Molecular Human Reproduction vol.2 no.9 pp. 651-658, 1996
Lectins binding on human sperm surface increase membrane
permeability and stimulate acrosomal exocytosis
Bruno Lassalle1 and Jacques Testart
INSERM Unit 355 and Institut Paris-Sud sur les Cytokines (IPSC), 32 rue des Carnets 92141 Clamart, France
1
To whom correspondence should be addressed
Cross-linked complexes formed between certain lectins and their specific multivalent carbohydrates and
glycoconjugates on the sperm surface were studied for their ability to modify sperm membrane permeability
and to induce the acrosome reaction. Wheat germ agglutinin (WGA), concanavalin A (Con A) and peanut
agglutinin (PNA) increased the proportions of human spermatozoa permeable to the impermeable propidium
iodide (31.9 compared with 13.8%, 38.4 compared with 18.4% and 72.7 compared with 18.9% respectively).
Removal of sperm surface sialic acid by neuraminidase treatment was a prerequisite for Con A and PNA
binding to the sperm surface. The percentage of permeable and acrosome-reacted spermatozoa was not
affected by sperm treatment with 500 mlU/ml Arthrobacter ureafaciens neuraminidase. WGA did not induce
the acrosome reaction, whereas PNA induced the acrosome reaction regardless of the sperm capacitation
status, allowing the proportion of acrosome-reacted spermatozoa to reach 27.7% of capacitated spermatozoa.
However, the ability of Con A to induce the acrosome reaction was limited to uncapacitated spermatozoa.
To test the physiological relevance of this study, uncapacitated human spermatozoa were incubated with
human zonae pellucidae and the permeability of spermatozoa bound to the zona surface was analysed
according to the time post-insemination. Two-thirds of spermatozoa bound to zona pellucida became
permeable to propidium iodide in the first 30 min post-insemination and almost all bound spermatozoa
became permeable to the impermeable dye after 60 min. Our results show that molecular interactions
between human zona pellucida and sperm surface increase the permeability of sperm membranes; the crosslinked complexes formed by PNA lectin and its specific multivalent carbohydrates and glycoconjugates on
the sperm surface were also able to increase sperm membrane permeability and to induce the acrosome
reaction. These results suggest a role for the saccharide moieties of sperm surface glycoconjugates in the
induction of the acrosome reaction.
Key words: acrosome reaction/capacitation/lectins/peanut agglutinin/receptor aggregation
Introduction
The capacitation event is considered to involve a series of
cellular and molecular changes that prepare the spermatozoon
for an exocytotic process, the acrosome reaction (reviewed
by Langlais and Roberts, 1985; Sidhu and Guraya, 1989;
Yanagimachi, 1994). Sperm capacitation has been viewed as
a reversible phenomenon that results in a net decrease in
negative surface charge, an efflux of membrane cholesterol,
and an influx of calcium ions between the plasma and the
outer acrosomal membranes (reviewed by Langlais and
Roberts, 1985). Alteration of the cholesterol content of the
erythrocyte membrane leads to changes in membrane properties
including ion permeability and membrane fragility (reviewed
by Langlais and Roberts, 1985). The acrosome reaction is
characterized by sperm membrane vesiculation, fusion of the
plasma membrane with the outer acrosomal membrane and
release of the acrosomal content (reviewed by Sidhu and
Guraya, 1989; Yanagimachi, 1994).
Human spermatozoa, especially those with normal morphology and an intact acrosome, bind to the zona pellucida (Liu
and Baker, 1992, 1994) where the acrosome reaction occurs
© European Society for Human Reproduction and Embryology
(Cross et ai, 1988; Tesarik, 1989; Lee et ai, 1992), probably
induced by the human zona pellucida glycoprotein ZP3 (Barrett
et al., 1993; Van Duin et ai, 1994). In the mouse, acrosomal
exocytosis seems to be initiated by the aggregation of sperm
molecules recognized by ZP3 glycoproteins (Leyton and
Saling, 1989). Whereas intact and solubilized human zonae
pellucidae (Cross et ai, 1988; Bielfeld et ai, 1994) have
acrosome reaction-inducing activity, neither ZP3 glycopeptides
derived from pronase-digested ZP3 nor isolated oligosaccharides are able to induce the acrosome reaction (Florman et ai,
1984; Leyton and SaJing, 1989; Bunch et ai, 1992). However,
when ZP3 glycopeptides previously bound to the sperm surface
were cross-linked by anti-ZP3 antibodies, induction of the
acrosome reaction was elicited to the same extent as that
occurring following exposure to whole zona pellucida (Leyton
and Saling, 1989). Macek et al. (1991) hypothesized that ZP3
induces the acrosome reaction by virtue of multiple spermbinding oligosaccharides bound to a protein core, which
are able to cross-link sperm surface receptors. Moreover,
aggregation of sperm surface receptors in the absence of zona
pellucida components may induce the acrosome reaction, as
651
B.Lassalle and J.Testart
has been reported for aggregation of progesterone receptors in
human (Tesarik and Mendoza, 1993) sperm surface galactosyltransferase (Macek et al, 1991) and proteinase inhibitor in
mice (Aarons et al., 1992), or sperm plasma membrane antigen
in ram (McKinnon et al, 1991). Receptor aggregation in
response to biological stimuli has been described in other
systems as an important mechanism for initiating signal transduction across the membrane (Schechter et al, 1979; Detmers
et al., 1987; Ratcliffe et al., 1992; Gold et al, 1994).
The aim of our work was to study changes in membrane
integrity and the occurrence of the acrosome reaction in human
spermatozoa consequent to the binding of specific lectins to
their respective receptors at the sperm surface. Propidium
iodide, a cellular DNA dye impermeable to the cell plasma
membrane, has been previously used to evaluate sperm membrane integrity (Harrison and Vickers, 1990; Gamer and
Johnson, 1995). Lectins are multimeric protein or glycoprotein
molecules bearing more than one binding site for their characteristic sugar ligands (reviewed by Koehler, 1981). They are
able to induce the aggregation of cell surface carbohydrate
receptors and to form distinct cross-linked complexes with
specific multivalent carbohydrates and glycoconjugates
(Bhattacharyya et al, 1989; Gupta et al, 1994). Certain
lectins have been reported to mimic typical signal transduction
mechanisms induced by epidermal growth factor (EGF) (Zeng
et al, 1995). Moreover, the role of carbohydrates in glycoprotein-hormone signal transduction has been established
(Sairam 1989). In this study, we used the three following
lectins: wheat germ agglutinin (Triticum vulgaris, WGA) which
recognizes both sialic acid (NeuNAc) and /V-acetylglucosamine
(GlcNAc) (Monsigny et al, 1980); peanut agglutinin (PNA)
which had a strong specificity for disaccharides with terminal
galactose (Gal) especially the D Gal a (1,3) D GalNAc
disaccharide (Pereira et al, 1976); and concanavalin A (Con
A) which recognizes mannose (Man) and glucose (Glc) and
exhibits a particularly high affinity for complex and high
mannose type glycopeptides on cell membrane (Brewer et al,
1985). WGA binds to terminal NeuNAc a (2,6) Gal/GalNAc
glycoconjugates on the plasma membrane of human motile
spermatozoa (Lassalle and Testart, 1994). The removal of
sialic acid from the human sperm surface increases spermzona pellucida recognition, probably by unmasking certain
sperm surface antigens implicated in the recognition mechanism (Lassalle and Testart, 1994). Binding sites for PNA and
Con A on the sperm surface are also unmasked by removal
of sperm surface sialic acid. The ability of these lectins to
alter sperm membrane integrity and to induce the acrosome
reaction was assessed taking into account the carbohydrate
specificity and biochemical characteristics of each lectin. To
test the physiological relevance of this study, uncapacitated
human spermatozoa were incubated with human zonae pellucidae and the permeability to impermeable propidium iodide
of spermatozoa bound to the zona pellucida surface was
analysed according to the time post-insemination.
Materials and methods
Media and chemicals
BM1 medium was obtained from Ellios Bio Media (91430 Igny,
France) and Percoll from Pharmacia Biotech S.A. (78051 Saint
652
Quentin en Yvelines, France). Arthrobacter ureafaciens neuraminidase
(EC 3.2.1.18), calcium ionophore A23187 and all lectins used in this
study were purchased from Sigma (38297 La Verpillere, France).
Sperm preparation and labelling with lectins
Ejaculated fresh semen samples were obtained with consent from
normal donors and liquefied for 30 min at 37°C and -0.6 ml of
samples was separated on a Percoll gradient (90/45%) as described
by Lassalle and Testart (1994). After centrifuging for 25 min at 400 g,
the 90% Percoll fractions were carefully separated and washed by
dilution with 5 ml phosphate-buffered saline (PBS) and then centrifuged at 600 g for 7 min. The pellets were resuspended in 1 ml B2
medium. Before treatment, motile and non-motile spermatozoa were
counted on a Malassez cell to verify the high (>80%) percentage of
motile spermatozoa recovered in die 90% Percoll gradient. Sperm
populations recovered in the 90% Percoll fraction after centrifugation
on a discontinuous Percoll gradient have a high proportion of normal
motile spermatozoa (Le Lannou and Blanchard, 1988) with intact
plasma membrane (Lassalle and Testart, 1994) and high fertilizing
ability (Hyne et al, 1983; Berger et al, 1985; Sapienza et al, 1993).
Preliminary studies (Lassalle and Testart, 1994) showed that <20%
of spermatozoa recovered in the 90% Percoll fraction bound PNA
and Con A lectins, whereas WGA binding occurred in at least 80%
of this sperm population. However, the proportion of spermatozoa
with binding sites for PNA and Con A reached >70% of the sperm
population after removal of sperm surface sialic acid with 500 mlU/
ml neuraminidase. Consequently, pretreatment of spermatozoa with
500 mlU/ml neuraminidase was performed before Con A or PNA
treatment to optimize the binding of these two lectins to the sperm
plasma membrane. Thus, sperm suspensions were either incubated
for 30 min at 37°C with 20 ug/ml WGA or preincubated for 30 min
at 37°C with 500 mlU neuraminidase/ml and then incubated for
30 min at 37°C with 20 ug/ml PNA or Con A.
When sperm neuraminidase or lectin treatments were performed
immediately after separation on the discontinuous Percoll gradient
and the washing procedure (<60 min after the semen liquefaction
step), spermatozoa were considered to be uncapacitated. Sperm
capacitation was obtained by a 24 h incubation of spermatozoa in B2
medium at room temperature (Lassalle and Testart, 1994).
Assessment of sperm acrosome reaction
Pisum sativum agglutinin (PSA) was shown to stain the acrosome
contents of permeabilized acrosome-intact spermatozoa (Centola
et al, 1990). Acrosome reaction was scored by using fluorescein
isothiocyanate-conjugated PSA (FTTC-PSA) (Cross et al, 1986;
Centola el al, 1990). Lectin-treated sperm suspensions were washed
twice in PBS to remove free lectin. Spermatozoa were permeabilized
after dropwise addition of 0.5 ml 95% ethanol directly onto the sperm
pellet followed by a 30 min incubation at 4°C. Ethanol was removed
by a minimum of two centrifugation cycles; 40 ul of FTTC-PSA
stock solution (100 ug/ml) was men added and mixed with the
resultant pellet. Sperm suspension (10 |il) was placed between slide
and coverslip for observation under a Leitz microscope equipped with
epifluorescence. Acrosome-reacted and acrosome-intact spermatozoa
were scored according to Cross et al. (1986). The percentages of
acrosome-reacted spermatozoa were estimated from at least 150
spermatozoa per preparation (Centola et al, 1990). A positive control
was carried out by inducing the acrosome reaction by incubating (30
min at 37°C) a sperm suspension with calcium ionophore A23187
[stock solution in dimethyl sulphoxide (DMSO) and frozen at -20°C]
at a final concentration of 20 |xM.
Human sperm acrosome reaction induced by lectins
Assessment of sperm plasma membrane permeabilization changes
Propidium iodide (PI), which binds to and stains cellular DNA, is
impermeable to the plasma membrane and therefore can only enter
and stain cells with permeable plasma membrane (Harrison and
Vickers, 1990; Garner and Johnson, 1995). PI has previously been
used to assess membrane integrity in mammalian spermatozoa and
has been shown not to stain intact spermatozoa (Harrison and Vickers,
1990). The number of spermatozoa permeable to PI among highly
motile sperm populations was scored. Stock solution (10 ul) of PI
(0.5 mg/ml in PBS, stored in the dark at -20°C) was added to 1 ml
of sperm preparation. Then 10 ul of the resulting suspension was
placed between slide and coverslip for observation under both
phase contrast and epi-fluorescence microscopy. This simultaneous
observation was obtained by maintaining the light intensity at a
sufficiently high level to score both non-fluorescent and fluorescent
spermatozoa under phase contrast microscopy and sufficiently low to
visualize the red fluorescence of Pl-permeant spermatozoa. Percentages of spermatozoa permeant to PI were estimated from at least 200
spermatozoa per preparation.
Effect of zona pellucida on the permeability of the sperm
plasma membrane to propidium iodide
Human oocytes remaining unfertilized 40-48 h after in-vitro insemination were stored at 4°C in ammonium sulphate solution for optimal
zona pellucida conservation (Yoshimatsu et al., 1988). At 2 h before
insemination, oocytes were thoroughly rinsed over a 90 min period
in four successive baths of PBS-bovine serum albumin (BSA) then
placed in B2 medium at 37°C before insemination.
To distinguish spermatozoa newly-bound to the zona pellucida
surface from those previously bound during the in-vitro fertilization
(IVF) procedure, spermatozoa recovered from the 90% Percoll fraction
were stained (15 min, 37°C) with 0.3 ml FITC solution (0.25 mg/ml
in PBS-glucose) according to the method of Liu et al. (1988). The
number of motile spermatozoa was assessed before and after FITC
staining. The sperm suspension was then washed twice with PBS and
the final pellet was resuspended in B2 medium before dilution to
obtain a concentration of 5X10 6 motile spermatozoa/ml. Human
zonae pellucidae were placed in the sperm medium for 10-120 min
before washing in PBS supplemented with 2% propidium iodide
stock solution (0.5 mg/ml PBS). Zonae pellucidae were placed
between slide and coverslip for observations performed under a Leitz
microscope equipped with epifluorescence. The numbers of green
(FITC) and red (PI) spermatozoa on each zona pellucida were
determined using extinction and emission filters appropriate to each
fluorescent probe. Spermatozoa previously bound during the IVF
procedure showed red fluorescence on the head with an absence of
green fluorescence on the tail. Spermatozoa bound to the zona
pellucida during the experimental period were those with green
fluorescence on the tail; among this latter population those spermatozoa which also showedredfluorescenceon the head were considered to
be permeable spermatozoa (Figure 1). The percentage of spermatozoa
permeable to PI after binding to the zona pellucida was estimated for
each zona pellucida. The mean number of bound spermatozoa,
the mean number of bound permeable spermatozoa and the mean
percentage of permeable spermatozoa per zona pellucida were calculated according to the time post-insemination.
Inhibition of the ability of PNA to induce the acrosome
reaction
Two inhibitors of protein kinase (PK), calphostin C (PKC inhibitor)
and KT 5720 (PKA inhibitor) (Calbiochem 92039, La Jolla, CA,
USA) have previously been used to inhibit the acrosome reaction
induced by PKA or PKC activators in human spermatozoa. The
optimal concentration of each inhibitor was found to be 100 nM
(Doherty et al., 1995). Stock solutions of calphostin C and KT 5720
were prepared by diluting 50 u.g in 0.5 ml DMSO and aliquots were
stored at 4°C. Each inhibitor was added (at the final concentration of
100 nM) 15 min before addition of PNA and was present during the
time of sperm incubation with PNA. Since the inhibitory properties
of calphostin C are most effective in the presence of light (Bruns
et al., 1991), an incandescent light was directed at all test tubes
during the time of incubation (Doherty et ai, 1995).
Statistical analysis
Data were analysed using Statview 4.0 software on a Macintosh II
si computer. Means (± SEM) were compared using Student's f-test
and Fisher's Protected Least Significant Difference (PLSD) test.
Results
Permeabilization of the sperm membrane to propidium iodide induced by zona pellucida
Two-thirds of zona pellucida-bound spermatozoa had become
permeable to propidium iodide 15-30 min post-insemination.
This proportion of permeable cells reached >89% after 45 min
in culture (Table I) whereas only 13.1% (± 7.3%) of unbound
spermatozoa present in the medium surrounding the zonae
pellucidae were permeable to propidium iodide.
Permeabilization of the sperm membrane to propidium iodide induced by lectins
Treatment of spermatozoa with 500 IU/ml neuraminidase had
no effect on their permeability to propidium iodide (22.3
compared with 18.6%, not significant; Table II). The percentage
of spermatozoa with membranes permeable to propidium
iodide showed a two-fold increase after treatment with either
Ca 2+ ionophore (27.7 compared with 14.5%, P <0.03),
20 |ig/ml WGA (31.9 compared with 13.8%, P <0.004) or
neuraminidase (20 (ig/ml) Con A (38.4 compared with 18.4%,
P <0.002; Table II). The percentage of spermatozoa with
permeable membranes reached 72.7% after treatment with
neuraminidase (20 (i.g/ml) PNA compared with only 18.9% of
spermatozoa {P <0.0001) in the control samples (Table II).
Acrosome reaction induced by lectins
The percentages of acrosome-reacted spermatozoa were not
affected by sperm treatment with 500 mlU/ml A. ureafaciens
neuraminidase (uncapacitated, 12.7 compared with 10.7%;
capacitated spermatozoa, 14.4 compared with 15.0%; Tables
m and IV). Percentages of uncapacitated spermatozoa with
reacted acrosome were significantly increased (as compared
with their own control) after sperm treatment with 25 (iM
calcium ionophore A23187 (14.3 compared with 7.3%,
P <0.0008) and with 500 mIU/ml neuraminidase followed by
20 ng/ml PNA (15.1 compared with 7.9%, P <0.0002) or 20
Hg/ml Con A (17.2 compared with 10.7%, P <0.006; Table III).
After capacitation (24 h at room temperature), the acrosome
reaction was ~2.5-fold increased for neuraminidase-PNA (27.7
compared with 10.3%, P <0.0003) and Ca 2+ ionophore-treated
spermatozoa (33.2 compared with 12.8%, P <0.0001) whereas
653
B.Lassalle and J.Testart
Figure 1. Effect of zona pellucida on the permeability of human sperm plasma membrane to propidium iodide. Spermatozoa previously
bound during the in-vitro fertilization (TVF) procedure show red fluorescence on the head with an absence of green fluorescence on the tail.
Spermatozoa bound to the zona pellucida during the experimental period are those with green fluorescence on the tail; among this sperm
population those with red fluorescence on the head are considered to be permeable spermatozoa. This photograph was a superimposition of
two epifluorescence images obtained after a dual recording procedure performed under a Biorad MRC-600 confocal laser scanning
microscope equipped with extinction and emission filters appropriate to each fluorescent probe. Bar = 20 u.m.
Table I. The ability of zona pellucida to increase the permeability of human spermatozoa to propidium iodide. The proportion of permeable spermatozoa is
documented according to the time post-insemination. Figures in parentheses are the numbers of zonae pellucidae used per experiment
Time post-insemination
15-30 min
31—45 min
46-60 min
>60 min
Mean sperm number bound per zona pellucida
15.6
± 5.1 ( 5 /
10.4
± 3 . 3 (5)b
65.9
± 7.4 (5) c J
27.6
± 6.1 (8)
23.5
± 5.6 (8)
82.4
± 4.4 ( 8 ) "
27.4
± 4.3 (12/
24.5
± 4.1 (12)
88.4
± 3.7(12/
35.0
± 4.3 ( 1 5 /
34.5
± 4.1 ( 1 5 /
98.9
± 0.4 (15)<u-f
Mean number of permeable spermatozoa bound per zona pellucida
Percentage of permeable bound spermatozoa
Means (± SEM) were compared using Fisher's PLSD test.
'P <0.03; bP <0.005; CP <0.01; dP = 0.0001; CP <0.001; 'P <0.008.
the acrosome reaction was not affected by sperm treatment
with neuraminidase-Con A (15.0 compared with 14.4%; Table
TV). Treatment of spermatozoa with 20 (ig/ml WGA had no
effect on acrosome reaction regardless of sperm capacitation
status when compared with untreated spermatozoa (7.5 versus
8.2% and 17.0 versus 13.1% respectively; Tables HI and IV).
The proportion of acrosome-reacted spermatozoa observed
after PNA sperm treatment was similar (27.7%) to that obtained
654
with acrosome reaction-inducing agents previously used in the
literature (Table V).
Effect of protein kinase A and C inhibitors on the
acrosome reaction induced by PNA lectin
The acrosome reaction induced by PNA lectin was not inhibited
by sperm pretreatment with 100 nM Calphostin C (23.9% of
acrosome-reacted spermatozoa), with 100 nM KT 5720 (27.1 %)
Human sperm acrosome reaction induced by lectins
Table 11. Ability of various treatments to increase the permeability of human spermatozoa to propidium iodide (values are means ± SEM). Figures in
parentheses are the numbers of ejaculates tested
Ca 2+ ionophore
Permeable sperm in control samples (%)
Permeable sperm in treated samples (%)
P values (Fisher's PLSD test)
Neuraminidase
A23187
WGA
neuraminidase
+ PNA
+ Con A
14.5 ± 1.4(5)
27.7 ± 2 . 0 (5)
P = 0.023
13.8 ± 1.0(5)
31.9 ± 5 . 9 (5)
P = 0.004
18.6 ± 1.7 (5)
22.3 ± 1.7 (5)
P = 0.96
18.9 ± 1.6(5)
72.7 ± 8.8 (5)
P = 0.0001
18.4 ± 2.0 (5)
38.4 ± 5.0 (5)
P = 0.0017
WGA = wheat germ agglutinin; PNA = peanut agglutinin; Con A = concanavalin A.
Table HI. Ability of various treatments to induce the acrosome reaction of uncapacitated human spermatozoa (values are means ± SEM) . Figures in
parentheses are the numbers of ejaculates tested
Ca 2+ ionophore
Acrosome-reacted spermatozoa in control samples (%)
Acrosome-reacted spermatozoa in treated samples (%)
P values (Fisher's PLSD test)
Neuraminidase
A23187
WGA
neuraminidase
+ PNA
+ Con A
7.3 ± 1.0 (4)
14.3 ± 2.4 (4)
P = 0.0008
8.2 ± 1.1 (4)
7.5 ± 1.2 (4)
P = 0.69
10.7 ± 1.0(3)
12.7 ± 1.3 (3)
P = 0.35
7.9 ± 1.0(5)
15.1 ± 1.4 (5)
P = 0.0002
10.7 ± 1.0(3)
17.2 ± 0.6 (3)
P = 0.006
WGA = wheat germ agglutinin; PNA = peanut agglutinin; Con A = concanavalin A.
Table IV. Ability of various treatments to induce the acrosome reaction of capacitated human spermatozoa (values are means ± SEM). Figures in parentheses
are the numbers of ejaculates tested
Ca 2+ ionophore
Acrosome-reacted spermatozoa in control samples (%)
Acrosome-reacted spermatozoa in treated samples (%)
P values (Fisher's PLSD test)
Neuraminidase
A23187
WGA
neuraminidase
+ PNA
+ Con A
12.8 ± 2.4 (4)
33.2 ± 5.8 (4)
P = 0.0001
13.1 ± 2.6 (4)
17.0 ± 3.8 (4)
P = 0.36
14.4 ± 2.2 (4)
15.0 ± 1.8 (4)
P = 0.89
10.3 ± 1.8 (4)
27.7 ± 2.6 (4)
P = 0.0003
14.4 ± 2.2 (4)
15.5 + 2.0 (4)
P = 0.79
WGA = wheat germ agglutinin; PNA = peanut agglutinin; Con A = concanavalin A.
or 0.8% DMSO (25.1%) when compared with control nonpretreated spermatozoa (28.1%).
Table V. The ability of specific agents to induce the acrosome reaction in
capacitated human spermatozoa. All spermatozoa used in these studies were
previously capacitated in different culture conditions
Acrosome reaction
inducing agents
Acrosome-reacted References
spermatozoa* (%)
Intact zona pellucida
46
Solubilized zona pellucida
24-56
Human follicular
25-40
Monoclonal antibody
CRC-10
Progesterone
fluid
29
8-25
Protein kinase A activators
22-33
(forskolin, dibutyryl cyclic
AMP)
Protein Idnase C activators
29-35
(diacylglycerols, phorbol ester)
Ca 2+ ionophore A23187
25-40
•Significantly different from their own controls.
Cross et ai, 1988;
Hoshi etai, 1993
Cross et ai, 1988;
Bielfeld et ai, 1994
SaragUeta et ai, 1994;
Thomas and Neizel,
1988; Yudin et ai, 1988
Garcia-Framis et ai,
1994
Tesarik and Mendoza,
1993; Tomiyama et ai,
1995; Benoff etai, 1995
Bielfeld et ai, 1994;
Doherty et ai, 1995
Bielfeld et ai, 1994;
Doherty et ai, 1995
Byrd etai., 1989; White
etai, 1990; Liu and
Baker, 1990
Discussion
In this study, we have shown that molecular interactions
between human zona pellucida and sperm surface molecules
increase the sperm membrane permeability to impermeable
propidium iodide. Changes in sperm membrane permeability
induced by the zona pellucida were previously observed in
mouse (Lee and Storey, 1989) and were described as an
intermediary stage leading to acrosome reaction (Lee and
Storey, 1989; Kligman et ai, 1991). The endpoint of the first
stage (S stage) in the zona pellucida-induced acrosome reaction
in mouse spermatozoa was characterized by an increase in
acrosomal H + and Ca 2+ permeability (Lee and Storey, 1989).
We observed that two-thirds of zona pellucida-bound spermatozoa were permeable to propidium iodide in the first 30 min
post-insemination and that by 60 min after sperm insemination,
almost all spermatozoa bound to the zona pellucida surface
had become permeable to propidium iodide. Our results
suggested a rapid alteration in the permeability of the sperm
membrane following binding to the zona pellucida.
In mouse, the interaction of ZP3 with the sperm surface
655
B.Lassalle and J.Testart
may occur in a multivalent process, and multiple interactions
are followed by receptor aggregation that may ultimately lead
to signal transduction and acrosomal exocytosis (reviewed in
Leyton and Saling, 1989 and in KJigman et al, 1991).
Moreover, aggregation of sperm surface receptors in the
absence of zona pellucida components were found to induce
the acrosome reaction. In our study, we have shown that crosslinked complexes formed between certain Iectins and their
specific multivalent carbohydrates and glycoconjugates on the
sperm surface were able to modify membrane permeability
and to induce the acrosome reaction. We observed that although
the Iectins WGA, Con A and PNA were able to increase the
proportion of permeabilized spermatozoa, the consequences
for the induction of the acrosome reaction varied from one
lectin to another. The binding of WGA to sperm surface
receptors containing terminal sialic acid did not induce the
acrosome reaction regardless of the sperm capacitation status.
Using capacitated spermatozoa, we observed that PNA
induced the acrosome reaction in the same proportions (27%)
as calcium ionophore A23187 (Tables I and II), human
follicular fluid, or solubilized human zonae pellucidae (see
Table V for references) whereas Con A was unable to induce
the acrosome reaction. Whereas the capacitation of spermatozoa seems to be a prerequisite process which leads to physiological acrosomal exocytosis, we observed that PNA and Con
A were able to induce the acrosome reaction in uncapacitated
human spermatozoa. However, removal of sperm surface sialic
acid by neuraminidase seems to be prerequisite for Con A and
PNA binding to the sperm surface. Sialic acid removal may
unmask Con A and PNA binding sites by exposing subterminal
sugar residues of sperm glycoconjugates. Sialic acid has been
shown to protect the spermatozoon and sperm surface antigens
from the immune system (Holt, 1980; Schauer, 1985; Toshimori
et al, 1991) and its removal from the sperm plasma membrane
could be considered to be one of the capacitation events
necessary to unmask certain sperm surface antigens implicated
in zona pellucida recognition (Lassalle and Testart, 1994). If
certain capacitation events were artificially induced by sperm
neuraminidase treatment, the effects of PNA and Con A
on the sperm acrosome reaction may comprise partiallycapacitated spermatozoa.
The biochemical characteristics of Iectins (including sugar
specificity, amino acid composition, molecular size and
valency) as well as the saccharide structure recognized by
each lectin or its biological effects on the cell (as mitogenicity
or cytotoxicity) or even its ability to induce signal transduction
(Pandolfino et al, 1983; Gupta et al, 1994, Rini, 1995) may
be responsible for its ability to induce sperm acrosome reaction.
PNA and Con A become mitogenic for human and rat
lymphocytes only after neuraminidase treatment, and binding
of these Iectins on the cell surface increases the free cytoplasmic
calcium concentration (Novogrodsky et al. 1975; Pereira et al.,
1976; Hesketh et al., 1983; Bijsterbosh and Klaus, 1986;
Dixon et al., 1987). Con A has been reported to mediate cell
cytotoxicity and apoptosis (Novogrodsky, 1975; Kulkami and
McCulloch, 1995). However WGA, a non-mitogenic lectin,
also causes an increase in free cytoplasmic calcium concentration and early stimulation of lymphocytes, although this
656
stimulation is self-aborted before DNA synthesis occurs
Hesketh et al., 1983). Pandolfino et al. (1983) suggested
that mitogenic Iectins are able to bind and cross-link more
membrane receptors than non-mitogenic Iectins.
In our results the proportions of uncapacitated spermatozoa
permeable to propidium iodide were higher than the proportions
of acrosome-reacted spermatozoa. Moreover, human zona
pellucida induced greater differences in the proportions of
either permeabilized or acrosome-reacted spermatozoa than
occurred with PNA. Changes in the sperm membrane permeability induced by zona pellucida in mouse were described as an
intermediary stage leading to the acrosome reaction (Lee and
Storey, 1989; Kligman et al, 1991). Mouse spermatozoa
previously incubated with zona pellucida to induce acrosome
reaction were blocked in a transition step (S stage) by addition
of a PKC inhibitor and possessed intact acrosomes; however,
the plasma and outer acrosomal membranes had started to lift
away from the sperm head (Kligman et al, 1991). According
to Lee and Storey (1989) permeabilization of the membranes
marking a transition step are probably due to pore formation
induced by punctate fusion of the plasma and outer acrosomal
membranes. The penetration of impermeable propidium iodide
across sperm membranes may suggest a loss of sperm membrane integrity. Changes in sperm membrane permeability
consequent to lectin binding to the sperm surface or to
attachment of spermatozoa to the zona pellucida surface could
be considered to be a preliminary step that may or may not
lead to acrosomal exocytosis.
Receptor aggregation in response to biological stimuli has
been described in other systems to initiate signal transduction
across the membrane (Detmers et al, 1987; Ratcliffe et al,
1992; Gold et al, 1994). The mechanisms by which aggregated
receptors transmit signal into the cell are not fully understood.
However, lymphocyte activation was induced after chemical
transformation of terminal sugar residues of glycoconjugates
on the cell surface or by lectin-inducing aggregation of cell
membrane glycoconjugates (Novogrodsky, 1975). It has been
reported that Con A-carbohydrate interaction can mimic typical
signal transduction mechanisms (Hesketh et al, 1983) and that
biosignalling via Iectins may involve modulation of protein
kinase activities (Zeng et al, 1995). The human sperm acrosome reaction seems to occur via the activation of at least two
signal transduction pathways, involving PKA and PKC (Rotem
et al, 1992; Doherty et al, 1995). We observed here that two
potent inhibitors of PKA (KT 5720) and PKC (calphostin C)
did not inhibit the acrosome reaction induced by PNA lectin.
From our results, we conclude that signalling events which
lead to acrosome reaction induced by PNA binding (i.e. crosslinking complexes formed between the lectin and the specific
multivalent glycoconjugates) on the sperm surface do not
involve the activation of protein kinase pathways.
The mechanism generally accepted as responsible for spermoocyte recognition involves saccharide moieties of zona pellucida glycoconjugates and complementary zona pellucida-binding proteins on the spermatozoa (Macek and Shur, 1988; Miller
and Ax, 1990). It has been suggested that the active binding
component is the glycan portion of ZP3 glycoprotein and that
the protein backbone functions, in part, to crosslink the glycan
Human sperm acrosome reaction induced by lectins
(review by Miller and Ax, 1990). Our results and those of
others (Macek et al., 1991; Aarons et ai, 1992; Tesarik and
Mendoza, 1993) confirm that aggregation of sperm surface
receptors per se (in the absence of zona pellucida components)
is able to induce the acrosome reaction. The use of lectins as a
tool for evaluating glycoconjugates in spermatozoa membranes
and acrosomal status may influence and distort results if
applied to live, unfixed spermatozoa. Therefore, histochemical
techniques using lectins should be limited to previously fixed
spermatozoa rather than live spermatozoa, although we accept
that fixation procedures may also modify membrane integrity.
Moreover, we have demonstrated that aggregation of certain
sperm surface glycoconjugates mediated via their glycan
component is able to induce the acrosome reaction.
References
Aarons, D., Battle, T., Boettger-Tong, H. and Poiner, G.R. (1992) Role of
monoclonal antibody J-23 in inducing acrosome reactions in capacitated
mouse spermatozoa- J. Reprod. Fertil, 96, 49-59.
Barratt, C.L.R., Andrews, P.A., McCann, C. et al. (1993) Recombinant human
ZP3 expressed in Chinese hamster ovary (CHO) cells is a potent inducer
of the human acrosome reaction. Hum. Reprod., 8 (Suppl. 1), 136,
Abstract 407.
Benoff, S., Rushbrook, J.I., Hurley, I.R. et al. (1995) Co-expression of
mannose-ligand and non-nuclear progesterone receptors on motile human
sperm identifies an acrosome-reaction inducible subpopulation. Am. J.
Reprod. Immunol, 34, 100-115.
Berger, T., Marrs, R. and Moyer, D. (1985) Comparison of techniques for
selection of motile spermatozoa. Fertil. Steril., 40, 268—273.
Bhattacharyya, L., Khan, M.I., Fant, J. and Brewer, C.F. (1989) Formation of
highly ordered cross-linked lattices between asparagine-linked
oligosaccharides and lectins observed by electron microscopy. J. Bio/.
Chem., 264, 11543-11545.
Bielfeld, P., Faridi, A., Zaneveld, L.J.D. and De Jonge, CJ. (1994) The zona
pellucida-induced acrosome reaction of human spermatozoa is mediated by
protein kinases. Fertil. Steril., 61, 536-541.
Bijsterbosch, M.K. and Klaus, G.G.B. (1986) Concanavalin A induces Ca 2+
mobilization, but only a minimal inositol phospholipid breakdown in mouse
B cells. J. Immunol., 137, 1294-1299.
Bleil, J.D. and Wassarman, P.M. (1983) Sperm—egg interactions in the mouse:
sequence of events and the induction of the acrosome reaction by a zona
pellucida glycoprotein. Dev. Biol., 95, 317-324.
Brewer, F., Bhattacharyya, L., Brown, R.D., Koenig, S.H. (1985) Interactions
of concanavalin A with a trimannosyl oligosaccharide fragment of complex
and high mannose type glycopeptides. Biochem. Biophys. Res. Commun.,
127, 1066-1071.
Bruns, R.F., Miller, F.D., Merrimen, R.L. et al. (1991) Inhibition of protein
kinase C by calphostin C is light-dependent. Biochem. Biophys. Res.
Commun., 176, 288-293.
Bunch, D., Leyton, L. and Saling, P. (1992) Sperm interaction with the zona
pellucida: role of a tyrosine kinase receptor for ZP3 in fertilization. In
Nieschlag, E. and Habenicht, U.F. (eds), Spermatogenesis, Fertilization,
Contraception: Molecular, Cellular and Endocrine Events in Male
Reproduction. Springer-Verlag, Berlin, pp. 367-380.
Byrd, W., Tsu, J. and Wolf, D.P. (1989) Kinetics of spontaneous and
induced acrosomal loss in human sperm incubated under capacitating and
noncapacitating conditions. Gamete Res., 22, 109—122.
Centola, G.M., Mattox, J.H. and Leary, J.F. (1990) Assessment of the viability
and acrosome status of fresh and frozen-thawed human spermatozoa
using single-wavelength fluorescence microscopy. Mol. Reprod. Dev., 27,
130-135.
Cross, N.L., Morales, P., Overstrect, J.W. and Hanson, F.W. (1986) Two
simple methods for detecting acrosome-reacted human sperm. Gamete Res.,
IS, 213-226.
Cross, N.L., Morales, P., Overstreet, J.W. and Hanson, F.W. (1988) Induction
of acrosome reactions by the human zona pellucida. Biol. Reprod., 38,
235-244.
Detmers, P.A., Wright, S.D., Olsen, E. et al. (1987) Aggregation of complement
receptors on human neutrophils in absence of ligand. J. Cell Biol., 105,
1137-1145.
Dixon, SJ., Stewart, D., Grinslein, S. and Spiegel. S. (1987) Transmembrane
signaling by the B subunit of cholera toxin: increased cytoplasmic free
calcium in rat lymphocytes. J. Cell Biol., 105, 1153-1161.
Doherty, CM., Tarchala, S.M., Radwanska, E. and De Jonge, CJ. (1995)
Characterization of two messenger pathways and their interactions in
eliciting the human sperm acrosome reaction. J. Androl, 16, 36-46.
Garcia-Framis, V., Martin-Lunas, E., lborra, A. et al. (1994) Characterization
of monoclonal antibodies specific for human sperm: effect of CRL-10 on
acrosome reaction. Res. Immunol, 145, 533-539.
Gamer, D.L. and Johnson, L.A. (1995) Viability assessment of mammalian
sperm using SYBR-14 and propidium iodide. Biol. Reprod., 53, 276-284.
Gold, M.R., Chiu, R., Ingham, RJ. et al (1994) Activauon and serine
phosphorylation of the p56lck protein tyrosine kinase in response to antigen
receptor cross-linking in B lymphocytes. J. Immunol, 153, 2369-2376.
Gupta, D., Arango, R., Sharon, N. and Brewer, C.F. (1994) Differences in the
cross-linking activites of native and recombinant Erythrina corallodendron
lectin with asialofetuin. Evidence for carbohydrate-carbohydrate interactions in lectin-glycoprotein complexes. Biochemistry, 33, 2503-2508.
Harrison, R.A.P. and Vickers, S.E. (1990) Use of fluorescent probes to assess
membrane integrity in mammalian spermatozoa. J. Reprod. Fertil, 88,
343-352.
Hesketh, T.R., Smith, G.A., Moore, J.P. et al. (1983) Free cytoplasmic calcium
concentration and the mitogenic stimulation of lymphocytes. J. Biol. Chem.,
258, 4876-^882.
Holt, W.V. (1980) Surface-bound sialic acid on ram and bull spermatozoa:
deposition during epididymal transit and stability during washing. Biol.
Reprod., 23, 847-857.
Hoshi, K., Sugano, T, Endo, C. et al. (1993) Induction of the acrosome
reaction in human spermatozoa by human zona pellucida and effect of
cervical mucus on zona-induced acrosome reaction. Fertil Steril, 60,
149-153.
Hyne, R.V., Stojanoff, A., Clarke, G.N. et al. (1983) Pregnancy from in vitro
fertilization of human eggs after separation of motile spermatozoa by
density gradient centrifugation. Fertil. Steril, 45, 93-96.
KJigman, I., Glassner, M., Storey, B.T. and Kopf, G.S. (1991) Zona pellucidamediated acrosomal exocytosis in mouse spermatozoa: characterization of
an intermediate stage prior to the completion of the acrosome reaction.
Dev. Biol, 145, 344-355.
Koehler, J.K. (1981) Lectins as probes of the spermatozoon surface. Arch.
Androl, 6, 197-217.
Kulkami, G.V. and McCulloch, C.A.G. (1995) Concanavalin A induced
apoptosis in fibroblasts: the role of cell surface carbohydrates in lectin
mediated cytotoxicity. J. Cell. Phys., 165, 119-133.
Lassalle, B. and Testart, J. (1994) Human zona pellucida recognition associated
with removal of sialic acid from human sperm surface /. Reprod. Fertil,
101,703-711.
Langlais, J. and Roberts, K.D. (1985) A molecular membrane model of sperm
capacitation and acrosome reaction of mammalian spermatozoa. Gamete
Res., 12, 183-224.
Lee, M.A. and Storey, B.T. (1989) Endpoint of first stage of zona pellucidainduced acrosome reaction in mouse spermatozoa characterized by acrosome
H + and Ca 2+ permeability: population and single cell kinetics. Gamete
Res., 24, 303-326.
Lee, M.A., Check, J.H. and Kopf, G.S. (1992) A guanine nucleotide-binding
regulatory protein in human sperm mediates acrosomal exocytosis induced
by the human zona pellucida. Mol. Reprod. Dev., 31, 78—86.
Le Lannou, D. and Blanchard, Y. (1988) Nuclear maturity and morphology
of human spermatozoa selected by Percoll density gradient centrifugation
or swim-up procedure. J. Reprod. Fertil, 84, 551-556.
Leyton, L. and Saling, P. (1989) Evidence that aggregation of mouse sperm
receptors by ZP3 triggers the acrosome reaction. / Cell Biol, 108,
2163-2168.
Liu, D.Y., Lopata, A., Johnston, W.I.H. and Baker, H.W.G. (1988) A human
sperm-zona pellucida binding test using oocytes that failed to fertilize
in vitro. Fertil. Steril, 50, 782-788.
Liu, D.Y. and Baker, H.W.G. (1990) Inducing the human acrosome reaction
with a calcium ionophore A23187 decreases sperm-zona pellucida binding
with oocytes that failed to fertilize in vitro. J. Reprod. Fertil, 89, 127-134.
Liu, D.Y. and Baker, H.W.G. (1992) Morphology of spermatozoa bound to
the zona pellucida of human oocytes that failed to fertilize in vitro. J.
Reprod. Fertil., 94, 71-84.
Liu, D.Y. and Baker, H.W.G. (1994) Acrosomal status and morphology of
657
B.Lassalle and J. Test art
human spermatozoa bound to the zona pellucida and oolemma determined
using oocytes that failed to fertilize in vitro. Hum. Reprod., 9, 673-679.
Macek, M.B. and Shur, B.D. (1988) Protein-carbohydrate complementarity
in mammalian gamete recognition. Gamete Res., 20, 93—109.
Macek, M.B., Lopez, L.C. and Shur, B.D. (1991) Aggregation of (J-1,4Galactosyltransferase on mouse sperm induces the acrosome reaction. Dev.
Biol., 147, 440-444.
McKinnon, C.A., Weaver, F.E., Yoder, J.A. et al. (1991) Cross-linking a
maturation-dependent ram sperm plasma membrane antigen induces the
acrosome reaction. Mol. Reprod. Dev., 29, 200-207.
Miller, D.J. and Ax, R.L. (1990) Carbohydrates and fertilization in animals.
Mol. Reprod. Dev., 26, 184-198.
Monsigny, M., Roche, A.C., Sene, C. et al. (1980) Sugar-lectin interactions:
how does wheat-germ agglutinin bind sialoglycoconjugates? European J.
Biochem., 104, 147-153.
Novogrodsky, A. (1975) Lymphocyte activation induced by modifications of
surface membrane saccharides. In Rosenthal, A.S. (ed.), Immune
Recognition. Academic Press, New York, pp. 43-61.
Novogrodsky, A., Lotan, R, Ravid, A. and Sharon, N. (1975) Peanut
agglutinin, a new mitogen that binds to galactosyl sites exposed after
neuraminidase treatment. J. Immunol., 115, 1243-1248.
Pandolfino, E.R., Namen, A.E., Munske, G.R. and Magnuson, J.A. (1983)
A comparison of the cell-binding characteristics of the mitogenic and
nonmitogenic lectins from lima beans. J. Biol. Chcm., 258, 9203-9207.
Pereira, M.E.A., Kabat, E.A., Lotan, R. and Sharon, N. (1976) Immunochemical
studies on the specificity of the peanut (Arachis hypogaea) agglutinin.
Carbohydrate Res., 51, 107-118.
Ratcliffe, M.J.H., Coggeshall, K.M., Newell, M.K. and Julius, M.H. (1992)
T cell receptor aggregation, but not dimensation, induces increased cytosolic
calcium concentrations and reveals a lack of stable association between
CD4 and the T cell receptor. J. Immunol, 148, 1643-1651.
Rini, J.M. (1995) Lectin structure. Ann. Rev. Biophys. Biomol. Struct., 24,
551-577.
Rotem, R., Gedelia, F.P., Homonnai, Z.T. et al. (1992) Ca2+-independant
induction of acrosome reaction by protein C in human sperm. Endocrinology,
131, 2235-2243.
Sairam, M.R. (1989) Role of carbohydrates in glycoprotein hormone signal
transducuon. FASEB J., 3, 1915-1926.
Sapienza, F, Verheyen, C , Tournaye, H. et al. (1993) An auto-controlled
study in in-vitro fertilization reveals the benefit of Percoll centnfugation
to swim-up in the preparation of poor-quality semen. Hum. Reprod., 8,
1856-1862.
SaragUeta, P., Lanuza, G., Miranda, P.V. et al. (1994) Immunoglobulins from
human follicular fluid induce the acrosome reaction in human sperm. Mol.
Reprod. Dev., 39, 280-288.
Schauer, R. (1985) Sialic acids and their role as biological masks. Trends
Biochem, Sci., 10, 357-360.
Schechter, Y, Hernaez, L., Schlessinger, J. and Cuastrecasas, P. (1979) Local
aggregation of hormone-receptor complexes is required for activation by
epidermal growth factor. Nature, 278, 835-838.
Sidhu, K.S. and Guraya, S.S. (1989) Cellular and molecular biology of
capacitation and acrosome reaction in mammalian spermatozoa In:. Rev.
Cytoi, 118, 231-280.
Tesarik, J. (1989) Appropriate time of the acrosome reaction is a major
requirement for the fertilizing spermatozoa. Hum. Reprod., 4, 957-961.
Tesarik, J. andMendoza, C. (1993) Insights into the function of a sperm-surface
progesterone receptor evidence of ligand-induced receptor aggregation and
the implication of proteolysis. Exp. Cell Res., 205, 111-117.
Thomas, P. and Meizel, S. (1988) An influx of extracellular calcium is required
for initiation of the human sperm acrosome induced by human follicular
fluid. Gamete Res., 20, 397^tll.
Tomiyama, T, Ohashi, K., Tsutsui. T. et al. (1995) Acrosome reaction induced
in a limited population of human spermatozoa by progesterone (Ca 2+ dependent) and ATP (Ca2+-independent). Hum. Reprod., 10, 2052-2055.
Toshimori, K., Araki, S., Oura, C. and Eddy, E.M. (1991) Loss of sperm
surface sialic acid induces phagocytosis. Arch. Androl., 27, 79-86.
Van Duin, M., Ploman, J.E.M., De Bret, I.T.M. et al. (1994) Recombinant
human zona pellucida protein ZP3 produced by Chinese hamster ovary
cells induces the human sperm acrosome reaction and promotes spermegg fusion. Biol. Reprod., 51, 607-617.
White, D.R., Phillips, DJvl. and Bedford, J.M. (1990) Factors affecting the
acrosome reaction in human spermatozoa. J. Reprod. Fertil., 90, 71-80.
658
Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neill,
J.D. (eds), The Physiology of Reproduction. Raven Press, New York, pp.
189-317.
Yoshimatsu, N., Yanagimachi, R. and Lopata, A. (1988) Zonae pellucidae of
salt-stored hamster and human eggs: their penetrability by homologous and
heterologous spermatozoa.Gam</« Res., 21, 115—126.
Yudin, A.I., Gottlieb, W. and Meizel, S. (1988) Ultrastuctural studies of the
early events of the human sperm acrosome reaction as initiated by human
follicular fluid. Gamete Res., 20, 11-24.
Zeng, F.Y., Benguria, A., Kafert, S. et al. (1995) Differential response of the
epidermal growth factor receptor tyrosine kinase activity to several plant
and mammalian lectins. Mol. Cell. Biochem., 142, 117-124.
Received on February 19, 1996; accepted on July 16, 1996