in the second dimension according to their molecular
weight. The 7-cm strips were all run simultaneously using an Ettan DALTsix gel
electrophoresis system (GE Healthcare), with three 7-cm strips run on each of
six 24-cm slab gels in the second dimension. The resulting gels were then
fixed overnight and proteins visualized using silver staining.
For each gel, the protein spots were quantified using Imagemaster 2-D Elite analysis software (GE Healthcare). The individual volume measurements (in relative units) for each protein were corrected for the total spot volume on each gel and were also normalized across gels using same reference gel. The spot volume measurements were exported into SAS (SAS Institute Inc, Cary, North Carolina) for analysis.
Liquid Chromatography-Tandem Mass Spectrometry Identification of Seminal Plasma Proteins
A preparative 2-D gel was run with 1 mg of total protein on a 24-cm
Immobiline DryStrip Gel (GE Healthcare) in the linear pH 3–10 range
using an extended electrofocusing protocol for 65 000 Volt-hours, and then it
was loaded onto a 12% SDS-PAGE slab gel. The resulting gel was fixed overnight
in 50% (vol/vol) methanol and stained using Bio-Safe Coomassie Blue (Bio-Rad
Laboratories). The protein spots of interest were manually excised and sent to
a mass spectrometry (MS) facility for further processing and identification
(Centre Genomique du Quebec, Sainte-Foy, Canada). Tryptic digestions of the
proteins were performed on a MassPrep liquid handling robot (Waters,
Mississauga, Canada) according to the manufacturer's specifications and using
sequencing-grade modified trypsin (Promega, Madison, Wisconsin).
Peptide extracts were separated by online reversed-phase nanoscale capillary liquid chromatography and analyzed by electrospray MS using an LTQ linear ion trap mass spectrometer (Thermo Electron, San Jose, California) equipped with a nanoelectrospray ion source (Thermo Electron). Peptide separation took place within a PicoFrit column BioBasic C18, 10 cm x 0.075 mm internal diameter (New Objective, Woburn, Massachusetts) with a linear gradient from 2%–50% solvent B (acetonitrile, 0.1% formic acid) in 30 minutes at 200 nL/min. Mass spectra were acquired using data-dependent acquisition mode (Xcalibre software, version 2.0). Each full-scan mass spectrum (400–2000 m/z) was followed by collision-induced dissociation of the 7 most intense ions. The dynamic exclusion function was enabled (30-second exclusion), and the relative collisional fragmentation energy was set to 35%.
Interpretation of Tandem MS Spectra
All tandem MS (MS/MS) samples were analyzed using Mascot (version 2.2.0;
Matrix Science, London, United Kingdom). The database used to search tryptic
peptides was the mammalian protein database Uniref_100_14_Mammalia_40674.
Fragment and parent ion monoisotopic mass tolerances were, respectively, of
0.5 Da and 2.0 Da. Iodoacetamide derivative of cysteine was specified as a
fixed modification and methionine oxidations were specified as variable
modifications. Two missed cleavages were allowed.
Criteria for Protein Identification
Scaffold (version Scaffold_2.1.03; Proteome Software Inc, Portland, Oregon)
was used to validate MS/MS-based peptide and protein identifications. Peptide
identifications were accepted if they could be established at greater than
95.0% probability as specified by the Peptide Prophet algorithm
(Keller et al, 2002). Protein
identifications were accepted if they could be established at greater than
95.0% probability and contained at least 2 unique identified peptides. Protein
probabilities were assigned by the Protein Prophet algorithm
(Nesvizhskii et al, 2003).
Proteins that contained similar peptides and could not be differentiated based
on MS/MS analysis alone were grouped to satisfy the principles of
parsimony.
Statistical Analysis
Differences between seminal plasma fractions and total protein and PSP-I
abundance for all 9 boars were analyzed using a repeated-measures analysis by
a mixed procedure of the Statistical Analysis System (SAS version 8.1; SAS
Institute). The fixed effects were time, fractions, and their interaction;
boar nested within fraction was used as the subject, and the boar group was a
random effect. AWN-I, OPN, and 2-D gel data were grouped for the 4 selected
boars within fertility class (the highest-fertility and the lowest-fertility
boars) and evaluated using a mixed procedure of SAS. The fixed effects were
time, fertility class, and their interaction; boar was used as the subject,
and the boar group was a random effect. In all statistical models, the
Kenward-Roger option was used to calculate the denominator degrees of freedom.
The variance-covariance matrix was chosen for each statistical model by an
interactive process wherein the best-fitting model was based on Schwarz
Bayesian criteria. Least square means and standard errors were generated and
separated using a pdiff adjusted by the Tukey option for significant, fixed
effects. All data are presented as least square means ± standard errors
of least square means.
The Insight procedure of SAS was used to evaluate the correlation between fertility in vivo (pregnancy rate, farrowing rate, total pigs born, and fertility index) and seminal plasma proteins (total protein content, PSP-I, AWN-I, osteopontin, and protein abundance in the 2-D gels).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Seminal Plasma Analysis
Different semen fractions (Sperm-Peak, Sperm-Rich, and Sperm-Free) have
been shown to result in differential fertility and sperm quality when sperm
are preincubated with these fractions independently. As a result, the specific
proteins, PSP-I, AWN-I, and osteopontin, were analyzed within these discrete
fractions to determine whether there were differences between fractions that
related to boar fertility in vivo. The seminal plasma proteome was restricted
to analysis within the Sperm-Rich fraction only, because this was the fraction
that was used for AI.
Seminal Plasma Evaluation: Total Protein and D-PSP-I
Total protein concentration and PSP-I abundance were analyzed across all 9
boars in the study within seminal plasma fractions. Their associations with
relative fertility were also established. For Western blotting, the specific
antiserum against PSP-I showed immunoactivity against both glycosylated
(PSP-I) and deglycosylated PSP-I (D-PSP-I) proteins in the range of
14–20 kDa and 12 kDa, respectively
(Figure 2). There were no
statistical differences between PSP-I and D-PSP-I, and thus D-PSP-I values
were chosen to represent the abundance for that protein throughout the
paper.
|
|
D-PSP-I relative abundance in the Sperm-Rich fraction was negatively correlated with pregnancy rate (r = –0.46, P = .005), farrowing rate (r = –0.43, P = .007), and fertility index (r = –0.42, P = .009). Sperm concentration and total protein concentration were negatively correlated when data of the Sperm-Peak and Sperm-Rich fractions were included in the model (r = –0.45, P = 0.0001), confirming an inverse relationship between sperm concentration and the amount of protein in seminal plasma.
|
|
Comparisons of seminal plasma fractions demonstrated that OPN-9 and OPN-12 concentrations were lower (P < .05) in the Sperm-Peak fraction than in the other 2 fractions (Table 2). Concentrations of AWN-1 and OPN-70 were lower (P < .05) in the Sperm-Peak fraction than the Sperm-Free fraction, whereas concentrations in the Sperm-Rich fraction were intermediate and not different from the other 2 fractions (Table 2). No difference in any measure of protein content among the highest-fertility and the lowest-fertility boars in the Sperm-Free and Sperm-Peak fractions was established (Table 3), whereas differences were found for the Sperm-Rich fraction in AWN-1 concentration, and the lowest-fertility boars had the highest amount of AWN-1, OPN-9, and OPN-12 per AI dose.
|
Relative abundance of OPN-70 in the Sperm-Peak fraction was negatively correlated with total born (r = –0.47, P = .02) and fertility index (r = –0.47, P = .03). However, in the Sperm-Rich and Sperm-Free fractions, AWN-1 and OPN relative abundance did not show any correlations with in vivo fertility. The estimated content of AWN-1, OPN-9, and OPN-12 per AI dose was negatively correlated with total born (r = –0.45, r = –0.42, r = –0.43, respectively; P < .05) and fertility index (r = –0.47, r = –0.49, r = –0.49, respectively; P < .05), and OPN-9 and OPN-12 were both negatively correlated with farrowing rate (r = –0.46, r = –0.44, respectively; P < .05).
2-D Gel Electrophoresis of Seminal Plasma Proteins
Figure 5A is a
representative gel showing 2-D separation of seminal plasma proteins for the
second collection from boar R-1. From a qualitative perspective, all seminal
plasma protein species were identified in both the highest-fertility and the
lowest-fertility boars. Quantification of the 42 numbered proteins established
differences (P < .05) among boars for proteins 7 (46 kDa, pI 6.9),
17 (10 kDa, pI 9.0), 22 (18 kDa, pI 9.2), and 24 (27 kDa, pI 7.6). One of the
lowest-fertility boars, R-1, had lower abundance of seminal plasma proteins 7,
17, and 22 (P < .05) than the other 3 boars, but there were no
differences in relative abundance (P > .05) of these proteins when
analyzed by fertility class.
|
|
When correlations with in vivo characteristics were considered, protein 4 (60 kDa, pI 6.5) was negatively correlated with farrowing rate (r = –0.66, P = .04) and with the fertility index (r = –0.66, P = .04), whereas protein 27 (22 kDa, pI 6.0) had a strong negative relationship with total piglets born (r = –0.77, P = .010). In addition, the semen characteristics and extended storage data collected and presented in Ruiz-Sánchez et al (2006) were also compared to the data from seminal plasma proteins from these boars, and sperm motility at day 7 of storage was also negatively correlated (r = –0.74, P = .037) with protein 27. The identity of protein 27 was confirmed to be PSP-I by MS techniques, whereas protein 4 did not reveal a positive identification (Table 4). In contrast, protein 26 (26 kDa, pI 5.9) tended to be positively correlated with pregnancy rate (r = 0.454, P = .09), farrowing rate (r = 0.450, P = .09), and fertility index (r = 0.481, P = .07). Protein 26 was confirmed to be glutathione peroxidase (epididymal androgen-related protein; GPX5), the only selenium-independent, epididymis-specific glutathione peroxidase, by MS (Table 4).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
In the context of the data reported from the present study, it is important to emphasize 2 key points. First, as described by Ruiz-Sánchez et al (2006), the quality of the ejaculates used in the present study (>80% progressive motility and >85% morphologically normal sperm) exceeded normal industry standards (>70% progressive motility and <30% abnormal sperm) for use in AI. Second, boar fertility was evaluated in vivo using a low-sperm dose (1.5 billion morphologically normal and motile sperm per AI dose) to avoid compensable traits that are masked if higher sperm doses are used. However, the differences observed with in vivo performance of these boars may have been more pronounced with an even further reduction of sperm dose, as shown in Tardif et al (1999). It is essential to recognize that these specific conditions may identify a different subpopulation of relatively less fertile boars than in previous studies.
In the present study, the collection of the ejaculates in discrete sequential fractions allowed further characterization of differences in seminal plasma proteins among these fractions, as previously reported by our laboratory (Zhu et al, 2000). The Sperm-Peak fraction containing the highest sperm concentration is usually the first tube collected, and sperm from this fraction have the highest fertilization ability in vitro. The Sperm-Peak fraction contained lower total protein than the other 2 fractions and lower concentrations of all specific proteins than the Sperm-Free fraction. One explanation for these differences could be the origin of the seminal plasma components with respect to the different accessory sex glands and their sequential contribution of secreted proteins to the ejaculate. The first secretions present in the ejaculate are produced by the prostate gland, which serves to clear the male urinogenital tract, followed by very high sperm concentrations in the first 10–15 mL of the Sperm-Rich fraction. The ejaculate then becomes increasingly diluted by seminal vesicle fluid in the next 30–70 mL of the ejaculate, until essentially sperm-free fluid is collected in the Sperm-Free fraction. Depending on the frequency of collection, a second, less concentrated Sperm-Rich fraction may be present, after which the ejaculate ends with a gel fraction being secreted from the bulborourethral glands. Prostate secretions contain lower concentrations of PSP-I and AWN-1 than seminal plasma of the complete ejaculate (Manásková et al, 2002). These results support our observations that the Sperm-Peak fraction, the first sperm fraction collected that contains mainly prostate secretions, had lower concentrations of PSP-I than the Sperm-Rich fraction. Similarily, Rodríguez-Martínez et al (2008) have also reported consistently higher protein concentrations in the sperm-rich compared with the sperm-peak fractions.
A second factor that could contribute to the protein differences between fractions is the variability in the concentration of sperm. The high concentration of sperm in the Sperm-Peak fraction would retain a higher proportion of proteins on their membranes, thus reducing the residual amount of total protein in seminal plasma. Metz et al (1990) reported that epididymal sperm adsorbed 14 pg of protein/sperm over a 10-minute period and that 82% of the proteins retained were low-molecular weight proteins. These authors also confirmed that low-quality sperm (sperm progressive motility of 50% to 30% after washing with Tyrode solution) adsorbed significantly less protein (3 pg of protein/sperm per 10 minutes). This relationship is consistent with our observations that sperm concentration and total protein concentrations were negatively related in the Sperm-Rich and Sperm-Peak fractions. Therefore, a combination of 3 factors—1) seminal plasma origin, 2) sperm quality (sperm ability to absorb seminal plasma proteins), and 3) sperm concentration—collectively determine the variation in total protein concentration among the seminal plasma fractions.
PSP-I and AWN-1 are members of the spermadhesin protein family isolated from seminal plasma and may play an important role during fertilization (Töpfer-Petersen et al, 1998; Yang et al, 1998; Manásková et al, 2000; Assreuy et al, 2002). PSP-I has multiple forms because of differences in its carbohydrate moiety (Rutherfurd et al, 1992; Nimtz et al, 1999). In the present experiment, glycosylated PSP-I species ranging from 20 to 14 kDa were identified, and a single deglycosylated form of PSP-I was present as a compact 12-kDa band, which was subsequently used for analysis of PSP-I abundance. Western blotting results revealed that PSP-I and AWN-1 were detected in all seminal plasma fractions analyzed, and the Sperm-Peak fraction contained the lowest concentration of these proteins. These results reflect the observed differences in total protein concentration and may be determined by the same 3 factors discussed previously.
OPN has been detected in both female (Johnson et al, 1999; Garlow et al, 2002) and male (Cancel et al, 1999; Rodríguez et al, 2000; Luedtke et al, 2002) reproductive tracts. OPN has been identified as a seminal plasma protein that is positively associated with fertility in the bull (55 kDa, pI 4.5) (Cancel et al, 1997, 1999) and the stallion (SP-1, 72 kDa, pI 5.6) (Brandon et al, 1999). In the present experiment, Western blot analysis with an antiserum against human OPN identified 3 immunoactive bands at 70, 12, and 9 kDa. A 70-kDa form of OPN has been reported in uterine flushings in the pig (Garlow et al, 2002) and appears to be homologous to SP-1, a stallion seminal plasma osteopontin (Brandon et al, 1999). Although the 70-kDa osteopontin species identified in the present study of boar seminal plasma is homologous to the bull (55 kDa) and stallion (70 kDa) OPN forms, the relative amount of OPN-70 did not differ among boars. Similarly, under the conditions used, none of the higher-molecular weight proteins nor those in the area of osteopontin (70 kDa, pI 4-6) in the seminal plasma proteome as analyzed by 2-D gels were associated with differences in fertility. The lack of an association between osteopontin abundance and in vivo fertility in our study is likely due to the use of boars with a narrow range of fertility, thus excluding the more infertile boars, which would likely reveal a higher variability in seminal plasma protein abundance. All of the ejaculates used in the present study exceeded normal industry standards for use in AI, and the differences in fertility in vivo were established using low numbers of sperm per AI dose. Because there were no major differences in the relative abundance of AWN-1, OPN-12, and OPN-9 in the ejaculates collected, differences in the concentration of these specific proteins in seminal plasma from the highest-fertility and lowest-fertility boars may largely reflect differences in total protein concentration and the sperm concentration of each ejaculate, which ultimately determines the amount of seminal plasma present after dilution of the ejaculate to 1.5 billion sperm per 50-mL AI dose.
The analysis of seminal plasma proteins using proteomics techniques provides important preliminary data on the abundance of specific proteins in seminal plasma without having to rely on existing antibodies for detection (Fouchécourt et al, 2000; Moura et al, 2007; Martínez-Heredia et al, 2008). This approach has also been successfully used to identify proteins that were associated with fertility in the stallion (Brandon et al, 1999), and the bull (Killian et al, 1993). In this study, analysis of 42 different proteins in the seminal plasma of the 2 lowest-fertility and 2 highest-fertility boars demonstrated that variations in the seminal plasma proteome showed clear associations with differences in boar fertility. Although 4 proteins were initially identified as differing among boars, none of these proteins were specifically related to fertility. Two of the proteins—protein 17 and protein 22—were located in the same molecular size range and pI as PSP-I, and preliminary immunoblots confirmed the presence of PSP-I in this area. Further analysis by MS confirmed these proteins as PSP-I. The significant differences observed in PSP-I between boars lacked associations with overall fertility. This suggests that the abundance of PSP-I appears to be related to the total protein concentration in the seminal plasma of these boars. Lower-fertility boar R-1 consistently had total and specific protein concentrations similar to the 2 higher-fertility boars, whereas the other lower-fertility boar, G-1, exhibited higher total and specific protein concentrations compared with all 3 boars.
Interestingly, protein 27 (20 kDa, pI 6.0), identified by 2-D gel analysis in the present study, showed a strong negative correlation (r = –0.7627, P = .01) to total litter size born but not to other fertility traits included in the overall fertility index. Protein 27 was also identified as PSP-I, which supports our hypothesis that PSP-I may limit fertility because of an inhibitory effect of these spermadhesins on sperm performance in a low-sperm insemination dose scenario. Specific seminal plasma components have been described as decapacitation factors in humans (Zhu et al, 2006), mice (Huang et al, 2007), and other species (as reviewed in Töpfer-Petersen et al, 1998) and are assumed to protect sperm from factors in the female tract that could trigger early capacitation, thus reducing the possibility of sperm-oocyte binding. These suggestions are consistent with the observations that preincubation of sperm from the Sperm-Peak fraction with seminal plasma from the Sperm-Free fraction reduced oocyte penetration rate in vitro (Zhu et al, 2000), whereas inclusion of seminal plasma in sperm samples sorted by flow cytometry increased the percentage of uncapacitated, acrosome-intact sperm and reduced oocyte penetration rate in vitro (Maxwell and Johnson, 1999). In the present experiment, total protein concentration of seminal plasma was negatively correlated with both zona pellucida penetration rate and number of sperm penetrating the zona, suggesting that an increase in total protein content produces a predominantly decapacitation effect. As a consequence, the increase in uncapacitated sperm could decrease fertilization rate in vitro. No differences were reported in sperm viability and mitochondrial activity when PSP-I was incubated with a highly diluted sperm sample (García et al, 2003). However, our unpublished observations have shown a negative correlation between the number of sperm attaching to each oocyte and PSP-I in seminal plasma, which is consistent with the observation in the present study that D-PSP-I abundance was negatively correlated with both in vivo and in vitro fertility. Direct evaluation of sperm capacitation state and the populations of specific seminal plasma proteins on the sperm membrane before and after capacitation is needed to better understand the specific functions of seminal plasma proteins during the fertilization process.
During natural mating and insemination, because the sperm peak fraction is the first part of the ejaculate and, arguably, the main source of the sperm that colonize the sperm reservoir in the oviduct, it is logical to assume that these sperm are not exposed to the seminal plasma that follows in the remainder of the ejaculate (Rodríguez-Martínez et al, 2008). The Sperm-Peak and P1 fractions exhibit positive effects on fertilization, and both have lower protein concentrations and PSP-I abundance, which suggests that these higher concentrations of certain proteins, especially the spermadhesins, have negative effects in an in vivo setting. The proteins may play an important role in sperm longevity as decapacitation factors; however, these proteins may also reduce fertility if in excess or if sperm are exposed to them for an extended period of time. This negative relationship between lower total protein and specific PSP-I, AWN-I, and OPN concentrations in the Sperm-Peak fractions and higher relative fertility needs to be further evaluated.
Consistent with the observed negative relationships between seminal plasma proteins and fertility, the relative abundance of protein 4 (60 kDa, pI 6.5) exhibited a strong negative correlation with both farrowing rate and the overall fertility index. The MS analysis failed to identify this protein because of low protein abundance and contamination with keratin. The identity of this protein is still under investigation.
In contrast, protein 26 (25 kDa, pI 5.9), as identified by proteomics techniques in the present study, tended to be positively associated with farrowing rate and the overall fertility index. Although we have previously speculated that this protein may be lipocalin-type prostaglandin D synthase (Foxcroft et al, 2008), this study confirmed its identity using MS as glutathione peroxidase-5. Glutathione peroxidase-5 is a selenium-independent free radical scavenger that is restricted to the male reproductive tract (Grignard et al, 2005). It has been shown to bind to the head of the spermatozoa in mice (Vernet et al, 1997) and is thought to protect the sperm plasma membrane from attacks by free radicals (Grignard et al, 2005). Other studies have detected another glutathione peroxidase, phospholipid hydroperoxidase (GPX4) in the seminal plasma of boars and are investigating its role in sperm quality (Dube et al, 2004; Bailey et al, 2005). Boar sperm are particularly sensitive to oxidative damage (Strzezek et al, 2005), which is of interest in situations when semen is to be extended for use in AI or for cryopreservation. The inclusion of seminal plasma during cryopreservation may serve to protect the sperm membranes, and the same concept may apply to the protection of sperm membranes by seminal plasma in extended semen. In Ruiz-Sánchez et al (2006), we demonstrated that the higher-fertility boars exhibit higher sperm motility in semen extended to day 7 and day 10 compared with the lower-fertility boars, suggesting that the this enzyme could play a role in the protection of sperm membranes and keep sperm motile in extended semen for longer periods of time.
The overall results presented in this study and the in vitro fertility performance of these boars in Ruiz-Sánchez et al (2006) provide strong evidence of substantial differences in boar fertility that cannot be identified by existing laboratory techniques used in commercial boar studs. However, inadvertent use of the relatively subfertile boars identified in this study would substantially affect breeding herd performance. The proteomic analysis reported in the present paper provides some of the first evidence of specific boar seminal plasma proteins that may mediate observed differences in semen quality. The combined results from this study confirm that there is no single test that can apparently predict sperm quality or boar fertility among groups of relatively fertile boars. Fertilization in mammals is a complex process, involving multiple interactions between the sperm and the seminal plasma components of the ejaculate. Furthermore, extensive interactions between components of the ejaculate and the female reproductive tract in vivo are very different to the conditions in which sperm maturation is carried out in vitro. Therefore, semen characteristics that favor successful in vitro fertilization may not necessarily favor optimal fertility when the same sperm are used for AI.
For artificial insemination purposes, either the first sperm-rich fraction or the whole ejaculate may be collected; however, in a natural mating scenario, the sperm would typically only come in contact with the sperm-rich fraction of the ejaculate (Foxcroft et al, 2008). Thus, it may become important when collecting semen to collect only the first sperm-rich fraction rather than the whole ejaculate, especially if the second fraction of the ejaculate may contain components that are inhibitory to overall boar fertility. Also, from a practical perspective, if the trend toward using lower sperm numbers for intrauterine insemination continues, it will be essential to identify boars that are deemed less fertile in this situation, if the benefit of using fewer, but higher indexed, boars is to be realized. Information on the balance of proteins that determine the rate of capacitation and those that ultimately allow effective sperm binding to the zona pellucida and to the oocyte will undoubtedly help to improve the selection of such high-impact boars. The results presented in this paper identify several seminal plasma proteins that merit further investigation as markers of ejaculate quality and boar fertility.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
* These authors contributed equally to this work. 
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Assreuy AM, Calvete JJ, Alencar NMN, Cavada BS, Rocha-Filho Melo
SC, Cunha FQ, Ribeiro RA. Spermadhesins PSP-I/PSP-II heterodimer and its
isolated subunits induced neutrophil migration into peritoneal cavity of rats.
Biol Reprod. 2002; 67: 1796
–1803.
Bailey JL, Tardif S, Dube C, Beaulieu M, Reyes-Moreno C, Lefievre L, Leclerc P. Use of phosphoproteomics to study tyrosine kinase activity in capacitating boar sperm. Kinase activity and capacitation. Theriogenology. 2005; 63: 599 –614.[CrossRef][Medline]
Brandon CI, Heusner GL, Caudle AB, Fayrer-Hosken RA. Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility. Theriogenology. 1999; 52: 863 –873.[CrossRef][Medline]
Calvete JJ, Ensslin M, Mburu J, Iborra A, Martinez P, Adermann K, Waberski D, Sanz L, Töpfer-Petersen E, Weitze KF, Einarsson S, Rodríguez-Martinez H. Monoclonal antibodies against boar sperm zona pellucida-binding protein AWN-1. Characterization of a continuous antigenic determinant and immunolocalization of AWN epitopes in inseminated sows. Biol Reprod. 1997; 57: 735 –742.[Abstract]
Cancel AM, Chapman DA, Killian GJ. Osteopontin is the 55-kilodalton fertility-associated protein in Holstein bull seminal plasma. Biol Reprod. 1997;57: 1293 –1301.[Abstract]
Cancel AM, Chapman DA, Killian GJ. Osteopontin localization in the
Holstein bull tract. Biol Reprod. 1999; 60: 454
–460.
Dube C, Beaulieu M, Reyes-Moreno C, Guillemette C, Bailey JL. Boar sperm storage capacity of BTS and Androhep Plus: viability, motility, capacitation, and tyrosine phosphorylation. Theriogenology. 2004; 62: 874 –886.[CrossRef][Medline]
Ekhlasi-Hundrieser M, Sinowatz F, Greiser De Wilke I, Waberski D, Töpfer-Petersen E. Expresion of spermadhesin genes in porcine male and female reproductive tracts. Mol Reprod Dev. 2002; 61: 32 –41.[CrossRef][Medline]
Erikson DW, Way AL, Chapman DA, Killian GJ. Detection of
osteopontin on Holstein bull spermatozoa, in cauda epididymal fluid and testis
homogenates, and its potential role in bovine fertilization.
Reproduction. 2007; 133: 909
–917.
Fisher LW, Stubbs JT III, Young MF. Antisera and cDNA probes to human and certain animal model bone matrix noncollagenous proteins. Acta Orthop Scand Suppl. 1995; 266: 61 –65.[Medline]
Fouchécourt S, Metayer S, Locatelli A, Dacheux F, Dacheux
JL. Stallion epididymal fluid proteome: qualitative and quantitative
characterization; secretion and dynamic changes of major proteins.
Biol Reprod. 2000; 62: 1790
–1803.
Foxcroft GR, Dyck MK, Ruiz-Sanchez A, Novak S, Dixon WT. Identifying useable semen. Theriogenology. 2008; 70: 1324 –1336.[CrossRef][Medline]
Garcia EM, Vázquez JM, Calvete JJ, Sanz L, Roca J, Parrilla I, Martínez EA. Effect of seminal plasma proteins PSP-I and PSP-II on viability and acrosomal status of highly diluted boar spermatozoa. Theriogenology. 2005; 63: 492 –493.
Garlow JE, Ka H, Johnson GA, Burghardt RC, Jaeger LA, Bazer FW.
Analysis of osteopontin at the maternal-placental interface in pigs.
Biol Reprod. 2002; 66: 718
–725.
Gerena RL, Irikura D, Urade Y, Eguchi N, Chapman DA, Killian GJ.
Identification of a fetility-associated protein in bull seminal plasma as
lipocalin-type prostaglandin D synthase. Biol Reprod. 1998; 58: 826
–833.
Grignard E, Morin J, Vernet P, Drevet JR. GPX5 orthologs of the mouse epididymis-restricted and sperm-bound selenium-independent glutathione peroxidase are not expressed with the same quantitative and spatial characteristics in large domestic animals. Theriogenology. 2005; 64: 1016 –1033.[CrossRef][Medline]
Hao Y, Mathialagan N, Walters E, Mao J, Lai L, Becker D, Li W,
Critser J, Prather RS. Osteopontin reduces polyspermy during in vitro
fertilization of porcine oocytes. Biol Reprod. 2006; 75: 726
–733.
Hao Y, Murphy CN, Spate L, Wax D, Zhong Z, Samuel M, Mathialagan N, Schatten H, Prather RS. Osteopontin improves in vitro development of porcine embryos and decreases apoptosis. Mol Reprod Dev. 2008; 75: 291 –298.[CrossRef][Medline]
Huang YH, Chen YH, Lin CM, Ciou YY, Kuo SP, Chen CT, Shih CM, Chang EE. Suppression effect of seminal vesicle autoantigen on platelet-activating factor-induced mouse sperm capacitation. J Cell Biochem. 2007;100: 941 –951.[CrossRef][Medline]
Johnson GA, Burgardt RC, Spencer TE, Newton GR, Ott TL, Bazer FW.
Ovine osteopontin: II. osteopontin and 
vβ3
integrin expression in the uterus and conceptus during the periimplantation
period. Biol Reprod. 1999; 61: 892
–899.
Keller A, Nesvizhskii AI, Kolker E, Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem. 2002; 74: 5383 –5392.[Medline]
Killian GJ, Chapman DA, Rogowski LA. Fertility-associated proteins in Holstein bull seminal plasma. Biol Reprod. 1993; 49: 1202 –1207.[Abstract]
Kwok SCM, Soares MJ, McMurtry JP, Yurewicz EC. Binding characteristics and immunolocalization of porcine seminal protein, PSP-I. Mol Reprod Dev. 1993; 35: 244 –250.[CrossRef][Medline]
Luedtke CC, McKee MD, Cyr DG, Gregory M, Kaartinen MT, Mui J, Hermo
L. Osteopontin expression and regulation in the testis, efferent ducts, and
epididymis of rats during postnatal development through to adulthood.
Biol Reprod. 2002; 66: 1437
–1448.
Manásková P, Balinova P, Kraus M, Tichá M, Jonáková V. Mutual interactions of boar seminal plasma proteins studied by immunological and cromatographic methods. Am J Reprod Immunol. 2003;50: 399 –410.[CrossRef][Medline]
Manásková P, Liberda J, Tichá M, Jonáková V. Aggregated and monomeric forms of proteins in boar seminal plasma: characterization and binding properties. Folia Biologica (Praha). 2000;46: 143 –151.
Manásková P, Liberda J, Ticha M, Jonakova V. Isolation of nonheparin-binding and heparin-binding proteins of boar prostate. J Chromatogr B Analyt Technol Biomed Life Sci. 2002; 770: 137 –143.[CrossRef][Medline]
Martínez-Heredia J, de Mateo S, Vidal-Taboada JM, Ballesca
JL, Oliva R. Identification of proteomic differences in asthenozoo-spermic
sperm samples. Hum Reprod. 2008; 23: 783
–791.
Maxwell WMC, Johnson LA. Physiology of spermatozoa at high dilution rates: the influence of seminal plasma. Theriogenology. 1999; 52: 1353 –1362.[CrossRef][Medline]
Metz KW, Berger T, Clegg ED. Adsorption of seminal plasma proteins by boar spermatozoa. Theriogenology. 1990; 34: 691 –700.[CrossRef][Medline]
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 –188.[CrossRef][Medline]
Nesvizhskii AI, Keller A, Kolker E, Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem. 2003;75: 4646 –4658.[Medline]
Nimtz M, Grabenhorst E, Conradt HS, Sanz L, Calvete JJ. Structural characterization of the oligosaccharide chains of native and crystallized boar seminal plasma spermadhesin PSP-I and PSP-II glycoforms. Eur J Biochem. 1999;265: 703 –718.[Medline]
Rodríguez CM, Day JR, Killian GJ. Osteopontin gene expression in the Holstein bull reproductive tract. J Androl. 2000;21: 414 –420.[Abstract]
Rodríguez-Martínez H, Iborra I, Martínez P, Calvete JJ. Immunoe-lectroscopic imaging of spermadhesin AWN epitopes on boar spermatozoa bound in vivo to the zona pellucida. Reprod Fertil Dev. 1998;10: 491 –497.[CrossRef][Medline]
Rodríguez-Martínez H, Saravia F, Wallgren M, Roca J, Pena FJ. Influence of seminal plasma on the kinematics of boar spermatozoa during freezing. Theriogenology. 2008; 70: 1242 –1250.[CrossRef][Medline]
Ruiz-Sánchez AL, O'Donoghue R, Novak S, Dyck MK, Cosgrove JR, Dixon WT, Foxcroft GR. The predictive value of routine semen evaluation and IVF technology for determining relative boar fertility. Theriogenology. 2006; 66: 736 –748.[CrossRef][Medline]
Rutherfurd KJ, Swiderek KM, Green CB, Chen S, Shively JE, Kwok SC. Purification and characterization of PSP-I and PSP-II, two major proteins from porcine seminal plasma. Arch Biochem Biophys. 1992; 295: 352 –359.[CrossRef][Medline]
Sanz L, Calvete JJ, Mann K, Schafer W, Schmid ER, Amselgruber W, Sinowatz F, Ehrhard M, Töpfer-Petersen E. The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa. FEBS Lett. 1992; 300: 213 –218.[CrossRef][Medline]
Sinowatz F, Amselgruber W, Töpfer-Petersen E, Calvet JJ, Sanz L, Plendl J. Immunocytochemical location of spermadhesin AWN in the porcine male genital tract. Cell Tissue Res. 1995; 282: 175 –179.[CrossRef][Medline]
Strzezek J, Wysocki P, Kordan W, Kuklinska M. Proteomics of boar seminal plasma-current studies and possibility of their application in biotechnology of animal reproduction. Reprod Biol. 2005; 5: 279 –290.[Medline]
Tardif S, Laforest JP, Cormier N, Bailey JL. The importance of porcine sperm parameters on fertility in vivo. Theriogenology. 1999; 52: 447 –459.[CrossRef][Medline]
Töpfer-Petersen E, Romero A, Varela PF, Ekhlasi-Hundrieser M, Dostalova Z, Sanz L, Calvete JJ. Spermadhesins: a new protein family. Facts, hypotheses and perspectives. Andrologia. 1998; 30: 217 –224.[Medline]
Vernet P, Faure J, Dufaure JP, Drevet JR. Tissue and developmental distribution, dependence upon testicular factors and attachment to spermatozoa of GPX5, a murine epididymis-specific glutathione peroxidase. Mol Reprod Dev. 1997;47: 87 –98.[CrossRef][Medline]
Xu X, Ding J, Seth PC, Harbison DS, Foxcroft GR. In vitro fertilization of in vitro matured pig oocytes: effects of boar and ejaculate fraction. Theriogenology. 1996; 45: 745 –755.[CrossRef][Medline]
Xu X, Pommier S, Arbov T, Hutchings B, Sotto W, Foxcroft GR. In
vitro maturation and fertilization techniques for assessment of semen quality
and boar fertility. J Anim Sci. 1998; 76: 3079
–3089.
Yamakawa K, Yoshida K, Nishikawa H, Kato T, Iwamoto T. Comparative
analysis of interindividual variations in the seminal plasma proteome of
fertile men with identification of potential markers for azoospermia in
infertile patients. J Androl. 2007; 28: 858
–865.
Yang WC, Kwok SC, Leshin S, Bollo E, Li WI. Purified porcine
seminal plasma protein enhances in vitro immune activities of porcine
peripheral lymphocytes. Biol Reprod. 1998; 59: 202
–207.
Zhu J, Massey JB, Mitchell-Leef D, Elsner CW, Kort HI, Roudebush WE. Platelet-activating factor acetylhydrolase activity affects sperm motility and serves as a decapacitation factor. Fertil Steril. 2006; 85: 391 –394.[CrossRef][Medline]
Zhu J, Xu X, Cosgrove JR, Foxcroft GR. Effect of seminal plasma from different fractions of individual ejaculates on IVF in pigs. Theriogenology. 2000; 54: 1443 –1452.[CrossRef][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
