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From the * Departamento de Medicina y
Cirugía Animal, Facultad de Veterinaria, Universidad de Murcia, Murcia,
Spain; and the Instituto de Biomedicina de
Valencia, CSIC, Valencia, Spain.
| Correspondence to: Juan M. Vázquez, Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Murcia, Spain (e-mail: vazquez{at}um.es). |
| Received for publication September 8, 2005; accepted for publication November 28, 2005. |
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Abstract |
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Key words: Boar seminal plasma, spermadhesin PSP-I/PSP-II, protective effect on sperm function, sperm survival, highly diluted spermatozoa
Spermadhesins are male secretory proteins detected so far in ungulates (pig, cattle, and horse) (Haase et al, 2005). In the pig, this family of proteins consists of 5 members—AQN-1, AQN-3, AWN, PSP-I, and PSP-II (Töpfer-Petersen et al, 1998)—and together they represent over 90% of the total boar seminal plasma proteins (Dostàlovà et al, 1994). The porcine spermadhesin genes are clustered on SCC 14q28–q29 (Haase et al, 2005). Porcine spermadhesins, 110- to 133-residue polypeptides built by a single CUB domain architecture (Romero et al, 1997), are synthesized by the epididymis and accessory glands (Ekhlasi-Hundrieser et al, 2002) and exhibit distinct sperm-coating and ligand-binding capabilities. Sequence variation, glycosylation, and their aggregation state of spermadhesins contribute to their specific pattern of biological activities (Calvete et al, 1993a,b; Dostàlovà et al, 1995a). AQN-1, AQN-3, and AWN coat the sperm surface at ejaculation and display zona pellucida glycoprotein-binding capability and affinity for ß-galactosides and heparin (Sanz et al, 1993; Dostàlovà et al, 1995b; Calvete et al, 1996a,b; Rodríguez-Martínez et al, 1998). These spermadhesins are thought to stabilize the plasma membrane over the acrosomal vesicle and are mainly released from the spermatozoal surface during capacitation (Sanz et al, 1993; Dostàlovà et al, 1994; Calvete et al, 1997). Moreover, AQN-3 has been reported to form part of a sperm motility-inhibitor factor complex (Iwamoto et al, 1995), and AQN-1 and AWN are sperm-associated acrosin-inhibitor acceptor proteins (Sanz et al, 1992). On the other hand, PSP-I and PSP-II are the most abundant boar seminal plasma proteins and form a non-heparin-binding heterodimer (Calvete et al, 1995) of glycosylated spermadhesins (Nimtz et al, 1999). Accumulating evidence points to a role for the PSP-I/PSP-II spermadhesin complex as an exogenous modulator of the uterine immune activity (Leshin et al, 1998; Yang et al, 1998), thus ensuring reproductive success. Hence, PSP-I and PSP-II are immunostimulatory for lymphocyte activity in vitro (Nimtz et al, 1999), and binding of PSP-I to lymphocyte has been demonstrated (Yang et al, 1998). Furthermore, the PSP-I/PSP-II heterodimer and its isolated subunits induce the recruitment of neutrophils into the peritoneal cavity of rats (Assreuy et al, 2002) and pigs (Rodríguez-Martínez et al, 2005). The neutrophil migration-inducing activity of PSP-I/PSP-II, and possibly of the PSP-II subunit, is mediated by the stimulation of resident macrophages, which release a neutrophil chemotactic substance (Assreuy et al, 2002), whereas PSP-I appears to act directly on neutrophils (Assreuy et al, 2003). The purpose of these proinflammatory and immunostimulatory activities would be to prevent possible infections of the lower reproductive tract and to provide a foreign cell-free uterine environment for the descending early embryos.
Besides their physiological functions in porcine reproduction, the heparin-binding and the non-heparin-binding spermadhesins exert opposite effects on spermatozoa that have been subjected to high dilution to mimic the conditions of sex sorting by flow cytometry (Centurión et al, 2003). Thus, whereas the pooled heparin-binding spermadhesins (AQN-1, AQN-3, and AWN) cause a concentration-dependent sperm membrane damage, the purified non-heparin-binding PSP-I/PSP-II spermadhesin complex contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 hours at physiological temperature (Centurión et al, 2003). The beneficial effect of the PSP-I/PSP-II heterodimer points to this spermadhesin as a candidate for an additive to improve the viability of highly diluted porcine spermatozoa (ie, flow cytometric sorting for chromosomal sex). In the present study, we sought to dissect the structural basis of the protective effect of boar spermadhesin PSP-I/PSP-II, its isolated PSP-I and PSP-II subunits, and their derived peptidic and glycan fractions on the sperm functionality by a time-course evaluation of the viability, the acrosomal status, the mitochondrial activity, and the motility of highly diluted boar spermatozoa.
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Materials and Methods |
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Preparation of Seminal Plasma
All experiments were performed with the seminal plasma from mature boars
that had previously sired offspring. Ejaculates were collected by the
gloved-hand method (Larsen,
1986). The seminal plasma was separated from spermatozoa by
centrifugation at 3800 x g for 15 minutes at room temperature
with a Heraeus Sepatech Megafuge (Osterode, Germany). The supernatants were
filtered sequentially through 10- and 1.2-µm filters and pooled.
Isolation of the Boar Seminal Plasma PSP-I/PSPII Heterodimer
The PSP-I/PSP-II heterodimer was isolated from the non-heparin-binding
fraction of boar seminal plasma by affinity chromatography on a
heparin-Sepharose column, equilibrated in 100 mM Tris-HCl; 150 mM NaCl; 5 mM
EDTA; and 0.025% sodium azide, pH 7.4, as previously described
(Calvete et al, 1995). The
identity and purity of the protein was assessed by N-terminal sequence
analysis with an Applied Biosystems 472 automated protein sequencer (Applied
Biosystems, Langen, Germany) and by MALDI-TOF mass spectrometry with an
Applied Biosystems Voyager DE-Pro mass spectrometer (Applied Biosystems) and a
saturated solution of sinapinic acid in 50% acetonitrile and 0.1%
trifluoroacetic acid (TFA) as the matrix. Protein concentration was determined
spectrophotometrically with the molar absorption coefficient (27 332
M-1 cm-1) determined by Menéndez et al
(1995) or by amino acid
analysis (after sample hydrolysis in 6 M chloride acid (HCl) for 24 hours at
106°C in evacuated and sealed ampoules) with a Beckman Gold Amino Acid
Analyzer (Beckman, Barcelona, Spain). Proteins were dialyzed against distilled
water and lyophilized.
Isolation of Boar Seminal Plasma PSP-I and PSP-II Subunits
The PSP-I and PSP-II subunits were purified from the heterodimer by
reverse-phase high-performance liquid chromatography (HPLC) on a Lichrocart
column (250 x 10 mm, RP-18, 7-µm particle size) (Merck, Germany)
eluted at 2 mL/min with a mixture of 0.1% TFA in water (solution A) and 0.1%
TFA in acetonitrile (solution B), first isocratically (10% B) for 5 minutes,
followed by 30% B for 10 minutes, 45% B for 45 minutes, 70% B for 15 minutes,
and 10% B for 15 minutes. The purified PSP-I and PSP-II subunits were dialysed
against distilled water and lyophilized. Purity and protein concentration were
determined as above.
Isolation of Peptides and Glycopeptides
To separate the peptidic and the glycan moieties, the 100 mg of the
PSP-I/PSP-II heterodimer were digested overnight at 37°C with trypsin by
using a 1:100 (wt/wt) enzyme:substrate ratio. Thereafter, the enzyme was
inactivated by heating at 100°C for 2 minutes, and the reaction mixture
was lyophilized. Completion of proteolysis was checked by sodium dodecyl
sulphate (SDS) polyacrylamide gel electrophoresis, reversed-phase HPLC, and
matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Nonglycosylated tryptic peptides were separated from glycopeptides by affinity
chromatography on a 5-mL Sepharose-Concanavalin A column (Amersham
Biosciences, Uppsala, Sweden) equilibrated and eluted with 20 mM sodium
phosphate, pH 7.4, 150 mM NaCl (phosphate-buffered saline [PBS]). ConA-bound
glycopeptides were eluted with equilibration buffer containing 100 mM
methyl-
-D-mannopyranoside. The glycopeptide fraction was desalted on a
C-18 Lichrosphere column (250 x 4 mm, 5-µm particle size) (Merck,
Germany), equilibrated, and washed with 0.1% TFA until the absorbance at 214
reached baseline level, followed by elution with 0.1% TFA and 50%
acetonitrile. Peptide concentration was determined by amino acid analysis.
Collection of Semen and Evaluation of Sperm Parameters
Sperm-rich fractions from fertile mature boars were collected by
gloved-hand method and extended to 30 x 106 sperm/mL in
Beltsville Thawing Solution (Pursel and
Johnson, 1975). Diluted sperm-rich fractions from 3 boars were
pooled and spermatozoa were separated from seminal plasma by centrifugation at
1200 x g (Megafuge 1.0 R, Heraeus, Germany) during 3 minutes.
To avoid sperm membrane damage caused by pipetting spermatozoa directly into
dilution medium (Maxwell and Johnson,
1999), the pellet was serially diluted in PBS to a final cell
count of 1 x 106 sperm/mL and incubated at 38°C
(Steri-Cult 200 incubator, Marietta, Ohio). Samples were taken at 0.5, 2, and
5 hours and analyzed for viability (membrane integrity), acrosomal status,
mitochondrial membrane potential, and motility.
Flow Cytometry
Flow cytometric analyses were carried out with a Coulter EPICS XL (Coulter
Corporation Inc, Miami, Fla) flow cytometer equipped with standard optics, an
argon-ion laser (Cyonics, Coherent, Santa Clara, Calif) performing 15 mW at
488 nm, and the EXPO 2000 software (Coulter Corporation). Subpopulations were
divided by quadrants, and the frequency of each subpopulation was quantified.
Nonsperm events (debris) were gated out based on the forward scatter and side
scatter dot plot by drawing a region enclosing the cell population of
interest. Events with scatter characteristics similar to sperm cells but
without reasonable DNA content were also gated out. Forward and sideways light
scatter were recorded for a total of 10 000 events per sample. Samples were
measured at flow rate of 
300 cells/s.
Flow Cytometric Assessment of Sperm Viability and Acrosomal Exocytosis
For an accurate estimation of the spermatozoa, the membrane and the
acrosome integrity was assessed simultaneously by flow cytometry using the
triple staining protocol described by Nagy et al
(2003). Briefly, 500 µL of
sperm samples (
500 000 cells) were incubated with 50 nM SYBR-14 (using a
100-µM stock solution in DMSO; component A of LIVED/DEAD Sperm Viability
Kit; Molecular Probes Europe, Leiden, The Netherlands), 0.5 µg/mL PE-PNA
(peanut agglutinin conjugated with phycoerythrin; Biomeda Corp, Foster City,
Calif), and 7.5 µM propidium iodide (PI). The samples were mixed and
incubated at 37°C in dark for 10 minutes before flow cytometric analysis.
SYBR-14 was measured with a 525-nm band pass filter, PI was collected with a
620-nm band pass filter, and PE-PNA was detected with a 575-band pass filter.
Viable spermatozoa with intact acrosome were defined as those stained only
with SYBR-14. Acrosome-reacted spermatozoa were defined as those stained with
SYBR-14 and PE-PNA. Spermatozoa stained with PI were classified as dead
cells.
Flow Cytometric Assessment of Sperm Mitochondrial Membrane Potential
JC-1
(5,5',6,6'-tetrachloro-1,1',3,3'tetraethylbenzymidazolyl
carbocianyne iodide; Molecular Probes Europe), a stain that differentiates
cells exhibiting high and low mitochondrial membrane potential
(Peña et al, 2003), was
used to assess the mitochondrial membrane potential of spermatozoa as
described in Martínez-Pastor et al
(2004) with slight
modifications. A total of 500 µL of sperm samples were incubated in a water
bath at 37°C in dark for 20 minutes with 0.2 µm JC-1, followed by flow
cytometric measurement through a 590-nm band pass filter. At low membrane
potential JC-1 exists as a green fluorescent monomer, whereas at higher
potentials JC-1 forms "J-aggregates" after accumulation in
mitochondria that emit a red-orange florescence at 590 nm
(Garner and Thomas, 1999;
Gravance et al, 2000).
Sperm Motility
Sperm motility was estimated by a computer-assisted sperm motility analysis
system with the software Sperm Class Analyzer (Microptic 2002, Barcelona,
Spain). Aliquots of 10 µL of semen samples (at 106
spermatozoa/mL) were placed in a warm (38°C) Makler chamber (Haifa,
Israel) and immediately transferred to the warm stage (38°C) of a Nikon
Labophot positive-phase contrast light microscope (Tokyo, Japan) equipped with
a 10 x objective and a monochrome video camera (Hitachi CCD model,
Chiba, Japan) connected to a personal computer. The program settings were as
in Centurión et al
(2003). Objective percentage
of motile spermatozoa was analyzed in at least 100 spermatozoa per sample.
Effect of Isolated PSP-I and PSP-II and Their Proteolytic Products on Sperm Functions
Diluted spermatozoa (1 x 106 sperm/mL PBS) were incubated
at 38°C for 5 hours with 0.75 mg/mL of either 1) native PSP-I, 2) native
PSP-II, 3) trypsin-digested PSP-I, 4) trypsin-digested PSP-II, or 5) 1.5 mg/mL
of trypsin-digested PSP-I/PSP-II heterodimer. Viability, acrosomal exocytosis,
mitochondrial membrane potential, and motility were assessed at 0.5, 2, and 5
hours of incubation. Controls were sperm incubated with 1.5 mg/mL of
heterodimer PSP-I/PSP-II or without added proteins.
Effect of the Peptidic and Glycan Fractions of PSP-I/PSP-II on Sperm Functions
Diluted spermatozoa (1 x 106 sperm/mL PBS) were incubated
at 38°C for 5 hours with either 1) 1.05 mg/mL of tryptic peptide fraction
depleted from glycopeptides by affinity chromatography on ConA-Sepharose (see
above) or 2) 0.5 mg/mL of ConA-retained glycopeptides. These amounts of
peptides and glycopeptides are equivalent to those released by proteolytic
digestion of 1.5 mg/mL of the PSP-I/PSP-II heterodimer. Viability, acrosomal
exocytosis, mitochondrial membrane potential, and motility were assessed at
0.5, 2, and 5 hours of incubation. Samples incubated with intact or
trypsin-digested PSP-I/PSP-II heterodimer as well as without proteins were
used as control.
Dose-Dependent Effect of the Peptidic Fraction of PSP-I/PSP-II on Sperm Functions
Samples of 1 x 106 sperm/mL in PBS were incubated at
38°C for 5 hours with decreasing concentrations of the peptidic fraction
of trypsinized PSP-I/PSP-II depleted from glycopeptides by ConA-Sepharose
chromatography. The following final concentrations were used: 1.05, 0.52,
0.23, and 0.10 mg/mL, which correspond to 1.5, 0.75, 0.33, and 0.15 mg/mL of
the native PSP-I/PSP-II heterodimer. Viability, acrosomal exocytosis,
mitochondrial membrane potential, and motility were assessed at 0.5, 2, and 5
hours of incubation. Sperm incubated with 1.5 mg/mL of native heterodimer or
without added proteins were used as controls.
For each experiment, the effects of the same batch of lyophilized proteins and their derived proteolytic products were assessed on 4 pools of spermatozoa collected on 4 different days. Each pool was made by mixing spermatozoa from 4 different boars. The same group of 4 boars was used on each day. Duplicate samples were performed for each treatment.
Statistical Analyses
All data editing and statistical analyses were performed in SPSS, version
11.5 (SPSS Inc, Chicago, Ill). Data were analyzed by analysis of variance
(ANOVA) by using the Hierarchical Linear Mix Model (MIXED) procedure according
to a statistical model including the fixed effect of treatment and of
incubation time and the random effect of replicate. To analyze data of sperm
viability, motility, mitochondrial activity, and acrosome status, percentages
were subjected to arcsine transformation before analysis. When ANOVA revealed
a significant effect, values were compared by using the Bonferroni test and
were considered to be significant when P < .05. Experiments were
replicated 4 times.
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Results |
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The same trend was observed regarding the percentages of spermatozoa with high mitochondrial membrane potential (Figure 1B). After 5 hours of incubation, the native PSP-I/PSP-II heterodimer, PSP-I, and PSP-II all preserved the mitochondrial activity in 75.5%, 68.1%, and 53.8% of the spermatozoa, though the PSP-I subunit showed a lower value (P < .05). For comparison, only 40.1% of spermatozoa retained this activity in the absence of added proteins.
Regardless of incubation time, the percentage of motile spermatozoa was higher (P < .05) in spermatozoa that were exposed to the native heterodimer (72.8% at 0.5 hours) or to the PSP-II subunit (75.5% at 0.5 hours) than that of control sperm (46.7% at 0.5 hours) and of spermatozoa incubated with the PSP-I subunit (39.6% at 0.5 hours) (Figure 1C). Moreover, after 2 hours of incubation, PSP-I had a marked detrimental effect on sperm motility, which dropped to undetectable values. This effect was not attributed to a membrane damage because the percentages of acrosome-reacted highly diluted spermatozoa at 0.5, 2, and 5 hours of incubation with the heterodimer or with PSP-I were not significantly different from that of control sperm (Table 1). On the other hand, the exposition of spermatozoa to PSP-II resulted in higher values (P < .05) of viable spermatozoa showing a reacted acrosome (Table 1).
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Effect of Glycopeptide and Peptide Fractions on Highly Diluted Boar Spermatozoa
The presence in the incubation medium of trypsin-digested PSP-I/PSP-II
heterodimer (Hdig), the glyco-peptide fraction of PSP-I/PSP-II (Glyc), and the
glycan-depleted tryptic-peptide fraction of PSP-I/PSP-II improved (P
< .05) the sperm viability compared with the control at all times tested
(Figure 3A). However, the
percentages of viable spermatozoa were significantly different among samples.
Thus, spermatozoa exposed to Hdig or Pept exhibited the highest values
(P < .05) of viable spermatozoa (72.4% and 82.2% at 5 hours,
respectively), which were not different from the percentage of spermatozoa
incubated with the intact heterodimer (76.6%). By contrast, spermatozoa
incubated with the glycopeptide fraction showed lower values during the 5
hours of incubation (51.3% at 5 hours), though they were significantly higher
(P < .05) than in the control by about 12%-15%. The same trend was
observed in the percentages of spermatozoa showing high mitochondrial membrane
potential (Figure 3B).
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At all incubation times, the percentage of motile spermatozoa was significantly higher in samples exposed to either native or trypsin-digested PSP-I/PSP-II heterodimer than in any other condition tested, including the control (Figure 3C). The time-dependent effect of incubating spermatozoa without added proteins (c) and in the presence of the PSP-I/PSP-II glycopeptide fraction (Glyc) were indistinguishable (Figure 3C). On the other hand, the percentage of motile spermatozoa after incubation in the presence of 1.05 mg/mL of the glycan-depleted tryptic-peptide fraction of PSP-I/PSP-II (Pept) markedly decreased with the incubation time from 68.7% at 0.5 hours to 29.4% at 2 hours and 4.1% at 5 hours (Figure 3C).
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Discussion |
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First, we studied the effects of the isolated PSP-I and PSP-II subunits on sperm functions. The PSP-II subunit mimicked the effect of the native heterodimer on maintaining sperm viable, motile, and with a high mitochondrial membrane potential (Figure 1). The effect of the PSP-I subunit was beneficial, though less pronounced than that of PSP-II on sperm viability and mitochondrial potential. However, it almost completely abolished sperm motility after 2 hours of incubation (Figure 1). Whether PSP-I might be affecting the glycolytic pathway of the spermatozoa is as yet unknown. This detrimental effect of PSP-I follows a similar time course and magnitude to the previously noticed sperm-immobilizing activity exerted by the pooled heparin-binding spermadhesins (Centurión et al, 2003). PSP-I is a glycosylated spermadhesin, and glycosylation indirectly modulates its ligand-binding properties. Thus, mannose-rich PSP-I glycoforms have been reported in the heparin-binding fraction of spermadhesins (Calvete et al, 1993b), whereas complex-type PSP-I glycoforms specifically associate with PSP-II into a noncovalent heterodimer (Calvete et al, 1995; Nimtz et al, 1999). The fact that the PSP-I glycoprotein isolated from PSP-I/PSP-II abolished the motility of highly diluted spermatozoa whereas the native heterodimer did not strongly suggested that this activity might be suppressed upon dimerization. Other binding activities impaired by complex formation are the heparin-binding capability and the mannose-6-phosphate recognition by PSP-II (Solis et al, 1998). Taken together, these results suggest a mechanism for the modulation of the ligand-binding properties of PSP-I and PSP-II.
To further dissect the effects of the PSP-I/PSP-II subunits on sperm functions, the native heterodimer and the isolated subunit were degraded with trypsin and the resulting peptide mixtures were checked for their activities on sperm parameters. The protective effect of the PSP-II on viability, mitochondrial membrane potential, and motility of highly diluted spermatozoa were not abolished by trypsin digestion, indicating that the conformation of the active epitope or epitopes was not destroyed by proteolysis. Similar results were observed when the PSP-I/PSP-II digestion mixture was analyzed. Moreover, the biological activity of the heterodimer was amplified after trypsin digestion compared with the native protein. In contrast, the negative effect of PSP-I on sperm motility was clearly attenuated. Hence, we may conclude that degradation of the native PSP-I/PSP-II exposes active epitopes, which remained hidden in the quaternary structure of the heterodimer or in the tertiary structure of its constituent subunits.
To evaluate the activity of the peptide and the glycan moieties of PSP-I/PSP-II, the tryptic peptide mixture of the heterodimer was fractionated by affinity chromatography on ConA-Sepharose. The nonbound (nonglycosylated peptides) and the bound (glycopeptides) fractions were used at 1.05 mg/mL and 0.5 mg/mL, respectively, which correspond to their calculated concentrations for a 1.5 mg/mL solution of native PSP-I/PSP-II heterodimer. This PSP-I/PSP-II concentration represents the optimal protective dose determined in previously reported experiments (Centurión et al, 2003). The data displayed in Figure 3 show that the peptide fraction retained the biological activity of PSP-I/PSP-II on the viability and mitochondrial activity of highly diluted boar spermatozoa, whereas the glycopeptide fraction exhibited a light positive effect, which was not different from the control. In addition, the glycopeptide fraction had no effect on sperm motility, whereas the peptide fraction abolished in a time-dependent manner the motility of viable spermatozoa (Figure 3C). This net detrimental activity may represent a composite of actions of the motility-impairing activities of native and trypsinized PSP-I and the positive effect of native and trypsin-degraded PSP-II (Figures 1C and 2C).
The deleterious effect of the tryptic peptide fraction of the PSP-I/PSP-II heterodimer followed a marked concentration-dependent pattern. Thus, decreasing the total peptide concentration from 1.05 to 0.1 mg/mL restored sperm motility to the same level observed with the intact heterodimer, and this effect lasted for at least 5 hours.
In conclusion, the subunits of the PSP-I/PSP-II heterodimeric disintegrin exert different activities on sperm functions. The beneficial effect of the native PSP-I/PSP-II on the functionality of highly diluted boar spermatozoa is largely preserved in its isolated PSP-II subunit and does not appear to require its glycan moiety.
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Acknowledgments |
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Footnotes |
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