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ABSTRACT

THE EFFECT OF ADENOSINE TRIPHOSPFATE ON THE
STIKULATION OF CLEAVAGE
IN THE EGGS OF RANA PIPIENS

 

by Ronald J. Pfohl

A series of experiments were conducted to determine
what effect the injection of adenosine triphosphate (ATP)

into parthenogenetically stimulated eggs of Rana pipiens

 

would have on the number which successfully cleaved to the
blastula stage.

In general, the experimental procedure involved the
injection into eggs of approximately 0.2-0.3 lambda quantities
of 1.61 X 10-3 M.ATP solutions in Niu-Twitty or Steinberg
buffer. In some cases the design of the experiments was
such that the injection process effected the activation of
eggs smeared with blood. In other cases the injection was
performed two hours after the eggs had been activated either
by fertilization or by smearing the eggs with blood and
pricking them with a fine glass needle.

The percentages of eggs which cleaved when injected
with ATP were compared with the percentages which cleaved
when injected with only the buffer solution. The results
obtained, though not conclusive, indicate that the ATP
injected into the eggs caused an increase in the numbers
which cleaved as compared with those simply injected with the

buffer medium.

Ronald J. Pfohl

The predominant effect of the ATP seems to stem from
an ability to aid the egg in overcoming the injury it has
sustained in the injection procedure. Utilization of the
high energy moiety of the ATP molecule and its possible sites
of action are discussed with reference to the mode of action

of ATP in the enhancement of cleavage.

THE EFFECT OF ADEIOSIKE TRIPHOSPHATE ON THE
S T IL‘U LAT I ON OF C LENA GE

IN THE EGGS OF RANA PIPIENS

 

By
Ronald J. Pfohl

A TEESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

EASTER OF SCIENCE

Department of Zoology

1962

ACKNOWLEDGEMENTS

The author wishes to express his sincere gratitude
to Dr. John R. Shaver for initiating this investigation and
for his continued confidence, interest, and encouragement
during the course of this study.

Thanks are also due Dr. Philip Clark and Dr. Lester
Wolterink for their constructive criticism in preparing the
final manuscript.

Finally, the author wishes to express his appreciation
to "mac" Henderson for her understanding and frequent

assistance on numerous occasions.

ii

TABLE OF COLTENTS
Page
LIST OF TABLES . . . . . . . . . . . . . iv

LIST OF APPEKDICES . . . . . . . . . . . . v

INTRODUCTION . . . . . . . . . . . . . . l
A. Parthenogenesis, With Special Reference

to the Eggs of Frogs l

B. A Review of Some Aspects of Cell Division 5

l. Sol-gel Transformations; Cell Cortex

in Cleavage 5
2. The Spindle in mitosis 8
3. Energetics of Cell Division ll

LmTERIALs AND METHODS . . . . . . . . . . . 21
RESULTS . . . . . . . . . . . . . . . 26
DISCUSSION . . . . . . . . . . . . . . 57
SUMKARY . . . . . . . . . . . . . . . 45
APPEHDICES . . . . . . . . . . . . . . 47

LITERATURE CITED . . . . . . . . . . . . 51

iii

Table
1A

1B

2A

2B

5A

5B

4A

4B

LIST OF TABLES

Pooled data from frogs III-IX; Activation
simultaneous with injection of Niu-Twitty
buffer (pH 7.0-7.5) or 0.1% ATP solution

(pH 600-605) 0 o o o o o o o o o o

Paired observations; Eggs smeared with
blood and injected with Niu-Twitty buffer

or with ATP; Values given are percentages

of eggs which developed to the blastula
Stage 0 O O O O O O O O O O O O

Pooled data from frogs X and XI; Niu-Twitty
buffer (pH 6.6) or 0.1% ATP solution (pH 6.6)
injected about 2 hairs after activation . .

Paired observations; Pricked blood-smeared
eggs and fertilized eggs injected with
Niu-Twitty buffer or with ATP two hours
after being activated; Values given are
percentages of eggs which developed to the
blastula stage . . . . . . . . . .

Pooled data from frogs XIII and XIV; Acti-
vation simultaneous with injection of
Steinberg's medium (pH 6.85) and 0.1% ATP
solution (pH 6.65) . . . . . . . . .

Paired observations; Eggs smeared with
blood and injected with Steinberg's medium
or with ATP; Values given are percentages of
eggs which developed to the blastula stage .

Pooled data from frogs XII and XIII; Stein-

berg's medium (pH 6.85) or 0.1% ATP solution
(pH 6.65) injected about 2 hours after acti-
vation . . . . . . . . . . . . .

Paired observations; Pricked blood-smeared
eggs and fertilized eggs injected with
Steinberg's medium or with ATP two hours
after being activated; Values given are
percentages of eggs which developed to the
blastula stage . . . . . . . . . .

iv

Page

28

28

50

50

33

53

55

55

Appendix

I

II

III

IV

LIST OF APPENDICES

Page

Data from frogs I-IX; Activation simul-

taneous with the injection of Niu-fwitty

buffer (pH 7.0-7.5) or 0.01, 0.1, or 0.5%

ATP solution (pH 6.0-6.5); Values in the

table indicate the numbers of eggs which

were treated and which cleaved to the

blastula stage in each category . . . . . 47

Data from frogs X and XI; Kiu-Twitty buffer

(pH 6.6) or 0.1% ATP solution (pH 6.6)

injected about 2 hours after activation;

Values in the table indicate the numbers of

eggs which were treated and which cleaved t0

the blastula stage in each category . . . 48

Data from frogs XIII and XIV; Activation
simultaneous with the injection of Stein-

berg's medium (pH 6.85) or 0.1% ATP solution

(pH 6.65); Values in the table indicate the
numbers of eggs which were treated and which
cleaved to the blastula stage in each

category . . . . . . . . . . . . 49

Data from frogs XII and XIII; Steinberg's

medium (pH 6.85) or 0.1% ATP solution (pH 6.65)
injected about 2 hours after activation;

Values in the table indicate the numbers of

eggs which were treated and which cleaved to

the blastula stage in each category . . . 50

INTRODUCTION

The aim of this investigation was to determine what
affect the injection of adenosine triphosphate (ATP) into
parthenogenetically stimulated frog eggs had on the number
which successfully cleaved to the blastula stage.

To familiarize the reader with the experimental material,
a brief discussion of parthenogenesis in the eggs of frogs
will be given. Following this, evidence concerning certain
sol-gel transformations in relation to the cell cortex and
to the spindle in cleavage and mitosis and their associations
with ATP will be discussed. The evidence supporting a role
for ATP in the process of cell division points, with some
reservations, to the high energy moiety of ATP as the probable
factor of functional significance. This review of the

literature serves as the basis for the study undertaken and

_reported on in this paper.

A. Parthenogenesis, With Special Reference 32 the Eggs

‘2: Frogs

 

 

Fertilization, as defined by Lord Rothschild (1956,
p. l), is "the incitement of an egg to development by a
spermatozgon, together with the transmission of male heredi-
tary material to the egg." That the male nucleus is not
always necessary for normal development is shown by the occur-
rence of natural or spontaneous parthenogenesis (i.e. devel-
Opment of the egg without fertilization) in such animals as

the aphids, butterflies, bees, silkworms, and certain of the

-1-

-2-
crustacea (Loeb, 1915, chapter V).

Observations on the occurrence of natural partheno-
genesis were the starting point for investigations upon
artificial parthenogenesis. It soon became apparent that a
great number and diversity of treatments, including puncture,
heat, cold, ultra-violet radiation, acids, bases, isotonic
salt solutions, hyper- and hypotonic solutions, fat solvents,
and some alkaloids, were capable of inciting the eggs of
some species of animals to develop. This array of agents
contrasts strikingly with the high degree of specificity in
fertilization and does not lend support to the idea that
the parthenogenetic agent represents the effective component(s)
of the spermatozBon. A number of theories, reviewed by
Tyler (1941), have been advanced to account for the same
response of the egg to a variety of agents.

The frog egg appears to be exceptional in that it has
been possible to incite it to develop completely only by the
use of one type of procedure, to be described below.

One may regard the initiation of parthenogenetic
development in the frog egg as involving two phases. The
first is an "activation" phase consisting of the separation
of the vitelline membrane with subsequent formation of the
perivitelline space, rotation of orientation, formation of
the grey crescent region, and completion of the second
maturation division. Many of the parthenogenetic agents
which successfully incite other eggs to deve10p are capable

only of stimulating the frog egg to undergo this first phase.

.—:5-

The events observed in this first phase may occasionally be
followed by so-called "abortive" cleavages which in effect
may involve one or two irregular cleavajes or superficial
indications of attempts at cleavage. Such events are not

generally considered to involve bona fide initiation of

 

cleavage, although migration of chromosomes and certain
sol-gel transformations may result.

Successfully inciting a frog egg to undergo further
parthenogenetic development therefore involves a second
phase. Bataillon (1911) used the terms "first factor" and
"second factor" to designate the components necessary to
initiate the two phases. The necessity of a "second factor"
for the initiation of zenuine cleavage in the frog egg was
established by Bataillon in 1912 (cited by Bataillon, 1929).
He showed that complete cleava e in the frog egg could be
accomplished by introducing a cellular element into the
cytoplasm at the time of activation. This may be achieved
simply by pricking the egg ("first factor") with a fine glass
needle in the presence of blood, lymph, or the brei of some
tissues ("second factor").

Bataillon believed that the "second factor" induces
the formation of a normal mitotic figure and must include
material from a nucleated cell. Einsele (1950), however,
demonstrated the cleavage-initiating capacity of supernatant
fluids which were obtained after centrifugation of homogenized
frog tissues and injected into virgin frog eggs. Shaver

(1953) substantiated the work of Einsele and carried the

-4-
analysis beyond that of previous workers by determining
which fractions of the cell were the most active. He accom-
plished this by homogenizing and differentially centrifuging
adult and embryonic frog tissues and injecting the fractions
thus obtained into the eggs. The large granule fraction,
possibly mitochondrial in nature, proved to be the most
effective in initiation of cleavage. When frog eggs were
injected with the large granule fraction obtained from frog
blood cell homogenates, 20.8% of the eggs cleaved to the
blastula stage. A maximum of 44.6% cleavage to blastulae
was obtained when the eggs were injected with the large
granule fraction obtained from the homogenate of early frog
gastrulae. In contrast to the findings of Bataillon (1919),
Shaver (1953) reported that injection of whole frog serum
into the eggs of frogs resulted in 11.7% cleaving to the

blastula stage. Since the "second factor" appears to be

principally concerned with the stimulation of bona fide

 

cleavage, Shaver (1955) preferred to call the active agent
(or agents) the "cleavage initiating substance" (013). The
chemical nature of the 013 in the cytoplasmic granules is
not yet known.

In this investigation, the eggs of frogs were incited
to develop by the usual technique, namely smearing the egg
lightly with frog blood and pricking them with a fine glass
:needle or micropipette. The percentage of eggs smeared with
'blood that one can normally expect to cleave to blastomeres

(of approximately the size attained by embryos in Shumway

-5-
stages 8 or 9) following artificial activation is generally
within the range of S-ZOK. Why only 15% of the eggs parthe-
nogenetically stimulated actually cleave is not well under-

"proper" astral system is

stood. Apparently, formation of a
necessary for segmentation of the egg to occur normally
(Herlant, 1913). Parthenogenesis in the egg of the frog
does not occur spontaneously in nature. It is likely that,
with the present procedures, the proper combination of a
number of factors, such as the condition of the egg and the
precise stimulation applied to it, occurs only in a small
percentage of attempts. Also, injury may be sustained by
certain eggs when punctured. It is remarkable, therefore,
that even 5-20% of the eggs should cleave. Obviously, then,
an increase in the proportion which undergo successful cleavage
may be the consequence of any of a number of possible mecha-
nisms.

On the basis of the evidence presented in the following
review, consideration was given to the possibility that ATP

added to the system might aid the egg in overcoming factors

which often hinder its cleavage.

B. A Review 2: Some Aspects 2: Cell Division

 

 

1. Sol-gel Transformations; Cell Cortex in Cleavage
The gelation of many protoplasmic systems is an endo-
thermic process involving volume increase. Gel systems of
such a nature may be referred to as type II according to the

Freundlich classification (Marsland and Brown, 1942; karsland,

1948).

The interconversion of protOplasm from the sol to

Cf

he gel state or vice verse is thought to play an important

 

part in cell division. Although the structure of protoplasmic
gels and the nature of the alterations thereof in sol-gel
transformations have not been clearly determined, X-ray
diffraction and electron microscopy studies and theoretical
considerations based on the physical properties of certain
gel systems sugrest that the gelation process probably repre-
sents the formation of a 3-dimensional network from fibrillar
units present in the system (Ferry, 1948; Kepac, 1950).
Gelation has been shown to be correlated with furrow
formation in the eggs of certain marine animals and in the

eggs of Rana pipiens (Marsland and Landau, 1954). With an

 

increase in temperature, a greater pressure is required to
block the first cleavage furrow (i.e. a temperature increase
favors gelation while a pressure increase favors solation).
Thus, when the "structural strength" (based on the relative
resistance to displacement of visible granules through the
gel in eggs of marine species) of the cortical gel systems
investigated by Marsland and coworkers falls to a certain
critical level, the furrowing ceases.

A number of theories have been advanced to explain
cleavage. They are critically analyzed by Wolpert (1960).
In the egg of a frog the new cell surface is formed dg'ngvo,
and the only theory directly applicable to this material is

that of Selman and Waddington (1955).

-7-
Using cine-film techniques, local vital staining
and serial sections, Selman and Waddihgton (1955) conclude
that the new unpigmented cortex, by which the daughter
blastomeres remain in contact after cleavage, is first formed
as a sheet of gel (in later stages they describe it as being
a double layer) which grows downward through the cytoplasm
from the animal toward the vegetal surface by a process
involving gelation at its lower edge. They suggest that
1) cortical gel is being synthesized before cleavage as an
additional layer beneath the pigmented cortex already exist-
ing, 2) a change from gel to sol occurs at the inner surface
of the pigmented cortex with subsequent transfer of the
material to the required region during cleavage, and 5) a
change from sol to gel takes place along the lower edge of
the new unpigmented cortex being formed ahead of the furrow.
They also conclude that the gel layer contracts immediately
after its formation, and in this way produces the "dipping
in" of the new furrow and all the observed surface movements.
It must be realized that the above description is
purely morphological. If the sol-gel transformations
described do indeed occur, and if the gelation is an endo-
thermic process involving volume increase, no explanation
is available, to the knowledge of the present author, with
regard to the nature of the energy transformations or
exchanges occurring in the synthesis of the gel and subse-

quent transition to the sol state, followed by gelation in

the furrow region. Does the temperature increase or pressure

-8-
applied to the eggs of 3. pipiens in the experiments of
Marsland and Landau (1954) affect the initial synthesis of

the gel or the later sol to gel transformation in the forma-
tion of the new cell surface? Furthermore, no adequate
description is given of the force vectors likely to be

involved in the contraction postulated by Selman and Waddirgton
(1955). Another process, possibly involving ATP, must be
invoked on the basis that if the gelation process itself
involves a volume increase as postulated, one would expect

the gelation process to result in elongation rather than

contraction of gel structures.

2. The Spindle in Mitosis

The preceding discussion has been concerned principally
with gel-sol transformations in the cell cortex. This pheno-
menon is thought to be of importance also in the establish-
ment of the mitotic apparatus. The initial development by
Hazia and Dan (1952) of a method for isolating the mitotic
apparatus of sea urchins has Opened new fields of investiga-
tion concerning this structure. decently, Nazia and coworkers
(1961) have utilized dithiodiglycol (DTDG) to obtain what are
possibly "native" mitotic apparatuses.

The mitotic apparatus has been defined (mazia and Dan,
1952) as the "ensemble of structures constituting the 'chromatic'
and 'achromatic' figures in the classical descriptions of
mitosis. It includes spindles, asters, centrioles, nuclei
(before breakdown) and chromosomal structures (after break-

<iown of the nuclear membrane)."

-9-

The mitotic apparatus has often been described as a
gel whose microsc0pically visible fibrous elements are
regions where the submicroscopic fibrils are condensed and
oriented (Kazia, 1955). The growth of the spindle or aster
may thus represent the transformation of some substance from
a disordered to an ordered condition. Observations on the
effects of antimitotic agents such as colchicine lead one to
the conclusion that the formation of the mitotic apparatus
is separable into two processes, a polymerization process
giving rise to a shapeless gel and a second process of con-
densing and orienting the elements of the gel into the fiber
system observed in the normal mitotic apparatus (Mazia, 1955).
It was originally thought that the molecules in the mitotic
apparatus were held together in polymers by disulfide
bridges, chemical bonds between sulfur atoms on neighboring
protein molecules (Kazia, 1955). More recent experience
with the isolated mitotic apparatus emphasizes its instabi-
lity and directs attention to weaker interactions that still
involve sulfur-containing groups. Hydrogen bonding has
played a prominent part in the theory of protein-to-pretein
interactions and must inevitably enter into our thinking
about the intermolecular linkages functional in the lateral
bonding which establishes the fibrous pattern of the mitotic
apparatus observable with the light microscope (Gross and
Spindel, 1960; Huggins, 1962). Inoué (1959) observed that
'the birefringence of the spindles in eggs of the annelid

CbmetOpterus increases with temperature in the range of 100

 

-10-
to 40°C. The effect is reversible and leads to the concep-
tion of the spindle as a gel in a temperature-sensitive
equilibrium. A thermodynamic analysis of this equilibrium
gives results which are in agreement with the proposed
lability of the spindle structure and which are consistent
with the idea that the orientation depends on very weak
bonding. That the material comprising the mitotic apparatus
may be a Freundlich type 11 gel is also indicated by the
observations of Pease (1941, 1946) and of marsland and
coworkers (1960) that high pressure causes the solation of
the spindle-aster complex.

he function of the spindle in the movement of chromo-
somes is a question still open to much debate. 0f the
various concepts postulated to account for chromosome move-
ment, the most credible ones are those which involve two
mechanisms. According to Ris (1945), the chromosomal fibers
attach the chromosomes to the proper spindle pole and con-
tract, drawing the chromosomes toward the poles. The spindle
fibers, extending the length of the spindle, apparently hold
the poles of the spindle apart and.elozgate in the second
half of anaphase. The two movements are complementary and
very probably alternate as the "motor" of chromosomal
movement.

In summary, the evidence presented suggests that there
are at least two sol-gel systems in the cell which concern

Us: the cell surface (cortex) and the spindle. The cleavage

Of the frog e00 is thought to involve the process of sol-gel

x) L)

-11-
transformations in the formation of a new cell surface.

The formation of the spindle presumably involves a sol—gel
transformation with subsequent orientation of the amorphous
gel into an ordered fibrous structure. The mechanics of
mitosis, still a matter of much controversy, are thought to
involve a contraction of chromosomal fibers and an elongation

of spindle fibers.

5. Energetics of Cell Division

Having briefly discussed some aspects of the mechanics
involved in cell division, our attention is now turned to
the basic metabolic pattern by which the cell provides energ
(protoplasmic gelations are endothermic) for the formation
of its essential gel structures.

The role of adenosine triphosphate in intermediary
metabolism is the subject of a vast literature. The energy
of ATP may be made available for different processes in the
cell (see Lipmann, 1941, and Lehninger, 1959), and it is the
natural choice as the important metabolite contributing
energy to the sol-gel cycle in cells.

Considerable evidence has been accumulated indicating
that ATP does play a prominent role in the process of gelation
and cell movement. Runnstrom, on the basis of gross obser-
vations and centrifugation experiments (Hunnstrom, 1949;
Runnstrom and Kriszat, 1950a,b) on unfertilized marine eggs,
concludes that the addition of ATP increases the rigidity

of the submicroscopic fabric of the cytoplasm, and suggests

-12-
that this increase is due to a reinforcement of the bonds
between the submicroscopic gel-forming molecular complexes.
mebrane formation, cleavage and further development of
fertilized eggs were improved upon addition of ATP.

As early as 1942, Marsland and Brown suggested that
the greater velocity of gelation and solation reactions
observed in protOplasmic systems as compared to methylcellu-
lose and gelatin systems might be due to the intervention
of an ATPase enzyme system which in itself may be modified
under various conditions of temperature and pressure.

Narsland, Landau, and Zimmerman (Landau, 33 31, 1955;
Marsland, gt_gl, 1955) showed that whenever the gel state
of cortical cytoplasm is "weakened" by lower temperature or
by higher pressure there is a corresponding fall in furrowing
strength (as determined by the amount of pressure or decrease
in temperature required to inhibit furrowing) and that when-
ever the "structural strength" of the cell cortex is forti-
fied by higher temperature, lower pressure, or by ATP added
to the medium about 50 minutes prior to furrowing, the
"furrowing strength" correspondingly increases. Furthermore,
the minimum pressures required to block furrows are distinctly
higher when ATP is added. At atmospheric pressure, the added
.ATP enabled the eggs to complete their furrowing at tempera-
tures ordinarily too low to allow for successful cleavage.
TPheir work indicates that the ATP system plays an important

13016 in the metabolism of the cell which determines when and

“finere gel structures are to be formed. Adenosine monOphos-

-15-
phate, adenosine, or inorganic phosphate, used in place of
ATP, were virtually ineffective. Thus the high energy
moiety of ATP is evidently important in the action observed.
The importance of ATP in cell division is further
emphasized by results obtained from studies of cell models.
Hoffmann-Berling (1960) and Weber (1955) have reviewed evi-
dence, most of it supplied by their own work, in support of
the role played by ATP in the mechanisms involved in active
movement. Similarities in the physical and chemical proper-
ties of extracted contractile proteins from sarcoma cells
and actomyosin were noted. The contraction of actomyosin
and the contraction of cell models prepared from fibroblasts
were shown to be dependent upon similar ionic strengths and
similar concentrations of Hg ions and ATP. Both were shown
to have ATPase activities. Contraction was initiated by ATP
(or TTP) only. AMP, creatine phosphate, other organic
phosphates and inorganic polyphosphates were ineffective,
thus emphasizing again the importance of the high energy
labile phosphate group. If ATP was in supraoptimal concen-
trations or if its hydrolysis was inhibited, elongation of
the models would occur. These workers observed, for example,
that in fibroblast models prepared from cells in early
anaphase and subjected to conditions which activate contractile
nmchanisms but exclude elongation mechanisms, the chromosomes
would move apart, apparently due to contraction of chromo-

somal fibers. In the second portion of anaphase, however,

the spindle bodies actively elongate. Active elongation of

-14-
the spindle bodies has been observed in grasshopper sperma-
tocytes by 361Ar (cited by Hoffmann-Berling, 1960). Spindle
elongation, brought about by the inner portions of the
spindle, is initiated by supraoptimal concentrations of ATP.
Thus ATP is thought to have a dual action on the contractile
protein structures. Elongation apparently involves the
binding but not the splitting of ATP, whereas upon splitting
(by contractile ATPases) the protein complexes contract. It
is of interest here that ATPase has recently been found in
association with the soluble fraction obtained from the
mitotic apparatuses of sea urchin eggs isolated by the DTDG
method (Mazia, Chaffee and Iverson, 1961).

Cytokinesis of the fibroblast models, as reviewed by
Hoffmann-Berling (1960), has likewise been shown to involve
an elongation and contraction under the influence of physio-
logical concentrations of ATP. AMP and inorganic phosphate
were ineffective. It is not clear how the contraction of
cell cortical material of the egg of an amphibian, for example,
might occur in such a way that it would aid in cytokinesis.
As shown by Selman and Waddington (1955), a rounding up of
the cells occurs prior to cleavage. Possibly a contractile
mechanism involving cell cortical material is important in
this phenomenon.

Further support for the importance of ATP in cell
division may be obtained from studies involving various
types of inhibitors.

Kersalyl acid (Salyrgan), a sulfhydryl-blocking

-15-
agent, is known to inhibit ATPase (see Weber, 1955). A
fibroblast model behaves like actomyosin when treated with
Salyrgan. Contraction, initially induced by ATP, is stopped
reversibly by addition of Salyrgan to the medium. Although
it has an inhibitory effect upon a wide variety of metabolic
enzymes in which catalytic activity depends, at least partly,
upon the pattern of -SH radicals in the protein moiety of
their structure, Zimmerman, Landau, and Marsland (Zimmerman,
‘33 El, 1957; Marsland, 1956; Landau, SE 31, 1954) believe
that its most specific effect is on the ATPase system. These
workers observed that treating marine eggs with Salyrgan
delayed and reduced the building up of the cortical gel
structure associated with the first mitosis. The furrowing
potency of the eggs was also lower.

An ther inhibitor which has been widely used is
2,4-dinitrophenol (DNP). This compound has been known for
some time to uncouple phosphorylation from oxidation (Loomis
andLipmann, 1948). Villee and coworkers (1949) reported
that DEP reduces the uptake of P52 and its subsequent incor-
poration into nucleic acids and phosphOproteins in fertilized
Arbacia eggs. These workers as well as a number of others
(MCElroy, 1947) have shown an increase in oxygen consumption
and an inhibition of mitosis upon addition of DNP. The
ability of an inhibitor such as DKP to increase respiration
of an egg above the normal level and simultaneously inhibit
cell division is believed to be due to the diversion of

oxidative energy from phosphOrylative synthesis to the combus-

-15-
tion of carbohydrate reserves (Hughes, 1952). Since biologi-
cal endergonic processes draw the energy they require princi-
pally from ATP, and not from oxidation, it is expected that
inhibition of ATP synthesis will arrest mitotic activity.

Brachet in 1954 (cited in Brachet, 1957, p. 177)
showed that cleavage of amphibian morulae is inhibited by
DTP. Clowes and coworkers (1950) demonstrated that the
concentration of a substituted phenol that will block oxidative
phosphorylation in cell-free particulate systems of Arbacia
will also inhibit cleavage in the intact egg. Barnett (1955)
was able to partially reverse inhibition of cleavage in sea
urchin eggs by addition of ATP, whereas addition of adenylic
acid, inorganic phosphate and perphosphate did not stimulate
division of the inhibited eggs. ATP was capable of completely
reversing cleavage inhibition by cyanide and anaerobiosis.
Kriszat and Runnstrom (1951) also were able to remove a DTP
block to cell division in marine eggs by adding ATP.

It thus seems apparent from studies on the effects of
DNP on cell division that the generation of phosphate bonds
forms a necessary link in the mechanism of cell division.

Sodium azide is known to inhibit transphosphorylation
and ATPase activity (Meyerhof, 1945). Krahl and coworkers
(1941) noted that sodium azide stOpped cell division in sea
urchin eggs. Spiegelman and Moog (cited in Brachet, 1950,

p. 169) found that a trace of azide will stop segmentation

of Rana pipiens eggs. They suggest that this effect is due

 

to the inhibition of an azide-sensitive apyrase. Earth and

-17-
Jaeger (1947) have shown that there exist at least three
apyrase-protein complexes in the frog egg which are capable
of hydrolyzing ATP, and which differ characteristically in
pH optima for activity as well as in degree of thermolability.
When the apyrase activity of the apyrase-protein fraction is
tested at successive stages of development, the enzymes are
found to differ in the periods of development at which they
attain maximum activity. Barth and Jaeger suggest that
apyrase-protein complexes are potential links between
energy-producing and energ -utilizing systems of specific
regions in the developing egg. Shaver, Subtelny, and Wania
(1952) have shown that inhibition of the development of frog
eggs with sodium azide at gastrulation also inhibits the
cleavage initiating capacity of granules isolated from homo-
genates of such blocked embryos.

The action of heparin on the cleavage of eggs presents
some indirect evidence for the role of ATP in cytokinesis.
D. Harding (1949, 1951) and Shaver (1949) have noted that
heparin or heparin-like polysaccharides inhibit to some
extent the activating effect of blood in frog eggs. Cell
division in tissue cultures is also inhibited by heparin
(see D. Harding, 1949). A number of workers cited by C.V.
Harding (1951) have shown the antagonistic effects of heparin
and ATP in the processes involved in fertilization and
cleavaje of marine eggs as well as structural changes in other

protoplasmic systems. The suggestion is made that heparin

might interfere with phosphate metabolism at the cell surface.

-13-

Contrary to the evidence thus far presented, some
workers question whether the ATP added to cell systems is
actually the effective agent. Adenosine triphosphate is a
large, highly polar molecule, and it is questionable whether
it can penetrate into the cell in significant amounts. On
the basis of experiments performed on sea urchin eggs,
Litchfield and Whiteley (1959) conclude that ATP neither
penetrates nor is adsorbed to the surface of fertilized eggs.
Wolpert (1961) recently retracted his original report (1958)
that ATP plays a role in the cleavage of the sea urchin egg.
He attributes his original results to artifact.

The situation may be such, as suggested by Lindberg
(1950) and Runnstrom and Kriszat (1950a), that the energy
from ATP in the surrounding medium is transferred across the
cell surface to the superficial layers of the cytoplasm.

Some indication that ATP may be of importance in cell
division is indirectly obtained from the behavior of mito-
chondria during cell division.

Mitochondria are cytOplasmic granules possessing a
complex and characteristic structure. They contain an array
of enzymes, including, in a specific way, the most important
oxidative enzymes. They are capable, if properly supplied
with soluble cofactors, ions, and substrates, of oxidative
phosphorylations. The mitochondrion is, in essence, a
cellular machine for converting energy liberated by oxida-
tion of the citric acid cycle substrates into the bond energy

of ATP. This appears to be a universal property common to

-19-
all mitochondria, regardless of their source (Green, 1960).

Honroy (1957) believes that activation of mitochondria
is one of the events of the activation of the egg. He gives
evidence for an increased activity of mitochondrial ATPase
as a result of fertilization.

Mitochondria have been observed to aggregate about the
spindle during cellular division (see De Robertis, gt El,

1960, pp. 172, 526-527).

Finally, Shaver (1955) has shown that the large gran-
ule fraction obtained upon differential centrifugation of
homogenates of frog gastrulae is the most active in initiation
of cleavage. This fraction is thought to consist predominant-
ly of mitochondria.

The preceding does not in itself constitute evidence
in support of the role of ATP in cell division or egg acti-
vation, but merely suggests that such a role is in accord
with observations on mitochondria.

This review has been corcerned with some of the evi-
dence supporting a functional role for ATP in the energetics
of cell division and structural changes in the cell in general.
Admittedly, the bulk of the evidence has been concerned with
animal material other than that of amphibians, but the evidence
which has been obtained directly from the eggs of amphibians
seems to this author to warrant the assumption that the
protoplasmic gel of the eggs of amphibians is of a type
similar to that of the cell models and marine eggs so com-

monly studied. Under this assumption, the results obtained

-go_
the present study on the effect of ATP on the initiation

cleavage in parthenogenetically stimulated frog eggs will

presented.

EE‘ERIAIS AND METHODS

Female frogs of the species Rana pipiens, obtained

 

from dealers in Vermont and Wisconsin, were induced to ovu-
late prior to the onset of breeding season by intracoelonic
injections of two or more anterior pituitary bodies from
frogs of the same species (Rugh, 1954). Eggs were extruded
onto clean glass slides. Following the treatment, experi-
mental or control, the slides with eggs were placed in
aerated tap water in finger bowls which were appropriately
labelled.

Since the purpose of this investigation was to deter-
mine the effect of ATP on the cleavage of parthenogenetically
stimulated frog eggs, it was necessary to employ a number of
controls in order to eliminate the possibility of other
factors being responsible for the results obtained. The
following controls were utilized for the eggs from every
frog used: fertilized eggs; unsmeared eggs, pricked; eggs
smeared with blood and pricked; injection of Niu-Twitty or
Steinberg buffer into unsmeared eggs; and injection of buffer
into eggs smeared with blood.

Eggs were inseminated at the start of an experiment
with a sperm suspension prepared by macerating two testes in
approximately 10 cc. of 1/10 full strength Holtfreter's
solution. One or two slides of eggs thus treated served as

a control to give an indication of the viability of the eggs.

Another fertilized control was generally prepared at the

-21-

-22-

termination of a particular experiment in order to deter-
mine whether the viability of the eggs remained at a
desirably high level throughout the experiment. If a ferti-
lized control showed less than 70% cleavage the results in
that experiment were usually discarded.

Throughout all other portions of the experiment parti-
cular care was taken to maintain sperm sterility. In the
unsmeared pricked control, eggs were punctured in the animal
hemisphere with micropipettes similar to those used in the
injections. Care was taken to avoid disturbing the area of
the maturation spindle. If any sperm, blood cells or other
tissue cells or debris were present which might initiate
cleavage, their presence should have been detected by this
control.

The control in which eggs smeared with blood were
punctured gave an indication of the responsiveness of the
eggs to artificial activation. In the instances where high
percentages (15% and 55%) of successful cleavages resulted
from this artificial activation, inspite of the low percentages
(50% and 53%, respectively) obtained in the fertilized controls
(see data from frogs VI and XIV in Appendices I and III),
the results were still retained.

The results of injecting ATP into eggs which had been
smeared with blood were compared with those obtained by
injecting ATP into unsmeared eggs as well as those obtained
by injecting smeared and unsmeared eggs with the buffer

solution. The injection of smeared eggs with buffer was

Iaaacessary since the control in which smeared eggs were
sszimply pricked is inadequate for the purpose of comparison

wm'ith the injected experimentals, due to the fact that injec-

1::ion of a substance into the egg may in itself have some

Cieleterious effect.

The disodium salt of adenosine triphosphate, obtained
ITrom Kutritional Biochemical Corporation (Cleveland, Ohio),
vvas dissolved in Eiu-Twitty solution or Steinberg's medium,
ishe pH of which was adjusted to about 7.0 prior to making
ishe ATP solution (Niu and Twitty, 1955; Steinberg, 1957).
Elm alkaline solutions, ATP is quickly converted to 5-adenylic
aacid and sodium pyrophosghate even in the presence of ice;

‘therefore solutions should not be allowed to become alkaline

:for more than a few seconds. If the pH of the ATP solution

is maintained below 7.4, the refrigerated solution should
'remain stable for about one week (yids Kerck Index of
Chemicals and Drugs, 1960, p. 21). The pH of the prepared
solutions was generally maintained between 6.0-7.0.

The injection apparatus consisted of a Luer Look 10 cc.
syringe fitted with a female Luer Slip adapter for the attach-
:ment of polyethylene tubing (I.D. 0.050" x O.D. 0.048"). The
Inicropipettes, attached to the other end of the polyethylene
'tubing, were hand-drawn from capillary tubing over a micro-
Iburner. The syringe and polyethylene tubing system was filled
vvith distilled water, constituting a simple hydraulic system.

Before the micropipettes were attached to this system

‘they'were filled with the solution to be injected. It was

-24-

found advisable to fill a number of pipettes with the
solutions and store them in vials before beginning the experi-
ment since during the course of making the injections the
pipettes may become clogged or the tips may be broken off.
Undue delay in filling another pipette while in the process

of injecting a slide of eggs caused the eggs to dry exces-
sively, with deleterious results.

Prior to attaching the filled micropipette to the
hydraulic system the plunger was withdrawn slightly so as to
provide an air space between the distilled water in the system
and the solution in the pipette. This air space was main-
tained at all times to avoid dilution and contamination of

he solution with the distilled water.

The amount of solution injected into the egg was
generally of the order of 0.2-0.3 lambda and was always kept
below a volume causing visible swelling of the eggs. Further-
more there was no assurance that all the injected solution
remained in the egg after the pipette was withdrawn.

Following activation many eggs will exhibit a super-
ficial wrinkling effect and some may undergo one or two
irregular cleavages and then cease dividing. To avoid con-
fusing true cell division with these aberrant forms the eggs
were observed at the blastula stage (Shumway stages 8-9) at
which time the blastomeres will give the embryo a character-
istic appearance (more than 64 cells in a total blastula).

Partial blastulae were counted as having cleaved, since the

blastomeres in similar cases have been shown by Frazier (1951)

-25-
tc>'be almost entirely nucleated.
Since the cleavage of the eggs was the only phenomenon

Inader investigation, the development of the embryos beyond

the blastula stage was not recorded.

RESULTS

The following three concentrations of ATP in Niu-
Twitty solution were initially tested: 0.01% (1.61 x 10’4
M); 0.1% (1.61 x 10‘3r); and 0.5% (8.05 x 10‘3m). Different
batches of eggs from each of the frogs III-V were injected
with the three concentrations of ATP. Combining the results
from the eggs of the three fr0gs, the injection of 0.01% ATP
solution into blood-smeared eggs resulted in 6.8% cleavage;
0.1% ATP, 9.6% cleavage; and 0.5% ATP, 7.6% cleavage. The
percentage of cleavages resulting from the control eggs which
were smeared with blood and injected with Niu-Twitty buffer
was 2.1%. The means of the differences between the percen-
tages 0f blood-smeared control eggs which cleaved when
injected simply with buffer and the percentages of blood-
smeared egcs which cleaved when injected with ATP at concen—
trations of 0.1% and 0.5% were significantly different from
0 at the 5% level. The 0.1% solution of ATP was selected
for the remaining injections.

In the first set of experiments the activation of the
eggs was effected at the time of the injection. The results
of injecting a 0.1% solution of ATP into the eggs of frogs
III-IX are shown in Appendix I, and the percentages of
cleavages calculated from the pooled data are shown in Table
1A. For cleavage to be initiated it was necessary to supply
another factor, in this case whole blood, prior to the injec-
tion. ATP by itself was incapable of initiating cleavage.

The 0.6% cleavage obtained in unsmeared eggs injected with

-25-

-27-

buffer, the 0.6% cleavage in unsmeared eggs injected with
ATP and the 0.2% cleavage in unsmeared eggs simply pricked
were probably due to contamination of the eggs with tissue
debris. .

The design of this experiment was such as to show any
difference in the number of cleavages resulting from the two
different treatments, namely, the injection of 1) Niu-Twitty
buffer and 2) ATP into eggs smeared with blood. Since the
eggs of different frogs may show some variation, genetic or
otherwise, and since the eggs from each frog were subjected
to both treatments, it was possible to apply a statistical
test to the mean of the differences between the percentages
of cleavages induced by the injection of buffer and the
percentages induced by the injecticn of ATP into the blood-
smeared eggs of each frog. This approach provides a more
sensitive test than one based on group comparisons in which
case the test is applied to the difference of the means of
the two groups (Snedecor, 1955; Fisher, 1950).

The percentage of cleavages for each group of eggs from
each frog and the differences between the two groups were
calculated from the data in Appendix I and are shown in Table
1B.

The mean difference, 3, is 6.88 and the standard error

of the mean difference, is 2.25. Using these figures we

83’
may calculate "Student's" t value and find t = 5.085. With
6 degrees of freedom, the table value at the 5% level is

2.447. Thus at the 5% level, on the basis of the figures

Table 1A: Pooled data from frogs III-IX; Activation simul—
taneous with injection of Niu-Twitty buffer (pH 7.0—
7.5) or 0.1% ATP solution (pH 5.0-5.5).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

-28-

Treatment
:3 :i s a a s
e P. a: L? L» a»
;: g m ‘° 3 2 Number of eggs Blastulae
g..- a) s: 0’ d' c-r ..
E,“ Q“ a '5' 3° 3° treated Number 23
m B o :3 :3
o. 0 a
m n o o
H m to *t
‘D 2”; z >
a m we re
e m
(D
D;
x i J . . 1.554. . ”1.4117 - 85:1“?
#3.... .1 X-.. x-.- _ - -H--...--.v--._-..--..._..--_ SQ.8_._._,-.___.,-,--__-. l . 1 0.2
x xj» 545 _ 76 15 9
X in; il-,_l_-lmM117§4 4 OLE
”ix“ 3” 986- 50 5.1
xi,“ x_.L..._ 71.5 . 4._ 0.6
.3- -léli l. -_lllgll 119 11-8
Table 1B: Paired observations; Eggs smeared with blood and
injected with Niu—Twitty buffer or with ATP; Values
given are percentages of eggs which developed to the
blastula stage.
, v-1-..q__ll.i.i .__
Frog Treatment Difference
Eggs smeared with blood
[will__ll.mll_.llllln-n_nl.llll_lfl__ll___
Inj. with NT [Inj. with ATP
:111 4.55 l 1.3.55..- l... “zeal.--“ .
V 0.67 10L62 9.95
VI 15.55 ; 15:11 1.56
VII 4155 1 12.90 8.55
VIII _12.94 : 11g20 -l.74
IX 2-19.... _ l 13.65 l_6-_.._.44__

 

-29-
given, we may assert that the difference between the percen-
tage of cleavages resulting from injection of buffer and the
percentage resulting from the injection of ATP into eggs
smeared with blood is significantly different from zero. One
cannot, however, conclude from this significant difference
that the injection of ATP does enhance the initiation of
cleavage, because the pH of the Niu-Twitty buffer used in the
control injections was not the same as the pH of the injected
ATP solutions. In preparing the 0.1% solution, the addition
of ATP to the Niu—Twitty buffer lowered the pH of the latter
as much as 1.5 pH units. Whereas the pH of the injected
buffer was generally 7.0-7.5, the pH of the ATP solution was
6.0-6.5. Maintainino the two solutions at the same pH would
have eliminated or established the possibility that the
acidity of the ATP solution was a factor contributing to the
results obtained.

In the second series of experiments, the Niu-Twitty
buffer and the 0.1% ATP solution were both maintained at
pH 6.6. In this series of experiments, observations were
made on the effect of injecting ATP into the eggs two hours
after they had been activated; in other words, after the
emission of the second polar body and before the first
cleavage division. During the first hour after activation
the egg is apparently in such a sensitive state that any
disturbance, such as puncturing, will stop cleavage. Acti-

vation was accomplished by one of three methods: pricking

Table 2A: Pooled data from frogs X-XI; Niu-Twitty buffer
(pH 6.6) or 0.1% ATP solution (pH 6.6) injected
about 2 hours after activation.

 

 

__.._._- -. - -. T ___ . ._-__.-..-

 

Treatment I

 

 

 

 

 

 

 

l 7 I ““7” ' ”fl—H W— ‘EA 7“ - "“77
‘ "1:1 7 '11 II} I31 I HI H
0 !H 02 on 5 d
a srh to :Q <u- L»
H. _I w O O I
H :o g ,0‘ cr c+ :
H I Q: I I—‘ !-‘° F“ I
N I m g o o o r r.
m 1 it 84 :3 5 Number of eggs Blastulae
Q. '. I
.' I T
g 9; m 8, 9., treated Number 33
I 0 s a A
I a m F3 is
I *3 to
I (D
. I 0»
‘ I
n- ----- I» ~I- ~~«—— - r —— — ——-— #~ -~ ——nL-—
insii_jilfii-- -1 _________ _-._, 1.564 l __i__. .1955_i_i_.av.e_i
x X _ _197 0*;
I I _ I _ i

 

I

I.

I

i

I

I

m
: f 'I-‘
' O

H

~J

 

I
I

 

>4
x><x
'H'
m
'H
m

_i

 

 

I

broil-l ICDfH'

blo'obio

OJ
(1
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IN
rm:
[L
Q

 

 

I I

 

 

 

 

 

 

34>:

>4

L

L

I

I

(\3

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0

HT

(1). ;
CflOCfiCDI—‘l—‘CDO

 

 

 

 

 

 

 

I
I. I
NI

I
I

_IL;LLQ:}239[;I;.W"I‘ ”41:7 ; i;

u.—-v——.44 *4.— »y—-—-.—_——- n-o-w —— ~.

Table 2B: Paired observations; Pricked blood-smeared eggs
and fertilized eggs injected with Niu-Twitty buffer
Or with ATP two hours after being activated; Values
given are percentages of eggs which developed to
the blastula stage.

._ ,_____._-._

'1]

H u

0 I
09 '

Treatment Difference}

 

 

..,. --_-___...____ -. - ___..- __ __ n..- -A... --- - i
Eggs smeared with blood and pricked I
p.._.-..._.‘-.... -__._.-_______.___._..__ . . ___..-___ ___ .. ___..- _- ___-_-- I

inj. with NT Inj. with ATP .

-X 7”}- ‘L"_-ifi.7éflfli 7“ :fiLSd;"'mm"

q}-—

776 Is 3—

x: - 6.47 .___,__.___.__._”_____,_1_61.88..~ 711.411

 

 

 

' "‘1

Eggs fertilized

_ . _4 I

Inj. with NT IfiInj. with ATP '

 

___—— n»-

 

 

 

X "" m IIP01.80:;“_“IMH“M_91.5Bubm“I“. 7"30{4éi_'1
ii; -ii_i 57.95WIHH-IHMHH_67.oeWM_

 

 

 

-30-

-51-
unsmeared eggs; pricking eggs smeared with blood; and
fertilization.

The eggs of frogs X and XI were used in this series,
and the percentages shown in Table 2A were calculated from
a summation of the results shown in Appendix II.

Upon examining the percentages of blastulae shown in
Table 2A, it is apparent that the injections of buffer and
ATP into unsmeared eggs two hours after being activated by
pricking had no appreciable effect. The 1.1% cleavage
obtained upon injection of buffer into unsmeared eggs was
probably due to contamination of the eggs with tissue debris.
The lower percentages of blastulae obtained where fertilized
eggs were injected with buffer and with ATP as compared to
the untreated fertilized control were very likely due to the
injury sustained by the eggs resulting from the injection
process. The percentages of cleavages of eggs from each
frog after each treatment are shown in Table 2B.

Testing the mean of the differences between the percen-
tages of cleavages of fertilized eggs injected with buffer
and of fertilized eggs injected with ATP shows that it is
not significantly different from zero at the 5% level.
Similarly, when testing the mean of the differences between
the percentages of cleaving eggs obtained by injecting buffer
and ATP into eggs smeared with blood and activated by pricking,
it was shown to be not significantly different from zero at
the 5% level.

An insufficient number of animals in this experiment

-52-
does not allow one to make any conclusive statements
concerning the effectiveness of ATP in enhancing the
cleavage initiation process. In the eggs of frog XI there
was an appreciable increase in the percentages of cleavages
when ATP was injected, particularly in the group activated
by pricking. It is quite possible that if the number of
animals had been larger the effect of ATP would have been
statistically significant since the standard error of the
mean would very probably have been considerably reduced.

In the next series of experiments, Steinberg's medium
replaced the Niu-Twitty solution. The Niu-Twitty solution
was not entirely satisfactory as a buffer because upon standing
its pH had a tendency to rise due to the breakdown of the
NaH005 component of the buffer into Na and 0H ions and C02
gas. Steinberg's medium is a solution having the same iso-
tonic salt combination as the Niu-Twitty solution but con-
taining a Tris-H01 buffer in substitution for the phosphate
and bicarbonate buffering system of the Niu-Twitty solution.

‘Groups of eggs from frogs XIII and XIV were treated
in a manner similar to those of frogs III-IX in the first
series of experiments except that the buffer solution used
was Steinberg's. The solutions of ATP prepared were of a
0.1% concentration. The pH of the Steinberg's medium to be
injected as the control was 6.85 while that of the ATP
solution (ATP dissolved in Steinberg's medium) was 6.65.
The results obtained are shown in Tables 5A and 5B. The

values given were compiled and calculated from the data in

Table 5A: Pooled data from frogs XIII and XIV; Activation
simultaneous with injection of Steinberg's medium
(pH 6.85) and 0.1%

 

 

 

 

 

 

 

 

Treatment
m w Diwltd +4 H
CD *3 to 109 :5 1'3
H F“ C? C? L5. L*
d o m im‘ 0 o
H- WI i to 0
H (I) I5:: 0":C'1' ct-
H Q .5 F41t* H
N am ! o ; O O
0 1 s :o :23 3
Q“ :0 ' OJ;
am: ll 0 O
:H 'm :Fs- m
:(D ‘ a} I
iDa'CD: (135:9
i 1 m: 3 !H3
: 012 gD-d
l .0 ! 3
i . Q. '
11_¢_.£ : : i
_sxn 1 g l
.1221}--.- .'. 1
a x ' “-x - 5‘
hi“ _ ix Q. :X i
. . l
-;-_fx ;X_;1-
1212--.- -._',- X-
_WTI. g-X-1i_ __X-

 

5

i
4
I

‘
.——.—.—-———-.~L ———.v—n-- 1W WNF‘n-h- ._ “-_—4—_———_ u... . - —- 1 -
I ‘
|
‘ ; ;
. 1
- .
l V ' ‘
‘ . ‘ A
‘ .
' \

——.—. —.v-_7»

ATP solution

treated

169
102

f'199“fw

454 ---_
116 __, 1
11431___1
_108__"N

Number of eggs

(pH 6.65).

 

 

 

 

Blastulae
Number %
" ' ' 555 1:: 77.2“
_-01111-_ Ow1_
1 _531. 22.4”
_ l _ 0.9“
1 51 18.5“
_1 01-- 0- _
1”,? -l§pl

Table 5B: Paired observations; Eggs smeared with blood and
injected with Steinberg's medium or with ATP; Values
given are percentages of eggs which developed to the
blastula stage.

 

 

 

 

 

Frog Treatment
FIEggs smeared with blood -
Inj. with SM Inj. with ATP
4XIII:;;_‘”"{, # 17.92 . v.17.so_ “11
_KIXWMM-1111_LN 19-05-1m1__m11_6.17 ,1WV1

-55-

 

__.. ‘1

'1

Difference [

“ E
s

_T i
_ _ _____i
_ - 0.12 11‘
J _-12_._es _fl;

 

 

Appendix III.

From Table SB we may calculate a mean difference of
-6.50 and a standard error of the mean difference of 6.58.
From these figures we obtain a "Student's" t value of
-1.019, which with 1 degree of freedom is not significantly
different from zero at the 5% level.

Finally, a series of eggs, smeared or unsmeared with
blood, were injected with Steinberg's medium or ATP solution
(0.1%) about two hours after activation by pricking or ferti-
lization. The pHs of the injection media were the same as
in the preceding series of experiments. The results, compiled
from the data in Appendix IV, are summarized in Tables 4A and
4B.

When comparing the results shown in Table 43 obtained
by injecting the control buffer and ATP into fertilized eggs,
a mean difference of 5.56 and a standard error of the mean
difference of 1.228 are calculated. These figures yield a
"Student's" t value of 2.756. A mean difference of 0.595
and a standard error of the mean difference of 2.185 are
calculated from the results obtained by injecting the control
buffer and ATP into the eggs two hours after they had been
smeared with blood and pricked. These figures yield a
"Student's" t value of 0.272. With 1 degree of freedom
neither of the mean differences obtained are significantly
different from zero at the 5% level.

The inadequate sample size again does not allow one

to make definite assertions concerning the effectiveness of

Table 4A: Pooled data from frogs XII ant XIII; Steinberg's
medium (pH 6.85) or 0.1% ATP solution (pH 6.65)
injected about 2 hours after activation.

.-._._._.__-_ . ._ . _____-.-___.__. - ”"“"Y"

 

Treatment

 

7—...._ r— —.o-1 .

m
m

p9ZIIIQJ9d

d)

PGHOTJd

 

Number of eggs g Blastulae

%_H_

treated ‘ Number

DGJBGQSUH 8

 

we JO UOIQOQFUI
div JO UOIqerUI

)
1
I
I
i
1
E

 

 

peaeems-pootq 8223

 

CR

1

‘1-” _-’__ " -_ -_ .. ‘-_---i _.--_._w__.-- __.-_.._-._ ___--- - _-1--.____ _
* “I , L 171.- _ . 51-11..
l-Weeo 170-_
1. 187... - ,-_H_- 1. -_M
1-L_111____112571 _-_H -‘1__16___
- xi 255 U“_ 1,-11501
- x _ 196 1” 0
X

 

__‘r .

l
l

 

----.Wv _ .—. _- “h... -—__..__.‘_. — _-__ _.

- (nu:
QOQGOQOOU'

 

 

 

 

I
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' ()1

 

 

1

——1"_

 

__.___--~_-_4 . ._...._

.11248- _ "_m_j_ 19

 

 

 

 

 

[<1 040301030)

[0

Table 4B: Paired observations; Pricked blood-smeared eggs
and fertilized eggs injected with Steinberg's medium
or with ATP two hairs after being activated; Values
given are percentages of eggs which developed to the
blastula stage.

9‘ . _ _.. . _ __ __ _, __._

Frog Treatment Difference

 

___“- __.---_-__.-.____._______-. ___. -. -_- ._ __-_.__ ___—__T

 

 

 

Eggs smeared with blood and pricked

 

 

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-55-

ATP in enhancing cleavage.

-56-

DISCUSSION

A number of experiments, the results of which have
been presented in the preceding section, have been performed
in an attempt to ascertain what effect, if any, the intro—
duction of ATP into unfertilized frog eggs would have on the
percentage which successfully cleaved to the blastula stage.
These experiments were suggested by evidence that ATP is
functional in cell division and structural alterations of
the cell (see marsland, 1956; Runnstrom and Kriszat, 1950a,b;
Hoffmann-Berling, 1960). The bulk of this evidence has been
gathered from such materials as marine invertebrate eggs and
fibroblast cell models. Some of the evidence mentioned was
obtained from amphibian material, however, and was of such
a nature as to suggest the presence of gel systems similar
to those in marine invertebrate egrs and cell models. The
lines of evidence indicating the presence of systems in the
eggs of amphibians which may utilize ATP in the process of
cell division are l) the demonstration by Marsland and
Landau (1954) that the gelation process in the eggs of E.
pipiens is an endothermic process, 2) the work of Selman

and Waddington (1955) which indicates that gelation is an

 

important process in the dg novo formation of the new cell
surface in the cleavawe of amphibian eggs, 5) the inhibition
of cleavage in amphibian morulae by DNP, which is known to
inhibit oxidative phosphorylation (see Brachet, 1957, p. 177),

and 4) the demonstration by piegelman and Moog (cited by

-57-

-58-

Brachet, 1950, p. 169) that sodium azide, an inhibitor of

transphosphorylation and ATPase activity, will stop segmen-

tation of 3. pipiens eggs. As might be expected, the

presence of enzymes catalyzing ATP hydrolysis in the eggs of

frogs has been shown by Barth and Jaeger (1947). Evidence

of a more indirect nature is gained from sodium azide

(Shaver, it 31, 1952) and heparin (Shaver, 1949; Harding,

D., l949, 1951) inhibition studies in relation to the cleavage

initiating substance in artificial parthenogenesis in amphi-

bians. Finally, the large granule fraction, presumably mito-

chondrial in nature, obtained upon centrifuging homogenates

of frog gastrulae, was found to have a high cleavage-initia-

ting capacity in experimental parthenogenesis (Shaver, 1955).

Althuigh the results of the present investigation indicate

that ATP pea 33 has no cleavage initiating capacity (0.6,

0.0, 0.0, 0.02 cleavage when unsmeared eggs are injected

with ATP; see Tables 1A, 2A, 5A, and 4A, respectively), it

is possible that the ATP produced by mitochondria may have

some function in increasing the number of successful cleavages.
Some experiments in this study were performed in such

a way that the injection procedure also effected the activa-

tion process (Tables lA,lB and 5A,5B), while in others the

injection procedure followed activation by pricking or by

fertilization by about two hours (Tables 2A,2B and 4A,4B).

In 1958, Wolpert reported that treatment of sea urchin eggs

with ATP ten minutes prior to cleavage resulted in the eggs

showing considerable delay and abnormalities in cleavage, or

-39-

resulted in the eggs failing to cleave at all. Such results
might be expected under the supposition that the eggs were
unable to regulate the ATP supply within such a short time
prior to cleavage and as a result a relaxation or elongation
would be induced by the supraoptimal concentrations of ATP
in a manner analogous to that described by Hoffmann-Berling
(1960) in cell model systems. When Niu-Twitty buffer solution
was used in the present study as a medium for the solution
of ATP, however, no apparent difference in the response of
the eggs to the injection of ATP at the time of activation
and two hours after activation was noted (Tables lA,lB and
2A,2B). Wolpert (1962) has since attributed his results to
artifact due to pH changes.

It is apparent from the results presented that the
injection of buffer into eggs smeared with blood had some
detrimental effect on the eggs. The percentages of eggs
cleaving to blastulae in this case (5.1, 6.9, 18.5, and 6.8
in series lA—4A) were always less than the percentages
obtained by simply puncturing smeared eggs (15.9, 8.1, 22.4,
and 29.8, respectively). With the exception of series 2,
the percentages of cleavages obtained upon injecting ATP
into the eggs (11.8, 15.7, 15.1, 7.7) were also less than
those obtained by the simple puncturing procedure, but, with
the exception of series 5, the percentages were more than
those obtained in the controls where buffer was injected
into smeared eggs. The results of injecting ATP into the

smeared eggs thus seemed to indicate that the ATP counter-

-43-
acted to some extent the detrimental effect of the injection
process.

As was pointed out in the presentation of results,
the sample sizes in the second, third, and fourth series

were too small for tests of statistical significance to be
meaningful. In the first series of experiments, the pH of
the injected control solution was in the alkaline range,
whereas the pH of the injected ATP solution was in the acid
range. As a result, one wonders if the statistically signi-
ficant data obtained in the first series of experiments are
a result of the acidity of the ATP solution or of some other
property inherent in the ATP molecule, such as its high
energy transfer potential.

Acidity plays a role in 8.8. Lillie's (1954) theory
of activatiOn, but this theory is based on observations on
artificial parthenogenesis in marine invertebrate eggs.

Sea urchin eggs have been successfully activated by acid
treatment (Loeb, 1915), whereas successful cleavage has not
been obtained through such treatment of frog eggs. Anderson
(1956) has proposed a general theory in which he suggests
that polyelectrolyte balance is important in sol-gel trans-
formations. According to this concept, a shift of the poly-
electrolytes toward polyanions premotes solation and a shift
toward polycations promotes gelation. A shift toward poly-
cations and hence toward gelation would be favored by a drop
in pH. However, this author is not aware of any evidence

indicating that acidity, per 33, would have any stimulatory

-41-
effect in cell division in the eggs of amphibians.

If the acid pH of the ATP solution were of signifi—
cance, one would expect the results obtained to be the same
when injecting an ATP solution and a buffer control of simi-
lar pH. It was noted, however, that in the second series of
experiments, with special reference to the eggs of frog XI,
where injections were made two hours after activation, the
ATP did show some positive effect in enhancing cleavage. The
percentage of fertilized eggs injected with ATP two hours
after fertilization which cleaved was about 16% greater than
the percentage of control eggs which cleaved when injected
with Niu-Twitty buffer two hours after fertilization. With
regard to the eggs of frog XI, which were injected two hogrs
after having been smeared with blooa and pricked, it was
found that the ratio of the percentage of eggs injected with

TP which cleaved to the percentage injected with Niu-Twitty
buffer which cleaved was greater than 2.5. A slight increase
in the percentage of cleavazes of those eggs injected with
ATP over the percentage of those controls injected with
Steinberg's medium is also apparent in the results recorded
in Tables 4A and 48.

On the basis of the literature summarized in the first
paragraph of this discussion, it is tempting to theorize
that ATP mediates in the sol-gel transformations involved
in the scheme of cell division in amphibian eggs as formu-

lated by Selman and Waddington (1955) and in the formation

and functioning of the mitotic apparatus. If one regards

-42-
the pH discrepancies of the first series of experiments in
the present investigation as insignificant, then the results
of this first series in addition to those of the second and
fourth series, though inconclusive, are not in contradiction
with the above conjecture, although the precise mode of
action is undefined.

If ATPase is present in the mitotic apparatus of
amphibian eggs as it is in the mitotic apparatus of the sea
urchin egg (ihzia, Chaffee, and Iverson, 1961), it is possible
that ATP injected into the interior of the egg may serve as
an added energy source for the functioning of the mitotic
spindle in chromosome movements. Rough calculations indicate
that the amount of ATP injected into the egg introduced added
energy of the order of 10'9 keel/egg. Calculations based on
labile phosphate determinations made by Barth and Jaeger (1947)
on early cleavage stages (Shumway stages 4-7) of 3. pipiens
indicate that the energy available in the form of ATP (or ADP)
is of the order of 10"8 to 10"9 kcal/egg. Thus the injection
of ATP as performed in this study is likely to involve a 50-
1003 increase in the energy available to the egg. Caution
is advisable, however, with regard to interpretations con-
cerning the affect of ATP, since the present study did not
investigate the possibility that AMP might be as effective
as ATP. This possibility must be eliminated before the
results obtained can be attributed to the high energy moiety

of ATP.

Although the literature reviewed in this paper does

-43-
indicate a role involving ATP in the sol-gel transformations
involved in cell cleavage and in the movements of the spindle,
good experimental data elucidating the nature of the mecha-
nisms involved are lacking. Lilewise, information has not
been obtained with regard to the mechanisms involving ATP in
the present investigation. Perhaps a systematic biochemical
and biophysical analysis of model gel systems might provide
some irteresting results with regard to the precise nature

of the role played by ATP.

In View of the above considerations, how can one
account for the results obtained in the third series of
experiments (Tables 3A and SB)? In this series the percen-
tages of eggs, particularly those of frog XIV, which cleaved
when injected with ATP were lower than the percentages which
cleaved when injected with Steinberg's medium (control).

The only explanation which seems feasible at this time
involves the assumption that the amount of ATP injected into
the eggs was in supraOptimal quantities. Supraoptiwal concen-
trations f ATP are known to cause motile protein structures
to eldngate (Hoffmann-Berling, 1960). 'Under these condi-
tions these same structures are unable to contract. Thus
the postulated contraction of the spindle might be inhibi-
ted. Furthermore, if contractiai is involved in the round-
ing up of a cell prior to cleavage (Selman and Waddington,
1955), supraOptimal concentrations of ATP may indirectly
prevent cleavare by inhibiting the general cell surface

contraction leading to the rounding up of the cell.

-44-
From this discussion it should be obvious that more
extensive data are needed before any conclusions can be drawn
with regard to the effectiveness of ATP in the enhancement
of cleavage initiation in frog eggs. The speculations put
forth in this discussion not only accentuate the limitations
of this investigation, but also offer a number of interesting
avenues of approach for the further investigation and clari-

fication of this problem.

SU IJLIARY

There is a considerable literature concerned with the
intracellular action of adenosine triphosphate. Mach of it
is particularly concerned with the role played by ATP in cell
division. On the basis of the previous work reviewed in this
paper, it was of interest to the present author to observe
what effect, if any, ATP injected into parthenogenetically
stimulated frog eggs would have on the number which success-
fully cleaved to the blastula stage.

The percentage of eggs in each experiment which cleaved
following injection with ATP (1.61 x 10‘5M) was compared
with a control batch from the same frog in which the eggs
were injected with Kin-Tuitty or Steinberg buffer. Phe
design of the experiments was such that in some cases (Tables
1 and 3) the injection of the buffer or ATP into eggs smeared
with blood effected the parthenogenetic activation whereas
in others (Tables 2 and 4) the injection was made approxi-
mately two hours after the eggs had been stimulated either
by fertilization or by pricking blood-smeared eggs with a
fine glass needle.

In all cases the percentage of control blood-smeared
eggs injected with buffer which cleaved was less than the
percentage of cleavages obtained by simply pricking blood-
smeared eggs. Similarly treated eggs, when injected with
ATP, also cleaved to a lesser extent than the pricked controls

in three out of the four series of experiments (Tables 1A,

5A, and 4A). Cleavages occurred in these ATP-injected eggs

-45-

-45-
more frequently, however, than cleavages in the eggs injected
with buffer in three of the four series of experiments (Tables
1A, 2A, and 4A).

These results seem to indicate that the ATP functioned
in some way in aiding the egg in overcoming injury it had
incurred as a result of the injection procedure.

The failure to maintain a proper pH in the control
injections in one series of experiments and the small sample
sizes in the subsequent three series of experiments prevent
the author from drawing any definite conclusions with regard
to the effectiveness of ATP in enhancing cleavage. There are
indications, however, that ATP may be important and with this

in mind, several possible modes of action for ATP are discussed.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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-50-

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Fisher, R. A. 1950. Statistical Kethods for Research
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