Retrospective Theses and Dissertations
1972
Gamete surface molecular components and their
functional roles in sperm-egg interactions of the
horseshoe crab, Limulus polyphemus L.
(Merostomata): an immunological and
biochemical approach
Rodney Cameron Mowbray
Iowa State University
Follow this and additional works at: http://lib.dr.iastate.edu/rtd
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Recommended Citation
Mowbray, Rodney Cameron, "Gamete surface molecular components and their functional roles in sperm-egg interactions of the
horseshoe crab, Limulus polyphemus L. (Merostomata): an immunological and biochemical approach " (1972). Retrospective Theses
and Dissertations. Paper 5943.
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MOWBRAY, Rodney Cameron, 1944—
GAMETE SURFACE MOLECULAR COMPONENTS AND THEIR
FUNCTIONAL ROLES IN SPERM-EGG INTERACTIONS OF
THE HORSESHOE CRAB, LIMULUS POLYPHEMUS L.
(MEROSTOMATA): AN IMMUNOLOGICAL AND
BIOCHEMICAL APPROACH.
Iowa State University, Ph.D., 1972
Zoology
University Microfilms, A XEROX Company, Ann Arbor. Michigan
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.
Gamete surface molecular components and their functional
roles in sperm-egg interactions of the horseshoe crab,
Limulus polvphemus L. (Merostomata):
An immunological
and biochemical approach
by
Rodney Cameron Mowbray
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Department:
Major:
Zoology and Entomology
Zoology (Embryology)
Approved;
Signature was redacted for privacy.
M of flajor Work
Signature was redacted for privacy.
For the Major Department
Signature was redacted for privacy.
For the Graduate College
Iowa State University
Ames, Iowa
1972
PLEASE NOTE:
Some pages may have
indistinct print.
Filmed as received.
U n i v e r s i t y M i c r o f i l m s , A Xerox E d u c a t i o n Company
iî
TABLE OF CONTENTS
Page
INTRODUCTION
1
Historical Background
2
Gamete Surface Interacting Substances
5
Chemotaxis
Sperm motility and respiration
Sperm agglutination
Sperm capacitation
Sperm-egg attachment and cell adhesion
Initiation of the acrosome reaction
Activation of the egg
Specificity of fertilization
6
7
8
9
10
13
14
16
Molecular Aspects of Sperm-egg Interactions
18
Egg substances involved in fertilization
Sperm substances involved in fertilization
Chemical inhibitors of gamete interaction
19
23
27
Immunological Aspects of Gamete Surface Interactions
30
Egg antigens
Role of egg antigens in fertilization
Sperm antigens
Role of sperm antigens in fertilization
31
33
37
39
Ljjnulus Fertilization
42
Statement of Problem
44
MATERIALS AND METHODS
47
Gamete Collection and Preparation
47
Antibody Preparation
48
Preparation of Univalent Antibody
48
Immunodiffusion and Immunoelectrophoresis
49
Design of Egg Section Treatment Experiments
50
iii
Page
Effects of Variables on Experiments
Duration of insemination time
Incubation temperature
pH
RESULTS
51
51
51
56
61
Inhibition of Sperm-egg Attachment with Bivalent Antibody
Sperm concentration
Duration of antibody treatment
Concentration of antibody
Effects of anti-heart antibody
Tissue specificity of anti-egg antibody
Inhibition of Sperm-egg Attachment with Univalent Antibody
Duration of treatment with antibody
Dilution of globulin
Inhibition of Sperm-egg Attachment by Chemical Treatment
of the Egg Surface
Effect
Effect
Effect
Effect
of
of
of
of
pH
enzymes
detergents
selected chemical reagents
Immunological Identification of Egg Surface Substances
Immunodiffusion analysis
Immunoelectrophoresis analysis
DISCUSSION
61
61
64
64
64
69
69
84
84
85
88
91
91
96
101
102
107
113
Methodology
113
Inhibition of Sperm-egg Attachment with Bivalent Antibody
115
Inhibition of Sperm-egg Attachment with Univalent Antibody
117
Inhibition of Sperm-egg Attachment by Chemical Treatment
of the Egg Surface
120
Immunological Identification of Egg Surface Substances
128
CONCLUSIONS
132
iv
Page
SUMMARY
133
LITERATURE CITED
135
ACKNOWLEDOIENTS
151
APPENDIX;
152
SOURCES OF REAGENTS
1
INTRODUCTION
"There is perhaps no phenomenon in the field of biology that touches
so many fundamental questions as the union of the germ cells in the act of
fertilization; in this supreme event all the strands of the web of two
lives are gathered in one knot, from which they diverge again and are
re-woven in a new individual life-history
Thus to consider the
antecedents and the consequents of the process of fertilization would be to
outline all of biology" (Lillie, 1919).
The sperm-egg interactions in metazoans follow a predictable, sequen­
tial pattern; (1) proximity, (2) contact and attachment, (3) sperm acrosome reaction, (4) membrane fusion, (5) egg activation, (6) penetration of
the sperm nucleus, and (7) pronuclear fusion (cf. Metz and Monroy, 1967).
These processes are well understood with respect to morphology and ultrastructure, however, the molecular picture of fertilization is still incom­
plete.
Several investigators (cf. Metz, 1967) have demonstrated that
gamete surface molecular components are involved in these processes, par­
ticularly in reference to sperm-egg recognition and physiological triggers
leading to the sperm acrosome reaction and egg activation.
One essential step in the gamete interactions is sperm-egg attachment.
In this study, the possibility that molecular components are involved in
sperm-egg attachment is explored with the gametes of the horseshoe crab,
Limulus polvphenius.
An ^ vitro system is used in which egg sections are
manipulated through various experimental operations and then inseminated
under controlled conditions.
Inhibitors to attachment, including specific
antibodies and various chemical treatments, along with several other
2
methods are used primarily to study egg surface molecular components and
their role in sperm-egg attachment.
The experimental procedures and
results are preceded by the following historical review of fertilization
and discussion of the immunological and molecular aspects of fertilization.
Historical Background
Among his many scientific observations, Leewenhoek, in 1677, was the
first to describe and recognize the significance of spermatozoa in semen.
This discovery aroused great interest among scientists and led to specula­
tion by Hartsoeker (1694) and others that each sperm cell enclosed a com­
plete, but minute, form of the respective species.
The spermists, as they
were called, flourished until the late eighteenth century, when Spallanzani
(1785) published the results of his excellent experiments on fertilization
with frogs and toads.
Spallanzani was opposed to the spermist theory and
designed many experiments to disprove this theory.
Spallanzani proved that
semen was necessary for the activation of embryonic development, however,
he developed the misconception, contrary to his experimental results, that
the seminal fluid and not the spermatozoa was the active agent.
Few advances were made after Spallanzani until the "New Theory of
Reproduction" was published by Prévost and Dumas (1824).
Based on exten­
sive fertilization experiments with frogs, these workers proved that sper­
matozoa were essential for fertilization and believed not only that the
sperm penetrated into the egg but also that it contributed to some manner
to the organs of the developing embryo.
This work stimulated extensive
experimentation in fertilization by other investigators, in particular
Kolliker (1856) and Bischoff (1847).
They believed that the sperm only
3
needed to come into contact with the egg for development to ensue, that is,
the essentiality of the sperm only depended on its ability to activate the
egg.
For the next quarter century, few advances were made in the theory of
fertilization.
Then during the late nineteenth century with improved
optics and cytological techniques, many of the problems of cell morphology,
including the observation of sperm organelles, wera he.lTig studied inten­
sively.
Hertwig (1876) and Fol (1876) independently observed and described
the details of sperm penetration and apparent pronuclear fusion in sea
urchins.
Also contributing greatly to the knowledge of the morphology of
fertilization, especially with reference to pronuclear dynamics and the
question of how egg and sperm produce a cell capable of division, was the
work of Van Beneden (1883) and Boveri (1887).
Waldeyer (1870), Benda
(1887), and Lenhossek (1898) not only determined the existence and origin
of the sperm acrosome but also believed that the acrosome played a major
role in egg penetration.
Early in the twentieth century, when the study of morphology of fer­
tilization was at a relatively advanced stage, many investigators began to
explore the physiology of this event.
One aspect intriguing early investi­
gators was the high degree of species specificity involved in sperm-egg
interactions.
This fundamental phenomenon of fertilization touched off
many cross-fertilization experiments (Gray, 1913; Vernon, 1900; Shearer,
DeMorgan, and Fuchs, 1913; Tennent, 1910; Balzer, 1910) which demonstrated
that species specificity existed on at least two levels.
specificity involving interactions of gamete surfaces.
First, external
Second, an internal
specificity involving the union of pronuclei and genetic compatibility.
4
Self-sterility, another problem closely related to species specificity
of fertilization, was examined by Morgan (1905, 1910, 1923) in the monecious
ascidian, Ciona.
Morgan found that the spermatozoa were unable to fertil­
ize eggs of the same Individual but successfully fertilized eggs of most
other members of the same species.
He concluded that this self-sterility
barrier resides in the egg chorion, since removal of this layer allows the
egg to be fertilized by spermatozoa of the same individual (Morgan, 1923).
In addition to the problem of fertilization specificity, gamete sur­
face substances and their effects on gametes of the opposite sex have also
been studied.
Lillie (1912, 1919), experimenting with sea urchin fertiliza­
tion, published the first accounts of these substances.
Lillie demon­
strated that the egg emitted a sperm agglutinating substance, which he
called fertilizin.
He proposed the function of fertilizin to be in the
activation of the egg and prevention of polyspermy.
In contrast, Loeb
(1901), working with artificial parthenogenesis in sea urchins, believed
that a lytic substance found in the sperm was responsible for egg activa­
tion.
Even with the early attempts of these investigators to describe the
roles of various gamete substances, the problem of egg activation is still
unresolved.
However, they generated considerable interest in the physio­
logical aspects of fertilization.
A more recent approach to the study of fertilization can be viewed as
a two-pronged attack;
first, the ultrastructure or morphology of sperm-egg
interactions and second, the molecular involvement of gamete surface inter­
acting substances.
Dan (1952, 1967) began the ultrastructural approach
with comparative descriptions of the acrosome reaction and correlation of
this phenomenon with penetration of the egg.
Many other investigators have
5
since pursued the diverse aspects of sperm-egg interactions.
Colwin and
Colwin (1967) and Austin (1968) have adequately reviewed this literature.
In general, these interactions can be described as follows:
the apical
portion of the sperm attaches to the egg outer surface, the sperm acrosomal
cap ruptures releasing its contents (usually containing lytic substances)
onto the surface of the egg, the acrosomal filament(s) protrude through the
egg investments, the acrosomal membrane fuses to the egg plasma membrane,
and the sperm nucleus passes into the egg cytoplasm.
The understanding of
these structural aspects greatly aids the molecular approach since specific
chemical events must be equated to morphological changes.
Although the molecular approach to fertilization began with Lillie
(1912), intensive investigation of the chemical and biological properties
of gamete surface interacting substances did not occur until much later.
As with the ultrastructural approach, many investigators have contributed
significantly to this approach.
Tyler (1948) and Metz (1967) have reviewed
the chemical and immunological investigations of sperm-egg interactions.
Essentially, these studies provide evidence for the participation of gamete
surface interacting substances in various fertilization events such as:
gamete recognition and adhesion, initiation of the acrosome reaction, egg
activiation, and sperm penetration.
These studies and others are discussed
in subsequent sections.
Gamete Surface Interacting Substances
Gamete surface substances are necessary for successful fertilization.
In order to understand the role and functions of these substances, they
have, for the purpose of this review, been separated into groups in which
6
their relationship to fertilization can be determined.
These groups are:
chemotaxis, sperm motility, sperm respiration, sperm agglutination, sperm
capacitation, sperm-egg attachment, initiation of the acrosome reaction,
egg activation, and specificity of fertilization.
Chemotaxis
The chemical attraction of one gamete towards another has to date only
been described in certain plants including bryophytes, pteridophytes, algae,
and in only one animal group, the coelenterates (cf. Machlis and RawitscherKunkel, 1967; Wiese, 1969). In most cases, the motile male gametes are
attracted by sessile female gametes.
The attraction mechanism depends on
the ability of the motile gamete to change direction in response to a chem­
ical gradient set up by the nonmotile gamete.
In the bracken, Pteridium
aguilinum L.. the chemotactic substance Is an organic acid (L-malic acid)
which is emitted by the archegonia (Pfeffer, 1884).
Pfeffer did not actu­
ally demonstrate the secretion of malic acid from the archegonia but
through a series of experiments showed that malate or a similar compound
was responsible for attracting the sperm.
No attempts have yet been made
to isolate the chemotactic substance from archegonia.
Chemotaxis in animal fertilization has been demonstrated in a few
hydroid coelenterates including Campanularia flexuosa (Miller, 1966). In
coelenterates, the attracting substance is produced by the ovarian tissue
and not the egg per se.
Nevertheless, the sperm attracting substances in
hydroids are highly species specific (Miller, 1966) and cinematographic
evidence definitely demonstrates that chemotaxis in hydroids is the direct
result of sperm turning movements towards the source of the chemical stimu-
7
lus (Brokaw, Goldstein, and Miller, 1970).
Although there is no strong
evidence of chemotaxis in other species, Nakano (1969) has suggested that
it occurs in fish. In Rhodeus and other species of fish, the spermatozoa
show a strong tendency to aggregate near the micropyle (Nakano, 1969).
Thus, chemotaxis has been demonstrated to be an important function of
egg surface substances.
To date, only sessile organisms with fixed female
gametes have been shown to have chemotaxis, but perhaps it is functional in
other situations as well.
Sperm motility and respiration
One probable function of soluble egg surface substances is to increase
sperm motility, an event which may increase the chance of sperm-egg colli­
sion and thus is important for successful fertilization (Metz, 1957).
The
occurrence of induced sperm motility by egg water has been observed in sea
urchins, annelids (Lillie, 1913), and molluscs (Tyler, 1940).
In sea
urchins, Hartmann £t ad. (1939) suggested that the echinochrome pigment in
the egg jelly is responsible for activating sperm motility.
Unfortunately,
this idea was disproved by Bielig and Dohrn (1950) who found no sperm acti­
vating action of echinochrome in buffered systems.
The phenomenon of sperm
motility activation was shown to be less specific when Tyler (1955) demon­
strated that sperm motility could be caused by egg fertilizin.
He sug­
gested that this activating action was due to its metal binding properties.
This agrees with the findings of Tyler and Rothschild (1951) that chelating
agents are effective in eluciting sperm motility.
Since sperm motility in sea urchins is activated by fertilizin, it
follows that fertilizin would also increase the sperm respiratory rate
8
(Metz, 1957). This is indeed true in some species, however, in certain
cases the increased motility is not always accompanied by an increase in
oxygen uptake (Metz, 1957). In comparing the agglutinating ability of fertilizin with the respiration stimulating ability, Spikes (1949) demon­
strated that exposure of fertilizin to ultraviolet light destroyed the
agglutinating ability as well as the stimulating affect on sperm respira­
tion.
However, heating fertilizin to 126° C enhanced its respiration
increasing activity but destroyed the sperm agglutinating activity.
These
investigations suggest that fertilizin may affect sperm respiration inde­
pendent of its agglutinating activity.
Sperm agglutination
Soluble egg surface substances have long been known to be involved in
sperm agglutination as demonstrated by Lillie (1912) with egg water of sea
urchins.
Metz (1957) suggested that these egg surface agglutinins are
important in sperm-egg attachment, prevention of polyspermy, and probably
other fertilization events.
Egg water agglutination of spermatozoa has
also been observed in annelids (Lillie, 1919), molluscs (Tyler, 1940;
von Medem, 1942), ascidians (Minganti, 1951), cyclostomes (Schartau and
Montaient!, 1941), fish (Hartmann, 1944; von Medem, Rotheli, and Roth,
1949), and amphibians (Glaser, 1914).
The sperm agglutinating substance in the egg water of sea urchins and
other species is called fertilizin (Lillie, 1919).
Agglutination results
from an antigen-antibody like reaction between fertilizin and the comple­
mentary substance antifertilizin located on the sperm surface.
Treatment
of egg water with an excess of spermatozoa lowers the agglutinating power.
9
thus demonstrating the presence of egg and sperm complementary substances
(Hathaway and Metz, 1961).
In addition, sea urchin sperm agglutination
reverses spontaneously and the disagglutinated spermatozoa are unable to
reagglutinate in the presence of fertilizin.
This reversal of agglutination
is due to the conversion of fertilizin to univalent, nonagglutinating
fragments (Metz, 1942).
Since the fragments are bound to antifertilizin
sites on the sperm surface, they prevent the spermatozoa from reagglutinating when fresh fertilizin is added to the suspension.
The probable func­
tion of the agglutination phenomenon is species specificity in gamete
attachment and prevention of polyspermy (Metz, 1967).
Sperm capacitation
Successful fertilization in many mammals requires that the spermatozoa
be in the female genital tract for a period of time before egg contact.
During this time, the spermatozoa undergo a physiological change, termed
capacitation, which primarily affects the acrosome allowing the release of
hyaluronidase, one of several enzymes necessary for sperm penetration of
the egg envelopes (Austin, 1965).
The mechanism of capacitation is not
fully understood but is believed to involve the removal of coating material
from the sperm acrosome (Piko, 1969).
Besides the release of enzymes,
this removal exposes the acrosomal membrane, thus enhancing the ability of
the sperm to penetrate the outer egg layers and to interact with the egg
surface.
Rabbit spermatozoa can be capacitated ijni vitro by treating with
p-amylase (Kirton and Hafs, 1965).
This suggests that capacitation may
involve the action of similar enzymes in the female genital tract.
10
Capacitation as described in mammals has not been observed in other
animal groups, although a similar process may exist in animals with inter­
nal fertilization or in animals that have female sperm storage capability.
One instance of sperm conditioning that has been compared to mammalian
capacitation is the effect of frog egg jelly on frog spermatozoa.
Frog
eggs which have had the jelly coat removed or coelomic eggs are usually
unfertilizable (Shaver, 1966). However, Shivers and James (1970) have
recently shown that jellyless eggs can be fertilized by spermatozoa pretreated with jelly coat material.
Therefore, the jelly interacts with the
sperm in a manner which enables the sperm to fuse with and activate the egg
(Shivers and James, 1970).
This process needs to be studied more inten­
sively before the term capacitation can be applied.
Therefore, we must
presently assume that capacitation is unique to mammals.
In conclusion, sperm capacitation involves the interaction of sperm
substances with the female reproductive tract (Piko, 1969).
This interac­
tion may be related to the removal of surface coatings on the sperm. In
masussls, a ccsplete elucidation of the physiological mechanism of capacita­
tion will require a reliable and precisely controlled method of ^ vitro
fertilization.
Sperm conditioning similar to capacitation, involving the
exposure of previously unexposed surface components, may exist in nonmammals.
Sperm-egg attachment and cell adhesion
The adhesion of one cell to another, including sperm-egg attachment,
undoubtedly involves complementary cell surface substances or some similar
molecular interaction (Metz, 1967; Humphreys, 1967).
One obvious example
11
of such molecular interaction is the previously described fertilizin-antifertilizin system.
Fertilizin occurs in the egg jelly or other egg invest­
ments and, therefore, functions partially in adhesion of the sperm to the
egg (Metz, 1967).
In addition, sperm-egg attachment is highly species
specific and can be measured in cross-fertilization experiments.
This has
been readily demonstrated among five species of decapod crustaceans
(Mowbray, Brown, and Metz, 1970), However, successful cross-fertilization
does occasionally occur and has been considered molecularly by Rothschild
(1956).
He suggests that sperm-egg attachment involves intermolecular
forces superimposed on specific complementariness of surface structure.
Furthermore, he believes that cross-fertilization can be explained on the
basis of a certain "rubberiness" of fit allowing sufficient van der Waals
forces to occur between sperm and egg surface proteins, thus resulting in
adhesion of the gametes.
Several theories on the mechanism of cell adhesion have been proposed,
including:
divalent cation stabilization of intercellular cement (Chambers
and Chambers, 1961), bonding between stsrically complementary surface
groups (Tyler, 1947; Weiss, 1947), calcium bridge bonding between cell sur­
faces (Steinberg, 1958), long range bonding between cell membranes (Curtis,
1962), and specific cell products acting at the cell surface (Moscona,
1962).
The most accepted theory is that of Moscona (1962), which is backed
up by a considerable amount of experimental evidence.
involve the
These experiments
vitro reassociation of artificially dissociated cells into
multicellular structures or aggregates (cf. Humphreys, 1967; Moscona, 1968).
Wilson (1907) originated this type of experimentation with his work on
species-specific aggregation of interspecific mixtures of dissociated
12
sponge cells.
The analysis of cell reaggregation was extended to the molec­
ular level by Moscona (1962) and Humphreys (1963) using cells from sponges,
embryos, and tissue cultures.
Humphreys (1963) devised a suitable chemical method for reversibly
dissociating sponge tissue using calcium and magnesium free sea water
(CMF-SW).
This method is an improvement over the previously used mechani­
cal and enzyme dissociation techniques because it does not damage cell sur­
face macromolecules.
The CMF-SW dissociated sponge cells reaggregate at
room temperature but not at 5° C or in CMF-SW, indicating that this type of
dissociation removes macromolecules from the cell surface which are
involved in cell adhesion.
Interestingly enough, the mechanically disso­
ciated cells are able to adhere at 5° C indicating that these cells still
possess the surface macromolecules necessary for adhesion.
Humphreys
(1967) concludes that the cells are able to synthesize new attachment sub­
stances at room temperature but not at 5° C.
Thus, the bonds which hold
the cells together Involve both macromolecules (aggregation factors or
ligands) and divalent cations.
Further studies on sponge aggregation factors indicate that they are
directly involved in cell attachment and aggregation, their adhesive
effects are species specific, they are inactivated by heating, their activ­
ity is destroyed by ^-amylase and protease but not DNase, Rnase, trypsin,
collagenase, hyaluronidase, or lysozyme (Moscona, 1968).
Biochemical tests
o
indicate that the aggregating activity is associated with a 20-25 A glyco­
protein particle (Margoliash e_t
, 1965).
Specific cell aggregation factors have also been demonstrated in other
tissue types.
Lillen (1968) found that supernatants of freshly dissociated
13
chick embryo neural retina cells enhanced reaggregation.
He also found
that low temperatures and cycloheximide prevented the synthesis of the
aggregation factor and that antibodies prepared against the factors inhib­
ited their aggregating activity.
Pessac and Defendi (1971) have demon­
strated the presence of acid mucopolysaccharide aggregation factors in var­
ious mouse, rat hamster, and human tissue culture cells.
In conclusion, although exceptions exist, cell adhhesion can be com­
pared to sperm-egg attachment.
Although probably a minor point, cell
adhesion involves the binding together of two or more morphologically and
chemically similar cells, whereas sperm-egg attachment involves the binding
together of morphologically dissimilar cells.
Quite possibly because not
enough information is available, the binding molecules involved in cell
adhesion appear to be a single molecular species (i.e. aggregation fac­
tors), whereas sperm-egg binding, at least in sea urchins, involves two
separate but complementary molecules (i.e. fertilizin and antifertilizin).
However, if the aggregation of sponge cells, for example, in the presence
of aggregation factors is similar to agglutination of gnermatozoa by fer­
tilizin, then sperm-egg attachment simply represents a special type of cell
adhesion.
Initiation of the acrosome reaction
Usually the sequential event following sperm-egg attachment is the
sperm acrosome reaction.
As with attachment, there is very strong evidence
that the interaction of sperm and egg surface substances is essential for
initiation of the acrosome reaction (Dan, 1967).
Initiation of the acro­
some reaction by egg water has been reported in echinoderms, annelids, and
14
molluscs (Wada, Collier, and Dan, 1956; Colwin and Colwin, 1955; Metz and
Morrill, 1955).
In Nereis, the incidence of acrosome reactions is usually
associated with the sperm agglutinating action of egg water (Metz and
Morrill, 1955). However, Dan (1954) found that egg water would agglutinate
spermatozoa but not initiate the acrosome reaction in the absence of cal­
cium.
Gregg (1971) provided further evidence that fertilizin acting
together with calcium is responsible for the initiation of the acrosome
reaction in sea urchins.
He proposed two possible mechanisms for this
process; (1) sperm agglutinated by fertilizin become activated and immedi­
ately undergo the acrosome reaction if sufficient calcium is present, or
(2) an acrosome inducing substance is formed as a result of fertilizin
induced agglutination; this substance, together with calcium, initiates the
acrosome reaction.
Thus it is reasonable to assume that soluble egg substances initiate
the sperm acrosome reaction in several species.
Although all the processes
of sperm-egg interactions cannot yet be categorized chemically, the initia­
tion of the acrosome reaction is definitely one of a series of nxïlecular
interactions which occur during the sperm-egg interaction.
Activation of the egg
The structural and functional changes which take place in the animal
egg during sperm penetration are referred to as egg activation.
The
changes can include alterations in the egg shape, migration of egg cyto­
plasm, resumption of meiotic activity, elevation of the fertilization mem­
brane, and various biochemical changes including an increase in respiratory
rate and initiation of protein synthesis (Monroy and Tyler, 1967).
How the
15
sperm includes activation of the egg is still unclear.
This has been a per­
plexing problem, since artificial parthenogenetic agents such as heat, cold,
acids, alkalis, detergents, and mechanical stimuli are also capable of acti­
vating the egg (Austin, 1965).
In addition, natural parthenogenesis is com­
mon in some animals such as aphids, rotifers, and various hymenopterans.
Lillie (1919) believed that fertilizin and antifertilizin were
involved in the activation of the egg.
Indeed, removal of the egg jelly
(fertilizin) from the sea urchin eggs lowers their fertilizability (Metz,
1957).
Antifertilizin treatment of eggs greatly reduces the fertilizabil­
ity of both normal and dejellied eggs (Tyler and Metz, 1955).
These
experiments show that both egg jelly fertilizin and fertilizin at the egg
surface are indeed involved in fertilization but do not necessarily indi­
cate that fertilizin participates in egg activation.
Again considering parthenogenetic activity, specific antiserum also
can initiate activation of sea urchin eggs.
For example, Perlmann (1954)
reported that treatment of sea urchin eggs with anti-egg homogenate serum
resulted in parthsncgsnstic activation.
This activation includes cortical
granule breakdown, formation of the fertilization membrane, and cleavage.
Perlmann (1959) attributed the activation to interaction between antibody
and a specific egg antigen (which he designated A antigen).
Unfortunately
others have not been able to duplicate these results, although Tyler (1959)
and Brookbank (1959) were able to obtain egg nuclear breakdown and an
increase in respiration following antibody treatment of sea urchin eggs.
Nevertheless, Perlmann's results are considered significant since they pro­
vide further evidence as to the molecular nature of the egg activation
mechanism.
16
Specificity of fertilization
Fertilization is highly species specific, that is, interspecies hybrid
crosses are usually not successful.
This specificity is due in part to
species differences in molecular structure of gamete surfaces and in this
respect has been studied on four different levels: (1) fertilizin-antifertilizin interaction, (2) initiation of the acrosome reaction, (3) sperm
acrosome lysins, and (4) membrane fusion.
The interaction between fertilizin and antifertilizin is species spe­
cific (Metz, 1967).
Lillie (1913) found that Arbacia (Echinodermata) egg
water strongly and irreversibly agglutinates Nereis (Annelida) spermatozoa
but that Nereis egg water had no effect on Arbacia spermatozoa.
With such
widely separated species, this failure of reciprocal cross-agglutination is
not surprising, since even with four closely related species of echinoids
Tyler (1949) found a high degree of specificity with sperm agglutination.
In these studies, he found that cross-agglutination failed to occur in most
cases.
However, when it did occur, cross-fertilization was also possible.
Indicating similar surface molecular compcncnts bctwesn the two species.
In a similar series of experiments, Metz (1945) found no cross-agglutina­
tion of spermatozoa between three species of asteroids (starfish). In
addition, Mowbray, Brown, and Metz (1970) demonstrated a high degree of
specificity in sperm-egg attachment between five species of decapod crabs.
Thus, in several species, specificity exists during early gamete interac­
tion.
A second specificity factor and possibly the most important with
reference to gamete surface interactions is the initiation of the acrosome
reaction.
In sea urchins, as the sperm comes into contact with the egg
17
surface or is treated with egg water, the first sperm morphological changes
are in the apical portion of the acrosomal complex.
Ultrastructural evi­
dence indicates that the sperm apical plasma membrane overlying the acro­
somal granule undergoes a change in permeability characteristics, expands,
partially disintegrates, and fuses with the acrosomal granule membrane (Dan,
Ohori, and Kushida, 1964).
Gregg (1971) demonstrated that induction of the
acrosome reaction in the sperm of Arbacia punctulata. Echinometra lucunter,
1
and Tripneustes esculentus by egg jelly (fertilizin) solutions of
Lytechinus variegatus and Eucidarls tribuloides was specific to the extent
that where cross-induction of the acrosome reactions occurred, they were
always preceded by sperm agglutination.
The third specificity system involves sperm acrosome lysins which dis­
solve egg enveloping materials aiding sperm penetration.
High sperm con­
centrations or sperm extracts of the same species are effective in dissolv­
ing egg membranes (Brookbank, 1958; Hauschka, 1964; Krauss, 1950; Tyler,
1939).
Spermatozoa and sperm extracts of the limpet, Megathura crenulata.
and the abalone, Haliotis cracherodii. will dissolve their
branes but cross-lysis does not occur (Tyler, 1939).
egg mem­
Similar results were
reported between the sand dollar, Mellita quinquiesporforata. and the sea
urchin, Arbacia punctulata (Brookbank, 1958).
Such lysin specificities
probably exist in higher animals since Katsh (1960) has demonstrated
hyaluronidase from different mammalian species to be immunologically dif­
ferent.
Besides these three specificity systems, others may eventually be
demonstrated, for example, gamete membrane fusion.
In most species, in
order for sperm penetration to occur, the sperm acrosomal membrane must
18
fuse with the egg plasma membrane.
Tyler (1949), using four species of
echinoderms, demonstrated that cross-fertilizability and cross-agglutination involves more than just the complementarity of fertilizin and antifertilizin.
In his experiments, the removal of egg jelly and the vitelline
membrane to expose the egg plasma membrane failed to extend cross-fertiliz­
ability in sea urchins to new species combinations (Tyler and Metz, 1955).
Although several variables exist, the fusion interactions of the sperm acro­
somal membrane and egg plasma membrane are probably also species specific.
As seen, sperm-egg interactions are species specific on several levels.
This specificity is at least partially due to molecular differences in
gamete surface structure.
However, this normal species specificity is
overcome if various abnormal conditions are used.
For example, if sperm
concentration, pH, age of gametes, and other variables are manipulated, the
chance of cross-fertilization is greatly increased.
Vernon (1900) was suc­
cessful in forming 29 echinoderm crosses out of a possible 56 combinations
by using these conditions.
Fertilization specificity must be functional in
nature, since the spasming seasons and habitats of many closely related
species overlap.
On the other hand, molecular specificity may not function
in preventing interspecies hybrids but may simply be a species character
resulting from the long separation of these species.
In any case, the
study of fertilization specificity has helped to understand the molecular
processes occurring during the sperm-egg interaction.
Molecular Aspects of Sperm-egg Interactions
Besides the specificity of various aspects of sperm-egg interactions,
considerable information has been obtained on the molecular events of
19
gamete surface substances and cheir interactions.
Most of these studies
have involved the use of immunological methods and have been supported or
supplemented by histochemical and biochemical methods including the use of
chemical inhibitors.
These studies are now considered with respect to
their importance in sperm-egg interactions.
Egg substances involved in fertilization
Egg jelly or fertilizins have been studied extensively in the animal
kingdom.
Fertilizin and complementary or associated substances (sperm anti-
fertilizin and cytofertilizin) have been best studied in sea urchins, in
which they are involved in sperm agglutination, initiation of the acrosome
reaction, and other events associated with sperm-egg interactions.
Some of
these aspects are briefly mentioned in the previous sections involving
specificity.
Fertilizin, a soluble substance commonly found in sea urchin egg water,
was once thought to be secreted by the unfertilized egg (Lillie, 1919).
However, Tyler and Fox (1940) demonstrated that egg jelly was the source of
fertilizin, indeed, that egg jelly is fertilizin.
It should be mentioned
though that some fertilizin is firmly bound to the egg surface (Tyler and
Metz, 1955).
Several investigators have chemically and physically charac­
terized sea urchin fertilizin (Runnstrom, Tiselius, and Vasseur, 1942;
Stern and Metz, 1967; Tyler, 1949; Vasseur, 1952).
They found it to be a
highly acidic hexosamine-free glycoprotein of molecular weight 300,000 con­
taining 20% amino acids and 80% polysaccharides with one sulfate group per
monosaccharide residue.
The amino acids are probably arranged as peptides
interspersed with branched chain carbohydrate (Tyler, 1949).
20
The most striking characteristic of fertilizin is its ability to
agglutinate spermatozoa, an effect attributed to a reaction similar to the
combination of antigen and antibody (Lillie, 1919). In this reaction, fer­
tilizin combines with sperm surface substance, antifertilizin, in a highly
species specific manner (Tyler, 1949) resulting in sperm agglutination.
In
sea urchins, but not other species (e.g. starfish, amphibians), the agglu­
tination is spontaneously reversible (Lillie, 1919), and the "reversed
spermatozoa" are not capable of reagglutination by fertilizin.
The dis-
agglutination is caused by conversion of multivalent fertilizin to a uni­
valent form (Tyler, 1948; Stern and Metz, 1967) which remains bound to the
sperm antifertilizin preventing reagglutination (Metz, 1967) and also
inhibiting the sperm from fertilizing the egg (Tyler, 1942).
With the for­
mation of univalent fertilizin, a large fragment is released which is incap­
able of further reaction witn spermatozoa (Hathaway and Metz, 1961).
These
authors suggest that this fragment is analogous to the Fc portion of the
antibody molecule which holds the combining entities together, further
demonstrating the antigen-âtîtibcdy nature of the fertilizin-antifsrtllizin
system.
Univalent fertilizin can also be prepared artificially by treat­
ment with hydrogen peroxide, ultraviolet light, or proteolytic enzymes
(Tyler, 1941; Metz, 1942). Univalent fertilizin prepared in this manner
yields four bands upon electrophoresis versus one band for the multivalent
form (Stern and Metz, 1967).
Absorption of the univalent fertilizin with
spermatozoa removes two of the four bands, indicating that only a portion
of the fertilizin molecule possesses the active site. The function of
univalent fertilizin and the disagglutination phenomenon still remains to
21
be elucidated, however, it is most likely involved in gamete attachment,
prevention of polyspermy, and initiation of the acrosome reaction.
Fertilizin, as is readily demonstrated, is certainly important in fer­
tilization, for it affects practically all aspects of sperm-egg interac­
tions.
For example, treatment of sea urchin eggs with sperm extract (anti-
fertilizin) reduces the egg fertilizing capacity (Tyler and Metz, 1955)
indicating the importance of free fertilizin sites for interaction with
spermatozoa.
In addition, the rate or length of time for fertilization to
occur after initial sperm-egg mixing increases with progressive removal of
fertilizin from the egg surface (Hagstrom, 1956) indicating that fertilizin
constitutes a barrier to fertilization and probably functions in prevention
of polyspermy.
Finally, as demonstrated in the experiments of Gregg (1971),
fertilizin acting together with calcium is responsible for initiation of
the acrosome reaction.
In searching for other molecular components involved in fertilization,
a substance capable of agglutinating homologous spermatozoa was isolated
from unfertilized jellyless sea urchin eggs by Motcnrara (1950).
Because of
the similarity to fertilizin, he called this agglutinin "cytofertilizin."
In addition, he found that cytofertilizin could be extracted from fertilized
jellyless eggs and even hatching blastulae (Motomura, 1953).
Gregg and
Metz (1966) also obtain this agglutinin in supernatants from acid dejellied
and trypsin-demembranated eggs following fertilization.
These authors pre­
fer to call this agglutinin or egg extract the "fertilization product"
instead of cytofertilizin since other substances such as cortical granule
components are also present.
Cytofertilizin agglutination of spermatozoa
is species specific (Hagstrom, 1956) and as with fertilizin reverses spon­
22
taneously (Gregg and Metz, 1966).
In addition, cytofertilizin appears to
increase sperm motility (Gregg and Metz, 1966). Electrophoretic separation
indicates that cytofertilizin has three components, two of which correspond
to similar components of fertilizin.
Cytofertilizin is also immunologi­
cally similar to fertilizin (Gregg and Metz, 1966). These workers, however,
have not been able to ascribe a definite role to it.
Another component operative in sea urchin fertilization is a sperm
binding protein which has been described by Aketa (1967).
This substance
which is extracted from the egg vitelline membrane is a conjugated protein
with carbohydrate and lipid moitiés and has a
value of 2.3 (Aketa,
Tsuzuki, and Onitake, 1968). The sperm binding capacity is reduced if the
protein is modified by reduction of disulfide bonds (Aketa and Tsuzuki,
1968).
The authors believe this loss of activity is due to conformational
changes, however, the participation of disulfide groups in bonding has not
been ruled out.
These studies are highly significant since they represent
attempts to analyze the specific role of one egg surface component in a
single fertilization ste-, 3perrr.=egg attachment.
Other studies in sperm-
binding protein utilizing inhibitors such as specific antiserum and
enzymes, are discussed later (see Chemical inhibitors of fertilization.
Egg antigens).
Egg jelly disulfide bonds have been studied recently with regard to
sperm capacitation (Wolf and Hedrick, 1971) and sperm penetration (Gusseck
and Hedrick, 1971). Mercaptoethanol-solubilized jelly as well as intact
jelly displayed sperm capacitating activity indicating that disulfide bonds
do not participate in sperm capacitation (Wolf and Hedrick, 1971).
The
fact that various disulfide-bond breaking reagents dissolve egg jelly led
23
Gusseck and Hedrick (1971) to postulate that disulfide bond breakage by
sperm substances may be essential in sperm penetration of the egg jelly and
fusion with the egg membrane.
No further proof of this hypothesis is yet
available.
Besides sea urchins, amphibian egg jelly has been shown to be essen­
tial for successful fertilization (Good and Daniel, 1943).
Chemical stud­
ies by Freeman (1968) on Xenopus laevis eggs demonstrate that the jelly
material is glycoprotein in nature, containing glucosamine, galactosamine,
and galactose.
Sialic acid and various amino acids were also found.
The
jelly is susceptible to digestion by papain and pepsin but not hyaluronidase (Freeman, 1968).
In addition, the egg jelly coats of many other
amphibians may be solubilized by alkaline solutions, ultraviolet light
(Rugh, 1962), and raercaptan solutions (Gusseck and Hedrick, 1971). The
latter indicates the presence of disulfide bonds in the jelly.
Although no
molecular comparison has been made between the egg jellies of amphibians
and sea urchins, both are glycoprotein in nature and both participate in
fertilization.
Egg substances, especially jelly coat materials, are functional in
sperm-egg interactions. They function in sperm-egg adhesion, sperm capacitation, initiation of the acrosome reaction, and other necessary interac­
tions with the penetrating sperm.
Sperm substances involved in fertilization
With the exception of antifertilizin, the study of sperm substances
has been limited primarily to the lysine found in the acrosome.
The bio­
chemistry of the sperm surface is somewhat difficult to examine because of
24
the relatively small surface area and problems with autodigestion upon
breakage of the acrosome.
Thus, most studies on the molecular level have
involved the immunological approach (see Sperm antigens).
The present dis­
cussion will be limited to sperm antifertilizin and sperm lysins.
A substance capable of neutralizing egg fertilizin can be extracted
from sea urchin sperm.
This substance, called antifertilizin, was first
extracted from sea urchin spermatozoa by Tyler and O'Melveny (1941).
Anti­
fertilizin is a heat-stable acidic protein with a molecular weight of
10,000 (Metz, 1957; Runnstrom, Tiselius, and Vasseur, 1942; Tyler, 1948).
With certain preparations, this low molecular weight protein is associated
with a larger particle sedimentable at 30,000 ^ (Metz and Kohler, 1960).
Sperm antifertilizin inhibits the action of fertilizin in egg water, agglu­
tinates homologous unfertilized eggs and causes the formation of a precipi­
tation layer on the surface of the egg jelly (Metz, 1967).
In addition,
removal of antifertilizin from sea urchin spermatozoa by acid treatment
lowers their fertilizing capacity (Tyler and O'Melveny, 1941). Lillie
(1919) suggested that the interaction of fertilizin with antifertilizin
results from the combination of specific complementary combining groups,
somewhat similar to the antigen-antibody reaction.
The complementary
interaction of fertilizin and antifertilizin on the gamete surfaces
strongly suggests that these substances play an Important role in fertili­
zation.
Two possibilities are sperm-egg attachment and initiation of the
acrosome reaction.
Bowen (1924) was first to recognize that the sperm acrosome contains
lysins (enzymes), and Tyler (1939) was successful in extracting a lysin
from the spermatozoa of the keyhole limpet, Megathura crenulata.
Since
25
that time, various studies have been made on the histochemical and biochem­
ical properties of lysins.
Sperm lysins are usually species-specific
(Brookbank, 1958) and tailored for the type of substrate they must act upon.
They are released by breakage of the acrosomal vesicle when the sperm makes
contact with the egg envelope (Dan, 1967) and, in general, most lysins act
singly (as in mammals) or in conjunction with the remainder of the acro­
somal complex to penetrate the egg envelope (Colwin and Colwin, 1967; Piko,
1969).
Evidence for the dissolution of egg envelopes by sperm lysins has been
presented by Tyler (1939), Krauss (1950), Berg (1950), and Hauschka (1963)
in molluscs; Brookbank (1958) and Hathaway, Warren, and Flaks (1960) in
echinoderms; Folakoski, Zaneveld, and Williams (1972), Stambaugh and
Buckley (1970), Hartree and Srivastava (1965) and Austin (1960) and others
in mammals.
The tough egg envelope of the mollusc Megathura crenulata is
easily dissolved with frozen-thawed sperm extracts or spermatozoa in a 1%
suspension (Tyler, 1939).
From this study, Tyler (1939) concluded that
lysins enable iridlvidual sperm to penetrate the egg surface although ether
investigations indicate that the lysins elicit a general softening effect
on the egg investments which ultimately allows one sperm to penetrate (cf.
Rothschild, 1956). The enzymatic nature of the lysin can be better under­
stood by examining the substrate upon which it acts.
Haino and Kigawa
(1966) have characterized the egg envelope of the gastropod Tegula pfeifferi
which is dissolved by homologous sperm lysin.
They have found that the
substrate is a glycoprotein containing 33% protein, 16% galactosamine, 11%
fucose, 10% sulfate, 7% glucosamine, and other residues.
In addition, the
lysin digestion product of this substrate has reducing ends which indicates
26
polysaccharase activity (Haino, 1965).
than one sperm lysin.
At least in mammals there are more
This is probably dependent on the number of egg
layers the sperm must penetrate (cf. Piko, 1969), for example, the egg
cumulus oophorus and zona pellucida.
With reference to mammals, sperm acrosome lysins (enzymes) which have
been found include:
neuraminidase, hyaluronidase, acid phosphatase,
p-N-acetylglucosaminidase, phospholipase A, protease, and arylsulphatase
(Allison and Hartree, 1970; Hartree and Srivastava, 1965). However, the
primary concern has been to characterize lysins affecting the cumulus
oophorus and the zona pellucida.
McClean and Rowlands (1942) discovered
that the cumulus oophorus of rat and mouse ova can be dispersed with
hyaluronidase extracted from homologous spermatozoa.
Austin (1948, 1960)
demonstrated that the individual mammalian sperm penetrates the cumulus
oophorus by the action of hyaluronidase on the intercellular matrix which
is composed of acid mucopolysaccharides such as hyaluronic acid.
Penetration of the zona pellucida was also thought to be due to the
action of hyaluronidase (Austin, 1951), however^ Srivastavaj Adams, and
Hartree (1965), Stambaugh and Buckley (1968, 1969), and others have pro­
vided evidence for a proteolytic zona lysin.
Srivastava, Adams, and
Hartree (1965) isolated a lipoglycoprotein complex from sperm acrosomes
which disperses the zona of rabbit eggs.
Treatment of eggs with this lysin
releases amino acids which indicates that it is a proteinase.
Others have
confirmed these results and purified the proteolytic enzyme, acrosomal
proteinase (Stambaugh and Buckley, 1968, 1969; Polakoski, Zaneveld, and
Williams, 1972).
Acrosomal proteinase is a dimer of molecular weight
55,000, has a pH optimum of 8.0, and can be inhibited by soybean trypsin
27
inhibitor (Zaneveld, Polakoski, and Williams, 1972).
Acrosomal proteinase
evidently acts on the mucoprotein component of the zona pellucida
(Stambaugh and Buckley, 1969). In addition to these biochemical studies,
Yanagimachi and Teichman (1972) have demonstrated proteinases in many mam­
malian and avian spermatozoa histochemically. Thus, clearly the mammalian
sperm acrosome possesses at least two distinct classes of lysins appropriate
for digestion of egg investments which differ in molecular structure.
In summary, the sperm possesses both external and internal substances
which interact with the egg surface during sperm-egg interactions.
The
external or surface substances function in such events as capacitation,
sperm-egg attachment, and initiation of the acrosome reaction.
The inter­
nal substances are lysins which aid the sperm in penetrating the egg invest­
ments.
Chemical inhibitors of gamete interaction
One means of studying the biochemical events of fertilization is with
th? use of chemical inhibitors.
Inhibitors to fertilization may be divided
into four classes; (1) the gamete surface interacting substances such as
fertilizin and antlfertilizin, (2) specific antlsera prepared against
gametes or gamete substances, (3) naturally occurring substances such as
Arbacla dermal secretion, and (4) chemical agents with well defined activi­
ties such as enzymes, oxidizing and reducing agents, detergents, and others.
The former two are considered in other sections (Gamete Surface Interacting
Substances; Immunological Aspects of Gamete Surface Interactions). The lat­
ter two groups are considered here,
28
Exposure of sea urchins to hypotonic solutions causes the release of
an integumental substance (dermal secretion) which inhibits fertilization
(Metz, 1960; Oshima, 1921; Pequegnat, 1948). Metz (1961) demonstrated that
dermal secretion binds irreversibly with eggs but not with spermatozoa,
thus its inhibitory effects are directed only against the egg.
Since fer-
tilizin does not neutralize the action of dermal secretion, this substance
must act on some other component of the egg (Metz, 1961).
The most likely
target is the vitelline membrane, since trypsin demembranated eggs are not
inhibited by dermal secretion (Metz, 1960).
Although totally unrelated to sea urchins, another naturally occurring
inhibitor to sea urchin fertilization is found in extracts of the marine
alga. Fucus (Harding, 1951; Runnstrom and Hagstrom, 1955).
Esping (1957)
provided evidence that this inhibitory activity is due to the presence of
polyphenols.
Branham and Metz (1960) demonstrated similar inhibitory
effects with commercial preparations of tannic acid.
Whatever the chemical
nature of the substance, it seems to act on the vitelline membrane, possi­
bly in a manner similar to dermal secretion (Metz, 1961).
Other naturally
occurring fertilization inhibiting substances which have been studied
include;
heparin, human H substance, chitin disulfuric acid, sialic acid,
flavanols, and germanin (cf. Metz, 1961).
The use of enzymes, oxidizing and reducing agents, and other reagents
as inhibitors has been avoided to a great extent because these substances
are often detrimental to the gametes or are altered by the gamete metabo­
lism (Metz, 1961).
Nevertheless, these agents, when applied in low concen­
trations, are valuable indicators of molecular participation in gamete
interaction.
For instance, treatment of sea urchin eggs with proteolytic
29
enzymes lowers their fertilizability (Tyler and Metz, 1955; Aketa, Onitake,
and Tsuzuki, 1972). This block to fertilizability is due to disruption of
the vitelline membrane but can be overcome by using very high concentra­
tions of spermatozoa (Aketa, Onitake, and Tsuzuki, 1972).
These authors
conclude that the vitelline membrane possesses essential sperm binding
sites that are at least partially destroyed or removed by the action of
trypsin.
Another example of enzyme action on gamete surface coverings was
reported by Soupart and Clewe (1965).
These workers treated the zona pel-
lucida of rabbit ova with neuraminidase also causing inhibition of sperm
penetration.
Evidently the sialic acid residues, removed during the enzyme
treatment, are necessary for normal sperm penetration.
Various oxidizing agents, reducing agents, and protein group reagents
have been examined as to their affect on the agglutinability of fertilizin.
Metz (1954) demonstrated that treatment of fertilizin with sulfhydryl
reagents (iodine, p-chloromecuribenzoate), amino group reagents (formalin,
nitrous acid, acetic anhydride, benzoyl chloride, dinitroflourobenzene),
and tyrosyl reagents (iodine, dinitroflourobenzene) all failed to affect
the sperm agglutinating power of fertilizin.
dithionite (Nag^SgO^) had no affect.
Also reduction with sodium
However, oxidation with periodate
(Runnstrom, 1935) and hydrogen peroxide (Metz, 1967; Tyler, 1941) does
destroy the sperm agglutinating action of fertilizin.
In the case of
hydrogen peroxide, the oxidation does not block the fertilizin active site
but only cleaves the molecule to the univalent form.
Thus, the specific
chemical groups of fertilizin involved in sperm agglutination do not seem
to involve sulfhydryl, disulfide, phenolic, or amino groups but may pos­
30
sibly involve the carbohydrate moiety since periodate oxidation destroys
the capacity of fertilizin to combine with sperm (Metz, 1967).
In another experiment testing the affects of specifically reactive
chemical compounds on fertilization, Kelly, Schuel, and Severin (1971)
found that treatment of sea urchin eggs with certain quaternary ammonium
drugs inhibits fertilization.
These drugs form stable complexes with sul-
fomucopolysaccharides which are abundant in the cortical granules of the
egg.
Thus, the authors believe that quaternary ammonium compounds such as
tetraethylammonium bromide interfere with the normal cortical reactions by
specifically combining with sulfomucopolysaccharides-t
To Summarize, various chemical inhibitors to fertilization, especially
those with specific reactivity, have been helpful in elucidating the chemi­
cal nature of gamete interacting substances.
As yet, no particular chemi­
cal groupings have been found to participate in fertilization, however, it
is clear that larger molecules such as the glycoprotein fertilizin are
necessary for fertilization.
Assuming that there is a definite biochemical
pathway associated with the sperm-egg interactions, then the use of spe­
cific chemical inhibitors is an important method in elucidating this path­
way.
Immunological Aspects of Gamete Surface Interactions
Several studies employing immunological methods have been undertaken
to study gamete antigens and determine their roles in fertilization.
Fol­
lowing is a review of the investigations of gamete antigens with special
reference to their involvement in fertilization.
31
Egg antigens
The antigenic structure of animal eggs has been investigated most
extensively in echinoderms and amphibians (cf. Tyler, 1959; Metz, 1967).
Egg antigens have been studied in relation to their function in fertiliza­
tion, egg activation, cleavage, and early development.
In addition, con­
siderable work has been performed on the oviducal deposition of egg jelly
antigens in amphibians.
These immunological investigations have not only
aided in understanding the molecular structure of egg surfaces but also the
function of the egg surface substances in fertilization.
Using immunodiffusion and immunoelectorphoresis, Perlmann (1953) and
Metz (1967) have demonstrated at least ten soluble antigens in unfertilized
sea urchin eggs.
Exposure of eggs to anti-egg antiserum causes precipita­
tion and contraction of the egg jelly layers, agglutination of the eggs
(Perlmann, 1954; Baxandall, Perlmann, and Afzelius, 1964), and often wrink­
ling, blistering, and cytolosis (Perlmann, 1954; Tyler and Brookbank, 1956).
In addition to these structural changes, anti-egg serum also has various
physiological effects such as parthenogenetic activation (Perlmann, 1954,
1959), increase in respiration (Tyler and Brookbank, 1956; Brookbank, 1959),
and inhibition of cleavage (Tyler and Brookbank, 1956). Perlmann (1959)
has chemically characterized a number of antigens involved in these
responses.
Of particular interest is the A-antigen involved in partheno­
genetic activation of the egg.
Treatment of sea urchin eggs with appropriate concentrations of either
anti-egg or anti-jelly sera at low temperatures induces parthenogenetic
activation and cleavage (Perlmann, 1954, 1959).
According to Perlmann
(1959), the egg activating action is attributed to the interaction of the
32
serum with a component extractable from the egg, the A-antigen, a heat
stable, water soluble carbohydrate.
Baxandall, Perlmann, and Afzelius
(1964) using immunoferritin labeling techniques demonstrated that A-antlgen
is present in a 0.1 ^-thick layer exterior to the plasma membrane.
Other
workers including Tyler (1959) and Brookbank (1959) were not able to con­
firm the parthenogenetic activation of sea urchin eggs with antiserum but
did note increases in respiration. Perlmann's findings provide evidence
for the role of a specific antigen in fertilization.
In amphibians, the antigens of the egg jelly are of special interest,
since the jelly layers are essential for fertilization (Good and Daniel,
1943),
Shivers (1965) has demonstrated by immunodiffusion at least five
egg jelly antigens in each of seven species of amphibians and, in Rana
plplens egg jelly, antigens have been localized in three distinct layers
(Shivers, 1962).
In additions to the egg jelly, antigens of the egg pro­
per have also been studied.
For example, Nace and Lavin (1963) demon­
strated an egg cortex antigen and another antigen shared by eggs, follicle
cells, and oviduct.
As can be seen, antigens are readily demonstrated in the eggs and egg
envelopes of various species.
However, the mere presence of these antigens
is of little significance unless definite functions can be ascribed to them.
Fortunately the unique properties of antibodies allow them to be used not
only as Indicator reagents but also as specific molecular inhibitors.
Hence, the use of antibodies allowed Tyler, Perlmann, Metz, and others to
study the dynamic involvement of gamete antigens in fertilization.
Follow­
ing are discussions of sea urchin and amphibian egg antigens and their
roles which have been determined by these investigators.
33
Role of egg antigens in fertilization
The function of egg surface antigens in fertilization has been inves­
tigated by using antibodies as inhibitors (cf. Perlmann, 1959; Metz, 1967),
since antibodies can be prepared with a very high degree of specificity for
certain egg surface niacromolecules, such as fertilizin.
Inhibition of fer­
tilization by combination of surface macromolecules with specific anti­
bodies is a direct measure of the degree of participation of these macromolecules in fertilization.
Sea urchin eggs treated with anti-egg antiserum become unfertilizable
(Perlmann, 1954). Perlmann (1954, 1959) has concluded that the fertiliza­
tion inhibiting action is due to the specific blockage of F-antigen located
both in the egg jelly and in the egg cortex. He suggests that the F-anti­
gen may constitute a specific sperm receptor site or be part of the corti­
cal mechanism (Perlmann, 1959).
Even though Perlmann's experiments demonstrate a high degree of speci­
ficity of anti-egg serum for F-antigen, this antiserum is undoubtedly
involved in certain types of cross-reactions.
These cross-reactions are
due to the bivalence of the antibody which induces such effects as precipi­
tation of the egg jelly layer, wrinkling of the egg surface, and cortical
damage (see Metz, 1967).
These side effects caused by the bivalent anti­
body leave open to question whether or not the fertilization inhibiting
action of the antibody is due to specific blockage of antigenic determi­
nants or to the formation of cross-linked molecular lattices (Shivers and
Metz, 1962; Metz and Thompson, 1967).
The formation of cross linkages
could act as a mechanical barrier to the sperm, possibly preventing its
interaction with other molecular components blocked or buried beneath the
34
lattice.
The validity of this reasoning is substantiated by ultrastruc­
tural studies on antibody treated eggs (Baxandall, Perlmann, and Afzelius,
1964; Metz, Cone, and Bryant, 1968).
These workers have demonstrated a
0.1 ix-thick electron-opaque layer at the surface of antibody treated eggs.
In addition, Metz and Thompson (1967) showed that bivalent antibody treated
sea urchin eggs recover fertilizability when post-treated with proteolytic
enzymes which remove the lattice barrier.
Indeed, electron micrographs of
antibody treated, protease post-tested eggs indicate the lack of a dense
layer (Metz, Cone, and Bryant, 1968),
These observations established that bivalent antibodies, by virtue of
their inherent cross-reactivity, are not always suitable for use as inhibi­
tors in cell interacting systems.
This fact was recognized early by Tyler
(1945) who developed a photooxidation method for converting bivalent anti­
bodies to the univalent, nonagglutinating, nonprecipitating form.
A more
convenient and perhaps more precise method of preparing univalent (3.5 S)
IgG antibodies (see Putnam, 1969 for review of antibody structure) was sub­
sequently developed by Porter (1959). Using this method to prepare univa­
lent fragments, Metz and Thompson (1967) undertook a thorough study of the
effects of univalent antibodies on sea urchin fertilization.
Univalent antibodies directed against eggs of the sea urchin
Lvtechinus variegatus failed to produce a number of morphological and
physiological effects elicited by normal bivalent (7 S) antibody (Metz and
Thompson, 1967). For example, univalent antibody did not form a precipita­
tion layer or cause egg wrinkling.
The only observable effect was the for­
mation of a faint hyaline layer in dejellied eggs.
Univalent antibody
treatment did, however, reduce the percentage of cleavage of eggs already
35
fertilized, although to a lesser extent than with bivalent antibody.
But
perhaps most significantly, the univalent anti-egg antibody did not affect
the fertilizability of egg (Metz and Thompson, 1967).
This latter observa­
tion was reaffirmed by Graziano and Metz (1967) with another species of sea
urchin.
These results answer two important questions, at least with
respect to sea urchin fertilization. First, the fertilization inhibiting
action of bivalent antibody is dependent on the formation of a cross-linked
lattice.
Second, essential fertilization antigens evidently do not exist
on the sea urchin egg surface as measured with this technique. In contrast,
univalent anti-egg antibodies have been used with some success in amphibians
(Shivers and Metz, 1962). These results, along with various bivalent anti­
body studies, are now considered.
Shaver and Barch (1960) demonstrated that eggs of the frog, Rana
pipiens. pretreated with anti-egg jelly serum have reduced fertizability.
This is a tissue-specific effect as demonstrated by various other studies.
These include experiments by Shaver, Barch, and Shivers (1962) which show
that absorption of anti-jelly serum with sperm and other tissues failed to
affect inhibition of fertilization.
However, the inhibiting effect is not
necessarily species specific since other experiments demonstrate that antijelly serum (Shivers, 1965) and anti-oviduct serum (Shaver, Barch, and
Umpierre, 1970) from heterologous species is inhibitory to Rana fertiliza­
tion.
Shaver (1966) interprets these results to mean that two different
jelly antigens may be involved in fertilization, those which are species
specific and those which are shared by other ranid species.
A recent study
by Shivers and James (1970) has demonstrated a specific role for one jelly
antigen, the conditioning or "capacitation" of spermatozoa.
These investi-
36
gators reported that treatment of frog eggs with anti-jelly serum does not
inhibit fertilization with "capacitated" spermatozoa (i.e. spermatozoa hav­
ing prior contact with dissolved jelly).
Presumably the antiserum blocks
essential jelly sites for capacitating spermatozoa.
Unfortunately, only
one study has been performed on amphibians where univalent antibodies were
used.
In this study, Shivers and Metz (1962) demonstrated that papain
digested, univalent anti-jelly antibody inhibits the fertilizability of
Rana pipiens eggs.
The authors believe that the loss of fertilizability is
due to direct blockage of essential sperm receptor sites.
Antigens located in the egg proper have also been implicated in
amphibian fertilization. If jellyless eggs are treated with antiserum
directed against various oocyte and embryo extracts and then reenveloped
with jelly, fertilization success is markedly reduced (Nace and Lavin,
1963).
The authors conclude that the antibody interferes with some sperm-
egg interacting process or possibly the egg activating mechanism.
non-jelly antigens may also be important In fertilization.
Thus,
Again these
results would be more conclusive had univalent antibodies been used.
In conclusion, bivalent antibody treatment of eggs is generally suc­
cessful in blocking fertilization.
This inhibition, however, may be due to
the formation of molecular cross-linkages rather than specific blockage of
Individual antigenic determinants.
This problem has been overcome with
univalent antibodies which do not inhibit fertilization in sea urchins but
are effective in the frog, Rana pipiens. In any case, univalent antibodies
directed against the egg surface appear to be good diagnostic reagents for
determining the role of certain egg surface macromolecules.
37
Sperm antigens
The antigens of spermatozoa have been studied widely in the animal
kingdom (cf. Metz, 1967; Edwards, 1969).
Sperm antigens are of Interest
not only from the standpoint of their relationship to antigens of other
body tissues but also with respect to their contribution to the structure
and functions of the gamete.
The latter is especially significant since
some sperm antigens have been shown to be important in fertility and
sterility.
Sperm antigens have been demonstrated in several invertebrate species
(cf. Metz, 1967, 1961).
The use of interspecies cross-agglutlnatlon-absorp-
tion, immunodiffusion, and Immunoelectrophoresis techniques have allowed
investigators to demonstrate a variety of surface and subsurface sperm anti­
gens.
Surface antigens are especially significant since they are thought
to function in initial sperm-egg interactions.
Extraction techniques have
demonstrated that certain antigens are soluble and that others are insolu­
ble or built into the membrane component of the sperm.
Tyler (1949) and
Kohler and Metz (1960) reported that the spermatozoa of various species of
sea urchins possess two or three common surface antigens. In addition,
four soluble antigens have been found in the sperm of the sea urchin,
Arbacia punctulata (Metz and Kohler, 1960).
These antigens have been
localized by various techniques and have been shown to display a unique
distribution pattern both in the head and tail and on the surface and sub­
surface of the sperm (Metz, 1967).
A similar analysis of spermatozoa from
several species of decapod crustaceans has demonstrated the presence of
constellations of interspecific and species specific surface and subsurface
antigens (Mowbray, Brown, and Metz, 1970).
Cooper and Brown (1972) have
38
shown that the sperm of the horseshoe crab, Llmulus polyphemus possesses at
least seven soluble antigens, five of which are surface antigens.
Thus,
several invertebrate species possess a number of sperm antigens which con­
tribute to the molecular structure of the sperm.
Sperm antigens have also received considerable attention in mammals.
Regional antigens, such as those demonstrated in sea urchin spermatozoa,
have also been found in various mammalian species (Edwards, 1969; Shulman,
1971).
With regard to localization and characterization of sperm antigens,
the work of Henle (1938) on bull sperm antigens led the way to future
investigations in this field.
Henle (1938) found sperm head-specific and
sperm tail-specific surface antigens identifiable by both agglutination and
complement fixation tests.
Other investigators have demonstrated sperm
antigens in various mammalian species:
Chinese hamster (Flko and Tyler,
1962), ram (Hathaway and Hartree, 1963), guinea pig (Hekman and Shulman,
1971), rabbit (Weil, 1960), and man (Hekman and Rumke, 1969). Mancini
et al. (1964) identified a specific antigen, hyaluronldase, in the bull
spersî acrcsoae by using specific anti-hyal«ronidase antibody with immuno­
fluorescence techniques.
As is well know, this important enzyme functions
in sperm penetration of the cumulus oophorus.
Recent findings indicate that mammalian spermatozoa are often coated
with substances from the epididymis and male accesory glands.
Many of
these spermatozoa coating substances are antigens and have been demon­
strated in the rabbit (Well, 1965), man (Well and Rodenburg, 1960), and the
bull (Hunter and Hafs, 1964). These sperm coating antigens are significant,
since they may function In sperm capacitation as discussed earlier.
Human
A and B blood group antigens (Edwards, Ferguson, and Coombs, 1964) and
39
bovine blood group J-substance (Stone, 1964) have been found on the
spermatozoa of secretor individuals.
Hekman and Rumke (1969) demonstrated
that one of the sperm coating antigens of human seminal plasma is antigenically related to lactoferrin, an iron-binding protein occurring in milk and
other external secretions.
Metz, Hinsch, and Anika (1968) reported that
rabbit seminal particles have at least one antigen in common with the sur­
face of the sperm.
This also may be a sperm coating antigen.
Thus, the antigens of spermatozoa from various animal species are
numerous and often associated with a particular region of the sperm such as
the acrosome or flagellum.
The surface antigens sometimes arise indepen­
dently, being absorbed by the sperm from seminal secretions.
In addition,
sperm antigens are often related to antigens of other tissues and common
sperm antigens have been demonstrated between species.
Role of sperm antigens in fertilization
Clearly, spermatozoa possess a number ^of regionalized antigens.
The
possibility exists that sperm antigens participate in one or more steps in
fertilization and since antibodies combine with these antigens, they must
block or interfere with their involvement in fertilization.
Accordingly,
several investigations, using antibodies as inhibitors, have attempted to
demonstrate the involvement of sperm antigens in fertilization.
Tyler and O'Melveny (1941) found that anti-sperm antibody treatment
lowered the fertilizing capacity of sea urchin spermatozoa.
They attributed
this inhibiting effect to the agglutination and mechanical trapping caused
by the multivalent antibody.
To avoid this difficulty, Tyler (1945)
developed a photooxidation method to convert multivalent antibody to the
40
nonagglutinating, univalent form.
Tyler (1946) found that such univalent
antibodies prepared against sperm extracts specifically lowered the fertil­
izing capacity of sea urchin (Lvtechinus pictus) and echiurold (Urechis
caupo) spermatozoa. In a similar experiment, Metz, Schuel, and Bischoff
(1964) demonstrated that univalent anti-whole sperm antibodies (prepared by
papain digestion) strongly reduced the fertilizing capacity of Arbacia
punctulata spermatozoa.
Metz (1967) provides evidence that the antigen
Involved is soluble, sedimentable at 30,000 jji and is present on the sperm
surface, but the mechanism of action and the fertilization step in which
the antigen is Involved is still unresolved.
Metz, Schuel, and Bischoff
(1964) suggested that antibody treatment could inhibit or prematurely ini­
tiate the acrosome reaction, preventing release of egg jelly lysln, mem­
brane fusion, or egg activation.
One of these possibilities was eliminated
when Fourtner and Metz (1967) determined that the sea urchin acrosome reac­
tion is unaffected by antibody pretreatment.
Recently, Mowbray, Brown, and
Metz (1970) demonstrated that one well defined fertilization step, namely
spcnn^cgg ottachuucnt, is inhibited in the dscspod crab,
by univalent anti-sperm antibody.
Thus several investigators have demon­
strated that sperm antigens of various Invertebrates play a definite role
in fertilization sperm-egg interactions.
The role of anti-sperm antibodies in relation to prevention of concep­
tion has been studied in various mammalian species.
In fact, natural iso­
immunization of either male or female with seminal antigens has been sug­
gested as a possible cause of infertility in mammals (Beer and Blllingham,
1971; Shulman, 1971).
Iso-immunization of males could result in aspermato­
genesis or other damage to spermatozoa and thus reduce fertility.
Semen
41
Iso-immunization of females also could result in circulating and/or local
secretory antibodies that would inhibit spermatozoa from reaching or other­
wise interacting with the ovum (Casida, 1961). Iso-immunization of females
with spermatozoa or semen plus adjuvant induces infertility in guinea pigs
(Katsh, 1959; Isojima, Graham, and Graham, 1959; Behrman et al.. 1963),
mice (McLaren, 1964; Edwards, 1964), rabbits (Behrman and Nakayama, 1965),
and cattle (Menge, 1967).
However, fertility in rabbits was not affected
by injection of seminal plasma alone (Weil and Roberts, 1965).
The point
at which antibody interferes with conception in the iso-immune system has
not been well documented, however, McLaren (1964) reported that iso-immuni­
zation of mice with spermatozoa resulted in fertilization failure and not
embryo death, as reported by others.
Another unresolved question is
whether or not there is local production of antibody in the reproductive
tract.
Kerr (1955) reported detectable antibody titers in vaginal and
uterine secretions but not in the serum of cows immunized interuterally
with Brucella organisms.
Interuterine injection of cattle with semen plus
adjuvant produced equivalent titers of antibody in vaginal mucus and serum
(Menge, 1967).
These results only suggest that antibodies may be synthe­
sized in the reproductive tract and do not constitute conclusive evidence.
The effects of jji vitro treatment of semen with antibodies have also
been studied to some extent in mammals.
The exposure of rabbit semen to
cattle anti-rabbit sperm serum resulted in failure of fertilization and
embryo mortality in rabbits (Kiddy, Stone, and Casida, 1959). Heifers
inseminated with bull semen pretreated with homologous anti-semen serum
failed to conceive (Menge £t al., 1962).
Both experiments utilized multi­
valent antibodies and thus are subject to the same criticism mentioned ear-
42
lier.
One account has been published, however, where univalent anti-sperm
antibodies were used as inhibitors to fertilization.
Metz and Anika (1970)
demonstrated failure of conception in rabbits inseminated with semen pretreated with univalent guinea pig anti-rabbit sperm antibodies.
The authors
suggest that the univalent antibody blocks some sperm antigen(s) that has a
role in penetration of the cervical mucus.
These experiments on mammals,
as well as those mentioned earlier on invertebrates, definitely show that
sperm antigens are involved in one or more fertilization steps,
Limulus Fertilization
Fertilization in the horseshoe crab, Limulus polyphemus, has been the
subject of a number of recent investigations (Brown and Humphreys, 1971;
Shoger and Brown, 1970; Cooper and Brown, 1972).
This species has been
selected for fertilization studies for several reasons.
Animals are easily
maintained in marine aquaria and viable gametes can be obtained throughout
the year.
Most significantly, the gametes are easily manipulated under a
variety of experimental conditions.
Finally, Limulus is a distant taxon
from those organisms in which fertilization has been studied most (i.e.
amphibians and echinoderms), thus represents a new species in which to sup­
port, enhance, or modify fertilization concepts.
In nature, Limulus normally spawns in the spring of the year at high
tide in shallow intertidal areas (Shuster, 1960).
The male attaches to the
posterior abdominal region of the female in an astplexus posture.
The
female excavates a nest into which are laid approximately 200 eggs.
The
male then inseminates the eggs by direct deposition of semen into the nest
(Kingsley, 1892).
Laboratory observations have shown that a large number
43
(10^-10^) of spermatozoa attach to each egg (Brown and Humphreys, 1971).
Further observations have proven that this polyspermic attachment is neces­
sary for normal development to ensue, since decreased concentrations of
2 4
spermatozoa (yielding 10 -10 spermatozoa per egg) drastically reduce the
percentage of developing eggs (Brown and Humphreys, 1971).
These authors
suggest two possibilities for the necessity of polyspermic attachment:
(1) to induce some chemical change in the egg surface, thus facilitating
the penetration of one sperm, or (2) to increase the probability of one
sperm penetrating a special sperm entry site.
Both the egg and sperm of Limulus have been studied ultrastructurally.
The mature egg is large (2.5 mm in diameter) and yolky, covered by an
envelope consisting of two layers:
a thin outer layer, the basement lamina
(5 ji), and a thicker inner layer, the vitelline envelope (35 p,) (Dumont and
Anderson, 1967).
The basement lamina is covered with electron dense gran­
ules and randomly perforated by perpendicular pores (Shoger and Brown,
1970).
There is no evidence of a micropyle.
The Limulus sperm has many
typical features including an apical acrosome, nucleus, mitochondrial region
and flagellum.
One unique structure is the preformed axial rod, a compo­
nent of the acrosomal complex.
The axial rod is coiled posterior to the
nucleus and extends through an intranuclear canal to the proximal border of
the acrosomal cap (Andre, 1963),
The acrosomal reaction in Limulus may be artificially induced by
treating spermatozoa with egg extracts (André, 1963; Shoger and Brown,
1970).
In the normal acrosome reaction, the axial rod pierces the acro­
somal cap and extends to produce a 40 ^ long acrosomal filament covered by
a membrane formed from the old acrosomal membrane.
The length of the axial
44
rod corresponds to the total width of the egg envelope.
During the sperm-
egg interaction, the acrosome reaction is initiated when the sperm makes
contact with the egg, whereupon the acrosomal vesicle opens and its con­
tents become bound to the basement lamina (Shoger and Brown, 1970). As the
acrosomal membrane fuses with the sperm plasma membrane, the axial rod
uncoils and extends (mechanically and/or enzymatically) through the base­
ment lamina and vitelline envelope to the plasma membrane of the egg.
The
axial rod is surrounded by a membrane derived from the acrosomal membrane.
Presumably the nucleus then passes through the tubular canal formed by the
axial rod.
Although polyspermic attachment is common in this species, it
is unlikely that a high number of sperm nuclei enter the egg, since this
would contradict previous findings in other species.
Statement of Problem
Gamete surface interacting substances have several important functions
in fertilization.
One means of examining these substances is to employ
antibodies directed against gamete surfaces as fertilization inhibitors.
Although the results thus far with anti-egg antibodies have been variable,
treatment with bivalent antibodies usually inhibits fertilization (Perlmann,
1959; Metz, 1967).
On the other hand, univalent antibodies have been found
to have no effect on sea urchin fertilization (Metz and Thompson, 1967;
Graziano and Metz, 1967) but do inhibit fertilization in Rana pipiens
(Shivers and Metz, 1962).
However, this latter result is questionable,
since recent experiments have demonstrated that univalent antibodies do not
45
inhibit fertilization in Rana pipiens^.
In addition, with these species
(i.e. sea urchins and amphibians), the fertilization step interfered with
is often impossible to determine.
Thus, in the two systems where anti-egg
antibodies have been employed, there exists a degree of uncertainty as to
their effectiveness and site of action.
Therefore, this problem needs
reinvestigation in another species.
The present study represents a new approach in two ways. First, all
experiments will concentrate on one specific step in fertilization, spermegg attachment.
Second, a new method has been developed for the quantita­
tive measurement of sperm-egg binding capacity.
The problem with many pre­
vious studies has been the lack of information concerning the site of
action of various inhibitors, including antibodies.
Even though these
inhibitors are highly specific in their activity, the parameter measured
was usually successful cleavage or embryo development.
Fertilization could
be inhibited in several possible steps (e.g. sperm-egg attachment, acrosome
reaction) but there would be no way of determining which step.
The present
investigation uses sperm-egg attachment aa a parameter for measuring spermegg interaction.
Since sperm-egg attachment is one of the earliest steps
in sperm-egg interactions and since gamete attachment is necessary for suc­
cessful fertilization, it is a valid parameter for fertilization studies.
Sperm-egg attachment is described in this context as the initial contact
Arakelian, H. K. Department of Zoology, Michigan State University,
East Lansing, Michigan. Effect of univalent antibody fragments of eggjelly serum on the fertilizability of Rana pipiens eggs. Personal communi­
cation. 1972.
46
and adhesion of gametes and probably does not include the cementing action
of the acrosomal contents.
The primary method employed in this investigation is the utilization
of anti-egg antibodies (bivalent and univalent) as inhibitors to sperm-egg
attachment.
This method is augmented by additional experiments involving
the use of enzymatic and chemical inhibitors to fertilization.
Thin sec­
tions of Limulus eggs were treated with these reagents and the degree of
sperm-egg attachment assessed by actual counts of bound spermatozoa.
In an
attempt to identify the egg substances involved in sperm-egg attachment,
additional tests were made utilizing immunodiffusion and Immunoelectro­
phoresis.
47
MATERIALS AND METHODS
Horseshoe crabs, Limulus polyphemus L., were obtained from the Supply
Department, Marine Biological Laboratory, Woods Hole, Massachusetts, and
from the Florida Marine Biological Specimen Company, Panama City, Florida.
In the laboratory these animals were maintained in an Instant Ocean System
at 15° C.
During the duration of these experiments, approximately 125
females and 75 males were used.
Gamete Collection and Preparation
Gametes were obtained by electrical stimulation, following a previ­
ously described procedure (Shrank et al., 1967).
Eggs were washed three
times in 25 ml of sea water and were immediately frozen, either in a 5%
gelatin solution or in sea water, and were then stored at -25° C. Freshly
collected "dry" semen was washed three times in 5 ml of sea water by centrifugation (300 Q).
One drop (0.05 ml) of the washed spermatozoa was
resuspended to make a 2.5% sperm suspension in 2 ml of sea water.
By
using a hemocytometer, the sperm count of a 2.5% resuspended sperm suspeng
sion was estimated at 2 x 10 spermatozoa/ml, a figure comparing favorably
with the sperm count of a 2.5% "dry" sperm suspension.
The sperm suspen­
sion was always freshly mixed before each experiment.
Eggs were prepared for experiments by sectioning at 12-15 p, with a
cryostat and mounting sections on glass microslides.
Egg sections adhere
well to glass and are able to withstand treatment with various reagent
solutions and washings.
In addition, they can be stored frozen for several
weeks but normally are used during the first week.
48
Antibody Preparation
Antiserum was prepared by injecting rabbits (New Zealand Whites) subscapularly with an emulsion consisting of whole eggs (300-400 eggs) or egg
envelopes (removed from eggs with watchmaker forceps), 0.85% saline (2 ml),
and complete Freund's adjuvant (1 ml).
The rabbits were injected similarly
again after one month and then were bled at weekly intervals for immune
serum harvest.
To assure that approximately equivalent amounts of antibody
were used in all experiments, the gamma-globulin fraction was separated
from the whole serum by triple precipitation with 33% ammonium sulfate
(Stelos, 1967). Protein determinations were made on the globulin fractions
using the biuret method (Gornall, Bardawill, and David, 1949), and the
antibody solutions were adjusted to equivalent globulin concentrations (IS­
IS mg protein per ml).
All globulin preparations were dialyzed against sea
water for 48 hr before use in diagnostic tests.
Preparation of Univalent Antibody
Univalent antibody (Fab fragments) were prepared by the method of
Porter (1959).
Each digest consisted of 50 mg gamma-globulin (IgG), 0.5 mg
2X crystallized papain (Sigma Chemical Co.), 0.1 M potassium phosphate,
0.01 M cysteine, 0.002 M disodium EDTA adjusted to pH 7.0.
Digestion was
carried out at 37° C for 8-12 houri and considered complete when the anti­
body no longer agglutinated homologo ; spermatozoa.
The digests were then
centrifuged at 13,000 K for 30 min and dialyzed overnight against 0.02 M
iodoacetamide in 1.5 M NaCl to assure partial removal and complete inactivation of the papain.
Dialyzation was continued another 36 hours against
sea water to remove the iodoacetamide.
The presence of active univalent
49
antibody was checked by two procedures: (1) agglutination of univalent
antibody treated spermatozoa by sheep anti-rabbit gamma-globulin, and
(2) inhibition of precipitin band formation between egg envelope extract
and bivalent antibody (Shivers and Metz, 1962). The latter involves prereaction of univalent antibody with egg extract on immunodiffusion plates
followed by inoculation of wells with bivalent antibody.
Immunodiffusion and Immunoelectrophoresis
Egg surface extracts for immunodiffusion and immunoelectrophoresis
were prepared by incubating 0.5 ml of whole eggs in 1 ml of sea water solu­
tion containing enzymes or other reagents (Table 1) at 37° C for one hour.
Egg envelope extracts were made by homogenizing isolated egg envelopes
(0.2 ml) in sea water (1 ml) in a glass homogenizer, then centrifuging at
5,000 jg[ for 20 min to remove insoluble material.
Extracts were sometimes
dialyzed against sea water to remove unwanted reagents.
The gel for immunodiffusion was prepared from 1% "Seakem" agarose in
0.85% saline with 0.2% sodium azide added as a preservative and 0.05% cad­
mium chloride added to enhance antigen-antibody precipitate formation
(Crowle, 1961).
Plates were prepared by pipetting 3 ml of hot agarose
solution onto glass microslides.
placed 3-4 mm apart.
to each well.
Wells were cut 6 iran in diameter and
Two drops (0.1 ml) of antibody or extract were added
Immunodiffusion was allowed to proceed for three days before
recording the resulting precipitin lines both photographically and by draw­
ings.
The gel for immunoelectrophoresis was prepared from 0.6% agarose in
Tris-citrate (0.02 M Tris-0.02 M sodium citrate) buffer, pH 8.6. Plates
50
were prepared by pipetting 3 ml of hot agarose solution on glass microslides (Grabar and Williams, 1953). Wells for extracts were cut 3 mm in
diameter and the antibody trough was 1 mm by 5 cm.
Samples were applied
and electrophoresis carried out at 300 v, 35 ma for one hour, then antibody
was added to the trough.
All plates were checked daily and recorded photo­
graphically and by drawings after three days.
Design of Egg Section Treatment Experiments
An experimental system was designed to measure the effects of anti-egg
antibody and other reagents (see Appendix for sources of reagents) on spermegg attachment.
This method involves the use of unfixed egg sections which
are mounted on glass microslides.
can be conveniently manipulated.
The conditions of any given experiment
These conditions include; (1) duration
of sperm-egg mixing (insemination), (2) temperature, (3) duration of treat­
ment with reagent, (4) pH of reaction medium, (5) concentration of reagent,
and (6) sperm concentration.
Except where variables are involved, the fol­
lowing conditions were used:
egg sections were ringed with petrolatum,
pretreated with two drops of antibody (15 rag protein/mi), other reagents or
controls for periods varying from 15 min to 10 hr depending on the experi­
ment at 23-26° C, washed thoroughly by dipping in three changes of sea
water, and then treated for 5 min with two drops of a 2.5% sperm suspension
(10^ spermatozoa) plus one drop of 0.004 M EDTA or of 0.025 M cysteine to
facilitate sperm motility (note:
egg sections pretreated with sea water,
antibodies, or other reagents frequently lose their normal ability to
elicit motility in spermatozoa).
Finally, these egg sections were washed
again and then wet mounted in 10% formalin in sea water and observed for
51
sperm attachment (Figure 1).
All experiments were performed at least three
separate times.
On a microslide containing about 20 egg sections, five sections were
selected and spermatozoa counted on approximately 1/5 (0.44 mm) of the egg
section circumference.
Bias was primarily shown in the case of antibody-
treated egg sections, where few if any spermatozoa attached. In these
cases, sections were always counted where some spermatozoa were attached.
The actual spermatozoa counted were those in one focusing field of a Zeiss
ML microscope using 40X objective lens and a lOX eyepiece.
When using a
2.5% sperm suspension, approximately 100 spermatozoa were counted in such a
field.
From this, the percentage of spermatozoa attached was determined by
dividing the average number of spermatozoa attached to treated egg sections
by the average number of spermatozoa attached to control egg sections.
Effects of Variables on Experiments
Duration of insemination time
To establish a reasonable time for spermatozoa to attach, the duration
of actual sperm-egg mixing was varied from 15 sec to 5 min (Figure 2).
When compared to maximum attachment, the percent of spermatozoa attaching
approach an optimum after 3 min. In all following experiments, 5 min of
sperm-egg mixing was used.
Incubation temperature
To determine the optimum temperature to perform sperm attachment
studies, a range of temperatures varying from 0° C to 45° C was used. In
each case, the egg sections and sperm suspension were allowed to equili­
brate for 1 hr and then mixed together.
The optimum temperature varies from
Figure 1. Egg sections before (la) and after (lb) the attachment of sper­
matozoa. In most experiments, two drops (0.1 ml) of a 2.5%
sperm suspension were placed on a mlcroslide containing about 20
egg sections. After 5 min, these sections were washed, fixed
with 10% formalin in sea water, and attached spermatozoa were
counted. Note the linear attachment of spermatozoa to the outer
edge of the egg envelope. X 280
53
Figure 2.
Duration to time needed for optimum sperm attachment. Untreated
egg sections were treated with two drops (0.1 ml) of 2.5% sperm
suspension for varying periods of time (15 sec to 5 min), then
were rinsed and prepared for observation. Time periods exceed­
ing 2 min showed optimum sperm attachment. The graph represents
the average of three separate experiments
55
100-
90 80-
1-70Z
LiJ
2 oO -
X
y 50
<
I- 40
<
3r 3 0 20-
10-
oI
0
I
I
r
1
2
3
TIME (MIN.)
I
\
4
5
56
s" C to 35" C (Figure 3).
IJased on these experiments, room temperature (23-
25° C) was considered satisfactory for all subsequent experiments.
£H
To establish the optimum pH required for maximum sperm attachment dur­
ing sperm-egg mixing, the pH of sperm suspensions was varied from 3 to 12.
A concentrated (20%) sea water suspension of spermatozoa was diluted 1:10
in pH adjusted sea water and applied to egg sections for a period of 5 min.
Acid sea water (pH 3 to pH 4) causes complete immobilization of spermatozoa,
thus resulting in no attachment to the egg surface (Figure 4).
Alkaline
sea water (pH 9 to pH 12) did not seem to hinder sperm attachment.
The
optimum pH for sperm-egg attachment ranges from 5 to 9. Unbuffered sea
water (pH 7.0) was used in all other experiments.
Figure 3.
Test demonstrating optimum temperature for maximum sperm attach­
ment. Egg sections and sperm suspensions were allowed to equil­
ibrate at each test temperature for 1 hr before mixing. Egg
sections were then treated with two drops of a 2.5% sperm sus­
pension for 5 min, then were rinsed and prepared for observation.
Note that optimum temperature range was from 5 C to 35 C.
Sperm attachment at each temperature was tested three times
58
100-
9080-
LU
2 60
X
^ 50H
<
H 40 ^ 30H
20-
10-
0 -
I
0
I
I
I
I
5
10
15
25
TEMPERATURE (®C)
35
45
Figure 4. Optimum pH required for sperm-egg attachment. Egg sections were
treated for 5 min with two drops (0.1 ml) of 2.5% sperm suspen­
sion adjusted to pH 3 through pH 12, then rinsed and prepared
for observation. The optimum pH ranges from 6 to 8
60
\
I
7
PH
8
9
10
1
1
11
12
61
RESULTS
Although egg surface substances (including antigens) have been shown
to participate in fertilization, their role in sperm-egg attachment has not
been adequately investigated.
Thus, several types of experiments were
designed to study the role of egg substances in sperm-egg attachment in
Limulus polyphemus.
These include; (1) inhibition of sperm-egg attachment
by treatment of the egg surface with bivalent antibody, (2) inhibition of
sperm-egg attachment by treatment of the egg surface with univalent anti­
body, (3) inhibition of sperm-egg attachment by chemical treatment of the
egg surface, and (4) immunological identification of egg surface substances.
Inhibition of Sperm-egg Attachment
with Bivalent Antibody
Experiments involving several variables were used to test the effec­
tiveness of bivalent anti-egg antibody as an attachment inhibitor.
These
tests were of primary importance in establishing the best method for use in
univalent anti-egg antibody experiments.
Sperm concentration
Sperm concentration was varied from a 33% suspension (dilution of
spermatozoa =1) to a 0.001% (1/64 dilution) to determine the effects on
the antibody treated egg sections (Figure 5).
All sperm concentrations
were prepared as resuspensions of washed cells.
The approximate number of
g
spermatozoa placed on each microslide varied from 10
10
spermatozoa (0.001%).
spermatozoa (33%) to
The percent of spermatozoa attaching on antibody
treated egg sections for all dilutions remained reasonably constant.
The
controls showed an expected gradual decrease in the percent of spermatozoa
Figure 5.
Effect of anti-egg antibody treatment of egg sections on sperm
attachment. Egg sections were treated for 4 hr with two drops
(0.1 ml) of globulin (15 mg protein/ml) and then were insemi­
nated with dilutions of spermatozoa (1 « 33% sperm suspension =
10^ spermatozoa/ml) and were prepared for microscopic observa­
tion. A gradual decrease in percent of sperm attachment is
initiated after a 1/4 dilution of spermatozoa. A 1/16 dilution
was used in all other experiments. SW, sea water treated egg
sections; C, control globulin treated egg sections (Rabbit No.
37-7); AE, anti-Limulus egg antibody treated egg sections (Rab­
bit No. 31-1). This graph represents the average of three sep­
arate experiments
53
'AE
,/
1
1/2
1/4
1/8
1/16
DILUTION OF SPERMATOZOA
1/32
1/64
64
attaching with decrease in dose per microslide.
The 2.5% sperm suspension
used in all later experiments represents a 1/16 dilution of spermatozoa.
Duration of antibody treatment
Before sperm attachment, egg sections were treated with anti-egg anti­
bodies at times varying from 30 min to 10 hr (Figure 6). As noticed with
anti-egg antibody treated eggs, a significant decrease occurred between 2
and 3 hr.
No significant decrease occurred with the control globulin or
with sea water treated egg sections.
Four hr treatment of egg sections
were used in all later experiments.
Concentration of antibody
To determine the effect of antibody concentration on inhibition of
sperm attachment, egg sections were treated with concentrations of anti-egg
antibody and of control globulin varying from 60 mg protein/ml (dilution of
globulin = 1) to 1.9 mg protein/ml (dilution of globulin 1/32). As may be
noticed (beginning with the first dilution), there is a steady increase
with increased antibody dilution in the percent of spermatozoa attaching
(Figure 7).
Since 15 mg protein/ml (1/4 dilution) was adequate for effec­
tive inhibition, this antibody concentration was used in all later experi­
ments.
Effects of anti-heart antibody
In order to test for the possibility that the blockage of sperm-egg
attachment was due to activity of ^aspecific antibody present in the antiegg globulin, the following experiment was performed.
Egg sections were
tested with antibody prepared against Limulus heart tissue (Figure 7).
Figure 6.
Effect of anti-egg antibody treated egg sections on sperm
attachment. Egg sections were treated with two drops (0.1 ml)
of globulin (15 mg protein/ml) or with sea water for duration of
time varying from 30 min to 10 hr. Slides were incubated in
moist chambers at room temperature. At appropriate times, the
treated egg sections were inseminated and were prepared for
microscopic observation. With use of anti-egg antibody, note a
significant decrease in percentage of sperm attachment arising
between 2 and 3 hr treatment times. SW, sea water treated egg
sections; C, control globulin treated egg sections (Rabbit No.
26-1); AE, anti-egg antibody treated egg sections (Rabbit No.
31-1). The graph represents the average of four separate exper­
iments
i
66
100-
9080-
H
w
z
^60-1
T
O 50<
<
'AE
40-
zr 3 0 2010-
I
0
12
3
4
T
5
T
6
T
7
T
8
i
"1
9
10
DURATION OF TREATMENT (HOURS)
Figure 7.
Effect of anti-egg antibody treatment of egg sections on sperm
attachment. Egg sections were treated with two drops (0.1 ml)
of various dilutions of globulin (1 • 60 mg protein/ml). After
4 hr, the sections were inseminated and were prepared for micro­
scopic observation. A gradual increase in the percentage of
sperm attachment was observed with decreasing antibody concen­
tration. A 1/4 dilution of globulin was used in all other
experiments. SW, sea water treated egg sections; C, control
globulin treated egg sections (Rabbit No. 37-7); H, anti-Limulus
heart antibody treated egg sections (Rabbit No. 47-2); AE, antiLimulus egg treated egg sections (Rabbit No. 31-1). This graph
represents the average of three separate experiments
68
100
90
#
80
70
w 60
2
5 50
^ 40
<
AE
30
/
20
10
.
/
J"
0
r
I
I
'
1/2
1/4
1/8
1/16
DILUTION OF GLOBULIN
69
When compared to egg sections treated with anti-egg globulin, the antiheart globulin has little or no effect on sperm attachment.
Tissue specificity of anti-egg antibody
To test the specificity of the anti-egg antibody for egg surface anti­
gens, the antibody solution was absorbed with three different Limulus tis­
sues including whole semen, heart muscle, and hepatic cecum.
Antibody
solutions (15 mg/ml) including controls were absorbed with equal volumes of
tissue overnight at 5° C.
Egg sections were treated with dilutions of
absorbed antibody and controls for 2 hr, then rinsed, treated with sper­
matozoa, and prepared for observation.
The results demonstrate that none
of the three absorbing tissues affect the ability of anti-egg antibody to
inhibit sperm attachment (Figures 8, 9, and 10).
These results mean that
the anti-egg antibody preparation is highly specific for egg surface anti­
gens.
Inhibition of Sperm-egg Attachment
with Univalent Antibody
Bivalent anti-egg antibody strongly inhibits sperm-egg attachment in
Limulus (Figures 5-10).
Possibly this inhibition is due to the formation
of a molecular lattice rather than site specific binding.
This possibility
was tested by utilizing anti-egg envelope antibody (AEE) in its univalent
form, which can only interact with one antigenic determinant at a time,
thus eliminating lattice formation.
Two types of experiments were per­
formed: (1) variation in the time of treatment with antibody (Figures 11
and 12) and (2) dilution of antibody (Figures 13 and 14).
Figure 8.
Effect of absorption of anti-egg envelope antibody (AEE) with
semen on sperm attachment inhibiting ability. AEE (15 mg pro­
tein/ml) and controls were absorbed with an equal volume of dry
semen (fresh semen, undiluted). Egg sections were treated with
two drops (0.1 ml) of the absorbed globulin for 2 hr. The egg
sections were then rinsed, treated with spermatozoa, and pre­
pared for observation. The sperm-attachment inhibiting action
of the absorbed antibody varies only slightly from the unabsorbed preparation. The results shown represent a summary of
three separate experiments. SW, sea water treated egg sections;
C, control globulin (Rabbit No. 70-2); S-C, control globulin
absorbed with semen; AE, anti-egg envelope antibody (Rabbit No.
62-3); S-AEE, anti-egg envelope antibody absorbed with semen
71
100
90
©•
S^^
80
^c
À-----
/
70
60
50
40
//
//
30
20
10
y'
/!
0
1/2
1/4
1/8
DILUTION OF GLOBULIN
1/16
Figure 9.
Effect of absorption of anti-egg envelope antibody with Limulus
heart tissue on sperm attachment inhibiting ability. AEE (15 mg
protein/ml) and controls were absorbed with an equal volume of
macerated Limulus heart tissue. Egg sections were treated with
two drops of the absorbed globulin for 2 hr. The egg sections
were then rinsed, treated with spermatozoa, and prepared for
observation. The sperm-egg attachment inhibiting action of the
absorbed antibody varies only slightly from the unabsorbed prep­
aration. The results shown represent a summary of three sepa­
rate experiments. SW, sea water treated egg sections, C, con­
trol globulin (Rabbit No. 70-2); H-C, control globulin absorbed
with heart tissue; AEE, anti-egg envelope antibody (Rabbit No.
62-3); H-AEE, anti-egg envelope antibody absorbed with heart
tissue
73
100 -
g^SW
,H-C
90r:-80-
^70-
UJ
2 60^50H
H-AEE
H
*- 40 <
0? 3 0 H
20-
10
oH
I
I
I
1/2
1/4
1/8
DILUTION OF GLOBULIN
1/16
Figure 10.
Effect of absorption of anti-egg envelope antibody with Limulus
hepatic cecum tissue on sperm attachment inhibiting ability.
AEE (15 mg protein/ml) and controls were absorbed with an equal
volume of macerated Limulus hepatic cecum tissue. Egg sections
were treated with two drops of the absorbed globulin for 2 hr.
The egg sections were then rinsed, treated with spermatozoa,
and prepared for observation. The sperm-egg attachment inhib­
iting action of the absorbed antibody varies only slightly from
the unabsorbed preparation. The results shown represent a sum­
mary of three separate experiments. SW, sea water treated
eggs; C, control globulin (Rabbit No. 70-2); HC-C, control
globulin absorbed with Limulus hepatic cecum tissue; AEE, antiegg envelope antibody (Rabbit No. 62-3); HC-AEE, anti-egg
envelope antibody absorbed with Limulus hepatic cecum tissue
75
'SW
-gA:
HC-AEE
AEE
/
/
jf""
0
1
1/2
1/4
1/8
DILUTION OF GLOBULIN
1/16
Figure 11.
Effect of anti-egg envelope antibody (AEE) on sperm-egg attach­
ment. Egg sections were treated for 1/2, 1, 2, and 4 hr with
two drops of globulin preparations (all were adjusted to 18 mg
protein/ml). After rinsing in sea water, the egg sections were
treated for 5 min with two drops of 2.5% sperm suspension plus
one drop of 0.025 M cysteine. Excess spermatozoa were then
washed off and sections were wet mounted in 10% formalin sea
water solution. Sperm counts were made at 400X on a Zeiss
phase-contrast microscope. SW, sea water treated eggs; C,
control globulin treated eggs (Rabbit No. 51-2); C Fab,
digested (univalent) control; H, anti-Limulus heart antibody
(Rabbit No. 47-2); H Fab, digested anti-heart antibody; AEE,
anti-Limulus egg envelope globulin (Rabbit No. 62-3); AEE Fab,
digested anti-egg envelope globulin
77
SW
Fab
H Fab
\
/AEE Fab
V
\
\
o
-i
r
1/2
1
1
2
I
3
DURATION OF TREATMENT (HOURS)
Figure 12.
Effect of anti-egg envelope antibody (A£E) on sperm-egg attach­
ment. Egg sections were treated for 1/2, 1, 2, and 4 hr with
two drops of globulin preparations (all were adjusted to 18 mg
protein/ml). After rinsing in sea water, the egg sections were
treated for 5 min with two drops of 2.5% sperm suspension plus
one drop of 0.025 M cysteine. Excess spermatozoa were then
washed off and sections were wet mounted in 10% formalin sea
water solution. Sperm counts were made at 400X on a Zeiss
phase-contrast microscope. SW, sea water treated eggs; C, con­
trol globulin treated eggs (Rabbit No. 70-3); C Fab, digested
control; H, anti-Limulus heart antibody (Rabbit No. 47-2);
H Fab, digested anti-heart antibody; AEE, anti-Limulus egg
envelope antibody (Rabbit No. 70-1); AEE Fab, digested anti-egg
envelope antibody
79
C Fab
H Fab
\
AEE Fab
2010-
AEE
:T.
0-
T
I
0
1/2
1
2
3
DURATION OF TREATMENT (HOURS)
1
4
Figure 13.
Effect of anti-egg envelope antibody on sperm-egg attachment.
Egg sections were treated with two drops of serially diluted
globulin preparations; 1 (18 mg protein/ml), 1/3, 1/9, 1/27,
and 1/81. After 4 hr of globulin treatment, the sections were
rinsed with sea water and were then exposed to two drops of
2.5% sperm suspension and one drop of 0.025 M cysteine for 5
min. Excess spermatozoa were washed off and the sections were
wet mounted in 10% formalin sea water solution. Sperm counts
were made at 400X on a Zeiss phase contrast microscope. SW,
sea water treated eggs; C, control globulin treated eggs
(Rabbit No. 51-2); C Fab, digested control globulin; H, antiLimulus heart antibody (Rabbit No, 47-2); H Fab, digested antiheart antibody; AEE, anti-Limulus egg envelope antibody (Rab­
bit No. 62-3); AEE Fab, digested anti-egg envelope antibody
81
CFab
100-1
H Fab
90 -
SW
80-
7060-
AEE
(J 50-
•AEE Fab
H 40o- 3 0 20-
0 -
I
1/3
I
1/9
I
1/27
DILUTION OF GLOBULIN
'
1/81
Figure 14.
Effect of anti-egg envelope antibody on sperm-egg attachment.
Egg sections were treated with two drops of serially diluted
globulin preparations; 1 (18 mg protein/ml), 1/2, 1/4, 1/8,
1/16. After 4 hr of globulin treatment, the sections were
rinsed with sea water and were then exposed to two drops of
2.5% sperm suspension and one drop of 0.025 M cysteine for 5
min. Excess spermatozoa were washed off, and the sections were
wet mounted in 10% formalin sea water solution. Sperm counts
were made at 400X on a Zeiss phase contrast microscope. SW,
sea water treated eggs; C, control globulin treated eggs (Rab­
bit No. 70-3); C Fab, digested control globulin; H, antlLlmulus heart antibody (Rabbit No. 47-2); H Fab, digested antiheart antibody; AEE, anti-Llmulus egg envelope antibody (Rab­
bit No. 70-1): AEE Fab. digested anti-egg envelope antibody
83
100-
C Fab
900 MWIMIIIMIIMIIMII»*—»t»oa
80-
1-70Z
LU
/
H Fab
/
60
X
u 50<
<
AEE Fab
/
40
X.
o- 30 H
/
-AEE
20-
10-
0 -
I
I
I
1/2
1/4
1/8
DILUTION OF GLOBULIN
1/16
84
Duration of treatment with antibody
Egg sections were treated with papain digested univalent AEE and
appropriate controls for periods of 1/2, 1, 2, and 4 hr.
A summary of data
from four experiments (Rabbit No. 62-3) is given in Figure 11 and from
three experiments (Rabbit No. 70-1) in Figure 12).
Sperm-egg attachment is
reduced significantly both by the bivalent and univalent AEE.
Normal and
immune (anti-heart) bivalent and univalent controls have little effect on
sperm-egg attachment.
The initial inhibitory effect of the univalent AEE
is not as great as the bivalent AEE.
This is probably due to the reduced
affinity of the univalent antibody for antigen (Klinman, Long, and Karush,
1967).
However, after 4 hr of treatment, the univalent and the bivalent
AEE have similar effects.
Dilution of globulin
Egg sections were treated for 4 hr with various dilutions of univalent
AEE and controls.
Data from experiments involving AEE from two separate
rabbits is summarized in Figure 13 and Figure 14.
At high concentrations
(1 = 15-18 mg protein/ml), both the bivalent and univalent AEE reduce
sperm-egg attachment to a much greater degree than controls.
centrations this inhibitory effect is not noticeable.
At lower con­
In this experiment,
the effect of the univalent AEE follows the bivalent AEE more closely than
in the previous experiment (Figure 13).
This is probably due to the long
4 hr treatment time, which allows the univalent affinity for antigen to
approach that of the bivalent form.
85
Inhibition of Sperm-egg Attachment by Chemical
Treatment of the Egg Surface
Treatment with enzymes or other reagents interferes with the ability
of the egg to interact normally with the sperm (Aketa, Onitake, and
Tsuzuki, 1972; Soupart and Clewe, 1965; Metz, 1954).
When enzymes or other
reagents with well defined activities are used, the chemical character of
the egg components participating in sperm-egg interactions can be deter­
mined.
Accordingly, this method was employed to investigate sperm-egg
attachment in Limulus.
Egg sections were treated (prior to insemination) with various enzymes
and other reagents to test their effect on sperm attachment.
stances used and their effects are summarized in Table 1.
The sub­
As noted, sev­
eral enzymes, detergents, and other reagents are capable of altering the
egg surface enough to impede sperm attachment.
The substances found to be
effective in inhibiting sperm-egg attachment were used in quantitative
dilution experiments (Figures 16-19). In addition, the substances which
were found to be effective in inhibiting sperm-egg attachment were used to
make egg extracts.
Egg surface extracts were prepared by incubating 0.5 ml
of whole eggs in 1 ml of sea water solution containing enzymes or other
reagents at 37° C for 1 hr. These egg extracts were tested for their
effect on sperm agglutination, sperm motility, and soluble antigen content
(results summarized in Table 1).
Most egg extracts were capable of mild
agglutination of spermatozoa, but few induced motility.
Evidently soluble
products are released upon treatment of the egg surface, which are capable
of agglutinating spermatozoa.
86
Table 1.
Effect of chemical treatment of egg sections on sperm-egg attach­
ment. Effects of egg surface extracts on sperm agglutination and
sperm motility. The number of soluble antigens in egg surface
extracts as determined by immunodiffusion analysis
Egg sections treated
for 15 rain with;&
collagenase (1 mg/ml)^
papain (1 mg/ml)
pepsin (1 mg/ml)
protease (1 mg/ml)
trypsin (1 mg/ml)
carboxy-peptidase A
(1 mg/ml)
phospholipase C (1 mg/ml)
phospholipase D (1 mg/ml)
lipase (1 mg/ml)
alkaline phosphatase (pH 9,
1 mg/ml)
acid phosphatase (pH 5,
1 mg/ml)
«-amylase (1 mg/ml)
Percent
sperm
attachment
(control»
100%)
Egg surface extracts
Percent
Agglutina­ motility
tion of
of
Precip­
sperma­
sperma­
itin
bands
tozoa^
tozoa
+f
0
0
0
0
0
1
2
1
2
2
40
100
0
-+
10
1
-
0
1
0
-+
0
2
100
60
+
0
1
5
0
0
0
0
-H-+
+
++
100
Length of treatment time. In all cases where 15 min of treatment was
not successful in blocking sperm attachment, 1 hr of treatment was used.
In all cases, the longer treatment time also failed to inhibit sperm-egg
attachment.
^Agglutination of spermatozoa notation
( - )No agglutination
(-+ )Doubtful agglutination
( + ) Weak agglutination
(-H- ) Moderate agglutination
(+44-) Moderate to strong agglutination
^Highest concentration of reagent used.
trations were tried first.
In most cases, lower concen­
87
Table 1. (Continued)
Egg sections treated
for 15 min with:
p-amylase (1 mg/ml)
hyaluronidase (1 mg/ml)
lysozyme (1 mg/ml)
RNase (1 mg/ml)
DNase (1 mg/ml)
Sarkosyl NL-97 (0.1%)
Triton X-100 (0.1%)
sodium dodecyl sulfate
(0.1%)
sodium deoxycholate (0.1%)
phenol (10%)
ether (100%)
ethyl alcohol (100%)
trichloro-acetic acid
(0.1 M)
mercaptoethanol (0.1 M)
cysteine (0.025 M)
N-ethyl-maleimide (0.1 M)
iodoacetamida (0.02 M)
mercaptoethanol +
iodoacetamide
cysteine + iodoacetamide
hydrogen peroxide (30%)
periodic acid (5 mg/ml)
hydroxylamine hydrochloride
(20%)
sodium borohydride (1 mg/ml)
tetraethyl ammonium bromide
(0.1 M)
Percent
sperm
attachment
(control=
100%)
Egg surface extracts
Percent
Agglutina- motility
tion of
of
Precipspermaspermaitin
tozoa
tozoa
bands
100
100
100
100
100
85
0
10
50
30
100
100
-
50
0
-
0
50
0
0
0
0
-
0
0
100
100
100
100
100
100
100
100
30
100
100
100
88
Table 1. (Continued)
Egg sections treated
for 15 min with:^
Percent
sperm
attachment
(controls
100%)
urea (8 M)
formalin (10%)
tannic acid (1 mg/ml)
phytohemagglutinin
sperm extract
sperm water®
40
0
0
100
0
100
sperm water (CMF-SW)^
egg envelope extract
egg water
alkaline egg surface
extract (pH 9)
100
Egg surface extracts
Percent
Agglutina­ motility
of
Precip­
tion of
sperma­
sperma­
itin
bands
tozoa
tozoa
-
4-M.
+++
20
0
0
2
0
0
50
25
1
2
0
2
Sperm extract made by homogenizing 0.2 ml whole semen in 0.8 ml sea
water with 0.05% Mase.
g
Sperm water made by allowing 0.5 ml whole semen to stand in 1 ml sea
water overnight.
^Sperm water made by allowing 0.5 ml whole semen to stand in 1 ml of
calcium-magnesium free sea water overnight.
Effect of pH
Egg surface substances involved in sperm attachment may be susceptible
to denaturation or dissolution by acid or base.
To test the effect of acid
and base on sperm-egg attachment, egg sections were pretreated for 15 min
with solutions varying in pH from 1 to 13.
ments are summarized in Figure 15.
The results of these experi­
Very acid conditions (pH 1 to pH 3)
and very alkaline conditions (pH 10 to pH 13) completely blocked the abil­
ity of spermatozoa to bind to the egg surface.
The egg was not severely
Figure 15.
Egg sections were treated with two drops of 3.3% NaCl solutions
ranging in pH from 1 to 13. The sections were treated for 15
min, rinsed, treated with two drops of a 2.5% sperm suspension,
and prepared for observation. The results of three experiments
are summarized in the graph. Note the low sperm attachment at
extreme acid and base conditions
90
~
5
I
6
r
7
PH
8
9
I
10
1
11
12
13
91
affected by treatment with solutions of pH 4 to pH 9.
attachment occurred between pH 5 and pH 8.
Optimum sperm
The sperm-egg attachment reduc­
ing effect at strong acid and strong base conditions is probably due to
hydrolysis of egg surface substances or other denaturing effects.
Effect of enzymes
Egg surface substances involved in sperm-egg attachment should be sus­
ceptible to hydrolysis by certain enzymes, depending on their molecular
makeup.
This possibility was tested by pretreating egg sections with dilu­
tions of various enzymes (for complete listing of enzymes used see Table 1),
then testing for sperm attachment.
The results of proteolytic enzyme
treatments are summarized in Figure 16 and other enzymes in Figure 17.
The
endopeptidases, pepsin, papain, trypsin, and protease (a pancreatic extract
containing tryptic and chymotryptic activity) as well as lipase and alka­
line phosphatase had the greatest effect on lowering sperm-egg attachment.
The exopeptidase, carboxypeptidase A, and RNase, DNase, and most carbohy­
drates tested had no effect on sperm-egg attachment.
These results indi­
cate that the sperm binding molecule on the egg surface is protein in
nature, possibly with a lipid moiety.
Effect of detergents
Surfactants such as detergents arm known to solublize proteins and
lipids.
"Hiey are especially effective in separating membrane bound molecu­
lar subunits.
Although Limulus egg envelope is not a membrane, it does
possess surface molecules which are capable of interacting with the sperm
plasma membrane.
Thus attempts were made to remove or alter egg surface
molecules involved in sperm attachment with a few selected detergents.
Egg
Figure 16.
Effect of proteolytic enzymes on sperm-egg attachment. Two
drops of proteolytic enzyme solutions (1 mg enzyme/ml) were
applied to egg sections for a 15 rain period. After rinsing
with sea water, the sections were treated with spermatozoa.
All enzymes were prepared in sea water except papain which was
prepared in phosphate buffer as described in the materials and
methods section. The experiment was repeated three times
93
COLLAGENASE
PAPAIN
!
t
I
I
I
PEPSIN-
/
I
t
!
^^TRYPSIN
»
î
!
I
!
I
t
!
PROTEASE
T
1/2
I
I
1/4
1/8
1/16
DILUTION OF ENZYME
1/32
Figure 17.
Effect of selected enzymes on sperm-egg attachment. Two drops
of enzymes solutions (1 mg enzyme/ml) in sea water were applied
to egg sections for 15 min. The sections were then rinsed with
sea water and treated with spermatozoa. The experiment was
repeated three times
95
#•>••••«•••§ #
100
sw
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90
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Y'
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•
JL
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jS 30
20
ALKALINE
PHOSPHATASE
10
/^LIPASE
O
I
1
I
I
I
1/2
1/4
1/8
:
1/16
DILUTION OF ENZYME
I
1/32
96
sections were pretreated with dilutions of detergents for 15 min, thor­
oughly rinsed, and then treated with spermatozoa.
All detergents tested
showed some effect on sperm-egg attachment (Figure 18), however, only
Triton X-100 totally abolished sperm attachment at all dilutions.
These
results suggest that molecular subunits, perhaps proteins or lipoproteins,
are involved in sperm-egg attachment.
Effect of selected chemical reagents
Specific chemical groups such as aldehydes, sulfhydryl, and amino
groups may contribute to the sperm binding properties of the egg surface.
Many of these groups can be blocked or altered by both specific and non­
specific reagents.
For example, free sulfhydryl groups can be blocked
specifically with iodoacetamide or be altered nonspecifically by precipita­
tion with trichloroacetic acid. In order to test for the involvement of
such chemical groups in sperm-egg attachment, various reducing agents,
oxidizing agents, fixatives, and other reagents were used to treat egg
sections prior to sperm-egg mixing (Table 1).
Those agents which signifi­
cantly reduced sperm-egg attachment were tested further in dilution experi­
ments (Figure 19).
The organic solvents, ether and alcohol, as well as the protein pre­
cipitating agent, trichloroacetic acid, did not affect sperm attachment
(Figure 19).
Treatment with other protein altering agents, 8 M urea and
phenol, did lower sperm-egg attachment.
Both the aldehyde blocking reagent,
hydroxylamine hydrochloride, and the mucopolysaccharide blocking drug,
tetraethylammonium bromide, showed no effect on sperm-egg attachment.
Treatment of egg sections with tannic acid and formalin did lower sperm
Figure 18.
Effect of selected detergents on sperm-egg attachment. Egg
sections were treated with two drops (0.1 ml) of detergent
solutions (0.1%) for 15 min, then rinsed and treated with sper
matozoa. DOC, sodium deoxycholate; SDS, sodium dodecyl sul­
fate
98
•nomvw*
100 n
90-
SARKOSYL /
80-
H
z
U 60
DOC
O 50
SDS
h- 40H
0? 30
20-
10
I Ki luN X-100
o1/2
T1/4
I
1/8
DILUTION
1/16
1/32
Figure 19.
Effect of various chemical reagents on sperm-egg attachment.
Egg sections were treated with sea water solutions (0.1 ml) of
urea (8 M), phenol (10%), periodic acid (5 mg/ml), formalin
(10%), tannic acid (1 mg/ml) for 15 min. After rinsing, the
egg sections were inseminated then prepared for observation.
The experiment was repeated three times
100
100-
9080-
FORMALIN
UREA ,
V
H- 70-
Z
^ 60-i
PHENOL
X
(J 50
1-40 4
<
30H
/ PERIODIC
ACID\
TANNIC
ACID
//
20-
10-
/ / /
/ /
r
/
0 -
1/2
I
I
1/4
1/8
DILUTION
1/16
1/32
101
egg attachment.
Reduction with the latter reagents followed by alkylation
with iodoacetamide also failed to affect sperm attachment. In addition,
alkylation with iodoacetamide or N-ethylmaleimide has no effect on sperm
attachment, thus ruling out the participation of both disulfide and sulfhydryl groups in sperm-egg attachment.
Oxidation of the egg surface with
hydrogen peroxide had no effect on sperm-egg attachment, but periodic acid
at relatively high concentrations (5 mg/ml) did lower sperm-egg attachment,
therefore, indicating the possible participation of carbohydrate in sperm
attachment.
The plant red cell agglutinin, phytohemagglutin, had no effect
on sperm-egg attachment.
Sea water extracts of sperm made by homogeniza-
tion lowered sperm-egg attachment significantly.
The latter result may be
due to either blockage of sperm receptor sites by soluble sperm molecules
or dissolution of egg components by sperm acrosome lysins.
Another possi­
bility, the binding of egg surface molecules by sperm nuclear DNA, has been
rules out by treatment of these sperm extracts with DNase.
Immunological Identification of Egg Surface Substances
Immunodiffusion tests involve the cross reaction of soluble antigens
and antibodies on a semisolid medium (Ouchterlony, 1968). Immunoelectro­
phoresis is a combination of electrophoresis and immunodiffusion (Grabar
and Williams, 1953).
Soluble proteins or other antigenic materials are
first separated in one dimension by electrophoresis then reacted with anti­
bodies in a second dimension.
Both immunodiffusion and Immunoelectro­
phoresis result in the formation of precipitin bands, each of which repre­
sents an immunologically homogeneous antigen which has reacted with its
corresponding antibody (Hirschfeld, 1968).
The antibodies used in this
102
study were prepared in rabbits against Limulus egg envelopes.
The antigens
were prepared by various extraction procedures using whole eggs and egg
envelopes.
Immunodiffusion analysis
All reagents which were effective in inhibiting sperm attachment
(Table 1) to egg sections were used to prepare egg extracts.
The extracts
were tested for the presence of soluble antigens by reaction with anti-egg
envelope antibody (AEE) in immunodiffusion tests (Figure 20).
Extracts
prepared by proteolytic digestion of the egg surface yield a number of sol­
uble antigens (Figures 20a, 20d, and 20e).
Protease, pepsin, collagenase,
and trypsin egg surface extracts have one antigen in common.
trypsin egg extracts possess an additional antigen.
Protease and
Other enzyme egg
extracts, including phospholipase C, lipase, of-amylase, and alkaline phos­
phatase also have at least one antigen (Figure 20b).
Egg surface extracts
made with detergents do not produce precipitin bands, but 8M urea egg sur­
face extracts possess two soluble antigens.
Egg water prepared by allow­
ing spermatozoa (0.5 ml) to stand in sea water (1 ml) for three days yields
two precipitin bands whereas homogenized egg envelope extracts produce only
one band (Figure 20d).
Egg surface extracts made from alkaline sea water
of pH 11 or above possess two soluble antigens (Figures 20d, 20e, and 21a).
Both bands of the pH 11 extract are shared with the alkaline phosphatase
extract (Figure 20e).
Finally, one band is shared between egg water, homog­
enized egg envelope extracts, pH 11 egg surface extracts, and egg surface
extracts made with several enzymes (Figures 20d and 20e).
These tests indi­
cate that the anti-egg envelope antibody (AEE) is indeed directed against
Figure 20.
Immunodiffusion tests on various egg surface extracts
a) A.
B.
C.
D.
E.
anti-egg envelope antibody (AEE)
collagenase egg surface extract
papain egg surface extract
protease egg surface extract
pepsin egg surface extract
b) A. AEE
B. phospholipase C egg surface extract
C. lipase egg surface extract
D. cv-amylase egg surface extract
E. alkaline phosphatase egg extract
c) A.
B.
C.
D.
E.
AEE
triton X-100 egg surface extract
sodium deoxycholate egg surface extract
sodium dedecyl sulfate egg surface extract
urea egg surface egg extract
d)
AEE
egg water
alkaline (pH 11) egg surface extract
trypsin egg surface extract
homogenized egg envelope extract
A.
B.
C.
D.
E.
e) A.
B.
C.
D.
E.
AEE
alkaline (pH 11) egg surface extract
protease egg surface extract
urea egg surface extract
alkaline phosphatase egg surface extract
f) A.
B.
C.
D.
E.
homogenized egg envelope extract
AEE
antl-Llmulus sperm antibody
anti-Limulus heart antibody
normal rabbit globulin
104
Figure 21.
Immunodiffusion tests on various egg surface extracts
a) A.
B.
C.
D.
E.
F.
G.
H.
anti-egg envelope antibody (AEE)
AEE
pH 3 egg surface extract
pH 5 egg surface extract
pH 7 egg surface extract
pH 13 egg surface extract
pH 11 egg surface extract
pH 9 egg surface extract
b) A.
B.
C.
D.
protease egg surface extract
AEE
alkaline phosphatase egg surface extract
AEE absorbed with whole eggs
c)
B.
C.
D.
urea egg surface extract
AEE
alkaline (pH 11) egg surface extract
AEE absorbed with whole eggs
d) A.
B.
C.
D.
protease egg surface extract
AEE
alkaline (pH 11) egg surface extract
AEE absorbed with whole eggs
e) A.
B.
C.
D.
homogenized egg envelope extract
AEE
egg water
AEE absorbed with whole eggs
A,
106
107
egg surface antigens which can be removed from the egg by enzymatic diges­
tion and other extraction procedures.
The cross-reactivity of the egg envelope extract was tested with anti­
bodies directed against other tissues (Figure 20f).
Precipitin bands did
not form with anti-Llmulus sperm antibody, anti-Limulus heart antibody, and
preinjection control globulin.
A few selected extracts were also tested with AEE absorbed with whole
eggs to remove antibodies specific for exposed surface antigens (Figures
21b-21e).
In all cases, absorption with whole eggs sufficiently reduces
the titer of AEE such that only one very faint band is formed.
These
results demonstrate that AEE combines with antigens located on the egg sur­
face.
These are the same antigens which can be removed by enzymatic treat­
ment.
Since enzyme and antibody treatment of egg sections reduce sperm
attachment and since extracts of the egg surface prepared by enzyme treat­
ment yield precipitin bands with same antibody, this is good evidence that
the precipitin bands represent sperm-egg attachment antigens.
Immunoelectrophoresis analysis
The same egg extracts that were studied with immunodiffusion were also
examined immunoelectrophoretically (Figures 22 and 23).
Electrophoresis of
egg extracts was carried out for 1 hr at 300 v and 35 ma. In each case,
the sample was reacted with AEE and AEE absorbed with whole eggs.
Immuno­
electrophoresis results of proteolytic enzyme egg surface extracts are
shown in Figure 22.
Protease egg extract yields two bands which are elec-
trophoretically and immunologically distinct (Figure 22a).
bands is totally lost with the absorbed AEE.
One of the
The trypsin extract yields
Figure 22.
Immunoelectrophoresis of selected egg surface extracts.
on right
a) X.
A.
B.
protease egg surface extract
AEE
AEE absorbed with whole eggs
b) X.
A.
B.
trypsin egg surface extract
AEE
AEE absorbed with whole eggs
c) X.
A.
B.
papain egg surface extract
AEE
AEE absorbed with whole eggs
d) X.
A.
B.
alkaline phosphatase egg surface extract
AEE
AEE absorbed with whole eggs
e) X. a-amylase egg surface extract
A. AEE
B. AEE absorbed with whole eggs
Anode
109
110
one fuzzy band which does not move far during electrophoresis (Figure 22b).
The trypsin band is lost when reacted with the egg absorbed AEE.
The
papain egg oxtract gives two distinct bands, one not moving at all during
electrophoresis and the other moving only slightly (Figure 22c).
In addi­
tion, a third very faint, extended band is formed with the papain extract.
Only one of the bands is lost when reacted with egg absorbed AEE.
The
alkaline phosphatase extract yields two distinct bands, one of which is
lost when reacted against the egg absorbed AEE (Figure 22d). Finally, the
Œ-amylase egg extract gives one band which is partially lost with the egg
absorbed AEE (Figure 22e).
A few other selected egg extracts were also tested with immunoelectrophoresis (Figure 23).
Egg water gives two bands, one electrophoretically
stationary and the other extended.
One is totally lost and the other par­
tially removed by absorption of the AEE with whole eggs (Figure 23a).
The
frozen-thawed, homogenized egg envelope extract gives one band which is
lost with the egg absorbed AEE (Figure 23b).
The alkaline (pH 11) egg
extract yields two immunologically identical bands, one of which is lost
by absorption of the AEE with whole eggs (Figure 23c).
The urea egg sur­
face extract yields three electrophoretically distinct bands, two of which
are similar immunologically (Figure 23d).
with t'gg absorbiMi AEE.
Two of the three bands are lost
Figure 23.
Immunoelectrophoresis of selected egg surface extracts.
on right
a) X.
A.
B.
egg water
AEE
AEE absorbed with whole eggs
b) X.
A.
B.
homogenized egg envelope extract
AEE
AEE absorbed with whole eggs
c) X.
A.
B.
alkaline (pH 11) egg surface extract
AEE
AEE absorbed with whole eggs
d) X.
A.
B.
urea egg surface extract
AEE
AEE absorbed with whole eggs
Anode
112
a
b
c
B=
=
—
113
DISCUSSION
As outlined in the introduction, antibodies are used as an investiga­
tional tool to study the physiological aspects of fertilization.
Previous
immunological investigations on sea urchins and amphibians (cf. Metz, 1967)
have demonstrated the presence of gamete antigens and have considered their
role in fertilization.
These studies indicate that egg antigens are
involved in fertilization but still leave some questions unanswered.
These
include such items as the discrepancy between the inhibitory effectiveness
of bivalent antibody versus univalent antibody and the site of action or
fertilization step being blocked by the antibody.
The current investiga­
tion attempts to deal with these and other questions.
Evidence is pre­
sented for the existence and participation of egg surface antigens in
sperm-egg attachment of the horseshoe crab, Limulus polyphemus.
Methodology
The sperm binding capacity of the egg surface was measured by using
egg sections.
Since variables such as temperature and pH affect this pro­
cedure, every attempt was made to reduce these variables to a minimum.
Consequently, several experiments were performed to determine the optimum
conditions under which to carry out subsequent experiments.
The optimum duration of sperm-egg mixing is 3 to 5 rain (Figure 2).
These times correspond with the normal time period of sperm motility,
although occasionally spermatozoa have been observed to remain motile for
30 min or more.
On untreated egg sections, large numbers of spermatozoa
3
4
(10 -10 ) attached to the outer surface of each egg section after 5 min of
114
sperm-egg mixing (Figure lb).
Five min insemination times were used in all
subsequent experiments.
Spermatozoa attach normally to egg sections over a relatively broad
temperature range (Figure 3).
tor.
Again, sperm motility is the limiting fac­
At low temperatures (below 5° C), the spermatozoa are sluggish or
nonmotile, and at high temperatures (above 35° C), motility is also
arrested.
High temperatures result in cell death, since the spermatozoa
are unable to recover their motility when the temperature is lowered.
Room
temperature (23-25° C) was used for all subsequent treatment experiments.
The pH of the insemination medium is important in two respects.
the acid-base balance may affect the viability of spermatozoa.
First,
Second, if
charge factors are involved in sperm-egg attachment, the incorrect pH will
affect the charge of the insemination medium and, consequently, lower
sperm-egg attachment.
Experiments indicate that spermatozoa remain viable
over a wide pH range (Figure 4).
Since cell death only occurs at extreme
acid conditions (pH 3 to pH 4), acid-base balance is not a factor under
normal conditions (pK 6 to pH S).
The charge of the insemination medium is
not a factor, since high sperm-egg attachment occurs over a wide pH range.
Evidently, attachment is not the result of charge interactions between egg
and sperm surfaces.
The current experiments performed on Limulus egg sections involve a
new approach to the study of sperm-egg interactions.
More specifically, a
direct monitoring and measurement of the activities of gamete surface com­
ponents during sperm-egg attachment.
This approach is especially practical
since judgement of attachment is based on a normal process where a large
number (and not just one) of spermatozoa actually attach.
Previous experi­
115
ments (such as those on sea urchin gametes) have relied on subsequent
cleavage of the eggs after treating them with various test reagents.
Such
an approach involves variables which occur after sperm-egg attachment (i.e.
egg activation, sperm penetration, fusion of the pronuclei).
Inhibition of Sperm-egg Attachment
with Bivalent Antibody
One of the problems of fertilization is to locate on gamete surfaces
binding sites or macromolecules which are necessary for proper sperm-egg
attachment to occur.
Such macromolecules determined with immunological
techniques could be "fertilization antigens" if blocking prevented sperm
attachment or had an effect on development.
As demonstrated by three different types of experiments, treatment
with bivalent anti-egg antibody reduces the extent of attachment of sperma­
tozoa to the egg surface of Limulus (Figures 5-7).
One explanation for
this is that the egg attachment sites for spermatozoa are antigenic and
are being masked by an appropriate antibody.
For example, inhibition of
fortilizatior. in sea urchins is caused by treatment of eggs with antiserum
directed against an egg sperm-binding protein (Aketa and Onitake, 1969).
These authors believe that the inhibition of fertilization is due to block­
age of specific antigenic sites by complementary antibody, since inhibition
does not occur with heterologous antibody (i.e. antibody directed against
the eggs of other species).
However, a second possibility is that the anti­
body is reacting with components of the egg surface to form a cross-linked
impermeable layer.
I
This latter effect has been demonstrated in sea urchin
hy F>.)xnndall, Perlmann, and Afzelius (1964) and Metz and Thompson
(i')67).
Metz (.967) relieves that such cross-linking could block essential
116
but unrelated sites through secondary steric effects or bury them through
tertiary effect such as folding or wrinkling of the cell surface.
One way
to test for this possibility is to employ univalent antibodies which are
not capable of forming cross linkages.
The formation of cross-linked lattices is due to the inherent biva­
lency of the 7S antibody but is not necessarily related to nonspecific
antigen-antibody interactions.
In the present study, the possibility that
the blockage of sperm attachment is due to nonspecific antigen-antibody
interactions has been ruled out. For example, treatment of eggs with anti­
body directed against Limulus heart tissue has no apparent effect on spermegg attachment (Figure 7).
In addition, experiments with anti-egg envelope
antibody absorbed with several Limulus tissues (including semen, heart
muscle, and hepatic cecum) have demonstrated a high degree of tissue speci­
ficity of the antibody for the egg surface antigens involved in attachment
(Figures 8-10).
To conclude, sperm-egg attachment is inhibited by pretreating egg sec­
tions with bivalent antibody.
This inhibition may be due to blockage of
specific sperm receptor sites on the egg surface or to the formation of a
molecular lattice which prevents sperm receptors from binding with the
sperm surface.
The inhibition, however, is not due to nonspecific antigen-
antibody interactions (i.e. tissue nonspecificity).
In any case, the
biviiLcnt untLbody experiments suggest the presence of sperm-egg "attachment
antigens" on the egg surface.
valent antibody experiments.
These results are substantiated by the uni­
117
Inhibition of Sperm-egg Attachment
with Univalent Antibody
Metz and Thompson (1967) demonstrated that univalent anti-egg antibody
does not reduce the fertilizing capacity of sea urchin eggs. However,
other studies with amphibian eggs (Shivers and Metz, 1962) and sea urchi "
sperm (Metz, Schuel, and Bischoff, 1964) demonstrated that univalent anti­
bodies are effective fertilization inhibitors.
Univalent antibodies have
also been used as inhibitors in other systems.
For instance, Beug ejt al.
(1970) successfully inhibited cell contact formation in the cellular slime
mold, Dictyostelium discoideum, with univalent (Fab) fragments or anti-cell
homogenate serum.
However, in yeast Brookbank and Heisler (1963) were
unable to inhibit the mating reaction in yeast with univalent antibody.
Thus, univalent antibodies have been used with mixed success to interfere
with cell surface interactions.
Nevertheless, the blockage of specific
cell receptor sites with univalent antibody is a promising technique for
the study of cell surface interactions.
Sperm-egg attachment in Limulus is inhibited by pretreating egg sec­
tions with univalent anti-egg envelope antibody (Figures 11-14). These
results not only enhance the findings with bivalent antibody but also elim­
inate the possibility that blockage is due to cross-linking between egg
surface antigens and anti-egg antibody.
Since univalent antibodies are
only capable of binding with one antigen at a time and since combination of
these antigens with AEE inhibits sperm-egg attachment, they are designated
"attachment antigens."
These results are significant since they demon­
strate the presence of receptor-like egg surface molecules ("attachment
antigens") which mediate sperm binding.
118
In considering the results of the antibody inhibition experiments, the
bivalent antibodies have a stronger inhibiting effect on sperm-egg attach­
ment than univalent antibodies (Figures 11-14).
This effect probably
results from two phenomena, both related to differences in reactivity of
the two types of antibody.
First, bivalent antibody is capable of forming
cross-linked molecular bridges (Metz and Thompson, 1967), whereas univalent
antibody combines only with individual antigenic determinants.
Thus the
bivalent form presents a more formidable barrier to sperm-egg attachment.
Second, bivalent antibody has a much greater affinity for antigen than do
univalent fragments (Klinman, Long, and Karush, 1967; Greenbury, Moore, and
Nunn, 1965).
Therefore, due to its greater binding energy, the bivalent
preparation is expected to be a more efficient inhibitor.
The results, as
shown with duration of antibody treatment (see Figures 11 and 12), reflect
these differences in antibody affinity.
Therefore, the inhibition of sperm
attachment by univalent antibodies, even though less effective than the
bivalent treatment, represents a significant reduction, enough to indicate
the involvement of individual antigenic determinants in sperm binding.
Longer treatment times (i.e. 4 hr or more) appear to overcome the dif­
ferences between bivalent and univalent antibody inhibition of sperm attach­
ment (Figures 11 and 12).
This is due to the longer duration of antigen-
antibody interaction, which allows more time for univalent fragments to
combine with free antigenic sites.
In addition, the number of bonds between
univalent antibody ond egg surface antigens probably increase with time.
This is the principle reason why four hr treatments were chosen for the
second experiment, antibody dilution (Figures 13 and 14).
119
As discussed in the introduction, univalent antibodies have been used
to demonstrate the presence of "fertilization antigens" in sea urchin sperm
(Metz, Schuel, and Bischoff, 1964; Tyler, 1946) and frog jelly (Shivers and
Metz, 1962). These studies, however, have not established at what step in
the sperm-egg interactions (i.e. sperm-egg attachment, initiation of the
acrosome reaction, membrane fusion, and sperm penetration) the antigens are
involved.
Their conclusions are only that the function of certain surface
antigens necessary for fertilization are being inhibited.
The present
study, however, was designed to detect egg surface antigens involved in one
particular fertilization event -- sperm-egg attachment.
The immunological
methods used in this study have readily demonstrated "attachment antigens."
The inhibition of their function by univalent antibody is significant
since: (1) they may represent a molecular sperm-egg recognition system
(cf. Mowbray, Brown, and Metz, 1970), (2) molecular evidence is provided to
support previous cytological findings that sperm-egg interaction is a sequen­
tial process, each step dependent on the successful completion of the previ­
ous stepj and (3) sperm and egg have comnlementmry or mutually reactive sur­
face molecules which must interact for sperm-egg attachment to occur.
The
latter is in agreement with previous observations on Limulus by Cooper and
Brown (1972).
Using absorption techniques combined with immunodiffusion,
these workers demonstrated that two sperm surface antigens react with mole­
cules on the surface of the egg.
Thus, "attachment ancigens" have been demonstrated on the egg surface
of Limulus.
How are these antigens related to the total event of sperm
,-)caut.\aC:;or.V
i)o direct. V..
want lines should further investigations of this problem
Til.
.Lv goal in understanding the molecular biology of
120
the sperm-egg interactions is to uncover some pathway or series of biochem­
ical events which take place as the sperm enters the egg.
Ideally, this
molecular pathway would correspond to the known ultrastructural events of
sperm penetration.
The demonstration of such molecular components as the
"attachment antigen" or "sperm binding protein" represents the beginning of
the elucidation of such a molecular pathway.
Needless to say, with an
event as complex as sperm penetration, the investigation of this problem
must be kept as simple as possible.
The egg section method used in this
study may represent the model system needed to investigate the molecular
events associated with sperm penetration.
Further investigations should
involve the demonstration, localization, and purification of molecular com­
ponents involved in various fertilization events.
Immunological methods
will be important in all phases of these operations.
Not only are anti­
bodies valuable in determining the biological activity of macromolecules
(i.e. antibodies used as inhibitors), but they also can serve as cytological markers, immunochemical monitors, and agents for isolating and purify­
ing fertilization antigens.
Inhibition of Sperm-egg Attachment by Chemical
Treatment of the Egg Surface
Treatment of Limulus egg sections with certain reagents prior to
insemination reduces sperm-egg attachment or binding.
These results demon­
strate that the egg substances involved in sperm binding are either being
removed, masked, or altered in some way such that spermatozoa are no longer
able to interact with them.
Indeed, immunodiffusion tests demonstrate that
sperm binding molecules are being removed from the egg surface with certain
treatments.
Thus, by analyzing the sperm-egg binding capacity of egg sec-
121
tlons subsequent to various chemical treatments, it Is possible to examine
the chemical properties of the sperm binding substance.
Trypsin treatment of sea urchin eggs reduces their fertillzability by
inhibiting sperm-egg bonding (Aketa, Onitake, and Tsuzukl, 1972).
Sperm-
egg bonding or attachment in Llmulus is also Inhibited by pretreating egg
sections with certain enzymes.
As a group, the proteolytic endopeptldases
are the most efficient inhibitors of sperm-egg attachment (Figure 16).
The
endopeptldases used in this study catalyze the hydrolysis of a variety of
peptide bonds.
These experiments demonstrate that proteins or peptides are
Involved in sperm-egg binding.
Since immunodiffusion tests demonstrate the
presence of antigens In proteolytic enzyme egg surface extracts (Figure 20),
the enzymes undoubtedly act to remove protelnaceous antigens from the egg
surface.
Other enzymes, particularly lipase, alkaline phosphatase, and
phosphollpase C, are also effective inhibitors of sperm-egg attachment
(Figure 17). Lipase and phosphollpase C catalyze the hydrolysis of link­
ages between glycerol and fatty acids and glycerol and phosphate, respec­
tively.
Alkaline phosphatase is a nonspecific phesphcaonoesterase %hich
catalyzes the hydrolysis of orthophosphorlc monoesters.
Thus, inhibition
by treatment with these enzymes indicates the Involvement of lipids or
phospholipids in sperm-egg binding.
These lipids may be closely associated
with proteins, since proteolytic enzyme treatment of egg sections also
inhibits sperm attachment.
However, most carbohydrases. Including
hynluronldasc and lysozyme, have little effect on sperm-egg attachment
(Table 1), indicating that carbohydrates are not involved in sperm binding.
The susceptibility of the egg surface to certain enzymes indicates the
presence of exposed macromolecules which function in sperm-egg binding.
122
These results lend support to the antibody inhibition experiments which
also demonstrate that exposed egg surface molecules (antigens) participate
in sperm-egg attachment.
These results also suggest that the egg surface
sperm binding molecule is lipoprotein in nature.
This compares favorably
with the work of Aketa, Tsuzuki, and Onitake (1968) who found both lipid
and carbohydrate moieties associated with the "sperm binding protein"
located in the vitelline membrane of sea urchin eggs.
The lipids associ­
ated with this protein include lecithin, lysolecithin, and several other
phosphatides, however, the involvement of these lipids in sperm binding has
not been demonstrated.
Treatment of egg sections with detergents (surfactants) prior to
insemination reduces sperm-egg attachment (Figure 18).
The ionic deter­
gents sodium deoxycholate, sodium dedecylsulfate, and Sarkosyl NL-97 are
less effective than the nonionic detergent Triton X-100.
This is not sur­
prising since nonionic detergents tend to be more surface-active than ionic
compounds (Hutchinson and Shinoda, 1967).
Surfactants are thought to dis­
solve proteins by forming water soluble micelles (McBain^ 1950)=
Recently,
Green (1971) demonstrated that Triton X-100 binds to proteins by forming
hydrogen bonds and hydrophobic bonds.
This suggests that the surfactant
competes with the normal inter- and intramolecular hydrogen and hydrophobic
bonds, causing an alteration of the normal tertiary and quaternary struc­
ture of the molecule and its relationship to surrounding molecules.
The
fact that spermatozoa no longer attach to detergent treated egg sections
indicates that the normal molecular surface structure of the egg has been
disrupted or removed.
Since detergent
buifaCc cXLïauLà do uoc forra
precipitin bands (Figure 20c), the antigens, if present in the extract, are
123
probably altered in such a way as to make them unreactive with antibody.
In any case, these experiments support other findings which show that mole­
cules, possibly lipoproteins, are involved in sperm-egg attachment.
Urea is another compound which interferes with the normal inter- and
intramolecular hydrophobic interactions of proteins (Whitney and Tanford,
1962).
Urea pretreatment of egg sections lowers sperm-egg attachment (Fig­
ure 19).
In addition, urea egg surface extracts yield two precipitin bands
with immunodiffusion (Figure 20c).
These findings strongly indicate the
removal by urea of essential sperm-binding molecules from the egg surface.
These binding molecules are antigenic and are probably protein in nature.
Treatment of egg sections with solvents such as ether and alcohol has
no effect on sperm-egg attachment (Table 1).
Alcohol and ether dissolve
lipids but not protein, thus indicating that free or exposed lipids on the
egg surface are not involved in sperm-egg attachment.
However, if present,
the lipids may be covalently linked to other molecules and, therefore,
insoluble in ether or alcohol.
Another organic solvent, phenol, which is
capable of dissolving proteins (Rendina, 1971), is also effective in inhib­
iting sperm-egg attachment, suggesting that proteins are altered or removed
from the egg surface.
Phenol egg surface extracts do not yield precipitin
bands, however, suggesting that the antigens, if present, are denatured.
The organic acid, trichloroacetic acid, which precipitates aqueous solu­
tions of proteins by forming salts with them (Rendina, 1971) does not
inhibit sperm-egg attachment, thus indicating either that trichloroacetic
acid does not react with egg surface bound proteins or that its interaction
with proteins does not inhibit their sperm binding properties.
124
Sperm-egg attachment in Limulus is inhibited subsequent to treatment
with the reducing agents formalin ancïrtannic acid (Figure 19).
Both of
these compounds are protein fixatives and are used in the tanning industry
(Pearse, 1968).
Formalin forms hydroxymethyl addition compounds with pro­
tein end groups and cross-links proteins by the formation of methylene
bridges (Pearse, 1968).
Almost all types of protein end groups can be
involved in these reactions.
Metz (1954) demonstrated that treatment of
fertilizin with formalin has no effect on its sperm agglutinating ability.
However, tannic acid does inhibit the sperm agglutinating power of fer­
tilizin in addition to its fertilization inhibiting effect on sea urchin
eggs (Branham and Metz, 1960).
Although tannic acid and formalin are rel­
atively nonspecific in their activity, the results obtained in this study
are consistent with other findings that proteins are involved in sperm-egg
attachment.
Treatment of Limulus egg sections with the oxidizing agent, hydrogen
peroxide, has no effect on sperm-egg attachment (Table 1).
Hydrogen perox­
ide oxidizes various chemical groupings including sulfhydryl groups
(Danielli, 1950).
It also attacks saturated fatty acids to form keto acids
(Mead, Howton, and Nevenzel, 1965).
Tyler (1941) demonstrated that hydro­
gen peroxide destroys the agglutinability of fertilizin but does not affect
the integrity of the active site which combines with sperm antifertilizin.
Thus, in Limulus also, oxidations of this nature do not interfere with egg
surface molecules involved in sperm binding.
However, periodic acid oxida­
tion of egg sections is effective in inhibiting sperm binding (Figure 19).
Periodate treatment cleaves carbon-carbon bonds of carbohydrates where
these carbon atoms have adjacent hydroxyl and amino groups yielding aide-
125
hydes (Thompson, 1966). Periodate is also capable of oxidizing exposed
Œ-amino alcohol groups of serine and threonine (Clegg, Clermont, and
Leblond, 1952). Thus the effect of periodic acid treatment of Limulus egg
sections may be due to alteration of proteins or carbohydrates.
This is in
agreement with recent cytochemical evidence which demonstrates the presence
of both proteins and carbohydrates in the outer portion of the Limulus egg
1
envelope .
Several specific blocking reagents were used to treat egg sections.
Two aldehyde blocking reagents, hydroxyalamine hydrochloride and sodium
borohydride (Pearse, 1968), were both ineffective in inhibiting sperm
attachment, thus indicating that free aldehydes do not participate in sperm
binding.
Treatment of egg sections with tetraethy1ammonium bromide which
specifically blocks sulfomucopolysaccharides (Kelly, Schuel, and Severin,
1971) does not affect sperm-egg attachment, ruling out the participation of
this class of molecules in sperm binding.
The blockage of free sulfhydryl
groups on the egg surface with iodoacetamide and N-ethylmaleimide has no
affect on sperm-egg attachment.
Also, cleavage of disulfide bonds by
cysteine and mercaptoethanol has no affect on sperm-egg attachment.
Thus,
these experiments demonstrate that sulfhydryl and disulfide groups do not
participate in sperm-egg binding.
In other studies, Metz (1954) found that
blockage of sulfhydryl groups with iodine did not diminish the sperm agglu­
tinating power of fertilizin, however. Wolf and Hedrick (1971) found that
^Bennett, J. Department of Zoology and Entomology, Iowa State Univer­
sity, Ames, Iowa. Ultrastructural and cytochemical studies on sperm-egg
interactions of the horseshoe crab, Limulus polyphemus. Personal communi­
cation. 1972.
126
chemical cleavage of disulfide bonds with mercaptoethanol resulted in dis­
solution of Xenopus egg jelly.
Wolf and Hedrick (1971) suggest disulfide
bond cleavage by spermatozoa as a possible mechanism of sperm penetration
in amphibians.
In conclusion, the chemical treatment experiments on Limulus egg sec­
tions indicate that egg surface substances participate in sperm binding.
The sperm binding properties of the egg surface can be impaired or
destroyed by various enzyme and chemical treatments.
In addition, some of
these treatments remove egg surface antigens which also are proposed to
participate in sperm binding.
Proteolytic and lipolytic enzymes are the
only specific reagents which are effective in destroying the sperm binding
capacity of the egg surface.
Other nonspecific reagents, mostly protein
dénaturants, also impair sperm binding.
Thus, the sperm binding molecule
is definitely protein in nature, with the possibility of possessing lipid
or carbohydrate moitiés.
Perhaps it is similar to the "sperm binding pro­
tein" found in sea urchin eggs, which is a conjugated protein (2.3 S) con­
taining both carbohydrate and lipid components (Aketa, 1967),
Although
unlikely, another possibility is that the Limulus sperm binding molecule
corresponds to sea urchin fertilizin, which is a polysaccharide-amino acid
complex (8.6 S) containing 20% amino acids and large amounts of fucose.
An additional topic is the sperm agglutinating property of egg surface
extracts (Table 1).
The strongest sperm agglutinating effects are elicited
by egg water and homogenized egg envelope extract.
A few of the enzyme egg
surface extracts, in particular those made with trypsin and protease, also
demonstrate the ability to agglutinate spermatozoa.
Sperm agglutination by
egg surface extracts can be considered as a form of sperm binding, very
127
closely related to sperm-egg attachment.
The agglutinin in the egg extract
may be similar to sea urchin fertilizin, although its effects are not as
strong and no disagglutination occurs.
Another, more likely, possibility
is that the agglutinin is similar to the sea urchin sperm-binding protein
demonstrated by Aketa (1967).
spermatozoa.
Acid precipitates of this protein will bind
In addition, spermatozoa will bind to bubbles of alkaline
(pH 8.2) sea water solutions of the protein (Aketa, 1967).
Similar experi­
ments with bubbles of an alkaline egg surface extract (pH 9) were attempted
with Limulus (not reported in results sections).
A few spermatozoa adhere
to the surface of the bubbles, but this attachment is probably due to sur­
face tension.
However, if whole Limulus eggs are rolled on a glass micro-
slide, a thin film of egg surface material firmly adheres to the glass.
Spermatozoa readily attach to this film and many undergo the acrosome reac­
tion.
Thus the sperm binding properties of the egg surface substances can
be demonstrated by sperm agglutination and by other experiments.
It is
evident that the sperm receptor or active site of the sperm binding sub­
stance is not destroyed when removed from the egg by various nseans.
How do egg extracts affect the ability of spermatozoa to attach to egg
sections?
Treatment of spermatozoa suspensions with egg envelope extracts
definitely retards the ability of the spermatozoa to attach to the egg sur­
face (not reported in results section). However, the extracts may affect
the spermatozoa in other ways besides just inhibiting the receptor sites.
For instance, the motility mechanism may be altered. In any case, further
experiments such as these with purified egg envelope substances will help
CO understand the mechanism of sperm-egg attachment.
128
Immunological Identification
of Egg Surface Substances
A number of soluble antigens have been demonstrated on the surface of
Limulus eggs with various imraunoprecipitin techniques (Figures 20-23).
These results concur with the antibody inhibition experiments which demon­
strate that egg surface antigens are involved in sperm-egg attachment.
Furthermore, these results are in accord with chemical inhibition experi­
ments which demonstrate that certain enzymes and reagents displace or
destroy necessary sperm binding substances on the egg surface.
With these
facts in mind then, the immunodiffusion analyses help to form a coherent
picture of the sperm-egg binding substance.
Proteolytic digestion of the egg surface as well as treatment with a
few other enzymes causes the release of soluble antigens which can be
detected with immunodiffusion tests (Figure 20).
One antigen is shared
between most of these enzyme extracts and with other extracts made with
urea, alkaline sea water, homogenized egg envelopes, or egg water (Fig­
ure 20).
This shared antigen is probably the same egg surface antigen that
is involved in sperm binding, since it is the only component shared in most
of the extracts made with reagents which reduce the sperm binding proper­
ties of the egg surface.
Once again, it would be instructive to consider
the sperm agglutinating ability of these egg surface extracts (Table 1).
The extracts which are most efficient in agglutinating spermatozoa are also
the ones which share the common antigen (Figure 20).
This constitutes fur­
ther evidence for the sperm binding properties of this antigen, which is
probably identical to the "attachment antigen" mentioned earlier.
129
Absorption of anti-egg envelope antibody with whole eggs reduces the
antibody titer to the extent that only faint precipitin bands form with
immunodiffusion tests (Figures 21b-21e).
This constitutes further evidence
that the precipitin bands formed with the unabsorbed antibody represent egg
surface antigens.
In those egg surface extracts which normally form two
bands, one of the bands is always totally lost when tested with absorbed
antibody.
The faint band that forms with the absorbed antibody appears to
be the "attachment antigen."
Evidently this faint band is not completely
absorbed because it is partially buried beneath the egg surface.
In any
case, this technique demonstrates the presence of two egg surface antigens,
one of which is probably involved in sperm binding.
Cooper and Brown (1972) compared Limulus egg antigens with sperm anti­
gens by using immunodiffusion techniques.
With anti-sperm serum, they
found that whole egg extracts share two antigens with whole sperm extracts.
These two antigens are located on the sperm subsurface and thus are prob­
ably not involved in sperm-egg attachment.
These two antigens are also egg
subsurface antigens (probably located in the yolk), since anti-sperm serum
does not form any bands against homogenized egg envelope extract (Fig­
ure 20f).
In another experiment. Cooper and Brown (1972) demonstrated that
absorption of sperm extracts with whole eggs removes two bands which nor­
mally form against anti-sperm antibody.
surface' antigens.
These two bands represent sperm
These results indicate the interaction of the egg sur­
face witli soliibif sperm antigens (Cooper and Brown, 1972).
Possibly these
.1 rc spi-rm ;.iir[;icc' attnchment antigens or "fertilization antigens" which
pare i i i p;, til' ; .1 some other sperm penetration event.
Preliminary results
cl.'r..oa6C^-acc that similar aosorptions of egg surface extracts with Limulus
130
semen fail to remove any of the bands normally formed against AEE (not
reported in the results section).
Thus, with this technique, these experi­
ments do not indicate a reaction between the sperm surface and egg surface
extracts.
Egg surface antigens can be separated electrophoretically (Figures 22
and 23).
These results enhance the information obtained by immunodiffusion
analysis.
Most of the antigens migrate toward the anode at pH 8.6, however,
a few antigens remain stationary.
(Figures 22c, 22d, 23d).
Some of the antigens form extended bands
These extended bands are due to molecules which
are electrophoretically heterogeneous but similar antigenically (Hirschfeld,
1968).
Even though electrophoresis adds another dimension to antigen iden­
tification, not many new antigenic specificities are uncovered.
Only the
papain and urea egg surface extracts yield a new band (Figures 22c, 23d).
Thus the egg envelope appears to be relatively homogeneous with respect to
antigen content.
The same degree of antibody removal by whole egg absorp­
tion is observed as with immunodiffusion tests.
appears as in immunodiffusion.
Also, the same faint band
The faint band is always associated with
the component that moves toward the anode during electrophoresis.
There­
fore, the position of this faint band is not only a good marker for deter­
mining the location of the "attachment antigen" but also enchances other
findings by adding a new dimension to the separation and characterization
of all egg surface antigens.
In conclusion, the immunoprecipitin experiments demonstrate the prescncr of soluble egg envelope antigens.
Absorption experiments demonstrate
thai these .mtigens are present on the egg surface.
Other experiments
reported in this study demonstrate that one or more of these soluble egg
131
antigens is involved in sperm-egg attachment.
Thus, indirect evidence indi­
cates that one of the soluble antigens is the "attachment antigen."
In the
future, these precipitin identification techniques will be extremely valu­
able in purification of the "attachment antigen."
Antigen-antibody precipi­
tates can be dissociated and separated chromatographically (Chase and
Williams, 1967).
In addition, the agar-gel precipitates themselves can be
injected into rabbits to produce specific antibody to a particular antigen
(Shivers and James, 1967).
After purifying the "attachment antigen," it
could be chemically degraded by various means and the specific antibody
used to identify the particular portion of the molecule involved in sperm
binding.
132
CONCLUSIONS
Experiments with egg sections reported in this study present strong
evidence that gamete surface substances participate in sperm-egg attachment.
Sperm-egg attachment is inhibited both by bivalent and univalent anti-egg
antibody which demonstrates that specific egg surface combining sites
("attachment antigens") are involved in sperm binding.
Thus, a single,
well defined fertilization step is inhibited, namely sperm-egg attachment.
The "attachment antigen" has been shown to be at least partially protein in
nature.
In addition, it is soluble in aqueous solutions, nondialyzable,
and can be identified by various immunological methods.
Other experiments
demonstrate that sperm-egg attachment can be inhibited by pretreating egg
sections with various reagents, including certain enzymes.
These results
establish that egg surface macromolecules, possibly lipoproteins, are
involved in sperm-egg binding.
These macromolecules are similar, if not
identical, to the "attachment antigen,"
133
SUMMARY
1.
A new method of studying sperm-egg interactions is introduced.
This
method involves the use of unfixed, thin egg sections which allows the
quantitative assay of sperm-egg binding.
2.
Various sperm attachment inhibition experiments were performed on
Limulus egg sections.
These experiments were designed to demonstrate
the presence of egg surface receptor macromolecules involved in spermegg attachment.
Additional experiments, involving immunoprecipitin
techniques were utilized to enhance and expand the egg section inhibi­
tion experiments.
3.
Pretreatment of egg sections with bivalent anti-egg antibody inhibits
the attachment of spermatozoa, indicating the presence of egg surface
antigens which participate in sperm-egg bonding.
The inhibiting
effects of the antibody are tissue specific.
4.
Papain digested, univalent (Fab) anti-egg envelope antibody also blocks
sperm-egg attachment demonstrating that individual antigenic determi­
nants participate sperm-egg attachment.
5.
Pretreatment of egg sections with proteolytic and lipolytic enzymes,
extreme alkaline and acid conditions, urea, certain detergents, sperm
extracts, and several other reagents significantly reduce sperm-egg
attachment.
These results demonstrate the presence of egg surface
macromolecules which are involved in sperm binding.
6.
Egg surface extracts made by enzyme treatment and other extraction
procedures yield one or two precipitin bands with immunodiffusion anal­
134
ysis of tests utilizing whole egg absorbed antibody indicates that
of the bands represents the "attachment antigen."
Antigens in egg surface extracts are separable with electrophoresis
These techniques yield a maximum of three egg envelope antigens.
135
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151
ACKNOWLEDGMENTS
I wish to express my appreciation to Dr. George Gordon Brown for sug­
gesting this project, for his continuing help and encouragement, and for
assistance in preparing this manuscript.
I am grateful for the advice and
assistance offered by Dr. James R. Redmond, Dr. David R. Griffith,
Dr. Merlin L. Kaeberle, and Dr. Bernard J. White.
I also wish to thank
G. Kutish, J. Bennett, S. Suleiman, C. Cooper, D. Sharpnack, J. Knouse, and
D. Moorman for their valuable suggestions and assistance.
Finally I wish
to thank my patient and devoted wife who has encouraged and supported my
study.
152
APPENDIX:
SOURCES OF REAGENTS
a-Amylase. Type II-A, from Bacillus subtilus.
Louis, Mo,
p-Amylase.
(cy-amylase-free)
Grade B.
Sigma Chemical Company, St.
Calbiochem, Los Angeles, California.
Carboxypeptidase A. 2X crystallized, from bovine pancreas.
Company, St. Louis, Mo.
Collagenase. Type I, from CjL. histolyticum.
Louis, Mo.
Sigma Chemical Company, St.
Deoxyribonuclease I. IX crystallized, from bovine pancreas.
cal Company, St. Louis, Mo.
Hyaluronidase. Type I, from bovine testes.
Louis, Mo.
Lipase (Steapsin).
Sigma Chemical
Sigma Chemi­
Sigma Chemical Company, St.
General Biochemicals, Chagrin Falls, Ohio.
Lysozyme (Muramidase). Grade I, 3X crystallized, from egg whiteChemical Company, St. Louis, Mo.
Papain. 2X crystallized, from papaya latex.
Louis, Mo.
Sigma Chemical Company, St.
Pepsin. Grade B, 3X crystallized, from porcine stomach mucosa.
Los Angeles, California.
Phosphatase, Acid.
fornia.
Grade B, from potatoe.
Grade B, from
Phospholipase D.
nia.
Grade B, from cabbage.
welchii.
Calbiochem, Los
Calbiochem, Los Angeles, Cali­
Calbiochem, Los Angeles, Califor­
Protease. Type VI, from Streptomvces griseus.
Louis, Mo.
Sigma Chemical Company, St.
Ribonuclease B. Type III-B, from bovine pancreas.
St. Louis, Mo.
Trypsin. Grade A, from bovine pancreas.
nia.
Calbiochem,
Calbiochem, Los Angeles, Cali­
Phosphatase, Alkaline. Grade B, from calf intestine.
Angeles, California.
Phospholipase C.
fornia.
Sigma
Sigma Chemical Company,
Calbiochem, Los Angeles, Califor­
153
Phytohemagglutinin P, Bacto.
Triton X-100.
Difco Laboratories, Detroit, Mich.
Sigma Chemical Company, St. Louis, Mo.
Sarkosyl NL-97.
Geigy Chemical Company, Chicago, 111.
Seakem Agarose.
Bausch and Lomb, Rochester, N.Y.