781
Development 102. 781-792 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Carbohydrate-binding properties of boar sperm proacrosin and
assessment of its role in sperm-egg recognition and adhesion during
fertilization
R. JONES, C. R. BROWN and R. T. LANCASTER
Department of Molecular Embryology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT,
UK
Summary
Recent evidence indicates that a carbohydrate recognition mechanism is involved in the early stages of
sperm-egg interaction in mammals. In this communication, we describe a potential zona-ligand molecule
on boar spermatozoa that has the capacity to recognize
and bind to carbohydrate moieties of zona pellucida
glycoproteins as well as neoglycoproteins, BSA-fucose
and BSA-mannose. The molecule has broad specificity for carbohydrate binding and there is a requirement for a polysaccharide structure or for 'clustering'
of saccharides on a protein backbone. The molecule
has been identified as proacrosin, the zymogen form of
the acrosomal protease acrosin. Strong similarities
exist between proacrosin and 'bindin', the lectin-like
protein that is thought to mediate sperm-egg adhesion
Introduction
Fertilization in mammals involves complementary
recognition and fusion between two specialized and
morphologically disparate cells, the sperm and egg
(reviewed by Yanagimachi, 1981). It is reasonable to
presume that the capacity of these cells to unite is
mediated by specific receptor molecules on or near
their limiting surfaces. The nature of these complementary recognition signals and the molecular details of the mechanisms involved are still poorly
understood although there is growing experimental
evidence that carbohydrate moieties on surface membrane glycoconjugates play a key role, as demonstrated in other systems in which cells interact and
adhere to one another (Frazier & Glaser, 1979). It has
been shown in a variety of species that lectins and
in echinoderms. An hypothesis is proposed for spermegg interaction in mammals in which proacrosin,
released during the early stages of the acrosome
reaction, mediates secondary or consolidated binding
of spermatozoa to the zona pellucida by virtue of its
carbohydrate-binding capacity. The localized proteolytic action of active acrosin on the zona enhances this
interaction in a manner analogous to the requirement
for trypsinization of erythrocytes before agglutination
by certain lectins. This hypothesis, which is supported
by evidence from in vitro fertilization experiments, is
discussed in relation to current concepts on sperm—egg
recognition.
Key words: proacrosin, zona ligand, spermatozoa, lectin,
carbohydrates, fertilization, boar, glycoprotein, adhesion.
hapten sugars, particularly L-fucose and fucose-containing glycan chains, are potent inhibitors of fertilization in vitro (Ahuja, 1982; Huang et al. 1982;
Peterson etal. 1984; Shalgi etal. 1986). The inhibitory
effects of these haptens appear to be directed towards
the sperm rather than the egg suggesting that they
may 'occupy' specific receptor sites on the sperm.
Shur and coworkers have proposed galactosytransferase as a potential receptor protein on mouse spermatozoa (Shur, 1986), the enzyme presumably mediating sperm-egg binding by forming a stable
complex with yV-acetylglucosamine residues on zona
glycoproteins. This would be consistent with current
evidence that 0-linked oligosaccharides on one particular zona glycoprotein (ZP3) possess sperm-binding activity (Wassarman, 1987). Allied to these
findings are those of Saling (1981) that, in the mouse,
782
R. Jones, C. R. Brown and R. T. Lancaster
inhibitors of trypsin-like enzymes can block fertilization in vitro, suggesting a role for proteases in the
recognition process.
Taken together, these observations imply the presence of a ligand molecule on spermatozoa that
recognizes carbohydrate components on the zona
pellucida and which may act in concert with a
protease to mediate sperm-egg adhesion. In fact, a
precedent for such a system is known from studies on
fertilization in echinoderms in which 'bindin', a lectin-like protein that is present within the acrosomal
granule, is thought to interact with fucose-containing
glycoproteins on the vitelline envelope surrounding
the egg (Vacquier & Moy, 1977). Protease activity has
been found associated with bindin in spermatozoa
from one species of sea urchin (Green & Summers,
1980).
Recently, a group of fucose-binding proteins with
molecular weights (Mr) of 17000 and 53000 were
described on boar spermatozoa (Topfer-Petersen et
al. 1985). The properties of the A/..53X103 component were very similar to those of a zona-binding
protein reported by us on boar spermatozoa (Brown
& Jones, 1987) and later identified as proacrosin, the
zymogen form of the acrosomal protease acrosin
(Jones & Brown, 1987). In this investigation, we
bring these two separate lines of work together by
demonstrating that proacrosin has the capacity to
interact with carbohydrate moieties on zona glycoproteins and neoglycoproteins. The evidence suggests
an important role for this molecule in sperm-egg
recognition and binding during the initial stages of
fertilization. In view of the widespread distribution of
proacrosin in mammalian spermatozoa, this mechanism may be common to all species.
Materials and methods
Chemicals
All chemicals were of the highest purity available commercially and were purchased from BDH Ltd, (Poole, Dorset,
UK) or Pharmacia Ltd, (Milton Keynes, Bucks, UK) or
Sigma (London). L25I-labelled Bolton & Hunter reagent
and [125I]NaI were supplied by Amersham International
(Amersham, Bucks, UK). Medium 199 was obtained from
Gibco Ltd, (Paisley, Scotland, UK). 125I-labelled fucoidin
(previously purified by gel filtration and ion-exchange
chromatography) was a generous gift of Dr G. M. W.
Cook, University of Cambridge.
Conjugation procedures
Bovine serum albumin (BSA, fraction V, Sigma Cat. No.
A-7638) was conjugated with OT-L(-)fucose or D(+)mannose using the cyanoborohydride method of Schwartz &
Gray (1977). The molar ratio of sugar to protein varied
between 10 and 15 to 1. Glycoproteins were iodinated using
the Iodogen method of Markwell & Fox (1978). Alternatively, BSA conjugated with a-L(—)fucose and fluoroceinisothiocyanate (FITC-BSA-fucose) was purchased from
Sigma and chromatographed on Sephadex G-25 before use.
Proteins were conjugated directly with FTTC (Isomer I,
Sigma) following the method of Johnson & Holborow
(1986). Total protein was measured by the method of
Bradford (1976) and carbohydrate by the phenol sulphuric
procedure (Dubois et al. 1956).
Spermatozoa
Ejaculated semen was collected from fertile boars, diluted
with an equal volume of PBS containing 2 mM-/?-aminobenzamidine (pAB) and spermatozoa washed by centrifugation
through 0-264 M-sucrose/2mM-pAB/10mM-Hepes pH7-4
(Harrison, 1976). The sperm pellet was resuspended in
0-25M-sucrose/2mM-pAB, the pH adjusted to 3-0 with
0-IN-HCI and the suspension incubated overnight at 4°C
(Polakoski & Parrish, 1977). Spermatozoa were pelleted by
centrifugation at 10000 g for 15min, the supernatant
removed (referred to hereafter as the pH3 extract) and
stored frozen at —20°C. In some experiments, pAB was
ommitted from the media. Spermatozoa from the cauda
epididymidis were collected by retrograde flushing of the
vas deferens with PBS and pH3 extracts prepared as
described above. Seminal plasma and epididymal secretions
were clarified by centrifugation at 10 000 g for 15 min at 4°C.
Electrophoresis and Western blotting
Proteins were separated on nonreducing SDS-PAGE
(Laemmli, 1970) and either stained directly with 005 %
(w/v) Coomassie blue R-250 or electroblotted onto nitrocellulose membranes (Towbin et al. 1979). Blots were
incubated with 3 % BSA (w/v) (Sigma Cat. No. A4503) in
PBS for 2h at room temperature to block nonspecific
protein binding, drained (i.e not washed) and then probed
with FTTC or 125I-labelled neoglycoproteins for a further
2h. In other experiments, a second blocking step was
introduced after incubation with 3 % BSA by exposing
blots to a variety of mono-, di-, tri, and polysaccharides for
lh at room temperature at concentrations described in
Results section. Unbound probe was removed by washing
three times in PBS and bound probe detected with u.v. light
at 254 nm or by autoradiography using preflashed Kodak XOmat S film. Blots and films were scanned using a Joyce
Loebl Chromoscan 3 densitometer equipped with u.v.
facility. Flat-bed isoelectric focusing of proteins was carried
out using dissociating conditions as described by O'Farrell
(1975) with ampholytes pH 3-10.
In vitro fertilization procedures
Eggs were collected surgically from the oviducts of 7- to 10month-old gilts that had been superovulated with a regime
of PMSG and hCG (Polge, 1982). Ejaculated spermatozoa
were capacitated overnight (Cheng, 1985) at 25°C, diluted
to 10* ml"1 in medium 199 and incubated for a further
30 min at 37°C in the presence or absence of various
oligosaccharides/inhibitors as described in Results section.
Groups of eggs (10-15) were added to 2 ml of the sperm
suspensions and incubation continued at 39°C for 4h in a
5 % CO^air mixture as described in detail by Cheng
Role of boar sperm proacrosin in fertilization
(1985). Spermatozoa that were loosely attached to the zona
were dislodged by repeated passage of eggs through a finebore pipette, the eggs mounted on glass slides and fixed
with 75% (v/v) ethanol/25% (v/v) acetic acid for 48 h.
Remaining spermatozoa (referred to as firmly-bound spermatozoa) were counted using phase-contrast microscopy
after staining of nuclei with 1 % (w/v) lacmoid in 45 %
(v/v) acetic acid.
Collection, iodination and solubilization of zona
pellucida glycoproteins
Zonae pellucidae were removed mechanically from ovulated pig eggs, iodinated directly with 125I-labelled Bolton
& Hunter reagent and solubilized by heating at 70 °C for
60min in 3mM-Na22CO3 pH90 (Brown & Jones, 1987).
Soluble glycoproteins were then used to probe Western
blots of sperm proteins as described in detail by Brown &
Jones (1987). Where appropriate, blots were also incubated
with a variety of carbohydrates after blocking with 3 %
BSA as described in Results section.
Fluorescence microscopy
Spermatozoa were washed in sucrose (see above) resuspended in PBS or 0-265 M-sucrose/10 mM-Hepes pH 7-4 and
either used as such (intact spermatozoa) or subjected to one
cycle of freezing and thawing (permeabilized spermatozoa).
Aliquots of sperm suspension were incubated for 30min
with 0-1% B S A t l m g m l " 1 fucoidin followed by the
addition of FITC-BSA-fucose for another 30min. Spermatozoa were washed twice in PBS or sucrose buffer and
viewed with a Zeiss phase-contrast microscope equipped
with epifluorescence illumination.
Other procedures
Protease activity was measured spectrophotometrically
using N-<*-benzoyl-L-arginine ethyl ester (BAEE) as substrate.
Results
Assessment of neoglycoprotein probes
Preliminary experiments showed that the synthesized
(125I-BSA-fucose and 125I-BSA-mannose) and commercial (FITC-BSA-fucose) neoglycoproteins gave
identical results when used to probe Western blots of
sperm proteins (cf. Fig. lc and li). Since, in future
experiments, we wished to investigate the distribution of these proteins on spermatozoa by fluorescence, the results presented below are those obtained with the same probe throughout i.e.
FITC-BSA-fucose. Preliminary experiments again
showed that uptake of the fluorescent probe on blots
was maximum after 2h incubation and that the
relative amounts bound were stoichiometric within
the limits of the experimental conditions used (results
not shown).
783
Identification of saccharide-binding proteins from
boar spermatozoa
When Western blots of proteins that had been
extracted from ejaculated spermatozoa at pH3 were
probed with FITC-BSA-fucose, strong binding of
the neoglycoprotein was detected over two components which migrated as a doublet and which had
average Mrof 53X103 (Figs lc, 2A). If the proteinase
inhibitor pAB (2 HIM) was omitted from the washing
and extraction media then the upper component of
the doublet disappeared and instead a M^SxlO 3
protein was detected in addition to the Mr53'x\Oi
(Fig. le). Weak binding of the probe was also found
over a protein at M r 67xl0 3 and over three or four
lower Mr proteins ranging from 18-24 xlO 3 . The
majority of these low MT proteins may be derived
from seminal plasma as: (i) they were present in small
amounts on blots of proteins in pH3 extracts of
spermatozoa taken from the cauda epididymidis
(Figs lg, 2A) and (ii) blots of seminal plasma showed
strong binding of FITC-BSA-fucose to proteins with
Mr of 18-24X103 (Figs Id, 2A). Seminal plasma also
contained a higher MT protein of 65 x 103 that bound
the neoglycoprotein probe. However, the A/..53X103
sperm components were never detected in seminal
plasma. Control blots probed with FITC-BSA
showed only weak background fluorescence
(Fig. lh). Inclusion of 2mM-pAB in the FITC-BSA-fucose solution had no effect on the binding of the
probe to any of the aforementioned proteins suggesting that protease activity was not involved in the
recognition process (Fig. If). Extensive washing
(30min with shaking) of blots with 0-1% NP-40 in
PBS pH7-2 or 1-OM-NaCl pH7-2 did not remove
significant amounts of bound probe; densitometric
analysis showed that fluorescence was reduced by
<10% relative to controls.
These results suggest that recognition and binding
of the FITC-BSA-fucose probe to specific sperm
proteins on Western blots was mediated through its
carbohydrate moiety. This view was supported by the
observation that if 3 % (w/v) ovalbumin (5 % carbohydrate) was substituted for BSA as the general
protein blocking agent on blots then binding of
FITC-BSA-fucose to the sperm M^SxlO3 doublet
proteins was reduced to background levels (results
not shown). Conversely, blocking with nonglycosylated proteins such as 3 % (w/v) carbonic anhydrase
or 3 % (w/v) soybean trypsin inhibitor had no effect
on uptake of the neoglycoprotein probe.
To obtain information on the specificity of the
M..53X103 sperm proteins for different carbohydrates, competition studies were carried out by
preincubating blots with a variety of saccharides
before probing with FITC-BSA-fucose. As shown in
Fig. 2B, uptake of the neoglycoprotein was inhibited
784
R. Jones, C. R. Brown and R. T. Lancaster
a
b
c
d
e
f
g
67-
m
53-
I
o
38-
18-
Fig. 1. Western blots of proteins previously separated by SDS-PAGE, in pH3 extracts of boar spermatozoa or seminal
plasma stained with Coomassie Blue R-250 (tracks a and b) or probed with FITC-BSA-fucose (tracks c-g) or 12iIlabelled BSA-mannose (track i). Tracks a and c, ejaculated spermatozoa. Tracks b and d, seminal plasma. Track e,
proteins extracted from ejaculated spermatozoa at pH3 in absence of 2mM-pAB. Track f, ejaculated spermatozoa
probed with FITC-BSA-fucose in presence of 2mM-pAB. Track g, cauda epididymidal spermatozoa. Track h,
ejaculated spermatozoa probed with FITC-BSA alone. Track i, ejaculated spermatozoa probed with 125I-labelled
BSA-mannose. Track j , ejaculated spermatozoa probed with l25I-labelled BSA alone.
by >90% if blots were incubated with 2mgml '
fucoidin (approx. 20 ^M assuming MTlxltf; a
branched heteropolysaccharide consisting mostly of
L-fucose sulphate residues but in addition containing
xylose, glucose, galactose and glucuronic acid: Medcalf & Larsen, 1977) or lOmgmF 1 dextran sulphate
(20//M assuming an M^SxlO5; a linear polymer of
a(l-6) linked glucose units). Low Mr dextran sulphate (Mr 5X103) did not compete at 20 ^M but
inhibited 80% at 20 IHM. Sulphation appears to be
important for the competitive activity of dextran
sulphate since dextran itself was not inhibitory. However, this cannot be due solely to charge effects of
sulphate groups as another sulphated polysaccharide
such as chondroitin sulphate (40mgml~') did not
compete. Polysaccharides such as mannan
(lOmgmF 1 ), inulin (50mgml~') amylopectin
(lOmgmP 1 ) and hyaluronic acid (lOmgml"1) were
ineffective as blocking agents (results not shown).
Also ineffective in this respect were a variety of
monosaccharides (0-2 M-L-fucose or -D-fucose or -Dgalactose or -D-mannose or -D-glucose), disaccharides
(0-2M-sucrose or -lactose and -cellobiose), a trisaccharide (0-2M-raffinose), amino sugars (0-2M-acetylglucosamine, or acetylgalactosamine) and a combination of 0-2 M-lactose + 0-2 M-L-fucose + 0-2 M-Dmannose.
As a further test of specificity, blots were probed
with an l25I-labelled BSA-mannose neoglycoprotein.
As shown in Fig. li, this probe bound strongly to the
M..53X103 proteins to produce a pattern identical to
that found with the FITC-BSA-fucose. 125I-labelled
BSA gave only background labelling (Fig. lj).
Therefore, although at present we cannot reach
any simple conclusion regarding the specificity of
carbohydrate binding by the sperm M ^ x l O 3 proteins, it would seem that there is preference for a
multivalent polysaccharide structure containing sulphated sugars (especially fucose and glucose) or for
multiple monosaccharides bound to a protein. In the
latter case, it may be that 'clustering' of sugars on a
protein backbone is the most crucial requirement for
binding and that this property can be fulfilled by a
variety of saccharides.
Characterization of the major carbohydrate-binding
proteins from boar spermatozoa
The major carbohydrate-binding proteins from ejaculated spermatozoa with approximate Mr 53 x 103 were
characterized further according to their biochemical
and cytochemical properties. Several features identified them as proacrosin, the zymogen form of the
acrosomal protease acrosin. First, purified boar
sperm proacrosin migrates on SDS-PAGE as a
Role of boar sperm proacrosin in fertilization
Mr X
67
I
53
1
785
10"3
38
I
18
1
S 01
u
•3.
o
E
o
Bo-05
C
o
a
10
67
1
53
I
38
1
18
1
O 2
Fig. 2. (A) Densitometric scans of pH 3 extracted
proteins on Western blots probed with
FITC-BSA-fucose. Ejaculated spermatozoa (
).
Cauda epididymidal spermatozoa (
). Seminal plasma
(
). (B) Densitometric scans of Western blots of pH3
extracted proteins from ejaculated spermatozoa not
preincubated (
) or preincubated with 2mgml ~ '
fucoidin ( • •
) or lOmgmF 1 dextran sulphate
(•
A) before addition of the FITC-BSA-fucose
probe. Blots preincubated with 40mgml~1 chondroitin
sulphate, lOmgrnP 1 mannan lOmgrnl"1 amylopectin or
lOmgmr 1 hyaluronic acid were identical to the control.
doublet with Mr53 and 55X103 (Polakoski & Parrish,
1977). These values are very close to those found in
this investigation for the carbohydrate-binding proteins. Also, as observed here and as reported by
Polakoski & Parrish (1977), the higher Mr component
of the doublet was only detected if protease inhibitors
were included in the media for washing and extraction of spermatozoa. Second, analysis of proteins in
pH3 extracts on two-dimensional gels revealed that
the major carbohydrate-binding proteins at
M ^ x l O 3 have a pi of >10-0 (results not shown).
Boar sperm proacrosin has a pH of 10-5 (MullerEsterl et al. 1980). Third, if the acrosomes of washed
spermatozoa were disrupted by freezing and thawing
followed by incubation of cells in 0-lM-Tris-HCl
pH8-4 at 0°C, then protease activity towards BAEE
20
Time (min)
40
90 180
Fig. 3. Activation profile of sperm proacrosin to acrosin
as measured spectrophotometrically against BAEE
substrate (
) and by SDS-PAGE followed by Western
blotting and probing with FITC-BSA-fucose. Track a,
Omin. Track b, lOmin. Track c, 40min. Track d, 180min.
substrate appeared rapidly (Fig. 3) and reached a
maximum after 20 min. Concomittant with the detection of enzymatic activity the M r 53xl0 3 doublet
proteins diminished in amount (as detected on blots
with FITC-BSA-fucose) whilst a M r 49xl0 3 component, and later a M^SxlO3 one, appeared. Both
the latter proteins retained their carbohydrate-binding capacity. This activation profile is highly characteristic of mammalian sperm proacrosin and boar
sperm proacrosin in particular (Polakoski & Parrish,
1977). Fourth, fluorescence microscopy of spermatozoa stained with FITC-BSA-fucose revealed that
carbohydrate-binding proteins were located within
the acrosome. Intact spermatozoa showed no detectable fluorescence (Fig. 4A) whereas 100 % of permeabilized spermatozoa were positive, the distribution depending on the ionic strength of the
medium. In PBS, medium fluorescence was uneven
over the head, frequently with a 'halo' around the
anterior tip of the acrosome (Fig. 4B), whereas in
0-264 M-sucrose/10 mM-Hepes pH7-4 the entire
sperm head was uniformly fluorescent (Fig. 4C). A
weak reaction was also seen on areas of the flagellum.
Harrison et al. (1982) have reported a similar effect of
ionic strength on the immunocytochemical appearance of proacrosin/acrosin in ram spermatozoa
stained with anti-acrosin antibodies. Incubation of
permeabilized spermatozoa with fucoidin (2 mg ml ~ ')
before addition of FITC-BSA-fucose blocked completely uptake of the fluorescent probe (Fig. 4D).
Recognition of sperm proacrosin by I25l-labelled
zona pellucida glycoproteins
The natural substrate for sperm proacrosin/acrosin in
vivo is considered to be the zona pellucida that
786
R. Jones, C. R. Brown and R. T. Lancaster
r*Fig. 4. Pairedfluorescenceand phase-contrast photomicrographs of washed boar spermatozoa stained with
FITC-BSA-fucose. (A) Intact spermatozoa. (B) Permeabilized spermatozoa suspended in PBS. (C) Permeabilized
spermatozoa suspended in 0-264M-sucrose/10mM-Hepes pH7-4. (D) As for C except for preincubated with 2mgml~'
fucoidin before addition of FITC-BSA-fucose. Bar, 5f«n.
surrounds the egg and, in this sense, the various
probes described above are highly artificial. Therefore, it was important to investigate if (i) glycoproteins from the zona pellucida would recognize and
bind to sperm proacrosin/acrosin in a manner similar
to that for FITC-BSA-fucose and (ii) if so, was
recognition mediated via their carbohydrate moieties.
As shown in Fig. 5a and as reported previously
(Brown & Jones, 1987) there was strong binding of
125
I-labelled zona glycoproteins to sperm proteins at
Mr53 and 67X103 with weaker recognition at MT, 38
and 18-24xlO3. The labelled probe could not be
displaced by washing blots with 0-1 % NP-40 or 1MNaCl but it could be prevented from binding if blots
were preincubated with fucoidin (2mgmP', Fig. 5b)
or dextran sulphate (lOmgmP 1 , Fig. 5c). However,
preincubating with a mixture of 0-2M-L-fucose,
+0-2M-mannose + 0-2M-lactose, or mannan (5mg
ml" 1 ), or chondroitin sulphate (40mgml~1) had no
effect on uptake of the 125I-labelled zona probe
(results not shown). These results, therefore, are very
similar to those obtained with the FITC-BSA-fucose
probe that a protein-carbohydrate recognition mechanism is involved in binding of 125I- labelled zona
glycoproteins to sperm proteins on Western blots. Pig
« 67-
° 53X
^•38-
18-*
Fig. 5. Autoradiograph of Western blots of pH3
extracted proteins from ejaculated spermatozoa probed
with 125I-labelled zona pellucida glycoproteins or I25Ilabelled fucoidin. Track a, zona probe alone. Track b,
blot preblocked 2mgml~' fucoidin followed by zona
probe. Track c, blot preblocked lOmgml"1 dextran
sulphate (MT5xl0?) followed by zona probe. Track d,
fucoidin probe alone.
Role of boar sperm proacrosin in fertilization
zona glycoproteins have been analysed extensively by
Dunbar et al. (1980) and Hedrick & Wardip (1987)
and found to contain on average 19 % carbohydrate.
/3-jV-acetylglucosamine, mannose, sialic acid and
fucose are prominant constituents. The glycoproteins
are also sulphated to approximately 2 % (Dunbar et
al. 1980) and display considerable charge heterogeneity (Urch, 1986).
It is possible that the A/..67X103 molecular species
that is recognized by 125I-labelled zona glycoproteins
in Fig. 5a represents the high molecular weight form
of boar proacrosin reported by Berruti (1985) who
considered it to be the true zymogen and ascribed to it
an Mr of 66X103. If so, then it differs from the
MJ.53X103 proacrosin form in having a greater affinity for the zona probe than for neoglycoprotein
probes (compare Fig. 5a with Fig. li). However,
further work is required to establish if there is any
primary relationship between these two zona-binding
proteins.
Recognition of sperm proacrosin by l251-labelled
fucoidin
The efficiency of fucoidin in blocking recognition of
proacrosin on Western blots by neoglycoproteins and
zona pellucida glycoproteins and cytochemically on
whole spermatozoa may be due to a generalized or
specific inhibition. To try and resolve this problem,
Western blots of sperm proteins were probed with
125
I-labelled fucoidin as described above for the zona
glycoproteins. As shown in Fig. 5d, the probe bound
strongly to M r 67xl0 3 and M^SxlO 3 components
and weakly to M^SxlO 3 and M..18X103 species.
Other major proteins on the blot (see Fig. la) were
not recognized by the fucoidin probe suggesting that
its inhibitory effects are specific and not generalized.
Effects of pretreatment of the zona probe with
acrosin on its capacity to recognize sperm proteins
on Western blots
Gwatkin et al. (1977) have reported that preexposure
of hamster zonae to soluble homologous acrosin
inhibits fertilization in vitro, implying that the enzyme
has a deleterious effect on sperm-binding sites. To
test this possibility using Western blotting techniques,
intact pig zonae were incubated for 60min with
0-5mgml~' active boar acrosin (a treatment that
causes limited proteolysis of the zona glycoproteins to
lower MT components; (Urch et al. 1985; Brown &
Cheng, 1986; Urch, 1986) the eggs washed free of
enzyme and the solubilized, labelled glycoproteins
were then used as probes as before. As shown in
Fig. 6, this treatment enhances the avidity of the zona
glycoproteins for the M.53X163 and Mr 18-24xlO3
sperm proteins. Densitometry of autoradiographs
from two separate experiments showed a twofold to
67
M, x icr 3
53
38
I
1
1
1
1
1
787
18
I
Fig. 6. Densitometric scans of autoradiographs taken
from Western blots of pH 3 extracted proteins probed
with 125I-labelled glycoproteins from normal zonae (
)
or from zonae pretreated with active boar sperm acrosin
(
). Results shown represent two separate
experiments. In each experiment, equal numbers of
zonae were incubated with 0-5mgml~' acrosin for 30min
or with medium alone. Zonae were then washed,
iodinated and equal numbers of ctsmin"1 (approx.
SxlO'ctsmin"1) used to probe Western blots under
identical conditions.
sevenfold increase relative to controls. Therefore,
these results suggest that proteolysis with acrosin
does not destroy sperm-binding sites on zona glycoproteins but instead appears to increase their affinity
for the complementary sperm ligand.
In vitro sperm-zona binding experiments
The capacity of sperm proacrosin/acrosin to interact
with carbohydrate moieties of natural and neoglycoproteins suggests that it may have a role in sperm-egg
recognition and binding in addition to any putative
action in assisting the sperm penetrate the zona. To
explore this possibility further under physiologically
relevant conditions, several of the aforementioned
compounds that inhibited or enhanced recognition on
Western blots were investigated for their effects on
binding of spermatozoa to eggs in a proven in vitro
fertilization system.
788
R. Jones,
C. R. Brown and R. T. Lancaster
Table 1. Binding of capacitated boar spermatozoa to the zona pellucida of homologous eggs; effects of
polysaccharides, protease inhibitors and pretreatment of gametes with acrosin or fucoidin
Reagent
Fucoidin
Final
concentration
in medium
1
Omgml"
0-5mgml-'
l-Omgrnl"1
Fucoidintreated eggs
Omgml" 1
Fucoidintreated sperm
Omgml" 1
0-5mgml~'
Mannan
Omgml" 1
lOmgrnl" 1
Chondroitin
sulphate C
Omgml" 1
lOmgrnl" 1
Soybean
trypsin inhibitor
pAB
Omgml" 1
Acrosintreated zonae
0 mgml" 1
0-5mgmr'
2-5mgmr1
OmM
0-05 mM
0-5 mM
O-Smgral"1
Total number of
eggs with
bound sperm
39/40
15/33
22/35
43/43
43/43
52/52
53/53
30/30
32/32
40/40
41/43
37/39
20/40
58/60
16/16
1/14
13/13
15/15
Mean number
of sperm per
egg
5-6
0-7"*
1-5"*
% Controls
100
13
27
54-2
54-4+
100
100
631
100-6***
100
159
73-4
79-4+
100
108
57-9
56-6+
100
98
5-4
1-0***
22-5
15-8+
0-1"*
100
19
100
70
0-4
11-2
21-5*
100
192
* Significantly different from controls, P< 0-05.
"'Significantly different from controls, P<0-001.
+ Not significantly different from controls, P<0-05.
Statistical analyses were carried out by a paired V test
As shown in Table 1, the presence of 0-5mgml ]
fucoidin in the fertilization medium reduced the
number of sperm binding by 80-90 % as did the
protease inhibitors, pAB (0-5 mM) and soybean trypsin inhibitor (2-5mgml~'). Mannan (lmgmP 1 ) and
chondroitin sulphate (lmgml" 1 ) on the other hand
had no significant effect on the number of spermatozoa that bound firmly to the zona whereas preexposure of eggs to boar acrosin (0-5 ing ml" 1 , under
the same conditions as described in the previous
section) enhanced sperm binding by 92%. In all of
these experiments, motility of spermatozoa was unaffected by the levels of reagents used.
To investigate the site of action of fucoidin in
blocking fertilization, capacitated spermatozoa or
cumulus-free eggs were exposed separately to the
polysaccharide (0-5mgml -1 for 30min) followed by
washing and incubation with the complementary
nontreated gamete in the usual manner. No significant inhibitory effects of this treatment were observed (Table 1) suggesting that fucoidin exerts its
effects at the point of sperm-egg recognition and
binding and not before it. This conclusion would be
consistent with the apparently specific interaction of
125
I-labelled fucoidin with proacrosin/acrosin
(Fig. 5d).
Discussion
This work has shown that a protein with an affinity for
polysaccharide residues on zona pellucida glycoproteins and on neoglycoproteins is localized within
the acrosome of boar spermatozoa and that this
protein has the properties of proacrosin. The evidence suggests a role for proacrosin in sperm-egg
recognition and adhesion during early stages of fertilization.
The important discovery by Vacquier & Moy
(1977) of a lectin-like protein (termed 'bindin') within
the acrosome of sea urchin spermatozoa gave considerable impetus to an early hypothesis that interaction of mammalian gametes was based on a proteincarbohydrate binding mechanism (reviewed by
Yanagimachi, 1981). This view has recently been
enhanced by direct experimental evidence that Olinked carbohydrates on one specific zona glycoprotein can act as a sperm receptor (Wassarman,
1987). The question then arises as to the nature of the
complementary carbohydrate-binding component on
spermatozoa.
In the mouse, one contender is surface-bound
galactosyltransferase (Shur, 1986) which, presumably, recognizes galactose/N-acetylglucosamine residues on zona glycoproteins to produce a polylactosamine complex that mediates attachment of
Role of boar sperm proacrosin in fertilization
spermatozoa. A similar enzyme-substrate complex is
envisaged for a trypsin-inhibitor-sensitive site on
mouse sperm (Saling, 1981; Benau & Storey, 1987),
although the interplay between this molecule and
galactosyltransferase is not clear at present. A third
candidate is a group of fucose-binding proteins recently described on boar spermatozoa (Topfer-Petersen et al. 1985). Fluorescence and ultrastructural
studies revealed that, although some of these proteins
were located on the plasma membrane overlying the
anterior tip of the sperm head, the number of fucosebinding sites increased substantially after induction of
the acrosome reaction (Friess et al. 1987). Our results
are very similar to these observations and taken
together are consistent with the view that the major
(i.e. Mr53xl03) carbohydrate-binding protein in
boar spermatozoa is intra-acrosomal and represents
proacrosin. Interestingly, FITC-fucoidin and FITClabelled zona glycoproteins have been shown to bind
to the head of acrosome-reacted guinea pig and rabbit
spermatozoa (Huang & Yanagimachi, 1984; O'Rand
& Fisher, 1987). The localization on boar spermatozoa of the minor components at MT18-24 xlO3 that
are also recognized by the neoglycoprotein probes is
not known at present but the possibility cannot be
excluded that they are found on the plasma membrane and that they may have a significant functional
role in the very early stages of sperm-egg attachment.
The observation that boar sperm proacrosin has the
capacity to interact with carbohydrate moieties of
glycoproteins raises some interesting parallels to the
bindin molecule in sea urchin spermatozoa. First,
both proteins are found within the acrosome and are
only exposed following the acrosome reaction. Thus,
they are targetted to their site of action and are
protected from spurious interactions with other cells.
Because of their intracellular location, low specificity
of recognition can be tolerated. Second, amino acid
and N-terminal sequence analysis show that bindin
and proacrosin are hydrophobic proteins with a
strong tendency to adhere to surface structures
(Vacquier & Moy, 1978; Fock-Nuzel et al. 1980).
Third, bindin and proacrosin have the capacity to
recognize polysaccharide residues on glycoproteins.
It must be stressed at this point that whilst bindin
meets many of the classical requirements of a true
lectin (Barondes, 1981), it would be premature to
refer to proacrosin in such terms in view of its
potential protease activity. Nevertheless, there are
striking similarities between bindin and proacrosin in
their affinities for carbohydrates. The haemagglutinating activity of bindin is strongly inhibited by
polysaccharides but poorly by monosaccharides
(Glabe et al. 1982). The rank order of their effectiveness (in terms of ^M-saccharide equivalent for 50 %
789
inhibition) was judged to be l:4:20:6700 for fucoidin,
xylan, dextran sulphate and L-fucose, respectively.
Mannan was a weak competitor relative to fucoidin in
direct binding assays. Essentially similar results were
obtained in the present work based on the ability of
polysaccharides to block recognition of FITC-BSA-fucose on Western blots. Fucoidin and dextran
sulphate were highly effective whereas mannan,
chondroitin sulphate and a wide variety of mono- and
disaccharides were unable to compete. We have also
found that pH3 extracts of either cauda epididymidal
or ejaculated spermatozoa will agglutinate trypsinized rabbit red cells (R. Jones, unpublished observations). At present we cannot attribute this to
proacrosin per se as the purified molecule has not
been tested but fucoidin and dextran sulphate were
able to prevent agglutination whereas free monosaccharides (0-1 M) were unable to do so.
In fact, dependence on tertiary structure is characteristic of many plant lectins, particularly the so called
class II lectins (Gallagher, 1984) which can be effectively inhibited only by linear or branched carbohydrate sequences and exhibit little or no affinity for
monosaccharides. Some lectins are also responsive to
'cluster effects' in which high local concentrations of
the complementary sugar on a protein backbone can
increase affinity constants several thousand fold (Gallagher, 1984). Examples of animal lectins with these
properties are also known, such as those described on
the vitelline membrane of the hen's egg (Cook et al.
1985) on mouse lymphocytes (Parrish et al. 1984) and
on teratocarcinoma cells (Grabel et al. 1981). It would
seem, therefore, that bindin and boar sperm proacrosin belong to an emerging class of proteins that have
strong affinity for multivalent polysaccharide chains.
The structural properties of these chains are of
primary importance for recognition and this can be
satisfied by polysaccharides of slightly different composition; hence, the apparent lack of sugar specificity.
Our discovery that proacrosin/acrosin binds to the
carbohydrate moiety of glycoproteins suggests that it
has an important role in sperm-egg recognition and
adhesion over and above any putative function it may
have in zona penetration. As a working hypothesis we
propose that, in the pig, the fertilizing sperm encounters the zona with either an intact plasma membrane
overlying the head or with an incipient acrosome
reaction. If the former, then loose attachment may
take place via surface-bound molecules (e.g. galactosyltransferase) but whatever the case a full acrosome reaction rapidly ensues. Zona glycoproteins
have been shown to stimulate the acrosome reaction
(O'Rand & Fisher, 1987). Released proacrosin/acrosin, which has been shown to relocate onto the
external surface of the acrosomal cap as the acrosomal matrix disperses (Shams-Borhann et al. 1979),
790
R. Jones, C. R. Brown and R. T. Lancaster
Acrosome
Zona
pellucida
Fig. 7. Diagram of proposed hypothesis for role of
proacrosin/acrosin in consolidating binding of boar sperm
to the zona pellucida. (1) Capacitated spermatozoa reach
the zona with either an intact acrosome (a) or incipient
acrosome reaction (b). (2) Full acrosome reaction takes
place with dispersal of acrosomal contents. Some
proacrosin/acrosin relocates onto the surface of the
acrosomal cap where it cross-links to carbohydrate
moieties of zona pellucida glycoproteins. This interaction
is enhanced by the limited proteolytic action of acrosin on
the zona, a process that may be regulated by the
endogenous acrosin inhibitor. (3) The sperm head is
sheared away from the acrosomal cap (which remains
behind on the zona surface) by forces generated from the
motile flagellum and penetration of the zona begins.
then interacts with carbohydrate chains on zona
glycoproteins to crosslink the cap firmly to the zona
pellucida. Consolidation of this binding is enhanced
by the localized proteolytic effect of acrosin on the
zona, thereby increasing the avidity of the protein-carbohydrate interaction. A mechanism of this sort
is well known from haemagglutination studies in
which the efficiency of agglutination of red cells by
lectins is increased (and in many cases is obligatory,
e.g. galaptin; Harrison et al. 1984) by trypsinization, a
treatment that is thought to make carbohydrates on
cell membranes more accessible to agglutinins by
perturbing surface charge and altering stereochemistry of attached proteins. An analogous situation may be required for 'agglutination' of spermatozoa to the zona. Subsequent to these events the
shearing forces generated by the motile flagellum
would detach the sperm head away from the acrosomal cap (which remains behind on the zona surface
as a 'ghost') and penetration of the zona would be
initiated. There is no evidence that proacrosin/acrosin is present on the inner acrosomal membrane
(Shams-Borhan et al. 1979) and hence the sperm head
would not be bound irreversibly to the zona. Fig. 7
summarizes this sequence of events.
This hypothesis is supported to an appreciable
extent by data from in vitro fertilization experiments.
Those polysaccharides that were most effective, e.g.
fucoidin, or ineffective, e.g. mannan, in blocking
recognition of glycoprotein probes to proacrosin on
Western blots were correspondingly successful or
unsuccessful in reducing sperm binding to eggs in
vitro. Likewise, predigestion of zonae with acrosin
increased the affinity of zona probes for proacrosin on
Western blots and doubled the number of sperm
binding in vitro. pAB was very potent in blocking
sperm-zona binding in vitro, suggesting that as in the
mouse a trypsin-inhibitor-sensitive site is involved in
recognition (Saling, 1981). The location of such a site
on boar sperm is not known so this result must be
interpreted with caution. However, it is not inconsistent with the view thatpAB either inhibits dispersal of
the acrosomal matrix (Harrison et al. 1982) (and
hence proacrosin would not be available for consolidated binding) or else it prevents proteolysis of the
zona by acrosin necessary to increase the number of
ligand sites available for binding. Answers to these
questions will only become available when we have
information on the exact status of the sperm acrosome at the first point of contact with the zona under
the conditions we have used in our in vitro fertilization system.
We are grateful to Drs F. L. Harrison, S. G. Gaunt and
R. A. P. Harrison for helpful discussions, Dr R. M. Moor
for his interest and encouragement and Mrs Linda Notton
for typing the manuscript. We thank Mr K. I. von Glos for
technical assistance and Professor D. H. Northcote for the
gift of BSA-mannose.
References
AHUJA, K. K. (1982). Fertilization studies in the hamster:
The role of cell surface carbohydrates. Expl Cell Res.
140, 353-362.
BARONDES, S. H. (1981). Lectins: their multiple
endogenous cellular functions. A. Rev. Biochem. 50,
207-231.
BENAU, D. A. & STOREY, B. T. (1987). Characterization
of the mouse sperm plasma membrane zona-binding
site sensitive to trypsin inhibitors. Biol. Reprod. 36,
282-292.
BERRUTI, G. (1985). Evidence of a high molecular weight
form of acrosin in boar acrosomal extract. Int. J.
Biochem. 17, 87-94.
BRADFORD, M. M. (1976). A rapid and sensitive method
for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Analyt.
Biochem. 11, 248-254.
Role of boar sperm proacrosin in fertilization
BROWN, C. R. & CHENG, W. T. K. (1986). Changes in
composition of the porcine zona pellucida during
development of the oocyte to the 2- to 4-cell embryo.
J. Embryol. exp. Morph. 92, 183-191.
BROWN, C. R. & JONES, R. (1987). Binding of zona
pellucida proteins to a boar sperm polypeptide of Mr
53000 and identification of zona moieties involved.
Development 99, 333-339.
CHENG, W. T. K. (1985). In vitro fertilization of farm
animal oocytes. PhD thesis. Council for National
Academic Awards. Institute of Animal Physiology,
Cambridge, UK.
COOK, G. M. W., BELLAIRS, R., RUTHERFORD, N. G.,
STAFFORD, C. A. & ALDERSON, T. (1985). Isolation,
characterization and localization of a lectin within the
vitelline membrane of the hen's egg. J. Embryol. exp.
Morph. 90, 389-407.
DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P.
A. & SMITH, F. (1956). Colorimetric method for
determination of sugars and related substances. Anal.
Chem. 28, 350-356.
DUNBAR, B. S., WARDRJP, N. J. & HEDRICK, J. K. (1980).
Isolation, physiochemical properties, and
macromolecular composition of zona pellucida from
porcine oocytes. Biochemistry 19, 356-365.
FOCK-NUZEL, R., LOTTSPEICH, F., HENSCHEN, A.,
MULLER-ESTERL, W. & FRITZ, H. (1980). N-terminal
amino acid sequence of boar sperm acrosin. Homology
with other serine proteinases. Hoppe-Seylers Z.
Physiol. Chem. 361, 1823-1828.
FRAZIER, W. & GLASER, L. (1979). Surface components
and cell recognition. A. Rev. Biochem. 48, 491-523.
FREISS, A. E., TOPFER-PETERSEN, E., NGUYEN, H. &
SCHILL, W.-B. (1987). Electron microscopy localization
of a fucose-binding protein in acrosome boar
spermatozoa by the fucosyl-peroxidase-gold method.
Histochemistry 86, 297-303.
GALLAGHER, J. T. (1984). Carbohydrate-binding
properties of lectins: a possible approach to lectin
nomenclature and classification. Bioscience Reports 4,
621-632.
GLABE, C. G., GRABEL, L. B., VACQUIER, V. D. &
ROSEN, S. D. (1982). Carbohydrate specificity of sea
urchin sperm bindin: a cell surface lectin mediating
sperm-egg adhesion. J. Cell Biol. 94, 123-128.
GRABEL, L. B., GLABE, C. G., SINGER, M. S., MARTIN, G.
R. & ROSEN, S. D. (1981). A fucan specific lectin on
teratocarcinoma stem cells. Biochem. biophys. Res.
Commun. 102, 1165-1171.
GREEN, J. D. & SUMMERS, R. G. (1980). Ultrastructural
demonstration of trypsin-like protease in acrosomes of
sea urchin sperm. Science 209, 398-400.
GWATKIN, R. B. L., WUDL, L., HARTREE, E. F. & FINK,
R. (1977). Prevention of fertilization by exposure of
hamster eggs to soluble acrosin. J. Reprod. Fert. 50,
359-361.
HARRISON, F. L., FITZGERALD, J. E. & CATT, J. W.
(1984). Endogenous /S-galactoside-specific lectins in
rabbit tissues. J. Cell Sci. 72, 147-162.
791
R. A. P. (1976). A highly efficient method for
washing mammalian spermatozoa. J. Reprod. Fert. 48,
347-353.
HARRISON,
HARRISON, R. A. P., FLECHON, J. & BROWN, O. R.
(1982). The localization of acrosin and proacrosin in
ram spermatozoa. J. Reprod. Fert. 66, 349-358.
HEDRICK, J. L. & WARDIP, N. J. (1987). On the
macromolecular composition of the zona pellucida from
porcine oocytes. Devi Biol. 121, 478-488.
HUANG, T. T. F., OHZU, E. & YANAGIMACHI, R. (1982).
Evidence suggesting that L-fucose is part of a
recognition signal for sperm-zona pellucida attachment
in mammals. Gamete Res. 5, 355-361.
HUANG, T. T. F. & YANAGIMACHI, R. (1984). Fucoidin
inhibits attachment of guinea pig spermatozoa to the
zona pellucida through binding to the inner acrosomal
membrane and equatorial domains. Expl Cell Res. 153,
363-373.
JOHNSON, G. D. & HOLBOROW, E. J. (1986). Preparation
and use offluorochromeconjugates. In Handbook of
Experimental Immunology, vol. I (ed. D. M. Wier),
pp. 28.1-28.21. London: Blackwell.
JONES, R. & BROWN, C. R. (1987). Identification of a
zona-binding protein from boar spermatozoa as
proacrosin. Expl Cell Res. 171, 503-508.
LAEMMLI, U. K. (1970). Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature, Lond. 227, 680-685.
MARKWELL, M. A. K. & Fox, C. F. (1978). Surfacespecific iodination of membrane proteins of viruses and
eucaryotic cells using l,3,4,6-tetrachloro-3a',6a'diphenyl-glycoluril. Biochemistry 17, 4807^1817.
MEDCALF, D. G. & LARSEN, B. (1977). Fucose-containing
polysaccharides in the brown algae Ascophyllum
nodosum and Fucus vesiculosus. Carbohydr. Res. 59,
531-537.
MULLER-ESTERL, W. S., KUPFER, S. & FRITZ, H. (1980).
Purification and properties of boar acrosin. HoppeSeylers Z. Physiol. Chem. 361, 1811-1821.
O'FARRELL, P. H. (1975). High resolution twodimensional electrophoresis of proteins. /. biol. Chem.
250,4004-4021.
O'RAND, M. G. & FISHER, S. J. (1987). Localization of
zona pellucida binding sites on rabbit spermatozoa and
induction of the acrosome reaction by solubilized
zonae. Devi Biol. 119, 551-559.
PARRISH, C. R., RYLATT, D. B. & SNOWDEN, J. N. (1984).
Demonstration of lymphocyte surface lectins that
recognize sulphated polysaccharides. J. Cell Sci. 67,
145-158.
PETERSON, R. N., RUSSELL, L. D. & HUNT, W. P. (1984).
Evidence for specific binding of uncapacitated boar
spermatozoa to porcine zonae pellucida in vitro. J. exp.
Zool. 231, 137-147.
POLAKOSKI, K. L. & PARRISH, R. F. (1977). Boar
proacrosin. Purification and preliminary activation
studies of proacrosin isolated from ejaculated boar
sperm. J. biol. Chem. 252, 1888-1894.
POLGE, C. (1982). Embryo transplantation and
preservation. In Control of Pig Reproduction (ed. D. J.
792
Ft. Jones,
C. R. Brown and R. T. Lancaster
A. Cole & G. R. Foxcroft). pp. 277-291. London:
Butterworths.
SALING, P. M. (1981). Involvement of trypsin-like activity
in binding of mouse spermatozoa to zonae pellucidae.
Proc. natn. Acad. Sci. U.S.A. 78, 6231-6235.
SCHWARTZ, B. A. & GRAY, G. R. (1977). Proteins
containing reductively animated disaccharides. Archs
Biochem. Biophys. 181, 542-549.
SHALGI, R., MATITYAHU, A. & NEBEL, L. (1986). The role
of carbohydrates in sperm-egg interaction in rats. Biol.
Reprod. 34, 446-452.
SHAMS-BORHAN, G., HUNEAU, D. & FLECHON, J.-E.
(1979). Acrosin does not appear to be bound to the
inner acrosomal membrane of bull spermatozoa. /. exp.
Zool. 209, 143-149.
SHUR, B. D. (1986). The receptor function of
galactosyltransferase during mammalian fertilization.
Adv. exp. Med. Biol. 207, 79-93.
TOPFER-PETERSEN, E., FRIESS, A. E., NGUYEN, H. &
SCHILL, W.-B. (1985). Evidence for a fucose-binding
protein in boar spermatozoa. Histochem. 83, 139-145.
TOWBIN, H., STAEHELIN, T. T. & GORDON, J. (1979).
Electrophoretic transfer of proteins from polacrylamide
gels to nitrocellulose sheets: procedure and some
applications. Proc. natn. Acad. Sci. U.S.A. 76,
4350-4354.
URCH, U. A. (1986). The action of acrosin on the zona
pellucida. Adv. exp. Med. Biol. 207, 113-132.
URCH, U. A., WARDIP, N. J. & HEDRICK, J. L. (1985).
Limited and specific proteolysis of the zona pellucida
by acrosin. J. exp. Zool. 233, 479-483.
VACQUIER, V. D. & MOY, G. W. (1977). Isolation of
bindin: the protein responsible for adhesion of sperm
to sea urchin eggs. Proc. natn. Acad. Sci. U.S.A. 74,
2456-2460.
WASSARMAN, P. M. (1987). The biology and chemistry of
fertilization. Science 235, 554-560.
YANAGIMACHI, R. (1981). Mechanisms of fertilization in
mammals. In Fertilization and Embryonic Development
in Vitro (ed. L. Mastroianni & J. D. Biggers),
pp. 81-182. New York: Plenum Press.
{Accepted 20 January 1988)