Molecular Human Reproduction vol.3 no.10 pp. 827–837, 1997
Induction of the human sperm acrosome reaction with mannosecontaining neoglycoprotein ligands*
Susan Benoff1,2,4, Ian R.Hurley1, Francine S.Mandel3, George W.Cooper1 and Avner Hershlag1
1Department of Obstetrics and Gynecology, North Shore University Hospital-New York University School of Medicine,
Manhasset, New York, 2Departments of Obstetrics and Gynecology and Cell Biology, New York University School of
Medicine, and 3Department of Research, North Shore University Hospital and Department of Environmental Medicine,
New York University School of Medicine
4To whom correspondence should be addressed at: North Shore University Hospital, 300 Community Drive, Boas-Marks
Biomedical Science Research Center-Room 125, Manhasset, New York 11030, USA
In the interest of classifying cases of male factor infertility, we have paid particular attention to the sugar
ligand binding properties of the human sperm surface and the functional capacity of the acrosome for
exocytosis—key parameters for assessing sperm fertilizing ability. Zona recognition and binding involve the
interactions of sperm surface mannose receptors (lectins) with mannose ligands on the zona pellucida. Sperm
surface mannose lectins can be visualized by their ability to bind a synthetic model zona ligand, fluorescein
isothiocyanate (FITC)-conjugated mannosylated bovine serum albumin (BSA) (Man–FITC–BSA). We now report
that Man–FITC–BSA biologically also mimics the effects of solubilized authentic human zonae, in that binding
of Man–FITC–BSA results in a time-dependent receptor aggregation and the induction of acrosome exocytosis
in capacitated sperm populations from fertile donors. In our assay, the addition of mM amounts of mannose
monosaccharide to Man–FITC–BSA increases the number of polyvalent mannose ligands bound by individual
spermatozoa and increases the rate of the acrosome reactions induced by Man–FITC–BSA, thereby increasing
specimen processing efficiency. We conclude that exposure of human spermatozoa to polyvalent mannose
ligands 1 D-mannose monosaccharide offers a new, convenient and readily available system to study sperm
capacity for induced acrosome loss.
Key words: induced acrosome loss/mannose receptor aggregation/monosaccharides/sperm–oocyte binding/
zona pellucida oligosaccharides
Introduction
The acrosome reaction is a specialized form of exocytosis
required for sperm penetration through the zona pellucida
(Overstreet and Hembree, 1976; Chen and Sathananthan, 1986;
Liu and Baker, 1993, 1994a,b, 1996b). The elucidation of the
cellular mechanisms inducing the acrosome reaction is of
particular relevance to the clinical evaluation of sperm function.
Human spermatozoa which are unable to undergo an acrosome
reaction will not fertilize oocytes following conventional
insemination in vitro (Overstreet et al., 1980; Liu et al.,
1989a,b; Liu and Baker, 1994b; Benoff et al., 1996). The goal
of the present study is to identify characteristics of human
sperm functionally important in acrosome exocytosis. These
characteristics should help classify cases of male factor infertility and, thus, could be used to predict the fertilizing potential
of sperm populations.
We have concentrated on the elucidation of the properties
of motile human spermatozoa able to acrosome react in
response to external stimuli for two reasons. Firstly, the
spontaneous acrosome reaction has been considered to be
*Presented in part at 51st Annual Meeting of the
American Society for Reproductive Medicine, Seattle,
Washington, October 7–12, 1995.
© European Society for Human Reproduction and Embryology
largely artifactual as in specimens of unknown fertility status
it was not affected by the presence of the cumulus complex,
the absence of extracellular calcium or low temperature (White
et al., 1990). However, the ability of human spermatozoa to
undergo an induced acrosome reaction is correlated with
in-vitro fertilization (IVF) outcome (Henkel et al., 1993; Calvo
et al., 1994; Parinaud et al., 1995; Benoff et al., 1996).
Secondly, although both acrosome-intact and acrosome-reacted
human spermatozoa can initiate binding to the human zona
pellucida (Morales et al., 1989), only acrosome-reacted spermatozoa have been observed to penetrate deep through the zona
(Sathananathan et al., 1982). A series of studies have now
indicated that the physiologically relevant acrosome reaction
of human spermatozoa occurs at the surface of the zona
pellucida (Cross et al., 1988; Tesarik et al., 1988; Tesarik,
1989; Coddington et al., 1990; Liu and Baker, 1990, 1994a,
1996a,b; Franken et al., 1991; Hoshi et al., 1993). We
postulated that the characterization of the acrosome reactioninducing residues in the human zona pellucida could lead to
the development of a new tool for the evaluation of sperm
function prior to IVF.
All mammalian species so far tested express three protein
families which make up the zona pellucida (Harris et al.,
1994). There is limited evidence that human spermatozoa can
827
S.Benoff et al.
directly bind to zona polypeptides (Whitmarsh et al., 1996),
that antibodies to a porcine zona protein epitope block binding
of human spermatozoa to homologous oocytes (Koyama et al.,
1996) and that abnormalities in the polypeptide backbone of
the zona’s sperm receptor may be contributory to fertilization
failure in vitro (Oehninger et al., 1996). It has, however, been
inferred that it is the species-specific glycosylation of the zona
proteins, and not the species-specific variation in protein
sequence, which is the primary regulator of sperm–zona
binding (Florman and Wassarman, 1985; Yurewicz et al., 1991;
Litscher et al., 1995; Benoff, 1997). This inference is supported
by lectin binding studies which indicate that the distribution of
carbohydrates on human and animal zonae differ significantly
(Shalgi et al., 1991; Maymon et al., 1994) and also helps
explain why human spermatozoa have limited affinities for
heterologous zonae (Bedford, 1977; Liu et al., 1991; Oehninger
et al., 1993). In contrast, the acrosome reaction-inducing
activity of soluble zona glycoproteins depends both on their
polypeptide conformation and glycosylation patterns (Florman
et al., 1984; Litscher and Wassarman, 1996; Liu and Baker,
1996a), possibly as the protein presents to the sperm-specific
oligosaccharide side chains in the form of polyvalent ligands
(Leyton and Saling, 1989).
Induction of human sperm acrosome exocytosis by oligosaccharide ligands on the zona surface appears to be a complex
phenomenon. Nevertheless, it is clear that sperm surface sugar
receptors are involved both in the initiation of acrosome
exocytosis (e.g. N-acetylglucosamine; Brandelli et al., 1994,
1995) and its prevention (e.g. fructose; Mori et al., 1993). The
best studied human sperm receptors for carbohydrates are
those for mannose ligands (Mori et al., 1989; Tesarik et al.,
1991; Benoff et al., 1992, 1993a, 1994a,b, 1995a,b, 1996;
Chen et al., 1995). By cloning and sequencing of the human
sperm surface mannose receptor, we have confirmed its homology with other human calcium-dependent (C-type) mannosebinding lectins (Benoff et al., 1992, 1993e, 1994a). Based on
two deduced amino acid sequences, the sperm mannose receptor is inferred to be a heterodimer consisting of two polypeptide
chains, each having two mannose carbohydrate recognition
domains. We have previously used this information to design
the protocols for the examination of sugar-induced acrosomal
exocytosis (Benoff et al., 1993c,d, 1995a, 1996). In our prior
studies we have used a brief (15 min) exposure of capacitated
human spermatozoa to polyvalent neoglycoprotein ligands and
monosaccharides to mimic the effects of sugar epitopes on the
zona surface which induce acrosomal loss. In this study, we
examined: (i) the concordance between mannose receptor
aggregation by mannose or antibodies to mannose lectins and
induction of the acrosome reaction; (ii) the time course of
acrosome exocytosis induced by polvalent mannose-containing
neoglycoproteins in the presence or absence of monosaccharides; and (iii) the effect of D-mannose on the numbers of
polyvalent neoglycoprotein ligands bound by each spermatozoon. The results of these studies have been informative for
developing an understanding of how polyvalent mannose
ligands and monosaccharide mannose can interact to influence
ligand binding affinities and the rate of acrosome loss.
828
Materials and methods
Media and chemicals
Modified Ham’s F-10 medium (Formula No. 90–8050PG) was
obtained from Gibco Laboratories (Grand Island, NY, USA). Unless
otherwise noted, all reagents were purchased from Sigma Chemical
Company (St Louis, MO, USA).
Human semen specimens
All protocols employing human subjects were reviewed and approved
by the Institutional Review Board of North Shore University Hospital.
Donors of known fertility participated after giving written informed
consent. The semen parameters of specimens from these donors were
within the normal ranges for morphology, motility and number based
on World Health Organization (WHO, 1992) criteria.
Semen preparation
Fresh semen specimens were collected by masturbation after 2–3
days of abstinence. Semen analysis was performed manually following
liquefaction. Sperm concentration, percentage motility and progression were assessed (WHO, 1992). Spermatozoa were then selected
for motility by a ‘swim-up’ method (Bronson et al., 1982). The
number of spermatozoa/ml was determined by haemacytometer count
after 1:20 dilution in counting fluid containing 2% phenol. Motility
in swim-up preparations ranged from 97 to 100%.
Untreated (‘fresh’ or uncapacitated) spermatozoa were prepared
for analysis by centrifugation (500 g for 8 min) to concentrate
spermatozoa. To induce capacitation, spermatozoa were pelleted,
resuspended in Ham’s F-10 containing 30 mg/ml charcoal delipidated
(Chen, 1967) human serum albumin (HSA) at a density of 123106
cells/ml and incubated for 16–20 h at 37°C in 5% CO2 in air. At the
end of incubation, spermatozoa were collected by centrifugation and
their motility was assessed by phase-contrast microscopy. Only
preparations with .80% motile spermatozoa were then used in the
experiments reported here.
Spermatozoa and oocytes are generally coincubated for a standard
16–20 h period as this time frame corresponds to the timing for
observation of the formation of pronuclei (Trounson and Gardner,
1993; Gianaroli et al., 1996). A similar 16–20 h incubation period
for assessment of sperm function was chosen for two practical
reasons. Firstly, fertile donor human spermatozoa exhibit a timedependent increase in surface mannose receptor expression which
reaches plateau levels by 18 h of incubation (Benoff et al., 1993b,
1996). Thus, this incubation period maximizes our ability to distinguish between fertile and infertile sperm populations (Benoff et al.,
1993c, 1996). Secondly, mannose receptor expression is observed to
rise starting at ~4 h of incubation (Benoff et al., 1993b). As a result
of patient scheduling, the time required to prepare the fresh sample
and to complete a surface labelling, severe constraints would be
placed on clinical laboratory personnel if they were to be required to
perform two mannose receptor assays on the same sample in a single
day. The prolonged incubation period enables routine use of this
protocol in clinical diagnosis.
Visualization of sperm surface D-mannose binding sites
To study the distribution of mannose binding sites on freshly isolated
and capacitated human spermatozoa, motile sperm populations were
surface labelled with 100 µg/ml fluorescein isothiocyanate-conjugated
mannosylated bovine serum albumin [Man–FITC–BSA; a neoglycoprotein prepared by reaction of a mannose-containing glycosidophenyl
isothocyanate with BSA (Monsigny et al., 1994; Sigma No. A7790)]
as previously described (Benoff et al., 1993b). Control reactions
contained 100 µg/ml FITC-conjugated BSA. In some experiments
Induction of human sperm acrosome reaction
Man–FITC–BSA was replaced with polyclonal goat serum prepared
against the human macrophage mannose receptor (anti-HMR antibodies; a generous gift of Dr Philip D.Stahl, Washington University,
St Louis, MO, USA), diluted 1:1000 in Ca21-supplemented buffer
and control aliquots were treated with pre-immune goat sera (Benoff
et al., 1992, 1993e). FITC-conjugated rabbit anti-goat immunoglobulin
(Ig)G at 1:16 dilution served as secondary antibody. With either
Man–FITC–BSA or anti-HMR antibodies, motility and viability (by
eosin Y dye exclusion) were assessed at the beginning and end of
the labelling protocol. In addition, in some experiments 75 mM
D-mannose monosaccharide (D-mannose) or 75 mM D-galactose
monosaccharide (D-galactose) was added to the Ca21-supplemented
buffer but not to the Ca21-free buffer.
Man–FITC–BSA and anti-HMR antibodies display similar distributions on the human sperm head (Benoff et al., 1993b,e). Binding was
enumerated as whole head plus midpiece (pattern II) or equatorial/
post-equatorial regions plus midpiece (pattern III) (Figure 1A) as
previously described (Benoff et al., 1993b,d, 1994b).
Evaluation of mannose ligand binding by spectrofluorometry
A new assay has been developed in order to quantify the intensity of
fluorescence of Man–FITC–BSA molecules bound to the sperm head.
Motile sperm populations were surface labelled with Man–FITC–
BSA as described above. Following post-labelling washes, labelled
spermatozoa were resuspended in 3 ml Ca21-free buffer (Benoff
et al., 1993b) and the fluorescence emission quantified for 5 s in a
Shimadzu RF5000U spectrofluorometer with monochromators set at
490 and 520 mm respectively, for excitation and emission. This assay
is particularly useful for assessing sperm mannose-ligand receptor
expression in specimens where ,13106 motile spermatozoa are
obtained.
Evaluation of acrosome status
Acrosome-intact and acrosome-reacted Man–FITC–BSA or antiHMR antibody labelled spermatozoa were ethanol permeabilized,
differentiated by reaction with 100 µg/ml rhodamine-labelled Pisum
sativum agglutinin (RITC–PSA; Vector Laboratories Inc, Burlingame,
CA, USA) in distilled water (Cross et al., 1986) and scored for the
presence or absence of acrosome content (Figure 1B) as previously
described (Benoff et al., 1993b, 1994b).
Statistical analysis
All statistical analyses were performed with the SAS/PC software
package (SAS Institute Inc, Cary, NC, USA). Statistical significance
was set at P , 0.05. In order to compare the rate of agreement
between topology of Man–FITC–BSA binding (pattern II versus
pattern III) and acrosomal status (intact versus reacted) in spermatozoa
from fertile donors, the Kappa statistic was used.
Repeated measures analyses of variance, or paired t-tests if only
two conditions were present, were used to compare the means of
the percentages of sperm binding Man–FITC–BSA or anti-HMR
antibodies under various test conditions (e.g. incubation time, added
monosaccharides).
The relationship between the percentage of incubated sperm binding
Man–FITC–BSA (patterns II 1 III combined) versus relative spectrofluorometric deflection (fluorescence intensity) was examined by
linear regression. The paired t-test was employed to compare the
fluorescence intensity of aliquots of incubated spermatozoa labelled
with Man–FITC–BSA in the presence or absence of 75 mM
D-mannose.
Figure 1. Photomicrographs of capacitated human sperm doublelabelled with fluorescein isothiocyanate-conjugated mannosylated
bovine serum albumin (Man–FITC–BSA) and rhodamine-labelled
Pisum sativum agglutinin (RITC–PSA). Labelled specimens were
viewed at 3400 with an Olympus microscope (Olympus Corp.,
Lake Success, NY, USA) equipped with epifluorescence optics and
photographed on 35 mm/400 ASA black and white film (Eastman
Kodak Co., Rochester, NY, USA). (A) The arrow indicates the
sperm surface labelled in pattern III with Man–FITC–BSA. The
remaining spermatozoa in the figure exhibit Man–FITC–BSA
binding in pattern II. (B) Examination of the corresponding RITC–
PSA labelling patterns of Man–FITC–BSA-labelled spermatozoa
shown in Part A demonstrates that pattern II spermatozoa are
acrosome intact whereas pattern III spermatozoa have undergone an
acrosome reaction (arrow).
Results
Human spermatozoa undergo an acrosome reaction
induced by polyvalent mannose-ligands
The topology of sperm surface binding sites strictly correlates
with the state of the acrosome in spermatozoa from fertile
donors. Irrespective of whether Man–FITC–BSA (n 5 25;
Figure 1A) or anti-HMR antibodies (n 5 26) were employed
to detect the distribution of mannose lectins on the human
sperm surface, 100% of spermatozoa exhibiting pattern II
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S.Benoff et al.
labelling were acrosome intact whereas 100% of spermatozoa
labelled in pattern III were acrosome-reacted (Figure 1B).
Kappa for each donor was 1.00, indicating complete agreement
between the distribution of mannose lectins and acrosome
status.
Both polyvalent mannose ligands and anti-HMR antibodies
have the potential to cross-link individual mannose lectin
heterodimers to one another. Thus, binding of either probe
could result in a topographical redistribution of sperm surface
mannose binding sites. To clarify the relationship between
mannose receptors distributed in pattern II versus pattern III,
we examined the effect of the time course of polyvalent
mannose ligand exposure on surface distribution of mannose
lectins. Motile fresh and incubated sperm populations were
surface labelled under standard conditions with 26.5 µM
mannose as a polyvalent ligand, Man–FITC–BSA, while varying the incubation period, e.g. 5 min, 15 min, 1 h and 2 h. A
total of 25 specimens from 12 fertile semen donors were
examined by this protocol and concordant results were
obtained. Figure 2 presents typical results obtained by the
repeated analysis of specimens (n 5 13) from one fertile
donor, who served as an intra- and inter-experimental control.
The Man–FITC–BSA surface labelling reaction with capacitated spermatozoa was complete within 15 min of incubation;
21.2 6 1.9% of spermatozoa exhibited surface mannose
binding sites (Figure 2A). No further increase in the percentage
of spermatozoa exhibiting head-directed Man–FITC–BSA
binding (pattern II 1 III combined) was detected upon further
incubation (Figure 2A). There was, however, a significant
time-dependent transition from pattern II to pattern III Man–
FITC–BSA binding (Figure 2B). At 5 min of incubation,
,30% of spermatozoa binding Man–FITC–BSA were labelled
in pattern III whereas by 2 h of incubation, .70% of
spermatozoa binding Man–FITC–BSA were labelled in
pattern III.
The transition from pattern II to pattern III Man–FITC–
BSA binding was accompanied by a corresponding timedependent increase in the percentage of spermatozoa which
had undergone acrosomal exocytosis as determined by double
labelling with RITC-PSA. The percentages of spontaneously
acrosome-reacted incubated spermatozoa in control aliquots
was 19.44 6 13.33%, consistent with prior observations (e.g.
White et al., 1990; Hershlag et al., 1997). At 2 h of incubation
in the presence of Man–FITC–BSA, the percentages of acrosome loss were significantly increased. Subtraction of baseline
spontaneous acrosome loss from the total loss indicates that
an acrosome reaction had been induced in 15.7 6 1.52% of
spermatozoa from 13 replicates from a single fertile donor
(Figure 2B), a value consistent with consensus normal values of
.10% for acrosome reaction inducibility (ESHRE Andrology
Special Interest Group, 1996). Mean motilities and viabilities
at the beginning and end of the incubation periods differed by
,10%. Thus, there was no significant loss of motility or sperm
viability associated with exposure to Man–FITC–BSA. These
data confirm that incubated, i.e. capacitated, human spermatozoa undergo an acrosome reaction when presented with micromolar amounts of mannose as polyvalent ligand.
In contrast, when freshly isolated spermatozoa were studied,
830
Figure 2. Examination of capacitated motile human spermatozoa
for the effect of D-mannose on the conversion of fluorescein
isothiocyanate-conjugated mannosylated bovine serum albumin
(Man–FITC–BSA) binding pattern II to pattern III. The results of
the repeated analysis of specimens (n 5 13) from one fertile donor
are presented. (A) Surface labelling is complete by 15 min of
incubation; no increase in the percentage of head binding of Man–
FITC–BSA was detected upon further incubation. The addition of
D-mannose to the pre-wash and labelling buffers has no effect on
the total percentage of sperm exhibiting head-directed Man–FITC–
BSA binding (patterns II 1 III combined). For the four points
examined, the P values ranged from 0.8921 to 0.9673 (not
significant). (B) Exposure of spermatozoa to polyvalent mannose
ligands (100 µg/ml Man–FITC–BSA containing 26.5 µM mannose)
over time induces the transition from pattern II to pattern III Man–
FITC–BSA binding (P ,0.006). The addition of D-mannose
significantly increases the rate at which the pattern II to III
conversion occurs (5 min, P ,0.0061; 15 min, P ,0.0001; 60 min,
P ,0.0045). This transition is complete by 2 h of exposure (0
versus 75 mM D-mannose; P 5 0.967, not significant) irrespective
of whether or not D-mannose was added.
neither the percentage of spermatozoa binding Man–FITC–
BSA in pattern III nor the percentage of acrosome loss
increased when the surface labelling reaction time was extended
from 5 min to 2 h (acrosome-reacted spermatozoa respectively,
6.0 6 1.96% versus 5.86 6 1.02%). These data indicate that
human spermatozoa normally acquire the ability to initiate an
acrosome reaction in response to mannose ligand exposure
following capacitation.
D-mannose accelerates polyvalent mannose-ligandinduced acrosome loss
To examine whether interactions between the four carbohydrate
recognition domains of the human sperm mannose receptor
could change the time course of conversion of pattern II to
pattern III, aliquots from the above described specimens
Induction of human sperm acrosome reaction
Figure 3. Examination of the effect on capacitated motile human
sperm populations from fertile donors (n 5 26) of D-mannose on
the conversion of pattern II to pattern III anti-human macrophage
mannose receptor (anti-HMR) antibody binding. Typical results are
shown. (A) The addition of D-mannose to the blocking and
labelling buffers has no effect on the total percentage of
spermatozoa exhibiting head-directed anti-HMR antibody binding
(patterns II 1 III combined) (P 5 0.95, not significant). (B) The
addition of D-mannose to the blocking and labelling buffers,
however, stimulated the conversion of anti-HMR antibody binding
pattern II to binding pattern III (P ,0.01).
(n 5 25) were surface labelled with Man–FITC–BSA in the
presence of D-mannose for 5 min, 15 min, 1 h or 2 h (Figure
2). D-mannose was added at 75 mM because including this
concentration of free sugar in the pre-wash and labelling
reaction buffers had no effect on the total percentage of
spermatozoa displaying head-directed Man–FITC–BSA binding (patterns II 1 III combined; Figure 2A). Our present
results confirm previous analyses (Benoff et al., 1993d).
The addition of D-mannose significantly increased the rate
at which the transition from pattern II to pattern III was
detected by exposure to Man–FITC–BSA (Figure 2B). These
data suggest that prior exposure of human spermatozoa to
D-mannose ‘primes’ or augments ligand binding when capacitated spermatozoa are subsequently exposed to Man–FITC–
BSA. The enhancing effect of the added monosaccharide on
the pattern III percentage was apparent even at the shortest
exposure time of 5 min (polyvalent mannose-ligand alone,
range: 21.5–27.6% versus polyvalent mannose-ligand 1 mannose monosaccharide, range: 42.9–48.9%). However, to ensure
that all available mannose binding sites have the potential to
participate in this conversion, we prefer a 15 min labelling
incubation (see Figure 2A).
Irrespective of whether or not the buffers included
D-mannose, the pattern II to pattern III transition was complete
by 2 h of incubation. Importantly, the addition of D-mannose
Figure 4. Examination of the ability of different monosaccharides
to prime the acrosome reaction initiated by polyvalent mannose
ligands. The results of analysis of specimens from four fertile
donors are presented. At all time points, there is a significant
difference between the three conditions (no monosaccharide added
versus 1D-galactose versus 1D-mannose, range of P values:
0.0004–0.0055) on the percentage of spermatozoa binding
fluorescein isothiocyanate-conjugated mannosylated bovine serum
albumin (Man–FITC–BSA) in pattern III as a fraction of total
binding (patterns II 1 III combined). No difference was detected
between no monosaccharide added versus 1D-galactose
(P: 0.0095–0.566, not significant). In contrast, the addition of
D-mannose dramatically increased the subpopulation (no
monosaccharide added versus 1D-mannose, P: 0.0069–0.0407;
1D-galactose versus 1D-mannose, P: 0.0051–0.0267).
to Man–FITC–BSA did not increase the final percentage of
spermatozoa exhibiting an acrosome reaction over that
observed with polyvalent Man–FITC–BSA alone (Figure 2B).
This supports other experimental data indicating that only a
small subpopulation of human spermatozoa are able to undergo
an acrosome reaction induced by zona ligands (Benoff et al.,
1995b, 1996).
Freshly isolated spermatozoa could not be stimulated to
undergo the pattern II to pattern III transition by exposure to
Man–FITC–BSA 1 D-mannose, nor did such exposure increase
the level of acrosome loss in that small percentage of motile
spermatozoa capable of binding Man–FITC–BSA immediately
after a 1 h swim-up. There was no difference between results
obtained using aliquots surface labelled for only 5 min and
duplicate aliquots labelled up to 2 h (control versus 1
D-mannose at 5 min respectively, 6.0 6 1.96% versus 4.71 6
1.51%; at 2 h respectively, 5.86 6 1.02% versus 6.09 6 1.73%).
These findings provide further support for the hypothesis that
the ability to undergo an induced acrosome reaction is acquired
during capacitating incubations.
To show that the D-mannose could stimulate acrosomal
exocytosis triggered by another polyvalent species capable of
cross-linking mannose lectins, the effect of D-mannose on the
topology of anti-HMR antibody binding was determined. All
aliquots were exposed to anti-HMR antibodies under standard
incubation conditions. Typical results are presented in Figure 3.
As observed for Man–FITC–BSA binding, the inclusion of
D-mannose in the pre-wash and labelling buffers had no effect
on the total percentages of spermatozoa exhibiting headdirected anti-HMR antibody binding (patterns II 1 III combined; Figure 3A). The addition of D-mannose to anti-HMR
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S.Benoff et al.
Figure 5. The density of fluorescein isothiocyanate-conjugated mannosylated bovine serum albumin (Man–FITC–BSA) binding on the
human sperm head examined by spectrofluorometry: (A) The determination of the fluorescence intensities of serial 1:2 dilutions of motile
spermatozoa from fertile donors (n 5 6) surface labelled with Man–FITC–BSA indicates that a linear relationship between cell number and
relative spectrofluorometric deflection of both freshly isolated, untreated human spermatozoa (Pearson correlation coefficient, r 5 0.998, P
,0.0001) and duplicate aliquots subjected to capacitating conditions (Pearson correlation coefficient, r 5 0.949, P ,0.0038). Furthermore,
spectrofluorometry readily illustrates the increase in Man–FITC–BSA binding which occurs following capacitation. (B) Unlabelled incubated
sperm and incubated sperm surface labelled with Man–FITC–BSA from fertile donors (n 5 6) were mixed to give varying percentages of
spermatozoa binding Man–FITC–BSA (patterns II 1 III) in a total number of 0.253106 spermatozoa. A linear relationship was detected
(Pearson correlation coefficient, r 5 0.937, P ,0.006). (C) Spectrofluorometric analysis of motile sperm from fertile donors (n 5 3) surface
labelled with Man–FITC–BSA in the presence of D-mannose versus control aliquots labelled with Man–FITC–BSA in the absence of added
monosaccharide indicates that the fluorescence intensity of monosaccharide-treated aliquots is increased by an average of 48.5 6 3.5% (P
,0.0089).
antibodies stimulated the conversion of pattern II to pattern
III binding (Figure 3B), again as observed with Man–FITC–
BSA. A simultaneous increase in acrosomal exocytosis was
also detected in specimens double-labelled with RITC-PSA
(not shown).
To examine whether monosaccharides other than D-mannose
could prime or stimulate the acrosome reaction induced by
polyvalent mannose ligands, aliquots of motile capacitated
spermatozoa populations from fertile donors (n 5 4) were
preincubated in buffers supplemented with D-galactose or
D-mannose. Substitution of D-galactose for D-mannose did not
832
alter the osmolality (390 mOsm) of the buffer. Preincubations
proceeded for 5 min, 15 min or 1 h prior to a 15 min
surface labelling with Man–FITC–BSA. Control aliquots were
incubated in Ca21-supplemented core buffer without any
added monosaccharide (Figure 4). Pre-exposure of human
spermatozoa to D-galactose did not change the percentage of
spermatozoa exhibiting head-directed Man–FITC–BSA binding (patterns II 1 III combined), confirming prior observations
(Benoff et al., 1993d). More importantly, exposure of incubated
spermatozoa to D-galactose had no effect on the distribution
of mannose-ligand binding sites nor did it affect acrosome
Induction of human sperm acrosome reaction
status. There was no stimulation of the transition of pattern II
to pattern III Man–FITC–BSA binding and there was no
increase in loss of acrosome content.
The human sperm acrosome reaction has been reported to
be influenced by the osmolality of the external medium, and
hyper-osmolal conditions (.449 mOsm) can inhibit ionophoreinduced acrosome loss (Bielfeld et al., 1993). However, the
osmolality of the D-galactose-containing buffer cannot account
for the failure of D-galactose to stimulate acrosome loss initiated
by mannose-containing neoglycoprotein binding. These data
indicate that the priming effect upon acrosomal exocytosis
resulting from pre-exposure of spermatozoa to monosaccharides is specific to the structure of the sugar.
Allosteric interactions between mannose carbohydrate recognition domains contribute to acrosome
loss
That polyvalent anti-HMR antibodies could more rapidly crosslink mannose lectins in the presence of D-mannose suggested
that the number of antigenic epitopes on the sperm surface
were increased by sugar exposure. In addition, we noted that
the intensity of sperm surface labelling with Man–FITC–BSA
was markedly increased when D-mannose was added to the
buffers employed in the pre-wash and labelling reactions,
confirming our prior observations (Benoff et al., 1993c,d). To
determine if these effects were due to an increase in mannose
binding sites, we have assayed the intensity of fluorescence of
Man–FITC–BSA molecules on the human sperm head by
spectrofluorometry (Figure 5).
Two sets of preliminary experiments were performed as
controls. In the first set, the relative spectrofluorometric
deflection produced by 1:2 serial dilutions of Man–FITC–BSA
surface label on fresh and incubated motile sperm populations
from fertile donors (n 5 6) was determined. This indicated
that the reagents employed are in the concentration range
where fluorescence intensity is directly proportional to occupied
binding site concentrations, and readily demonstrated the
increase in mannose-ligand binding which occurs following
capacitation (Figure 5A). No apparent artifacts are detected
by varying the number of cells analysed (Figure 5A).
In the second control series, unlabelled sperm populations
were mixed with Man–FITC–BSA surface labelled populations
up to a combined total number of 0.253106 spermatozoa. The
resultant percentage of spermatozoa exhibiting head-directed
Man–FITC–BSA binding was determined by microscopic
inspection. In all samples tested (n 5 6), fluorescence emission
was proportional to the percentage of spermatozoa binding
Man–FITC–BSA as determined by microscopic inspection of
stained slides (Figure 5B).
This assay was then employed to examine the relative
fluorescence intensity of incubated sperm surface labelled with
Man–FITC–BSA in the presence of D-mannose versus control
aliquots (n 5 3) (Figure 5C). The fluorescence emission of
D-mannose-treated aliquots increased by an average of 48.5 6
3.5% at all cell concentrations tested. Thus, in the presence of
non-competing levels of D-mannose which leaves unchanged
the total number of spermatozoa binding Man–FITC–BSA on
their surface, the number of Man–FITC–BSA molecules bound
by individual spermatozoa is increased. This increased fluorescence emission detected fluorometrically supports the notion
that allosteric interactions are occurring between multiple
mannose binding sites on the same heterodimeric sperm surface
lectin, making more binding sites on each spermatozoon
accessible to polyvalent mannose ligands.
Discussion
The ESHRE Andrology Special Interest Group (1996) recently
reviewed four areas of advanced diagnostic andrology previously referred to as ‘research tests’. The recommendations for
standardization of sperm function testing are of particular
relevance to the current study. Consensus points included
elimination of the analysis of the spontaneous acrosome
reaction as a direct diagnostic test or in the zona-free hamster
oocyte penetration test. Rather, it was concluded that both
clinical tests be performed with sperm population induced to
acrosome react with the ionophore A23187. However, because
ionophores bypass membrane mechanisms which regulate
calcium influx into spermatozoa (e.g. Goodwin et al., 1997),
false positive results can often be obtained (Benoff et al., 1996).
During this ESHRE meeting, Tesarik suggested that recombinant human zona pellucida glycoproteins (rhuZP) will serve
as agonist in the ‘perfect acrosome reaction test’ (ESHRE
Andrology Special Interest Group, 1996). After prolonged
incubation the unglycosylated polypeptide backbone of rhuZP
synthesized in a coupled in-vitro transcription/translation system is capable of inducing the human sperm acrosome reaction
(Whitmarsh et al., 1996). However, glycosylation of rhuZP
plays an important role in induction of the acrosome reaction
(Barratt et al., 1994; Van Duin et al., 1994) and rhuZP
synthesized in cell lines capable of fully glycosylating the
expressed protein rapidly induces high levels of acrosome loss
(Brewis et al., 1996). Large scale production of a biologically
active rhuZP, required for routine use in clinical assessment
of the ability of spermatozoa to undergo an acrosome reaction,
is not currently feasible (Whitmarsh et al., 1996). In this
report we demonstrate that polyvalent mannose ligands and
monosaccharide mannose serve as a cost effective alternative
for rhuZP that can be readily and immediately applied by
many laboratories.
It is now generally agreed that the acrosome reaction is a
receptor-mediated event (ESHRE Andrology Special Interest
Group, 1996). It is therefore significant that the structure of
the human sperm mannose-ligand receptor has at least two
features critical for its interaction with the zona pellucida and
induction of the acrosome reaction. First, the active site of the
human sperm mannose-ligand receptor is a C-type lectin,
sharing a common sugar recognition motif with a large family
of similar molecules (Drickamer, 1989). Ca21-dependent mannose-specific lectins of human somatic cells have been reported
to be ‘core-specific’, i.e. recognizing not only the terminal sugar
but also those within the core region of the oligosaccharide
(Summerfield and Taylor, 1986). These observations suggest
that mannose need not be the terminal residue of zona
glycoconjugates for sperm mannose lectins to serve as zona
receptors. Second, both components of the human sperm
833
S.Benoff et al.
mannose-ligand receptor dimer contain two carbohydrate
recognition domains. This duplication suggested that mechanisms for sperm recognition of zona pellucida mannose residues
rely on cooperative interactions between multiple mannose
recognition domains, which individually have a weak affinity
for monosaccharides. Competition experiments have demonstrated that the human sperm mannose receptor has only low
affinity for D-mannose monosaccharide. A 10 000-fold excess
monosaccharide as compared with D-mannose linked to BSA
was required to achieve equivalent (.70%) inhibition of Man–
FITC–BSA binding (Benoff et al., 1993d). Similar findings
have been reported for other animal lectins and their corresponding ligands (e.g. Lee and Lee, 1987; Lee et al., 1992;
Taylor et al., 1992; Taylor and Drickamer, 1993) and for the
purification of the lectins by affinity chromatography (Lennartz
et al., 1987). These structural features were the background
for our experiments.
The present study was prompted by our observation that
exposure of capacitated motile sperm populations from fertile
donors to polyvalent mannose ligands and D-mannose for
only 15 min markedly increased the relative percentages of
spermatozoa binding Man–FITC–BSA in pattern III (Benoff
et al., 1993c,d). As observed in animal models (Leyton and
Saling, 1989; Beebe et al., 1992), ligand-initiated receptor
aggregation appears to be an early step in the induction of the
human sperm acrosome reaction. In our fertile donors, there
was a 1:1 concordance between sperm binding Man–FITC–
BSA in the equatorial/post-acrosomal segment of the head
(pattern III) and spermatozoa which had undergone acrosomal
loss. This direct correspondance had hitherto been implied
rather than demonstrated in our previous publications (Benoff
et al., 1993c,d). Clearance of the acrosomal plasma membrane
of integral membrane proteins facilitates acrosomal exocytosis.
However, in the spermatozoa from infertile men where Ca21
influx into spermatozoa is blocked by anti-hypertension medications, we consistently find that pattern III Man–FITC–BSA
binding occurs on acrosome-intact spermatozoa, i.e. in the
absence of an acrosome reaction (Benoff et al., 1994b, 1995a;
Hershlag et al., 1995; Goodwin et al., 1997). These data
indicate that Ca21 influx (Thomas and Meizel, 1988) is not
obligatory for mannose receptor movement between contiguous
regions of the plasma membrane. These observations also
demonstrate that mannose lectin translocation seems to be
required for the induction of an acrosome reaction, but does
not seem to be the only determinant.
We report that binding of polyvalent mannose ligands
appears to induce an allosteric change in either the mannose
lectin or in some super-molecular complex of which mannose
lectin forms a part (e.g. with the non-nuclear progesterone
receptor; Benoff et al., 1995b). We also report that the
monosaccharide and the polyvalent form of mannose together
increase the rate at which the conformational change occurs
in mannose lectins on the sperm surface. In either case, the
allosteric change exposes additional mannose binding sites.
Similar observations have been made in related systems
(Lee and Lee, 1987; Taylor et al., 1992; Weis et al.,
1992). Interaction between multiple carbohydrate recognition
domains markedly contributes to high affinity binding of
834
polyvalent ligands to other mammalian mannose-specific
lectins (Lee et al., 1992; Taylor et al., 1992). These
observations support our conjecture based on partial primary
sequences of the mannose receptor that binding of mannose
by one carbohydrate recognition domain results in a change
in the accessibility of the remaining carbohydrate recognition
domains to external ligands. More importantly, this mechanism is consistent with data on the kinetics of the human
sperm acrosome reaction induced by increasing doses of
solublized human zona pellucida glycoproteins (Henkel et al.,
1995) wherein a sigmoidal curve was obtained. The shape
of the curve provides evidence of cooperativity and indicates
that human spermatozoa must bind multiple zona ligands in
order for exocytosis to occur.
The acrosome reaction stimulated by mannose-containing
neoglycoproteins shares five additional characteristics with
the zona pellucida-induced acrosome reaction: (i) induction
of the acrosome reaction by polyvalent mannose ligands
requires prior capacitation of sperm populations. Human
spermatozoa acquire the ability to acrosome react in response
to intact or solubilized human zonae after 6–8 h capacitation
in vitro (Overstreet and Hembree, 1976; Hoshi et al., 1993).
Mannose lectins appear on the sperm surface with a similar
time course (Benoff et al., 1993b); (ii) preliminary studies
suggest that after mannose receptors have redistributed to
the equatorial/post-acrosomal region of the sperm head,
signal transduction proceeds via activation of protein
phosphokinases (Benoff, 1997; A.Jacob and S.Benoff, unpublished observations) and Ca21 influx through a voltagedependent Ca21 channel (Goodwin et al., 1997; Jacob et al.,
1997). Interaction at fertilization of a human sperm tyrosine
kinase with the zona pellucida has previously been reported
(Burks et al., 1995) and activation of voltage-dependent
Ca21 channels is required for zona pellucida-induced
acrosomal exocytosis (Florman et al., 1992; Florman, 1994);
(iii) the time course of the mannose-initiated acrosome
reaction, wherein maximum percentages of acrosomal
exocytosis are observed by 2 h of incubation, is consistent
with ultrastructural reports of acrosome loss and sperm
penetration into intact zonae (McMaster et al., 1978;
Sathananthan et al., 1982; Chen and Sathananthan, 1986;
Cross et al., 1988; Liu and Baker, 1994a); (iv) we have
shared our protocols for oligosaccharide-stimulated induction
of the acrosome reaction with others who independently
have confirmed these results (Amin et al., 1995). Stages in
the zona pellucida-mediated acrosome reaction may be
monitored using a chlorotetracycline (CTC) fluorescence
assay (Lee et al., 1987; Kligman et al., 1991). The CTC
and mannose methods were compared and found to produce
equivalent results: both loss of CTC fluorescence over the
sperm head and the pattern II to pattern III Man–FITC–
BSA binding transition reflect the formation of hybrid
vesicles of the plasma and outer acrosomal membranes,
normally observed by electron microscopy (e.g. Chen and
Sathananthan, 1986); (v) the maximum percentages of
spermatozoa undergoing acrosome loss following mannose
challenge are similar to those obtained with solublized zonae
Induction of human sperm acrosome reaction
(e.g. 11–24%; Cross et al., 1988; Bielfeld et al., 1994; Liu
and Baker, 1996a).
In all aspects tested, the mannose-initiated acrosome
reaction of human spermatozoa mimics the acrosome reaction
induced by authentic zonae pellucidae. In the accompanying
report, we demonstrate in prospective analysis that our new
protocols will be of value for routine use to screen patients
prior to an IVF cycle in order to select appropriate treatment
(conventional insemination versus intracytoplasmic sperm
injection (ICSI) (Benoff et al., 1997). We have defined a
measure which allows discrimination between specimens of
reduced or normal fertility even when ,15% of spermatozoa
can be induced to acrosome react and which is independent
of sperm morphology (Benoff et al., 1997). We conclude
that the methods reported in this paper can be used to
better define the timed sequence of receptor regulated events
leading to acrosome exocytosis.
Acknowledgements
Appreciation is expressed to Bayard T. Storey for stimulating
discussions and encouragement, to Barbara Napolitano and Nina
Kohn for additional statistical input, and to James W.Overstreet,
Leslie O.Goodwin and Asha Jacob for critical reading of the
manuscript. We also acknowledge Michele Barcia and Heather
M.Bennett for their technical assistance.
Supported in part by National Institutes of Health Grant No.
ES06100 to S.B.
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Received on February 14, 1997; accepted on July 27, 1997
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