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From the Biotechnology and Germplasm Laboratory, Beltsville Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland.
| Correspondence to: Dr Julie A Long, USDA, ARS, ANRI, ABBL, BARC-East, Bldg 200, Room 120, Beltsville, MD 20705 (e-mail: julie.long{at}ars.usda.gov). |
| Received for publication September 20, 2007; accepted for publication March 17, 2008. |
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Abstract |
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-mannose/-glucose, - and β-galactose,
-fucose, - and β-N-acetyl-galactosamine, monomers
and dimers of N-acetyl-glucosamine, and
N-acetyl-lactosamine. Potential changes in these carbohydrates during
in vitro semen storage at 4°C were evaluated using males of both high- and
low-sperm–mobility phenotypes. Changes in carbohydrate residues were
quantified by flow cytometry analysis using a battery of 14 fluorescein
isothiocyanate–labeled lectins in combination with control (sialylated)
or neuraminidase-treated (nonsialylated) sperm. Sperm were evaluated at 0, 2,
4, 8, 12, and 24 hours of storage. For control sperm, 4 different patterns of
lectin binding were observed over time: 1) increased mean fluorescence
intensity (MnFI) at 2 hours (Griffonia simplicifolia lectin-I [GS-I])
and 8 hours (Ricinus communis lectin-I [RCA-I]) that remained
elevated during storage; 2) increased MnFI at specific time points (Limax
flavus lectin [LFA], 2 hours; Artocarpus integrifolia lectin
[jacalin] and succinyl Triticum vulgare lectin [sWGA], 8 hours;
Galanthus nivalis lectin [GNA], 12 hours) followed by decreasing MnFI
during the remainder of the 24-hour storage period; 3) increased MnFI only at
the 24-hour time point (Lotus tetragonolobus lectin [lotus] and
Arachis hypogaea lectin [PNA]); and 4) no changes in MnFI during the
24-hour storage period (Erythrina cristagalli lectin [ECA], GS-II,
Pisum sativum lectin [PSA], Glycine max lectin [SBA], and
Wisteria floribunda lectin [WFA]). For nonsialylated sperm, increased
binding of ECA, GS-II, SBA, and WFA was observed at variable time points; only
Canavalia ensiformis lectin (Con A) and PSA remained unchanged during
storage. Differences between mobility phenotypes existed for lectins Con A,
GS-II, LFA, PSA, SBA, and sWGA, with sperm from low-mobility males exhibiting
higher MnFI than high-mobility males throughout 24 hours of storage. We
concluded that the observed increases in lectin binding during semen storage
indicate an augmentation of nonsialylated terminal residues, which could alter
sperm antigenicity and negatively impact fertility. Further, spermatozoa from
low-mobility males may have higher antigenicity even before semen storage.
Other possible functional implications are discussed.
Key words: Lectin, artificial insemination, glycoprotein, sperm physiology
The sperm glycocalyx represents the primary interface between the sperm cell and its environment and is a critical component for sperm maturation, sperm transport, and sperm-egg interaction in mammals (Diekman, 2003). The functional significance of the poultry sperm glycocalyx is not well defined. It has been demonstrated that rooster spermatozoa acquire surface-associated glycoproteins during maturation within the male duct system that remain associated with spermatozoa in the female tract (Morris et al, 1987). Impaired fertility has been associated with alterations in the carbohydrate content of rooster spermatozoa (Froman and Engel, 1989). In particular, sialic acid is required for sperm passage through the hen's vagina (Steele and Wishart, 1996) and for sequestration in the hen's sperm storage tubules (Froman and Thursam, 1994). N-acetyl-D-glucosamine, localized on the perivitelline layer of the chicken ovum, is necessary for sperm-egg interaction (Robertson et al, 2000). Although these data pertain to chickens, the possibility exists for similar functional implications of surface carbohydrates in turkey spermatozoa. Further, the loss and/or alteration of surface glycoproteins during semen storage also may impact the ability of turkey spermatozoa to traverse the hen's reproductive tract and recognize/bind to the ovum.
We recently characterized the carbohydrate composition of the turkey sperm
glycocalyx in fresh semen from males with average-mobility semen by means of
flow cytometry assessment of lectin binding
(Peláez and Long,
2007). The glycocalyx of turkey spermatozoa is extensively
sialylated and contains residues of -mannose/-glucose, -
and β-galactose, -fucose, - and
β-N-acetyl-galactosamine, N-acetyl-lactosamine, and
monomers and dimers of N-acetyl-glucosamine in variable amounts. Our
aim here was to determine if these surface carbohydrates are altered during
conventional in vitro semen storage at 4°C for 24 hours. Because the
majority of terminal carbohydrate residues are masked by sialic acid, we used
a neuraminidase treatment to detect differences in unmasked residues over
time. Finally, we compared the carbohydrate composition of semen from high-
and low-sperm–mobility phenotypes because males exhibiting high mobility
indices have a competitive advantage with respect to paternity
(Donoghue et al, 1998).
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Materials and Methods |
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Prior to initiation of the study, males were screened at 28, 32, and 40 weeks to determine their sperm mobility phenotypes. Sperm mobility is a quantitative trait of semen quality originally described in the fowl (Froman and McLean, 1998).
It has been well established that this parameter fits a normal distribution within a population of individual males, allowing the characterization of individual males with either low- or high-sperm mobility as classified by 1 standard deviation above or below the population mean. We assessed turkey sperm mobility according to the methods of King et al (2000) using a 1 x 109/mL sperm suspension, 6% (wt/vol) Accudenz solution (Accurate Chemical & Scientific Corp, Westbury, New York), and microreader photometer (IMV Microreader; IMV International Co, Minneapolis, Minnesota) to measure optical density at 597 nm after 1 minute of equilibration. After characterization, 6 males (3 high mobility and 3 low mobility) were randomly chosen for the study and assigned to the respective phenotype groups.
Semen Collection and Processing
Semen was collected manually (Burrows
and Quinn, 1937) from each male twice per week, with a minimum
48-hour interval between the 2 collections. Immediately after collection,
semen was pooled within the designated mobility group and diluted 1:1 with
Beltsville Turkey Semen Extender II (Continental Plastics Corp, Delavan,
Wisconsin). Aliquots of diluted semen from each mobility group were
transferred to a 24-well culture plate (0.5–1 mL/well). The plate was
covered loosely with aluminum foil, placed on an orbital shaker (125 rpm), and
maintained at 4°C for 24 hours. Seminal plasma was removed from 150-µL
aliquots of fresh (0 hours, nonstored) and stored (2, 4, 8, 12, or 24 hours)
semen by centrifugation (400 x g; 5 minutes). Sperm pellets
were resuspended in the buffer appropriate for either lectin staining
(control) or neuraminidase treatment followed by lectin staining
(neuraminidase-treated [NT] samples).
Lectin Staining and Neuraminidase Treatment
Fourteen fluorescein isothiocyanate (FITC)–conjugated lectins (EY
Laboratories Inc, San Mateo, California) were used to detect residues of 9
carbohydrate groups: 1) -galactose (Griffonia simplicifolia
lectin-I [GS-I], Artocarpus integrifolia lectin [jacalin]); 2)
β-galactose (Ricinus communis lectin-I [RCA-I], Arachis
hypogaea lectin [PNA]); 3) -mannose/-glucose (Canavalia
ensiformis lectin [Con A], Pisum sativum lectin [PSA],
Galanthus nivalis lectin [GNA]); 4) N-acetyl-glucosamine
monomers (GS-II); 5) N-acetyl-glucosamine dimers (succinyl
Triticum vulgare lectin (sWGA]); 6) - and
β-N-acetyl-galactosamine (Glycine max lectin [SBA],
Wisteria floribunda lectin [WFA]); 7) -fucose (Lotus
tetragonolobus lectin [lotus]); 8) sialic acid (Limax flavus
lectin [LFA]); and 9) N-acetyl-lactosamine (Erythrina
cristagalli lectin [ECA]). The specificity of these lectins for the
respective carbohydrate group was previously demonstrated
(Peláez and Long,
2007). For all experiments, lectins were used at concentrations of
100 µg /mL in Tris buffer (TBS; 0.05 M Tris, 0.15 M NaCl [pH 7.6]). Lectins
GS-I, GS-II, Con A, and PSA were prepared in TBS containing 1 mM
CaCl2 and 1 mM MgCl2.
Control sperm were resuspended in TBS to a concentration of 2.5 x 109 sperm/mL. A 2.5-µL aliquot of each sperm suspension was added to a 62.5-µL volume of lectin solution to yield a final concentration of 100 x 106 sperm/mL. Spermlectin mixtures were incubated protected from light for 30 minutes at room temperature. After incubation with lectins, samples were washed twice by centrifugation (700 x g; 5 minutes), and sperm pellets were resuspended in the appropriate buffer (eg, TBS with or without cations). Ten microliters of the final resuspension of each sample was diluted in 0.5 mL of the appropriate buffer and counterstained with 12 µM propidium iodide (PI; Molecular Probes, Eugene, Oregon) for a minimum of 5 minutes at room temperature.
Because the turkey sperm glycocalyx is extensively sialylated (Peláez and Long, 2007), neuraminidase treatment was used to detect changes in masked carbohydrates for all lectins (except the sialic acid–specific lectin LFA). After removal of seminal plasma, pelleted spermatozoa were resuspended in TBS (pH 6.0) to a final concentration of 1 x 109 sperm/mL and incubated for 30 minutes at 37°C with 1 IU neuraminidase (type V; Clostridium perfringens; Sigma-Aldrich, St Louis, Missouri)/109 sperm. After an initial centrifugation (700 x g; 5 minutes), sperm were resuspended in TBS (pH 6.0) and washed once, followed by 2 centrifugations in TBS (pH 7.6). The final pellet was used immediately for lectin staining as described above.
Flow Cytometry Assessment of Lectin Binding
A Coulter Epics XL-MCL flow cytometer (Coulter Corp, Miami, Florida)
equipped with a single 488-nm excitation source was used for all analyses.
Forward and side scatter gating were used to select single sperm from clumps
and debris. The fluorescence from FITC-stained and PI-stained spermatozoa was
collected in FL1 (525-nm bandpass) and FL3 (620-nm bandpass) fluorescence
detectors, respectively. Because cells with intact plasma membranes preclude
lectins from binding to internal structures, only FITC fluorescence signals
generated by PI-negative cells were considered in the analysis. The mean FITC
fluorescence intensity/cell (MnFI) of the viable sperm population was recorded
from the FL1 detector output as an indicator of lectin binding. The entire
experiment (ie, semen collection, sample preparation, and flow cytometry
assessment) was repeated 6 times with a minimum 1-week interval using the same
semen donors.
Statistical Analysis
All analyses were conducted using STATISTICA software for Windows (version
7; StatSoft Inc, Tulsa, Oklahoma). P < .05 was considered
significant. Data were first tested for differences between the MnFI values of
control and NT samples to elucidate the lectins in which an effect of
treatment on the fluorescence intensity exists. MnFI values obtained for each
of the 14 lectins (n = 36 [6 time points x 6 replicates]) within
mobility phenotype (high or low mobility) were subjected to separate 1-way
analyses of variance (ANOVAs). If the test showed a significant effect of
treatment, the rest of the analyses were conducted separately on either
control or NT samples. If, on the contrary, a significant effect of treatment
did not exist in any of the 2 mobility phenotypes, data from both control and
NT samples were combined. Differences in the MnFI values obtained throughout
the incubation period (time effect) and between semen mobility phenotypes
(mobility phenotype effect) were tested using 2-way ANOVA. The model
considered "mobility phenotype" as a fixed factor and
"time" as a fixed factor with repeated measures. In the absence of
significant interactions, when a significant effect of the mobility phenotype
was observed, the effect of time was studied separately on each mobility
sample (high- or low-mobility semen). Post hoc comparisons were made using the
Student-Newman-Keuls test (Carrasco,
1995).
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Results |
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.05) between control and NT spermatozoa from the low-mobility
phenotype males (Table 1).
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The main effects of time and mobility phenotype on lectin binding are shown in Table 2. Significant changes (P < .05) in the MnFI occurred over time for 9 of the 14 lectins incubated with control spermatozoa. For lectins GS-II, WFA, and ECA, the time effect was observed only for NT spermatozoa (P < .05). The mobility phenotype effect was only significant (P < .05) for lectins Con A, PSA, and SBA. The interaction "time x mobility group" was not significant for any sample.
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For most lectins, the changes detected in fluorescence intensity over time were related to elevated MnFI values. For control spermatozoa, higher MnFI (P < .05) was noted as early as 2 hours after storage (GS-I, 0.84 ± 0.08; LFA, 1.45 ± 0.03) compared with initial samples at 0 hours (GS-I, 0.65 ± 0.04; LFA, 1.17 ± 0.04). Also in control samples, the first significant MnFI increases for lectins sWGA and jacalin occurred at 8 hours of storage, whereas GNA binding increased at 12 hours compared with 0-hour samples (P < .05). For NT samples, most MnFI values were higher at the 8-hour time point compared with those of fresh semen, including lectins GS-II, ECA, SBA, and RCA (P < .05), whereas lectins GS-I and PNA had increased MnFI at the 12- and 24-hour time points, respectively (P < .05). The MnFI were not different between control and NT samples for lectins RCA or lotus, which increased at the 8- and 24-hour time points, respectively (P < .05). No changes in MnFI over time were apparent for lectins Con A or PSA (P < .05). Lectins SBA, WFA, and PNA also exhibited similar MnFI at each time point but only for control samples (P < .05).
Four basic patterns of lectin binding were observed over time when stored semen was compared with fresh semen: 1) increased MnFI (P < .05) between 2 and 8 hours (GS-I and RCA-I) that remained elevated for the remaining time points (Figure 1A); 2) incremental elevations in MnFI that peaked (P < .05) at specific time points (LFA, 2 hours; jacalin, 8 hours; GNA and sWGA, 12 hours) and subsequently decreased during the remaining time points (Figure 1B); 3) increased (P < .05) MnFI values only at the 24-hour time point (lotus and PNA; Figure 1C); and 4) no changes in MnFI (P > .05) throughout the 24-hour storage (ECA, GS-II, SBA, and WFA; Figure 1D). Seven lectins exhibited different binding patterns over time between control and NT spermatozoa. For NT samples, the MnFI values of lectins ECA and SBA followed pattern 1 (Figure 2), whereas lectin WFA exhibited pattern 2 and lectin PNA followed pattern 3 (P < .05) for NT spermatozoa. Conversely, the MnFI values of NT spermatozoa incubated with lectins GNA, jacalin, and sWGA did not change over time (P > .05).
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Discussion |
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The abundance of virtually all membrane surface carbohydrates was altered during the 24-hour storage period at 4°C. We focused first on the nontreated samples because those represented the physiological state of spermatozoa during in vitro semen storage. In general, 4 patterns of quantitative changes were observed in nontreated spermatozoa: 1) increased MnFI at specific time points that remained elevated during storage, 2) increased MnFI at specific time points followed by decreasing MnFI during the remainder of the 24-hour storage period, 3) increased MnFI only at the 24-hour time point, and 4) no changes in MnFI during the 24-hour storage period. Changes in the abundance of N-acetyl-glucosamine and N-acetylgalactosamine residues over time were detected only after terminal sialic acid residues were removed with neuraminidase, and the timing of those changes corresponded mainly to pattern 2. The physiological significance of changes for those 2 carbohydrate groups could be considered less relevant than those observed for control sperm; however, it is possible that terminal sialic acid residues are cleaved or lost prior to sperm/egg binding in the female reproductive tract because carbohydrate moieties other than sialic acid are known to be required for gamete recognition (Robertson et al, 2000). Thus, alterations in the abundance of carbohydrate groups that are temporarily masked by sialic acid may be physiologically relevant. We recently demonstrated that localization of some carbohydrate groups to specific morphologic regions on turkey and chicken spermatozoa was possible only after terminal sialic acid residues were removed (Peláez and Long, 2007). In particular, lectins GS-II and SBA (corresponding to specific configurations of N-acetyl-glucosamine and N-acetylgalactosamine residues) were not visualized on turkey spermatozoa prior to neuraminidase treatment but were localized to the acrosome and acrosome/head regions, respectively, upon removal of terminal sialic acid residues. Given the regional localization of these carbohydrates, it seems plausible that the particular glycoconjugates apparently masked by sialic acid have an important functional role in fertilization. It is important to note that the observed alterations in carbohydrate abundance over time in the current study likely were not a result of sialic acid residues being lost and/or cleaved during in vitro semen storage. The main evidence for this assertion is that levels of sialic acid on the sperm surface did not decrease until the 24-hour time point, whereas increased abundance of some carbohydrate groups occurred as early as 2 hours after the start of semen storage.
Of particular interest is the finding that lectins differentially bound to spermatozoa from males exhibiting either the high- or low-mobility phenotype, which indicates that the glycocalyx composition of these 2 phenotypes is dissimilar and is suggestive of functional implications for both fresh and in vitro stored semen. It has been shown for both turkeys and chickens that males characterized with the high-mobility phenotype provide superior fertility rates compared with those of low-mobility males (Donoghue et al, 1998; Birkhead et al, 1999). In fact, mobility is the only sperm attribute shown to be directly correlated with fertility outcome in poultry (Froman et al, 1999; King et al, 2000). In chickens, mobility phenotype is determined by mitochondrial function (Froman and Kirby, 2005), which depends on the extent of intracellular Ca2+ uptake (Froman et al, 2006). Although a similar mechanism is likely for turkey mobility phenotype, our findings implicate additional distinguishing factors at the molecular level of the sperm plasma membrane. In our study, when differences were found between mobility phenotypes with respect to lectin binding, the MnFI values were consistently higher in the low-mobility males, indicating a higher content of the specific carbohydrate. Interestingly, the increased abundance of some mannose/glucose, N-acetyl-glucosamine, and N-acetylgalactosamine glycoconjugates in the plasma membrane of spermatozoa from low-mobility males was evident in fresh as well as in vitro stored semen. In mammals, differences in plasma membrane glycoconjugates have been found in spermatozoa exhibiting different rates of motility (González-Chábarri et al, 1994), thereby supporting the hypothesis that glycocalyx characteristics have physiological significance with respect to poultry sperm mobility phenotype.
In the light of these findings, the major question arising from the current study is the functional significance of the quantitative changes described. Carbohydrates of biological fluids and cell surfaces play a crucial role in reproduction. Evidence is accumulating that carbohydrate groups on the sperm surface regulate early gamete interactions (Diekman, 2003). The role of specific sperm surface glycoproteins in the process of fertilization and sperm physiological events, such as capacitation and the acrosome reaction, has been reported for a wide range of species and taxa (Kopecny and Flechon, 1987; Veselsky et al, 1992; Bérubé and Sullivan, 1994; Lassalle and Testart, 1996; Evans, 1999; Yu et al, 2002; Srivastav et al, 2004). Changes in the sperm glycocalyx, such as the molecular remodeling of plasma membrane components during capacitation of mammalian sperm, subsequently affect sperm function (Eddy, 1988). In particular, glycocalyx modifications occurring during mammalian spermatozoa maturation in the epididymis are believed to confer fertilizing ability to the spermatozoon (Eddy, 1988). Moreover, the characteristics of changes occurring in the glycocalyx of boar spermatozoa during capacitation and acrosome reaction have implications for fertility (Jiménez et al, 2002). Based on the available literature, it seems reasonable to suggest that the modifications of the turkey sperm glycocalyx observed in our study could have functional repercussions that affect the fertility of in vitro stored semen. We also can speculate that known differences in fertility between high- and low-mobility phenotype males may be mediated, in part, through differences in glycocalyx composition. For example, the levels of N-acetyl-galactosamine clearly differed between spermatozoa from high- and low-mobility phenotype males, suggesting an important functional role for this carbohydrate and associated glycoconjugate(s). We are planning studies to identify the glycoconjugates that were altered during in vitro storage of turkey semen to determine the functional significance of these poststorage carbohydrate modifications and increase our current knowledge on how storage protocols affect the fertility of turkey spermatozoa.
Until more is known regarding the relationship between alterations of glycocalyx and functionality of spermatozoa in poultry species, the experimental data reported here permit only speculation about the potential negative impact of an increased proportion of nonsialylated sugar residues on the fertility of semen. The increased MnFI values observed for most lectins during storage indicate that more binding occurs as a result of changes in the availability of sugar residues over time. Because sialic acid residues do not decrease during incubation, the observed increases in lectin binding probably do not result from a phenomenon of shedding this terminal sugar and unmasking of subterminal residues. Rather, new components could be incorporated into the glycocalyx from free carbohydrates existing in the seminal plasma, and/or enzymatic transformations could operate in the existing sugar moieties over the incubation period. The seminal plasma of poultry species is known to contain free sugar residues as well as the specific enzymes (glycosidases, glycosyl transferases) for driving these processes (Ahluwalia and Graham, 1966; Droba and Droba, 1987, 1992; Droba and Dzugan, 1993). The net increase in unmasked sugar residues would increase the antigenicity of spermatozoa and would have negative consequences for the storage of sperm in the female reproductive tract (sperm storage tubules) because sperm selection in the vagina of poultry species is based on immunologic recognition of surface antigenicity (Steele and Wishart, 1992), and proper masking of sugar residues by sialic acid is essential for ensuring the transvaginal migration of spermatozoa (Steele and Wishart, 1996). In the chicken, it has been demonstrated that artificially increasing the antigenicity of sperm results in fewer sperm reaching the storage tubules and a lower proportion of fertile eggs (Froman and Thursam, 1994). If a similar sialic acid selection mechanism applies to the turkey, then increased antigenicity as a result of cold semen storage would negatively impact fertility. Further research is needed to verify whether incorporation/transformation of glycocalyx sugar residues occurs as a result of the availability of free carbohydrates and/or enzymes from the seminal plasma during storage of turkey semen. If so, improvements in the storage protocol such as removal of seminal plasma or addition of molar concentrations of sialic acid to the extender medium could be proposed.
In conclusion, quantitative changes occur in the carbohydrate content of turkey sperm surface glycocalyx during a 24-hour period of storage at 4°C. The pattern of changes varies among sugar residues, with increased rates of lectin binding being observed during the incubation period. Of special interest is the fact that sperm glycocalyx is not considerably altered until after 6 hours of storage. Sperm of low-semen–mobility phenotype appear to contain higher amounts of some sugar moieties compared with the high-mobility phenotype and also present differences in the pattern of changes with regard to the latter. Increases in the rate of lectin binding reveal an augmentation of nonsialylated terminal residues that could modify sperm antigenicity, eventually impacting fertility negatively. Other potential functional implications of the carbohydrate modifications reported here are not known and warrant investigation. The mechanism responsible for changes is also a clear area for further research. Previous work on the effects of cold storage on turkey sperm plasma membrane has mainly concerned the lipid component (Douard et al, 2000, 2003, 2005). To the authors' knowledge, this is the first report that other important functional constituents are also affected.
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Acknowledgments |
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Footnotes |
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