IMMUNOBIOLOGICAL STUDIES OF THE SPECIES-SPECIFICITY

OF EGG JELLIES OF THE FROG

BY

Charles Alex Shivers

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies
of Michigan State University of Agriculture and
Applied Sciences in Partial Fulfillment
of the Requirements

for the Degree of
DOCTOR OF PHILOSOPHY

Department of Zoology

1961

Approved Li/z (WK/w Q? Sikm AJ'-\_,$Z/\"’

ABSTRACT

IMMUNOBIOLOGICAL STUDIES OF THE SPECIES-SPECIFICITY

OF EGG JELLIES OF THE FROG

By Charles Alex Shivers

Antisera were prepared by injecting lyophilized egg-jelly

material from four species of frogs (Rana pipiens, 3. clamitans,

 

 

‘3. sylvatica, and 3. catesbeiana) into rabbits. Using the

 

Ouchterlony techinque it was determined that all four species
have common components as well as a number of specific components.
By adsorbing anti-jelly serum of each species with jelly from
the other species, the specific components of each species
were identified. It was also determined that the jelly
components of g. pipiens are, in general, restricted to the
oviducal tissues and not found in other body tissues of the
frog.

Eggs of Rana pipiens were treated with antisera against both
homologous and heterologous jellies prior to insemination
with normal spermatozoa of 5. pipiens. Treatments with both
heterologous and homologous antisera resulted in satistically
significant inhibition of the fertilizability of the eggs.
The inhibitory effect of heterologous antiserum could be
removed by treating it with either homologous or heterologous

jellies. The inhibitory action of the homologous antiserum

Charles Alex Shivers
could be removed only by treating it with homologous jelly.
These results indicate that the specific components of anti-
jelly serum of B. pipiens are inhibitory, as are the common
components in the heterologous anti-jelly serum. The specific
components of the heterologous sera are non-inhibitory.

The effect of antisera prepared against jelly material of
other Amphibian genera has also been tested. In cases (ggfg)
where a cross-reaction was observed between these sera and
jelly of 3. pipiens, inhibition was also observed. No
inhibitory action was seen with anti-jelly sera that did not

cross-react with the jelly of_3.jpipiens (Ambystoma).

 

 

IMMUNOBIOLOGICAL STUDIES OF THE SPECIES-SPECIFICITY

OF EGG JELLIES OF THE FROG

BY

Charles Alex Shivers

A THESIS

Submitted to the School for Advanced Graduate Studies
of Michigan State University of Agriculture and
Applied Sciences in Partial Fulfillment
of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Department of Zoology

1961

x f‘. 4"
I; T..." L?/ Li?"

' J -. 0 _ ‘3)
f -./ /c’»~ ,1

ACKNOWLEDGEMENTS

The author wishes to express his sincere thanks to Dr.
J. R. Shaver for his interest, direction and encouragement
during the course of this investigation. Grateful thanks are
also due to Drs. G. B. Wilson, F. A. Fennell, and P. J. Clark
for their able assistance while serving on the author's
guidance committee. Thanks are also due to Dr. S. H. Barch
for her interest and assistance during the course of this
study.

Acknowledgement is made to the National Institutes of
Health for the predoctoral fellowship (GE-11,123).

Special recognition is made to my wife, Sara, whose

encouragement and sacrifices have made this study possible.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . . .

LIST OF PLATES . . . . . . . . . . . . . . . . . . .

LIST OF TEXT FIGURES . . . . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . . . . . . . . .

MATERIALS AND METHODS . . . . . . . . .

RESULTS . . . . . . . . .
A. Analysis of anti-jelly sera . . .

1. Analysis of antisera prepared against
jelly of Rana pipiens

2. Analysis of antisera prepared against
jelly of Rana clamitans

3. Analysis of antisera prepared against
jelly of Rana sylvatica

4. Analysis of antisera prepared against
jelly of Rana catesbeiana

5. Analysis of antisera prepared against
jelly obtained from another genus of
Anura and another order of Amphibia
(Urodela)

 

 

 

Effects of antisera on the fertilizability of
eggs of Rana pipiens . . . . . . . . . . . . .

 

1. Effect of treating eggs with antisera
prepared against the jelly of Rana pipiens

2. Effect of treating eggs with antisera
prepared against the jelly of Rana clamitans

3. Effect of treating eggs with antisera
prepared against the jelly of Rana sylvatica

4. Effect of antisera prepared against egg-
jellies of another Anuran genus (Bufo) and
of a Urodele species (Ambystoma)

5. Effects of antisera prepared against
fertilizin of eggs of Arbacia (Echinodermata)

6. Effects of treating eggs with antisera
prepared against homogenates of organs of
adult Rana pipiens

 

 

 

 

iii

Page

vi

vii

ll
18

l8

18
22
24

24

25

45

46
48

50

51

52

53

Page

C. Effect of treating eggs of Rana pipiens
with fractions of anti-jelly sera . . . . . . . 6O

 

DISCUSSION
A. Analysis of antigenic components
represented in anti-jelly sera by means

of agar-diffusion technique . . . . . . . . . . 63

B. Analysis of the effect of anti-jelly sera

on fertilizability of eggs . . . . . . . . . . 72

C. General considerations and speculations . . . . 77
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . 80
LITERATURE CITED . . . . . . . . . . . . . . . . . . . 83

iv

LIST OF TABLES

 

Table Page

1 Percentages of cleavages in batches of eggs

treated with serum dilutions of 1:1 prior

to insemination with normal spermatozoa . . . . 59
2 Cross-reactions between jellies as determined

by the Ouchterlony technique . . . . . . . . . 69
3 The presence of specific components in

anti-jelly sera as determined by the

technique of adsorption . . . . . . . . . . . . 7O
4 Tissue—specificity of Rana pipiens jelly

components as determined by the Ouchterlony

technique . . . . . . . . . . . . . . . . . . . 7l

Plate

LIST OF PLATES

Representative photographs of Ouchterlony
plates and results observed on eggs after
treatments with anti-jelly serum . . . .

Diagrams

of Ouchterlony plates. Analysis

of species-specificity of antisera prepared
against Rana pipiens jelly . . . . . . . .

 

Analysis
prepared

Analysis
prepared

Analysis
prepared

Analysis
prepared

Analysis
prepared

Analysis
prepared

Analysis
prepared

of species-specificity of antisera
against Rana clamitans jelly . .

 

of species-specificity of antisera
against Rana sylvatica jelly . . .

 

of species—specificity of antisera
against Rana catesbeiana jelly . .

 

of tissue-specificity of antisera
against Rana pipiens jelly I .

 

of tissue-specificity of antisera
against Rana pipiens jelly II

 

of tissue-specificity of antisera
against Rana pipiens jelly III . .

 

of specificity of antisera

against jellies from another Anuran

genus and a genus of another Amphibian

(Urodela)

vi

Page

28

3O

32

34

36

38

4O

42

44

LIST OF TEXT FIGURES

 

 

Figure Page

1 Species-specificity of inhibitory effect of

anti-jelly sera on fertilization of eggs of

Rana pipiens . . . . . . . . . . . . . . . . . 56
2 Tissue-specificity of inhibitory effect of

antisera on fertilization of eggs of Rana

pipiens . . . . . . . . . . . . . . . . . . . . 57
3 Table of significant differences between

treatments of eggs of Rana pipiens . . . . . . 58
4 Results obtained after treating eggs of

Rana pipiens with fractionated serum . . . . . 62

 

vii

INTRODUCTION

At fertilization, the egg is incited to undergo a number
of changes that lead to the development of a new individual.
It has been suggested that the initiation of these changes
is due to reactions that occur between the surface of the egg
and the spermatozoon which allows the latter to penetrate the
egg. The mechanism of activation of the egg by the spermatozoan
has been the central problem in the study of fertilization.
This problem may be approached experimentally in a number of
ways but it seems that the one most likely to produce results
would be a study of the role of interacting substances of the
sperm and egg.

Many attempts have been made to extract interacting
substances from eggs and spermatozoa. The first serious
study of this nature was done by F. R. Lillie on the isoag-
glutinins in eggs of the sea urchin, Arbacia, and of the
annelid, Nereis.

Lillie (1913) showed that sea water which had been in
contact with sea urchin eggs caused the agglutination of
spermatozoa of the same species. When eggs were kept for
several days in sea water they ceased to produce the agglu-

tinating substance and their capacity for fertilization was

2

also diminished. Lillie concluded that this agglutinating
substance of the egg, which he called "fertilizin," was
indispensable for the process of fertilization. It was
suggested by Lillie that this agglutination reaction is very
similar to an antigen-antibody reaction, and that fertilizin
of the egg reacts with "anti-fertilizin" present in the
spermatozoon.

More recently, antigen-antibody like reactions have
been postulated to account for a number of different kinds
of specific intercellular reactions. For example, Spiegel
(1954a, b, and 1955) has been able to interfere with the
reaggregation of disaggregated cells by the use of species-
specific antibodies in a way that would indicate a role of
specific surface antigens. This worker found that antiserum

prepared against embryos of Rana pipiens inhibits the

 

reaggregation of dissociated ectodermal cells of the homologous
species. The antiserum had no effect on the reaggregation of
dissociated cells of a species of Triton. Spiegel concluded
that the adhesion of homologous cells was due to the

presence of surface configurations with the reciprocal
structural relationship of antigen and antibody. Tissue
incompatibility (Woerdeman, l955,and Billingham and Sparrow,
1955) and tissue affinity studies (Holtfreter, 1947a, b) also

indicate the importance of cellular interactions.

3
The most thoroughly investigated cells with reference to
interacting substances are the spermatozoa and eggs of species

of Echinodermata. Studies of these substances have been given

 

considerable attention in the last few years (for recent
reviews see Tyler, 1955, and 1959; Rothschild, 1956;

Metz, 1957; Runnstrom et. al., 1959). Tyler's work seemingly
supports the "fertilizin theory" of F. R. Lillie and further
suggests that fertilizin is identical with the jelly-coat
material of the sea urchin egg.

Conflicting and divergent interpretations have been
suggested for the role of interacting substances of the
gametes in fertilization (Perlmann, 1959; Tyler, 1959;
Hagstrom, 1956). There can be no doubt at the present time
that these substances are operative in the fertilization
reaction, and that they occur in the gametes of a number of
species of animals, particularly among the Echinoderms (cf.
Tyler, 1948, 1957, and 1959; Bishop and Tyler, 1956; Bernstein,
1952).

Before one can understand completely the mechanisms of
the process of fertilization, one must know the molecular
.structure of the surface of both spermatozodn and egg, and the
ultrastructual patterns of the interacting molecules.

Chemical analysis of the fertilizin from eggs of the sea

urchin has shown it to be composed of mucopolysaccharides

4
(glycoproteins) with a high sulfur content (Tyler, 1949;
Tyler and Fox 1940; Vasseur, 1948). However, very little is
known chemically about the groups present in fertilizin
molecules which specifically react with antifertilizin. In
this connection, studies of specific antibodies used in
conjunction with other biochemical and biological techniques,
have made possible the analysis of biologically active materials
of complex mixtures, which cannot be distinguished by other
means at the present time.

The results of a thorough examination of the effects of
specific antibodies produced in rabbits against various
materials from eggs, spermatozoa, embryos, and adults of
various species of Echinoderms, have been reported by Tyler
and co—workers (1959) in California, and by Perlmann and co—
workers (1959) in Sweden. In general, these investigators have
demonstrated that treatment of eggs with antisera produced
against eggs and various egg materials of the sea urchin,
results in inhibition of fertilization, cleavage, and develop-
ment of the embryo.

Perlmann and co-workers (for references see Perlmann, 1959)
report finding at least four different antigenic components

in eggs of Paracentrotus lividus. These workers have attributed

 

certain specific activities such as jelly precipitation,

activation, cortical damage, etc., to antisera prepared against

5

these components, as measured by the effects on eggs treated
with the antisera. These components have been designated as
(1) J-antigen--that component which is involved in the pre-
cipitation of the jelly layer on treatment with antisera against
an egg surface antigen. (2) A-antigen——that component which
is concerned with the artificial activation of the egg which
occurs after treatment with antisera prepared against an egg
surface antigen. (3) C-antigen-—probably located in the cortex.
Cortical damage results when the egg is treated with anti—
bodies against this component. (4) F—antigen--probably the
sperm receptor antigen of the egg cortex. Perlmann interprets
these results as indicating that these antigenic components
have a particular role in connection with the attachment of
the sperm and activation of the egg in normal fertilization.

The problems of species- and tissue-specificity of the
fertilization reaction has been studied by Lillie (1921),
Tyler and co-workers (see Tyler, l959,for references), and
by Perlmann (1959). Tyler (1949) has demonstrated that there
is considerable cross-agglutination of spermatozoa of Echino-
derm species by antisera prepared against sperm extract of
other species. However, antibodies formed against anti-'
fertilizin of the sperm react with other tissues from the
homologous species but not with the same tissues of foreign

species. Considerable cross-reaction was also seen in the case

6
of the blockage of cleavage of fertilized eggs after treatment
with antiserum prepared against fertilizin, in the different
species tested (Tyler, 1959). Thus, the specificity of the
fertilizin-antifertilizin reaction does not, in itself,
account for the specificity of fertilization since a number
of other reactions are possibly involved in fertilization.
However, Tyler has pointed out that where cross-fertilization
occurs, cross-agglutination also generally occurs. Evidence
for tissue-specificity of fertilizin comes mainly from work
on sperm agglutination, it having been determined that no
cellular types other than the sperm or egg yield fertilizin
or antifertilizin. The conclusion drawn from these results
is that both tissue- and species-specificity of fertilization
are based, in part, on the specificity of the fertilizin-
antifertilizin reaction.

It is quite surprising that very few studies have been
performed on the role of the jelly-coat_material of eggs of
Amphibians, as compared to the rather extensive studies done
on jelly of eggs of sea urchins and other Echinoderms.

There can be no doubt as to the importance of the jelly
envelope around eggs of Amphibians, since eggs without their
jellies, either as they normally occur before passage down the
oviduct, or after removal of jelly from uterine eggs by

artificial means, are not fertilizable (Batallion, 1919;

Rugh, 1935; Kambara, 1953; Tchou-Su and Wang, 1956; Shaver and
Barch, 1960; Subtelny and Bradt, 1961). The latter workers
have shown that coelomic (jellyless) eggs of the frog are
capable of normal cleavage before passage down the oviduct,
if artificial activation is accomplished by pricking with a
glass needle and the subsequent transfer of blastula nuclei.
Batallion (1919) demonstrated that unfertilized, jellyless
eggs were capable of development after the parthenogenetic
inoculation of a cellular element. The fact that the egg is
capable of undergoing development prior to the deposition of
the jelly, only after artificial stimulation, suggests that
the jelly is important in the initial steps in fertilization.
Kambara (1953) and Tchou—Su and Wang (1956) have shown that
jellyless eggs of the toad are fertilizable when artificially
supplied with jelly.

Kambara (1953) noted that eggs of the toad (Bufo vulgaris
formosus) become increasingly more fertilizable as they pass
down the oviduct, and that the so-called "C layer" (the second
layer of jelly to be laid down) was indispensable for fertili-
zation. However, in the results presented by this worker, it
was not possible to determine whether a maturation factor is

involved, since the eggs possibly do mature during the sojourn

in the oviduct. Eggs of Rana pipiens (Shivers, unpublished

 

results) are not fertilizable immediately upon reaching the

8

uterus but become increasingly more fertilizable after remaining
in the uterus for some time, i.e., several hours, suggesting
a maturation factor.

It has been well established that unfertilized eggs of
the frog become increasingly less fertilizable as they stand
in fluid, which has been attributed, in part, to the swelling
of the jelly layers which begins immediately upon contact with
the fluid (cf. Rugh, 1951). Changes in the surface of the
egg (Tyler, 1955) and breakdown of cortical granules (Motomura,
1952), perhaps reflecting initial phases of activation, have
been observed in eggs of other Amphibians and other vertebrates
which may contribute to the loss in fertilizability. The
loss of antigenic components from the jelly material or the
egg almost immediately after immersion in water (Shivers,
unpublished results) may also account for the decrease in
fertilizability.

Both physical and chemical mechanisms have been used in
an attempt to explain the role of the jelly in the process
of fertilization of Amphibians. Kambara (1953) came to the
conclusion that no chemical substance present in jelly of toad
eggs is responsible for fertilization, since eggs deprived of
their gelatinous coatings were not fertilizable even after
being covered with homogenized jelly. On the other hand, this

worker noted that denuded eggs became fertilizable after being

covered with gelatine or agar. Thus, Kambara was led to
believe that the jelly layers of the toad egg served as a
mechanical foothold for the sperm in the penetration of the
egg and that gelatine or agar could serve as a substitute for
the jelly in this capacity.

Observations made by other workers have suggested that the
jelly coating of eggs of Amphibians operates in the same
manner as the fertilizin of the sea urchin egg (Bernstein,
1952). Bernstein (1952) was unable to detect the presence of

an agglutinating substance in the egg water of Rana pipiens,

 

but reports the irreversible agglutination of sperm of Rana

clamitans by egg water of the homologous species. The fact

 

that sperm agglutinating substances were not observed in non-
ovulated, jellyless eggs, suggests that the substances were
located in the jelly layers. Sperm agglutinating substances
have also been reported in a number of other vertebrates (cf.
Bishop and Tyler, 1956).

Chemical studies made on the egg-jelly material of several
species of Amphibia have shown it to be very similar to the
jelly—coat of the sea urchin egg, e.g., the egg jelly of Rana
is composed primarily of mucopolysaccharide or glycoprotein
(Folks, Grant, and Jones, 1950). According to Minganti (1955),
the principal difference between the egg jelly of Amphibians

and the jelly—coat of Echinoderm eggs is the presence of

10
hexosamine in the former.
Shaver and Barch (1960) have studied the effects of

treating eggs and spermatozoa of the frog, Rana pipiens, with

 

antisera prepared against the jelly—coat material of the
homologous species. These workers have shown that pre-treatment
of gametes with the anti-jelly serum resulted in significant
inhibition of cleavage of the egg, implying an interference
in the union of the gametes. Also, it was shown that the
inhibitory effect of the anti-jelly serum could be completely
removed by adsorption of the antiserum with jelly—coat material
prior to treating the gametes.

The objectives of the present investigation were: (1) to
characterize by serological methods, the components present

in egg-jelly of Rana pipiens, both as to tissue— and species-

 

specificity; (2) to test the effects of treating eggs of Rana
pipiens, prior to normal fertilization, with antisera prepared
against the jelly material from the homologous and heterologous

species; (3) to show the effect of treating eggs of Rana pipiens

 

with antisera that had been previously adsorbed with jelly from
the heterologous and homologous species;(4) to test the effects
of treating eggs of Rana pipiens with antisera prepared against
organ extracts of the adult frog; (5) to test the effect of

treating eggs of Rana pipiens with antisera prepared against

 

egg-jellies from other taxa of Amphibia.

MATERIALS AND METHODS

Jelly coat material was mechanically removed from mature

unfertilized eggs of four species of frogs (Rana pipiens, R.

 

clamitans, R. sylvatica, and R. catesbeiana), after allowing

 

 

 

the gelatinous material to hydrate completely in distilled
water. The jelly was washed several times with distilled
water prior to lyophilization. Antigens were prepared by
blending ten mg of the lyophilate with one m1 of physiological
saline solution, 0.85%, buffered at pH 7.4 with Sorenson's
phosphate mixture, to Which sodium ehtyl mercurithio-salicylate
(Merthiolate, Lilly) was added in a proportion of one part
per 10,000.

Tissues that were to be used as antigens were removed
from the adult frog, weighed and homogenized, either in a
glass homogenizer or a Virtis homogenizer, model 23. The
homogenization medium used for these tissues was the buffered
saline solution described above. The homogenate was centri-
fuged at 1200 x g and the supernatant fluid was used as the
antigen. Practically all representative tissues have been
tested.

Blood for control serum was drawn from the marginal ear
vein of large rabbits prior to the injection of the antigen.

In no case was a cross-reaction observed between rabbit control

11

12
serum and any frog tissues tested. All serum was dialyzed
against one—tenth full strength Holtfreter's solution for 48
hours and frozen until ready for use.

Production of antisera has employed the use of the Freund
adjuvant technique. The adjuvant mixtures are available in
two types, complete and incomplete, from Difco Laboratories,
Detroit, Michigan. The complete adjuvant mixture is composed
of mannide monooleate, 1.5 m1; parrafin oil, 8.5 m1; and 5 mg

of killed and driedeycobacterium butyricum. The incomplete

 

adjuvant mixture lacks the Mycobacterium butyricum. Each

 

rabbit was injected via the subscapular route with 1.5 ml

of emulsion prepared by mixing equal volumes of complete
adjuvant mixture and antigen. One week later, a second injection
of 1.5 ml of emulsion was given, this time using equal volumes
of incomplete adjuvant mixture with the antigen.

Four weeks after the second injection, serum from a
bleeding made from the ear vein of the rabbit was tested for
the presence of antibodies by the use of the Ouchterlony
technique. If the presence of antibodies was indicated, the
animal was bled twice more on successive weeks from the ear
vein before being exsanguinated by cardiac puncture.

Ouchterlony plates were prepared by heating a 1% solution
of "Ionagar" (obtained from Consolidated Laboratories, Inc.)

using the buffered physiological saline solution, mentioned

13
previously, as the solvent. Prior to pouring the heated agar
into Petri dishes of 90 mm diameter, strips of filter paper,
approximately one inch long and one-half inch wide, are folded
over the lip of the "male" half of the dish, the ends of the
Istrips touching the floor of the dish. The strips were moistened
thoroughly with distilled water before adding the still warm
agar, 20 ml per dish. After the agar was completely cooled,
various arrangements of wells were cut and floored with one
drop of hot agar. Plates were then covered and stored, bottoms
up, in the refrigerator at 40C until ready for use.

Each well was refilled with antigen or antiserum until a
.total of 0.75 ml had been given, an attempt being made to re-
fill wells with successive applications before the well dried.
Advancing zones of antigens and antisera diffuse into the agar;
in the area between the wells where they met, antiserum
components which were complementary to antigen components,
were indicated by a line of precipitation. When the plate
was fully developed, usually after 10 days at room temperature
(18 to 200C), a drawing was made of the plate, with all the
lines, and the preparation was photographed for a permanent
record.

Adsorption of antisera was accomplished by treating them
with various dilutions of the inhibiting antigen in glass tubes

for 24 hours at 40C, care being taken to see that antiserum

14

sites were completely saturated with complementary antigen sites.
An equal volume of antiserum and antigen was sufficient to
remove or neutralize complementary sites of the antiserum in
each case. (Adsorption with larger amounts of antigen did
not noticeably change the patterns of the precipitates.)
Plates were then run in the usual way using these adsorbed
antisera.

Antisera were prepared against the egg jelly material from
species of another Anuran genus (Bufo americanus and Bgfg
marinus) as well as from another order of Amphibia (Ambystoma

maculatum), in the same manner as that described for the species

 

of Rana. An antiserum against a preparation of fertilizin

from the eggs of Arbacia punctulata (Eichnodermata), kindly

 

supplied by Dr. C. B. Metz, was also available.

In the experiments to be reported, gametes of Rana pipiens,

 

obtained from commercial dealers in vermont, were used for
egg treatments. The eggs employed were obtained by artificially
inducing ovulation by the injection of pituitary glands
obtained from adult frogs (Rugh 1934).
Normal sperm suspensions were prepared by macerating whole
testes, obtained from previously pithed frogs, in one-tenth
full strength Holtfreter's solution. Batches of 30-50 eggs
were stripped out on 1 x 3 inch glass slides and flooded with

the sperm suspension. Insemination was accomplished by treating

15
eggs for 10 minutes. After the excess sperm suspension was
poured off, eggs were placed in large volumes of aerated tap
water and cultured at room temperature (18-200C). Results
were recorded in all cases as the percentages of eggs that
cleaved. The number of cleaving eggs was counted immediately
after fertilization and again at 8-32 cell stage (Shumway
stages 5-7). Results of both the Ouchterlony studies and of
treatment of gametes are from experiments in which sera pooled

from at least three different rabbits were employed.

Egg treatments: Eggs were treated with serum diluted to

 

1:1 with one-tenth Holtfreter's solution. After treatments
of two minutes, the serum was decanted from the eggs, which
were washed with a large volume of Holtfreter's solution to
remove any unreacted antibodies. Immediately after washing,
the eggs were inseminated with normal spermatozoa. Control
batches of eggs were treated with the Holtfreter's solution
and washed before insemination. Adsorption of the various
sera prior to egg treatment was accomplished by the same
method as described in connection with the Ouchterlony plate
procedure.

In one experiment an attempt was made to remove a non—
specific inhibitory effect of normal rabbit serum on the
spermatozoa and eggs of R. pipiens. The technique used in

this procedure was the "Rivanol" method of fractionation of

16
serum as suggested by Horejsi and Smetana (1956). For
separation of the gamma globulins and albumins, 3.5 parts of
a 0.4% Rivanol solution (ethoxy-diamino acridine lactate) was
added to one part serum. The pH was adjusted to 8.0 by the
addition of NaHCO3. The bright yellow precipitate which
formed (albumin—Rivanol complex) was separated from the super—
natant fluid (globulin fraction) by decanting the latter.
Unreacted Rivanol was removed from the filtrate by adding an
excess of activated charcoal. The charcoal-Rivanol complex
was then removed by filtering the solution through Whatman's
No. 1 filter paper. The filtrate was lyophilized until dry,
resuspended in the original volume of fluid, dialyzed against
one-tenth full strength Holtfreter's solution for 48 hours,
and frozen until ready for use.

In the spring of 1960, an attempt was made to see if the
fertilizability of uterine eggs could be affected by anti-
jelly serum treatments prior to the deposition of the jelly-
coat material. Female_§. pipiens were injected with whole
pituitary glands for the induction of ovulation. Several
hours later a series of injections, using 3. pipiens anti-
jelly sera, was started. Eight mls of the sera, diluted 1:1
with Holtfreter's solution, were injected into the body cavity
at two hour intervals. Each frog was checked before the

injections of the sera were started to make sure that no eggs

17
were present in the uterus at the time of the first injection.
Injections of the anti—jelly sera were continued until
ovulation was completed, which was about six hours. Eggs

from these injected females were then inseminated with

normal spermatozoa.

RESULTS

A. Analysis of Anti:jelly Sera

 

(1) Analysis of antiseraAprepared_against

 

jelly of R. pipiens.

 

Species-specificity: Plate 1, Figure 1 shows the result

 

of reacting rabbit control serum, i.e., serum drawn from the

rabbit prior to the injection of antigen into the rabbit, with

 

jelly material from three species of frogs (Rana pipiens. R.

sylvatica, and R. clamitans). No cross-reactions were observed

 

 

between rabbit control serum and any of the frog tissues tested.

Plate 1, Figure 2 shows the result of reacting antisera
prepared against jelly of R. pipiens with homogenates of jelly
material from the same three species of frogs. Drawings made
immediately after the completion of plates will be used
instead of photographs, since it is very difficult to get
lines of precipitation to show up distinctly in photographs.
Plate 2, Figure 7 is a drawing made of the Ouchterlony plate
using anti—jelly serum of g, pipiens against jelly homogenates
of the three species described in Plate 1, Figure 2.

As shown by Plate 1, Figure 2 and Plate 2, Figure 7, the
jelly material of R. pipiens contains a number of specific

components. These specific components are indicated by

18

19
lines of precipitation between the antiserum well and the well
containing the jelly material of R. pipiens. A component that
is found in jellies of all three species is indicated by a
continuous line formed opposite all three antigen wells. The
jelly of R. pipiens also contains a component in common with

one found in the jelly of R. clamitans that is not present in

 

the jelly of R. sylvatica (Plate 2, Figure 7).

 

If the anti-jelly serum against 3. pipiens is treated with

an equal volume of jelly of R. clamitans for 24 hours, the

 

common component between all three species is removed (Plate 2,

Figure 8). The component in common with jelly of R. clamitans

 

which is not present in the jelly of R._§y1vatica is also

 

removed by treatment with jelly of R. clamitans. The specific

 

components for jelly of R. pipiens are not removed by treatment

with jelly of R. clamitans.

 

Treatment of antiserum against the jelly of R. pipiens

 

with jelly of_R. sylvatica removes only the component that is
common between all three species (Plate 2, Figure 9). The A
component of the jelly of R. pipiens that is common with a
component found in the jelly of_R. clamitans is not removed by

treatments with jelly of R. sylvatica. Jelly material of_R.

 

catesbeiana, on the other hand, removes a component from anti—
jelly serum of R. pipiens that is common to R. pipiens, R.

clamitans, and_R. catesbeiana, as well as the component shared

 

 

20

between R. pipiens and R. clamitans (Plate 2, Figure 11),

 

leaving only the specific components for the jelly of R. pipiens.
All components were removed by treating the anti-jelly sera of
.3. pipiens with the homologous jelly (Plate 2, Figure 10).

Anti-jelly sera of R. pipiens were also treated with homo—
genates of various adult tissues from the homologous species
prior to the running of the plate. Adsorption with these
tissues failed to remove any of the jelly components (see
Plate 8, Figure 38).

No cross-reaction was observed between the jelly material

of R. pipiens and antiserum against the egg-jelly of Ambystoma

 

maculatum (Plate 9, Figure 42), nor does treatment of anti-

 

jelly serum of R. pipiens with jelly of Ambystoma remove any

 

of the jelly components of R. pipiens (Plate 2, Figure 12).

The jelly of a toad species, Bufo americanus, does have a

 

component that is common with a component of the jelly of R.

pipiens (Plate 9, Figure 41). Anti-jelly serum of Bufo marinus,

 

on the other hand, does not have components in common with the
jelly of R. pipiens (Plate 9, Figure 43). Anti-jelly sera of
.3. pipiens were treated with the jelly from these species of
lgufg and then reacted with the jelly of the species 0f.§§£§~

The jelly material of Bufo americanus was the only one to

 

remove components from anti-jelly sera of B. pipiens. No

cross-reaction was observed between fertilizin prepared from

21
from eggs of the sea urchin, (Arbacia) and anti-jelly serum of

_R. pipiens (Plate 9, Figure 44).

Tissue-specificity: A series of reactions was performed

 

to test the tissue-specificity of the jelly components of R.
pipiens (see Plates 6, 7, and 8). Anti-jelly sera of R.
pipiens were reacted with homogenates of various adult tissues
from the homologous species. It was determined by testing
practically all representative tissues of the frog that the
jelly components were, in general, restricted to the oviducal
tissues (Plate 7, Figure 34). Cross—reactions were obtained
between the anti-jelly sera and homogenates of mature oviduct
’(Plate 7, Figure 34), as would be expected since the jelly
layers are deposited on the egg during their sojourn in the
oviduct. When anti-jelly serum of R. pipiens is adsorbed with
homogenized oviduct prior to reaction with jelly of R. pipiens.
no lines appear subsequently on plates, indicating that the
source of all jelly components must be in the oviduct.

Other components are present in the oviduct homogenate,
however, that do not react with the anti-jelly sera. These
components may be demonstrated in the oviduct by treating anti-
oviducal sera with an equal volume of jelly material and re-
acting this treated serum with an homogenate of oviduct (Plate
6, Figure 31). Some of these non—jelly components of the

oviduct react with anti-ovary sera (Plate 6, Figures 28 and 31).

22
Indeed, almost alltissues tested cross-react with anti-ovary
sera with the exception of the jelly material. In some
instances, however, a very weak reaction has been observed
between certain anti-jelly sera and ovarian homogenates.
This reaction may be due to contamination of the jelly by
egg material which occurs during the process of removing the
jelly from the egg.

One component of anti—jelly serum of_R. pipiens cross-
reacts with a serum component of blood of R. pipiens (Plate
7, Figure 36). This component seems to be species-specific,
i.e., the component does not react with serum from the other

species of Rana. Anti-jelly serum of 3. clamitans on the other

 

hand, contains components that cross-react with blood serum
which are not species—specific, i.e., the components react with
serum from the homologous species as well as with serum from

.5. pipieng and R. catesbeiana (Plate 7, Figure 37). Anti-jelly

 

serum of R. catesbeiana reacts with serum from the homologous

 

species but not with serum from_§. pipiens or R. clamitans

 

(Plate 7, Figure 38).

(2) Analysis of antisera prepared againstyjelly

 

of g. clamitans.

 

 

Reactions obtained with anti-jelly sera of R. clamitans show

 

that egg-jelly of R. clamitans contains components in common

 

23
with components found in the jelly material of the species of
Rana tested (Plate 1, Figure 3 and Plate 3, Figure 13).

Jelly of R. clamitans has a number of components that are

 

specific for this species and a component in common with one
found in jelly of R. pipiens which is not present in the jelly

of R. sylvatica (Plate 3, Figure 13). There are also components

 

in common with ones found in jelly of_§. catesbeiana,

 

which are not found in either of the jellies of_3. pipiens

or R. sylvatica (Plate 3, Figure 18).

 

The configuration in anti-jelly serum of_R. clamitans

 

complementary to the component shared by the Species of Rana
was removed by treating this antiserum with jelly material
from any of the species used (Plate 3, Figures 14, 15, 16, and
17). Specific components represented in anti-jelly serum of

.5. clamitans could be removed only by adsorbing with the

 

homologous jelly material.

Tests for the tissue-specificity of jelly components of
.3. clamitans have been attempted with the same results being
observed as were seen in the comparable situation in R. pipiens,
i.e., the jelly components are restricted primarily to the

oviducal tissues. However, tissues of R. clamitans have not

 

been tested as thoroughly as have the tissues of B. pipiens.

24
(3) Analysis of antisera_prepared against jelly

of R. sylvatica.

 

 

Common components were observed between jelly material of
all species of Rana tested when the anti-jelly serum of R.

sylvatica was used (Plate 4, Figure 19) and these components

 

could be removed by treating the antiserum with jelly material
from any of the species of Raga_(Plate 4, Figures 20, 21, 22,
and 23).

The species-specific components represented in the anti-

jelly serum of R. sylvatica were removed only by treating with

 

the homologous jelly material (Plate 4, Figure 22). A com—
ponent represented in anti-jelly serum of R. sylvatica that was
common with one found in jelly of R. pipiens could be removed

by treating with the homologous jelly or jelly of 5. pipiens.

(4) Analysis of antiserayprepared against
16111 of AW~
Jelly components common to three species of Rana represented
in antiserum against the jelly of R. catesbeiana could be
removed by treating with any of the jellies of Rana (Plate

5, Figures 24, 25, 26, and 27). Other components in the jelly

of.3. catesbeiana were common with components found in R.

 

clamitans, but were not found in 3. pipiens or 3._§ylvatica.

 

Complementary configurations for this component could be

25

removed from the anti-jelly sera of R. catesbeiana by either

of the two former jellies. Jelly of R. sylvatica or R. pipiens

 

would not remove this component. The specific components for

'3. catesbeiana could be removed only by treating with the jelly

 

of R. catesbeiana (Plate 5, Figure 27).

 

(5) Analysis of antisera prepared against_jelly

obtained from another genus of Anura

 

and a genus of another order

 

(Urodela; Ambystoma).

 

 

(a) Anti-jelly serum of Bufo americanus contains

 

configurations against a number of components that are specific
for this species, as well as against components common with

ones found in the jelly material from Bufo marinus. A cross-

 

reaction was also observed between anti-jelly serum of Bufo

americanus and jelly material from all the species of Rana

 

(Plate 9, Figure 41).

(b) Anti-jelly serum of Bufo marinus represents a

 

number of components that are specific for this species, as
well as components in common with ones found in the jelly of

Bufo americanus. No cross-reactions were observed between

 

the anti-jelly serum of Bufo marinus and the jelly from the

 

Species of Rana (Plate 9, Figure 43).

(c) Anti-jelly serum of Ambystoma maculatum represents

 

26

a number of specific components. No cross—reaction was observed
between anti-jelly serum of the Urodele (Ambystoma) and jellies
of the Anurans, Bg§g_and ggfg (Plate 9, Figure 42). However,
the reciprocal combination (Plate 9, Figure 41) did produce
a reaction.

(d) Jelly (fertilizin) of Arbacia punctulata (Echinodermata)
contains a specific component. No cross—reaction was observed
between antiserum against fertilizin of the sea urchin and

jellies of the Urodele or Anuran (Plate 9, Figure 44).

27

Plate 1

Representative Photographs of Ouchterlony Plates and Results

Observed on Eggs after Treatments with Anti-jelly Serum.

Figure 1 Ouchterlony Plate No. 383.

control serum drawn from rabbits prior to the
injection of antigen.

SJ -.5. sylvatica jelly

PJ -_5. pipiens jelly

CJ -.3. clamitans jelly

C

 

 

TaFigure 2 Ouchterlony Plate No. 203.

8A -.R. pipiens anti-jelly serum
-.3. sylvatica jelly

-.R. pipiens jelly
-_R. clamitans jelly

 

WNH

 

Figure 3 Ouchterlony Plate No. 200.

 

 

29A- 5. clamitans anti—jelly serum
‘ l - _R_. pipiens jelly

2 ”.B- clamitans jelly

3 -‘§.<§ylvatica jelly

 

Figure 4 Ouchterlony Plate No. 300.

 

 

SA -‘5. sylvatica anti-jelly serum
1 — 3. _sylvatica jelly

2 - B, pipiens jelly

3 —‘R. clamitans jelly

 

Figure 5 - Eggs treated with rabbit control serum. For ex-
planation see text.

Figure 6 — Eggs treated with anti—jelly serum of R. pipiens
‘ in the same dilution as used for results presented
in Text Figure l & 2, i.e., a dilution of 1:1.
For explanation see text.

28

Plate 1

   

Figure 1 Figure 2

 

Figure 3

   

Figure 5' Figure 6

Diagrams of Ouchterlony Plates. Analysis of species—specific-
ity of antisera prepared against E; pipiens jelly.

used were.R. pipiens jelly (PJ), 3. clamitans jelly (CJ), 3.

29

Plate 2

 

sylvatica jelly (SJ), and R. catesbeiana jelly (FJ).

 

Figure

Figure

Figure

Figure

Figure

Figure

10

11

12

 

Ouchterlony Plate No. 203.

PA - Antiserum prepared against 3. pipiens jelly.

Ouchterlony Plate No. 208.

PA + CJ -_3. pipiens anti-jelly
with R. clamitans jelly.

 

Ouchterlony Plate No. 220.

PA + SJ -.3. pipiens anti—jelly
with 5. sylvatica jelly.

Ouchterlony Plate No. 302.

PA + PJ -.3. pipiens anti—jelly
with g. pipiens jelly.

Ouchterlony Plate No. 335.

PA + FJ -'R. pipiens anti—jelly
with R. catesbeiana jelly.

Ouchterlony Plate No. 375.

PA + AJ “.3- pipiens anti-jelly
with Ambystoma maculatum jelly.

serum

serum

serum

serum

serum

inhibited

inhibited

inhibited

inhibited

treated

Antigens

’\/

 

Figure 7

 

 

—\
%;

Figure 9

 

G

 

 

 

Figure 11

30

Plate 2

 

 

Figure 10

E,
/6\

Figure 12

 

GD

31

Plate 3

Analysis of species-specificity of antisera prepared against
‘3. clamitans jelly. Antigens used were 3. pipiens jelly (PJ),

.3. clamitans jelly (CJ), 5. sylvatica (SJ), and R. catesbeiana

 

jelly (FJ).

Figure 13 Ouchterlony Plate No. 200.

CA - antiserum prepared against 3. clamitans
jelly.

Figure 14 Oucterlony Plate No. 360.

B, clamitans anti-jelly serum inhibited withgg.
pipiens jelly.

 

Figure 15 Ouchterlony Plate No. 207.

CA + SJ -.3. clamitans anti-jelly serum inhibited
with R. sylvatica jelly.

 

Figure 16 Ouchterlony Plate No. 324.

CA + CJ --3. clamitans anti-jelly serum inhibited
with R. clamitans jelly.

Figure 17 Ouchterlony Plate No. 373.

CA + FJ -Ԥ. clamitans anti-jelly serum inhibited
with R. catesbeiana jelly.

 

Figure 18 Ouchterlony Plate No. 360a.

CA + PJ --3. clamitans anti-jelly serum inhibited
with_§, pipiens jelly.

 

 

Figure 13

 

Figure 15

 

Figure 17

32

Plate 3

 

 

360

Figure 14

Figure 16

{a

@1—

//.

Figure 18

33

Plate 4

Analysis of species—specificity of antisera prepared against

_3. sylvatica jelly. Antigens used were 3. pipiens jelly (PJ),

 

3.

Figure

Figure

Figure

Figure

clamitans jelly (CJ), and R. sylvatica jelly (SJ).

 

 

19

20

21

23

Ouchterlony Plate No. 300.

SA — antiserum prepared against 3. sylvatica jelly.

 

Ouchterlony Plate No. 303.

SA + PJ --3. sylvatica anti-jelly serum inhibited
with R. pipiens jelly.

 

Ouchterlony Plate No. 322.

SA + CJ -_3. sylvatica anti-jelly serum inhibited
with 3. sylvatica jelly.

Ouchterlony Plate No. 342.

SA + FJ -.R. sylvatica anti-jelly serum inhibited
with R. catesbeiana jelly.

300

F

Figure 19

igure 21

34

Plate 4

Figure 23

303

Figure 20

Figure 22

35

Plate 5

Analysis of species-specificity of antisera prepared against

‘3. catesbeiana jelly. Antigens used were 3, pipiens jelly

 

(PJ), 5. clamitans jelly (CJ), and R. catesbeiana jelly (FJ).

Figure 24

Figure 25

Figure 26

Figure 27

 

Ouchterlony Plate No. 361.

FA — antiserum prepared against 3. catesbeiana
jelly.

 

Ouchterlony Plate No. 370.

FA + PJ -.3. catesbeiana anti-jelly serum inhibited
with R. pipiens jelly.

 

Ouchterlony Plate No. 371.

FA + CJ -‘5. catesbeiana anti-jelly serum inhibited
with R. clamitans jelly.

 

Ouchterlony Plate No. 369.

FA + FJ -.R. catesbeiana anti-jelly serum inhibited
with R. catesbeiana jelly.

 

36

Plate 5

361 370
0—“ ==

0\

Figure 24 Figure 25

 

Figure 26 Figure 27

37

Plate 6

Analysis of tissue-specificity of antisera prepared against

Figure

Figure

Figure

Figure

Figure

Figure

28

29

30

31

32

33

B, pipiens jelly I.

Ouchterlony Plate No. 285.
1 - Ovary homogenate

2 - _R. pipiens jelly

3 - Oviduct homogenate

4 - Anti-ovary serum

Ouchterlony Plate No. 286.

1 — Ovary homogenate
2 -.R. pipiens jelly
3 - Oviduct homogenate

4 - Anti-oviduct serum

Ouchterlony Plate No. 287.
1 -.§. pipiens jelly

2 — Ovary homogenate

3 - Oviduct homogenate

4 - Anti-ovarian serum inhibited with R. pipiens
jelly

Ouchterlony Plate No. 309.
l - Ovary homogenate

2 - B, pipiens jelly

3 - Oviduct homogenate

4 — Anti—oviduct serum inhibited with R. pipiens
jelly

Ouchterlony Plate No. 310.

l - Ovary homogenate

2 -75, pipiens jelly

3 - Oviduct homogenate

4 - Anti-oviduct serum inhibited with ovary
homogenate

Ouchterlony Plate No. 374.

l - Ovary homogenate

2 -_§. pipiens jelly

3 - Oviduct homogenate

4 -_3. pipiens anti-jelly serum inhibited with

ovary homogenate

38

 

Plate 6
285. 286
00
%\0
Figure 28 Figure .02:\\\
287. .309
—-—
‘ .:\.
Figure 30 Figure 31

 

Figure 32 Figure 33

39

Plate 7

Analysis of tissue-specificity of antisera prepared against

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

R. pipiens jelly II.

Ouchterlony Plate No. 284.

1

2
3 -
4

Ovary homogenate

-.R. pipiens jelly

Oviduct homogenate

-.§. pipiens anti—jelly serum

Ouchterlony Plate No. 386.

'U'U

J—

m x w 3 o
I

R.

A -‘R. pipiens anti-jelly serum

pipiens jelly

Ovary homogenate
Muscle homogenate
Brain homogenate
Kidney homogenate
Heart homogenate

Ouchterlony Plate No. 387.

PA -
FS -
CS -
PS -
PJ -

F’

lewlgulgvl

pipiens anti—jelly serum
catesbeiana serum
clamitans serum

pipiens serum

pipiens jelly

Ouchterlony Plate No. 388.

CA -
FS -
PS -
CS -
CJ -

?°

.5.
.3
3.
_13_.

clamitans anti-jelly serum
catesbeiana serum

. pipiens serum

clamitans serum
clamitans jelly

Ouchterlony Plate No. 389.

Same as Plate No. 387(Figure 36) except 3.
catesbeiana anti-jelly serum and R. catesbeiana
jelly were used.

 

40

Plate 7

386

o
\
on

Figure 34 Figure 35
387
6) a

Figure 36 Figure 37 l
89

.”.

Figure 38

0,
/.

 

 

 

 

388

I 0

 

 

 

 

41

Plate 8

Analysis of tissue-specificity of antisera prepared against

3, pipiens jelly III.

Figure 38 Ouchterlony Plate No. 376.

PA + H —.R, pipens anti-jelly serum inhibited
with heart homogenate.

FJ -'§. catesbeiana jelly

PJ -.5. pipiens jelly

CJ -.3. clamitans jelly

 

 

Figure 39 Ouchterlony Plate No. 378.

- Oviduct homogenate
Heart homogenate
Ovary homogenate
Anti-heart serum

vwaH
II

Figure 40 Ouchterlony Plate No. 377.

Oviduct homogenate

Heart homogenate

Ovary homogenate

Anti-heart serum inhibited with R. pipiens
jelly

waH
II

376
.71: T

T—

/O

 

Figure 38

Figure 40

42

Plate 8

Figure 39

43

Plate 9

Analysis of specificity of antisera prepared against jellies

from another Anuran genus and a genus of another Amphibian

Figure 41

Figure 42

Figure 43

Figure 44

(Urodela).

Ouchterlony Plate No. 346.

BA - Bufo americanus anti-jelly serum
BJ - Bufo americanus jelly
MJ - Bufo marinus jelly

’AJ - Ambystoma maculatum jelly

 

SJ -.3. sylvatica jelly

PJ -.3. pipiens jelly
CJ -.3, clamitans jelly

 

Ouchterlony Plate No. 347.

Same as Plate No. 346 (Figure 41) except
Ambystoma maculatum anti-jelly serum was used.

Ouchterlony Plate No. 382.

Same as Plate No. 346 (Figure 41) except Bufo

marinus anti-jelly serum was used.

Ouchterlony Plate No. 351.

Same as Plate No. 346 (Figure 41) except Arbacia
jelly and Arbacia anti-jelly serum were used.

45

B. The Effect of Antisera on the Fertilization of

Eggs of R. pipiens.

 

When eggs were treated with anti-jelly serum, two effects
were observed: (1) A visible precipitation layer forms around
the outer layer of jelly (Plate 1, Figure 6); the extent of
the jelly precipitation was dependent on the concentration of
antiserum used. (2) Eggs, which normally adhere together in
a mass (Plate 1, Figure 5), became separated.

The results obtained after inseminating eggs that had
been previously treated with antisera in 1:1 dilutions are
presented in Table 1 (summarized in Text Figures 1 and 2).

All together, the eggs of 25 females were treated with anti-
sera pooled from at least three different rabbits. The eggs
from a female were not used more than once in testing each
series of treatments.

Three sets of controls were employed in each series of
treatments. (a) A test of the quality of the eggs by insemi-
nating untreated eggs with normal spermatozoa (Column anp,
Table 1, and Bar anp, Text Figure l). (b) A test of the
effect of treating eggs for two minutes with one-tenth full
strength Holtfreter's solution and washing with a large volume
of the Holtfreter's solution prior to insemination with normal

spermatozoa (Column N, Table l, and Bar N, Text Figure 1).

46

In all subsequent treatments with antisera, eggs were washed
with a large volume of the Holtfreter's solution prior to
insemination to remove any unreacted antibodies. (c) A test
of the effect of treating eggs with control rabbit serum in
the same dilution as antiserum, prior to inseminating with
normal spermatozoa (Column C, Table l, and Bar C, Text Figure 1).

Percentages of cleaving eggs were calculated and these
values were transformed into arcsin equivalents in accordance
with the statistical test employed, i.e., an analysis of
variance. The values obtained were compared by means of a

sequential Q procedure as described in Statistical Methods

 

by G. W. Snedecor, Sections 10 and 11 (see Text Figure 3 for
table of significant differences). Although variations in
reSponse of the eggs of different females were significant,

the statistical design renders this difference irrelevant.
There was no significant difference between the fertilizability
of untreated eggs and those treated with the Holtfreter's
solution or with control serum, i.e., no significant difference
was observed between any of the control treatments (Bar anp,

N, and C, Text Figure 1).

(1) Effect of treating eggs with antisera pre-

 

pared against the jelly of R. pipiens.

 

The fertilizability of eggs after treatment with antiserum

47
prepared against jelly of R. pipiens was lower by a very
significant degree from that of eggs treated with control
serum (Bar PA, Text Figure 1). Therefore, there is a sub-
stance in the anti-jelly serum which significantly inhibits
the fertilization reaction, or some post-fertilization phenomena
leading to cleavage.

In order to determine if the inhibiting substances in
anti-jelly serum of_§. pipiens could be removed by adsorbing
with the homologous jelly material, experiments were performed
in which an equal volume of the jelly material was reacted
with an equal volume of the anti—jelly serum. Adsorption was
continued for 24 hours at 40C. After removal of the jelly
by centrifugation, the antiserum was used to treat eggs in the
same manner as with the control serum and the unadsorbed
antiserum, described above. Bar PA + PJ, Text Figure 1,
represents the values obtained after insemination of eggs,
treated with antiserum against the jelly of R. pipiens
adsorbed with the homologous jelly. It may be concluded that
the inhibitory material in the non-adsorbed anti-jelly serum
is removed or neutralized by the jelly material.

Experiments were also made in which anti—jelly serum of

Br pipiens was adsorbed with the jelly material of R. clamitans

 

and R. sylvatica in the same manner as that described above.

Bars PA + CJ and PA + SJ, Text Figure 1, represent the values

48

obtained after treating eggs with anti-jelly serum of R. pipiens
which had been adsorbed with the jelly material from these
other species. Since no significant differences were observed
between treatments with anti—jelly serum of R. pipiens
adsorbed with these heterologous jelly materials and the
unadsorbed antiserum of R. pipiens, it may be concluded that
the heterologous jelly material did not remove the inhibitory
substance. These results suggest the presence of species-
specific inhibitory substances in anti-jelly serum of_R.
pipiens.

The anti-jelly serum of R. pipiens was also adsorbed with
the jelly material from Ambystoma maculatum, which also failed

to remove the inhibitory material (Bar PA + AJ, Text Figure l).

(2) Effect of treating eggs with antisera_prepared

against the jelly of R. clamitans.

 

 

Bar CA, Text Figure 1 represents the values obtained after
treating eggs of R. pipiens with antiserum against the jelly
of R. clamitans in the same manner as described for treatments
with the anti-jelly serum of R. pipiens. These results are
significantly lower than the values obtained in treatments with
the control serum, which indicates the presence of inhibitory
substances in this heterologous anti-jelly serum. The values

obtained in treatments with anti-serum against the jelly of

49

.3. clamitans were significantly higher than the values obtained

 

in treatments with antiserum against the jelly of R. pipiens;
however, this difference may be explained either by assuming
that the components represented in the anti-jelly serum of B.

clamitans, which are common to this species and to R. pipiens,

 

were less concentrated in the antiserum of R. clamitans than

in the antiserum of R. pipiens, or by assuming that more inhi-
bition is produced by the species-specific components represented
in the anti-jelly serum of 3. pipiens, or by both possibilities.

Adsorption of anti—jelly serum of 3. clamitans was performed

 

with the jelly material of R. clamitans, R. pipiens, and R.

 

sylvatica (Bars CA + CJ, CA + PJ, and CA + SJ, Text Figure 1).

 

Values obtained by treating eggs prior to normal insemination

with antiserum against the jelly of R. clamitans adsorbed with

 

these three jelly materials were not significantly different
from that obtained by treating eggs with control serum. Thus,
the inhibitory substances found in anti-jelly serum of R.

clamitans were removed by treating with the homologous as well

 

as the heterologous jelly material. If the anti-jelly serum

of R. clamitans contains species-specific inhibiting components,
it may be concluded that these components have no effect on

the eggs of R. pipiens. The Ouchterlony plate studies do

indeed indicate that the anti-jelly serum 0f.§- clamitans does

 

contain Species—specific components.

50

(3) Effect of treating eggs with antisera prepared

 

against the jelly of R. sylvatica.

 

 

Bar SA, Text Figure 1, represents the values obtained after
treating eggs of R. pipiens with anti-jelly serum of R.

sylvatica. These values are significantly lower than those

 

obtained by treating eggs with the control serum, and signifi-
cantly higher than obtained after treatment with anti-jelly
serum of_R. pipiens. These results demonstrate that the anti-

jelly serum of R. sylvatica contains inhibitory substances, as

 

did the anti-jelly serum of R. clamitans, but that these

 

inhibitory substances differ qualitatively and/or quantitatively
from those found in the anti-jelly serum of_R. pipiens, since
there is a significant difference between the effect observed

due to treatment with anti-jelly sera of 59 sylvatica and of

 

_R. pipiens.

Adsorption of the anti-jelly serum of R. sylvatica was

 

performed with jelly materials of R. sylvatica, R. pipiens,

 

and R. clamitans (Bars SA + SJ, SA + PJ, and SA + CJ, Text

 

Figure 1). Values obtained by treating eggs with anti-jelly

serum of_R. sylvatica adsorbed with these three jelly materials,

 

were not significantly different from those obtained after
treating eggs with control serum. Thus, the inhibitory sub-

stance found in anti-jelly serum of R. sylvatica was removed

 

.by treating the serum with either the homologous or heterologous

51
jelly materials. Again Ouchterlony plate studies have shown

that anti-jelly serum of R. sylvatica contains species-specific

 

components; however, these components have no effect on the
fertilizability of eggs of_R. pipiens. Also these results,

plus the fact that the values obtained by treatments with

 

anti-jelly sera 0f.§- sylvatica and of R. clamitans of eggs

 

of_§. pipiens do not differ significantly may be interpreted
as indicating that the inhibitory substance of these two anti-
jelly sera are the same. Indeed the Ouchterlony plate studies
have demonstrated that these two anti-jelly sera contain

common components.

(4) Effect of Antisera_prepared against eggijellies

of another Anuran genus (Bufo) and of a

 

Urodele species (Ambystoma).

 

 

Bar AA, Text Figure 1, represents the values obtained by

treating eggs with antiserum against egg-jelly of Ambystoma

 

maculatum. These values are not significantly different from

 

the results obtained by treating eggs with control serum, which

demonstrates that anti-jelly serum of Ambystoma contains no

 

components that are inhibitory to fertilization of eggs of
g, pipiens.
Eggs were also treated with anti-jelly serum of B. pipiens

that had been previously adsorbed with the jelly material of

52

Ambystoma in the same manner as described for the adsorption
with the jelly material of species of_§ana. Results obtained
were not significantly different from those obtained by
treating eggs with unadsorbed anti-jelly serum of R. pipiens
(PA + AJ, Text Figure 1). Thus, none of the inhibiting materiifil
of the anti-jelly serum of R. pipiens was removed by adsorbing
with the jelly material of Ambystoma. In the Ouchterlony plate
studies, no cross—reaction was observed between these two
jelly materials.

Results obtained by treating eggs with anti—jelly serum

of Bufo americanus were significantly lower than the results

 

of treatments with control serum and significantly higher
than the results of treatments with antisera prepared against
jelly of the species 0f.§ifli (Bar BA, Text Figure 1). Cross-
reactions were observed between anti—jelly sera of Bgfg

americanus and jelly material of R. pipiens in the Ouchterlony

plate studies.

(5) Effects of antiseragprepared against fertilizin

of eggs of Arbacia (Echinodermata).

 

Experiments employing anti—jelly serum against fertilizin
of eggs of Arbacia punctulata have indicated that this serum
does not cause any inhibitory effect on eggs of R. pipiens.

Owing to the fact that insufficient numbers of egg samples from

53
different females were tested for statistical analysis, the
effects of antiserum against fertilizin are not included in

Text Figure l.

(6) Effect of treating eggs with antiseragprepared

 

against homogenates of organs of

 

adult_§. pipiens.

 

Bar HA, Text Figure 2, represents the values obtained
after treating eggs with antisera prepared against heart.
These values are significantly lower than those obtained after
treatment with control sera, but higher than after treatment
with anti—jelly sera. However, in Ouchterlony tests, these
antisera do not display any components in common with the
jelly material. Pretreatment of the anti-heart serum with
heart material did not completely remove all of the inhibiting
substances from the serum (Bar PA + H Text Figure 2). However,
pretreatment with the jelly material removed the inhibitory
substances present in anti-heart serum (Bar HA + PJ, Text
Figure 2). The heart material, on the other hand, does not
remove the inhibitory substance from the anti—jelly serum
(Bar PA + H, Text Figure 2).

Experiments employing anti-ovary serum (Bar CA, Text
Figure 2) were performed, with no inhibitory effect being

observed, since these values are not significantly lower than

54
those obtained by treating with control serum. Also, the
ovary material did not remove the inhibitory substance of
the anti-jelly serum (Bar PA + 0, Text Figure 2). Cross—
reactions were not usually observed between the anti-heart

or anti-ovary sera and jelly on the Ouchterlony plates.

55

Legend for symbols used in test figures 1, 2, and 3;

table 1;

treatments of eggs of Rana pipiens prior to insemination.

anp
N
C

PA

PA +
PA +

PA +

PA +
PA +

PA +

CA +

SA

SA +

SA +

SA +

HA
HA +
HA +

CA

CA +
OA +

PJ
CJ

SJ

CJ

PJ

SJ

SJ

PJ

CJ

PJ

PJ

 

untreated eggs

one-tenth full strength Holtfreter's solution

rabbit control serum

anti-jelly
anti-jelly
anit-jelly
clamitans

anti-jelly
sylvatica

anti-jelly
anti-jelly

 

 

serum
serum
serum

serum

serum
serum

of
of
of

of

of
of

Ambystoma maculatum

 

anti-jelly

anti—jelly
anti—jelly
clamitans
anti-jelly
pipiens
anti-jelly
sylvatica

 

 

anti—jelly
anti-jelly
sylvatica
anti-jelly
pipiens
anti-jelly
clamitans

 

serum

serum
serum

serum

serum

serum
serum

serum

serum

of

of
of

of

of

of
of

of

of

anti-jelly serum of

antiserum
antiserum
antiserum
pipiens

antiserum
antiserum
antiserum
pipiens

antiserum

against

heart

lac lw Inc

In:

Isa Isa

law

Is: I3:

Inc

In:

3.
‘5.
3.

3.

pipiens
pipiens
pipiens
pipiens
pipiens
pipiens

pipiens

clamitans

 

clamitans

 

clamitans

 

clamitans

 

sylvatica

 

 

sylvatica

sylvatica

 

sylvatica

 

jelly
jelly

jelly

heart
jelly

ovary

jelly
jelly

jelly

jelly
jelly

jelly

Ambystoma maculatum

 

against
against

against
against
against

against

heart
heart

ovary
ovary
ovary

jelly

homogenate

of
of

of

of

of

of

of

of

of

of

adsorbed with heart
adsorbed with jelly

+ ovary

adsorbed with jelly

of Bufo americanus

 

_3. pipiens
3.

3.

3.

3.

of‘g.
of_3.

56

bU+ bm+ bm+ bm+ bm+ bU+ b£+ bm+b0+ bm+

£m ££ £m £m £m £m £0 £0 £0 £0 £m .flm £m £m £m 0 Z mmcz

 

 

 

 

m.ma
mJln m.m._..

 

 

 

 

 

¢.HN

 

n.mN

 

 

 

 

 

 

 

 

m.om

 

 

 

 

o.mm

m.©m

m.om H.om o.mm ¢.mm

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.poonmEm mummfius£ Eoum sumo MC th® m.mm

 

‘

 

coaumasesuo£ .msmflmflm.msmm mo mmmm mo coaumuflafluuom
so whom maamhusps£ mo uommmm wuouflnfl£sH mo huflUHMHUmmmlmmHmem

a mudbflm uxma

mm

om

m5

OOH

HDVAVHTO INHO 33d

CL EAV A GE

1

.—

PER CEl‘IT

57

Text Figure 2

Tissue-specificity of Inhibitory Effect of Antisera on
Fertilization of Eggs of Rana pipiens.
Accumulation of Data from All
Antisera Employed.*

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100

92.3 92-7

84.1
76.0 77.5
75 69.0
50
41.9
25
10.6
6.7

0 -

anp N 0 PA PA PA HA HA HA OA

+0 +H +H +PJ

*Values for anp, N, C, and PA are the same as on Text
Figure l.

58

Text Figure 3
Table of Significant Differences Between Treatments

of Eggs of Rana pipiens.

 

£m

X

X

X

X

 

bm+£O

 

O+£O

XX X

 

£0

 

hm+£m

 

m+£m

X X

 

X X X X

X X X X

X X X X

X X X X

 

X.X X X X X X

X X X X X

X X X X X XX X

 

Immutam

X)(

p
L

 

bm+£m

X

X X X X

 

bm+£m

 

£m

.X

xxxxxxxxxxx

XDC

 

)6

X

 

hm+£0

X X

X X

 

 

hm+£0

 

bU+£U

xxx )cbcxxxxxfixxx

X X X k X X X X X X X XX X

X X X X X X X X X X

xxx

XXX XXXXXXXXXXX

 

£0

X

X X X

X X.X

X X X X XX X X X X X X X X X

 

O+£m

X

X X

 

b£+£m

)(X X

X}(X

X X X

)(X X X X

 

m+£m

X

)(X X X

X X X X X

 

hm+£m

 

bU+£m

X X X XX X

X‘X X XX X

X X

xngxx
XXXXXX

X X X)(X X

X X X XICX

X

X

X

X)(X X X X

 

bm+£m

X

X

X

X

X

 

£m

X X X X X

XXXXXXXX

X

X

X

X

X

X

X

X

X

 

 

X X X

 

,mmsz

 

XXXXX

X X X X X

Xx

XxxxXxxxxxxxxxxxfi

 

 

anp

 

 

 

PA

 

PA +PJ X X

PAj-CJXXX

PA +SJ X X X

 

 

 

PA+H XX

 

PA +AJ X x X

 

PA +0 X X X

 

CA

 

CA +CJ

 

CA +PJ

 

CA +SJ x x

 

SA

 

SA +SJ

 

SA +PJ

 

SA +CJ

 

 

 

+H

 

 

HA +PJ if X

[(x x xxxxxxxx xxx

 

0A +0 )(

 

0A +PJ L

Lxxxxx X xxxxxx XXXX xx x

Significant differences at 0.05 level.

 

 

BA

 

X:

 

59
Table l

Percentages of Cleavages in Batches of Eggs Treated with Serum

 

Dilutions of 1:1 Prior to Insemination with Normal Spermatozoa

 

 

 

Female PA PA PA PA PA PA CA CA CA SA SA SA HA 8A 0A 0A
No anp N C PA + PJ + CJ +SJ + H + AJ + 0 CA + CJ +‘PJ + SJ + SJ + PJ + CJ AA HA H PJ 0A + O + PJ BA
14 100.0 88.0 85.0 5.7 77.7 10.5 12.7 5.8 19.5 6.0 7.5 75.0 89.3 84.2 8.3 85.7 64.8 75.5 77.5 72.7 88.5 84.2 75.6 86.2 77.7 37.9
16 89.8 96.6 91.4 0.0 93.4 5.7 4.2 13.6 13.6 2.5 7.8 73.8 82.1 77.2 14.2 81.8 71.4 46.1 88.2 64.5 76.9 77.0 84.6 75.6 75.0 27.2
18 97.7 100.0 100.0 16.2 93.6 8.8 4.7 11.6 9.7 15.3 10.0 75.0 96.9 78.5 25.0 100.0 94.1 90.0 87.0 48.1 87.0 93.1 84.6 86.2 96.2 61.9
19 100 0 96.4 94.1 6.0 70.5 2.8 19.5 0.0 7.4 6.6 17.1 100.0 94.5 84.3 27.0 93.5 97.2 86.1 73.8 25.8 85.7 66.6 85.2 96.4 84.3 88.0
20 97.5 100 0 96.0 0.0 86.9 13.3 8.0 4.6 6.5 8.0 23.2 92.1 74.4 73.6 22.2 89.2 94.2 84.0 93.6 22.9 91.8 81.0 70.4 80.0 89.1 67.7
21 76.9 95.3 92.3 5.4 69.2 14.2 8.3 13.7 12.8 3.3 32.1 88.2 89.1 72.9 32.3 91.4 76.1 83.3 70.5 34.6 74.3 62.8 78.9 75.4 67.3 74.0
22 96.0 100.0 72.9 7.3 75.0 5.2 10.7 5.2 6.5 12.1 25.0 77.5 90.0 65.2 28.5 79.0 88.2 82.3 84.8 29.7 55.8 66.6 63.6 85.0 84.4 75.0
23 100 0 84.0 74.3 5.0 75.4 6.5 9.6 23.0 14.8 6.2 10.2 89.0 87.7 94.8 52.0 84.6 93.3 68.7 86.8 39.5 65.1 80.4 85.2 90.4 76.4 64.2
24 100.0 97.7 97.4 2.3 95.3 2.2 18.7 9.5 15.9 12.8 11.9 95.1 100.0 81.4 22.6 90.1 87.8 90.6 97.8 39.6 88.0 93.3 79.1 97.9 85.4 85.1

5 97.9 95.1 96.0 3.6 80.9 6.3 25.5 3.2 22.6 21.6 72.2 90.2 91.6 95.3 33.3 100.0 94.2 86.2 97.9 53.0 92.1 84.2 80.3 96.4 95.6 64.9
26 93.9 96.9 97.7 8.1 87.1 6.0 27.0 13.3 13.3 0.0 16.6 93.7 87.5 82.1 45.4 96.5 96.4 100.0 96.9 44.4 84.8 88.8 76.0 95-8 71.4 66.6
28 96.8 94.7 46.1 0.0 79.1 40.0 15.3 37.5 3.4 0.0 0.0 91.8 75.7 66.6 12.1 80.0 94.9 82.0 62.2 38.2 43.9 71.0 52.6 80.9 74.5 56.4
29 75.9 77.8 64.0 13.3 70.0 3.4 0.0 4,7 0.0 0.0 12-1 65.8 85.0 72.9 7.3 86.5 91.5 76.0 69.4 21.8 16.1 93.1 51.2 76.7 86.8 43.9
31 45.7 73.3 70.5 3.3 68.7 2.9 5.7 O=O 000 5-7 9-1 9093 83.3 67.8 0.0 86.6 84.6 94.2 92.0 42.6 42.0 82.0 80.0 100.0 90.7 78.0
32 100.0 100.0 64.3 0.0 71.7 6.7 7.3 5.7 2.3 0-0 9-1 79-5 76.3 69.2 7.5 91.9 90.6 93.3 78.8 40.0 46.0 84.2 66.6 83.3 86.5 53.6
34 100.0 92.5 77.1 3.1 91.9 33.3 19.0 12.2 9.1 6-8 3109 93-0 97.5 87.8 26.9 91.1 90.2 88.1 90.7 59.2 76.5 86.1 66.6 87.9 90.2 55.9
36 92.4 91.3 95.8 3.8 97.8 18,6 5:3 8o5 1895 1-9 35.5 95-4 91.1 80.7 16.9 92.8 97.6 95.4 83.3 75.7 76.3 69.0 81.2 77.7 82.7 68.4
37 83.8 88.0 81.2 11.1 77.3 22.2 18.6 15.9 22.8 3.0 41-3 lOO-O 90.3 91.8 41.9 82.7 87.5 83.9 77.5 59.1 71.1 65.8 78.7 81.2 71.0 76.0
38 100.0 92.1 77.1 4.4 94.0 36.6 7.6 1,9 23.1 6-3 35-4 98-2 96.0 86.8 33.3 94.1 92.8 95.3 90.6 41.5 82.5 88.3 82.0 76.3 86.0 71.0
39 89.4 100.0 94.7 27.2 94.6 48.7 45.8 10.9 29=4 21.8 26-8 93-3 100.0 86.2 48.0 97.0 94.6 100.0 89.5 32.4 82.8 76.9 97.0 89.5 94.1 73.0
40 97.7 79.4 92.5 6.6 88.6 20.0 17.5 9.7 601 O~0 25-8 93-7 95.2 97.8 21.2 97.3 100.0 97.2 73.3 16.6 57.1 76.7 93.7 74.1 100.0 62.2
41 94.1 97.7 85.3 10.4 88.0 18.7 15.2 15.2 22,9 l4-9 20-4 97-4 87.7 86.9 28.8 91.9 94.1 82.2 93.2 37.1 57.9 75.0 85.7 86.6 90.6 70.8
42 100.0 100.0 89.5 7.3 97.5 18,0 25-6 17:9 21~2 14-5 l2=5 97~7 91.5 87.5 22.2 92.5 97.5 97.8 87.1 38.5 76.3 93.3 82.5 84.3 87.9 60.0
43 87.1 89.4 70.8 0.0 88.4 16.0 14.2 11.7 5.0 5.1 28-2 82-9 84.8 65.1 12.8 92.1 96.9 92.5 88.9 32.2 57.1 87.9 78.8 88.2 96.5 21.9
44 96.2 90.9 97.4 16.6 83.9 17.5 16.7 10.6 6.6 4.8 l3-3 82~0 88.2 76.9 54.2 84.3 95.1 91.7 43.7 40.0 50.0 33.3 77.4 84.4 97.0 83.7

2': 92.3 92.7 84.1 6.7 83.8 15.3 14.5 10.6 12.5 7.1 21.4 88-4 89.0 80.5 25.7 90.1 90.5 86.5 83.0 41.9 69.0 76.0 84.0 85.4 85.5 63.5

 

 

 

 

 

60

C. Effect of Treating Eggs of_3. pipiggg with

 

Fractions of Anti-jelly Sera

 

Experiments employing serum fractionated by the "Rivanol"
method have been made to test the effect of the globulin
fraction on eggs of 3. pipiens. Text Figure 4 represents
the values obtained by treating eggs with the globulin fraction
of serum after separation of albumins by the Rivanol technique.
Eggs from seven females were used in the same manner as that
described for treatments with whole serum. No statistical
analysis was performed on the results; therefore, conclusions
drawn are based strictly on percentages of cleavages. The
results obtained by treating eggs with fractionated control
serum (Bar PCR, Text Figure 4) are considerably higher than
the values obtained with the unfractionated control serum (PC).
Thus, it may be concluded that the difference observed between
fractionated and unfractionated serum is due to albumins
present in the serum.

Results obtained by treating eggs with fractionated anti-
serum prepared against the 3333 jelly materials are presented
in Text Figure 4: PAR = Globulin fraction of anti-jelly serum
of 3. pipiens, CAR = Globulin fraction of anti-jelly serum of

'3. clamitans, and SAR = Globulin fraction of anti-jelly serum

 

of 3. sylvatica. These results indicate that inhibitory

 

61
materials are still present in the globulin fractions of the
anti-jelly sera.

Comparisons of fractionated and unfractionated anti-jelly
sera by agar diffusion studies have demonstrated that some
antibodies are lost during the fractionation procedure.

For this reason Whole serum was used instead of the Rivanol

fractionated serum.

PER CENT CLEAVAGE

anp

PCR

PC
PAR
CAR

SAR

62
Text Figure 4

Results Obtained after Treating Eggs of R. pipiens
100 with Fractionated Serum

94%
90 90%

 

 

80
77%

 

7O
70%

 

6O

50

4O
41%

 

30
31%

 

20
19%

 

10

 

 

 

 

 

 

 

 

 

anp N PCR PC PAR CAR. SAR

- untreated eggs inseminated with normal spermatozoa.

- eggs treated with one-tenth Holtfreter's solution
prior to insemination.

- eggs treated with globulin fraction of control
serum prior to insemination.

- eggs treated with unfractionated control serum prior
to insemination

- eggs treated with globulin fraction of anti-jelly
serum 0f.§- pipiens prior to insemination.

- eggs treated with globulin fraction of anti-jelly
serum of_3. clamitans prior to insemination.

- eggs treated with globulin fraction of anti-jelly
serum of 3. gylvatica prior to insemination.

 

 

63

DISCUSSION

A. Analysis of Antigenic Components Represented in

 

Anti-jelly Sera by Means of Agar-diffusion

 

Technique.

 

Results obtained by the modified agar—diffusion technique
by Ouchterlony have been presented for determining antigenic
components of frog egg—jelly materials. It became apparent
rather quickly that components of the jelly materials were
quite strong antigenically and that the jelly material of each
species of 3333 contained a number of antigenic components.
For a summary of cross—reacting components in jellies of

different species of Amphibia, as determined by serological

 

methods, see Tables 2 and 3, pages 69 and 70.

One of the difficulties encountered with this type of
immunological method is the different responses of individual
rabbits to complex antigen preparations, especially tissue
preparations. However, this differential response of different
rabbits to the jelly antigens is not nearly as great as that
for antigens represented in other tissues of the frog. A
strong titer of antibodies was obtained in each rabbit injected
with egg jelly.

The results of injecting some fifty rabbits with jelly

materials and of running several hundred Ouchterlony plates

64

on the resulting antisera make it appear that each species of
Amphibia tested has a number of components in the egg jelly

which are species-specific. In addition to species-specific

components, it has been demonstrated that the jelly from the

eggs of each of the species of 3333 contains a component that
is common with a component found in the jelly of the eggs of

each of the other species of_§333 tested (3. pipiens, 3.

sylvatica, 3. clamitans, and 3. catesbeiana).

 

 

 

Results have been presented (Table 3, page 70) which indicate
that jelly of eggs of 3. pipiens contains a component in

common with a component found in jelly of eggs of 3. clamitans

 

that is not present in jelly of eggs of 3. sylvatica; jelly of

 

eggs of_3. clamitans contains a component in common with a

 

component found in the jelly of eggs of 5. catesbeiana that is

 

not present in the jelly of either 3. pipiens or g. gylvatica

 

eggs; jelly of 3. clamitans also contains a different component
that is common to one found in the jelly of 3. pipiens which

is not found in the jelly of 3. sylvatica; jelly of 3. sylvatica

 

contains a component that is common with one found in the
jelly of_3. pipiens that is not present in jelly of_3.

clamitans. The fact that components could be demonstrated
to be common between the jellies of two different species
and not present in a third species, does not rule out the

possibility of the difference being quantitative and not

65

qualitative. These components may be present in the jellies
of the other species in such small quantities that they would
not be visible as precipitates on the agar plates.

The jelly of another species of Anuran, Bufo americanus,

 

 

was analyzed and found to contain components in common with
components present in the jelly of each of the species of

Bana. However, the jelly material of Bufo marinus does not

 

contain components in common with the species of Rana but

does contain components in common with Bufo americanus.

 

The exact meaning of the species-specificity of jelly
antigens as determined serologically, in connection with the
biological role of these components in the process of fertili-
zation, is limited to speculation at the present time, as
will be suggested in the next part of the discussion. What
few chemical studies that have been performed on amphibian
egg jellies (Folkes, Grant & Jones, 1950; Minganti, 1955)
have indicated a close chemical similarity between them and
the fertilizin of the sea urchin egg, which is rather well
analyzed. If the jelly material of the frog egg plays a role
in fertilization similar to that postulated for fertilizin of
the sea urchin egg, then this might be the basis for the
rather high degree of specificity of fertilization, in Amphibia.

The specificity of fertilization leads one to believe that
there are specific molecular patterns on the surface of gametes

which interact in order for fertilization to occur. Since this

66
specificity in frogs is not absolute but allows a certain degree
of cross-fertilization between species, it would seem that the
union of gametes of different species would depend on the
degree of similarity between surface layers of gametes of
species which are capable of cross—fertilization. One cannot
assume, however, that the identification of common components
in the jelly-coatings of eggs of different species necessarily
indicates the capability of cross-fertilization between these
species. For example, a crossureaction between the anti_jelly

serum of Bufo americanus and the jelly of 5, pipiens does

 

not necessarily mean that these Anurans are capable of hybridi-
zation. Cross-fertilization tests employing European species
Of.§E£2 and 5333, were done by G. Hertwig (cited by Bataillon
and Tchou-Su, 1929), in which sperm penetration occurred.

It should be pointed out that where cross-fertilization occurs
between the different species of 3333 tested in this experiment,
common components have also been observed between the jellies.
It is quite possible that the spermatozoa of one of two or

more species which have egg—jelly antigens in common would
actually be capable of penetrating the surface of the egg of
the other species without causing rotation or cleavage of the
egg. However, the specificity of fertilization may indeed be
greater than the specificity of serological cross-reactions.

It has been suggested that a relationship might exist between

67

the composition of the jelly and the ecological conditions where
the eggs develop (Moore, 1940; Minganti, 1955). No attempt was
made to compare the jellies of Amphibians of the same species,
from different geographical areas.

In connection with cross-fertilization between the species of
3333, an observation made during this study should be pointed
out here. According to Rugh (1948), Moore (1955) and others,

3. clamitans eggs do not undergo cleavage when inseminated with

 

.3. pipiens spermatozoa. In the spring of 1960, it was noted

that eggs of 3. clamitans inseminated with spermatozoa of 3.

 

pipiens (both obtained from a commercial dealer in Wisconsin)
did undergo cleavage up to the blastula stage. The reason for
the discrepancy between this observation and that made by other
workers is not clear at the moment.

The tissue-specificity of the egg-jelly components of Amphibia
closely parallels the tissue-specificity of fertilizin of the
sea urchin egg, i.e., the jelly components of the species of
.3333 are in general restricted to the oviducal tissue. (For
summary of reactions showing the tissue-specificity of anti-jelly
sera of 3. pipiens see Table 4, page 71.) Results have been
presented in which anti-jelly serum reacted with a component
present in frog blood serum. Unless special attention is given
to the washing of tissues in order to remove all blood, a cross-

reaction is always possible between the anti-jelly serum and the

68
body tissue being tested.

The question arises as to whether the specific and common
components of the jelly visualized by the reaction on the agar
plates with the complementary configuration in the antiserum
against the jelly, are actually different molecules present
in the jelly materials, or different reactive sites on a
single large molecule. Immunochemical research has not pro—
vided much information about size, number, or structure of
substances eliciting the immune response in rabbits (cf.
Tyler, 1957). Since very little is known about the chemical
nature of the jelly, it is not easy to decide whether the
components represent different molecules or merely different
reactive sites on a large complex molecule. However, adsorption
techniques employed in which anti-jelly sera of different
species were adsorbed with heterologous jellies present
evidence which would suggest that more than one molecule is

involved.

69

Table 2 Cross—reactions between Jellies as Determined
by the Ouchterlony Technique.

 

 

Jelly Materials

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

E

.0 53
3 m
m H
5 5 S
(U U Q 4.)
m m c m m m U
C U m E U 5 C
m m .a -H -H s s
C P P Q m H -H m

m -a m m s m H
H E > o O E m m
m H u u m E -H
" U 51 8 "’ o 6 U
._. m
Anti jelly Sera 51 g E fl
ml MI “I m| 4 m m a
3, pipiens + + + + — + — _
._. clamitans + + + + — — - _
‘3 sylvatica + + + + + + — -
‘3. catesbeiana + + + _ + — _ 1 -
Ambystoma maculatum - — — + + _ _ _
Bufo americanus + + + + + + + +
Bufo marinus - - - - _ + + _
Arbacia punctulata - - - — — _ _ +

+ = presence of line (or lines)

in Ouchterlony plates.

- = absence of line in Ouchterlony
plate.

Table 3

70

The Presence of Specific Components in Anti-jelly

Sera as Determined by the Technique of Adsorption.

 

 

Jelly Materials

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

'0!

c

m m m

s u -a

m m -H o

s p u p

m H m m

H E. > 0)

m H u

a H a m

Jellies Used 0 m U

Anti-jelly Sera for adsorption ml m1 3, g,

_3. pipiens _3. pipiens - - — 0

.5. pipiens _3. clamitans + - — —

‘3. pipiens ‘3. sylvatica + + - 0

‘3. pipiens .3. catesbeiana + - 0 -

_3. clamitans .3. clamitans - - - —

.3. clamitans Br pipiens - + - +
.3. clamitans .3. sylvatica + + -

_3. clamitans ‘3. catesbeiana - + - —

.3. sylvatica .3. sylvatica - - - O

.3. sylvatica .3. pipiens - - + -

_3. sylvatica .3. clamitans + - + -

.3. sylvatica .3. catesbeiana - - + 0

.3. catesbeiana .3. catesbeiana - - 0 -

B, catesbeiana .3. pipiens - + 0 +

.3. catesbeiana ‘3. clamitans - - 0 +

+ = presence of lines in the Ouchterlony

plate which

appeared Specific.

- = absence of specific lines in
the Ouchterlony plate.

0 = no test made.

71

 

 

 

 

Table 4 Tissue-specificity of 3. pipieng Jelly Components
as Determined by the Ouchterlony Technique.
Antigens
*6

>. >. :3 .u if a
H H "O 3.4 c: °r-i
. . 3 g 'g m B m
Antisera Adsorption b o 0 £3 M a
Jelly — + +? + - — -
Ovary — - + + + + +
Oviduct - + + + O O 0
Heart - _ + + + + +
Kidney - - + 0 + + +
Jelly Ovary + - + 0 0 0
Jelly Oviduct — - - 0 0 0
Ovary Jelly - + + O 0 0
Ovary Oviduct - + - 0 0 0
Oviduct Jelly - + + 0 0 0
Oviduct Ovary + — + O O 0

+ = presence of lines in Ouchterlony plates.

absence of lines in Ouchterlony plates.

0
N

no test made.

72
B. Analysis of the Effect of Anti-jelly Sera on

Fertilizability of Eggs.

It has been demonstrated that antisera prepared against the
jelly material of each of the species of 3333 has a significant
inhibitory effect on the fertilizability of eggs of 3. pipiens.
However, the adsorption of antiserum, prior to treatment of
the egg, with jelly of heterologous origin, indicates that the
specific components represented in anti—jelly serum of 3.
pipiens decrease the fertilizability of eggs of 3. pipiens,
whereas the specific components of egg-jellies of heterologous
species do not elicit production of inhibitory antibodies.
Consequently, it may be concluded that the inhibition observed
in treating eggs of_3. pipiens with the unadsorbed heterologous
anti—jelly sera was due to components common to all three
species of 3333. Subsequent experiments, employing treatments

of eggs of 3. §y1vatica with adsorbed and unadsorbed homologous

 

and heterologous anti-jelly sera have produced similar results.
Obviously it would be desirable to know specificially

what is being affected by these anti-jelly sera treatments.

As pointed out previously in this paper, Shaver and Barch (1960)

have demonstrated that the antiserum against jelly of 3. pipieng

has an inhibitory effect on the fertilizing capacity of

spermatozoa and the fertilizability of eggs of the homologous

73'
species. Due to the technique employed by these workers, they
were unable to determine whether the inhibition observed,
resulted from an effect on the spermatozodn or egg, or both,
in the treatment of the gametes. In the experiments reported
here, eggs were washed subsequent to the antiserum treatments
to remove any unreacted antiserum. Thus, it may be concluded
that the inhibition observed was due to a block in fertiliza-
tion produced by a reaction between the jelly or the egg and
the anti-jelly serum and not to an effect directly on the
spermatozoa.

An attempt was made to determine if the effect of anti-
jelly serum treatments was on the jelly layers or on the
surface of the egg by injecting anti-jelly serum of 3. pipiens
into the body cavity of female frogs during the process of
ovulation. By this procedure eggs were in contact with the
anti-jelly serum prior to the deposition of the jelly coat.

A total of 24 mls of antiserum was injected into each of six
female frogs. At the completion of ovulation, eggs fertilized
and cultured in the normal way, were observed for cleavage.

No inhibition of fertilization was observed in any of the

eggs tested. Also, if the inhibition is due to a reaction
between the egg or the surface of the egg and the anti-jelly
serum, one would expect anti-ovary serum to have a greater

effect than the anti-jelly serum since the jelly components

74
are restricted to the oviducal tissues. However, no inhibititni
was observed due to the treatments with anti-ovary sera. These
results further implicate a reaction between the jelly and
anti-jelly serum as the blocking mechanism.

In conclusion it would seem that the inhibition observed
may be attributed to a reaction between the jelly and comple—
mentary configurations produced in the globulin fraction of
rabbit serum against antigenic components present in the
jelly-coat material, since treatments with control serum
Show no inhibition. Also the inhibitory effect could be
completely removed by adsorbing the anti—jelly serum with the
homologous antigen, prior to treatment of eggs.

The question arises as to whether the reactions observed
on the Ouchterlony plates actually represent the-inhibitory
substances involved in the egg treatments. The fact that a
close correspondence exists between the precipitation reaction
of the jelly antigens with the complementary configurations in
the antiSerum observed on the agar plates, and inhibitory:
effects observed in egg treatments,indicates that the anti-
body component of the precipitate on the agar plates is
actually responsible for the inhibitory effect. In other
words, where a precipitation was observed between an antiserum
and the egg-jelly of_3. pipiens, an inhibition was also

observed. Another experiment which demonstrates this fact is

75
a case where antisera prepared against the jelly of another

genus of Amphibia (Ambystoma) failed to react with jelly of

 

.3. pipiens, inhibition was not observed. In cases where an
anti-jelly serum prepared against the jelly of another Anuran
(3339) did react with the jelly of 3. pipiens, inhibition was
also observed.

The inhibitory effect observed in treating eggs of 3.
pipiens with antiserum prepared against a homogenate of heart
of the adult frog cannot be explained on this basis since no
cross-reaction was observed between the anti-heart serum and
the jelly on the agar plates. Also, since treatments with
anti-ovary serum fail to inhibit fertilization, it is not
likely that the effect of the anti—heart serum is on the
surface of the egg. A resolution of the uncertainty as to
the location of the reactive sites might be accomplished if
jelly-less, uterine eggs were available, but the technical
difficulties of doing this makes the procedure unfeasible.
Experiments employing antisera prepared against various other
adult tissues of the frog (kidney, brain, testis, and liver)
have Shown that these antisera also result in slight inhibitory
effects. The fact that practically all the inhibitory effect
of the anti-heart serum could be removed by adsorbing the
antiserum with either a heart or jelly homogenate, suggests

that the inhibitory effect may be due to non-precipitating

76
components present in the anti-heart serum, or to a component
common between jelly and blood serum represented in the anti—
serum.

Although the problem of tissue-specificity of the inhibitory
effect is not completely clear at the moment, it is believed!
that a study employing antibodies labeled with fluorescent
dyes would be helpful in demonstrating what reactions account
for the inhibition observed in treatments with antisera
employed in this study and where the reactions occur.

Recent observations of Hathaway and Metz (1961) suggest
the possibility of isotopic labeling of surface components
in order to identify the sites of interaction of complementary

configurations of the spermatozoa and eggs.

77

C. General Considerations and Speculations

 

The purpose of this section is to try to fit obervations
presented here into the broader framework of cellular inter-
actions, from a speculative point of view. Two possible
consequences of reaction systems such as have been demonstrated
in this study might be: (1) a species—isolating mechanism
representing a barrier to fertilization between Species such
as the patterns of antigen-antibody reactions analyzed above;
(2) the origin of tissue—specificity of cells on both phylo-
genetic and ontogenetic levels.

(1). Interacting substances between cells have been shown
to exist by a number of workers. The most thoroughly investi-
gated cells exhibiting such substances are gametes of species
of Echinoderms and Amphibians. It has been shown that these
substances are mucopolysaccharide in nature and that they have
some relationship to the process of fertilization. Workers
have long suspected that the tissue-and species-specificity of
fertilization is due to specific components which are present
on the surface of the gametes. Indirect evidence for this
specificity has come mainly from work on gametes of the
Echinoderms, by studies of the effect of "egg water" on the
spermatozoa. The presence of species-specific components in

egg-jellies of Amphibians has been directly demonstrated in

78
this study, and offers concrete evidence for what has been
postulated to occur in gametes of Echinoderms. These specific
components could serve as the basis for an isolating mechanism
at the time of fertilization. Such a mechanism would be
necessary to prevent hybridization and gene-transfer between

Species whose breeding areas and periods coincide.

(2). Considering the similarity of the chemical make-up
and immunological characteristics of interacting substances
of gametes and of surface components of cells making up tissues,
it is interesting to speculate on the phylogenetic and onto-
genetic origins of tissue-specificity.

For example, a well-known case of Specificity of cellular

types is that observed in Pneumococcus, where genetically

 

different strains exist which are marked by the presence or
absence of a polysaccharide sheath. It is also quite common
to find colonies of certain algae which are enclosed in a
gelatinous matrix which is probably mucopolysaccharide in
nature (for example Volvox and Anaboena). One could speculate
that in the course of evolution, cells of Similar structure
and function became enclosed or bound together by such a
matrix or gelatinous sheath. In this respect, Volvox would
represent a primitive tissue differentiation, inasmuch as
"somatic" cells may be distinguished from "reproductive"

cells. It is very interesting that wherever cells are bound

79
together as a unit, the intercellular matrix is often repre-
sented by a gelatinous or hyaline material.

From an ontogenetic point of view, the first evidence one
sees of cells being bound together byEImatrix (in the Amphibian,
for example) occurs during cleavage of the egg. At cytokinesis
the hyaline layer, beginning with first cleavage, follows the
contours of the daughter cells, so that each cell is enclosed
within this layer. It is interesting to analogize the dividing
egg with a colony of cells bound together by hyaline inter-
cellular matrices. Holtfreter (1943 and 1948) and Lewis (1949)
have emphasized the importance of the surface gel layer (matrix)
in gastrulative movements of cells in the Amphibian and fish
embryos. Grobstein (1955) has suggested that intercellular
matrices may play an important part in induction and that one
way in which these matrices might interact is by molecular
complementariness. The rather high degree of specificity which
governs the aggregative properties of cells (see Holtfreter,
1943 and 1948; Spiegel, 1954a, b, and 1955) suggests a role
for interacting substances in cellular adhesion and the latter
worker suggested that the adhesion of these cells is due to
an antigen-antibody like reaction which is similar to that
postulated to occur in the egg-jellies of the Amphibian.

Thus, one is struck by the fundamental similarity of the

mechanisms which, in development, first insure species-Specifi-

ni+v aha Tn+or are racnnneik1n Fnr +iccna_cnaniFin4+n

80

SUMMARY

1. Antisera were prepared against jelly-coat material of

eggs of several species of Rana (3. pipiens, 3. clamitans,

 

3, sylvatica, and 3, catesbeiana) and other species of Amphibia

 

 

(Bufo americanus, Bufo marinus, and Ambystoma maculatum).

 

 

 

Serological characterizations as to species- and tissue—
specificity of antigenic components found in these jellies
have been presented.

2. Analysis of the jelly material tested showed that the
jellies of each species contain a number of species-specific
components. Common components were observed in the jelly of
Species belonging to the same genus (either_3333 or 3339).

In certain cases common components were observed between

species of different genera (Bufo americanus and each of the

 

species of 3333).

3. Experiments employing antisera prepared against homo—
genates of adult frog organs of 3. pipiens have shown that the
jelly components are restricted in general to the oviducal
tissues, which demonstrates the tissue—specificity of the
jelly components.

4. Results of treating unfertilized eggs of_3. pipiens
with both heterologous and homologous anti-jelly sera, prior
to insemination with normal spermatozoa, have been presented.

Treatments of eggs of_§. pipiens with anti-jelly sera of each

81
of the species of 3333_resulted in a significant decrease in
the fertilizability of the eggs. In cases where a cross-
reaction was observed between the jelly of 3. pipiens and the
anti-jelly sera of a different genus (23:9), inhibition of
fertilization was also observed. No inhibition was observed
in cases where a cross-reaction failed to take place between
the jelly of_3. pipiens and anti-jelly sera of a genus of a

different order (Ambystoma).

 

5. By the technique of adsorption it was demonstrated
that treatments of eggs of 3. pipiens with specific components
represented in the anti-jelly serum of_§. pipiens and with
the common components represented in the anti-jelly serum of
each of the species of 3333, decrease the fertilizability of
the eggs. Treatments with species-specific components repre-

sented in anti-jelly sera of heterologous species (either Rana,

 

.2339, or Ambystoma) did not decrease the fertilizability of the
eggs.

6. Treatments of eggs of 3. pipiens with anti-organ sera
of the adult frog resulted in significant inhibitory effects
in the fertilizability of the eggs. The possibility of this
effect being due to non—precipitating components in the jelly
has been presented.

7. The results of these studies of specificity of anti-

genic components of the jellies are discussed in connection

82
with the possible role of these components in the process
of normal fertilization. Some possibilities concerning
the nature of the inhibitory effect observed in treatment

of eggs with anti—jelly sera are discussed.

83

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