AMER. ZOOL., 14:487-493
(1974).
Gamete Interaction During Fertilization in Campanularia—The Female
Epithelial Cell Surface
MICHAEL G. O'RAND
Department of Biology, Temple University, Philadelphia, Pennsylvania 19122
and Marine Biological Laboratory, Woods Hole, Massachusetts 02543
SYNOPSIS. Prior to fertilization in Campanularia flexuosa a capacitation-like interaction
occurs between the spermatozoa and the epithelial cells that lead to and surround the
eggs. Trypsin treatment of egg packets (eggs surrounded by epithelial cells) prevents
the capacitation-like action and results in loss of fertilizability. Following treatment,
the normal fuzzy surface coat of the epithelial cells is reduced from 200 A to 50 A.
Even with increased amounts of sperm the fertilization rate of trypsin-treated eggs in
the egg packets never reaches normal levels. This indicates loss of available epithelial
cell surface interaction sites. Epithelial cell surface material is solubilized by incubation
of egg packets in calcium- and magnesium-free sea water. Treatment of trypsinized egg
packets with the solubilized material increases the fertilization rate. The possibility
that the necessary capacitation-like interaction is mediated by the cell surface is
discussed.
Cnidarian and particularly hydrozoan
reproductive systems have received considerable attention during the past century. A major concern of earlier workers
was the origin and development of sex cells
(Desor, 1851; Agassiz, 1862; Allman, 1863;
De Varenne, 1882; Weismann, 1883, 1888;
Hargitt, 1913). In Laomedea flexuosa
(Campanularia flexuosa) Allman (1863)
described the arrangement and development of the gonophores, each of which
bears one oocyte along the blastostyle. He
further described oocyte maturation and
germinal vesicle breakdown and noted
(1863, p. 404) spermatozoa swimming in
the cavity of the female gonophore at the
time of germinal vesicle breakdown. Following fertilization, the eggs develop to the
planula stage which breaks out of the
gonophore and leaves the gonangium.
This work is part of a thesis submitted in partial
fulfillment of the requirements for the degree of
Doctor of Philosophy, Temple University. Current
address: Institute for Molecular and Cellular Evolution 521 Anastasia Ave., Coral Gables, Florida
33134.
I thank Dr. R. L. Miller for his advice and
criticism during the course of this study and Dr.
C. B. Metz for reading the manuscript. Supported
in part by NIH grant HD 04543 to Dr. R. L. Miller.
Weismann (1883) also described sex cell
development in various hydrozoa, including the cell layers surrounding the maturing oocyte and their relationship to the
blastostyle in C. flexuosa. Hargitt (1913)
showed the position of the maturing
oocytes within the entire immature gonangium (C. flexuosa) in a drawing of a
histological section. However, none of
these early workers studied the fertilization
process directly.
Maturation of the C. flexuosa gonangium
is concomitant with the maturation of the
oocytes within the gonangium. Only after
maturation is it possible for sperm to enter
the gonangium and reach the egg. Although
the development of the female gonangium
has been described (Allman, 1863; Berrill,
1950), the final maturation stages have
been reported only briefly (Miller, 1969).
The method by which the spermatozoa,
after entering the female gonangium, subsequently reach the egg has only recently
been considered (O'Rand, 1971, 1972a;
O'Rand and Miller, 1974).
Once the spermatozoon has reached the
funnel of the female gonangium, it may
swim directly to the proximal end of the
funnel or adhere to the funnel epithelium
487
488
MICHAEL G. O'RAND
by either head or tail (O'Rand, 1971,
1972a). From the funnel the spermatozoon
proceeds to the egg surface via passageways through the epithelial cell layers of
the female (O'Rand, 1971, 1972a; O'Rand
and Miller, 1974). During the spermatozoon's passage from funnel to egg, two important events occur: (i) the membranebounded vesicles in the apical and lateral
regions of the sperm head are lost
(O'Rand, 1971; O'Rand and Miller, 1974),
and (ii) a capacitation-like interaction between sperm and epithelial cell takes place
(O'Rand, 1972i>). This interaction is apparently an essential step in the reproductive process. Without it spermatozoa are
incapable of fertilizing eggs. The capacitation-like action of the epithelial cells on
the sperm may be prevented by various
chemical treatments. Spermatozoa exposed,
to such treated cells do not fertilize eggs,
but addition of untreated epithelial cells
renders spermatozoa capable of fertilization
(see O'Rand, 19726, for details). Among
the various chemical agents which inhibit
fertilization, trypsin treatment of egg packets (eggs surrounded by epithelial cells)
has been shown (O'Rand, 1972ft) not to
damage the egg itself. This paper concerns
the effects of trypsin treatment on the epithelial cell surface.
LOSS OF FERTII.IZABIUTY IN TRYPSIN
TREATED EGG PACKETS
Trypsin-treated (0.75%) egg packets
which had been exposed to spermatozoa
were examined by electron microscopy to
determine the effect of the trypsin treatment (for methods of electron microscopy
fixation, see O'Rand and Miller, 1974).
Epithelial cells of trypsin-treated packets
appeared unaltered in fine structure except for the loss of surface coat material.
Epithelial cells of normal (untreated) egg
packets have a fuzzy surface coat in electron micrographs which is approximately
200 A thick (Fig. 1). This surface coat
was reduced to approximately 50 A following trypsin treatment (0.75%, 1 hr,
15 C, pH 7.8; Fig. 2). The fuzzy surface
coat had not returned to its original thickness in egg packets fixed for electron microscopy even at 72 hr following trypsin
treatment (Fig. 3). Addition of spermatozoa to egg packets 24 hr after recovery from
the trypsin treatment did not increase the
fertilization rate (O'Rand, 1972«).
Membrane-bounded 640-700 A vesicles,
located in the apical and lateral regions of
the sperm head, decrease in number during sperm penetration of the female gonangium (O'Rand, 1972a; O'Rand and
Miller, 1974). Membrane-bounded vesicles
were also observed in sperm in trypsintreated egg packets fixed for electron microscopy. Vesicle counts (see O'Rand and
Miller, 1974, for methods of vesicle counting) of spermatozoa in trypsin-treated egg
packets (2.8 vesicles per sperm per section)
did not differ significantly from the counts
in untreated packets (2.1 vesicles per
sperm per section). The number of vesicles per sperm per section present in either
treated or untreated egg packets was approximately the same as the number present in sperm found in the epithelial passageways in vivo (O'Rand and Miller,
1974).
As well as having a normal complement
of vesicles, electron micrographs show that
sperm in trypsin-treated packets adhere
normally to the female epithelial cells
(O'Rand, 1972«). Further examination of
preserved trypsin-treated packets revealed
that there were probably fewer sperm adhering to female epithelial cells per unit
volume. A calculation (sperm/volume sectioned) of the number of sperm found in
one untreated packet and in one treated
packet showed 17.6 X 103 sperm/mm3 and
9.5 X 103 sperm/mm3 respectively.
To compensate for an apparent reduction in the number of adhering sperm in
trypsin-treated packets, increasing amounts
of sperm were given to eggs in trypsintreated packets. Figure 4 shows the effect
of increasing amounts of sperm on fertilization in egg packets taken from freshly
collected gonangia. In egg packets treated
with trypsin (0.75%) the fertilization rate
IT.
..
FIG. 1. A Campanuluria flexuosa egg packet
epithelial cell showing a fuzzy surface coat (arrow)
approximately 200 A thick. x49,500.
FIG. 2- A C. flexuosa egg packet epithelial cell
after the egg packet was treated with trypsin
(0.75%, 1 hr, 15 C, pH 7.8). The fuzzy surface coat
is now approximately 50 A thick (arrow), x 80,000.
FIG. 3. A C. flexuosa egg packet epithelial cell after
72 hr recovery from the trypsin treatment. The
fuzzy surface coat (arrow) has not returned to its
original thickness (Fig. 1). x 76,500.
490
MICHAEL G. O'RAND
100
(15,104/120)
(3,14/22)
(3,16/25)
(11,186/312)
(12,130/221)
(3,7/20)
19,4/91)
10
SPERM/m
11 ]2
x 10
FIG. 4. In vitro fertilization of C. flexuosa eggs
with increasing numbers of spermatozoa. Egg
packets were taken from freshly collected colonies
and either treated with trypsin (Q—_Q) or not
treated (%
£ ) . The egg packets were then
washed, given spermatozoa and incubated in millipore filtered sea water at 15 C, pH 7.8. a, individuals,
fertilized eggs/total number of eggs.
increases until it reaches the 46% level at
800,000 sperm/cc. Eggs in untreated packets also show an increasing fertilization
rate until the 87% level at 400,000
sperm/cc. Untreated packets begin at the
59% level since sperm are already present
in freshly collected packets. In treated
packets the fertilization rate begins at less
than 5% since the sperm already present are destroyed by trypsinization (see
O'Rand, 19726). The decreases in the
fertilization rates at high sperm concentrations may be due to abnormal cleavages resulting from polyspermy. If eggs
undergoing such abnormal cleavages did
not develop to the elongate embryo stage
(used as a measure of the fertilization rate,
O'Rand, 19726), then they would not be
counted as fertilized eggs.
From the data in Figure 4 it appears that
in trypsin-treated packets the low rate of
fertilization may be partially compensated for by increasing the number of
spermatozoa.
However,
trypsinization
(0.75%) has probably removed enough of
some necessary epithelial cell material to
prevent complete recovery of the potential
for the capacitation-like action on sperm.
Recovery of sperm fertilizing capacity can
be achieved either completely by addition
491
FERTILIZATION IN CAM PAN U LA RI A
of untreated epithelial cells (O'Rand,
19726) or partially by the fertilization
enhancing factor (see below). Thus, loss
of fertilizability in trypsinized egg packets
may be due to the inability of sperm to
adhere to or come in contact with the intact surface of the female epithelial cells
surrounding the eggs as would normally
occur.
A SOLUBLE FACTOR THAT ENHANCES SPERM
FERTILIZING CAPACITY
Incubation of egg packets in increasing
concentrations of trypsin decreased the
fertilization rate of sperm (O'Rand, 19726)
and removed epithelial cell surface material. Likewise, incubation in calciumand magnesium-free sea water (CMFSW)
also decreased the fertilization rate of
sperm. This suggests removal (e.g., solubilization) of the epithelial cell surface
material responsible for the capacitationlike action on sperm. Such solubilization
might be comparable to removal of the
hyaline layer from sea urchin eggs (Tyler
and Metz, 1955) and the organic factor
responsible for sponge cell adhesion
(Humphreys, 1963).
To examine for such a soluble factor,
egg packets of gonangia from either freshly
collected or laboratory cultured colonies
were incubated in CMFSW at 4 C for 4 hr
on a gyratory shaker. The egg packets
were then removed by centrifugation at
2500 rpm for 30 min at 4 C and the Ca2+
concentration of the supernatant adjusted
to that of normal sea water. This supernatant increased the fertilization rate in
trypsinized egg packets (Fig. 5). In three
experiments the average fertilization rate
(1.5 X 105 sperm/mm3) increased from
25% in trypsinized egg packets (10 individuals; 18/72 fertilized eggs) to 48% in
comparable trypsinized egg packets in the
presence of the supernatant factor (15 individuals; 68/142 fertilized eggs). Extraction
of this factor by cold CMFSW suggests
that it may be a surface molecule but until
further studies are performed to characterize the factor such a conclusion must remain tentative.
DISCUSSION
00,
(15,
1&72)
68/112)
EGGS +
EGGS +
EGGS +
SPOT
TRYPSIN +
TRYPSIN +
SPERM
SPERM +
FACTOR
FIG. 5. In vitro fertilization of C. flexuosa in
trypsinized egg packets. Eggs were fertilized in the
presence or absence of the soluble fertilization enhancing factor, a, individuals, fertilized eggs/total
number of eggs.
The trypsin treatment (0.75%) reduced
the fuzzy epithelial cell surface coat from
approximately 200 A to 50 A. Although
cell migration inside empty funnel perisarc
(O'Rand, 19726) may indicate some recovery from the trypsin treatment by the
epithelial cells, the fertilization rate did
not recover in trypsin treated packets inseminated 24 hr after treatment. The surface coat material was not replaced by the
cells after as much as 72 hr after removal
from the trypsin. Thus, the removal of
epithelial cell surface material may be responsible for the reduced fertilizability of
sperm in trypsinized egg packets. Such a
conclusion is further strengthened by the
observation in histological sections (for
electron microscopy) of trypsin-treated egg
packets that the ability of spermatozoa to
adhere to female epithelial cells is greatly
reduced, although adherence appears normal when it does occur.
The removal of surface coat material by
492
MICHAEL G. O'RAND
0.75% trypsin can be partially overcome
by fertilization with increasing numbers of
spermatozoa (Fig. 4). Similar experiments
were conducted on sea urchins by Tyler
and Metz (1955). They were able to show
that although 0.05% trypsin treatment reduced the percentage of fertilized eggs this
could be overcome completely by addition
of sufficient spermatozoa. Since increasing
concentrations of trypsin reduce the percentage of fertilized eggs in C. flexuosa,
presumably at low trypsin concentrations
this could be overcome with additional
sperm. However, at 0.75% it appears that
enough of the epithelial cell surface component has been removed to prevent normal fertilization even when large numbers
of spermatozoa are employed.
From the data presented above, the
essential spermatozoan-female epithelial
cell interaction appears to involve the
spermatozoon and a component of the epithelial cell surface. The effects of trypsinization indicate that a female epithelial cell
surface protein, or some component connected to a protein, must interact with the
spermatozoa. This interaction probably
occurs between bursts of active motility by
the spermatozoon inside the female gonangium (O'Rand, 1972a). The partial or
complete removal of the surface component by chemical treatments results in loss
of fertilizability to the system. However,
the surface component in the form of untreated epithelial cells can be added to
treated packets to restore fertilizability. A
soluble form of the surface component also
can restore fertilizability to the treated
packets. The results (Fig. 5) show an enhancement of the fertilization rate of eggs
in trypsinized egg packets, but not complete recovery. This was probably due to
a relatively low titer of the component allowing only a small number of sperm to
be affected. Further studies will be necessary to find the conditions that will increase the component's effectiveness in restoring fertilizability and to characterize
the molecule (s) responsible.
The role of cell surface components in
mediating intercellular interaction has
been demonstrated in a number of cases
(see, for example, Roth et al., 1971; Buck
et al., 1971). The glycoprotein fertilizin
has long been thought to be a component
of the egg membrane and antifertilizin a
component of the sperm membrane (see
Tyler and Tyler, 1966, for a review).
Roseman (1970) has suggested that in
intercellular adhesion the opposing cell
surfaces have complementary enzymes and
substrates which are carbohydrates and
glycosyltransferases. Cell aggregation factors which are either glycoprotein (Humphreys, 1965) or acid mucopolysaccharide
(Pessac and Defendi, 1972) have been
found in several cell types and are probably components of the cell surface. In fact,
many cell types are covered with a coat of
mucopolysaccharide (Rambourg and Leblond, 1967). It is not unreasonable,
therefore, that gamete interaction with female reproductive tissues may be mediated
by mucopolysaccharides or glycoproteins.
The events that occur prior to sperm-egg
interaction in C. flexuosa indicate the importance of spermatozoan behavior within
the female reproductive tract. In Campanularia at least three steps in the fertilization process occur prior to sperm-egg
contact. The first step would be species
specific chemical attraction of the homologous sperm to the female gonangium
(Miller, 1966). The second would be
spermatozoan vesicle loss within the female gonangium which is apparently at
least generically specific (O'Rand, 1972«).
The third step which may or may not be
separate from vesicle loss is the capacitation-like action of the female epithelial
cells on the spermatozoa. The final step in
the fertilization process would be spermegg contact. It is at the level of sperm-egg
contact that the fertilizin-antifertilizin system would operate. The fertilizin-antifertilizin system has been well characterized in a number of invertebrates with
external fertilization (see Tyler and Tyler,
1966, for a review) and may also exist in
mammals (Bishop and Tyler, 1956). The
events of the fertilization process as studied in Campamdaria may also serve to
regulate fertilization in other invertebrates
with internal fertilization.
FERTILIZATION IN CAMPANULARIA
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