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From the Biotechnology and Germplasm Laboratory, Beltsville Agricultural Research Service, US Department of Agriculture, Beltsville, Maryland.
| Correspondence to: Dr Julie A. Long. USDA, ARS, ANRI, BGL, BARC-East, Bldg 200, Rm 120, Beltsville, MD 20705 (e-mail: JLONG{at}anri.barc.usda.gov). |
| Received for publication December 29, 2005; accepted for publication November 9, 2006. |
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Abstract |
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Key words: Turkey, chicken, semen, glycoconjugate, lectin
Like the glycocalyx of somatic cells, the glycocalyx of spermatozoa is critical for many specific cell functions. A notable difference between the glycocalyx of somatic cells and that of spermatozoa is the depth of the carbohydrate layer, with a maximum thickness of 60 nm reported for guinea pig spermatozoa (Bearer and Friend, 1990). Moreover, the sperm cell glycocalyx appears to be extremely complex in both composition and organization compared to other cell types. For example, the glycocalyx of mammalian oocytes contains 3 or 4 different families of glycoproteins (Yanagimachi, 1988), whereas that of human spermatozoa is estimated to contain between 50 and 150 different glycoconjugates, which are segregated into different functional domains rather than being homogeneously distributed on the cell surface (Schröter et al, 1999). The composition of the mammalian sperm glycocalyx also is dynamic, with the original carbohydrate layer being extensively modified during the transit through the epididymis and the female reproductive tract (Eddy, 1988). The sperm glycocalyx represents the primary interface between the male gamete and its environment, and, although specific functions have not been completely determined, is known to be involved with immunoprotection in the female genital tract, acquisition of fertilizing ability, acrosome reaction, and early gamete interactions (Schröter et al, 1999; Diekman, 2003).
Most of the data for sperm glycocalyx pertaining to composition or
functionality has been derived from mammalian species. A limited number of
studies have been conducted with other taxa, such as marsupials
(Cooper et al, 2001),
invertebrates (Perotti and Pasini,
1995), and fishes (Rojas and
Esponda, 2001) documenting the composition, distribution, and/or
functional implications of surface membrane glycoconjugates. In contrast, few
data regarding the composition, spatial distribution, or function of the avian
sperm cell glycocalyx have been reported. It is known that the chicken sperm
glycocalyx contains residues of sialic acid
(Froman and Thurston, 1984),
as well as 
-glucose and/or -mannose
(Bakst and Howarth, 1977).
Based on what has been reported for other species, however, the avian sperm
glycocalyx should contain a complex diversity of carbohydrates that are
critical for sperm function. Currently, the only functions associated with the
avian sperm glycocalyx are that terminal sialic acid residues are necessary
for chicken spermatozoa to traverse the vagina and become sequestered within
the sperm storage tubules in the hen's reproductive tract
(Froman and Engel, 1989).
Before specific poultry sperm cell functions can be attributed to the
glycocalyx, the carbohydrate content and distribution need to be delineated.
Accordingly, our objective here was to characterize the types of sugar
residues comprising the glycocalyx of poultry spermatozoa. Specifically, a
battery of fluorescein isothiocyanate (FITC)-labeled lectins were used in
combination with flow cytometry and epifluorescent microscopy to determine the
types and distribution of carbohydrate residues present on the plasma
membranes of both turkey and chicken spermatozoa.
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Methods |
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± SD range) from each species
were randomly assigned to 1 of 3 groups (3 males/group).
Semen Collection and Processing
Semen was collected manually (Burrows
and Quinn, 1937) from both turkeys and roosters on a weekly basis,
pooled within the designated group, and diluted 1:1 with Beltsville Poultry
Semen Extender II (Continental Plastics Corp, Delavan, Wis). Seminal plasma
was removed using the Accudenz washing method
(McLean et al, 1998). Briefly,
diluted semen (500 µL, turkey; 750 µL, chicken) was gently layered on
top of the discontinuous gradient (12% Accudenz, 5 mL; 30% Accudenz, 0.5 mL)
and centrifuged (1250 x g; 4°C) for 25 minutes with a
gradual stop (ie, without using the brake). Spermatozoa present at the
interface between the Accudenz layers were recovered and aliquoted for
immediate lectin staining or neuraminidase treatment followed by lectin
staining.
Lectins, Inhibitory Sugars, and Neuraminidase Treatment
Seventeen FITC-conjugated lectins (EY Laboratories Inc, San Mateo, Calif)
were used to detect residues of 7 carbohydrate groups: 1) galactose (GS-I,
Jacalin, RCA-I, PNA), 2) glucose and/or mannose (Con A, PSA, GNA), 3)
N-acetyl-glucosamine (GS-II, succinyl-WGA [s-WGA], STA), 4)
N-acetyl-galactosamine (SBA, WFA), 5) fucose (Lotus, UEA-I), 6) sialic acid
(LFA, LPA), and 7) N-acetyl-lactosamine (ECA). The specific carbohydrate
affinity and inhibitory sugar of each lectin is shown in
Table 1. For all experiments,
lectins were used at a concentration of 100 µg /mL in Tris buffer (TBS;
0.05 M Tris, 0.15 M NaCl; pH 7.6). GS-I, GS-II, Con A, PSA, and UEA-I were
prepared in TBS containing 1 mM CaCl2 and 1 mM MgCl2.
LPA was prepared in TBS containing 10 mM CaCl2, pH 8.0. Sperm
processing methods for lectin staining were dependent upon the type of
fluorometric analysis (flow cytometry or microscope) as detailed below
(experiments 1–3).
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The specificity of lectin binding was confirmed using competitive inhibition with free sugars for all lectins except LFA and LPA. Lectins were preincubated with specific inhibitory carbohydrates (Sigma Chemical Co, St Louis, Mo; except lactose and fucose, EY Laboratories) for at least 1 h prior to incubation with spermatozoa. The molar concentration of all inhibitory sugars except chitin ranged from 100 to 400 mM, depending upon the success of lectin inhibition and the method of fluorometric assessment. Because of the polymeric structure of chitin, the precise molar concentration of this sugar could not be determined. Instead, an iso-osmotic solution was prepared by dissolving 10 µg of chitin in 1 mL of TBS, and the inhibitory action of the sugar was evaluated at 3 concentrations (2.5, 5, and 10 µg/mL). The specificity of binding for lectins recognizing sialic acid was confirmed using a neuraminidase treatment to enzymatically remove terminal sialic acid residues.
Terminal sialic acid residues also are known to mask other sugar residues (Cooper et al, 2001); therefore, neuraminidase treatment was used to verify the presence of masked carbohydrates. Spermatozoa recovered from the Accudenz gradient were resuspended in TBS, pH 6.0, to a final concentration of 1 x 109 sperm/mL and incubated with 1 IU neuraminidase (Type V, from Clostridium perfringens; Sigma) for 30 minutes at 37°C. Because chicken spermatozoa were estimated to contain a maximum of 0.44 µmol sialic acid per 109 cells (Froman and Engel, 1989), and 1 IU of neuraminidase liberates 1 µmol sialic acid per minute at pH 5.0 and 37°C (unit definition for this enzyme), the conditions used would be satisfactory enough to remove terminal residues of this sugar from the sperm surface. After an initial centrifugation (700 x g, 5 minutes), spermatozoa were resuspended in TBS, pH 6.0, and washed once, followed by 2 centrifugations in TBS at a pH of 7.6. The final pellet was used immediately for lectin staining as described below (experiments 1–3).
Experiment 1: Flow Cytometry Assessment
Semen from each group of males was assessed twice for a total of 6
replicates. Nontreated and neuraminidase-treated spermatozoa were resuspended
in TBS to a concentration of 2.5 x 109 sperm/mL. A 5-µL
aliquot of each sperm suspension was added to a 120-µL volume of lectin
solution to yield a final concentration of 100 x 106
sperm/mL. A control sample was prepared by diluting nontreated spermatozoa in
TBS only, and neuraminidase-treated spermatozoa also were diluted in
lectin-sugar mixtures to study the specificity of binding. All samples
contained 100 x 106 sperm/mL in a final volume of 125 µL.
Samples were incubated for 30 minutes at room temperature and protected from
light. After incubation, they were centrifuged (700 x g, 5
minutes) 4 times and the pellets were resuspended in the appropriate buffer
(eg, TBS with or without cations, pH 7.6 or 8.0). 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, Ore) for a minimum of 5 minutes at room temperature.
A Coulter Epics XL-MCL Flow Cytometer (Coulter Corporation, Miami, Fla) equipped with a single 488-nm excitation source was used for all analyses. Forward and side scatter gating were used to select single spermatozoa from clumps and debris. The fluorescence from FITC-stained and PI-stained spermatozoa was collected in FL1 (525 nm band pass) and FL3 (620 nm band pass) 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 to determine lectin binding–related changes in the population.
Experiment 2: Microscopic Assessment of Spermatozoa Stained in Suspension
Spermatozoa from each group of males were assessed once for a total of 3
replicates. Nontreated and neuraminidase-treated spermatozoa were resuspended
in TBS to a concentration of 1 x 109 sperm/mL. A 2-µL
aliquot of each sperm suspension was added to 18 µL of either lectin
solution or lectin-sugar mixture to yield a final concentration of 100 x
106 sperm/mL. Samples were incubated for 30 minutes at room
temperature protected from light. After incubation, a 5-µL aliquot was
placed on a slide, fitted with a coverslip, and examined using a Zeiss
Axioskop microscope (Carl Zeiss Inc, Thornwood, NY) equipped with an
excitation filter (450–490 nm) and a barrier filter (LP520). Individual
spermatozoa were evaluated for the presence/absence of fluorescence over the
acrosome, nuclear region of the head, midpiece, or tail at a magnification of
1000x. Only morphologically normal, whole spermatozoa were assessed. For
clarity of presentation, binding that was observed in the nuclear region of
the head is referred to as binding in the head region, and is not inclusive of
the acrosomal region.
Experiment 3: Microscopic Assessment of Fixed Spermatozoa Stained on Slides
The experiment was conducted in triplicate with each replicate using
spermatozoa from a different group of males. Nontreated and
neuraminidase-treated spermatozoa were resuspended in 1 mL of 4% (wt/vol)
paraformaldehyde in TBS (pH 7.2) and fixed for 30 minutes at room temperature.
The fixed sperm suspension was then further diluted (1:20–1:30) with
fixative and a 10-µL aliquot was spread onto a glass slide (1 slide/lectin)
and allowed to air-dry. Following air-drying, a blocking solution (5% wt/vol
bovine serum albumin in TBS) was applied to prevent nonspecific background
staining. Once complete evaporation of the blocking solution was observed
(room temperature), selected areas on the slide were covered with 20 µL of
either the lectin solution or the lectin-sugar mixture. Slides were held at
room temperature for 30 minutes protected from light. After incubation, slides
were washed with gentle agitation (3 times, 1.5 minutes each) in the
appropriate buffer for each lectin and allowed to air-dry protected from
light. Slides were then mounted with ProLong Gold antifade reagent (Molecular
Probes) and kept at room temperature until examination (within 2 days of
staining). Individual sperm cells were evaluated as described for experiment
2.
Statistical Analysis
Values of MnFI obtained in the flow cytometry assessment of lectin binding
were compared using the Mann-Whitney U Test. Differences within a species
between the control and each of the lectin-incubated samples of nontreated
spermatozoa were evaluated to determine the presence (P < .05) or
absence (P > .05) of sugar residues in the intact glycocalyx.
Nontreated and neuraminidase-treated samples also were compared within a
species to determine possible masking of carbohydrate residues by sialic acid.
Species comparisons were made for neuraminidase-treated spermatozoa to study
differences in carbohydrate content. Selective abolition of binding was
evaluated by comparing the values obtained in the presence and absence of
inhibitory sugars within lectins. All comparisons were made using STATISTICA
software for Windows (release 4.5, 1993; StatSoft, Inc, Tulsa, Okla).
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Results |
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Control samples (eg, spermatozoa incubated without lectins) emitted low levels of fluorescence (MnFI average: 0.17 ± 0.02, turkey; 0.13 ± 0.01, chicken). For non–neuraminidase-treated turkey spermatozoa, 4 of the 15 lectins showed high (P < .05) MnFI (LPA, 1.68 ± 0.53; LFA, 2.36 ± 0.60; Con A, 2.52 ± 1.05; RCA-I, 38.42 ± 9.70) compared to control samples. Similarly, 5 of the 13 lectins incubated with non–neuraminidase-treated chicken spermatozoa showed high (P < .05) MnFI (LPA, 3.96 ± 1.66; LFA, 2.98 ± 1.21; Con A, 2.88 ± 0.67; s-WGA, 4.07 ± 1.35; RCA-I, 54.58 ± 33.70) compared to control samples. The MnFI for the remaining lectins incubated with non–neuraminidase-treated spermatozoa was low (range: 0.20–0.35, turkey; 0.23–0.73, chicken) but still significantly different (P < .05) from control values with the exception of GS-II (0.21 ± 0.03) and Lotus (0.20 ± 0.05) in turkey samples.
The MnFI values for nontreated and neuraminidase-treated turkey and chicken
spermatozoa are shown in Tables
2 and
3. Because the enzyme
neuraminidase cleaves terminal sialic acid residues, the MnFI for
neuraminidase-treated spermatozoa was lower (P < .05) than for
nontreated spermatozoa incubated with LPA or LFA. Treatment of both turkey and
chicken spermatozoa with neuraminidase increased (P < .05) the
MnFI for all remaining lectins except s-WGA in chicken spermatozoa. Lectins
RCA-I, WFA, and ECA yielded the highest MnFI values for both turkey and
chicken neuraminidase-treated spermatozoa (Figure). Similar MnFI (P
> .05) were detected for most lectins incubated with either
neuraminidase-treated turkey or chicken spermatozoa (Figure). Exceptions
included sWGA and ECA, which had higher MnFI (P < .05) in chicken
spermatozoa than in turkey spermatozoa, and WFA, which had higher MnFI
(P < .05) in turkey spermatozoa than in chicken spermatozoa
(Figure).
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Experiment 2: Microscopic Assessment of Spermatozoa Stained in Suspension
Fluorescence data for spermatozoa stained in suspension with lectins are
shown in Table 4. The
proportion of stained cells varied among lectins; however, virtually all
spermatozoa were stained after incubation with RCA-I, WFA, LFA, and LPA, as
well as with s-WGA in chicken spermatozoa only. Binding was typically seen as
spots of fluorescence in the indicated regions. Additionally, sperm
agglutination was observed in some samples as a further indication of lectin
binding. With a few exceptions, lectin binding was not observed in
morphologically normal turkey or chicken spermatozoa unless the cells were
pretreated with neuraminidase. RCA-I was the most notable exception in that
nontreated turkey and chicken spermatozoa were highly agglutinated and
displayed bright fluorescence over the entire cell, the intensity of which
increased after neuraminidase treatment. The specificity of RCA-I binding was
confirmed by the complete absence of fluorescence in sperm cells incubated
with RCA-I + 100 mM lactose. Nontreated and neuraminidase-treated chicken
spermatozoa also exhibited fluorescence over the entire cell after incubation
with s-WGA that was abolished in the presence of 2.5 µg/mL chitin.
Nontreated turkey spermatozoa incubated with Con A exhibited
carbohydrate-specific binding over the acrosome and over the entire cell after
neuraminidase treatment.
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Similar staining patterns were observed for neuraminidase-treated turkey and chicken spermatozoa incubated with GNA, GS-II, WFA, ECA, or Lotus, and the lectin binding was specifically inhibited by the competing sugars (Table 4). Fluorescence was detected exclusively over the acrosome region after incubation with GNA and GS-II, whereas binding sites for WFA and ECA were distributed all over the cell. Lotus lectin binding also was observed over the entire cell; however, the degree of Lotus binding differed slightly in that chicken spermatozoa were clearly stained in the acrosome and the remaining morphological regions were covered by a cloud of diffused fluorescence, whereas binding in turkey spermatozoa was observed over the whole surface as spots of fluorescence. The staining patterns with SBA also differed between species; with chicken spermatozoa fluorescing over the entire cell, whereas binding was limited in the acrosome and head regions of turkey spermatozoa.
Sperm cellular debris (ie, detached heads or tails) frequently was stained, even in the absence of lectin binding in morphologically normal spermatozoa. For example, with GS-I, PNA, and Jacalin, normal spermatozoa were basically unstained in both nontreated and neuraminidase-treated samples; however, a large percentage of detached acrosomes and whole cells with decondensed heads were observed showing homogeneous and bright fluorescence.
Experiment 3: Microscopic Assessment of Fixed Spermatozoa Stained on Slides
In contrast to the binding patterns observed for spermatozoa stained in
suspension, some measure of fluorescence was observed for each lectin when
spermatozoa were fixed, air-dried, and incubated with lectin, regardless of
neuraminidase treatment. For example, GS-I bound to the acrosomal and head
regions of all turkey and chicken spermatozoa observed on slides, whereas
binding was minimal (observed only in abnormal cells and cellular debris) when
this lectin was incubated with spermatozoa in suspension. A similar staining
pattern was observed for Jacalin and PNA with the slide technique, although
considerably fewer cells overall exhibited fluorescence. More importantly,
Jacalin and PNA binding observed in fixed cells were not abolished by the
respective inhibitory sugars, and the inhibitory sugars for GS-I only reduced
fluorescence intensity at high molar concentrations (400 mM). Similarly, Con
A, GNA, PSA, ECA, and Lotus all were observed to bind the head region of
nontreated turkey and chicken spermatozoa, and this binding was not inhibited
by any of the competitive sugars. Binding patterns associated with LPA and LFA
were different despite the fact that both have affinity for sialic acid. LPA
stained only the head region, whereas LFA binding was observed over the whole
sperm surface. Surprisingly, neuraminidase-treated spermatozoa also were seen
stained, and changes in the staining characteristics of these cells were not
evident.
Binding was observed in the head region of turkey spermatozoa stained on slides with ECA irrespective of neuraminidase treatment, and this fluorescence was not abolished by the inhibitory sugar. For chicken spermatozoa, neuraminidase treatment increased the occurrence of ECA binding from only the head region to the entire cell. In this case, however, binding in the acrosome, midpiece, and tail were abolished by the inhibitory sugar lactose. For turkey spermatozoa, a similar shift in the RCA-I, SBA-, and WFA-binding patterns occurred between nontreated and neuraminidase-treated spermatozoa, and the fluorescence of all regions except the head also was inhibited in the presence of competing sugars. Regions stained by GS-II included the acrosome and head regions in nontreated turkey spermatozoa but only the head region in nontreated chicken spermatozoa. Binding extended to acrosome in the neuraminidase-treated chicken spermatozoa, with no change being observed in turkey. For both species, abolition of binding occurred only in the acrosome region with the inhibitory sugar.
The lectin s-WGA displayed different fluorescent intensity in nontreated and neuraminidase-treated spermatozoa. In general, binding was observed over acrosomal and head regions in a few cells with the majority unstained. The intensity of fluorescence was low in nontreated spermatozoa, and although neuraminidase treatment slightly increased the number of stained spermatozoa and the intensity of fluorescence, no change in binding sites was observed. Fluorescence intensity was clearly reduced or not observed when spermatozoa were incubated with lectin-sugar mixtures.
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Discussion |
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Flow cytometry assessment of lectin binding revealed that the glycocalyx of
turkey and chicken spermatozoa in its physiological state (eg, not
enzymatically treated with neuraminidase) is composed mainly of
glycoconjugates containing ß-galactose, -mannose/-glucose,
and sialic acid as terminal saccharides, based on the high fluorescence
intensity values obtained with RCA-I, Con A, LFA, and LPA. Some lectins that
recognize terminal ß-galactose (PNA), -mannose (GNA), and
-glucose and/or -mannose (PSA) residues, however, provided low
fluorescence intensity despite the fact that they recognize the same broad
carbohydrate groups as RCA-I and Con A. This apparent conflict in carbohydrate
identification is the result of the highly specific binding sites recognized
by each lectin. It is known that lectins with a same nominal sugar binding
specificity (eg, fucose-binding lectins, galactose-binding lectins) can react
quite differently with complex sugar structures
(Leathem and Brooks, 1998).
For example, PNA can react strongly with a disaccharide known as the T-antigen
[Gal(ß1,3)GalNAc]. Factors affecting binding site access, such as steric
configurations, are important for lectin binding
(Koehler, 1978), and subtle
differences in binding specificity within carbohydrate groups also exist. For
example, unlike most mannose-specific lectins, GNA does not react with
glucose, and PSA does not recognize branched mannose structures. These
examples illustrate the possibility that a diversity of glycoconjugates
containing unique saccharide structures or sequences exists in the sperm
surface, and a wide spectrum of lectins is necessary to properly characterize
glycocalyx composition.
Few residues of -galactose, N-acetyl-galactosamine,
N-acetyl-lactosamine, and fucose, as well as terminal monomers of
N-acetyl-glucosamine (also dimers in turkey spermatozoa) were detected by flow
cytometry in non–neuraminidase-treated spermatozoa, initially suggesting
that these carbohydrates were minor components of the glycocalyx. Enzymatic
removal of terminal sialic acid residues with neuraminidase, however,
demonstrated that this sugar was masking the majority of the carbohydrate
residues in the sperm glycocalyx. Although ß-galactose and
-mannose/-glucose residues were still abundant in
neuraminidase-treated spermatozoa, residues of N-acetyl-galactosamine,
N-acetyl-lactosamine, and N-acetyl-glucosamine became predominant carbohydrate
residues for both species after terminal sialic acid residues were removed.
Interestingly, the extent to which sialic acid masked the carbohydrate
residues varied. Residues recognized by s-WGA in the chicken, for instance,
did not appear to be significantly sialylated, and only a few of those
recognized by Con A were masked. The extent of sialylation for the rest of the
residues was directly related to their relative abundance in the
glycocalyx.
Sialic acids act as masking agents on antigens, receptors, and other recognition sites of the cell surface (Schauer, 1985); therefore, the observation that a considerable amount of sperm surface saccharides were coated with sialic acid molecules is not surprising. This phenomenon has been observed in other species (Cooper et al, 2001), and it appears that the amount of sialic acid in sperm cells is considerably higher than that in somatic cells (Diekman, 2003). In mammalian spermatozoa, this masking of terminal sugars seems to be an integral step during sperm maturation, as sialic acid groups appear on the sperm surface as spermatozoa proceed through the epididymis (Holt, 1980). The occurrence of a similar mechanism for maturation of poultry spermatozoa needs to be verified, in part, because of the lack of a subdivided epididymal structure in the avian reproductive tract and known differences in posttesticular changes between mammalian and avian spermatozoa (Esponda and Bedford, 1985). Regardless of the mechanism for sialic acid deposition, the functional significance of the extensive sialylation of the sperm glycocalyx may be similar for both mammalian and avian species. Removal of surface sialic acid residues may increase antigenicity of chicken spermatozoa in the vagina of the hen, resulting in their destruction by an immunologically-based sperm-selection mechanism (Steele and Wishart, 1996); a similar selective mechanism has been proposed for mammalian spermatozoa (Holt, 1980). If this also applies to the turkey species, then the high extent of sialylation observed in the poultry sperm glycocalyx could be explained on the basis of that immunoprotective effect of sialic acid. Moreover, the different levels of sialylation observed among sugar residues may be related to differences in the antigenicity of the glycoconjugates.
The most remarkable differences in glycocalyx composition between the 2 species were observed for N-acetyl-glucosamine dimers (s-WGA) and N-acetyl-galactosamine (WFA), with the former being more prevalent in chicken spermatozoa and the latter more prevalent in turkey spermatozoa. Another interesting species difference is that chicken spermatozoa contained higher levels of N-acetyl-lactososamine (ECA) than turkey spermatozoa.
Other aspects of poultry sperm glycocalyx composition can be discerned
based on binding specificity. For instance, neither turkey nor chicken sperm
glycocalyx appeared to contain N-acetyl-glucosamine oligomers, as specific
binding for STA could not be demonstrated. Likewise, distinctive
-linkages also exist within fucose residues, as Lotus, rather than
UEA-I, was the only fucose-binding lectin that specifically bound to the sperm
membrane. Also interesting was that PSA (-mannose/glucose) and Jacalin
(-galactose) did not bind specifically to the chicken sperm membrane,
whereas low but specific binding of these lectins was evident in turkey
spermatozoa.
The 2 techniques used for microscopic determination of lectin-binding sites gave quite different staining patterns, most notably with non–neuraminidase-treated spermatozoa. This dichotomy has been reported for other species when comparing lectin binding patterns of unfixed and fixed spermatozoa (Kallajoki et al, 1985; Navaneetham et al, 1996), as well as when comparing binding patterns after different methods of fixation (Gabriel et al, 1994). It has been suggested that procedures like fixation and air-drying disrupt the sperm plasma membrane and thereby expose acrosomal and other intracellular glycoconjugates, which results in false positive glycocalyx lectin binding (Kallajoki et al, 1985); paradoxically, a number of publications have based glycocalyx characterizations on methods employing fixed and/or air-dried spermatozoa. In our study, we compared the 2 methods of staining poultry spermatozoa for our own verification of the suitability of using fixed, air-dried cells for microscopic localization of lectin binding. The fact that a majority of lectins appeared to bind to spermatozoa when stained on slides but not when stained in suspension is consistent with the idea that intracellular lectin binding could be occurring. Moreover, this binding was not easily inhibited and showed clear inconsistencies with the flow data (ie, changes in staining characteristics after neuraminidase treatment were not evident for most lectins; s-WGA hardly stained a few cells in nontreated chicken spermatozoa), as well as inconsistencies in some binding patterns. For example, no sialic acid residues appeared to exist over the midpiece or tail according to LPA binding pattern with the slide method; however, it is clear that the carbohydrate actually was present in those morphological regions, as neuraminidase treatment exposed masked carbohydrates detected by ECA, SBA, WFA, and RCA-I. Therefore, lectin staining of fixed, air-dried cells most likely does not represent the true surface binding pattern for poultry spermatozoa.
Data from neuraminidase-treated spermatozoa stained in suspension revealed
that the entire cell surface was coated with sialic acid and that certain
glycoconjugates were segregated into membrane domains. In particular,
glycoconjugates containing terminal monomers of N-acetyl-glucosamine, as well
as -mannose residues recognized by GNA, appeared to be distributed only
over the acrosomal region in both species. For turkey spermatozoa,
N-acetyl-galactosamine residues recognized by SBA were localized over the
entire head region, including the acrosome. Segregation into domains was less
evident for other carbohydrates, which seemed to be distributed over the whole
surface. The distribution of Con A- and Lotus-recognized residues over certain
areas of the chicken spermatozoa was not clear and requires further study
using techniques of superior resolution. In general, lectins that demonstrated
binding in unfixed spermatozoa also were delineated by flow cytometry.
However, binding sites for the lectins s-WGA, Jacalin, and PSA, which
exhibited low fluorescence intensities in conjunction with flow cytometry
analysis of turkey spermatozoa, were not evident during microscopic
evaluation. Further studies are thus also necessary to clarify the absence of
binding sites observed for these lectins in turkey spermatozoa, as well as for
GS-I and PNA in both species.
From a comparative standpoint, the use of lectins to identify glycoconjugates in unfixed, mature/ejaculated spermatozoa from other species highlights the diversity found in the sperm glycocalyx. For example, although sialic acid and galactose residues clearly were abundant in the glycocalyx of poultry spermatozoa, these major carbohydrate groups are not surface components of Drosophila spermatozoa (Perotti and Pasini, 1995). Conversely, fucose residues did not appear to be a major component of the poultry sperm glycocalyx, whereas the glycocalyx of human spermatozoa has been reported to contain 30% fucose (Calzada et al, 1994). Similar to poultry, the glycocalyx of rabbit spermatozoa does not contain UEA-I–recognized fucose residues, but has been reported to contain sialic acid, N-acetyl-glucosamine, N-acetyl-galactosamine, galactose, glucose, and mannose (Nicolson and Yanagimachi, 1972). Also similar to chicken spermatozoa, boar and ram spermatozoa exhibited bright fluorescence when incubated with lectins RCA-I and s-WGA; however, unlike poultry spermatozoa, the glycocalyx of porcine and ovine spermatozoa demonstrated high fluorescence after incubation with the lectins STA and Jacalin (Ashworth et al, 1995). Ram spermatozoa also differ from poultry spermatozoa by exhibiting higher fluorescence intensity after incubation with the lectin PNA (Magargee et al, 1988). In addition to the species-specific variability in the number of carbohydrate residues (as indicated by the level of fluorescence intensity) the distribution of carbohydrates over the sperm surface also varies among species (Magargee et al, 1988; Ashworth et al, 1995; Perotti and Pasini, 1995), including chicken and turkey spermatozoa. For example, SBA recognized N-acetyl-galactosamine residues along the entire length of neuraminidase-treated chicken spermatozoa, but these same terminal carbohydrates were found only on the acrosome and head regions of turkey spermatozoa. Taken together, these data indicate that the carbohydrate content of sperm glycocalyx is highly species-specific, and suggests that delineation of the physiological basis for this diversity among species and taxa may provide new insight about sperm function.
In summary, the glycocalyx of turkey and chicken spermatozoa is extensively
sialylated and contains residues of -mannose/-glucose, -
and ß-galactose, -fucose, - and
ß-N-acetyl-galactosamine, and N-acetyl-lactosamine, as well as monomers
and dimers of N-acetyl-glucosamine in variable amounts. Sugar residues
specific for STA and UEA-I do not appear to exist in either of the 2 species,
whereas those for PSA and Jacalin appear to only exist in turkey spermatozoa.
Chicken spermatozoa are considerably richer in dimers of N-acetyl-glucosamine
and residues of N-acetyl-lactosamine than turkey spermatozoa, whereas turkey
spermatozoa contain a higher amount of WFA-recognized N-acetyl-galactosamine
residues. Monomers of N-acetyl-glucosamine and glycoconjugates containing
GNA-recognized mannose residues appear to be clearly restricted to the
acrosomal region in both turkey and chicken spermatozoa.
In the light of the findings reported here, it becomes evident that the glycocalyx of poultry spermatozoa is generally similar to that of mammalian spermatozoa in that 1) it contains the same types of carbohydrates reported for mammals, 2) there is a phenomenon of sialylation in terminal sugars with apparently the same functional purpose, 3) the membrane is structurally subdivided into regional domains, and 4) species specificity exists with regard to composition and distribution of carbohydrates. Known physiological differences existing between mammalian and avian spermatozoa, such as the fertilizing ability of testicular avian spermatozoa (Howarth, 1983) without a requirement for capacitation (Howarth, 1970), suggest that even similar glycoconjugates may have widely differing functional relevance. In mammalian spermatozoa, for example, glycoconjugates recognized by the WGA lectin have been shown to participate actively in the phenomenon of capacitation (Mahmoud and Parrish, 1996); however, the role of WGA-recognized glycoconjugates in poultry spermatozoa would not be expected to be associated with this physiological event. Likewise, N-acetyl-galactosamine residues appear to play a role in the development of hyperactive motility in mammalian spermatozoa (Bergerson et al, 1994; Kawakami et al, 2002), whereas hyperactivation is not observed in poultry spermatozoa. Characterizing the carbohydrate residues in the turkey and chicken sperm glycocalyx represents the first step in our investigation. Studies currently are underway to determine the functional significance of these carbohydrates with respect to conventional semen storage methodologies.
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