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Biology of Reproduction 62, 1010-1015 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Physiological State of Bull Sperm Affects Fucose- and Mannose-Binding Properties1

Irma Revaha,b,c, Barend M. Gadellab,c, Frits M. Flescha,b, Ben Colenbranderb, and Susan S. Suárez2,a

a Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 b Department of Basic Sciences, Division of Biochemistry and c Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In cattle, sperm are stored in a reservoir in the caudal isthmus of the oviduct until the time of ovulation approaches. Bull sperm are trapped in the reservoir by binding to fucosylated molecules on the oviductal epithelium. Capacitated sperm lose binding affinity for the epithelium; therefore this study was undertaken to determine whether this occurs because capacitated bull sperm lose binding affinity for fucose. BSA conjugated to {alpha}-L-fucopyranosylphenyl isothiocyanate and fluorescein isothiocyanate (fuc-BSA-FITC) was used in conjunction with flow cytometry to monitor the capacity of bull sperm to bind fucose. Dead sperm were identified using ethidium homodimer and were excluded from analysis. BSA-FITC conjugated with mannose (man-BSA-FITC) and BSA-FITC were used as controls. When examined by epifluorescence microscopy, motile bull sperm that exhibited labeling by any of the probes were fluorescent over the acrosomal region of the plasma membrane. By flow cytometry, labeling of live sperm was greatest for sperm that had been washed in TALP medium and probed with fuc-BSA-FITC (mean ± SD:167 ± 6.0 relative fluorescence units, collected in logarithmic mode). Labeling by fuc-BSA-FITC was lower in unwashed sperm (60 ± 2.7) and in washed sperm with seminal plasma added back (56 ± 8.0). Labeling was also reduced by centrifuging washed sperm through a Percoll step gradient (103 ± 6.3) and by capacitating washed sperm in medium containing 10 µg/ml heparin (50 ± 4.4). BSA-FITC labeling was barely detectable in all treatments. Man-BSA-FITC produced little labeling of washed sperm (22 ± 0.6), as expected; however, intense labeling appeared over the acrosomal region of sperm incubated under capacitating conditions (128 ± 21.6). It was concluded that removal of seminal plasma exposes fucose-binding sites, which are then lost or modified during capacitation, thereby allowing the release of sperm from the reservoir. At that time, mannose-binding sites are revealed or activated, which might serve to bind sperm to the zona pellucida.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
At the time of insemination in many eutherian mammals, millions of sperm are released into the reproductive tract. Only thousands pass through the uterotubal junction into the oviductal isthmus, where they form a reservoir [1, 2]. The sperm reservoir forms in species such as cattle [3], sheep [4], pigs [5, 6], hamsters [7, 8], rabbits [9, 10], guinea pigs [11], and mice [12, 13]. As the time of ovulation nears, release of sperm commences, and a few reach the ampulla where fertilization takes place [1, 14].

In several mammalian species, sperm are trapped to form the reservoir by being bound to the oviductal epithelium. The interaction takes place between the sperm plasma membrane overlying the acrosome and the surface of the mucosal epithelium, often via the cilia [2, 13, 15, 16]. Binding has been found to be mediated by specific carbohydrate ligands in various species. Sialic acid is involved in mediating sperm-oviductal epithelium binding in hamsters [17]. In the horse, galactose is involved [18]. In the case of cattle (Bos taurus), there is evidence that a molecule on the surface of sperm binds to a fucose-containing ligand on the epithelium. Fucose blocks binding of bull sperm to bovine oviductal epithelium in vitro [19]. Fucose-specific lectins have been used to demonstrate that fucose is densely distributed on the surface of bovine oviductal epithelium; furthermore, pretreatment of oviductal epithelium with fucosidase reduces sperm binding [19]. This evidence strongly suggests that fucose is involved in bovine sperm binding to oviductal epithelium.

There is evidence that release of sperm from the reservoir is brought about by changes associated with capacitation and hyperactivation. Capacitation involves changes in the sperm that enable it to undergo the acrosome reaction when exposed to specific stimuli. Hyperactivation is a form of motility characterized by increased flagellar beating amplitude and asymmetry. It is seen in sperm recovered from the site of fertilization (reviewed by Yanagimachi [20]). Smith and Yanagimachi [15] reported that hamster sperm that had undergone both capacitation and hyperactivation in vitro did not bind to epithelium when infused into hamster oviducts. With use of transillumination to study motile sperm within oviducts removed from mated mice, it was noted that only hyperactivated sperm detached from epithelium [21]. Lefebvre and Suarez [22] demonstrated that uncapacitated bull sperm bound to oviductal epithelium; however, after the sperm were capacitated by incubation with heparin, binding was significantly lower. More recently, Fazeli and colleagues [23] demonstrated that when uncapacitated and capacitated boar sperm were added to porcine oviductal epithelium in culture, uncapacitated sperm preferentially bound to the epithelium.

The aim of this study was to investigate whether capacitation causes a loss in the binding affinity of bovine sperm for fucose. A loss of fucose-binding affinity would explain, at least in part, why capacitated sperm do not bind to epithelium in vitro and why, in vivo, sperm would be able to release from the epithelium and advance through the oviduct toward the oocyte. Flow cytometry was used in these studies, in conjunction with a fucosylated fluorescent probe, to evaluate the fucose-binding properties of uncapacitated and capacitated bull sperm. Techniques have been developed previously by others for evaluation of various properties of live bull sperm by flow cytometry [24–28].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media and Chemicals

BSA conjugated with both {alpha}-L-fucopyranosylphenyl isothiocyanate and fluorescein isothiocyanate (fuc-BSA-FITC), or with both {alpha}-D-mannopyranosylphenyl isothiocyanate and fluorescein isothiocyanate (man-BSA-FITC), or with fluorescein isothiocyanate alone (BSA-FITC) were purchased from Sigma Chemical Co. (St. Louis, MO). Ethidium homodimer-1 (EthD-1) was purchased from Molecular Probes (Eugene, OR). All other biochemicals were purchased from Sigma, unless noted otherwise.

A modified Tyrode's balanced salt solution (TALP) was used as medium for sperm incubations. The medium consisted of 99 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.35 mM NaH2PO4, 10 mM Hepes (Calbiochem-Novabiochem Corporation, La Jolla, CA), 2 mM CaCl2, 1.1 mM MgCl2, 21.6 mM sodium lactate, 1.1 mg/ml sodium pyruvate, 6 mg/ml BSA (fraction V-FA free, Sigma A6003), and 1 µg/ml gentamicin or 10 µg/ml penicillin-streptomycin (pH 7.4, 290 mOsm/kg). To capacitate sperm, 10 µg/ml heparin was added to TALP [29]. TALP was sterilized through a 0.22-µm filter and was equilibrated before use in a humidified atmosphere at 39°C with 5% CO2.

Sperm Preparation

For experiments involving flow cytometric analysis, fresh semen was obtained from three different fertile Holstein Friesian bulls housed at the School of Veterinary Medicine, Utrecht University, The Netherlands. The bull housing facility was located next to the laboratory, so only a few minutes passed between the time of semen collection and sperm preparation. Sperm concentration and percentage motility were assessed before sperm were prepared for analysis. Sperm were prepared from the semen according to the following treatments: 1) whole semen, 2) sperm washed twice in TALP (1 ml semen in 4 ml TALP, centrifuged at 700 x g for 10 min at 27°C), 3) TALP-washed sperm centrifuged through a discontinuous Percoll density gradient (0.5 ml sperm layered over 1 ml 35% Percoll in TALP, over 1 ml 70% Percoll in TALP; centrifuged at 700 x g for 30 min at 27°C), or 4) TALP-washed sperm incubated in capacitation medium containing TALP and 10 µg/ml heparin for 4 h [29].

Seminal plasma was collected by centrifuging undiluted semen twice at 600 x g for 10 min. After each centrifugation, only the top half of the supernatant was saved. Seminal plasma was aliquoted (500 µl) and frozen at -20°C until used. It was thawed and equilibrated with a 5% CO2 atmosphere at 39°C 30 min before addition to TALP-washed sperm.

For a second set of experiments, in which fluorescently labeled sperm were visually assessed, semen was generously donated by Genex Cooperative, Inc. (Ithaca, NY). Fresh semen from three different fertile bulls was diluted 1:5 with TALP shortly after collection and transported to the laboratory at room temperature in an insulated container.

Fluorescent Labeling of Sperm

All fluorescent labels were equilibrated to 39°C before being added to sperm. Sperm (400 µl of 15 x 106 cells/ml) were labeled with 50 µl PBS containing 10 µg fuc-BSA-FITC, man-BSA-FITC, or BSA-FITC (as control). Then, 25 µl of 20 µM ethidium homodimer in PBS (EthD-1; a DNA stain) was added to identify dead sperm in all treatments [30]. In cases in which seminal plasma was added back to washed sperm, the sperm were incubated with seminal plasma at 20% of the total volume for 30 min at 39°C and then labeled as described above. Labeled sperm were immediately analyzed in a flow cytometer (FACScan; Becton Dickinson, San Jose, CA) with 100-mW argon laser. The cells were excited at 488 nm, and FITC fluorescence was detected using a 530/30-nm band-pass filter (FL1) while EthD-1 fluorescence was detected using a 620-nm long-pass filter (FL3). Fluorescence data were collected in logarithmic mode, while forward- and side-scattered light (FSC and SSC) data were collected in linear mode. The amount of fuc-BSA-FITC as well as BSA-FITC fluorescence was detected at FL1 settings of 340 mV, whereas man-FITC was detected at a more sensitive mode of 570 mV. The flow cytometer was calibrated routinely using green and red fluorescent beads (Molecular Probes Europe, Leiden, The Netherlands). Data for 10 000 events were collected per analysis. At the light scatter settings for FSC (E00) and SSC (400 mV), the sperm events were recognizable as presenting a typical L-shaped scattering profile. Non-sperm events (mostly small particles, < 3% of total events) were gated out before data were subjected to further detailed analysis. Data were stored using Becton Dickinson software and analyzed on WinMDI version 2.1.4 (J. Trotter; free software, e-mail: trotter@scripps.edu). Boundaries were drawn to delineate dead (EthD-1 positive) and live (EthD-1 negative) sperm subpopulations. The mean FITC fluorescence (FL1) of the live populations (that is, sperm that excluded the EthD-1 dye) was calculated from histogram peaks (95% subpopulation range). Data were expressed as the mean FITC fluorescence of live sperm ± SD. For FACS analyses, experiments were repeated with sperm prepared from three different bulls.

Labeling patterns were determined and the results of the FACS analysis were confirmed by using direct visual assessment. Sperm were labeled with fuc-BSA-FITC, man-BSA-FITC, or BSA-FITC as described above; then 200 motile sperm were assessed for labeling. A Zeiss Axiovert (Carl Zeiss, Thornwood, NY) epifluorescence microscope was used with a fluorescein filter set, x40 plan neofluor objective, and a stage warmer set to 39°C. In addition, images of labeled sperm were acquired using a Bio-Rad (Richmond, CA) MRC600 Kr/Ar mixed gas laser scanning confocal microscope. For fluorescein, a 488DF10 excitation filter was used with a 510DCLP dichroic mirror and a 515OG barrier filter. For EthD-1, a 647DF10 excitation filter was used with a 660DCLP dichroic mirror and a 680EFLP barrier filter. The objective was a x63 Zeiss Plan apochromat, N.A. 1.4.

Statistical Evaluation

Data were examined by ANOVA [31]. Post hoc multiple comparisons were made using Tukey's honestly significant difference procedure [32]. The software used was Minitab 10.5 Xtra for Power Macintosh (State Park, PA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
When viewed by epifluorescence microscopy, live sperm that had been washed with TALP medium for removal of seminal plasma labeled with fuc-BSA-FITC over the acrosomal region of the head (Fig. 1). Under all treatment regimes, immotile, presumably dead sperm showed EthD-1 fluorescence over the entire head. When observed directly, dying sperm first exhibited EthD-1 fluorescence in the head near the base of the flagellum. Under all conditions, the BSA-FITC control labeled sperm only faintly.



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  		FIG. 1. Confocal fluorescence images of sperm washed in TALP medium and labeled with fuc-BSA-FITC and EthD-1. Dead sperm exhibit red fluorescence over the entire head. Fuc-BSA-FITC labeling can be seen as green fluorescence in the acrosomal region of the head. Yellow is where red and green overlap. x1500

The mean fluorescence values for live sperm labeled with fuc-BSA-FITC, as determined by flow cytometric analysis (FACS), are illustrated in Figure 2A. Labeling was faint for live sperm in whole semen and increased significantly after washing. However, after washed sperm were passed through a Percoll gradient, the intensity of labeling decreased. Washed sperm incubated for 4 h under capacitating conditions exhibited even greater diminishment of label (Figs. 2A and 3). When TALP-washed sperm were incubated in seminal plasma before labeling, the labeling was similar to that achieved with whole semen, although the percentage of live sperm decreased. Preincubation of washed sperm with fucose significantly reduced labeling (Fig. 2A).



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  		FIG. 2. Mean fluorescence signals from live sperm; i.e., sperm that excluded EthD-1. Means ± SD for three replicates, with approximately 10 000 sperm analyzed per sample. Bars labeled with different letters indicate significant differences between the treatments (P < 0.05). Inserts indicate percentage of live sperm per treatment. A) Sperm labeled with fuc-BSA-FITC. B) Sperm labeled with man-BSA-FITC. Semen: unwashed sperm, labeled in whole semen; wash: sperm washed in TALP medium; wash/s: washed sperm with seminal plasma added back; perc: washed sperm passed through a two-step gradient of Percoll; capac: washed sperm incubated under capacitating conditions; fuc: labeling blocked with free fucose; BSA: labeling of washed sperm with BSA-FITC; cap/s: sperm incubated under capacitating conditions with seminal plasma added back; c-BSA: sperm incubated under capacitating conditions and labeled with BSA-FITC.

When sperm were probed with man-BSA-FITC (Figs. 2B and 4), only faint labeling was observed on live sperm in whole semen, TALP-washed sperm, and Percoll-washed sperm. However, the intensity of labeling was dramatically increased on sperm incubated under capacitating conditions. The region labeled with man-BSA-FITC resembled that of fuc-BSA-FITC; i.e., sperm were labeled over the acrosomal area. Mean fluorescence readings for all sperm considered to be alive showed a significant increase in intensity with Percoll washing and an even greater increase with capacitation (Fig. 2B). Because man-BSA-FITC was originally intended as a negative control, testing of labeling specificity by blocking with mannose was not employed initially. Later, it was determined that preincubation with 31 mM mannose reduced man-BSA-FITC binding by 30–65%, and the results varied substantially among bulls. However, mannan, a polymer of mannose, was found to agglutinate sperm that had been capacitated for 4 h using dibutyryl cAMP and 3-isobutyl-1-methylxanthine, according to the method of Galantino-Homer et al. [33]. Using this method, sperm can be capacitated without becoming agglutinated, as they do when heparin is used. Sperm that were incubated for 4 h in the absence of inducers of capacitation showed only minimal agglutination after the addition of mannan (data not shown).

Visual assessment of the labeling of motile sperm yielded results similar to those of the FACS analyses. Specific labeling of both fuc-BSA-FITC and man-BSA-FITC was over the acrosomal region of the sperm head. For fuc-BSA-FITC-labeling, 79 ± 4% of TALP-washed motile sperm were labeled. BSA-FITC faintly labeled 11 ± 3% of the TALP-washed motile sperm. For sperm that had been incubated for 4 h in capacitating medium, man-BSA-FITC labeled 65 ± 2% and BSA-FITC labeled 10 ± 1%. We had previously established that these conditions produced capacitation, by in vitro fertilization, lysophosphatidyl choline-induced acrosome reactions, and chlortetracycline labeling [22]. Dead sperm were labeled by fuc-BSA-FITC and man-BSA-FITC in the postacrosomal region and the rostral ridge of the acrosome.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results obtained in the present study suggest that there is a fucose-binding molecule on the plasma membrane overlying the acrosome of bovine sperm that is covered, outcompeted, or inactivated by a component of seminal plasma. As seminal plasma is removed by washing of sperm with TALP, the molecule is made available for labeling with fuc-BSA-FITC. Centrifugation through a two-step Percoll gradient appeared to remove the molecule from the plasma membrane or destroy its binding capacity, thus explaining the reduction in labeling when compared to that of TALP-washed sperm. Similarly, de Maistre et al. [34], using various lectin probes to evaluate surface characteristics of human sperm, found that the sperm surface was modified by centrifugation through Percoll.

When washed sperm were incubated under capacitating conditions, fuc-BSA-FITC labeling decreased even more, suggesting that the fucose-binding molecule is lost or modified during capacitation. These findings provide an account for the observation that binding to oviductal epithelium is decreased by capacitation [15, 22, 23]. At capacitation, the fucose-binding molecule must be either lost or modified to allow release of sperm from the epithelium.

Labeling of sperm in whole semen, TALP-washed sperm, and Percoll-washed sperm with man-FITC-BSA was faint. This had been predicted, because mannose does not block bovine sperm binding to oviductal epithelium in vitro [35]. However, after incubation in capacitating medium, a mannose-binding site was evidently exposed or activated. There are reports indicating that mannose is involved in human sperm binding to zona pellucida [36, 37]. Perhaps a similar mechanism exists for bovine sperm and eggs. Analysis of the N-linked carbohydrate chains of bovine zona pellucida glycoproteins indicated that the major neutral oligosaccharide chain is high in mannose and contains mannose in terminal positions at the nonreducing end [38]. Some authors have suggested that uncapacitated bull sperm do not bind to the zona pellucida and that zona pellucida-binding affinity is generated during the process of capacitation [39]. In this light, it is interesting to see that uncapacitated sperm have only low affinity for mannose while capacitation led to a marked increase in affinity for mannose. Nevertheless, the function of the mannose-binding site on bull sperm remains to be established.

When seminal plasma was present, as it was in the unwashed sperm and in washed sperm to which seminal plasma had been added back, labeling with fuc-FITC-BSA was faint. These data suggest that there is a coating of seminal plasma components that cover the fucose-binding molecule on sperm. Coating could be achieved by fucosylated components in the seminal plasma (free sugars, glycopeptides, glycoproteins, or glycolipids) that bind specifically to the fucose-binding site on sperm. Alternatively, seminal plasma could contain a soluble form of the sperm surface molecule that acts as a competitive inhibitor by binding to the fuc-BSA-FITC in solution. Re-addition of seminal plasma killed a significant percentage of sperm; however, only live sperm were included in the analysis of labeling. Furthermore, use of unwashed sperm yielded labeling results similar to those with adding back seminal plasma, and motility was high in these samples. Some components of seminal plasma are known to rapidly damage washed bull sperm [40]; therefore, these components may account for our observation of decreased survival of washed sperm treated with seminal plasma.

On the basis of these results, we propose the following scenario for fertilization in vivo. Uncapacitated sperm lose some coating by the time they enter the oviduct, thereby revealing a fucose-binding molecule. These sperm form a reservoir by binding to the epithelium via fucose expressed on the epithelial surface. As the time of ovulation approaches, sperm become capacitated, and their capacity to bind to the epithelium is lost because of removal or alteration of the fucose-binding molecule. Capacitated sperm can no longer bind to the oviductal epithelium and are released to swim up toward the ampulla. At the same time, a mannose-binding site, which was masked or otherwise inactivated, is revealed on the sperm surface, making it available for binding sperm to the zona pellucida or inducing the acrosome reaction. It may be advantageous to inactivate the mannose-binding site until shortly before oocyte contact, in order to guard against premature acrosome reactions or inadvertent sticking to various surfaces of the female reproductive tract.



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  		FIG. 3. Example of flow cytometric dot plots for fuc-BSA-FITC labeling for one bull. Each dot represents a single sperm. The green fluorescence on the x-axis presumably represents staining with fuc-BSA-FITC, while the red fluorescence on the y-axis represents staining with EthD-1. Sperm whose EthD-1 fluorescence values fell above the horizontal line were considered to be dead and were excluded from analysis of labeling. For Figure 2, mean FITC fluorescence was determined for all of the sperm whose EthD-1 label was below the threshold. Note that the fluorescence labeling plots are logarithmic. A) Washed sperm labeled with fuc-BSA-FITC. B) Fuc-BSA-FITC labeling of washed sperm incubated under capacitating conditions for 4 h



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  		FIG. 4. Example of flow cytometric dot plots for man-BSA-FITC labeling of one bull. Each dot represents a single sperm. The green fluorescence on the x-axis presumably represents staining with man-BSA-FITC, while the red fluorescence on the y-axis represents staining with EthD-1. Sperm whose EthD-1 fluorescence values fell above the horizontal line were considered to be dead and were excluded from analysis of labeling. For Figure 2, mean FITC fluorescence was determined for all of the sperm whose EthD-1 label was below the threshold. Note that the fluorescence labeling plots are logarithmic. A) Washed sperm labeled with man-BSA-FITC. B) Man-BSA-FITC labeling of washed sperm incubated under capacitating conditions for 4 h. Note that the vertical line is located to the right of the line shown in Figure 3. The reason is the more sensitive settings of FL1 at 570 mV for man-BSA-FITC compared to 340 mV used for fuc-BSA-FITC


   ACKNOWLEDGMENTS
 
We are grateful for the technical assistance of Dr. Ana Alcaraz and Ms. Kathleen P. Kyle in determining specificity of man-BSA-FITC binding.


   FOOTNOTES
 
First decision: 5 October 1999.

1 This project was supported by USDA NRICGP project number 32738 to S.S.S. and by the School of Veterinary Medicine, Utrecht University, The Netherlands. Back

2 Correspondence: S.S. Suárez, Department of Biomedical Sciences, T5-006 Veterinary Research Tower, Cornell University, Ithaca, NY 14853. FAX: 607 253 3541; sss7{at}cornell.edu Back

Accepted: November 22, 1999.

Received: July 16, 1999.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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