Journal of Andrology, Vol. 16, No.

1, January/February 1995
Copyright @ Amencan Society of Andrology

Expression of Mannose-Binding Sites on Human

Spermatozoa and Their Role in Sperm-Zona Pellucida Binding

JITH-SHYAN CHEN, GUSTAVO F. DONCEL,t CRISTINA ALVAREZ,t

AND ANIBAL A. ACOSTAt

From the *Depa?fnt of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan,
R.O.C.; tThe Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia
Medical School, Norfolk, Virginia; and the Hospital Universitari “La Fe” H. Maternal, Servicio de Reproduccion,
Avd. Campanar 21, 46009 Valencia, Spain.

ABSTRACT: A D-mannosylated
albumin (DMA) neoglycoprotein was ing capacitation, appearing on an average of 20% of the sperm after
assessed to validate experimentally
a probe capable of detecting overnight incubation. They also increased, especially the bar pattern,
mannose-binding sperm receptors involved in human sperm-egg following calcium ionophore treatment. Nearly all of methanol-fixed
interaction. DMA specifically blocked zona binding of swim-up human spermatozoa displayed the fluorescent label at the head level. Con-
spermatozoa in a concentration-dependent manner. While no con- comitant assessment of sperm membrane integrity and DMA fluo-
siderable effect was observed on sperm-zona initial contact, almost rescent patterns revealed that DMA fluorescence coincided mostly
50% of spermatozoa bound to the zona during a 2-hour period with permeabilized or altered sperm plasma membrane. In condu-
detached from it when DMA was introduced in the incubation me- sion, DMA is a suitable probe to identify human sperm mannose-
dium. DMA inhibition was evident when 10% fetal bovine serum, but binding sites crucially involved in sperm-zona interaction. These sites
not 3.5% human serum albumin (HSA), was used as Ham’s FlO appear to require free calcium concentrations to operate, and their
medium supplementation. This may be due to the amount of free expression changes with capacitation and acrosome reaction. Pre-
calcium in the medium since addition of 40 mM CaCI2 to F10-HSA cise location on human spermatozoa, however, warrants further
restored DMA inhibition. Furthermore, the higher the calcium con- investigation.
centration in the incubation buffer, the greater the DMA blockage of Key words: Sperm receptors, sperm-egg interaction, carbohy-
sperm-zona binding. Unfixed sperm presented fluorescent DMA la- drate-binding sites, sperm capacitation, acrosome reaction.
bel over the entire acrosomal area (cap pattern), or concentrated at J Androl I 995;16:55-63
the equatorial segment (bar pattern). These patterns increased dur-

rtilization is a complex process during which the added to the coincubation medium (Ahuja, 1982; Shalgi
spermatozoon undergoes a cascade of events prior to et al, 1986; Oehninger et al, 1991). Zona-binding sperm
its eventual fusion with the oocyte plasma membrane. proteins may act as lectins or enzymes. Several macro-
One crucial step in this process is the recognition and molecules such as galactosyltransferase (Macek and Shur,
interaction between complementary molecules present on 1988), trypsin-like protease (Saling, 1981), sialytransfer-
the sperm and the zona pellucida (ZP). In different mam- ase (Dun et al, 1977), and fucosyltransferase (Ram et al,
malian species, it has been demonstrated that sperm sur- 1989) were identified in mouse spermatozoa. A novel a-D-
face carbohydrate-binding proteins mediate gamete rec- mannosidase, with different characteristics from other
ognition by binding to complex glycoconjugates of the ZP mannosidases previously described, e.g., acrosomal and
(Macek and Shur, 1988; O’Rand et al, 1988). Addition- hepatic, was isolated from rat sperm plasma membranes
ally, numerous studies have reported that some carbo- and characterized (Tulsiani et al, 1989). All these proteins
hydrates competitively inhibit sperm-zona binding when have been suggested to be involved in some aspect of
sperm-egg recognition.
Mannosidase activity was also found in human sperm
Correspondence to: Dr. Gustavo F. Doncel, The Jones Institute for plasma membranes (Tulsiani et al, 1990). Furthermore,
Reproductive Medicine, Department of Obstetrics and Gynecology, it was reported that pretreatment of human spermatozoa
Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia
with the monosaccharide r-mannose inhibited sperm
23507.
Received for publication December 20, 1994; accepted for publication penetration through the zona (Mon et al, 1989).
August 26, 1994. From a clinical standpoint, expression of mannose-

55
56 Journal of Andrology . Januaiy/Februaiy 1995

binding sites on human spermatozoa has been recently related control. In some experiments, in order to determine the
correlated with sperm fertilizing potential (Tesarik et al, effect of DMA on the zona, hemizonae were preincubated with
1991; Benoff et al, 1 993a). Human spermatozoa that fail DMA at 300 ig/ml, washed, and finally coincubated with sper-
matozoa for 4 hours.
to fertilize mature oocytes under in vitro fertilization (IVF)
To establish
the role of Ca2 in DMA inhibition of sperm-
conditions may lack zona receptors or bear defective ones.
zona binding, various media were used including Ham’s FlO +
Aiming to detect these alterations on subfertile sperm, we
3.5% HSA, Ham’s FlU + 3.5% HSA + 40 mM CaC12, and
have studied the involvement of several molecules in
MgCl/Hepes buffer (containing 30 mM Hepes, pH 7.0, 150 mM
sperm-zona interactions (Doncel et al, 1993). The main NaC1, 10 mg/mi bovine serum albumin [BSA], and 0.5 mM
goal of the present study was to validate experimentally MgCI2) + 2 or 20 mM Cad2.
a probe capable of detecting mannose-binding sperm re- Short time (15-minute) incubations of spermatozoa with
ceptors involved in human sperm-egg interaction. hemizonae were employed to assess DMA effect on gamete initial
contact. To study DMA action on gamete secondary binding,
sperm and zonae were allowed to interact for 2 hours in Ham’s
Materials and Methods Fl 0+ 3.5% HSA. Then, hemizonae were washed and transferred
to droplets of sperm-free MgCl/Hepes buffer with 20 mM CaC12
Source of Spermatozoa (Ca2 buffer) (control) or the same Ca2 buffer containing DMA
at 200 g/ml (test). ci-Glucosamide-albumin was also used as a
Semen samples were provided by healthy proven-fertile donors.
nonspecific control in the latter experiment.
After liquefaction, each semen specimen was diluted with an
equal volume of Ham’s FlO medium (GIBCO, Grand Island,
Sperm Motion Parameters
New York) supplemented with 3.5% human serum albumin
(HSA) (Sigma Chemical Co., St. Louis, Missouri) or 10% v/v Sperm aliquots were incubated for 2 and 4 hours in supple-
fetal bovine serum (FBS) (GIBCO). Sperm were washed twice mented Ham’s FlO or Ca2 buffer containing DMA at 300 g/
by centrifugation at 400 x g for 7 minutes and 5 minutes, re- ml, and sperm motion parameters were assessed with the help
spectively to remove seminal plasma. The resulting pellet was of a computer-assisted semen analyzer (Cell Soft#{174},Labsoft Di-
overlaid with 0.3 ml Ham’s FlO containing 3.5% HSA or 10% vision of Cryo Resources, Ltd., New York, New York). Motility
under 5% C02, and the motile
v/v FBS for 1 hour at 37#{176}C fraction was visually checked on all control and test droplets from the
swimming up in the supernatant was retrieved. The samples used binding experiments.
in any given experiment, e.g., zona binding or fluorescent lo-
calization, belong to different donors. The number of donors/ Acrosomal Status
samples (n) employed per experiment is indicated in the cor- Spermatozoa were incubated for 5 or 18 hours in Ham’s Fl 0 +
responding figures or tables. 3.5% HSA at 37#{176}C
under 5% CO2 in water-saturated air, cen-
trifuged out of the medium, and then resuspended and incubated
Sperm-Zona Binding Assay for 90 minutes in Ca2 buffer containing 200 or 400 ag/mI DMA,
Swim-up sperm from fertile donors were preincubated for 30 400 Mg/mI DMA with 100 mM D-mannose, or 10 MM calcium
mm in 1 00-id droplets of Ham’s Fl 0 + 10% v/v FBS (control) ionophore A23 187 (10 mM stock solution in dimethyl sulfoxide
or medium containing different concentrations (100, 200, and diluted 1:1,000 in Ca2 buffer; Calbiochem, La Jolla, California).
300 ag/mi) of n-mannosylated albumin (DMA) (Sigma Chemical After the incubations, sperm were washed, spotted on glass slides,
Co.) (test). Subsequently, the hemizona assay was used to de- and fixed with methanol. Their acrosomal status was assessed

termine potential inhibition of sperm-zona binding (Burkman

using fluoresceinated Pisum sativum agglutinin (FITC-PSA; Sig-
et al, 1988). Briefly, salt-stored prophase I human oocytes from ma Chemical Co.) according to the method described by Cross
an IVF program were microbisected and matching hemizonae and co-workers (1986). Since PSA affinity for a-o-mannosyl res-
idues of glycoproteins could interfere with PSA determination
incubated for 4 hours in control or test droplets containing 5 x
l0 motile sperm/mi. One zona was used per sperm sample, i.e., of acrosomal status in experiments containing DMA, additional

one zona for each experimental replicate. After the incubation experiments were run to rule out this possibility. Five sperm
period, hemizonae were washed extensively, and sperm tightly samples from different donors were stimulated with 10 iM

bound to the outer surface of the ZP were counted. The hemizona A23 187 and then incubated with Ca2 buffer (control) or Ca2
index (HZ!) was obtained as follows: buffer containing 200 Mg/mI DMA (test). Acrosomal status was
assessed using FITC-PSA. No differences could be found in pat-
terns or percentages of acrosome-reacted sperm between control
= no. of sperm bound in test sample 100
no. of sperm bound in control sample and test samples (43.2 ± 1.6 and 42.8 ± 1.1, respectively).
Experiments were also carried out in which DMA was washed
Ruorescent Localization of Sperm
off after the 30-minute preincubation. D-Glucosamide-albumrn
DMA-Binding Sites
(Sigma Chemical Co.) at 300 g/ml was used as nonspecific
To localize the sperm DMA-binding
sites, a fluoresceinated DMA
(FITC-DMA) (Sigma no. A7790) was used. Sperm were studied
§ Concentrations given in percentages are w/v unless otherwise indi- immediately after swim up (Ti) or incubated in supplemented
cated. This does not apply for gases, which are all expressed in % v/v. Ham’s FlO 5% CO2 for 5 (T6) or 17 (T18) hours.
at 37#{176}C, At
chen et al . Mannose-Binding Sites on Human Sperm 57

fresh-mounted in a drop of regular buffer (not antiquenching)

150
and observed immediately. DMA fluorescent patterns were dif-
ferentially identified on motile or immotile spermatozoa. To
confirm these results, sperm were simultaneously incubated with
FITC-DMA as described above, and a supravital stain, Hoechst
33258 (Sigma Chemical Co.), at I Mg/mi (Cross et al, 1986). On
every spermatozoon analyzed, the uptake of each marker was
‘-4
evaluated using the appropriate fluorescence filters. A sequential
combination ofFITC-DMA labeling and a hypoosmotic
sweffing
test (HOST) was also performed. Briefly,afterstaining
the sperm
with FITC-DMA, they were washed and resuspended. A 100-pl
aliquot was then transferred to 1 ml of hypoosmotic medium,
containing 7.35 g sodium citrate and 13.51 g fructose in 1 L of
too 200 300 3000 300G 300HZ
distilled water (Jeyendran et al, 1984), and incubated at 37#{176}C
and 5% CO2 for 1 hour. Finally, fluorescent patterns were dif-
0-mannosyl at ed albumin ( uq/mL)
ferentially recorded on spermatozoa with or without curled tail.
FIG. I. Inhibition of human sperm-zone binding by DMA. Ham’s FlO As described, “coiled” tails would be indicative of intact sperm
+ 10% v/v FBS was used. Test sperm (n = 4/mean) were preancubated membranes.
with DMA for 30 minutes and then further coincubated with the hemizona
for 4 hours. 300 W: DMA (300 pg/mI) was washed off prior to sperm-
zona coincubation. 300 G: 300 pg/mI of D-glycoslated albumin was used
Statistical Analysis
Instead of DMA. 300 HZ: Hem,zonae were preincubated with DMA (300 In Figure 1, comparison among the inhibitory effect (HZIs) of
pg/mi), then washed, and finally coincubated with sperm. HZls for 100, various DMA concentrations (100,200, and 300) was done using
200, and 300 pg/mI are statistically different (P = 0.003). HZIs of 300W,
the ANOVA test. For comparisons between 300 W or 300 G,
300 G, and 300 HZ are statistically different from 300 HZI (all P = 0.02).
or 300 HZ and 300, a Mann-Whitney test was used. This test
was also used to compare appropriate pairs in Figures 2-4 and
the end of incubation, sperm were washed twice in MgCI/Hepes Table 1. In Table 2, changes in percentages of fluorescent patterns
buffer + 20 mM CaCl2 (Ca2 buffer) and labeled with FITC- were analyzed using the ANOVA test. Selected pairs were then
DMA 200 Mg/mi for 15 minutes at 37#{176}C
under 5% CO2. Sub- compared using post-tests with the Bonferroni’s correction. The
sequently, sperm were washed twice with MgCI/Hepes buffer level of significance chosen for all tests was P 0.05.
without calcium, spotted onto glass slides, briefly air-dried and
mounted with antiquenching buffer. Some experiments were done
on methanol-fixed spermatozoa to determine probe recognition Results
of internal binding sites. Determination of fluorescent patterns
was performed at 1,000 x magnification using an epifluorescence Inhibition of Human Sperm-Zona Pellucida Binding
microscope (Nikon). At least 200 sperm per specimen were eval-
A D-mannosylated albumin (DMA) neoglycoprotein
uated.
blocked the tight binding of swim-up human spermatozoa
In competition experiments, spermatozoa
and were pretreated
to human zonae in a concentration-dependent manner
incubated with different concentrations mM, of r-mannose (50
(Fig. 1). The DMA was preincubated with test sperm for
100 mM, 200 mM, or 400 mM) and FITC-DMA (200 pg/mI).
To observe potential changes in the DMA-binding patterns 30 minutes and then kept in the sperm-zona coincubation
after the acrosome reaction, spermatozoa were treated with 10 medium during the entire interaction. When DMA was
pM Ca2 ionophore A23 187 for 1 hour at 37#{176}C,washed, and preincubated with sperm but washed off prior to sperm-
then labeled with FITC-DMA. Sperm acrosomal status was eval- zona coincubation, its inhibitory effect was lost, even when
uated either on a different aliquot or on the same sample using the maximum assayed concentration (300 pg/ml) was tried.
a double-labeling technique with rhodamine-conjugated PSA The same lack of activity was observed when, instead of
(TRITC-PSA, Sigma Chemical Co.). In this latter case, after the the sperm, the hemizonae were preincubated with DMA.
FITC-DMA step, sperm were washed, fixed with methanol, in-
In regard to specificity, a D-glycosylated albumin was
cubated with TRITC-PSA, again washed, and finally mounted
assayed under the same conditions that proved to be max-
with an antiquenching buffer (100 mg O-phenylenediamine in
imally inhibitory for DMA. No blocking activity could
10 ml phosphate-buffered saline + 90 ml glycerol, pH 8.6 with
carbonate/bicarbonate buffer). Observation was performed using
be detected in this case.
the appropriate filters for each fluorochrome. The above experiments were run employing Ham’s FlO
as medium supplemented with 10% v/v FBS. Interest-
Sperm Membrane Integrity ingly, when FBS was replaced by 3.5% HSA, the DMA
To assess sperm membrane integrity and mannose-binding sites inhibitory effect disappeared (Fig. 2). In order to verify
concomitantly, motility of the sperm samples was carefully pre- if, as demonstrated for mannose-binding receptors in oth-
served. Sperm were centrifuged only once per washing step, at er cell systems (Pontow et ai, 1992), free Ca2 was required
lower speed (290 x g) for shorter time (5 minutes). They were to facilitate ligand-receptor interaction, Ham’s FlO sup-
58 Journal of AndroJogy Janua,y/Februarj 1995

rio rio Duller Butter initial Secondary Secondary C

3.5% NSA 3.5% NSA 1.0% BSA 1.0% BSA
40mMCo 2Co 2OmMCa FIG. 3. DMA-mediated inhibition of inItial (30-minute) and secondary
(120-minute) sperm-zona Interactions. For testing initial binding, sperm
FIG. 2. Influence of Ca#{176}-
concentration on DMA-mediated sperm- were preincubated with DMA (200 pg/mI) and then further coincubated
zona binding Inhibition. Sperm were preincubated with DMA (200 pg/mI) with hemizonae for 30 mInutes. For secondary binding, sperm were coin-
for 30 minutes and further coincubated with hemizonae for 4 hours. cubated with hemizonae for 2 hours, then hemlzonae were transferred
Different medium supplements and Ca2* concentrations were used. HZI to a sperm-free medium containing DMA at 200 pg/mI. Secondary 6:
of FlO + 3.5% HSA (n = 10) Is statistically different (P = 0.007) from D-glycosylated albumIn was used Instead of DMA. All n =4; inItial or
HZI of 200 paJmI in FlO + 10% FBS (Fig. 1); the other three variants, n secondary 6 versus secondary (P = 0.05).
4; F10/HSA #{247}
40mM Ca#{176}versus F1O/HSA (P= 0.008); buffer/BSA,
2mM Ca#{176} versus 20mM Ca#{176} (P = 0.02). All buffer/medium containing
extra Ca2’ were significantly different from Fl 0/HSA without extra Ca#{176}. siderable inhibition was observed when the sperm-zona
interaction was mostly limited to the initial contact (Fig.
3). Conversely, almost 50% of spermatozoa bound to the
plemented with 3.5% HSA and 40 mM CaC12 was used. zona during the 2-hour incubation detached from itwhen
DMA-mediated sperm-zona binding inhibition was thus DMA was introduced in the incubation medium. This
recovered. The influence of Ca2 concentration on such effectwas not seen when n-glucosamide-albumin was em-
activity was confirmed by the utilization of a MgClIHepes ployed.
buffer with 2 or 20 mM CaCl2. As predicted, the higher
Ca2 concentration permitted a stronger DMA-induced Sperm Motion Parameters
inhibition. Alterations of sperm motility can be the cause of sperm-
As for the aforementioned experiments, we knew that zona binding impairment. To rule out this possibility,
DMA was able to impede sperm-zona interaction in a motility was visually checked on all control and test drop-
4-hour incubation. However, in order to ascertain wheth- lets from the binding experiments. No noticeable differ-
er this inhibition was primarily effected through the initial ences were detected. To support this finding in a more
sperm-zona binding or through secondary interactions, objective and detailed manner, swim-up spermatozoa were
the incubation time was modified. In an attempt to assess incubated with DMA for 2 and 4 hours at 37#{176}C
and var-
initial binding, only a 15-minute sperm-zona interaction ious motion parameters were assessed with a computer-
was allowed, whereas to study secondary binding, zonae assisted semen analyzer (CASA). No significant differ-
were incubated with sperm for 2 hours and then trans- ences were found comparing sperm samples with or with-
ferred to a sperm-free medium containing DMA. No con- out DMA at any time (Table 1).

Table 1. E ifect of n-mannos ylated albumin on motion parameters*

Time after
swim-up DMA in Velocity ALH Motility
(hours) medium (pm/second) Uneanty mean (pm) B/CF (Hz) (%)
0 - 104.6 ± 6.8 5.39 ± 0.68 4.47 ± 0.24 17.60 ± 0.31 94.6 ± 4.4
2 - 119.9 ± 17.1 4.67 ± 0.54 5.20 ± 1.14 18.01 ± 1.02 85.5 ± 8.7
+ 120.5 ± 17.2 4.80 ± 0.81 5.05 ± 0.76 17.21 ± 0.92 87.6 ± 7.5
4 - 107.3 ± 14.2 4.54 ± 0.68 4.80 ± 0.70 19.48 ± 0.82 81.5 ± 4.7
+ 98.9 ± 13.1 4.35 ± 0.68 4.81 ± 0.25 16.56 ± 0.13 82.6 ± 5.9
* Data represent mean ± SE, n = 3. Sperm were Incubated with DMA (200 pg/mI) for 2 and 4 hours. ALK: amplitude of lateral head displacement;
BICF: flagellar beatJcross frequency. No statistical differences between medium with or without DMA for any of the motion parameters.
Chen et al ‘ Mannose-Binding Sites on Human Sperm 59

DMA did not modify this result. Ca2 ionophore treat-

a ment (10 iM in Ca2 buffer) used as positive control
V
a substantially increased the percentage of acrosome-react-
V.,

ed sperm, both at 5 and 18 hours of incubation.

#{149}0
a,

V
C

Ruorescent Localization of DMA-Binding

E
Sites on Spermatozoa
0
To localize DMA-binding sites in unfixed swim-up sperm,
0

FITC-DMA was used. Two main fluorescent patterns were

seen at the head level (Fig. 5). One covering the entire
acrosomal area (cap pattern) and the other with the label
concentrated at the equatorial segment (bar pattern). Flu-
orescent labeling of neck andlor tail was present alone
Post -Swi m Up
and in combination with the described head patterns in
FIG. 4. Acrosomal status of sperm coincubated with DMA. Sperm (n some spermatozoa. Staining of the entire spermatozoon
=8 except for 400 with or without o-mannose, n =4) were preancubated or neck and tail only was considered nonspecific and
for 5 and 18 hours in Ham’s FlO + 3.5% HSA and further challenged
with Ca2 buffer 4, DMA 200 pg/mI 4, DMA 400 pg/mI 4, DMA 400 grouped as “others” in Table 2.
pg/mI plus 100 mM mannose 4, or A23187 10pM (tl for 90 minutes. The incidence of FITC-DMA head patterns, cap and
A231 87 is the only treatment statistically different from Ca2 buffer (P =
bar, increased with the incubation time of swim-up sperm
0.02).
samples of fertile donors (Table 2). After 5 hours of in-
cubation in Ham’s FlO + 3.5% HSA at 37#{176}C,13.1 ±
1.5% of the sperm were head-labeled, indicating a signif-
Acrosomal Status of Spermatozoa icant increase compared to aliquots studied right after
Incubated with DMA swim-up (6.4 ± 0.81%). Spermatozoa bearing these pat-
The sperm acrosome reaction is thought to be a receptor- terns represented around 20% of the sperm incubated for
mediated event whose main natural triggers are ZP pro- 18 hours under capacitating conditions. Negative sperm
teins, especially ZP3 (Wassarman, 1988; Saling, 1991). decreased while pattern “others” did not show any sig-
To determine the potential of DMA for inducing an ac- nificant changes.
rosome reaction, swim-up sperm were capacitated for 5 In an attempt to verify specificity of the head patterns,
or 18 hours in Ham’s FlO + 3.5% HSA at 37#{176}C,
and increasing concentrations of D-mannose (50-400 mM)
further challenged with Ca2 buffer containing 200 and were preincubated with the sperm and kept during the
400 cg/ml of DMA. The neoglycoprotein did not induce incubation with FITC-DMA (Table 2). Although head-
a statistically significant increase in the percentage of ac- labeled sperm diminished, no complete blockage could
rosome-reacted sperm when compared to corresponding be obtained. At 400 mM D-mannose, still 10% of the
controls (Fig. 4). Addition of 100 mM D-mannose during sperm were head positive.
preincubation and incubation of sperm with 400 ig/ml To study the location of DMA-binding sites after the

FIG. 5. Ruorescent localization of DMA-binding sites on spermatozoa. Unfixed sperm with the label (HTC-DMA) covering the entire acrosomal
area (cap pattern; Fig. 5b) or concentrated at the equatorial segment (bar pattern; Fig. 5d). Methanol-fixed membrane-permeabilized sperm show the
label in a disperse pattern all over the head with a brighter band at the equatorial segment (Fig. 5f). Neck and tail were also fluorescent. Corresponding
phase-contrast pictures (Fig. 5a,c,e) are included (sperm magnification: 600 x).
60 Journal of Andrology January/Februaty 1995

Table 2. Fluorescent localization of DMA-binding sites on spermatozoa’

Incubation
Fluorescent patterns (%)
time Mannose
(hours) (mM) n Bar Cap Negative Otherst Bar + Cap

1 None 14 1.8 ± 0.4 4.7 ± 0.5 89.0 ± 1.6 4.2 ± 0.8 6.4 ± 0.8
6 None 14 4.8 ± 0.8 8.3 ± 0.9 82.2 ± 1.9 4.7 ± 0.6 13.1 ± 1.5
50 2 5.6 ± 0.9 8.0 1.9
± 81.5 ± 2.7 4.9 ± 0.7 13.6 ± 2.8
100 8 3.9 ± 1.1 4.5 ± 0.8 89.2 ± 1.8 2.3 ± 0.4 8.4 ± 1.5
200 10 3.2 ± 0.6 5.1 ± 0.5 88.7 ± 0.9 3.0 ± 0.3 8.3 ± 0.8
400 6 3.5 ± 1.2 3.8 ± 0.9 90.3 ± 1.8 3.2 ± 0.8 7.3 ± 1.5
18 None 14 7.8 ± 1.1 11.5 ± 2.0 75.2 ± 4.1 5.5 ± 1.2 19.3 ± 3.1
50 2 5.7 ± 0.7 11.6 4.1
± 75.9 ± 3.3 6.9 ± 1.5 17.3 ± 4.7
100 8 4.7 ± 0.8 8.0 ± 1.7 84.0 ± 2.5 3.2 ± 0.6 12.8 ± 2.1
200 10 4.9 ± 0.9 6.6 ± 1.1 84.6 ± 2.2 3.8 ± 0.7 11.5 ± 1.7
400 6 5.7 ± 1.3 4.8 ± 0.9 87.4 ± 1.6 2.9 ± 0.6 10.5 ± 1.4
* Data represent mean ± SE. Sperm were labeled with FITC-DMA (200 pg/mI) in Ca2 buffer (20 mM Cad2) with or without D-mannose for 15
minutes 5 hours or 17 hours after swim-up (total incubation time: 1, 6, and 18 hours,
immediately, respectively). Spermatozoa were incubated in
Ham’s FlO + 3.5% HSA at 37’C, 5% Co2.
t Staining of neck and tall or the entire spermatozcon. Except for Others, the bar and the cap patterns significantly Increased their incidence along
the incubation time (Bar: P = 0.0001; Cap: P = 0.003; Negative: P = 0.004; Bar + Cap: P = 0.0003). The most significant changes occurred between
1 and 18 hours (P < 0.01). The addition of mannose produced some inhibition of FITC-DMA binding; however, it did not significantly change the
Incidence of the fluorescent patterns.

acrosome reaction has taken place, swim-up sperm were dence were changed when spermatozoa treated with Ca2
treated with Ca2 ionophore (10 NM), incubated with ionophore and then fixed were studied.
FITC-DMA, fixed, and finally stained with TRITC-PSA. Aware that plasma membrane integrity and, therefore,
This double labeling allowed for a sequential determi- sperm viability could be confounding factors for DMA-
nation of DMA patterns and acrosomal status on the same binding site location and incidence, experiments using
spermatozoon. As shown in Table 3, the bar pattern FITC-DMA and a positive identification of viable sperm
strongly associated with acrosome-reacted sperm. Such were run.
good correlation, however, could not be found between Capacitated sperm after 18 hours of incubation were
cap pattern and acrosome-intact sperm. Five percent of labeled with FITC-DMA while motility was carefully pre-
acrosome-intact and 10% of acrosome-reacted swim-up served. A fresh drop of the suspension was placed on a
spermatozoa treated with Ca2 ionophore bore the cap prewarmed slide, cover-slipped, and observed under an
pattern in double-staining experiments. In sperm samples epifluorescence microscope. Sperm head DMA-fluores-
labeled only with FITC-DMA, there was always an in- cent patterns could not be detected on any motile sperm
crease in both bar and cap patterns after the induction of (Table 4). Conversely, 14% of the immotile population
acrosome reaction with Ca2 ionophore. displayed these patterns.
To find out if this observation could be due to the In another set of experiments, a HOST, which assesses
recognition of an internal molecule by FITC-DMA, meth- plasma membrane integrity through the coiling of the tail
anol-fixed (membrane-permeabilized) sperm were stud- of sperm subjected to hypoosmotic medium, was run to-
ied. Almost all of them were fluorescent at the head level gether with the DMA fluorescence. Again, no head pat-
displaying a cap pattern with a brighter band at the equa- terns were observed on sperm bearing intact plasma mem-
torial segment (Fig. 5). Neither this pattern nor its mci- branes (coiled tails) (Table 4).

Table 3. Influence of acrosorne reaction (AR) on the fluorescent patterns of DMA-binding sites’

Ca2 buffer A 23187

DMA
AR AR
fluorescent
pattern Negative Positive Negative Positive
Bar 0.37 ± 0.22 2.61 ± 0.43 1.47 ± 0.35 36.60 ± 3.41
Cap 3.20 ± 0.37 1.38 ± 0.20 4.95 ± 0.73 9.87 ± 1.80
Negative 90.80 ± 0.41 1.27 ± 0.53 39.60 ± 3.31 3.43 ± 1.50
Others 0.32 ± 0.18 1.01 ± 0.35 1.47 ± 0.34 3.41 ± 0.53
* Data represent mean percentages ± SE, n = 6. Swim-up sperm were treated with Ca2 ionophore (10 pM) in Ca2 buffer or with buffer alone,
incubated with FITC-DMA (200 pg/mI) for 15 minutes, fixed, and stained with TRITC-PSA.
Chen et al . Mannose-Binding Sites on Human Sperm 61

Table 4. Influence of sperm membrane integrity on DMA- though only few oocytes were assessed, each of them with
fluorescent patterns’ low numbers of bound sperm.
Sperm Since ZP carbohydrates are presented to the sperm in
membrane a defined spatial arrangement, most likely in special clus-
DMA-fluoresce nt patterns (%)
ters, we chose to use the mannose coupled to a backbone
assessment Bar Cap Negative Others
of albumin in an attempt to resemble these conditions.
Motility In order to be inhibitory, the neoglycoprotein DMA
Motile 0.0 ± 0.0 0.0 ± 0.0 52.6 ± 5.7 0.0 ± 0.0 had to be present in the coincubation medium (Ham’s
Immotile 8.1 ± 0.5 6.1 ± 0.5 30.5 ± 5.7 2.7 ± 0.4
FlO + 10% v/v FBS). No activity was detected when
HOS test DMA was preincubated with either sperm or zonae and
Positive 0.6 ± 0.3 0.6 ± 0.2 57.1 ± 4.1 0.4 ± 0.2 then washed off. Mori and co-workers (1989) also con-
Negative 7.4 ± 1.1 5.8 ± 0.6 25.6 ± 3.7 2.5 ± 0.3
cluded that pretreatment of oocytes with D-mannose did
Hoechst 33258 not block penetration of untreated spermatozoa.
Negative 0.0 ± 0.0 0.0 ± 0.0 83.9 ± 1.4 0.0 ± 0.0 Surprisingly, when fetal serum was replaced by HSA as
Positive 6.1 ± 0.6 4.3 ± 0.4 0.4 ± 0.1 5.3 ± 0.7
medium supplement, the DMA inhibitory effect was lost.
* Data represent mean ± SE; motility, n = 8; HOS test, n = 6; Hoechst The possibility existed that the sperm mannose-binding
33258, n = 12. Sperm incubated under capacitating conditions for 18
sites would be similar to the mannose receptor of mac-
hours were labeled with FITC-DMA (200 pg/mI) for 15 minutes and flu-
orescent patterns were assessed. Concomitantly, sperm membrane per- rophages and hepatic endothelial cells, which binds mon-
meability was evaluated through motility, hypoosmotic swelling test osaccharides but displays much higher affinity for mul-
(HOST), or staining with Hoechst 33258.
tivalent oligosaccharides in the presence of free calcium
(Pontow et al, 1992; Taylor and Drickamer, 1993). There-
fore, to overcome the chelating effect of 3.5% HSA, 40
Finally, the supravital stain Hoechst 33258, a DNA mM CaCl2 was added to the medium. The percent binding
marker that cell membranes are impermeable to, was used was significantly reduced revealing a recovery of the DMA
concomitantly with FITC-DMA. No DMA head patterns inhibitory activity. To confirm this finding, a MgClJHepes
could be seen in Hoechst 33258-negative sperm (Table buffer with lower protein content that had been success-
4). fully utilized with the same probe was used (Benoff et al,
1 993a). Again, the higher the calcium concentration, the
greater the sperm-zona binding inhibition.
Discussion Alteration of sperm motility can be the cause of binding
impairment in sperm-zona binding assays. With this in
Several authors have demonstrated the crucial partici- mind, we checked motility of coincubation-remaining
pation of ZP glycoproteins and zona sperm receptors in sperm in all experiments herein reported. Moreover, mo-
the mammalian fertilization process (Yanagimachi, 1981; tion parameters were analyzed with a computerized sys-
Swenson and Dunbar, 1982; Wassarman, 1987; Saling, tem after various times of sperm-DMA incubation. No
1989). Yet, the nature of the sperm carbohydrate-binding significant changes were observed with respect to controls.
component remains elusive. To localize the mannose-binding sites on sperm, a flu-
In this work, we have shown evidence that a mannose- oresceinated DMA was used. The same probe had been
enriched neoglycoprotein (DMA) specificallyinhibits, in previously employed on human sperm (Tesarik et al, 1991;
a concentration-dependent manner, human sperm-zona Benoffet al, 1993a), but results concerning incidence and
binding. type of fluorescent patterns were not in agreement.
Mannose, as well as fucose, galactose, N-acetylglucos- In our experiments and in accordance with the binding
amine, and sialic acid are prominent constituents of stud- assay findings, when Ham’s Fl 0 + 3.5% HSA was used
ied mammalian ZPs (Shalgi et al, 1986; Wassannan, 1988; as medium for FITC-DMAJsperm incubation, the re-
Mon et al, 1989; Yurewicz et al, 1991; Tulsiani et al, sulting fluorescent patterns were expressed in a low per-
1992). D-Mannose proved to be an effective inhibitor of centage of the spermatozoa (data not shown). In addition,
sperm-zona binding in rats (Shalgi et al, 1986). In turn, fluorescence was faint and inconsistent. This could have
an a-i>mannosidase activity was demonstrated in rat and been the case in Tesarik’s experiments. Conversely, when
human sperm plasma membranes (Tulsiani et al, 1989, a buffer containing 20 mM calcium was used, a defined
1990). bar and cap patterns, similar to those observed by Benoff
Using various lectins with different specificities, Mori and co-workers, were seen at the sperm head level.
and co-workers (1989) reported the presence of D-man- Consistently with above-mentioned results, 100 mM
nose in human ZP. They also demonstrated a role for the D-mannose completely blocked sperm fluorescence when
monosaccharide in the sperm-zona binding process al- incorporated to FITC-DMA-containing Ham’s FlO.
62 Journal of Andrology January/Febrary 1995

However, only partial inhibition could be achieved when ules for mannose-binding receptors has been recently sug-
even higher concentrations of the monosaccharide were gested (Benoff’ et al, l993b). They would externalize dur-
used in an FITC-DMA calcium-supplemented buffer. ing capacitation in association with membrane cholesterol
Sperm-ZP interaction progresses through a cascade of efflux and spontaneous acrosomal loss. These sperm man-
events involving multiple binding steps. From our data nose receptors would be similar to those already described
changing the incubation time and conditions of the HZA, in human macrophages, which belong to calcium-depen-
it seems that the DMA probe would preferentially block dent animal lectins with C-type carbohydrate-recognition
secondary binding, that taking place after 2 hours of sperm- domains (Taylor et al, 1990).
zona coincubation. A similar probe where mannose is Although displaying different requirements and kinetics
replaced by glucosamine did not interfere with such bind- (Tulsiani et al, 1990) both human sperm acrosomal and
ing, endowing specificity to the DMA effect. This type of surface a-i>mannosidases are inhibited by D-mannose and
binding would involve sperm receptors relocated or ex- could well represent binding sites for DMA. The authors
posed during the acrosome reaction, which are responsible have clearly suggested that a-i>.mannosidase activity may
for binding to secondary ligands on the ZP, e.g., ZP2 (Bleil have a role in the interactions of mammalian gametes.
et al, 1988) during sperm penetration. DMA could block Another possibility, however, would be the existence
sperm penetration as well. In this regard, Mori et al (1993), of intra-acrosomal mannose-binding proteins with lim-
confirming earlier results, have recently reported that the ited surface expression (Tesarik et al, 1990). Proacrosin,
monosaccharide D-mannose inhibits zona penetration the zymogen form of the acrosomal protease acrosin, would
while it does not affect the spontaneous acrosome reaction fit within this category. It has already been described as
in human spermatozoa. a potential zona-ligand molecule on boar spermatozoa
Using sperm capacitated during 6 hours or overnight (Jones et al, 1988). Proacrosin has a definite requirement
incubation in Ham’s Fl 0 + 3.5% HSA and a rhodamine- for a polysaccharide structure or for “clustering” of sac-
conjugated PSA for acrosomal status assessment, we were charides on a protein backbone. It recognizes carbohy-
unable to detect any acrosome reaction-inducing effect of drate moieties of ZP glycoproteins as well as neoglyco-
DMA. Addition of 100 mM D-mannose to DMA-con- proteins like BSA-fucose and BSA-mannose.
taining calcium-supplemented challenging buffer neither As in our experiments with human sperm, fluorescein-
facilitated nor affected this fact. The same sperm popu- ated neoglycoproteins labeled 100% of permeabilized boar
lation typically responded to the calcium ionophore spermatozoa. Fluorescence and ultrastructural studies us-
A23 187. ing fucosylated probes revealed that, although some of
Mannose-binding sites were located at the sperm ac- these proteins were located on the plasma membrane
rosomal area in a disperse manner (cap pattern) or con- overlying the anterior tip of the sperm head, the number
centrated at the equatorial segment (bar pattern). As with of fucose-binding sites increased substantially after in-
other carbohydrate-binding sites and other better-char- duction of the acrosome reaction (Friess et al, 1987). This
acterized sperm antigens (Myles and Primakoff, 1984; could be an explanation for our results showing an un-
Saxena et al, 1986; Lopez and Shur, 1987; Cowan et al, expected increment in the incidence of cap pattern in the
1987), the bar pattern incidence increased after calcium sperm treated with Ca2 ionophore. Since the surface den-
ionophore treatment. Interestingly, the cap pattern also sity of receptors would be low, it is also understandable
increased after such treatment. that the DMA could not stimulate the acrosome reaction.
The possibility that acrosome-reacting sperm would al- Using sperm extracts from various mammals including
low the probe to enter and recognize cytoplasmic/acro- humans, and [‘25I]BSA-fucose and mannose, Jones (1989)
somal mannose-binding sites was tested using fixed sperm demonstrated that proacrosin is the major protein species
immediately after swim-up and after overnight incuba- recognized on western blots. The author proposed a hy-
tion. Almost all of them presented fluorescence at the head pothesis for sperm-egg interaction in mammals in which
level. proacrosin, released during early stages of the acrosome
Verification of plasma membrane integrity in experi- reaction, mediates secondary or consolidated binding of
ments using unfixed spermatozoa was then warranted even spermatozoa to the ZP by virtue of its carbohydrate-bind-
under the assumption that previous FITC-DMA local- ing capacity. Again our observation that DMA prefer-
ization experiments had been carried out with “good mo- entially blocks secondary binding would support this
tility” samples. No fluorescent head patterns could be mechanism in the human system.
detected on motile, or HOST-positive, or Hoechst 33258- From our experiments, it is clear that mannose-binding
negative sperm when techniques were combined to assess receptors on sperm are critically involved in human sperm!
DMA fluorescence and membrane integrity concomi- egg interaction. It would be important, however, to de-
tantly in overnight-incubated spermatozoa. termine their exact location to better understand the phys-
The existence of sperm subplasmalemmal storage mod- iological role they play.
Chen et al Mannose-Binding Sites on Human Sperm 63

Myles DO, Primakoff P. Localized surface antigens of guinea pig sperm

Acknowledgment migrate to new regions prior to fertilization. J Cell Biol 1984;99:
The authors express their appreciation to Ms. Pauline M. Clynes of the 1631-1641.
Jones Institute for Reproductive Medicine for her excellent editorial Oehninger 5, Clark OF, Acosta AA, Hodgen GD. Nature of the inhibitory
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