Download the manipulations of magromolecular substances during fertilization

A M . ZOOLOGIST, 3:109-126(1963).
THE MANIPULATIONS OF MAGROMOLECULAR SUBSTANCES
DURING FERTILIZATION AND EARLY DEVELOPMENT
OF ANIMAL EGGS
Ai-BERT TYLER
Division of Biology, California Institute of Technology
Pasadena
In considering the role of various macromolecular substances in the developmental and other physiological processes
of fertilized eggs and embryos, it is customary to think largely in terms of transactions within the cell. Ordinarily, there
is a tendency to assume arbitrarily that
macromolecular substances do not enter
cells except under unusual circumstances,
or in special form such as viruses, transforming DNA, or spermatozoa, or in certain
specialized cells that are termed phagocytes. Where intercellular influences of
large molecular substances, such as the
protein hormones, are known, one is inclined a priori to think of these in terms
of interactions at the surface of the cell
rather than in terms of penetration into
the intimate interior of the cell.
In recent years the general outlook has
altered considerably. This change is largely due to the work and writings of the
late Professor A. M. Schechtman, to whom
this symposium and this paper are dedicated. For example, in experiments with
chickens, he demonstrated that large molecular substances were incorporated in
essentially unaltered form into the growing oocyte. This was basic to the concept, developed since the early experiments of Hevesy and Hahn (1938) that
much of the material of the mature egg
is not manufactured by the growing oocyte
itself but is supplied to it in essentially
"finished" form from other cells (cf Tyler,
1955). Schechtman, also, speculated about
the possible developmental role of such
naturally-supplied substances, and others
The preparation of this paper and the original
experiments of the author reported herein have
been supported by grants from the National Institutes of Health, U.S. Public Health Service (RG6965 and H-3103) and from the National Science
Foundation (GB-28).
that might be introduced artificially,
speculations which, in a sense, bear fruit
in experiments reported in several articles
of this symposium.
Through the activities of Holter (1961)
and his associates and many others, the
incorporation of large molecular substances into various kinds of cells (by
mechanisms such as pinocytosis) and under various conditions, has been amply
documented.
One may, then, consider the role of
macromolecular substances in various developmental processes in terms of both
intercellular and intracellular transactions.
The papers of the present symposium provide a number of examples of the discovery of new embryological phenomena
and their analysis (first-order, or more detailed) in such terms.
I shall present here a brief account of
some pertinent current work of our laboratory. This work deals largely with interactions that are termed immunological in
that they involve (a) immunologically
produced antibodies against various antigenic constituents of eggs and embryos, or
(b) naturally occurring substances whose
interactions are of the "immunologic"
type that depend on mutually complementary molecular configurations. The dis-.
tinction between (a) and (b) essentially
disappears if one accepts the clonal selection concepts of antibody formation proposed by Burnet (1959), Lederberg (1959),
and Talmage (1959). They propose, in
effect, that there is no such thing as an
immunologically produced antibody, in
the sense of a novel substance that the
cell manufactures under instructions, direct or indirect, of the introduced foreign
antigen. Rather, it is thought that antigen
simply stimulates proliferation of those
(109)
110
ALBERT TYLER
cells of an organism that are normally
producing a substance (antibody) capable
of preferential reaction with the antigen.
I shall not attempt to discuss here the
relative merits of this theory, as contrasted
with the instructional (template) types of
theories originally proposed by Breinl and
Haurowitz (1930), Mudd (1932) and Pauling (1940) and more recently developed
by Karush (1961). I wish only to remark
that the clonal selection theories do not
eliminate the assumption of template
mechanisms, but simply restrict these to
the "DNA makes RNA makes Protein"
part of the process.
The current work that I shall report
also deals with another kind of macromolecular substance, now known as Messenger RNA, that has molecular configurations complementary both to correlative
DNA and correlative protein. Many years
ago I (1940, 1946) had demonstrated the
occurrence, within single cells (sea-urchin
eggs), of macromolecular substances capable of interaction with one another in
the manner of antigen with antibody, and
I had adduced from the literature evidence of other reactions that could be
interpreted in the same way (cf Tyler,
1956, 1957). On this basis I had proposed
a "Natural Auto-Antibody Concept" to
express this feature of the interrelations
of the macromolecular constituents of cells
and its relation to growth and differentiation. The concept stated simply that the
macromolecular constituents of cells bore
the same relationship to one another as
did antigen with antibody, and that this
came about by one serving as the basis
for the construction of another, in the
same way as antibody globulin was considered to have a structure regionally complementary to specific structures of the
substances present at the site of synthesis
of the globulin. As expressed at that time
antibody production was considered a special case of the general manner in which
macromolecular substances are manufactured by cells.
The "Natural Auto-Antibody Concept"
was a brief predecessor of what is now
encompassed by "Template Theories," the
development of which is largely due to
Pauling (1948, 1954). In modern terms
it is generally expressed as a unidirectional
influence of DNA on the structure of
(Messenger) RNA which, in turn, specifies
the structure of protein. The Auto-Antibody Concept did not specify restrictions
as to direction nor as to the chemical nature of the specific macromolecular substance that could serve as a template.
SPECIFIC INTERACTING SUBSTANCES OF EGGS
AND SPERM
The union of egg and sperm in the
initial steps of the process of fertilization
has been interpreted (Tyler, 1957, 1959,
1961a, b, 1962a) in terms of interactions
of certain substances carried on and in
the respective gametes. F. R. Lillie (1913)
applied the term fertilizin to a spermagglutinating material obtained from eggs.
Fertilizin and the complementary antifertilizin from sperm are now considered
to represent the specific receptor substances
located in the plasma membranes of egg
and sperm respectively. Fertilizin also extends, in attenuated form, into a gelatinous coat in those species of animals whose
eggs possess such a coat. These substances
interact in antigen-antibody-like manner
and the species-specificity of this interaction, along with the fact that these substances are detectably present only in the
gametes, largely accounts for the speciesand tissue-specificity of fertilization.
In addition to the specific adherence of
sperm to egg by virtue of combination of
fertilizin and antifertilizin of the respective plasma membranes, this interaction
can also lead to incorporation of the sperm
into the egg. A general scheme for this
has been previously presented (Tyler,
1959), and its current version is represented in Fig. 1. The scheme proposes
that, following initial local attachment,
the progressive union of the plasma membranes of egg and sperm draws the sperm
into the egg, by a sort of pinocytotic process, while the fused membranes break
down as they enter into the egg cytoplasm.
FERTILIZATION AND EARLY DEVELOPMENT
The diagrams represent species in which
the entire sperm enters the egg, but requires only slight modification for those
cases in which the tail is left out.
As the diagrams illustrate, there is a
vitelline membrane that overlies the plasma membrane of the egg. However, as
was earlier inferred and is now substantiated by electron miscroscopic observations,
the vitelline membrane is perforated by
microvillous extensions of the plasma
membrane. It is with the fertilizin on
protruding tips of these microvilli that
the sperm is considered to make the first
effective contact that attaches it to the
eggDissolution of the intervening portion
of the vitelline membrane is accomplished
by a membrane-lysin contained in the
acrosome of the sperm. The opening up
of the acrosomal pocket is considered also
to be a result of the stresses set up by
the progressive fusion of the plasma membranes through interaction of the receptors, fertilizin and antifertilizin.
This scheme also offers an explanation
for the establishment of the block to polyspermy by a retraction of other microvilli
after the initial effective contact has been
made with the fertilizing sperm. Also, it
can account for the refertilizability of
mechanically demembranated fertilized
eggs (Tyler, Monroy, and Metz, 1956)
through the persistence of fertilizin on the
plasma membrane. While there may be
some observations that do not seem to be
consistent with the scheme in its present
form, such as the microvilli that remain
protruding through the vitelline membrane in eggs of Mylilus that have evidently been fertilized (Dan, 1962), information on which to base any further substantial modifications is not available at
present.
Besides the three kinds of substances
that have been mentioned there is also
an antifertilizin that occurs within the
egg (Tyler, 1940, 1946). The finding of
such a substance, complementary to the
fertilizin of the surface of the egg suggested the concept of Natural Auto-Antibodies, mentioned above. With respect to
111
processes of fertilization, however, there
are no experiments yet on the basis of
which one might reasonably assign a role
to this material. Possibly it is involved
in the activation process by interaction
with the fertilizin of the plasma membrane
that disappears as it involutes with that
of the sperm. This could possibly account
for the ability of eggs to be activated
mechanically, for example, by puncture
with a glass needle. One may speculate
also on the basis that it may be ribonucleoprotein, but again experimental evidence is lacking.
Experimental intervention with fertilization by manipulations of these substances,
antibody-production against them in rabbits, and their chemical and physical
properties, have been described in earlier
publications (cf Metz, 1961a, b; Tyler,
1961a, b). Some further consideration will
now be given to problems of activation
and later development.
ACTIVATION OF THE EGG
I reviewed the subject of artificial parthenogenesis some time ago (1941) and,
while there have been a number of interesting developments pertaining mainly to
chromosomal aberrations in higher animals (cf Beatty, 1960), there has been no
real advance in our knowledge of the
physico-chemical basis for the process of
activation.
Considerable interest attaches to the
reports of Perlmann (1956, 1957, 1959;
Perlmann and Perlmann, 1957) that unfertilized sea-urchin eggs can be activated
parthenogenetically by means of rabbit
antisera prepared against egg-materials. He
has indicated that the effective antibodies
are directed against a specific "activation
antigen" located in the egg. One can
formulate a number of attractive hypotheses on this basis. For example, such an
"activation antigen" might be an inhibitor
for the ribosomes (see below) and its
neutralization could then permit proteinbiosynthesis and other developmental activities to proceed. Other attempts (Tyler,
1959; Tyler, Seaton, and Signoret, 1961)
to obtain parthenogenetic activation of
ALBERT TYLER
a
i-c
Perivitelline Space
ine Layer
FIG. 1. Diagram of union of sperm and egg based
on fertilizin-antifertilizin interaction and specific
pinocytosis. a, b, c and d represent successive stages.
Outer surface of egg is toward top in each figure.
The reactive chemical groups of antifertilizin (specific receptor substance of sperm) are shown as
knob-shaped structures on the sperm. Those of
fertilizin (specific receptors of egg) are shown as
cup-shaped structures on the plasma membrane
(which protrudes as microvilli through pores in
the vitelline membrane in the unfertilized egg) and
on the micelles of the gelatinous coat (which is an
attenuated extension of the plasma membrane).
These react by virtue of complementary configuration, as in antigen-antibody reactions, and this proceeds in rapid, zipper-like fashion, so that the several microvilli, that make initial contact with the
sperm, extend over the surface of the sperm, drawing the latter into the egg. As this occurs the acrosomal vesicle opens up (assumed to result from
stresses set up by fertilizin-antifertilizin interaction)
and liberates lysin which dissolves the portion of
FERTILIZATION AND EARLY DEVELOPMENT
113
the vitelline membrane enclosed within the microvilli that are attached to the sperm. As the sperm
is, thus drawn into the egg, the fused plasma membranes of sperm and egg break down and disappear
starling at the innermost portion, thus exposing
the sperm nucleus to early interaction with the egg
c) topi asm.
Rapid retraction of the microvilli elsewhere,
along with elevation of the vitelline membrane,
causes the receptors on the plasma membrane to
become inaccessible to additional sperm (block to
polyspermy). Mechanical removal of the vitelline
(fertilization) membrane permits ready refertilization. The granules, that are released from the cortical vesicles in the plasma membiane, serve to
"tan" the inner surface of the vilelline membrane,
to elevate the latter by osmotic action, and, in time,
to help cover up the receptors on the plasma membrane.
sea-urchin eggs by similar and other kinds
of antisera have not been successful.
According to Perlmann's reports, most
batches of eggs are not readily activatable
by treatment with antiserum, and it is
necessary to test many sea-urchins to obtain eggs that respond well. The attempts
to confirm the reports were done on a
large scale so as to take such factors into
account, but obviously one can attribute
the lailures of activation by this method
to unsuitability of either the batches of
eggs, or of the particular species of seaurchins. Also there were undoubtedly differences in the specific conditions of the
experiments. On the other hand the
changes that are reported by Perlmann
are mostly not in the category that one
would clearly designate as a type of parthenogenetic activation; many may be
ordinary cytolytic types of changes. Possibly some represent parthenogenetic activation occurring on occasion in some eggs
in response to a constituent present in the
antiserum—a constituent that may be unrelated to the antibodies against sea-urchin
material. As is well-known, sea-urchin eggs
respond parthenogenetically to a large variety of simple chemical substances and
physical agents. On the whole, then, one
cannot consider the existence of an "activation-antigen" to have been securely
established.
While we find no clear developmental
changes upon treatment of unfertilized
eggs with antisera prepared against eggmaterials, cytolytic effects are commonly
obtained. Such effects can be obtained
with antisera that are prepared against
purified fertilizin (Fig. 2).
There have been two previous reports
of stimulating action on cell proliferation
by specific antisera. One was by Bogomolets (1943) using an antiserum prepared
against reticulo-endothelial cells; this reportedly stimulated cell growth and activity,
and had many remarkable therapeutic
properties when used in low doses. Attempts by Pomerat (1945, 1946, 1949) and
others to confirm some of the claims have
been unsuccessful. The other report was
by Weiss (1947) to the effect that there is
a relative stimulation of growth of the
homologous organ when hens' eggs receive
at an early stage of incubation, rabbit
antisera prepared against adult liver or
kidney. More recently, however, Weiss
(1953, 1955) has indicated that the effect
was more likely due to hemorrhage caused
by vascular damage, rather than to a
stimulation of growth by the antisera.
A report of another kind of stimulating
effect of antibodies is cited and analyzed
by Nace (1955) in his concise review of
investigations of development in the presence of antibodies. This refers to the work
of Asai and Umeda (1929) indicating that
ciliary activity of isolates of pharyngeal
epithelium of Rana esculenta is accelerated by a rabbit antiserum produced
against saline extracts of this tissue. On
examination of this report one would
agree with Nace's conclusion that the effect may be only an apparent case of
stimulation by antibody.
In summary it can be stated that, while
it seems theoretically possible that specific
antibodies might stimulate various cellular processes, directly or by neutralization
of an inhibitor, clear-cut demonstrations
of such effects are lacking.
114
ALBERT TYLER
FIGS. 2-5. Photomicrographs of eggs of Lytechinus pictus. Magnif. 200 x. Unfertilized eggs
placed in rabbit antiserum versus fertilizin (Fig. 2) and in control (pre-injection) serum (Fig. 3)
for 8 hours at 20°C. Fertilized eggs placed in rabbit antiserum versus fertilizin (Fig. 4) and in
control (pre-injection) serum (Fig. 5) at 15 minutes after fertilization for 2 hours at 20°C.
FERTILIZATION AND EARLY DEVELOPMENT
115
later stages corresponding to the extent of
dilution.
Our current concept of the arrangement
Effectiveness of fertilizin-antisera. In con- of the surface layers of the unfertilized
trast to the lack of evidence of cell stimu- and fertilized egg (Fig. 1) provides a basis
lation by antibodies there is an extensive for understanding the effectiveness of ferliterature dealing with cytotoxic effects. tilizin in engendering the formation of
Reference to some of this has been made antibodies that are inhibitory to the ferearlier (Tyler, 1957, 1959; cf Latta, 1959). tilized egg. Fertilizin is no longer thought
Experiments with sea-urchin eggs have to represent simply the material of the
permitted some further analysis. As one gelatinous coat of the egg; nor is it a part
might expect, antisera that are prepared of the vitelline membrane (which elevates
in rabbits against whole egg homogenates as the fertilization membrane). As a comcan block the development of fertilized ponent of the plasma membrane fertilizin
eggs. Somewhat more surprising was the remains available for reaction with specific
finding (Tyler and Brookbank, 1956a. b; antibodies in the fertilized egg. Generally,
Tyler, 1957, 1959) that antisera prepared in these experiments the fertilization memagainst purified fertilizin could also block brane is mechanically removed shortly
development of the fertilized egg, and after it elevates, since this membrane rethat they were generally more effective tards to some extent the access of the
than were antisera prepared against whole antibodies to the plasma membrane. The
egg homogenates, against sperm-materials, presence of fertilizin as a component of
or against various tissues of the adult sea- the plasma membrane of the fertilized
eggs is consistent also with the discovery
urchin.
(Tyler, Monroy, and Metz, 1956) that
A typical example of blocking of cell mechanically demembranated eggs can be
division of fertilized sea-urchin eggs by ex- refertilized.
posure to an antiserum obtained from a
It is, then, no longer surprising that
rabbit immunized with homologous fertiantisera
against fertilizin can block divilizin is shown in Fig. 4. The eggs were
sion
of
the
fertilized eggs. In fact this
placed in the antiserum shortly after fertilization. As illustrated, there were no represents the first instance, in experidistinctive cytolytic changes occurring dur- ments with animal cells, in which such
ing the approximately two hours that the cytotoxic antibodies have been engendered
eggs were in antiserum, while the control with an antigen that is readily prepared
eggs (Fig. 5) had reached the four to eight in highly pure form (electrophoretically
cell stages. Distinct cytolytic changes do, and ultracentrifugally homogeneous, and
however, generally appear after another specifically absorbable by homologous
two hours or so, depending upon the anti- sperm) and has been chemically characserum. Also, it is not necessary for the terized to a large extent (a glycoprotein,
eggs to remain in the antiserum in order the molecules of which are comprised of
for cleavage to be blocked. An exposure two kinds of sugars, some fourteen amino
to the usual antisera, used full strength, acids, and sulphate in approximately equal
for the equivalent of one-fourth of the amounts; and have weights of the order
time of a division cycle (about 15 minutes of 300,000 with axial ratios of 20:1).
in most species of sea-urchins) suffices to
Additional questions of special interest
block subsequent cleavage. When eggs are concern antibodies against other egg-conplaced in dilutions of antisera that permit stituents and the relation of the cleavageinitial cleavage the divisions are delayed, block effect in sea-urchins to other cytoare usually abnormal in form with sepa- toxic systems that have been studied.
ration and frequent disintegration of blasIneffectiveness of antibodies against intomeres, and complete block sets in at ternal constituents of the egg. As noted
INHIBITION OF CLEAVAGE AND DEVELOPMENT
WITH ANTISERA
116
ALBERT TYLER
TABLE 1. Reduction of cleavage-blocking action of anti-egg-homogenate-sera following absorption
with fertilizin-lreated sperm in the sea-urchin Lytechinus pictus.
Experiment
A
B
C
Serum
absorbed with
unabsorbed
control sperm
fertilizin-treated sperm
unabsorbed
control sperm
fertilizin-treated sperm
unabsorbed
control sperm
fertilizin-treated sperm
Percentage of first division at I14 to \\/2 hours after fertilization
in eggs placed iin following dilutiions of antiserum
at 10-15 minutes
2x
4x
8x
16x
32x
64x
128x
25Gx
0
0
5
0
0
0
0
0
2
0
3
50
5
0
15
0
0
15
1
3
80
15
0
50
0
25
80
15
2
80
50
70
80
80
80
80
80
80
80
80
80
80
20
15
90
50
50
95
80
80
95
90
80
95
0
1
65
3
20
75
60
70
85
75
80
90
80
90
90
85
90
90
above, it has been found that antisera
against whole egg homogenates can block
cleavage of sea-urchin eggs. This occurs
also when the antigen has been prepared
from eggs in which the fertilizin of the
gelatinous coat has been removed. However, such preparations are still likely to
contain fertilizin-antigen derived from the
plasma membrane. Possibly, then, cleavageblock by such antisera may be due simply
to the antibodies against fertilizin rather
than to those directed against other constituents of the egg.
This has been examined in some absorption experiments that have been
previously reported in abstract (Tyler,
Seaton, and Signoret, 1961). The results
indicate that antibodies directed against
internal constituents of the egg are not
particularly effective, or probably not at
all effective, in blocking cell division. The
principal experiments consist in absorbing such antisera with fertilizin. For
technical reasons (since fertilizin solutions
gelate at relatively low concentrations and
this limits their use for absorbing antisera) the absorption was done with sperm
that had reacted with fertilizin and were
thus coated with it. Absorption with such
sperm greatly reduces the cleavage-blocking action of anti-egg antisera, whereas
the control, untreated sperm have no such
effect. Table 1 illustrates an experiment
of this type. In most other experiments,
some of which are controlled also by absorption of anti-fertilizin antisera, it ap-
pears that complete absorption of the
fertilizin-antibodies was not effected. While
the use of fertilizin-coated sperm has some
advantages, it too has its limitations since
large quantities must be employed and,
when densely packed and centrifuged,
even in normal sera, may release some
materials that interfere with development
of the eggs.
The most reasonable conclusion from
the present data is that antibodies directed against internal constituents of the
egg are ineffective in blocking development. In fact, it appears that eggs may
develop in the presence of large quantities
of antibodies directed against internal constituents. The antibodies evidently do not
manage to get inside the egg, or the cells
of the embryo, in sea-urchins. This appears to be the general experience with
other kinds of organisms but it is not
usually clear if one is dealing with antibodies directed against surface constituents
or internal constituents. However, the
clearest example probably would be that
of antibodies directed against viruses. It
is well-known (Loffler, Henle, and Henle,
1962) that virus-neutralizing antibodies are
ineffective against virus once it has entered the cell.
Exploration of the extent to which this
general situation may apply to developing
organisms, and of methods for inducing
cells of developing embryos to take in
antibodies, denotes important areas of investigation for the further analysis of de-
117
FERTILIZATION AND EARLY DEVELOPMENT
TABLE 2. Effect of complement on the blocking action of rabbit antisera vs fertilizin of
Lytechinus pictus.
Eggs placed in solutions at 15 minutes after fertilization
Per cent cleaved at 60 minutes after fertilization
Rabbit serum dilution
1/10
1/20
1/40
1/80
Guinea pig serum*
1/5
fresh
heated
fresh
heated
80
80
20
20
80
80
50
50
80
80
Normal rabbit serum (JC 7 A)
fresh
heated
75
75
Antiserum vs fertilizin (JC 7 B)
fresh
heated
0
0
Normal rabbit serum (JC 2 A)
Antiserum vs fertilizin (JC 2 BC)
1/160
80
80
80
80
80
80
80
80
80
80
80
80
80
80
75
80
80
80
80
80
80
80
80
80
15
15
50
50
80
80
80
80
80
80
• Guinea pig serum was reconstituted lyophilized preparation (Hyland Labs) used at a dilution of 1/20; complement titer 1/160 for 50% hemolysis of sensitized 2.5% sheep RBC.
velopmental processes and of the experimental control of development in specific
ways by appropriate manipulations with
these macromolecular reagents. As mentioned in the introduction, the growing
oocyte possesses incorporating-ability, and
as indicated below, the ripe unfertilized
egg may still retain it to some degree. It
is not unreasonable to expect that under
appropriate conditions various cells of the
developing embryo may exhibit this property. Since tissue-specific antigens appear
to be primarily subsurface constituents of
cells, the importance of the solution to the
penetration problem is further evident.
Complement and cleavage-block. In most
cytotoxic systems of most vertebrates the
cytolytic effects have generally been found
to be promoted by, or in many cases to
require, the cooperation of a set of components of the serum, termed "complement" (cf Osier, 1961; Winn, 1962, for
current reviews). In the earlier experiments with sea-urchin eggs it was found
that the heated rabbit antisera were effective in blocking cleavage and in inducing
the subsequent cytolytic changes. This has
been investigated further in this laboratory by Dr. H. Timourian in experiments
that also involved testing effects of unheated and heated guinea-pig serum on
the blocking action of a heated rabbitanti-fertilizin-serum.
Table 2 presents the part of an experiment of this type that contains data for
the effect on the first division. The results
for the quantitative examination of effects
on later development are similar. It is
clear, also, that added complement had
no effect on the titers of the antisera with
respect to these effects.
This does not, however, mean that
chemical components related to "complement" may not be involved in the action
of the rabbit antisera on sea-urchin eggs.
There is, in fact, some indication that the
egg itself may provide a component of
this sort. Some years ago, in an investigation (Tyler, 1942) of the possibility that
the fertilizin-antifertilizin reaction might
fix "complement," it was found that fertilizin itself was anticomplementary, binding C'4 of guinea pig "complement."
Reaction with antifertilizin released the
bound C'4 in quantitative fashion. The
experiments indicated a similarity in some
properties between antifertilizin and C'4.
It is possible, then, that the subsurface
antifertilizin of the egg might, in effect,
be supplying the equivalent of an essential
component of complement.
PROTEIN BIOSYNTHESIS IN SEA URCHIN EGGS
Incorporation of amino acids into protein before and after fertilization. The rate
of incorporation of labelled constituents
into protein by intact eggs of sea urchins
increases considerably upon fertilization.
This was shown originally in the experiments of Hultin (1950) with N1B-labelled
118
ALBERT TYLER
ammonia, and of Hoberman, Metz, and
Graff (1952) with deuterium. Hultin
(1952) also obtained evidence that incorporation of N15 glycine and alanine into
protein increased after fertilization but the
results were obscured by possible changes
in permeability. Nakano and Monroy
(1958) overcame this difficulty by injecting
S35 methionine into the body cavity of the
female, thus preloading the eggs and effectively demonstrating that the increased incorporation upon fertilization was due to
an intrinsic change in activity rather than
to an increase in permeability.
Hultin and Bergstrand (I960) found
that the difference is exhibited also by cellfree amino-acid-incorporating systems prepared from unfertilized and fertilized eggs.
By exchanging cell sap and particulate
fractions of homogenates Hultin (1961)
obtained evidence that the increased activity is largely due to modifications of the
ribosomes. More recently, Nemer (1962),
Tyler (1962b), and Wilt and Hultin
(1962), upon following-up the discovery of
Nirenberg and Matthaei (1961) on the
E. coli cell-free system, reported that polyuridylic acid greatly stimulated incorporation of phenylalanine in homogenates of
sea-urchin eegs. The preparations from
unfertilized eggs responded as well as, or
better than, those from fertilized eggs or
later embryos. From this it would appear
that, in contrast to Hultin's (1961) view
of essentially inert ribosomes that are activated upon fertilization or artificial parthenogenesis, the inactivity of the ribosomes of the unfertilized egg is primarily
attributable to lack of messenger RNA.
A further test of this proposition has
been undertaken in this laboratory, principally by Mr. Paul C. Denny,1 with homogenates prepared from non-nucleated seaurchin eggs before and after artificial parthenogenetic treatment. We have also explored other features of the cell-free preparations from sea-urchin eggs. I have also
initiated some experiments on the effects
of polyribonucleotides on amino-acid incorporation and development of the intact
eggs, In these and certain other experi-
ments on ion-composition of the medium
we have been joined by Dr. Hector Timourian.- A brief account of some of these
experiments is given here.
Stimulation of phenylalanine-incorporation in homogenates by polyuridylic acid.
Methods. The tests were performed with
the sea urchins Lytechinus pictus and
Strongylocentrotus purpuratus. The cellfree suspensions were prepared with a
motor-driven, Potter all-glass, or glass and
teflon, homogenizer. In some experiments
the homogenization was done by sonication. The homogenization- and incubationmedia were modifications of those employed by Nirenberg and Matthaei (1961) and
by Hultin (1961). The TCA-precipitation
and purification of proteins were done as
described by Siekevitz (1952), with additional solution in N/l NaOH, reprecipitations and extraction with hot TCA and
lipid solvents so as to obtain zero-time
(t:l) radioactivity values close to that of
the background.
The final solutions were dried on filter
paper and radioactivity measured in a
scintillation counter with counting efficiencies near 50%. Some experiments were
performed by the method of Mans and
Novelli (1961) in which the incubation
mixtures are placed directly on filter
papers which are then all run through the
various extraction procedures in a single
beaker. Protein determinations were done
by the biuret method of Ellman (1962).
Results. Table 3 shows the results
of an experiment in which polyuridylic
(poly U) acid is added to cell-free preparations of unfertilized eggs and of embryos
at the hatching blastula stage (ca. 500
cells) of the sea urchin Lytechinus pictus.
The set of preparations termed Homogennte was made by use of the Potter homogenizer. The other set {Sonicate) was made
by exposure of the egg-suspension (in an
ice-bath) to ultrasonic waves (40 kc, 500watt Cavitator Mark I of Mettler Electronics Corp., Pasadena) for just enough time
to reduce all cells to a suspension of particles. The tests were done in duplicate
as indicated in the table.
119
FERTILIZATION AND EARLY DEVELOPMENT
TABLE 3. Influence of polyuridylic acid on incorporatioti of C"-L-phenylalanine into protein with
homogenates and sonicates of eggs and embryos of Lytechinus pictus.
Counts per minute (minus t0; blanks = 37, 40, 36, 34)
Without poly U
With poly U
Increase
Homogenates of: Unfertilized eggs
Blastulae (just hatching)
Sonicates of:
Unfertilized eggs
Blastulae (just hatching)
53,56 avg55
(to = 50)
274,261 avg268
(to = 73)
34,40 avg37
(to = 79)
123, 124 avg 124
(t« = 67)
987, 916 avg 952
897
903, 891 avg 897
629
849, 888 avg 869
832
1049, 1014 avg 1032
908
Incubation mixtures = 0.225 ml homogenate or sonicate (derived from 1.5 X 105 eggs in 0.01
M tris, 0.01 M MgAc, 0.275 M KC1), 0.025 ml of Reaction Mixture (0.8 ml M/8 PEP; 0.1 ml of
0.0038 M C"-L-phenylalanine at 9.8 curies/mole; 0.1 ml M / 1 0 ATP, with or without poly U at
0.08 molar calculated as uridylic acid).
Both types of preparations gave similar
large increases in the incorporation of
phenylalanine into protein upon the addition of polyuridylic acid. With the homogenate the increase in activity averaged
somewhat less for the blastulae than for
the unfertilized eggs. In the case of the
sonicates the figures for the poly U-induced
increases are more closely alike when the
unfertilized eggs are compared with the
embryos.
An interesting feature of the sonicates,
as indicated in Table 3, is that the endogenous activity of the preparations can be
reduced considerably while the ability of
the ribosomes to respond to poly U remains as high as in the homogenates, or
higher. Unfortunately, the exposures are
not readily reproducible and many preparations have shown considerably reduced
ribosomal activity, which evidently means
that inactivation of ribosomes proceeds
soon after cell destruction by this method.
Four other sets of experiments of this
type have given similar results. The polyuridylic acid-stimulated average increase
in uptake of phenylalanine for the preparations from the fertilized eggs and from
the embryos was within about 20% of the
average for the unfertilized eggs. This differs somewhat from the results reported
by Nemer (1962) who found the poly Ustimulated incorporation of phenylalanine
by preparations from blastulae to be less
than half that obtained with preparations
from unfertilized eggs of Arbncia punctulata.
It seems unlikely that the difference in
results represents species differences. Nemer mentions that absolute levels of response to poly U seem to depend upon
the batch of unfertilized eggs used, but
that the relative decreases in the stimulation, upon fertilization and development,
occur regularly regardless of batch. With
regard to the latter statement it does not
seem reasonable to me to express, as Nemer
does, the poly U-stimulated incorporation
of phenylalanine into protein as a multiple
of the value obtained without poly U and
then to conclude that since this ratio decreases markedly upon fertilization and development, this means that the ability of
the ribosomes to respond decreases correspondingly. It seems to me that the comparisons for this purpose should be simply
with respect to the absolute amount of the
poly U-stimulated increase in incorporation of phenylalanine into protein. On
this basis, as noted above, Nemer's results
still show a lower activity on the part of
blastulae, preparations from which exhibit
about half the activity of those from unfertilized eggs. In our experiments the differences are considerably less and, for the
present, our findings do not demonstrate
any substantial falling-off, with development, in ribosomal activity with respect to
120
ALBERT TYLER
their ability to be stimulated by poly U.
Apart from this, the results of the poly
U experiments on preparations from seaurchin eggs, in the three different laboratories, agree in showing that the unfertilized egg contains a considerable number of
ribosomes capable of responding to poly U.
The data on incorporation of labelled phenylalanine into protein may give a misleading impression of the extent of the
response. Since it is a homopolymeric protein—namely, polyphenylalanine (Nirenberg and Matthaei, 1961—that is presumably formed upon addition of poly U in
the presence of the C14 phenylalanine, this
would be expected to show much greater
radioactivity than the ordinary sea-urchin
egg proteins produced in response to endogenous messenger RNA, in which the labelled phenylalanine would presumably
represent a small fraction of the different
amino acids present in the molecules.
In any case it appears that in preparations from unfertilized eggs, and also from
developing embryos, most, or many of the
ribosomes are "unprogrammed" or can be
"reprogrammed." One could then attribute,
as does Nemer (1962), the increase in activity upon fertilization primarily to an initiation of synthesis of new messenger RNA
and its attachment to the ribosomes, rather
than to a neutralization of some inhibitor
of ihe ribosomes or other alteration in
properties of the ribosomes, as Hultin
(1961) proposed. There is evidence (Gross
and Cousineau, 1963) that new RNA is
produced shortly after fertilization and this
would accord with the above-stated view.
Artificial activation of non-nucleated
fragments. This proposition is being explored further in this laboratory mainly
by Mr. Paul Denny. In these experiments
sea-urchin eggs are separated into nucleated (light) and non-nucleated (heavy)
fragments by centrifugation in tubes containing sucrose-sea-water layers of different
densities. As the eggs elongate and separate into two fragments under the influence
of the centrifugal force, the heavy fragments form a band on the surface ot the
lowermost sucrose-sea-water laver and the
TABLE 4. Incorporation of C'-L-valine into protein
of liomogenates of artificially activated eggs of
Strongylocentrotus purpuratus.
Counts per minute*
Fragment
Untreated
Treated t
Activation
non-nucleated
19
17
98
92
50%
50%
nucleated
19
91
100%
14
(to = 50)
94
100%
* 0.075 ml packed egg-fragments; background =
32 cpm.
t One minute in 5 X 10"3 M butyric acid in sea
water.
light fragments collect on an upper layer.
Generally, when properly performed, the
procedure yields suspensions of the two
kinds of fragments that are negligibly crosscontaminated. The suspensions are then exposed to butyric acid or other parthenogenetic agents, and homogenates prepared
for tests of their ability to incorporate
amino-acid into protein.
At present there are four sets of experiments in which the conditions appeared
satisfactory with respect to absence of extraneous influences and in which the eggs
responded reasonably well to the artificial
activation. Tn all of these the homogenates
prepared from the treated non-nucleated
fragments were more active than those from
the untreated non-nucleated fragments with
respect to the incorporation of labelled
amino acid into protein under the influence of the endogenous messenger RNA.
Data of one of these experiments, performed jointly, is given in Table 4. In this
experiment an approximately five-fold increase in incorporating activity was obtained. Since the radioactivities for the
preparations from the untreated eggs were
low (as is usual) with respect to the t0
values (near background), the data can
only be taken to give an order of magnitude of the response. The data in the table
also show a response to artificial activation
of the same order on the part of the nucleated fragments. However, the percentage
of eggs that responded visibly (by membrane elevation) to the artificial activation
in the case of the norv-nucleated fragments
FERTILIZATION AND EARLY DEVELOPMENT
121
was only about half of that obtained with sibly by release of hydrolytic enzymes of
the nucleated fragments. It would appear the lysosomes.
then that the latter are not as well enPossibly, also, the DNA that has been
dowed as are the activated non-nucleated reported (cf Brachet, 1960) to be present
fragments with one or another of the com- in the cytoplasm of the unfertilized egg
ponents of the protein-synthesizing system. may play a role in the initiation of protein
In other experiments with the sea urchin synthesis. However, there are considerable
Lytechinus pictus, apart from the stimula- differences in the amounts of cytoplasmic
tion resulting from butyric acid treatment DNA that different workers have reported
of the non-nucleated fragments, prepara- to be present, even in one and the same
tions from the latter (untreated) consis- species. Also, there is considerable uncertently showed higher incorporating activity tainty about the nature of the cytoplasmic
than did those from the nucleated frag- DNA and whether it really is a proper
DNA at all. This is, then, an area that
ments.
requires considerable further exploration,
In general, the results of the experiments the results of which should provide inforwith non-nucleated fragments would tend mation of great value not only with respect
to favor Hultin's (1961) view of blocked to the initiation of development but for
ribosomes being responsible for the in- the analysis of subsequent ontogenetic
activity of the unfertilized egg. In another changes.
similar experiment, Hultin (1961) exEffect of polyuridylic acid on amino acid,
plored the possibility of activating the
microsomes from unfertilized eggs in vitro incorporation by intact eggs and embryos.
by various agents. He obtained some ac- Since, as noted at the beginning of this
tivation which was relatively modest com- article, macromolecules of various kinds
pared with that shown by microsomes from have been known to get into cells of varthe fertilized, or parthenogenetically acti- ious types, it seemed worthwhile to examine the possibility that the synthetic
vated, eggs.
There appears, then, to be evidence in polyribonucleotides might enter sea-urchin
favor of both hypotheses that have been eggs at various stages of development. The
advanced to account for the initiation of fact that free nucleic acids of various
active protein synthesis upon fertilization. viruses (both RNA- and DNA-types) can
The poly U experiments support the view infect cells is further encouragement for
that messenger RNA becomes available. this kind of investigation.
The effect of polyuridylic acid was exThe experiments with non-nucleated fragments, and possibly also Hultin's with rib- amined in five sets of experiments, all of
osomal preparations, support the view that which showed a marked stimulation of inan inhibitor of the ribosomes is removed. corporation of phenylalanine into protein
Naturally, one might also formulate com- on the part of the intact unfertilized eggs.
binations of these hypotheses along with In the fertilized eggs and embryos, howother modifications involving changes, ever, poly U was generally somewhat inupon fertilization, in distribution of var- hibitory.
ious ions or other constituents, or in the
The results of one of these sets of exaggregation of the ribosomes that are periments is given in Table 5. The unknown, from experiments on cell-free sys- fertilized eggs had been freed of their gelatems to affect protein biosynthesis. Also, tinous coat by repeated washing. One
one should take into account changes in fourth of the egg suspension was fertilized
protein degradation, since after the initial on the previous evening and allowed to
small supply of free amino acids and pep- develop to the hatching blastula stage,
tides is utilized, the newly synthesized pro- while the remainder was kept in the cold.
teins must be obtained from the partial An aliquot of one-third of the suspension
or complete splitting of yolk proteins, pos- was removed and washed to provide the
122
ALBERT TYLER
u
TABLE 5. Influence of polyuridylic iacid on the incorporation of C -phenylalanine into protein in
whole eggs and embryos of Strongylocentrotus purpuratus.
Unfertilized
eggs
— poly U
+ poly U
605
768
8,485
6,235
(to = 43)
Counts per minute (blanks = 47,44)
De-membranated
2-4-cell stages
2-4-cell stages
20,726
20,584
13,298
17,634
(t. = 38)
20,166
23,110
11,404
15,073
(to = 44)
"Hatching"
blastulae
26,614
25,264
27,197
22,095
(t0 = 3 8 )
Incubated 2 hours at 20°C; suspensions contained in 2 ml ca. 4 X 10* eggs or embryos, 0.18
of C"-L-phenylalanine and 1.67 nig polyuridylic acid.
unfertilized egg samples. The remainder
was fertilized and divided into two equal
suspensions one of which was mechanically
de-membranated (passage through a pipette with a narrow bore at the tip within
two minutes after insemination). Care was
exercised to insure uniform distribution of
eggs among the four different lots and in
the incubation tubes.
Incubation was terminated by addition
of an equal volume of 10% trichloracetic
acid (TCA), and the egg-material was
washed and extracted thoroughly with hot
and cold TCA and lipid solvents, dissolved
in N/l NaOH and re-precipitated as described above. The effectiveness of this
washing procedure is indicated by the fact
that contents of the tubes (t0) that received
TCA at the start of incubation gave radioactivity values that were essentially the
same as the background. The differences
shown by some of the duplicate tubes can
be attributed mainly to losses of material
occurring during the washing procedure.
In the unfertilized eggs of this experiment, poly U caused a marked stimulation
of incorporation of phenylalanine into protein. The difference between duplicates is
relatively small. The other four experiments likewise show a stimulating effect
on the unfertilized eggs, the increase in
rate of incorporation ranging from 3- to
15-fold.
The data for the fertilized eggs and embryos of this experiment show a decrease
which is more marked in the earlier stages
than in the blastulae. The results of the
other experiments accord with this except
for one that showed a 40% increase in eggs
that were exposed at 30 minutes after fertilization.
One of the experiments was run also
with labelled valine (Table 6). As the
data show, in the unfertilized eggs poly U
stimulates incorporation of valine into protein almost to the same extent as it does
that of phenylalanine. In the fertilized egg
the inhibiting effect of poly U is manifested for both ami no acids.
Temporary unavailability of supplies of
poly U has prevented the performance of
many other obvious tests concerning the
mechanism of its action here. If it is acting by getting into the egg then it is not
simply specifying the incorporation into
protein of the single amino acid, phenylalanine, for which it contains the code.
One would have to assume that, in that
situation, it could interact with endogenous nucleotides and other constituents of
the cell so as to form additional messenger
RNA's specifying other amino acids.
On the other hand the poly U might
exert its effect by action on the surface of
the cell, so as, for example, to alter its
permeability to some constituents. The
effect could not, however, be explained as
an alteration of permeability to the phenylalanine and the valine since experiments
with the homogenates have shown that the
activity of preparations from unfertilized
eggs is low even when there is complete
access to the amino acids.
If poly U is operating by an effect on
permeability in these experiments it would
seem more reasonable to consider changes
in various ions known to affect activity of
homogenate systems. As an example of
123
FERTILIZATION AND EARLY DEVELOPMENT
TABLE 6. Influence of polyuridylic acid on the incorporation of amino acids into protein in whole
unfertilized and fertilized eggs of Stiongylocentrotus purpuratus.
Counts per minute
C'-L-phenylalanine
Unfertilized eggs
Fertilized eggs
-polyU
+polyU
1,260
1,008
(to = 48)
(t. = 47)
15,635
29,759
(bk — 39)
(bk = 39)
7,808
7,795
(to = 45)
(to = 44)
14,372
12,131
(bk=35)
(bk = 34)
C"-L-valine
- poly U
-fpoly U
2,240
1,499
8,765
7,921
26,470
25,672
(t0 = 38)
(t0 = 35)
14,896
15,286
(to = 41)
(to = 40)
Incubated li/| hrs at 20°C; fertilized eggs started 15 minutes after insemination. Mixtures
contained in 2 ml 1.1 X 105 eggs; 0.5 fiC of the labeled amino acids and 1.67 mg polyuridylic acid.
this, experiments were performed (mainly
by Dr. H. Timourian) in which the magnesium ion concentration of the medium
is varied (Table 7). As the data show, incorporation of amino acid into protein
falls off sharply as the Mg+ + concentration
is raised. The optimum in this experiment
is less than no added Mg+ + .
In the unfertilized eggs that have been
stimulated by poly U to incorporate amino
acid into protein at a greatly accelerated
rate there are no visible signs of activation
even after several hours in the medium.
These eggs can still be fertilized and can
develop normally to the blastula stage.
Fertilization can take place in the presence
of the added poly U and phenylalanine.
Detailed investigations of effects on later
development have not been made yet, but
preliminary examinations of treated cultures show greater frequencies of abnormal
development starting at the late blastula
stage.
CONCLUDING REMARKS
The present article has given an account
of some experiments dealing with interactions of certain macromolecular substances, both naturally occurring and synthetic, in developmental processes. The
interactions are primarily of the type characterized by complementarity in configuration of regions of the reactant molecules, as
originally exemplified by antigen-antibody
reactions. A general term "alleloplasts"
was proposed some time ago by my col-
league Dr. Sterling Emerson, to designate
such substances as fertilizins and antifertilizins, blood group antigens and antibodies, or other such naturally occurring
systems, as well as the immunologically
produced antibodies and homologous antigens. The DNA-RNA-protein relationship
would fit also into this category.
Primarily interactions of alleloplasts are
characterized by a high degree of specificity and thus provide a basis for the specificity, both with respect to tissues and species, of the biological processes in which
they may be involved. This has been illustrated by the scheme for sperm to egg
attachment, a scheme that involves interaction of the respective receptors, fertilizin
and antifertilizin. The scheme also includes a possible mechanism whereby the
sperm is drawn into the egg by continuation of the same process. However, as yet,
there is no indication that these alleloplasts
TABLE 7. Influence of Mg** on incorporation of C'1L-valine into protein of an homogenate of fertilized
(1 hr) eggs of Strongylocentrotus purpuratus.
^moles of added Mg++
Counts per minute
.0*
0.5
1.0
5.0
10.0
20.0
300, 315
236, 243
214, 230
57, 64
11, 17
8, 15
* Eggs themselves contain 12 micromoles per ml.
Homogenate is 1 vol eggs to 2.3 vols solution. Incubation mixture in millimoles per ml: 0.04 Tris,
0.075 sucrose, 0.23 KC1, 0.01 PEP, 0.001 ATP,
0.OOOO75 C"-L-valine (4.8 C/mole); nineteen amino
acids (0.00005) and 20 micrograms PEP kinase.
124
ALBERT TYLER
are involved in the further processes in the
initiation of development of the egg.
In the present article the action of immunologically produced antibodies on unfertilized and fertilized eggs is discussed
particularly with respect to experiments on
sea-urchins performed in this laboratory
and elsewhere. Reports of activation of
unfertilized eggs by certain antisera, corresponding to a so-called "activation-antigen" have not been substantiated. Instead
cytolytic and inhibitory effects have been
described in experiments with unfertilized
and fertilized eggs. With respect to the
latter, an account has been given of experiments showing that cleavage and development can be blocked by antisera prepared against purified fertilizin but not by
antisera containing only antibodies against
internal constituents of the cell. The limitations that this imposes on attempts to
influence development in specific ways are
discussed, and it is suggested that at present specific antibodies may find their most
effective use as analytical tools for help in
unravelling the components of specific
processes in differentiation.
Finally, experiments on the initiation
of increasingly active protein biosynthesis
in sea-urchin eggs have indicated that the
inertness of the unfertilized egg can be
largely attributed to lack of messenger
RNA for programming the ribosomes, but
that there may also be present a ribosomal
inhibitor. Use of the synthetic polyribonucleotides, as well as the isolation of endogenous messenger RNA from various tissues, now offers ways of exploring the programming of the protein-biosynthetic system in various regions during development. This, combined with the use of
specific antibodies and controlled modifications of development by methods long
known to experimental embryologists,
opens further opportunities for attack on
many of the important problems of development.
The finding of a stimulating action of
polyuridylic acid on intact unfertilized
eggs is too recent to permit much speculation other than to remark that if such
substances are actually able to get into the
cell, as would be one reasonable interpretation of the results presented here, then
this opens up further powerful means for
the analysis of problems of differentiation
and the modification of development in
highly specific ways.
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