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From the Department of Biochemistry and Molecular and Cell Biology, School of Veterinary Medicine, University of Zaragoza, Zaragoza, Spain.
| Correspondence to: J. A. Cebrián Pérez, Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, C/ Miguel Servet, 177, 50013 Zaragoza, Spain (e-mail: pcebrian{at}unizar.es). |
| Received for publication November 10, 2004; accepted for publication March 10, 2005. |
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Abstract |
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Key words: Immunocytochemistry, capacitation, acrosome reaction
The seminal plasma of mammals is a complex biological mixture of various fluids in the male reproductive tract. It contains several proteins (Mann and Lutwak-Mann, 1981), some of which are adsorbed onto the surface of ejaculated sperm (Leeuw de et al, 1990; Metz et al, 1990; Desnoyers and Manjunath, 1992; Amann et al, 1999). Many of these proteins are secretory products of the seminal vesicle (Huarte et al, 1987; Aumuller et al, 1988; Chandonnet et al, 1990), an accessory reproductive gland in most male mammals, where they accumulate in the lumen after puberty. Some of the adsorbed proteins maintain the stability of the membrane until the process of CA in the female genital tract (Cross, 1996), when their removal is a prerequisite for fertilization (Desnoyers and Manjunath, 1992). Seminal plasma plays an important role in improving ram sperm viability (Ashworth et al, 1994; Maxwell et al, 1997), maintaining spermatozoa motility in the bull (Baas et al, 1983) and the ram (Graham, 1994), and increasing the resistance of boar spermatozoa to cold-shock damage (Pursel et al, 1973; Berger and Clegg, 1985). A sperm surface receptor for a seminal plasma protein that increases sperm motility and viability has been found in rabbit spermatozoa (Minelli et al, 2001a,b).
It is well known that low temperatures alter the function of spermatozoa (Watson, 1981). Cold shock results in the destabilization of sperm membranes and impairment of sperm function, and it is also well known that ram spermatozoa are more sensitive to cold-shock stress than are that of other species such as the bull, rabbit, and man (Watson, 1981; Holt and North, 1984; Fiser and Fairfull, 1989). It has been suggested that cold shock induces premature sperm CA (Ashworth et al, 1994; Fuller et al, 1994; Watson, 1995; Perez et al, 1996) and that the main role of seminal plasma is to maintain the spermatozoa in a decapacitated state (Cross, 1996).
In a previous work, we have shown that the adsorption of seminal plasma proteins on cold-shocked ram sperm plasma membranes modifies the functional characteristics of damaged spermatozoa, restoring them to a state similar to that of live cells (Barrios et al, 2000). Fractioning of ram seminal plasma proteins by exclusion chromatography provided 3 fractions that could reverse the cold-shock effect. The restoring capacity appeared to be due to 1 major protein band of about 20 kd (Barrios et al, 2000), and we have shown recently that this effect is related to the inhibition of tyrosine phosphorylation of sperm proteins during CA (Pérez-Pé et al, 2002). Moreover, we have proved that adding seminal plasma proteins to the medium before cold treatment has an immediate beneficial effect on sperm survival (Pérez-Pé et al, 2001). These results prompted us to further investigate the nature of the ram seminal plasma components that are responsible for maintaining sperm membrane functionality. In this study, we present data to demonstrate that 2 proteins of about 14 (P14) and 20 (P20) kd from ram seminal plasma can prevent cold-shock membrane damage. Since the decapacitating role is frequently demonstrated by protein release from the membrane at CA (Desnoyers and Manjunath, 1992), we have quantified these proteins and analyzed their distribution on the surface of fresh spermatozoa and the changes induced by CA and the acrosome reaction (AR). In addition, according to a partial analysis of the primary structure of both proteins, there was no significant similarity between P20 and protein sequences collected in the databank, so this protein is probably still undescribed. A hypothetical function for P14 is discussed in terms of the homology between this protein and bovine PDC-109 (Esch et al, 1983).
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Materials and Methods |
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A seminal plasma–free sperm population was obtained by a dextran/swim-up procedure (García-López et al, 1996) performed at 37°C using a medium without Ca2Cl and NaHCO3 (Pérez-Pé et al, 2002). For thermal shock, aliquots of approximately 1 x 106 cells obtained after the swim-up process were incubated for 5 minutes at 25°C, transferred to 5°C for 10 minutes, and then replaced at 25°C for a further 5 minutes.
Evaluation of Semen Samples
Sperm concentration was calculated in duplicate with a Neubauer chamber
(Marienfeld, Germany).
Cell viability (membrane integrity) was assessed by fluorescent staining with carboxyfluorescein diacetate and propidium iodide (Sigma Chemical Co, St Louis, Mo) (Harrison and Vickers, 1990). The cells were then examined under a Nikon fluorescence microscope, and the numbers of propidium iodide-negative (membrane intact) spermatozoa and propidium iodide–positive (membrane damaged) spermatozoa per 100 cells were estimated and recorded. At least 200 cells were counted in duplicate for each sample. Results are expressed as the percentage of membrane-intact spermatozoa ± standard deviation.
Obtaining Seminal Plasma Proteins
Whole seminal plasma proteins and fraction 6 (F6) isolated by exclusion
chromatography in Sephacryl-100 were obtained as described previously
(Barrios et al, 2000). To
separate the F6 component bands as much as possible, we used a 15% to 20%
preparative, nondenaturing, gradient polyacrylamide gel. Two lanes were loaded
with the same F6 sample in identical conditions, and we cut and stained one of
them with Coomassie blue to use as a reference to cut the separated bands from
the other lane. Thus, 2 major components of this fraction with an approximate
relative molecular weight of 14 (P14) and 20 (P20) kd were recovered from the
gel (by cutting and mincing) and obtained by electroelution in a 422
ElectroEluter (BioRad, Hercules, Calif) under nondenaturing conditions
(without sodium dodecyl sulfate [SDS]) for 6 hours. The proportion of P14 and
P20 varied during the year. The medium value was 11.6 ± 3.1 (% P14) and
88.2 ± 3.2 (% P20).
Protein concentration was determined according to the method described by Bradford (1976), and samples were stored at -20°C.
Assessment of Seminal Plasma Protein Effect
The protective effects of the 8 protein fractions obtained from 2 exclusion
chromatography columns (Barrios et al,
2000) were analyzed by incubating 1.4 mg of each fraction with
sperm samples (106 sperm in 500 µL) for 30 minutes at 20°C
before coldshock treatment to assist with the adsorption of protein onto the
surface of the sperm
(Pérez-Pé et al,
2001). A control containing bovine serum albumin (BSA) was used.
Sperm membrane integrity was analyzed immediately after cold-shock treatment,
since a previous study of ours has demonstrated that no differences were found
after 1 hour of incubation
(Pérez-Pé et al,
2001). F6 had the highest ability to prevent cold-shock membrane
damage (data not shown). Therefore, the whole F6 and both P14 and P20 bands
isolated from this fraction, as described above, were diluted with the swim-up
medium, and their protective effect on spermatozoa was evaluated. Results were
expressed as the percentage of membrane-intact spermatozoa (ie, propidium
iodide negative). The data were compared using analysis of variance. Post hoc
comparisons were made using Tukey's significant difference test. The software
used was GraphPad InStat (San Diego, Calif).
Induction of CA and AR
Aliquots of approximately 1 x 106 cells obtained after the
swim-up process
(García-López et al,
1996) were diluted to 0.5 mL with BSA-depleted swim-up medium to
avoid premature CA of the control sample. In vitro CA was performed by
incubating the samples (containing 5 mg/mL of BSA) for 4 hours at 39°C in
a humidified incubator with 5% CO2 in air, as reported previously
(Pérez-Pé et al,
2002). The control was incubated under these conditions without
the addition of BSA. The chlortetracycline assay was used to assess the CA
state as described (Pérez-Pé
et al, 2002) and was based on the technique developed by Ward and
Storey (1984).
For the ionophore-induced AR, calcium ionophore A23187 was dissolved in dimethylsulfoxide (DMSO) and added to 20 µL of raw semen diluted 1:200 (2 x 107 cells/mL) with HEPES glucose buffer (149 mM NaCl, 2.5 mM KCl, 10 mM glucose, 20 mM HEPES, and 3 mM CaCl2). The pH was adjusted with NaOH to 7.4 (Shams-Borhan and Harrison, 1981). The final concentration of A23187 was 1 µM and 0.3% DMSO. DMSO was added to control tubes without ionophore—conditions that were shown not to have a protective effect (data not shown). The samples were incubated at 39°C for 1 hour, after which we assessed the acrosomal status and cell viability.
Extraction of Proteins
Sperm proteins were extracted (Harayama
et al, 1999) by centrifuging 4 x 107 spermatozoa
in a microfuge at 600 x g for 8 minutes at room temperature.
The pellet was resuspended with 300 µL of phosphate-buffered saline (PBS)
and centrifuged again. The pellet was resuspended with 100 µL of PBS and
100 µL of extraction buffer (125 mM Tris-HCl, 4% SDS, 10%
ß-mercaptoethanol, 20% glycerol, and 0.02% bromophenol blue) and, after
incubation at 100°C in a sand bath for 4 minutes, was centrifuged again at
7500 x g for 15 minutes at 4°C. The supernatant was
recovered and, after adding 10% of a protease and phosphatase inhibitor
cocktail (Sigma), was stored at -20°C.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis
SDS-polyacrylamide gel electrophoresis (PAGE), gel staining, and
densitometric analyses were carried out as previously described
(Barrios et al, 2000).
For Western blot analysis, polyclonal antibodies were raised against the whole F6 and the purified P14 and P20 bands by rabbit immunization with 500 µg of F6 and 300 µg of P14 and P20 protein in Freund complete adjuvant. After 15 days, they were reimmunized with the same amount of antigen dissolved in Freund incomplete adjuvant. The antiserum was obtained 15 days after the second immunization by the centrifugation of 10 to 20 mL of blood from each rabbit and purification by protein G affinity chromatography.
Immunoblots were carried out with 25 to 100 µg of protein using 7% to 22% SDS-PAGE and then transferred for 2 hours onto a polyvinylidine difluoride (PVDF) membrane with a Hoe-fer TE70 Semiphor Semidry-Transfer Unit (Pharmacia-Biotech, Uppsala, Sweden). Nonspecific sites on the membranes were blocked for 1 hour with 5% BSA in blocking buffer (10 mM Tris-HCl, pH 8; 120 mM NaCl, 0.05% Tween). The proteins were immunodetected by incubating for 3 hours with the polyclonal antibodies diluted at 1:3500 (F6) and 1:500 (P14 and P20) in blocking buffer that contained 0.17% BSA. After exhaustive washing, the blots were incubated with a secondary goat antirabbit alkaline phosphatase–conjugated immunoglobulin G (IgG; Sigma) at a dilution 1:30 000 for 2 hours. After 4 washings of 5 minutes each, the membranes were incubated with 66 µg/mL of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 111 µg/mL of nitro blue tetrazolium (NBT) in 0.19 M Tris, 1 mM MgCl2 until color appeared. The image was scanned, and a densitometric analysis was carried out. The Gel Doc System with Molecular Analyst software (BioRad) was used to quantify the changes in intensity of various bands. The second antibody alone was used as a control to rule out the possibility of nonspecific binding to the transferred proteins.
Immunocytochemistry
Glass coverslips that were 13 mm in diameter coated with
poly-L-lysine were used for the immunolocalization of P14 and P20
in control, capacitated, and acrosome-reacted sperm. Aliquots of 159 µL of
1 mg/mL of poly-L-lysine in PBS were loaded on coverslips
previously washed with ethanol and incubated for 20 minutes at room
temperature. After drawing off the poly-L-lysine solution with a
pipette, the coverslips were washed with distilled water, air dried, and
sterilized with a 254-nm ultraviolet light for 15 minutes. Sperm samples
containing about 0.5 x 105 cells in 300 µL of medium were
incubated for 10 minutes with 4% form-aldehyde, placed on the coverslips, and
centrifuged for 8 minutes at 400 x g in a Jouan BR4 centrifuge
(DEC Inc, Lorton, Va) with a Jouan S20 microtiter rotor. After being washed
twice (5 minutes each) with sterile PBS, nonspecific binding sites were
blocked with 5% BSA in PBS (300 µL) for 30 minutes at 37°C and washed
again with sterile PBS. Samples were exposed to 250 µL of the primary
antibodies (1:75 anti-P20 and 1:150 anti-P14) in PBS/1% BSA for 1 hour at
37°C. After being washed 3 times with PBS, samples were stained for 2
hours in the dark at room temperature with 250 µL (1/1000) Alexa fluor
488–conjugated goat anti-rabbit IgG (Molecular Probes, Leiden, The
Netherlands) in PBS/1% BSA, washed as above and dried at room temperature, and
mounted over 1 mg/mL p-phenylendiamine in 90% glycerol
(Oriol and Mancilla-Jimenez,
1983). To rule out any nonspecific binding to the sperm surface, a
control using the second antibody alone was performed.
Sperm were evaluated with an E-400 Nikon epifluorescent light microscope (Tokyo, Japan) interfaced with a Sony digital power HAD camera (Carson, Calif), and fluorescence images were photographed (40x and 100x).
NH2-Terminal Sequencing
Aliquots of 5 to 10 µg of purified P14 and P20 were used for
NH2 terminal sequencing on an Edman automatic sequencer (Protein
Chemistry Service, CBM Severo Ochoa, Madrid, Spain) with the reagents and
methods recommended by the manufacturer. The amount of the sample limited the
number of cycles and subsequently the number of sequenced amino acids.
Homology was analyzed using the Expasy Molecular Server and BLAST software
(Altschul et al, 1990).
Glycosylation Analysis
Proteins separated by SDS-PAGE were transferred onto a PVDF membrane, and
the glycosylated proteins were visualized with an immunoblot-based
glycoprotein detection kit (ECL Glycoprotein Detection System, Amersham
Biosciences, Barcelona, Spain) using a streptavidine-alkaline-phosphatase
conjugate (BioRad). Transferrine was included in 1 well as a positive control.
For the negative control, 1 lane was cut from the membrane and incubated as
the rest of the membrane but without the periodate treatment.
Phosphorylation Analysis
Proteins that were separated by SDS-PAGE and transferred onto a PVDF
membrane were analyzed to detect phosphorylation on their Tyr, Ser, or Thr
residues. The blots were blocked as described above and incubated with the
appropriate dilutions for each monoclonal antibody (Sigma): 1:3000
(antiphospho-tyrosine, mouse IgG1 clone PT 66); 1:1000 (antiphospho-serine,
mouse IgG1 clone PSR 45, and -threonine, mouse IgG2b clone PTR 8) for 2 hours
at 25°C. After an exhaustive washing, the membranes were incubated for 1
hour with a secondary goat anti-mouse alkaline phosphatase–conjugated
IgG (Sigma) diluted 1:2500 in blocking buffer and then washed and incubated
with the reagents BCIP and NBT, as described above, to develop the colored
reaction. The images were scanned, and densitometric analysis was carried out.
The specificity of the antiphosphoserine and -threonine antibody was checked
by using Flavodoxin from Chlorella (negative control
[Peleato et al, 1994], a kind
gift from Dr L. A. Inda) and 
-bovine casein (positive control, Sigma).
Further, the second antibody alone was used as a control to rule out
nonspecific binding to the transferred proteins.
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Results |
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Content of P14 and P20 on the Ram Sperm Membrane
To investigate whether the protective effect of P14 and P20 was related to
the decapacitation role played by some seminal plasma proteins, we carried out
a comparative analysis of the changes that in vitro CA and the AR had on the
quantity of both of these proteins on the surface of ram spermatozoa. Proteins
from the sperm membrane were extracted with detergent, and the contents of P14
and P20 were analyzed by Western blotting
(Figure 2). CA had a greater
effect on the quantity of P20 that was released from the membrane once the
process was begun, with about 37% remaining after 5 hours of incubation under
capacitating conditions (Figure 2C and
D). However, nearly 70% of P14 persisted on the sperm membrane
after 5 hours of incubation (Figure 2A and
B). The induction of the AR accounted for an additional release of
both proteins, with not more than 35% remaining on the acrosome-reacted sperm
(Figure 2B and D, lane 6).
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Immunolocalization of P14 and P20 on the Sperm Surface
Since CA and the AR modified the content of both protein bands on the sperm
membrane, we investigated the changes induced by these processes on the
distribution of P14 and P20 on the cell surface.
Immunocytochemical localization and relative distribution analysis of P14 and P20 on control sperm samples showed that both proteins are localized on several surface domains. The percentage of differently immunostained subpopulations is shown in Figure 3A. The main sperm subpopulations in the control samples showed P14 labeling on the entire cell surface (34%) (Figure 3B1, pattern W) as well as on the acrosomal region (Figure 3B1, pattern A), either alone (26%) or with additional labeling on the equatorial and/or the flagellum (27%) regions. Other minor subpopulations showed immunofluorescence on the entire flagellum (Figure 3B1, pattern T) or head (Figure 3B1, pattern H). Similarly, the major subpopulations in the control spermatozoa had P20 labeling at the acrosomal region (about 46%) (Figure 3B2, patterns A-T and A-E) and the entire cell surface (31%) (Figure 3B2, pattern W). A minor subpopulation showed fluorescence at the postacrosomal region and the intermediate piece (Figure 3B2, pattern P). These results were highly specific, because replacing the antiserum with preimmune serum eliminated protein detection.
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Sequence Analysis
Automated Edman degradation of P14 and P20 gave reliable data, which
indicated Asp as the N-terminal residue of both proteins, and 34 and 29 amino
acids were obtained for P14 and P20, respectively. The N-terminal sequence of
P14 is:
NH2-D-D-E-L-T-R-D-K-S-S-E-E-S-H-E-D-E-E-C-V-F-P-F-T-Y-Y-D-D-R-H-F-D-C-T-.
This sequence was performed twice until amino acid 35 was reached and at least
4 times for the first 10 amino acids. Sequencing cycles at 9 and 33 did not
produce any amino acid peaks, and they were putatively assigned as
cysteine.
Comparative sequence analysis (Expasy Molecular Server) showed a relationship between P14 and several proteins and showed the highest homology with PDC-109 (also called BSP-A1/A2 or SFP1-Bovin), a protein secreted by the seminal vesicles that modulates bovine sperm CA (Manjunath and Sairam, 1987; Thérien et al, 1997; Gwathmey et al, 2003). Alignment of the N-terminal amino acid sequences of P14 and PDC-109 (Protein Data Bank) showed that P14 contains a part of the Fibronectin Domain Type II, from amino acid 19 to at least 34 (49 to 64 of 134 amino acids contained by PDC-109). This domain has been conserved during evolution, as it is found in the proteins of the cytoskeleton and extracellular matrix of eucaryotic, metazoan, and chordate organisms and is mainly involved in collagen linkage.
For the N-terminal sequencing of P20, although we had sampled only the 20.5-kd prominent band, a slight contamination with the 22-kd protein could have occurred, probably due to the presence of hydrophobic domains (shown by partition in a 2-phase system with Triton X-114 [data not shown]), which could account for their tendency to aggregate. Another possibility is that both bands correspond to the same protein with a different degree of glycosylation. Therefore, we also found small peaks (very low concentration) of a minor band sequence. The main sequence that certainly corresponded to the major 20.5-kd protein band was NH2-D-E-P-L-P-D-V-Y-D-V-L-G-M-L-C-C-T-W-S-Y-Y-Y-A-D-Q-G-G-P-P- (this sequence was performed twice). Sequencing cycles at 15 and 16 did not produce any amino acid peaks, and they were putatively assigned as cysteine. This N-terminal sequence did not appear to be homologous with any known protein in the current ProteinBank databases.
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Biochemical Characterization
The analysis of the sequenced fragments of both proteins showed no possible
sites of glycosylation, although P14 was predicted to possibly be bound to
N-acetyl glucosamine. We found that P14, separated in the
polyacrylamide gel from F6 and transferred onto a PVDF membrane, did not
contain any carbohydrate residue, which indicates that it is not a
glycoprotein. However, P20 showed an intense labeling of glycosylation
(Figure 6A).
The prediction of phosphorylation of both N-terminal sequenced fragments showed that P14 could have 3 residues of serine and 2 of threonine that could be phosphorylated, while P20 could have only 1 phosphorylated tyrosine. In an attempt to further characterize these proteins, the components of F6 obtained in the presence of 200 µM of sodium orthovanadate, a powerful phosphatase inhibitor (Gordon, 1991; Viñals et al, 2001), were separated by SDS-PAGE and transferred onto a PVDF membrane, and phosphoproteins were immunodetected using anti-phosphotyrosine, anti-phosphoserine, and anti-phosphothreonine antibodies. Only P14 was phosphorylated at serine and threonine residues (Figure 6B).
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Discussion |
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We have already shown that whole seminal plasma proteins and one of its fractions separated by exclusion chromatography (F6) can repair cold-shock damage in ram spermatozoa (Barrios et al, 2000). In the present work, we prove that the addition of F6 before cold-shock treatment prevents sperm membrane injury and that 2 protein bands of approximately 14 and 20 kd are responsible for this protective effect.
The adsorption of specific components of seminal plasma onto the surface of ejaculated sperm has been reported in several studies (Metz et al, 1990; Desnoyers and Manjunath, 1992; Manjunath et al, 1993; Amann et al, 1999). These sperm-coating components could play an important role in CA and/or sperm transport (Cross, 1996). Some of these components could stabilize the membrane during critical time frames, whereas others ultimately could contribute to exocytosis (membrane loss).
Results of this investigation indicate that whole ram seminal plasma proteins (>3 kd) and the F6 obtained by exclusion chromatography (Barrios et al, 2000) are adsorbed onto the sperm surface and significantly improve cold-shocked sperm survival. The beneficial effect of the inclusion of seminal plasma proteins into the cold-shock medium is specific for such proteins, since the viability of control samples with only BSA as a supplement was strongly decreased as a consequence of the cold-shock treatment. Moreover, it was necessary to add BSA to samples that did not contain plasma proteins to avoid cells sticking to the tube wall during cold-shock, which significantly affected results.
We have shown that the addition of either F6 or either of the 2 protein bands isolated from this fraction (P14 or P20) before cold shock increased sperm resistance to damage. Analysis of the quantity of P14 and P20 on the surface of ram spermatozoa indicated that their protective effect could be related to their membrane stabilizing effect. We could hypothesize that this protective effect is related to the decapacitating role reported for some seminal plasma proteins, which can be deduced by protein release from the membrane at CA (Desnoyers and Manjunath, 1992). That P20 is released from the membrane once CA begins could suggest a decapacitating role for this protein, where its partial loss could contribute to the onset of CA. Conversely, the induction of the AR had a greater effect on the quantity of P14 that was released than P20. About 35% of each protein persisted on acrosome-reacted sperm, suggesting that both proteins are also involved in subsequent processes as gamete interactions.
To investigate further the physiological function of P14 and P20, we analyzed how their surface distribution was affected by the in vitro induction of CA and the AR. The results of the immunodetection of P14 and P20 on the sperm surface before and after the induction of these processes confirm that the induced membrane alterations account for a decrease in the quantity as well as the migration and redistribution of both proteins. Cytochemical observations showed that the sperm surface has several binding sites for P14 and P20. The main location of both proteins in the control samples (acrosomal membrane and flagellum) could suggest that they contribute to the regulation of CA and/or the AR and motility, respectively. The observation, according to the Western blot analysis of protein content and the immunocytochemical distribution study, that P20 was highly affected by CA would suggest the decapacitating role of this protein, as its loss occurs at the beginning of the CA. That CA and the AR induce the migration of both proteins to the equatorial and postequatorial region supports our previous observation of that some proportion of P14 and P20 still remains on the sperm surface after both processes and could be related to their role in gamete interactions.
The process of CA has been described as a series of largely uncharacterized cellular and molecular events that occur within the female reproductive tract and is required for the AR and fertilization (Yanagimachi, 1994). During the AR, the apical plasma membrane of the sperm head starts to fuse with the underlying outer acrosomal membrane at multiple sites, resulting in the dispersal of the acrosomal content (Saling et al, 1979). The equatorial plasma membrane area of the sperm head is not modified (Curry and Watson, 1995) because it does not participate in the fusions with the outer acrosomal membrane and seems to be the specific site involved in the oolemma interaction (Clark and Koehler, 1990; Yanagimachi, 1994; Flesch and Gadella, 2000). This remodeling could be the main reason for the redistribution of P14 and P20 reported in this study during CA and the AR. In vitro capacitated and acrosome-reacted ram sperm displayed a change in the cellular distribution of both proteins, more significant for P20, from the acrosome to the equatorial and post-equatorial regions in a large population of sperm. This protein redistribution could contribute to the exposition and binding of recognition residues, or the proteins could even act as a direct intermediate in fusion with the zona pellucida. These results are consistent with reports in several species regarding the redistribution of some proteins (Yanagimachi, 1994; Yudin et al, 1998; Meyers and Rosenberger, 1999; Cohen et al, 2000a,b; Lalancette et al, 2001) and decapacitating factors (Yanagimachi, 1994) during CA or the AR. Together, these results suggest that the protective effect of P14 and P20 is related to their decapacitating role. Moreover, their role in gamete interaction cannot be ruled out, as both proteins remain on the sperm surface after the in vitro induction of CA and the AR.
Although P14 and P20 have a similar capacity to protect sperm, the results of the comparative study of their amino acid sequences show that they do not seem to be homologous and appear to be different proteins. The P14-sequenced fragment showed a high identity with several seminal plasma proteins from different species, particularly bovine PDC-109 (Esch et al, 1983) (also called BSP A1/A2) (Manjunath and Sairam, 1987) and GSP-14/15 kd (goat seminal plasma protein, related to the bull seminal protein [BSP] family) (Villemure et al, 2003). PDC-109 has been reported to be involved in a first step toward the stabilization of the sperm membrane (Greube et al, 2001) and, subsequently, to participate, in the female tract, in CA by releasing cholesterol and binding high-density lipoprotein (HDL) and heparin (Thérien et al, 1998, 2001; Gwathmey et al, 2003).
Interestingly, the P14 sequenced fragment shares a high homology from amino acid 19 to 34 (CVFPFTYYDDRHFDCT) with 49 to 64 of PDC-109 (CVFPFVYRNRKHFDCT), which is a conserved domain in several proteins called FN2 (Fibronectin Domain Type II) (Greube et al, 2001). This is a collagen-binding domain that binds to different extracellular matrix and cytoskeleton components to stabilize the extracellular matrix and determine the shape of the cell and cytoskeleton organization. The FN2 domain is also found in proteins from different tissues (Plucienniczak et al, 1999) and the seminal plasma of different species such as the bull BSP family (Thérien et al, 1995), stallion HSP-1 (Calvete et al, 1995), boar pB1 (Calvete et al, 1997), and goat (Villemure et al, 2003), and it has been suggested that this domain plays a similar biological role in all cases because of the observed structural relationships.
The FN2 domain binds choline phospholipids and heparin and promotes the binding of the seminal plasma protein to the sperm membrane upon ejaculation (Desnoyers and Manjunath, 1992; Moreau et al, 1998; Muller et al, 1998). We could hypothesize that P14 takes part in the protein structure surrounding the spermatozoa in a way similar to that of fibronectin, stabilizing membrane phospholipids, and the cytoskeleton. The P14 protective effect might be due to the association of the protein with the sperm membrane in a way that would be instrumental in the production of a protective barrier, which might also involve the cytoskeleton. Additionally, we obtained evidence that P14 is partially released from the sperm membrane during CA, as has been reported for BSP proteins (Thérien et al, 2001), and is redistributed over the sperm surface. The implications of this migration are significant, and we suggest that P14 is involved in sperm CA and gamete interaction, a first step in the stabilization of the sperm membrane, and participates in the subsequent CA in the female reproductive tract by binding HDL and heparin in a way that is similar to BSP proteins (Thérien et al, 1998, 2001; Gwathmey et al, 2003).
That this protein is phosphorylated in its serine and threonine residues suggests a role in the regulation of the fertilization process, possibly through its dephosphorylation by membrane protein phosphatases.
Since the P20-sequenced fragment was not homologous with any reported protein, no information can be deduced about its possible function. By immunochemical analysis, we obtained evidence that P20 is a glycosylated protein and that it continues to reside, partially, on the sperm surface after CA and the AR. These results suggest a role in sperm CA and zona pellucida binding, as noted.
In conclusion, we have shown that 2 seminal plasma proteins of apparent molecular weights of 14 and 20 kd can protect ram spermatozoa against cold shock, and we suggest that these proteins are involved in the fertilization process. Further studies on the functional role of P14 and P20 would provide a better understanding of the molecular events of CA and reproductive mechanisms in rams.
The availability of these proteins could assist in the formulation of improved diluents for preserving ram spermatozoa during freezing or storage. Adding P14 and P20 to the semen extender could protect ram semen against cold shock during storage, allowing the storage of spermatozoa for extended periods and the improvement of cryopreservation methods.
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Acknowledgments |
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Footnotes |
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References |
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Amann RP, Hammerstedt RH, Shabanowitz RB. Exposure of human, boar
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Ashworth PJC, Harrison RAP, Miller NGA, Plummer JM, Watson PF. Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma. Reprod Fertil Dev. 1994; 6: 173 -180.[CrossRef][Medline]
Aumuller G, Vesper M, Seitz J, Kemme M, Scheit KH. Binding of a major secretory protein from bull seminal vesicles to bovine spermatozoa. Cell Tissue Res. 1988; 252: 377 -384.[Medline]
Austin CR. Observations on the penetration of the sperm into the mammalian egg. Aust J Sci Res B. 1951; 4: 581 -592.
Austin CR. The "capacitation" of the mammalian sperm. Nature. 1952;170: 326 .[CrossRef][Medline]
Baas JW, Molan PC, Shannon P. Factors in seminal plasma of bulls that effect the viability and motility of spermatozoa. J Reprod Fertil. 1983;68: 275 -280.
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) and capacitated (
) samples
were assessed for different labeling patterns (%) using polyclonal antibodies
to P14 or P20. A indicates acrosomal; E, equatorial; W, whole cell surface; T,
flagellum; H, head; A-T, acrosomal and tail; A-E, acrosomal and equatorial;
and P, postacrosomal. Mean ± SEM of 3 experiments.
*P < .001.
), and acrosome-reacted (
) samples were assessed for
different labeling patterns (%) using polyclonal antibodies to P14 or P20. A
indicates acrosomal; E, equatorial; W, whole cell surface; T, flagellum; H,
head; A-T, acrosomal and tail; A-E, acrosomal and equatorial; and P,
postacrosomal. Mean ± SEM of 3 experiments. *P <
.001.