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From the * Department of Medicine and Surgery,
Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain; the
Institute of Biomedicine, C.S.I.C., Valencia,
Spain; and the Division of Comparative
Reproduction, Faculty of Veterinary Medicine and Animal Science, Swedish
University of Agricultural Sciences (SLU), Uppsala, Sweden
| Correspondence to: Juan M Vazquez, Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad de Murcia, 30071 Murcia, Spain (e-mail: vazquez{at}um.es). |
| Received for publication May 8, 2006; accepted for publication June 18, 2006. |
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Abstract |
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Key words: Reproductive tract, semen, sperm, seminal plasma, sperm capacitation
Either whole SP or specific SP components of low molecular weight affect survivability of boar spermatozoa, depending on how long they are exposed to the PSP-I/PSP-II (Centurión et al, 2003). However, there is an advantage to using an isolated protein instead of whole SP in order to avoid the inherent variability that exists in SP composition between males or ejaculates (Killian et al, 1993; Asworth et al, 1994; Zhu et al, 2000). Previous studies, using various in vitro fertilization treatments, indicated that the PSP-I/PSP-II heterodimer does not sustain its influence for long periods during incubation (Caballero et al, 2004b). This suggests that the heterodimer may either lose its action by blockade or degradation or perhaps by attaching in a loose manner to the plasmalemma, thus losing influence over time. In any case, there is a need to establish the pattern of attachment and the influence of incubation on this pattern, as well as to elucidate whether there is any relationship between the presence of the protein, and its relative concentration, and the protective role in vitro.
The present study examined the relationship between the protective effect of the spermadhesin PSP-I/PSP-II and its binding pattern on the membrane surface of boar spermatozoa subjected to extreme dilution conditions similar to those used for flow cytometric high-speed sorting as performed for chromosomal sex separation.
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Materials and Methods |
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The PSP-I/PSP-II heterodimer was isolated from the non-heparin-binding fraction of SP by size-exclusion chromatography on a 2000 x 5 cm Sephadex G-50 column equilibrated in 50 mM Tris-Hcl, 150 mM NaCl, 1 mM EDTA, 0.025% sodium azide, pH 7.4 (Calvete et al, 1995). The identity and purity of the protein preparation was assessed by N-terminal sequence analysis (using an Applied Biosystems 492 automated protein sequencer; Applied Biosystems, Langen, Germany) and MALDI-TOF mass spectrometry using an Applied Biosystems Voyager DE-Pro mass spectrometer. A saturated solution of sinapinic acid in 50% acetonitrile and 0.1% trifluoroacetic acid was used as matrix. Protein concentration was determined spectrophotometrically using a molar absorption coefficient (27 332 M–1 cm–1) as determined by Menéndez et al (1995) or by amino acid analysis (after sample hydrolysis in 6 M HCl for 24 hours at 106°C in evacuated and sealed ampoules) using a Beckman Gold Amino Acid Analyser (Beckman, Barcelona, Spain). Proteins were dialyzed against distilled water, lyophilised, and stored at –20°C until used.
Preparation of Anti-PSP-II Antibodies
The PSP-I and PSP-II subunits were purified from the isolated PSP-I/PSP-II
heterodimer by reverse-phase HPLC as described by Calvete et al
(1995). Polyclonal anti-PSP-II
monospecific antibodies were obtained by immunizing female rabbits with
subcutaneous inoculations of 0.5 mg of highly purified PSP-II heterodimer in
0.5 mL of PBS emulsified with 1.5 mL of Freund complete adjuvant. The animals
were inoculated twice at intervals of 5 weeks after the first immunization
with 0.25 mg of the antigen. Two weeks after the last administration, the
rabbits were bled through the ear vein and the blood sera were tested for anti
PSP-II heterodimer activity by dot-blot ELISA and Western blot.
Semen Handling, Incubation With the Heterodimer PSP-I/PSP-II, and Evaluation of Sperm Parameters
Sperm-rich ejaculate fractions were collected from 3 mature Pietrain boars
of proven fertility using the gloved-hand method. Shortly after collection,
the semen was extended in Beltsville Thawing Solution (BTS;
[Pursel and Johnson, 1975]) to
30 x 106 spermatozoa/mL, and the spermatozoa were evaluated
for motility. Only ejaculates showing >80% progressive sperm motility were
used. The extended ejaculates were pooled and centrifuged (1700 x
g for 3 minutes), the supernatant discarded, and the final sperm
pellet re-extended in PBS to a final concentration of 1 x 106
spermatozoa/mL.
One million spermatozoa, extended as described above, were preincubated without or with 1.5 mg/mL of PSP-I/PSP-II (from the same batch of lyophilized proteins) at 38°C. After 0.5, 5, or 10 hours of preincubation, the sperm plasma membrane, acrosome integrity, mitochondria membrane potential, motility, and binding patterns of PSP-I/PSP-II were evaluated.
Plasma membrane and acrosome integrity were simultaneously assessed by flow cytometry, using the staining protocol described by Nagy et al (2003). For flow cytometric analysis, 50 nM of SYBR-14 working solution (100 µM stock solution in DMSO; component A of LIVE/DEAD Sperm Viability Kit; Molecular Probes Europe, Leiden, The Netherlands), 0.5 µg/mL of PE-PNA (PhycoErythrin-conjugated PeaNut Agglutinin, Biomeda Corp. Foster City, Calif) solution (1 mg/mL stock solution), and 7.5 µM of PI solution (propidium iodide, 1.5 mM stock solution in PBS) were added to 500 µL of sperm suspension. The samples were incubated at 37°C in the dark for 10 minutes. All analyses were performed by analytical flow cytometry using an EPICS XL (Coulter Corporation Inc, Miami, Fla) equipped with standard optics, an argon ion laser (Cyonics; Coherent, Santa Clara, Calif) with 15-mW laser power at 488 nM, and EXPO 2000 software (Coulter Corporation). Forward and side scatter were recorded for a total of 15 000 to 25 000 events per sample, and only sperm-specific events, which appeared in a typically L-shape scatter profile, were positively gated for the analysis. The SYBR-14 fluorescence was measured through a 525-nm band pass filter, PI fluorescence was collected through a 635-nm band pass filter, and PE-PNA fluorescence was detected through a 575-nm band pass filter. Membrane intact spermatozoa with intact acrosomes were defined as those stained only with SYBR-14.
The JC-1 probe (5,5',6'6-tetrachloro-1,1',3,3'tetraethylbenzymidazolcarbocianyne iodide; Molecular Probes Europe) was used to assess mitochondrial membrane potential of spermatozoa as described by Martinez-Pastor et al (2004), with slight modifications. Briefly, 0.2 µM of JC-1 working solution (3.8 mM stock solution) was added to 500 µL of sperm suspension. Then, samples were incubated at 37°C in darkness for 10 minutes and analyzed immediately on the flow cytometer (see above). Fluorescence emission of JC-1 monomers and JC-1 aggregates were detected, respectively, in FL1 and FL2 using 520-nm or 590-nm band pass filters.
Sperm motility was estimated with a computer-assisted motility analysis system. Samples (5 µL containing 1 x 106/mL spermatozoa) were placed in a warm (38°C) Makler chamber (Haifa, Israel) and immediately transferred to the warm stage (38°C) of a Nikon Labophot microscope (Kanagawa, Japan) equipped with a 10x contrast phase objective and a monochrome video camera (Hitachi CCD, Tokyo, Japan) connected to a personal computer. The sperm analysis was performed using the Sperm Class Analyzer software (Microptic, Barcelona, Spain). The program settings were as follows: frame rate, 25 HZ; search radius, 11.5 µm; minimum track points, 7 frames; and threshold straightness, 75%. Two replicates per sample were examined with at least 100 spermatozoa recorded per replicate for each parameter evaluated. Motion parameters were: percentage of motile spermatozoa, curvilinear velocity (VCL, µm/s), straight-line velocity (VSL, µm/s), and amplitude of lateral head displacement (ALH, µm).
PSP-I/PSP-II Heterodimer Immunocytochemistry
Immunolocalization of the PSP-I/PSP-II heterodimer was studied by both
light and scanning electron microscopy (SEM) levels. Sperm suspensions
preincubated either without or with PSP-I/PSP-II as described above were
smeared onto polylysine-coated glass slides and air-dried for 24 hours. Smears
were fixed in 1% paraformaldehyde in PBS for 10 minutes, rinsed in PBS, and
incubated with a rabbit monospecific polyclonal antibody against PSP-II (1:500
in PBS) for 120 minutes at room temperature. Samples were rinsed several times
in PBS and incubated with Auroprobe EM GAR G10 (10 nm gold labeled goat
anti-rabbit IgG; Amersham Biosciences, Uppsala, Sweden) for 100 minutes. After
washing in PBS and distilled water, a silver enhancement kit (RPN 491;
Amersham) was applied for 10 minutes. Finally, the smears were washed in
distilled water and air-dried. To provide positive controls, samples of
epididymal spermatozoa were smeared, air-dried, and fixed as above, incubated
with the PSP-I/PSP-II heterodimer for 30 minutes, and subjected to the same
protocol as above. Negative controls were obtained by omission of the primary
antibody.
For evaluation by light microscopy level, incubated samples were photographed with a Nikon Microphot-FXA light microscope (Chibe, Japan) with at least 100 spermatozoa being evaluated per sample at 400x magnification. For evaluation by SEM level (6000x magnification), portions of the glass slides with labeled spermatozoa were cut out, mounted onto metal chucks, critical-point coated with gold-palladium for 15–30 seconds, and examined using a JEOL JSM-6320F SEM (JEOL, Japan), operated at 5 kV.
Statistical Analysis
All data editing and statistical analyses were performed in SPSS version
11.5 (SPSS Inc, Chicago, Ill). Data were subjected to arcsine transformation
and analysed by ANOVA using the MIXED procedure according to a statistical
model including the fixed effects of heterodimer presence and the random
effect of replicate. When ANOVA revealed a significant effect, values were
compared using the Bonferroni test and were considered significant at
P less than .05.
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Results |
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Sperm motility was better maintained in presence of the PSP-I/PSP-II heterodimer than in controls (P < .05), even considering a significant (P < .05) decrease of motility being observed after 10 hours of incubation (Figure 1C).
The displacement speed of the incubated spermatozoa (as VCL or VSL) as well as the ALH showed changes over time and following the addition of PSP-I/PSP-II to the incubation medium (Figure 2A through C). The addition of PSP-I/PSP-II heterodimer reduced all 3 variables after 0.5 hours of incubation when compared to controls (P < .05). However, from 5 hours of incubation onwards, the variables equilibrated to levels similar to controls.
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The SEM evaluation was performed to confirm the immunolabeling and its temporal localization on the plasma membrane head domains. The SEM disclosed several binding patterns which are presented in Figure 4: pattern a: PSP-I/PSP-II heterodimer overlying the entire acrosome domain (Figure 4a); pattern b: PSP-I/PSP-II heterodimer covering the acrosome domain with additional labeling on the postacrosomal area and a negative equatorial segment (Figure 4b); pattern c: slight immunolabeling of the postacrosomal area (Figure 4c); and pattern d: no immunolabeling of spermatozoa (Figure 4d). As expected, negative controls were totally immunonegative (Figure 4e), while positive controls had silver aggregates over the whole spermatozoon (Figure 4f). The PSP-I/PSP-II heterodimer was mainly located in the acrosomal area (>90%) of highly extended spermatozoa incubated either with or without heterodimer at 0.5 hours. During the incubation, there was a temporal redistribution of the pattern of immunolabeling over time, from pattern a to pattern d. While most of the spermatozoa incubated without PSP-I/PSP-II (66%) showed pattern b after 5 hours, an increase in patterns c and d (26 and 30%, respectively) was observed after 10 hours of incubation. However, most spermatozoa incubated with PSP-I/PSP-II displayed pattern a throughout the 10-hour incubation period (Table).
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Discussion |
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Recently, we have identified the non-heparin-binding spermadhesin PSP-I/PSP-II heterodimer as one of the SP components responsible for such beneficial effect on boar spermatozoa (Centurión et al, 2003; Caballero et al, 2004b). However, to the best of our knowledge, whether this effect relates to the association of the heterodimer with the sperm plasma membrane has not been studied.
To evaluate the binding pattern of the PSP-I/PSP-II heterodimer on the
spermatozoa, a rabbit monospecific polyclonal antibody against PSP-II was
produced. In boar seminal plasma, spermadhesin PSP-II has been found
exclusively associated into a heterodimeric complex with PSP-I, whereas PSP-I,
which exists in excess over PSP-II, is present also as a noncomplexed
molecular species (Calvete et al,
1995). Thus, the anti-PSP-II antiserum represents a specific
immunochemical to detect the PSP-I/PSP-II complex. The immunocytochemistry
clearly revealed that PSP-I/PSP-II heterodimer is located mainly on the
acrosomal region of the head sperm and that the immunolabeling was being lost
over time. Addition of exogenous PSP-I/PSP-II heterodimer caused, as expected,
almost 100% of the spermatozoa to become immunolabeled. Contrary to a previous
report suggesting that the PSP-I/PSP-II complex lacked sperm-binding activity
(Calvete et al, 1995), the
present results show that a high subpopulation of highly extended boar
spermatozoa (71%) did not entirely lose their adsorbed PSP-I/PSP-II coating in
samples of highly extended ejaculated spermatozoa where no extra PSP-I/PSP-II
was added (eg, controls). Similar results have previously been reported in
boar spermatozoa in relation to the spermadhesins AQNs and AWN, which coat the
sperm membrane after ejaculation in almost 100% of the spermatozoa but were
progressively lost during incubation under capacitation conditions
(Dostálová et al,
1994). In our experiment, the proportion of immunolabeled control
spermatozoa dropped below 50% after 10 hours of incubation. However, the
proportion of spermatozoa incubated in the presence of PSP-I/PSP-II
heterodimer remained in about 80% positively labeled with the immunostain
(P < .05). This indicates that 1) the heterodimer binds to the
sperm membrane head domains before or at ejaculation and 2) the binding is
intense enough to last for several hours in a large proportion of spermatozoa,
even following an extreme dilution of the sperm suspension. However, the
binding is probably not strong enough to remain unless an additional source of
the heterodimer is present. Lastly, the highly extended, long-incubated
spermatozoa are able to bind additional heterodimer and maintain
functionality. Regarding functionality, while the percentage of membrane
intact spermatozoa as well as the percentage of spermatozoa presenting
appropriate mitochondrial activity were maintained during the incubation
process, the percentage of progressively motile spermatozoa was always lower
than that of membrane intact spermatozoa (and also below the proportions of
immunolabeled spermatozoa). Differences among motility and mitochondrial
activity have been reported earlier and probably reflect differences in sperm
activity or the ability of the measuring methods
(Windsor, 1997;
Centurión et al, 2003).
It has been recently hypothesized that mitochondria provide the sperm head and
the midpiece with ATP (by oxidative phosphorylation) in order to maintain
different gradient processes over the plasma membrane that are important for
sperm survival. However, the ATP needed for sperm motility seem to be produced
by glycolysis (anaerobic ATP production) by enzymes located in the fibrous
sheath of the tail (Silva and Gadella,
2006). Although interesting as a hypothesis, there is a need for
experimentally based proof for these
assumptions.
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Although a higher percentage of motile cells was seen in the exogenous PSP-I/PSP-II group than in controls, the addition of PSP-I/PSP-II during incubation decreased their ALH when compared with control group. These results do not suggest that spermatozoa in PBS were hyperactivated, but indicate that the widths of the sperm heads' movement were significantly higher than those of spermatozoa in PSP-I/PSP-II. Consequently, the sperm motility should be more preserved in spermatozoa incubated in presence of PSP-I/PSP-II as the percentage of motile cells showed.
The percentages of acrosome-reacted and immunonegative spermatozoa were similar during the first stages of incubation. These results might indicate that the PSP-I/PSP-II heterodimer is linked to the acrosome while it remains intact. Nevertheless, it should be noted that when spermatozoa were incubated for 10 hours, a higher number of immunonegative spermatozoa than of acrosome-reacted spermatozoa was found. Moreover, in some of the spermatozoa, the PSP-I/PSP-II was partially (pattern b) or totally (patterns c and d) removed from the acrosome-intact sperm head membrane. These findings suggest that the removal of the heterodimer from the sperm acrosome domain occurs earlier than the disruption of the acrosome and could, therefore, be related to remodeling of the sperm surface, as it occurs during the destabilization of the plasma membrane (Tulsiani et al, 1997). This hypothesis is in agreement with the fact that PSP-I/PSP-II seems to be removed from the sperm surface when spermatozoa are subjected to in vitro capacitation treatments (Caballero et al, 2004b). These data would suggest that, at least in vivo, PSP-I/PSP-II is released from the spermatozoa before reaching the ovulated oocytes and may not have the chance to play a role in gamete interaction (as do other SP proteins, such as the AWN [Rodríguez-Martínez et al, 1998]). However, the in vivo role of the heterodimer is yet to be determined.
The SEM study showed that PSP-I/PSP-II is a surface-adsorbed protein located principally on the acrosomal sperm head domain of ejaculated boar spermatozoa (pattern a). The PSP-I/PSP-II seems to migrate to the postacrosomal domain during the incubation process (patterns b and c), being finally lost from the sperm surface (pattern d). This redistribution is consistent with previous results in several species where it has been related to the processes of sperm capacitation and acrosome reaction (Kamaruddin et al, 2004; Barrios et al, 2005). However, before establishing a causal relationship between surface localization and temporal distribution to sperm capacitation, more studies are required. In any case, the electron microscopy showed an overwhelming population of spermatozoa (97%) with pattern a after 0.5 hours of incubation, which corresponded to samples showing the best sperm viability, motility and mitochondrial activity. Likewise, there was a clear trend toward redistribution to pattern d after 10 hours of incubation in a spermatozoa population (30%) whose viability, mitochondrial activity, and particularly motility were lowest.
Related to the protective effect of the heterodimer on boar spermatozoa, it could be argued that the PSP-I/PSP-II heterodimer would be responsible for the stabilization of the sperm membrane, thus counteracting destabilizing phenomena that lead to membrane deterioration, opening of the acrosome, and eventually cell death. Obviously, identifying such an additive for sperm suspensions resulting from flow cytometry should help to improve the efficiency of new biotechnological processes. Moreover, there is no negative influence of the heterodimer on the capability of fresh-extended boar spermatozoa to penetrate in vitro the homologous oocytes pre-exposed to low doses of PSP-I/PSP-II (Caballero et al, 2004b).
In conclusion, the protective effect of the PSP-I/PSP-II heterodimer on highly extended spermatozoa seems to last for at least 10 hours and could be related to the adhesion of the heterodimer to the acrosome domain of the sperm head plasma membrane. Although these results may indicate a stabilizing effect of the heterodimer on the fluidity of the sperm membrane and perhaps a decapacitating postejaculation role, further research is needed in order to clarify the causal link between the binding patterns of the PSP-I/PSP-II and the capacitation status of the spermatozoa.
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Footnotes |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) (1.5 mg/mL in
phosphate-buffered saline) on the percentage of membrane intact spermatozoa
with intact acrosome (A), mitochondrial activity (B) and
progressive motility (C) of ejaculated boar spermatozoa diluted to a
concentration of 1 x 106 spermatozoa/mL after 0.5, 5, and 10
hours of incubation at 38°C. Controls (
) were sourced from the same
spermatozoa but incubated in the absence of PSP-I/PSP-II heterodimer. Data are
the mean ± SEM of 3 independent experiments. Different letters indicate
significantly different values (P < .05).
