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BOR - Papers in Press, published online ahead of print April 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.016527
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BIOLOGY OF REPRODUCTION 69, 640–646 (2003)
DOI: 10.1095/biolreprod.103.016527
© 2003 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Influence of Porcine Spermadhesins on the Susceptibility of Boar Spermatozoa to High Dilution1

Fernando Centurion3,4, Juan M. Vazquez2,4, Juan J. Calvete5, Jordi Roca4, Libia Sanz5, Inmaculada Parrilla4, Eva M. Garcia4, and Emilio A. Martinez4

Departamento de Medicina y Cirugía Animal,4 Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain Instituto de Biomedicina,5 C.S.I.C., Valencia, Spain


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effect of heparin-binding and non-heparin-binding spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa at the high dilution (300 000 sperm/ml) to which sperm are exposed during the process of sex sorting by flow cytometry was investigated. Incubation of spermatozoa with heparin-binding spermadhesins caused a time- and dose-dependent decrease in the percentage of functional spermatozoa. The percentage of viable spermatozoa incubated at 38°C with heparin-binding spermadhesins diluted in PBS (1 mg/ml) dropped from 75% (0.5 h) to 4% (5 h), whereas the percentage of viable spermatozoa incubated in PBS without proteins (control) decreased from 85% (0.5 h) to 19% (5 h). Addition of non-heparin-binding PSP-I/PSP-II spermadhesin to the PBS resulted in a concentration-dependent increment of the percentage of viable cells (65% after 5-h incubation), with maximum effect at 1.5 mg/ml. The heparin-binding spermadhesins totally suppressed sperm motility and mitochondrial activity after 5 h of incubation. The same parameters of sperm incubated in the presence of 1.5 mg/ml of PSP-I/PSP-II were 50% and 58%, respectively, and the percentages of control sperm displaying motility and mitochondrial activity were 21% and 26%, respectively. Moreover, the viability, motility, and mitochondrial activity all decreased on incubation of spermatozoa with mixtures of PSP-I/PSP-II and heparin-binding spermadhesins as the concentration of the latter increased. We conclude that PSP-I/PSP-II and the heparin-binding spermadhesins exert antagonistic effects on the functionality of highly diluted boar spermatozoa. The finding that PSP-I/PSP-II contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 h at physiological temperature points to its potential use as an additive for sperm preservation, specifically of highly diluted, flow-sorted spermatozoa for sex preselection.

assisted reproductive technology, male sexual function, sperm, sperm motility and transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Survival of ejaculated boar spermatozoa in seminal plasma alone is limited to a few hours [1]. To prepare semen for fertilization, dilution in an appropriate extender is necessary [1]. The precise role of the components of extenders (sugars, proteins, a range of additives, etc.) in preserving the integrity and the fertilizing potential of spermatozoa is still far from being understood, and the composition of the diluents has been improved by more or less empirical studies since the critical breakthrough in cryopreservation of sperm that occurred more than 50 yr ago [2].

The formulation of semen extenders appropriate to the species to overcome the detrimental effects of the extreme dilution (1000–2000-fold) of the spermatozoa that occur during the flow-cytometric sorting for preselection of sex is also a subject of current discussion in reproduction technology. An increase of the percentage of cell death, along with changes in the metabolic activity, motility, and fertilizing ability of spermatozoa have been documented (for review, see [3]). The effect of dilution has been linked to the removal of seminal plasma factors that contribute to the stabilization of the sperm membranes. The protective properties of seminal plasma have been reported [4, 5], and increasing evidence from a number of species supports the view that dilution triggers a capacitation-like state in spermatozoa, culminating in premature acrosome reactions. Consequently, from 1% to 10% homologous seminal plasma is routinely added to the collection medium during the flow-cytometric sorting of spermatozoa [6].

The seminal plasma, the fluid in which mammalian spermatozoa are suspended in semen, is a complex mixture of secretions originating from the testes, epididymis, and the male accessory reproductive organs. Seminal plasma contains factors that influence the fertilizing ability of spermatozoa and exert important effects on the female reproductive physiology [7–9]. Thus, the seminal plasmas of a variety of mammalian species contain both decapacitation factors, which prevent inappropriate acrosome reactions, and proteins that, on binding to the sperm surface, enhance the fertilizing potential of spermatozoa [10, 11]. Mounting evidence suggests that the concerted action of positive and negative regulatory seminal plasma factors modulates the capacitation state of mammalian spermatozoa. The biological effects of these seminal plasma factors on sperm function are complex and not well understood, in part because of the high variability in the composition of the seminal plasma between species, between males within the same species, and between ejaculates of homologous males [4, 12–14].

Porcine seminal plasma contains nonestrogenic factors that protect spermatozoa, thereby producing a suitable population of viable spermatozoa that eventually reaches the site of fertilization [14]. In this species, the bulk (>90%) of the seminal plasma proteins belong to the spermadhesin protein family [15–17], a group of proteins built by a single CUB domain architecture [18]. In the pig, the spermadhesin protein family comprises five members (AQN-1, AQN-3, AWN, PSP-I, and PSP-II). Sequence variation, glycosylation, and the aggregation state of spermadhesins contribute to their distinct biological activities [19–22]. AQN-1, AQN-3, and AWN are heparin-binding and sperm-coating molecules that appear to stabilize the plasma membrane over the acrosomal vesicle and are mainly released during capacitation [23–25]. AQN-3 forms part of a sperm motility-inhibitor factor complex [26]. AQN-1 and AWN are sperm-associated acrosin-inhibitor acceptor proteins [27]. AWN has been characterized as a sperm surface-associated lectin thought to mediate sperm-zona pellucida interactions at fertilization [15–17, 28]. On the other hand, PSP-I and PSP-II [29], the major proteins of boar seminal plasma, accounting for more than 50% of the total proteins, form a non-heparin-binding heterodimer [21] of glycosylated spermadhesins [30], which displays proinflammatory and immunostimulatory activities that appear to modulate immune responses in the porcine uterine environment, thereby contributing to the reproductive success of the species [31–33].

The aim of the present study was to evaluate the biological effects of the heparin-binding and the non-heparin-binding (PSP-I/PSP-II) spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa subjected to the extreme dilution condition to which sperm are exposed during the process of sex sorting by flow cytometry.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection and Preparation of Seminal Plasma

All experiments were performed with the seminal plasma from four mature boars that had previously sired offspring. Sperm-rich ejaculate fractions (100 ml) were obtained using the gloved-hand method [34]. The seminal plasma of each boar ejaculate was separated from spermatozoa by centrifugation at 3800 x g for 15 min at 20°C using a Heraeus Sepatech Megafuge, Osterode, Germany). The supernatants were filtered sequentially through 10- and 1.2-µm filters and pooled.

Isolation of Heparin-Binding Proteins

To isolate the fraction of heparin-binding proteins (HBP), the seminal plasma from the fresh ejaculates collected and prepared as described above was further clarified by centrifugation at 12 000 x g for 15 min at room temperature, followed by affinity chromatography [23]. To this end, 50 ml of seminal plasma were applied to a 40-ml heparin-Sepharose CL-6B (Amersham Pharmacia Biotech., Uppsala, Sweden) column and washed at a flow rate of 6 ml/h with 20 mM phosphate buffer (pH 7.3), containing 150 mM NaCl, until the absorbance at 280 nm reached baseline level. The heparin-bound proteins were eluted with a NaCl gradient (0.15–3 M) in the same buffer at a flow rate of 6 ml/h and an increase in NaCl concentration of 0.015 M/min. Proteins were dialyzed against distilled water, lyophilized, and stored at -20°C until use.

Isolation of Spermadhesin PSP-I/PSP-II Heterodimer

The PSP-I/PSP-II heterodimer was isolated from the non-heparin-binding fraction of seminal plasma by size-exclusion chromatography on a 2000- x 5-cm Sephadex G-50 column equilibrated in a solution (pH 7.4) of 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and 0.025% sodium azide [21]. The identity and purity of the protein preparation was assessed by N-terminal sequence analysis (using an Applied Biosystems 472 automated protein sequencer, Langer, Germany) and MALDI-TOF mass spectrometry (using an Applied Biosystems Voyager DE-Pro mass spectrometer and a saturated solution of sinapinic acid in 50% acetonitrile and 0.1% trifluoroacetic acid as the matrix). Protein concentration was determined spectrophotometrically, using the molar absorption coefficient (27 332 M-1 cm-1) as determined by Menéndez et al. [35] or by amino acid analysis (after sample hydrolysis in 6 M HCl for 24 h at 106°C in evacuated and sealed ampoules) using a Beckman Gold Amino Acid Analyzer (Beckman, Barcelona, Spain) [36].

Collection of Semen and Evaluation of Sperm Parameters

Sperm-rich ejaculate fractions were obtained using the gloved-hand method [34] twice per week in a sterilized measuring flask. Spermatozoa were counted in a Burker Chamber (Superior, Marienfeld, Germany) at a 1:100 dilution in saline solution as recommended by the manufacturer. Shortly after collection, the semen samples, extended in Beltsville Thawing Solution (BTS) [37] to 30 x 106 spermatozoa/ml, were evaluated for membrane integrity, motility, and mitochondrial activity as described below, and only those exhibiting no apparent sperm abnormalities (motility, >80%; total sperm per ejaculate, >20 x 109; acrosomal abnormalities, <10%; abnormal sperm morphology, <15%) [38] were used. To remove the BTS and the seminal plasma, spermatozoa were washed once in PBS by centrifugation at 400 x g for 5 min. The final pellet was serially diluted in PBS to a final sperm count of 0.3 x 106 spermatozoa/ml and used in subsequent studies with seminal plasma proteins. Sequential dilutions were performed, because it has been shown that pipetting spermatozoa directly into dilution medium is more detrimental to sperm membranes than the gradual mixing of dilution medium with spermatozoa [3].

The viability (membrane integrity) of spermatozoa was determined using the fluorescent probe carboxyfluorescein diacetate (CFD; Sigma Chemical Co., Alcobendas, Spain) by a modification of the method of Harrison and Vickers [39]. Briefly, 20 µl of 0.46 mg/ml of carboxyfluorescein diacetate in dimethyl sulfoxide and 20 µl of formaldehyde (2.5 mg/ml in water) were added to 1 ml of BTS containing 0.3 x 106 spermatozoa to achieve a final concentration of 1.7 mM formaldehyde and 20 mM carboxyfluorescein diacetate. After 10 min at 30°C, the stained sperm suspensions were observed at 400x magnification using a Nikon (Tokyo, Japan) Eclipse 800 microscope with epifluorescence illumination. Spermatozoa were scored for the percentage of cells showing green fluorescence along their whole length (intact sperm).

Sperm motility was estimated with a computer-assisted motility analysis system. Sperm samples (10 µl at 0.3 x 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 equipped with a 10x phase objective and a monochrome video camera (Hitachi CCD model, Hitachi, Chiba, Japan) connected to a personal computer. The sperm analysis was performed using the software Sperm Class Analyzer (SCA, Barcelona, Spain). The program settings were as follows: frames rate, 25 Hz; search radius, 11.5 µm; minimum track points, seven frames; and threshold straightness, 75%. At least 100 spermatozoa were analyzed per sample. Motion parameters were percentage of motile spermatozoa, curvilinear velocity (VCL; time-average velocity of the sperm head along its actual trajectory), straight-line velocity (VSL; time-average velocity of the sperm head along a straight line from its first position to its last position), and dance (DNC; curvilinear velocity multiplied by amplitude of lateral head displacement [40]).

Rhodamine 123 (R 123; Sigma) was used as a probe of mitochondrial membrane potential. Sperm (0.3 x 106 sperm/ml) were stained by incubation for 15 min in 1 µg/ml of R 123 and 4 µg/ml of propidium iodide (Sigma). Sperm were examined under an Eclipse 800 (Nikon) with epifluorescence illumination. Only spermatozoa showing green fluorescence in their midpieces were considered to be positive for mitochondrial activity.

At least 200 spermatozoa were analyzed per duplicate sample (100 spermatozoa/sample) for each parameter evaluated.

Effect of Spermadhesins on Highly Diluted Boar Spermatozoa

All experiments were performed with the same batch of lyophilized proteins and with four pools of spermatozoa collected on four different days. Each pool was made by mixing the sperm of four different boars. Boars among days were the same. Duplicate samples were done for each treatment.

The effect of the non-heparin-binding PSP-I/PSP-II spermadhesin heterodimer, the HBP, and the mixture of them on the motility, membrane integrity, and mitochondrial activity of highly diluted boar spermatozoa was evaluated. To this end, washed spermatozoa (3 x 105 sperm/ml in PBS) were incubated at 38°C for 0, 2, or 5 h with 1) 1.5 mg/ml of PSP-I/PSP-II, 2) 1.12 mg/ml of PSP-I/PSP-II and 0.25 mg/ml of HBP, 3) 0.75 mg/ml of PSP-I/PSP-II and 0.5 mg/ml of HBP, 4) 0.38 mg/ml of PSP-I/PSP-II and 0.75 mg/ml HBP, 5) 1 mg/ml of HBP, and 6) without added proteins (control).

To study the effect of the concentration of non-heparin-binding PSP-I/PSP-II spermadhesin heterodimer on the motility, membrane integrity, and mitochondrial activity of highly diluted boar spermatozoa, samples of 3 x 105 sperm/ml in PBS were incubated at 38°C for 0, 2, or 5 h with increasing amounts (0.15, 0.3, 1.5, 7.5, and 15 mg/ml) of PSP-I/PSP-II and without added proteins (control).

Data Analysis

All experimental data correspond to the mean ± SEM. Analysis of variance (ANOVA) was carried out using the general linear model procedure implemented in the SPSS 9.0/PC statistics package (SPSS, Inc., Chicago, IL). A P value of less than 0.05 was considered to be statistically significant. When ANOVA revealed a significant effect, values were compared by Tukey honest-significant-difference test adjustment for multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dose- and Time-Dependent Effects of PSP-I/PSP-II and HBP on Sperm Functions

Figure 1A shows the time-course decrease of the percentage of viable (CFD-positive [CFD+]) spermatozoa exposed to high dilution. Approximately 75% of highly diluted (0.3 x 106/ml) spermatozoa were viable after 0.5-h incubation at 38°C, and this value significantly decreased to 19% at 5 h. Addition of the isolated non-heparin-binding spermadhesin PSP-I/PSP-II heterodimer to a final concentration of 1.5 mg/ml resulted in a greater percentage of viable cells at each incubation time period assayed; that is, 65% of spermatozoa were scored CFD+ after 5-h incubation. On the other hand, the addition of HBP at a final concentration of 1 mg/ml had a markedly detrimental effect on the viability of spermatozoa, with only 4% CFD+ cells following 5 h of incubation. Moreover, the negative effect of the HBP was also evidenced by exposure of the highly diluted spermatozoa to mixtures of PSP-I/PSP-II and HBP. Figure 1A shows the time-dependent viability decrease of spermatozoa incubated with increasing concentrations of HBP.



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  		FIG. 1. Effect of spermadhesins on the viability, motility, and mitochondrial activity of highly diluted spermatozoa. Shown are the effects of the percentage of mixtures of PSP-I/PSP-II and heparin-binding spermadhesins on spermatozoa displaying fluorescence after incubation with CFD (A), the percentage of motile spermatozoa evaluated by computer-assisted motility analysis system (B), and the percentage of spermatozoa displaying fluorescence after incubation with R 123 (C). Spermatozoa were diluted to 0.3 x 106 sperm/ml and incubated for 0.5, 2, or 5 h at 38°C in PBS containing 1.5 mg/ml of PSP-I/PSP-II (column a), 1.125 mg/ml of PSP-I/PSP-II and 0.25 mg/ml of HBP (column b), 0.75 mg/ml of PSP-I/PSP-II and 0.5 mg/ml of HBP (column c), 0.375 mg/ml of PSP-I/PSP-II and 0.75 mg/ml of HBP (column d), and 1 mg/ml of HBP (column e). A control sample without added proteins is also shown (column f). Columns represent the mean ± SEM (error bars) of two separate determinations of four samples per treatment evaluated in duplicate. *P < 0.05 compared to the control, #P < 0.05 compared to the previous point within the same incubation time series

The same trend was observed regarding the motility and mitochondrial activity of highly diluted spermatozoa incubated with spermadhesins (Fig. 1, B and C, respectively). After 5 h of incubation at 38°C, 1.5 mg/ml of PSP-I/PSP-II in the incubation medium preserved the motility and mitochondrial activity in 50% (Fig. 1B) and 58% (Fig. 1C) of spermatozoa, respectively. Under the same conditions, only 20–25% of spermatozoa retained these activities in the absence of added proteins, and these figures dropped to 0% and 2%, respectively, when 1 mg/ml of HBP was included in the incubation medium (Fig. 1, B and C). In addition, and similar to what happened with sperm viability, mixtures of PSP-I/PSP-II and HBP displayed detrimental effects on both the motility and the mitochondrial activity of highly diluted spermatozoa as a function of the increasing concentration of the heparin-binding spermadhesins (Fig. 1, B and C).

The motility characteristics VCL, VSL, and DNC were all sensitive to both the incubation time and the addition of PSP-I/PSP-II or HBP to the incubation medium (Table 1). A time-dependent increase in VCL, VSL, and DNC was observed when highly diluted spermatozoa were incubated in the presence of PSP-I/PSP-II (1.5 mg/ml). On the other hand, lower velocities were recorded as a function of increasing concentrations of HBP (Table 1). In particular, concentrations of HBP greater than 0.5 mg/ml completely abolished sperm motility as measured by VCL, VSL, and DNC (Table 1).


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  		TABLE 1. VCL, VSL, and DNC of highly diluted boar spermatozoa (0.3 x 106 spermatozoa/ml) as a function of the incubation time at 38°C and of the presence or absence (control) of protein mixtures containing increasing ratios of PSP-I/PSP-II and HBP.a

Dose- and Time-Dependent Effects of Increasing Concentration of PSP-I/PSP-II on Sperm Functions

Increasing the amount of PSP-I/PSP-II in the incubation medium of highly diluted spermatozoa slightly enhanced the number of viable cells (CDF+) in a concentration-dependent manner, reaching maximal effect at 7.5 mg/ml in samples incubated at 38°C for 5 h (Fig. 2A). The percentage of spermatozoa with intact mitochondrial activity was also enhanced by increasing the concentration of PSP-I/PSP-II in the incubation medium, with maximal effect at 7.5 mg/ml (Fig. 2B). On the other hand, concentrations of PSP-I/PSP-II greater than 1.5 mg/ml significantly decreased the percentage of motile cells (Fig. 2C). Incubation of spermatozoa with 15 mg/ml of PSP-I/PSP-II for 5 h completely suppressed their motility (Fig. 2B and Table 2). The sperm motility characteristics (VCL, VSL, and DNC) were sensitive to both the incubation time and the increase of the PSP-I/PSP-II concentration. A time-dependent increase of VCL, VSL, and DNC was observed at 1.5 mg/ml of PSP-I/PSP-II, but lower velocities were recorded as a function of increasing concentrations of PSP-I/PSP-II (Table 2). In particular, at the concentration of PSP-I/PSP-II found in boar seminal plasma (15 mg/ml), sperm motility was completely abolished.



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  		FIG. 2. Effect of spermadhesin PSP-I/PSP-II on the viability, motility, and mitochondrial activity of highly diluted spermatozoa. Shown are the effects of the percentage of increasing concentration of spermadhesin PSP-I/PSP-II on the percentage of spermatozoa displaying fluorescence after incubation with CFD (A), the percentage of motile spermatozoa evaluated by computer-assisted motility analysis system (B), and the percentage of spermatozoa displaying fluorescence after incubation with R 123 (C). Spermatozoa were diluted to 0.3 x 106 sperm/ml and incubated for 0.5, 2, or 5 hours at 38°C in PBS containing the following final concentrations mg/ml of purified spermadhesin PSP-I/PSP-II: 0.15 mg/ml (column a), 0.30 mg/ml (column b), 1.5 mg/ml (column c), 7.5 mg/ml (column d), and 15 mg/ml (column e). A control sample without added PSP-I/PSP-II is also shown (column f). Columns represent the mean ± SEM (error bars) of two separate determinations of four samples per treatment evaluated in duplicate. *P < 0.05 compared to the control, #P < 0.05 compared to the previous point within the same incubation time series


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  		TABLE 2. VCL, VSL, and DNC of highly diluted boar spermatozoa (0.3 x 106 spermatozoa/ml) as a function of the incubation time at 38°C and of the presence or absence (control) of increasing concentrations of isolated spermadhesin PSP-I/PSP-II.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Extending the viability of highly diluted spermatozoa is of interest for sperm preservation, particularly when artificial insemination with flow-cytometric, sex-preselected spermatozoa is intended [3, 41, 42]. It has been repeatedly documented that even a gradual dilution of sperm (as in the flow-cytometric sorted spermatozoa) induces changes in motility pattern, short life span, and loss of the fertilizing ability of spermatozoa [3, 43]. The efficiency of the sex sorting of spermatozoa by flow cytometry allows 10–15 million sorted spermatozoa to be obtained per hour with a purity of 85–90% [41]. This means that for employing deep intrauterine insemination as the best technique available to inseminate these spermatozoa, sorting times of approximately 10 h are required. Every 3–4 h, the highly diluted, cell-sorted spermatozoa are concentrated. Therefore, we used a time window of 0–5 h to evaluate the effects of added proteins.

In line with previous reports, our control data show that, in the absence of added seminal plasma proteins, dilution to 0.3 x 106 spermatozoa/ml, which represents the dilution rate that spermatozoa must undergo on flow-cytometric cell sorting for sex preselection, reduced their viability (~50%) (Fig. 1A), motility (~60%) (Fig. 1B and Table 1), and mitochondrial activity (~55%) (Fig. 1C) after 5 h of incubation at physiological temperature. The beneficial effects of the addition of whole seminal plasma to the collection media of flow-cytometric sorted spermatozoa of several species has been reported [4, 6, 44]. These investigations and the high variability in the seminal plasma composition between males within the same species as well as between ejaculates of homologous males [12–14] prompted us to evaluate the effect of specific protein fractions (heparin-binding and non-heparin-binding spermadhesins, which together represent >95% of the total porcine seminal plasma proteins) on the physiology of homologous spermatozoa. To this end, porcine seminal plasma was fractionated by affinity chromatography on heparin-Sepharose, and the non-heparin-binding PSP-I/PSP-II spermadhesin heterodimer was further purified by gel filtration chromatography. The working concentrations of HBP and of PSP-I/PSP-II (1.0 and 1.5 mg/ml, respectively) represent the concentrations determined in 10% of seminal plasma [21, 24; unpublished results], which in turn represent the protective dose of seminal plasma routinely added to the collection medium of flow-cytometric sorted spermatozoa [6]. Our data clearly show that the HBP and PSP-I/PSP-II spermadhesins exert opposite effects on the viability, motility, and mitochondrial activity of highly diluted spermatozoa. The addition of the HBP had a markedly time- and concentration-dependent detrimental effect on these parameters, whereas PSP-I/PSP-II preserved the membrane integrity, motility, and mitochondrial activity in a greater number of spermatozoa in comparison with control samples. The deleterious effect of the HBP was consistently noticed in sperm samples incubated with mixtures of PSP-I/PSP-II and HBP as a function of the HBP concentration. Increasing the concentration of PSP-I/PSP-II alone in the dilution medium also caused a deleterious effect on sperm. In particular, spermatozoa incubated with 15 mg/ml of PSP-I/PSP-II for 5 h retained a high degree of viability (Fig. 2A) but were immotile (Fig. 2C and Table 2), although more than 20% of live spermatozoa showed R 123 fluorescence (Fig. 2B). A similar percentage of immotile spermatozoa showing R 123 fluorescence has been reported earlier [45], and Windsor and White [46] have demonstrated that ram spermatozoa treated with rotenone, a potent inhibitor of mitochondrial function, displayed high fertility rates on laparoscopic insemination. The mechanism by which the inhibition of mitochondrial function may have extended the survival of spermatozoa deserves further investigation.

As a whole, our results indicate the existence of an optimal PSP-I/PSP-II concentration of approximately 1.5 mg/ml for enhancing the viability, motility, and mitochondrial activity of highly diluted boar sperm for at least 5 h of incubation. These results are in agreement with studies showing that the addition of 10% boar seminal plasma to highly diluted spermatozoa after sorting improved the percentage of live, motile, and acrosome-intact boar spermatozoa, whereas lower (5%) and higher (20%) concentrations were detrimental to the survival and membrane integrity of the spermatozoa [43]. Moreover, it has been reported that incubating boar spermatozoa with seminal plasma from the sperm-rich fraction produced better in vitro fertilization results than when spermatozoa were incubated with the seminal plasma from other fractions of the ejaculate [13]. Ongoing results from our own laboratories show that PSP-I/PSP-II is the predominant spermadhesin in the sperm-rich fraction, whereas the HBP represent the major proteins in the postspermatic fraction of the ejaculate.

Delineating the physiological functions of individual spermadhesin molecules is a complex matter, both because spermadhesin genes are expressed at different locations along the porcine male genital tract [47] and because, with the exception of PSP-II, which quantitatively forms heterodimers with PSP-I, each spermadhesin exists in different isoforms and/or aggregation states that modulate their binding properties [19–22]. Hence, although the net effect of the HBP mixture appears to be deleterious for in vitro preservation of highly diluted spermatozoa, no extrapolation should be made to the in vivo situation. It is worth noting that the PSP-I/PSP-II spermadhesin heterodimer interacted only weakly with sperm membrane components, because it was not found on the surface of in vitro-capacitated boar sperm [21]. The weak binding of PSP-I/PSP-II to the noncapacitated sperm membrane along with its reported receptor-mediated stimulatory effect of macrophages [31–33, 48] indicate that its effect on sperm physiology may involve signaling events, rather than membrane-structure alterations, via interactions with specific sperm surface receptor(s). Initial experiments using isolated PSP-I and PSP-II show that the sperm function-preserving activity of the heterodimeric spermadhesin appears to be linked to the PSP-II subunit. This suggests that the PSP-II activity may not be associated to its glycan moiety, because both subunits of the PSP-I/PSP-II complex share the same glycans [30]. On the other hand, a subset of the heparin-binding spermadhesins remained tightly bound to the sperm membrane of in vitro-capacitated spermatozoa [15, 23, 24] and, among them, low levels of spermadhesin AWN have been demonstrated on live ejaculated boar sperm [49] and on spermatozoa bound in vivo to the zona pellucida [28]. Ongoing research in our own laboratories indicate that the HBP, but not the PSP-I/PSP-II heterodimer, cause concentration-dependent sperm membrane damage. These data, in conjunction with the results presented here, suggest that the in vitro deleterious effect of the heparin- and membrane-binding spermadhesins might be linked to a membrane-perturbing mechanism. Factors present in both the seminal plasma and the sow's genital tract, including heparin-like glycosaminoglycans, may modulate the extent of the spermadhesin-induced membrane remodeling process, leading to the physiological sperm capacitation state, which cannot be mimicked by incubation with purified HBP. Clearly, further detailed investigations are needed to disclose the physiological function(s) of, and the mechanisms used by, the different spermadhesins. Moreover, besides their in vivo participation in porcine reproduction, studies with purified proteins are required to evaluate the possible biotechnological potential of the distinct spermadhesins comprising the heparin-binding fraction.

In conclusion, the major finding of the present study is that the HBP and PSP-I/PSP-II spermadhesins exert opposite effects on spermatozoa that have been highly diluted to mimic the conditions of flow cytometry. The beneficial effect of the purified PSP-I/PSP-II points to this spermadhesin as a clear candidate for an additive to improve the viability of spermatozoa sorted for sex preselection in the pig.


   FOOTNOTES
 
1 Supported by grants AGL2001-0471 (DGICYT), RZ01-019 (INIA), and BMC2001-3337 (DGICYT) (Madrid, Spain). Back

2 Correspondence: Juan M. Vazquez, Departamento de Medicina y Cirugia Animal, Facultad de Veterinaria, Universidad de Murcia, 30071 Murcia, Spain. FAX: 34 968 367069; vazquez{at}um.es Back

3 Current address: Universidad del Yucatán, Merida, México Back

Received: 21 February 2003.

First decision: 14 March 2003.

Accepted: 14 April 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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