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The effects of estradiol-17B on cleavage, nucleic acid metabolism, and protein synthesis in embryos of the sea urchin, Strongylocentrotus purpuratus
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The effects of estradiol-17B on cleavage, nucleic acid metabolism, and protein synthesis in embryos of the sea urchin, Strongylocentrotus purpuratus
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THE EFFECTS OF ESTRADIOL-1 ?B ON CLEAVAGE,
NUCLEIC ACID METABOLISM, AND PROTEIN SYNTHESIS IN
EMBRYOS OF THE SEA URCHIN, STRONGYLOCENTROTUS PURPURATUS
Weldon B ^Jolley
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Biology)
August 1959
UNIVERSITY O F S O U T H E R N CALIFORNIA
G R A D U A T E S C H O O L
U N IV E R S I T Y P A R K
L O S A N G E L E S 7 . C A L I F O R N I A
This dissertation, written by
;...........WeldQn„BJ ,.„Jo.lley........
under the direction of hXs...Dissertation Com
mittee, and approved by all its members, has
been presented to and accepted by the Graduate
School, in partial fulfillment of requirements
for the degree of
D O C T O R OF P H I L O S O P H Y
Dean
Date.......AUGUST. ,..125.2.
DISSERTATION C O M M ITTEE
..
ACKNOWLEDGMENTS
I wish to express my appreciation to Dr. Walter
E. Martin and Dr. Lyell J. Thomas, Jr. for their helpful
suggestions and for the use of their facilities, without
which this work could not have been accomplished. I
also extend my thanks to the other members of my
committee, Dr. John L. Webb, Dr. Norman T. Mattox, and
Dr. Robert M. Chew.
I am grateful to Dr. Clifford V. Harding and
Dr. Robert M. Chew for their encouragement and advice
during the early years of my graduate work.
I wish to express my appreciation to Mr. Joseph
W. Bamberger and Mr. Louis W. Stearns for their help
and association.
My sincere gratitude to my wife, daughters, and
parents for helping me during this endeavor.
TABLE OP CONTENTS
PAGE
INTRODUCTION........................................... 1
Historical Review............ * 1
Occurrence of estrogens ..................... 3
Effects of estrogens in mammals........... • A
Action of estradiol on metabolism ...... 6
Estradiol effects on lipids 9
The effect of estradiol on nucleic acids
and proteins.............................. 11
The effect of estradiol on oxygen con
sumption and carbohydrate metabolism . . . 13
The effect of estradiol on nucleic acids
and proteins ...... ................. 14
Nucleic Acid and Protein Metabolism in Sea
Urchin Embryos• ................... ..... 14
Nucleic acids ................................ 14
Protein and amino acids.................. 16
Work of Others Concerning the Action of
Estradiol on Embryos* ........ ....... 18
STATEMENT OP PROBLEM AND METHOD OP ATTACK .......... 21
MATERIALS AND METHODS............................... 24
Materials . . . . • • • • • e . . . ••.*<> 24
Culture procedure • ••••••• 24
V
PAGE
Removal of lipids ............. 26
Extraction of the nucleic acids.......... .. 26
Chromatography of purines...............« . o 28
Estimation of the bases................. • ? 28
Isotope analysis a ............... 29
Protein and amino acid analysis........... 29
Experimental ........................... 30
Effect of prolonging cleavage ........ 30
Effect of time of treatment............ 30
Glycine-1-C^ and glycine-2-C-1 -^ Incorpo
ration « . . . ........................... 31
Effect of adenosinetriphosphate........... 31
Comparison of estradiol and testosterone. . „ 31
RESULTS........................................... 33
DISCUSSION......................................... A8
SUMMARY........................................... 55
LITERATURE CITED...................................58
APPENDIX........................................... 67
LIST OF TABLES
TABLE PAGE
I. Effect of Prolonged Delay of Cleavage
Due to Low Temperature on RNA and
DNA Concentration....................... 40
II. Effect of Estradiol Treatment on Total
Nucleic Acid Content of Embryos............ 41
III. Effect of Estradiol Treatment on the
Incorporation of Glycine-2-C-^ into
the Purine Bases of DNA.................... 42
IV. Effect of Estradiol on the Level of
Activity of the Free Amino Acids,
Glycine and Serine....................» 43
V. Effect of Estradiol Treatment on the
Incorporation of Glycine-l-C1^ and
Glycine-2-C-^ Into Purine Bases of
RNA and DNA................................. 44
VI. Effect of Simultaneous Treatment with
Adenosinetriphosphate and Estradiol
on the Level of DMA and RNA, and the
Incorporation of Glycine-l-C^ into
the Purine Bases and Protein . ...... 45
VII. A Comparison of the Effect of Estradiol
and Testosterone on the DNA Content and
vii
TABLE PAGE
Incorporation of Glycine-l-C^^
into Protein..................................^6
VIII« A Comparison of the Effect of Estradiol
and Testosterone on the Incorporation
of Glycine-1-C^ into the DMA and
RNA Purine Bases..............................4?
LIST OP FIGURES
FIGURE PAGE
1. The Difference Between the Ultraviolet
Absorption Spectra of the Acid Soluble
Fraction of Embryos Immediately After
Fertilization and After Eighteen Hours
at 5° Centigrade............................... 39
INTRODUCTION
The steroids are a group of fat-soluble compounds
which contain the perhydrocyclopentenophenanthrene
nucleus. These compounds comprise a wide range of
naturally occurring substances among which are the plant
and animal sterols, bile acids, steroid hormones, the
cardiac glycosides, sapogenins, toad venoms, and some
minor groups#
The estrogens are steroid hormones which can be
chemically characterized by the steroid nucleus in which
the A ring is an unsaturated phenolic group. Physiologl
cally these compounds have been characterized by their
ability to condition and maintain the mammalian female
accessory sex organs such as the uterus, vagina, and
mammary gland.
The following is a study of the effects of one of
the estrogens, estradiol-17B, on the nucleic acid
metabolism, protein synthesis, and cleavage during the
early biastula stage In developing embryos of the sea
urchin, Strongylocentrotus purpuratus.
Historical Review
A number of very comprehensive reviews have
appeared concerning the general and specific aspects of
the action and interaction of the estrogen hormoneso
It is the purpose of this section to present an
historical review of only those works which hear an
Immediate relationship to the experiments of this
dissertation*
Although there were many previous investigations
which Indicated that there were substances concerned with
decreased sexual activity and atrophy of the female
secondary sex characters following ovariectomy, one of the
major contributions to the understanding of estrogen
activity came when Allen and Doisy (1923) published their
technique for the detection of estrogens* This ingenious
method, still in use today, utilizes the stratification
and comificatlon of vaginal epithelium of ovariectomized
rats and mice as a means of detecting the presence and
the relative activity of these compounds.
Aschelm and Zondek (1927) reported that female
pregnancy urine showed the presence of large quantities of
estrogenic material* However, the first successfully
isolated estrogen, estrone, was crystallized in a pure form
independently and almost simultaneously by Butenandt (1929)
and Doisy et al. (1929)• Shortly thereafter, Marrian
(1930) Isolated the placental hormone, estrlol. The first
preparation of estradiol from a natural source was per
formed by MacCorquodale et al, (1936) from an extract of
four tons of sow ovaries* A-estradiol, a less potent
sterioisomer, has subsequently been isolated by Hirschmann
and Wintersteiner (1939)• At the present time several
other natural estrogens, with closely related structures
but decreased activity, have been Isolated. The names of
these naturally occurring compounds are 16-ketoestrone,
equilen, hippulin, equilenin, and 17B-dlhydroequilenin«
Occurrence of estrogens. Doisy et al* (1929),
Westerfield et al, (1938), Beall (19^0), Huffman et al*
(19^0), and Girard et al. (1932) have shown estrogens to be
present in a great diversity of mammalian tissues such as
the ovary, testes, adrenals, and placenta* Also, a number
of Inactive estranediols from urine have been isolated*
Marker (1938) has suggested that these estranediols are
reduction products of the biologically active estrogens.
Muhlbock (1937) showed that about twenty to fifty
per cent of the estrogenic material in the blood plasma
exists in the bound or conjugated form. Szego and
Roberts (19^6) have stated that available evidence
indicates that these hormones are secreted into the blood
in the free form but that they rapidly become conjugated.
Fractionation of the blood plasma by the method of Cohn
et al. (19^6) has shown most of the estrogen to be in the
B^-Lipoprotein (fraction III-O). Additionally, Szego and
Roberts (19^6) state that the conjugated form may be that
of a glucuronide* There is some evidence from the work
of Veldhuls (1953) of the presence of unconjugated estrone
or estradiol in human plasma*
Although it is not known whether all of the
substances isolated act as hormones, Bullough (1955) a*id
Sharrer (1941) have shown the presence of estrogenic
substances in extracts made from protozoans, coelenter-
ates, worms, molluscs, crustaceans, and Insects. Donahue
and Jennings (1940) and Hagerman .et al* (1957) have
Isolated estrogens from the echlnoderms.
The widespread distribution of the estrogens is
further shown by their presence in plants* Legg et al,
(1950) have detected the existence of high concentrations
of estrogens in various grasses and clover during the
early spring. Similar substances have been detected in
plants ranging from the yeasts to pussy willows.
Bradbury and White (195*0 » a review of estrogens
in plants, discuss the occurrence of a compound structur
ally unrelated to the steroidal estrogens * This
substance, an isoflavon, is called genistein* It
manifests estrogenic activity in animals and has been
identified In numerous plants*
Effects of estrogens in mammals. Since the most
pronounced physiological effect of the estrogens occurs
in the uterus, this organ has been the object of invest!-
5
gations of numerous papers. However, it should be pointed
out that the uterine effect is only one of many effects
on mammalian physiology. Pincus (1955) has reviewed the
action of estrogens on ovary, vagina, uterus, oviducts,
anterior pituitary, and some minor areas.
McLeod and Reynolds (1938) have shown that within
one hour after treatment of the ovariectomized rat with
estradiol there is a generalized hyperemia of the uterus.
Astwood (1938) and others have observed that the hyperemia
is followed by an imbibition of water which reaches a
maximum during the fourth to sixth hour of estrogen treat
ment. During the period of water Imbibition, the uterine
electrolytes are also undergoing changes in concentration.
Spazlani and Szego (1957) detected an Increase in the
uptake of radioactive sodium and potassium.
Spaziani and Szego (1958) have published results
which show an Increase in the release of histamine from
uterine tissue following estrogen treatment. They
attribute part of the increased permeability of the
uterus to the Increased histamine level of the blood.
They also mentioned the possibility of permeability
changes which might be associated with alteration of the
surface properties of monomolecular layers of protein in
the presence of estrogens.
In an attempt to determine whether the shifts in
uterine water and electrolytes were accompanied by changes
in the structure of epithelial cells, Nilsson (1958)
studied the uterine epithelium of the spayed mouse before
and after the administration of estradiol,, Electronmicro-
graphs showed a tremendous increase in the size and
number of spaces of the a-cytomembranes in the cytoplasm
of estradiol-treated animals,, Since the number of
projections along the luminal surface of the uterine
epithelium occurred during the same period as that in
which water was Increasing, Nilsson correlated these two
phenomena.
Action of estradiol on metabolism„ Szego and
Roberts (1953) have pointed out that an understanding of
the mechanism of action of any hormone must eventually
come from the chemical reaction in which it participates.
The action of estradiol seems to be highly diversified.
Therefore, to facilitate presentation of this section,
the material will be divided into two parts: Part A will
be concerned primarily with the influence of estradiol on
the metabolic processes of the uterus. Part B will In
clude information obtained from studies involving non-
uterlne tissues and cells.
Part A.
The first six-hour period, Mueller et al.(1958)
have called the Induction phase. It has been shown that
during this phase there is little or no accumulation of
either ribonucleic acid (RNA), deoxyribonucleic acid,
(DNA) or protein. These same authors have further de
lineated two additional periods during which specific
changes are occurring. The second period, six to twenty-
four hours, is referred to as the RNA accumulation phase.
The second period Is characterized by a rapid accumula
tion of RNA, a vast spreading out of the endoplasmic
reticulum, and an increased synthesis of protein. The
third phase or DNA aynthesis phase, occurs between forty
and seventy-two hours after administration of estradiol.
The DNA production is quite marked during this phase,
Szego and Roberts (1953) have found that oxygen
consumption and glucose utilization Increase In the rat
uterus after exposure to estradiol. Walaas and Walaas
(1952) demonstrated an Increase in both aerobic and an
aerobic glycolysis upon addition of estradiol. They
further demonstrated that the increased glycolysis was
proportional to the Inflow of carbohydrate. Glycogen
synthesis and uptake were increased by estradiol but
pituitary extract, thyroxin, epinephrine, or adrenal
extract had no effect. The above mentioned results
prompted Walaas and Walaas (1952) to localize the
action of estradiol at the hexokinase reaction in which
8
glucose, in the presence of adenoslnetrlphosphate and
hexokinase, Is phosphorylated to give glucose-6-phosphate*
This theory was further substantiated when they observed
increased deposition of glycogen in the liver of fasting
ovarlectomized rats under prolonged administration of
estradiol. Moreover, Zondek and Shlomo (19^7) had pre
viously shown that estradiol increased glycogen in the rat
uterus. Bullough (1955)> using epidermal mitotic activity
as an indicator, found that estrone action was specific
for the glucokinase reaction and that the fructokinase re
action was not affected by estrogen.
Bever et al. (1956) have contributed an additional
possible role of estradiol in glycolysis which links
estradiol activity with the enzyme system catalizing the
oxidation of lactate and the reduction of pyruvate in the
final reductive phase of glycolysis. These authors have
shown that estradiol increases the activity of reduced
diphosphopyridine nucleotide oxidase in the reaction
involving lactic dehydrogenase and reduced diphospho
pyridine nucleotide oxidase. Similar results have been
obtained by Villee and Hagerman (1958) who have shown an
in vitro stimulation of a placental estrogen-sensitive
enzyme involving both diphosphopyridine and triphospho-
! pyridine nucleotide and lsocitric dehydrogenase as
i
follows:
DPN+ + estradiol DPNH + estrone
TPNH + H+ + estrone --^ TPN+ + estradiol
TPN+ + is oc it rate ---^ TPNH + H+ + CC>2 +
a-ke t oglutarate
Estradiol effects on lipids . One of the first
observations of the Influence of estradiol on lipids was
made by Bloor et al. (1930) who recorded an Increase in
the phospholipid, lecithin, in the uterus of the sow at
the time of uterine elaboration. Borell (1952) has re
ported an Increase in the uptake of P^2 in the lipid
extract of rabbit uterus in response to estrogen adminis
tration. Nicoll and Snell (1955) have claimed that there
is an increase in the glycogen content of the uterine
cells in ovarlectomized guinea pigs following injections
of estradiol benzoate.
Mueller (l95*0 found a 250 per cent increase in the
incorporation of radioactive glycine when uterus was
treated with estradiol. In the same study he showed no
increase in respiration during the experimental period.
This indicates that the first metabolic changes are
apparently independent of any effect which estradiol may
have on respiration.
It appears that one of the effects of estradiol on
lipid metabolism in the uterus is centered around mobi
lization of at least one step, and possibly many steps,
10 !
in the utilization of one-carbon fragments.
Mueller and Herranen (1956) have shown that
estrogen-pretreated uterine segments take up increased
amounts of glycIne-2-C1^. Similarly to the glycine data,
they have shown that the incorporation of labeled
serine^-C^ is Increased in the treated segment. They
have interpreted these results to signify that the action
of the hormone must have been related to the transfer of
one-carbon molties since it is known that glycine and part
of serine are Incorporated into the nucleic acid purine
bases. Furthermore, Davis _et al. (1956) have given
additional support to this idea by the use of aminopterln
which has been shown to interfere with the exchange of
one-carbon compounds via the folic acid system. Their
studies have shown that the simultaneous administration
of aminopterln and estradiol abolishes the effect of
estrogen on Increased uptake of the label in the phospho
lipids. Mueller and Herranen (1956), with the use of a
large non-labeled formate pool have minimized the effects
of estradiol on lipid metabolism as shown by incorporation
of labeled glycine.
In an attempt to associate hormone and enzyme
activity, Mueller (1957) investigated the enzyme, serine
aldolase, that catalyzes the reversible interconversIon
of glycine and serine. By measuring the amount of glycine
11
that was converted to serine in rat uterine homogenates,
he found that serine aldolase activity was stimulated
by estradiol pretreatment0
The effect of estradlol on nucleic acids and
proteins. Telfer (1953) and. Mueller et al* (1958) have
shown that estradiol stimulates the production of RNA and
DNA in uterine preparations, and Mueller et al. (1958)
have shown that estradiol was involved in the metabolism
of one-carbon precursors* These findings were also
reflected in an increased incorporation of labeled glycine
in the purine bases, adenine and guanine. However, in
studies on estrogen-treated uteri which had been incubated
with C-^02 it was discovered that the incorporation in
pyrimidine had also been increased. From these experi
ments on the uptake of labeled glycine and carbon dioxide
and a subsequent study which showed an Increased incorpo
ration of preformed adenlne-8-C^ into the nucleic acids,
they have concluded that estrogen treatment seems to
stimulate a generalized increase in nucleotide synthesis.
An increased dry weight following estrogen treat
ment as reported by Szego and Roberts (1953) suggests that
estradiol might be implicated in some regulatory mechanism
in protein synthesis. Mueller et al. (1958) have observed
an increased incorporation of labeled amino acids into
the protein of pretreated uterine segments. Other studies
12
have shown quite conclusively that a specific enzyme is
Involved in the activation of each amino acid. These
enzymes are specific for the naturally occurring 1-amino
acid isomer. Another important aspect of this work is the
demonstration of an Increased level of a number of amino-
activating enzymes. A time plot of this enzyme response
reveals that the enzyme activity actually precedes the
time in which RNA and protein are accumulating.
Mueller et al. (1958), Zamecnik and Keller (195*0»
Hoagland et al. (1956), DeMoss and Novelli (1956), and
others have proposed the following diagrammatic repre
sentation of the reactions which are probably Involved
in protein synthesis:
H 0 Soluble H 0
I I I Enzyme I J
R— C — C + ATP .- . . R — G — G + PP
II I \
H2N OH NH2 AMP Enzyme
(active amino acid)
Active amino acid + soluble RNA RNA-amino acid
H 0 H
Mlcrosomes I I I
RNA-amino acid + Peptide—NH2____GTP — Peptide-N—G-C—R
\
NH2
The primary site of estradiol as a regulator of protein
synthesis would be to stimulate the production of soluble
enzyme.
13
Part B.
The effect of oxygen consumption and carbohydrate
metabolism 0 McDonald and Latta (1956) observed that
slices of human prostatic adenoma treated with high levels :
of natural or synthetic estrogens caused a significant
inhibition of aneroblc glycolysis. Low concentrations of
the hormone occasionally stimulated aneroblc glycolysis.
This inhibition is exactly opposite to the effects of
estradiol on uterine tissues as shown in Part A. However,
prostatic adenoma has, as have other neoplasms, an
extremely high aneroblc glycolytic rate. Barron and
Huggins (19^) have reported that prostate gland of
castrate and diethylstllbestrol-treated dogs showed
increased aerobic carbohydrate metabolism, but aneroblc
glycolysis remained unchanged.
Dirschel et al. (1952) were unable to observe any
effect of estradiol on the carbohydrate metabolism of
mouse liver homogenates or cardiac muscle. Gurdy et al.
(1952) reported a similar finding but noted a decrease in
oxygen consumption of liver homogenates in the presence
of the hormone and those substrates which are oxidized by
coenzyme I and II.
Aldraan .et al, (1951) have shown that certain
phosphate esters of estradiol inhibit the action of rabbit
kidney alkaline phosphatase. Meyer and McShan (1950)
studied the inhibition of the synthetic estrogen,
diethylstllbesterol, on the succlnoxldase system of brain,
adrenal, pituitary, kidney, and heart tissue. They
compared the inhibition observed with a number of other
compounds containing phenolic groups. These comparative
studies indicate that the amount of inhibition observed is
dependent upon the number of phenol groups present,
although pure phenol had no influence on this enzyme
system. Furthermore, when cytochrome was replaced with
brilliant cresyl blue, no inhibition was observed.
Therefore, it appears that the major inhibition is through
cytochrome oxidase and there is no direct effect of the
hormone on succinic dehydrogenase.
The effect of estradiol on nucleic acids and
proteins« Campbell _et al. (1953) have shown an increase
in the amount of ENA-protein and RNA-protein in rat livers
of animals receiving daily administration of estradiol.
Mandel et al. (1952) have reported that estradiol in
creased RNA and protein in pullets.
Nucleic Acid and Protein Metabolism in
Sea Urchin Embryos
Nucleic acids. Brachet (1957) has pointed out that
many early conclusions about the nucleic acids in sea
urchin embryos were incorrect since the methods of analysis
were inadequate and the knowledge of nucleic acid
chemistry was Insufficient. Therefore, only recent
studies will be reviewed.
Elson et al. (195^) have estimated the amount of
RNA and DNA in sea urchin embryos, and they have shown
that there is an initial rapid decrease in RNA content
immediately after fertilization. The RNA concentration
soon returns to a level slightly higher than that of the
unfertilized egg, and it remains at this level until a
second rise occurs Just before the onset of gastrulation.
Vlllee et al. (1949) and Schmidt, Hecht, and Thannhauser
(1948) , using incorporation as an index of DNA and
RNA activity, showed that during the first twelve hours
of development most of the incorporation occurs in the
DNA. Abrams (1951) observed that the DNA purines have
approximately ten times the activity of the purines of
RNA. Unpublished results obtained by the author have also
shown greater activity in the DNA purines.
Agrell and Persson (1956), have shown that DNA
synthesis begins when synchronization of mitosis is lost.
They maintain that most of the DNA that is used by the
nuclei up to this time comes from cytoplasmic DNA stores.
They were also able to detect changes in the free
nucleotides during this same period. The concentration
of DNA present in the egg at the time of fertilization
16 |
amounts to five to fifteen times the diploid content.
This excess would allow cleavage to proceed for a short
time before new synthesis was required.
The similarity between the nucleic acid concentra
tion in sea urchin embryos during the early stages of
development and the fact that the nucleic acid concentra
tion of sea urchin embryos bears a resemblance to that of
various other embryos during the early stages of develop
ment has been noted by Brachet (1957) •
Marshak and Marshak (195**» 1955) have concluded
that unfertilized eggs of the sea urchin contain no DNA„
However, Burgos (1955) a^d Brachet (1957) have indicated
some serious errors in techniques and some questionable
assumptions in the Marshaks' work.
Protein and amino acids . In a chromatographic
study of the sea urchin, Strongylocentrotus purpuratus.
Berg (1950) found nine free amino acids. Glycine was the
most abundant with a large concentration of alanine.
Alanine, lysine, and glutamine were observed to decrease
during embryonic development•
Kavanau (1953) has made a very extensive study of
the early developmental stages of embryos for the content
of Individual amino acids in the free, peptide, and
protein form. One group of free amino acids, histidine,
isoleucine, leucine, methionine, phenylalanine, threonine,
17
tyrosine, and valine, was shown to decrease in concentra
tion during early cleavage. This group of amino acids
then increased to a peak value at the time of hatching
which was followed by a sharp drop in the mesenchyme-
blastula stage. During gastrulatlon only minor changes
were noted. A second group, arginine, glutamic acid, and
lysine, decreased in concentration from the time of
fertilization to the gastrula stage. Glycine, which
comprised the bulk of the free amino acids, showed a
•*»
continuous decrease down to the late mesenchyme-blastula.
In a different species, Paracentrotus llvidus.
Kavanau (195*0 showed that changes in protein and non-
protein amino acids occur in complementary cyclic waves.
Furthermore, it was shown that yolk-protein breakdown and
new protein synthesis follow known respiratory changes«
Marked synthesis occurs during periods of respiratory
increase.
Hultln (1957) was able to show a correlation
between the incorporation of C^-formate into the purines
of RNA and protein-bound serine. This is consistent with
the close interrelationship between the metabolism of
serine and the introduction of formate into purines which
has been noted In other systems.
18
Work of Others Concerning the Action of
Estradiol on Embryos
Tondury (1942) reported results of estradiol upon
the early cleavages of Triton embryos . He showed that the
hormone affects mitosis by forming chromosomal bridges
and disrupting chromosomes from the spindle, Brachet
(1955) Has cited a similar work of Cagianut who reported
that estradiol affects the distribution of RNA in am
phibian eggs. Yeast RNA was shown to work antagonisti
cally and promote cleavage.
Agrell (195*0 > working with the sea urchin,
Psammechlnus mlliarls, observed that estradiol produces
cleavage disturbances in concentrations as low as
2 x 10“6 molar. Higher concentrations of the hormone,
10-40 x 10“^ molar, completely inhibited cellular
division. However, even though cellular division had been
suppressed, nuclear division continued and thus syncytia
were observed. Lower concentrations of the hormone tended
to produce hypoploidy and higher concentrations of the
hormone produced hyperploidy. Agrell (1955) measured the
concentration of RNA and DNA in the embryo up to the sixth
hour of development, and he found that estradiol reduced
the amount of DNA present, but the hormone had a negli
gible effect on RNA concentration.
Runnstrom and Krlzat (195*0 also studied the
cleavage Inhibition in Psammechlnus miliaris. They have
shown that the highest sensitivity of embryos to the
hormone occurs during the two to eight-cell stage» Once
the microraeres have formed, the period of sensitivity
seems to decrease,, They further demonstrated that the
addition of .003 molar adenosinetriphosphate Improves the
cleavage of eggs exposed to estradiol. In eggs which were
exposed for a longer period of time to lower concentra
tions of estradiol, they observed Irregular cleavage i^ith
a tendency to form unequal cells. They also reported
that exposure in sulphate-free sea water enhanced the
Inhibitory effects on cleavage.
Agrell and Persson (1956) demonstrated that
estradiol acts as a substrate activator for DNA-ase. The
authors showed that estradiol treatment of DNA-protein
from the sperm of Paracentrotus llvldus in the presence of
DNA-ase resulted in a separation of the nucleic acid and
protein. They propose the following explanation for the
effect of estradiol on cleavage: Early in development,
DNA may form an integral part of the cytoplasmic structure
in the form of DNA-protein. Splitting of this structural
nucleoprotein by estradiol should result in a decreased
viscosity of the cytoplasm, and the separation of the
nucleic acid and protein should increase the number of
sulfhydryl groups exposed which should cause a dissocl-
20
at ion of the asters and spindles. All of these changes
would readily affect cleavage.
Hagerman (1956) cultured gravid ovaries of Arbacla
punctulata and Asterlas forbesl in a culture medium of sea
water containing estradiol, pyruvate, and glucose. He
observed that the estradiol had no effect on oxygen
consumption, glycogen utilization, or lactate production.
Hagerman concluded from these data that the dlphospho-
pyridine-llhked lsocitrlc dehydrogenase was not present
or, if it was present, it was not functional at this
stage of development.
STATEMENT OP PROBLEM AND METHOD OP ATTACK
As discussed in the previous section, estradiol
inhibits cleavage in many embryos, However, no intensive
investigation has been made concerning the actual cellular
mechanism involved. Therefore, the problem of investigat
ing the effects of this hormone on nucleic acid metabo
lism and protein synthesis might be of value in eluci
dating the mechanism by which cell cleavage is inhibited.
The sea urchin embryo, during early cleavage,
seemed particularly well suited for this investigation
since it undergoes relatively rapid division with a
minimum of growth. Furthermore, culturing is relatively
easy, and there is less yolk material than is found in
many embryos. Since the number of metabolic processes
which are occurring in many tissues are minimal or
completely inactive during the early cleavage, the in
terpretation of the action of estradiol would not be as
difficult as has been the case in many other studies•
Isotopically labeled glycine was chosen as a
metabolite since it is Incorporated into the purine bases,
adenine and guanine, which play Important roles in nucleic
acid and protein synthesis. Furthermore, Kavanau (1953)
has shown that the most abundant amino acid in the sea
urchin embryo is glycine and therefore, it should not
22
introduce any abnormal culture conditions since only a
comparatively small amount of glycine was used in the
culture mediumo Moreover, the use of an amino acid which
can be labeled in two positions extends the range of in
vestigation without undue complications«
After preliminary investigations had shown that
estradiol did affect the incorporation of glycine, experi
ments varying the time of exposure to estradiol were
planned.
Since energy is required for cell division, another
set of experiments was planned to test the effect of
adding a high energy compound, adenoslnetrlphosphate,
during the estradiol treatment.
In addition to the useful information which might
be obtained to explain the action of estradiol in
inhibiting cleavage, it was also hoped that the informa
tion obtained might be useful in helping to explain some
of the carcinostatic or carcinogenic properties of this
hormone.
In an attempt to synchronize cleavage and minimize
the error of a system in which many stages of cell
division may possibly be present, experiments were planned
to determine the effect of low temperature for various
time periods on cleavage delay. This delay in cleavage
increased synchronous development and decreased the
23
variability of the results obtained. Furthermore, it was
hoped that this low temperature period would help to
equilibrate the cellular contents of eggs taken from
ovaries of sea urchins whose exact physiological state
could not be predetermined.
MATERIALS AND METHODS
Materials. All of the commonly used chemicals in
this study were of chemically pure or analytical reagent
gradee The sources of special chemicals are given in the
following list:
The resins and all of the standards, such as the
purine bases and amino acids, were obtained from the
California Corporation for Biochemical Research, Los
Angeles, California,
The Isotopes, glyclne-l-C1^ and glycine-2-C-^ were
obtained from Nuclear Corporation, Chicago, Illinois,
The estradiol for the preliminary experiments was
obtained from the Sherlng Corporation, All subsequent
experiments used estradiol from the California Corporation
for Biochemical Research,
Culture procedure. Eggs from the sea urchin,
Strongylocentrotus purpuratus. were used in all experiments
except experiments $06 and 507 in which eggs of
Strongylocentrotus franc is canus were used. The technique
of Harding and Harding (1952) was used to obtain the eggs
and to fertilize them with the exception that potassium
chloride was omitted since It was found that in this
species the release of the eggs from the ovaries occurred
25
quite readily in sea water which had been chilled to 8°
centigrade. Only those cultures were used in which
approximately 100 per cent of the eggs were fertilized.
After several washings with chilled sea water to
remove excess sperm, the embryos were suspended in sea
water by gentle agitation and several trays were filled
with an equal amount of the suspension. This method
distributed the embryos in each culture dish so that the
variation in the recovered dry weight of embryonic
material was approximately five per cent or less. The
embryos were cultured in flat glass trays which were
gently rocked at an accurately controled temperature. The
temperature of the cultures was maintained at 7 to 9°
centigrade for the first nine hours after fertilization
and thereafter the temperature was raised to 13° centi
grade .
The embryos were collected at the termination of
the culture period by mild centrifugation in a refriger
ated centrifuge. The embryonic mass was washed twice with
cold sea water followed by appropriate centrifugation.
The material was then quickly frozen in a mixture of dry
ice and alcohol and immediately lyophllized. The lyo-
philized material was either stored at -20° centigrade or
the lipids, acid soluble nucleotides, and nucleic acids
were immediately extracted for analysis as outlined below.
26
The lipids were always removed shortly after performing the
experiments since it was felt that the remaining powder was
subject to less decomposition after the lipids were removed.
Removal of lipids. The lipids were removed by
homogenizing an aliquot of the dried powder with 95 per
cent ethyl alcohol, and centrifuged. The centrifuged
material was extracted two additional times with alcohol.
It was then extracted two times with a 3=1 mixture of
ethyl alcohol and ether. The remaining material was
washed twice with ether, centrifuged, and air dried. This
powder, lyophilized and lipid free, was used for the
extraction of the nucleic acids.
Extraction of the nucleic acids. The method used
for the extraction of the nucleic acids, RNA and DNA, was
that of Schmidt and Thannhauser (19^5) as modified by
Schneider (19^5)- The acid soluble nucleotides were
removed by homogenizing the lipid-free powder with 10
per cent trichloroacetic acid. The extracted residue was
washed two times by centrifugation and resuspension in
5 per cent trichloroacetic acid. All of the above steps
were carried out at 0° centigrade. The nucleic acids were
removed by solubilizing the residue in 1.0 N. potassium
hydroxide for eighteen hours. The RNA was obtained by
jchilling the solution to 0° centigrade, and neutralizing
27
It with an appropriate volume of hydrochloric acid and
five per cent trichloroacetic acid. The residue was
redlssolved and reprecipitated to insure removal of the
RNA. The residue, which consists of DNA and protein, was
heated for twenty minutes in five per cent trichloroacetic
acid to free the DNA. The protein was removed by
centrifugation.
An aliquot of each RNA and DNA fraction was used
to determine total amounts of the respective nucleic
acid present. The orcinol method of Nejbaun (1939) was
used to determine the RNA present. Seibert's (19^-0)
dlphenylamlne reaction was used to determine the amount
of DNA present. The amount of each nucleic acid was
determined from standard curves which had been prepared
from purified samples.
The RNA and DNA fractions were hydrolyzed one
hour in a solution made 1.0 N. with respect to
hydrochloric acid. The solution which contained the
liberated purine bases was neutralized with ammonium
hydroxide and the purine bases were precipitated as their
silver salts by the addition of silver nitrate. The
purines were regenerated from the precipitate by boiling
the silver purine in 1.0 N. hydrochloric acid for fifteen
minutes. The free purines were then chromatographed.
28
Chromatography of purines. The purine "bases were
separated, by the method of Cohn (1949)# Dowex 50 X 8
columns, .8 X 6,0 centimeters were used in the separation.
Guanine was eluted with 1.0 N. hydrochloric acid. The
elution of adenine was made by increasing the normality
of the elutant to 2.0 N. hydrochloric acid. The
effluent from a given peak was combined and the acid
removed by lyophillzatlon.
Estimation of the bases. A known volume of 0.5 N.
acetic acid was added to each sample and the optical
density was determined in a Beckman Model DU spectro
photometer. Purity of the sample was checked by re
chromatography and by measuring the optical densities
at 250, 260, and 280 millimicrons wave length. The ratios
250/260 and 280/260 were used as criteria for purity.
Purified samples of guanine and adenine gave the follow
ing ratios:
guanine 250/260 1.37 guanine 280/260 .84
adenine 250/260 .76 adenine 280/260 .37
The amount of purine present was determined from
its molecular extinction coefficient calculated from
standard curves made from pure samples of each base. The
following values were obtained and are similar to those
published by Chargaff and Davidson (1955):
29
E 260 X 10“3 guanine = 8.0, E 260 X 10"3
adenine ~ 13oO
Isotope analysis. Planchets containing Infinitely
thin layers of the base were made by pipetting known
amounts of the sample on aluminum planchets. The
planchets were dried and the radioactivity level was
determined by counting the sample in a windowless gas
flow counter. Duplicate samples were counted for each
determination*,
Protein and amino acid analysis. The trichloro-
acetlc-precipltated protein samples were solubilized in
warm concentrated formic acid according to the method of
Stein (1937). The solution was then placed on planchets,
dried under a heat lamp, and counted as mentioned above.
The amino acids were separated and purified by the
method of Moore and Stein (195*0 • Protein samples were
hydrolyzed in 5*0 N. hydrochloric acid. The hydrolysates
were chromatographed on .9 x 150 centimeter columns of
Dowex 50 X 4, 200 to 400 mesh. The eluted peaks were
compared with separations of a synthetic mixture of known
amino acids. Furthermore, the separations were Identical
with those obtained by Moore and Stein.
The procedure for the quantitative determination
of the amino acids was essentially that of Yemm and
30 '
Cocking (1955) • The ninhydrin color obtained by this
technique was read in a Model DO Beckman Spectrophotometer
at 540 millimicrons, Standard ninhydrin color curves
were prepared from pure samples•
I
Experimental
Effect of prolonging cleavage. In this preliminary
study, three trays of eggs were cultured at 5° centigrade.
One tray, 53^» was collected and lyophilized Immediately
after fertilization. Analyses were made for total acid
soluble material, total nucleic acids present, and
protein. Experiments numbered 535 sltkL 536 were treated
exactly as culture 53^ except the embryos remained in the
culture chamber for a period of eighteen hours and
twenty-four hours respectively. Aliquots were removed and
allowed to equilibrate at room temperature. Microscopic
examination was made to determine if the embryos were still
viable and also to determine if there was any alteration
in cleavage time.
Effect of time of treatment. Experiments numbered
513-522 were planned in which the embryos were cultured in
the presence of 3.0 X 10“^ molar estradiol and
glycine-2-C^ for times ranging from eight to twenty-six
hours. The effect of placing the embryos in a medium
containing estradiol immediately after fertilization or
31 1
after two cleavages had occurred was studied. Control
cultures, with no estradiol present, were identical in
all other aspects.
Glyclne-l-C-*-^ and glycine-2 - Incorporation.
Cultures numbered 610 and 612 were exposed to 3 X 10“3
molar estradiol nine hours after fertilization. Cultures
numbered 609 and 6ll served as controls. The embryos had
undergone only two cleavages at the end of this period.
Four hours prior to the termination of the culture,
glycine-l-Cl^ was added to cultures 609 and 610, while
glycine-2-C1^ x^as added to 611 and 612.
Effect of adenosinetriphosphate. Cultures
numbered 63^ and 635 were grown in the presence of
3 X 10“3 molar estradiol from the time of fertilization to
the end of the culture. Adenosinetriphosphate, .001
molar, was added to culture numbered 635• The culture
numbered 636 served as a control. Four hours prior to
termination of the culture period, glycine-l-C^ was added
to each culture.
Comparison of estradiol and testosterone. Cultures
numbered 615 and 616 were grown in the presence of
3 X 10“3 molar estradiol and testosterone respectively.
Culture numbered 617 served as control. Glyclne-l-C^
was added four hours prior to termination of the culture
periodo
RESULTS
It was shown In experiments 53^» 535» and 536 that
the egg did not lose its viability even after a very
prolonged period during which the temperature was kept at
5° centigrade and no cleavages occurred. However, when
the embryos were returned to room temperature, the next
cleavages occurred very rapidly. Some cleavages occurred
within ten minutes after the temperature was raised to
15° centigrade.
Table I shows the nucleic acid level after the
various time intervals. It is quite evident from these
data that there was no change in the total amount of RNA
present. However, the DNA content was decreased by about
six per cent. Although this is a slight decrease, it
does indicate a change.
The acid soluble nucleotide fraction was examined
to see if any apparent change occurred In this fraction.
Figure 1 shows a curve on which is plotted the difference
between the ultraviolet absorption spectra of the acid
soluble fraction of the embryos which were frozen immedi
ately after fertilization, and the cultures kept for
eighteen hours at 5° centigrade. Small peaks are shown
at 2k2, 250, 260, 266, and 279 millimicrons. Since this
curve was plotted from the differences in absorption
34
spectra at two different times, it probably Indicates a
decrease in concentration of those compounds which have
maxima..at„ the respective wavelengths* It is interesting
to note that xanthine, hypoxanthine, guanine, adenine,
and thymine have absorption maxima at or very near these
wavelengths.
Table II presents the data of the preliminary
studies showing concentration of the nucleic acids of
control and estradiol-treated embryos. The results are
expressed as micrograms of the nucleic acid contained in
one gram of lyophillzed lipid-free powder. The lipid-free
powder was used since it had been noted in previous
studies that the lipid content of different batches of
eggs varied considerably and, therefore, the usual
expression of concentration as a function of the embryonic
dry weight did not seem reliable. Table II clearly shows
that there is little or no effect on the RNA concentration
while the DNA concentration Is reduced In each experi
mental culture.
Plates I, II, and III are photomicrographs which
show the effects of estradiol on cleavage in sea urchins,
Strongylocentrotus purpuratus and Strongylocentrotus
franciscanus. During the first four cleavages, incomplete
cleavage furrows are easily discernible in those embryos
cultured In the presence of estradiol. Furthermore, In
35
addition to the irregular cleavage, there seems to be a
large number of unequal cells formed. In some embryos
single blastomeres are protruding from the group of
apparently normal blastomereso Plate III, a later stage,
shows the increased separation of the individual blasto
meres in the estradiol-treated embryos. It should be
noted that even though some of the experimental embryos
appeared to cytolyze, nearly all of them reached the
swimming stage.
The effect of estradiol treatment on the incorpora
tion of glyclne-2-Gl^ into the purine bases of both DNA
and RNA is recorded in Table III. These results show an
Increase in the relative specific activity of each of the
purines of the experimental cultures. The source of the
original estradiol used is mentioned in the Materials and
Methods section. This estradiol was obtained in tablet
form and had to be extracted from its binder before it
was used. Since no actual assay was made, it could well
be the case that the estradiol in the culture used in these
experiments was considerably less than 3 X 10“3 molar.
The results of the effects of estradiol on the
level of activity of the free amino acids, glycine and
serine, are recorded in Table IV. The Increased relative
specific activity for each amino acid parallels the
increased activity of the purine bases as reported above.
36 !
Table V summarizes the effect of estradiol treat
ment on the incorporation of glycine-l-C-^ and
glyc ine—2— into the bases of DNA and RNA. A depression
in the incorporation of glycine is evident in glycine
containing the label in the number one position. Further
more, the depression occurs in both RNA and DNA. The
data show that there is an increase in the uptake of
glycine, or fragment thereof, in the adenine and guanine
when glyclne-2-C-*-^ was the precursor. There was relative
ly little change in the activity of the bases of RNA with
glyc ine-2-C^ „
Although the results are not shown in Table V,
the level of activity of the protein fraction from each
experiment was measured. These results show a depression
of thirty-eight per cent in relative specific activity of
the protein in the estradiol-treated embryos grown in the
presence of glycine-l-C1^ while the protein of the
* | h
estradiol-treated embryos cultured with glycine-2-Cx^
shows an increased activity of fifty-five per cent.
The results of an attempt to relieve the inhibition
of estradiol by the addition of ATP are shown in Table VI.
A consistent Increase in the relative specific activity of
the purine bases of DNA occurred when ATP was added. The
guanine activity of the RNA fraction actually exceeds the
activity of the control RNA guanine. The protein fraction,
37 !
i
In culture 635 containing ATP, shows a twelve per cent
increase in specific activity over the estradiol-treated
culture, 63^0
Microscopic examination did not show the charac
teristic estradiol cleavage delay in culture 635 which
contained ATP. Furthermore, culture 635> s-s shown in
Table VI, actually contained 10 per cent more DNA than
the control culture, 636, and twenty-one per cent more
DNA than culture 63^ which contained only estradiol.
Microscopic study on the comparative effects of
estradiol and testosterone showed the following differ
ences at the end of nine hours culturing:
(1) The control embryos were at the sixteen-
cell stage.
(2) Seventy per cent of the testosterone-treated
embryos were in the four-cell stage. A few of the embryos
had reached the eight-cell stage. Some of the blastomeres
appeared very large and may have actually been syncytial.
(3) Eighty per cent of the estradiol-treated
embryos had not cleaved. The remaining twenty per cent
had cleaved once.
Differences in the effect of these two hormones
on DNA concentration and protein activity are shown in
Table VII. Estradiol treatment caused a decreased ENA
concentration. This decrease also occurred in the second
38
estradiol culture which was allowed to continue until the
embryos appeared to be at the same stage of development
as the control culture at eighteen hours . The
testosterone-treated embryos show an Increased concentra
tion of DNA at the end of the culture periods#
The level of Incorporation of Isotope into the
protein fraction was decreased by approximately the same
percentage with either estradiol or testosterone.
The data showing the comparative effects of
estradiol and testosterone on the Incorporation of
glycine-l-C1^ into the purine bases are shown in
Table VIIIe However, the relative specific activity of the
ENA adenine of both experiments is not shown. The spectral
ratios, 250/260 and 280/260, used to determine purity of
the sample gave values that are not acceptable for adenine.
It appeared that some contamination occurred during the
removal of the hydrochloric acid In which adenine was
eluted from the chromatograph column. The available data,
as shown in Table VIII, Indicate a decreased incorporation
in the ENA and RNA purines in both the cultures which
contained estradiol and testosterone. The depression was
not as great in the testosterone-treated embryos.
Optical density X 10“3
39
230 --
220 --
210
200 ■■
190
180
170
160
140
130
120
110
280
Wave length in millimicrons
FIGURE 1
THE DIFFERENCE BETWEEN THE ULTRAVIOLET
ABSORPTION SPECTRA OF THE ACID SOLUBLE
FRACTION OF EMBRYOS IMMEDIATELY AFTER
FERTILIZATION AND AFTER EIGHTEEN HOURS
AT 5° CENTIGRADE
TABLE I
EFFECT OF PROLONGED DELAY
OF CLEAVAGE DUE TO LOW TEMPERATURE
ON RNA AND ENA CONCENTRATION
Experiment JT DNA * RNA
gm LLFP* gm LLFP*
(1) 535
5.2 x io3 50.3 x io3
(2) 536
4.9 X 103 50.6 x io3
(3) 537
4.9 x iq3 49.8 X 103
(1) At time of fertilization
(2) After eighteen hours at 5° centigrade
(3) After twenty-six hours at 5° centigrade
* LLFP = lyophilized lipid-free powder
TABLE III
EFFECT OF ESTRADIOL TREATMENT ON THE
INCORPORATION OF GLYCINE-2-C^ INTO THE
PURINE BASES OF ENA
Experiment Guanine Adenine
No.
513
C
77
X
10”3
22 X 10-3
51^
E 636 X io“3
152
X 10 “3
521 c
53
X 10 “3
75
X io-3
522 E 140 X 10-3
106 X io-3
515
c
31
X
10 “3 —
*516
E
877
X 10 “3 ----
51?
C
03
0 \
Ol
H
X
10“3
450 X
10 “3
*518 E 2490 X
io~3
380 X 10 ”3
C = Control
E = Experimental, treated with 3 X 10“3
molar estradiol
* = Estradiol added after embryos were in
eight-cell stage
Relative specific activity expressed as
counts/ralnute/raicromole/microgram DNA
TABLE IV
EFFECT OF ESTRADIOL ON THE LEVEL OF ACTIVITY OF
THE FREE ANINO ACIDS, GLYCINE AND SERINE
Experiment
Number
Serine Glycine
513 C
1970 10420
514 E 2250 11600
517 C
— —
*518 E
— —
521 C
4370 9070
522 E 5050 10400
C = Control
E = Experimental, 3 X 10“3 molar estradiol
added at time of fertilization
* = Estradiol added nine hours after
fertilization
Relative specific activity of glycine and
serine expressed as counts/minute/mlcromole
TABLE V
EFFECT OF ESTRADIOL TREATMENT ON THE INCORPORATION OF GIXCINE-l-C1^
AND GIXCINE-2-C14 INTO PURINE BASES OF RNA AND ENA
Experiment
Number
DNA
Adenine Guanine Adenine
RNA
Guanine
609 C glycine-l-C1^
765
2803 220
367
610 E glycine-l-C^ 274 800
157
274
611 C glycine-2-C1^ 102 1214 256
353
612 E glyclne-2-C1^ 1013 4249 102 143
C = Control
E - Experimental
Relative specific activity expressed as counts/minute/micromole
TABLE VI
EFFECT OF SIMULTANEOUS TREATMENT WITH ADENOSINETRIPHOSPHATE AND ESTRADIOL
ON THE LEVEL OF ENA AND RNA, AND THE INCORPORATION OF GIZCINE-l-C^
INTO THE PURINE BASES AND PROTEIN
Fraction 634
Adenine
635
Adenine
636
Adenine
634
Guanine
635
Guanine
636
Guanine
DNA
1855
2126
2565 3857
518 6 5722
RNA
905
1180 1360
417
1100
815
Fraction 634
635
636
Protein 58 65 67
i DNA
gm LLFP
/RNA
gm LLFP
6 .9 1 x io3
52,9 X 103
8.4 X io3
48.6 X io3
7.7 X io3
48.3 X io3
LLFP = Lyophilized lipid-free powder
Relative specific activity of adenine and guanine expressed as
counts/minute/micromole
Relative specific activity of protein expressed as counts/minute/milligram
634 - Estradiol
635 - Estradiol and ATP
636 = Control
46
TABLE VII
A COMPARISON OF THE EFFECT OF ESTRADIOL AND
TESTOSTERONE ON THE ENA CONTENT AND
INCORPORATION OF GLYCINE-l-C^ INTO PROTEIN
Culture Culture
Time
(hours)
Y DNA
gm LLFP*
Protein
Activity
Control 18
5.75 X 103 80.3
Estradiol (1) 18 2,45 X io3 4?
Es tradlol (2) 30 4.44 X 103 106
Tea tos terone (1) 18 8.11 X 103 48
Tea tost erone (2) 20 18.66 X 103 48
* » lyophilized lipid-free powder
Relative specific activity of protein expressed
as counts/mlnute/railligram protein
TABLE VIII
A COMPARISON OF THE EFFECT OF ESTRADIOL AND TESTOSTERONE
ON THE INCORPORATION OF GLICINE-l-C1^ INTO THE DNA AND RNA PURINE BASES
Culture
Adenine
ENA
Guanine Adenine
RNA
Guanine
Control
1695
6141 350 750
Estradiol (1) —
955 171
216
Estradiol (2) —
3279 596 601
Testosterone (1) — 1070 222 272
Testosterone (2) 2036
335 239
Relative specific activity of adenine and guanine expressed
as counts/minute/micromole
D IS C U S S IO N
It is apparent from the data that embryos cultured
at 5° centigrade did not synthesize ENA. Furthermore,
no cellular division occurred. These two results seem to
agree with current concepts of cell division and ENA
synthesis. Cohn and Volkin (1957) have stated that most
of the recent evidence continues to support the hypothesis
that DNA is formed only incidental to cell division and
is presumably metabolically inert thereafter.
The increased synchrony of cleavage of embryos
subjected to decreased temperature before the culture
period deserves some discussion. Swann (1957)» in a
review of the control of cell division, has pointed out
that temperature shocks have been shown to increase
synchrony in many microorganisms and in tissue cultures.
The mechanism by which this is accomplished is far from
being understood.
In the sea urchin, Strongylocentrotus purpuratus.
it appears that part of the synchronization is probably
Involved with DNA synthesis. It was shown that embryos
undergoing prolonged exposure to cold temperature had a
decreased concentration of acid soluble material, presum
ably nucleotides or nucleosides of hypoxanthine, adenine,
guanine, and thymine. Furthermore, the DNA concentration
^9
was reduced. One possible explanation of the synchronized
development might be given. Those cells which are in an
advanced state in the process of cell division may actually
be set back or, they may be held in some particular stage
of cell division. The prolonged period in which the
embryos are exposed to the decreased temperature might
allow for an equilibrium state to develop in which the
chemical reactions would proceed to that point of the
particular energy level which the culture temperature
permits.
It has been proposed by many workers that most of
the DNA utilized In the initial cleavage comes from
cytoplasmic ENA. Agrell and Persson (1956) hypothesized
that the nuclear ENA arises from a carefully controlled
breakdown of the cytoplasmic DNA. These products must
then be reconstituted to form the nuclear ENA.
Since the amount of ENA remained constant after
the eighteen-hour period, the Initial decrease could
well represent the amount necessary, along with the
decreased acid soluble material, for the synthesis of
ENA for the first cleavages. This would necessitate the
existence of some compound which was not acid soluble
and which would not give a positive reaction (ENA) to
the dlphenylamine test.
This part of the Investigation was done only to see
50
if the embryo would be viable after prolonged exposure
to decreased temperature. Therefore, no additional effort
was made to study the mechanism involved. Further work
v/ould be necessary before any valid conclusions could be
made.
It has been shown in all of the experiments con
ducted that estradiol inhibited cleavage during the early
stages of development of the sea urchin. These results
are in agreement with those obtained by other workers pre
viously cited.
In the early experiments the concentration of
estradiol administered was not known. However, it is
presumed that the concentration was below 3 X 10“3 molar.
At this concentration, there was an Increase in the uptake
of labeled glycine in all of the purine bases when
estradiol was present. There was also an increased
activity In the free amino acids, serine and glycine.
These data are in agreement with the results of Mueller
et al. (1958) who have shown that estradiol stimulates the
metabolism of one-carbon fragments through the folic acid
system.
The use of a relatively high concentration of
estradiol, 3 ^ 10“ *3 molar, caused some very interesting
changes in the metabolism of the embryos. The remainder
of the dissertation discussion will be concerned with the
51
results of these experiments.
In the estradiol-treated embryos, in which
glycine-1-C1^ was used as the purine precursor, the
characteristic depression of activity occurred in the
protein and the purine bases of DNA and RNA. However,
in the estradiol-treated embryos containing glycine-2-cl^
as a precursor, the specific activity of the purine bases
of ENA was markedly increased. This finding further
substantiates the concept of the sensitivity of the one-
carbon transfer system to estradiol.
A proposed method by which the DNA purines might
have increased specific activity is given. Estradiol
might initiate the reactions by stimulating the enzyme
serine aldolase. Serine aldolase catalyzes the reaction
by which a one-carbon fragment is condensed with glycine
to form serine. Serine has been shown to contribute one
carbon to 5-&mino-4-imidazole-carboxamide ribotide and
form 5~fcrmamido-4— imldazole-carboxamlde ribotide. The
uformamido" compound in turn undergoes ring closure
giving rise to the purine nucleotide, inosine monophos
phate, which is a precursor of the purine nucleotides.
The increased activity in the DNA purines seems consistent |
with these reactions.
The decrease in the specific activity of the RNA j
purine bases does not seem consistent with the increase In
52
the DNA purines. However, all of the experiments have
shown that RNA is undergoing very slow synthesis during
this stage of development. Moreover, the cytoplasmic DNA
could contribute purine components if it were being
degraded as has been proposed. These purines from the
cytoplasmic DNA would be available for RNA synthesis and
would tend to decrease the specific activity of RNA
purines.
Agrell and Persson (195&) have demonstrated that
estradiol does indeed cause a degradation of sea urchin
sperm DNA.
In light of the increased activity of the IMA
purines, the decrease in the total amount of DNA and the
inhibition of cleavage would not seem to be dependent upon
some block in purine synthesis but rather, the inhibition
of these two processes must be the result of some other
mechanism.
In a further attempt to investigate the inhibition
of cleavage, ATP was added to a culture of estradiol-
treated embryos. Microscopic examination showed that there
was no delay in cleavage. Furthermore, an increase in the
DNA concentration was shown. These findlngs, along with
i the observed Increase in the relative specific activity
of the protein fraction and the purine bases of RNA, offer
! evidence that ATP had decreased the inhibition in part,
53
if not completelyo
Scott and Engel (1957) have shown that steroids
are capable of forming complexes with purine bases and
with compounds which contain purines * If this were to
occur with estradiol and some high energy-containing
nucleotide such as ATP, it seems probable that the energy
reservoir of the cell could be diverted to other processes
than those which bring about cleavage in the embryo.
Swann (1957) suggests that energy partitioning processes
are involved in normal embryonic development.
A regulatory function of estradiol at many loci
is possible when the total number of purine-containing
compounds is considered. Many of these purine nucleotides
serve as cofactors in the chemical reactions that occur
within the cell.
Although no permeability studies were made, it was
observed that the estradiol-treated embryos were slightly
larger than the control embryos. Buteow (1959), Spazianl
and Szego (1958), and others have shown that estradiol
increases the permeability of different cells. Therefore, j
it is entirely possible that the action of estradiol might
be to alter the movement of substances across the cell
membrane.
In an attempt to determine if the action of
| estradiol was a specific or a general action that any
54
steroid hormone would produce, a comparative study was
made between the effects of testosterone and estradiol
on the sea urchin embryos« It was shown that the effect
of estradiol and testosterone is apparently not the same
on such embryos. Testosterone did not cause as extensive
delay in the cleavage as did estradiol. Moreover,
testosterone-treated embryos showed an increased level
Of DNA.
SUMMARY
lo The effect of estradlol-17B on cleavage,
nucleic acid metabolism, and protein synthesis in embryos
of the sea urchin, Strongylocentrotus purpuratus, was
studied.
2. Increased synchronization of development
occurred in embryos subjected to 9° centigrade for nine
hours.
3. Prolonged exposure to cold did not decrease
viability, but did change the ultraviolet absorption
spectra of the acid soluble fraction.
A. DNA concentration was decreased and cleavage
was inhibited in estradiol-treated embryos.
5. The utilization of one-carbon fragments in
purine synthesis, as shown by incorporation of
glyclne-l-C^ and glycine-2-G^, was stimulated by
estradiol.
6. Adenosinetriphosphate decreased the inhibition
of estradiol-treated embryos.
7. Testosterone did not cause the marked delay
in cleavage as did estradiol. Moreover, testosterone-
treated embryos showed an Increased level of DNA.
8. A mechanism was proposed whereby estradiol
might stimulate the enzyme serine aldolase and thus
increase the utilization of one-carbon fragments in the
synthesis of the nucleic acid purine bases.
9o The possibility of a regulatory function of
estradiol in embryonic development by the formation of
complexes with compounds which contain purines was dis
cussed.
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L_
APPENDIX
PLATE I
Embryos of the sea urchin,
Str ongy loc entr o tus franc is canus.
after nine hours of development*
Photograph 1. Control embryos#
Photograph 2# Estradiol-treated
embryos# Magnification about
150 X.
PLATE I
PLATE II
Embryos of the sea urchin,
Strongylocentrotus purpuratus.
after fifteen hours of develop
ment, Photograph 1, Control
embryos. Photograph 2, Estradiol-
treated embryos• Magnification
about 150 X,
70
PLATE II
PLATE III
Embryos of the sea urchin,
Strongyloc entrotus francIscanus«
after twenty hours of develop-
ment» Photograph 1, Control
embryos. Photograph 2. Estradiol-
treated embryos. Magnification
about 150 X.
72
PLATE III
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Jolley, Weldon Bosen
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The effects of estradiol-17B on cleavage, nucleic acid metabolism, and protein synthesis in embryos of the sea urchin, Strongylocentrotus purpuratus
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1959-08
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Martin, Walter E. (
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