/. Embryo/, exp. Morph. Vol. 30,1, pp. 243-256, 1973
Printed in Great Britain
243
Early cyclical changes in polyamine synthesis
during sea-urchin development
By CAROL-ANN MANEN 1 AND DIANE H. RUSSELL 2
From the Department of Zoology, University of Maine, and
National Institutes of Health, National Cancer Institute,
Baltimore Cancer Research Center
SUMMARY
The polyamines, putrescine, spermidine, and spermine, undergo dramatic cyclical variation
in both synthesis and accumulation during the early cleavage stages of sea-urchin development. Ornithine decarboxylase activity (putrescine synthesis) in developing Strongylocentrotus
purpuratus exhibits maxima at •£• and 2 h after fertilization; increases in ornithine decarboxylase activity appear to correspond to the first and second S phases. Putrescine-stimulated
S-adenosyl-L-methionine decarboxylase (spermidine synthesis) and spermidine-stimulated
S-adenosyl-L-methionine decarboxylase (spermine synthesis) activities reflect rises during
prophase-metaphase of the first and second divisions in two species of sea urchins. Cyclical
changes in the concentrations of these three amines were evident also. In general, there were
drops in the levels of the amines prior to cleavage. These rapid declines in polyamine concentrations may reflect (.1) selective degradation or (2) selective secretion.
INTRODUCTION
Polyamines have not been studied extensively in marine invertebrates. There
have been two reports of the presence of spermine in such, one in an echinoderm,
Echinarachinius mirabilis (Ogata & Komada, 1943), and the other in the tunicate
Cionia infest inalis (Ackermann & Janka, 1954). We have studied polyamine
biosynthesis and accumulation in several species of sea urchins (Manen &
Russell, 1973). Gametes of sea urchins as well as their adult tissues contain high
amounts of spermine and relatively low amounts of putrescine and spermidine.
This is in contrast to the polyamine patterns exhibited by other major groups.
For instance, bacteria and amphibians contain substantial amounts of putrescine and spermidine, with spermine either absent or present only in trace
amounts (Tabor & Tabor, 1964; Russell, Snyder & Medina, 1969). Spermidine
and spermine, present in similar amounts, are the major polyamines detectable
in mammalian tissues, with putrescine present in low amounts (Tabor & Tabor,
1964). However, the putrescine concentration increases rapidly when tissues
1
Author's address: University of Maine, Department of Zoology, Orono, Maine, U.S.A.
Author's address: National Institutes of Health, National Cancer Institute, Baltimore
Cancer Research Center, Laboratory of Pharmacology, Baltimore, Maryland 2.12.1.1, U.S.A.
2
16-2
244
C.-A. MANEN AND D. H. RUSSELL
L-ORNITHINE
0
ORNTTHINE
DECARBOXYLASE
CH2
I
CH2
|
CH,
'
NH +
3
NH
*
LH
>
<-H 2 -CH 2 -CH 2 -
PUTRESCINE
(DIAMINOBUTANE)
+
Decarboxylation CH 2 -CH 2 -CH 2 -NH 3 +
and
From
propylamine
SAM
transfer
CH 2 -CH 2 -CH 2 -NH 3 +
From SAM +
-NH 2 + -(CH 2 )3-NH3 + NH 3 + -(CH 2 ) 4 -NH 2 + -(CH 2 )3-NH 3 +
Decarboxylation
and
SPERMINE
. .
SPERM1DINE
propylamine
transfer
Fig. 1. Schematic polyamine biosynthetic pathway.
undergo growth processes, i.e. in regenerating rat liver (Dykstra & Herbst,
1965;Janne & Raina, 1968), in cardiac hypertrophy (Russell, Shiverick, Hamrell
& Alpert, 1971; Feldman & Russell, 1972), and in appropriate mammalian
tissues after hormonal stimulation (Pegg & Williams-Ashman, 1968; Russell &
Snyder, 1969; Janne & Raina, 1969; Russell, Snyder & Medina, 1970; Russell
& Taylor, 1971; Russell & Potyraj, 1972).
The polyamine biosynthetic pathway in sea urchins appears to be similar to
those reported for yeast (Coppoc, Kallio & Williams-Ashman, 1971), amphibians (Russell, 1971), and mammals (Pegg & Williams-Ashman, 1969; Feldman,
Levy & Russell, 1972), and differs from the bacterial systems (Tabor & Tabor,
1964). In general, the precursor for polyamine synthesis is ornithine. The decarboxylation of ornithine results in the formation of putrescine (Fig. 1). Spermidine and spermine are formed from putrescine by the addition of one or two
propylamine moieties respectively (Fig. 1). In bacteria, the conversion of
putrescine to spermidine involves two separate enzymic reactions (Tabor &
Tabor, 1964). The first is the enzymic decarboxylation of S-adenosyl-L-methionine to form carbon dioxide and 5'-deoxy-5'-S'-(3-methylthiopropylamine)
sulfonium adenosine (decarboxylated S-adenosyl-L-methionine). This 5-adenosyl-L-methionine decarboxylase required Mg 2+ and contains pyruvate as a
prosthetic group. A propylamine transferase then catalyzes the formation of
spermidine from putrescine and a propylamine molecule which derives from
decarboxylated S-adenosyl-L-methionine. In sea urchins as well as in mammals, at
least in crude homogenates, there appears to be a coupling of the decarboxylase
Cyclical changes in polyamine
synthesis
245
and transferase. Decarboxylated S-adenosyl-L-methionine cannot be separated
as a free intermediate, and putrescine or spermidine are required to accept the
propylamine molecule (Manen & Russell, 1973, and data in this paper).
In contrast to the bacterial system, metal ions are not required nor is pyruvate
known to be a cofactor. There may be a pyridoxal phosphate requirement
(Feldman et al. 1972). We have reported that the pathway in sea urchins, like
the mammalian system, is stimulated by putrescine or spermidine, does not
exhibit metal requirements and there is coupled decarboxylation and transfer
function. Although there is controversy as to whether purification of S-adenosylL-methionine decarboxylase leads to uncoupling of these two functions (Janne
& Williams-Ashman, 1971; Janne, Schenone & Williams-Ashman, 1971;
Feldman, et al. 1972), the important consideration here is the coupling in crude
homogenates. The rate-limiting step in spermidine or spermine synthesis
appears to be the activity of -S-adenosyl-L-methionine decarboxylase. Therefore
the most accurate estimates of spermidine and spermine synthesis can be
obtained from the measurements of putrescine-stimulated S-adenosyl-Lmethionine decarboxylase and spermidine-stimulated S-adenosyl-L-methionine
decarboxylase respectively.
In a study of changes in polyamine biosynthesis and accumulation during
sea-urchin development, we found that during development (i.e. to gastrulation) the spermidine concentration increased markedly, with little change in
either putrescine or spermine concentration. However, prior to fertilization
spermine concentration can be as much as tenfold above that of spermidine.
After gastrulation both spermine and spermidine were elevated. Enzyme
activity patterns paralleled the changes detected in the levels of polyamines with
the exception of ornithine decarboxylase activity.
In this paper we report on studies of the changes in activities of the polyamine
biosynthetic enzymes and in the levels of polyamines of sea-urchin eggs within
the first 4 h after fertilization. Essentially, synchrony of cell division is exhibited
at least through the first two cell cycles. Sharp drops in polyamine synthesis
occur prior to cell division, along with marked drops in pool sizes. Therefore,
cyclical variations exist in early polyamine synthesis as well as in other early
biochemical events that have been studied in sea urchins (Mano, 1970; Lovtrup
& lverson, 1969). Labelling experiments reported herein verify the biosynthetic
pathway in sea urchins, and studies of partially purified S-adenosyl-L-methionine
decarboxylase substantiate the coupling of decarboxylation and transfer activities in both spermidine and spermine formation. The discrepancy stated earlier
in this paper between ornithine decarboxylase activity and putrescine levels in
sea urchins is explained by the very low Km for putrescine exhibited by partially
purified S-adenosyl-L-methionine decarboxylase.
246
C.-A. MANEN AND D. H. RUSSELL
MATERIALS AND METHODS
Materials
Ripe Lytechinus pictus and Strongylocentrotus purpuratus were obtained from
the Pacific Bio-Marine Supply Co., Venice, California. Spawning was induced
by injection of 0-55 M-KCI. Eggs were collected and washed in filtered artificial
sea water. Only those cultures with at least 90 % normal development were used
in experiments. [Carboxyl-14C]5'-adenosyl-L-methionine (7-7 mCi/mM), [1-14C]DL-ornithine (11-9 mCi/mM) and [l-4-14C]putrescine dihydrochloride (20-29
mCi/m.M) were obtained from New England Nuclear.
Preparation of enzyme solutions
The material was homogenized in 4 vol. of 005 M sodium-potassium phosphate buffer, pH 7-2, containing 0-1 HIM dithiothreitol. These enzymes are
inhibited by Tris buffer, as are the mammalian enzymes. The homogenate was
centrifuged at 20000# for 20 min, the pellet discarded, and the supernatant used
in the assays. Since the supernatant after centrifugation at 100000 g for 90 min
gave the identical enzyme activities, the 20000# supernatant was used routinely.
Protein was determined by the Lowry method (Lowry, Rosebrough, Farr &
Randall, 1951) with bovine serum albumin as the standard.
Assay for ornithine decarboxylase activity
Ornithine decarboxylase activity was determined by measuring the liberation
of 14CO2 from [l-14C]ornithine as described previously (Russell & Snyder, 1968;
Russell & Snyder, 1969). Although the substrate concentration (0-1 HIM as
L-ornithine used routinely) was non-saturating, the same changes were noted
when excess ornithine (2 HIM) was used as substrate in some experiments.
Assay for putrescine-stimulated S-adenosyl-L-methionine decarboxylase activity
Enzyme activity was determined by measuring the liberation of 14CO2 from
[carboxyl-14C]5'-adenosyl-L-methionine as previously described (Pegg &
Williams-Ashman, 1969). Unless otherwise stated, incubation mixtures consisted of 0-1 mM [carboxyl-^CJS-adenosyl-L-methionine, 25-50 HIM sodiumpotassium phosphate buffer (pH 7-2), 50 /*M pyridoxal phosphate, 2-5 mM
putrescine, and 0-1-0-15 ml (3-5 mg protein) of enzyme solution in 0-2 ml. When
the formation of [14C]spermidine from unlabelled S-adenosyl-L-methionine
and [l,4-14C]putrescine was estimated as previously described (Russell &
McVicker, 1972), there was a stoichiometric relationship between the amount
of [14C]-spermidine formed and the evolution of 14CO2 when [carboxyl-14C].Sadenosyl-L-methionine was added. Therefore, 14CO2 evolution from [carboxyl14
C]5'-adenosyl-L-methionine was used routinely as a measure of the spermidine
biosynthetic rate.
Cyclical changes in polyamine synthesis
247
Assay for spermidine-stimulated S-adenosyl-L-methionine decarboxylase activity
This assay is identical to that for putrescine-stimulated S-adenosyl-L-methionine decarboxylase except 5 mM spermidine was added instead of 2-5 mM
putrescine. Again, there was a stoichiometric relationship between the amount
of [14C]spermine formed and the evolution of 14CO2 when [carboxyl-14C]-Sadenosyl-L-methionine was added. Therefore, 14CO2 evolution from [carboxyl"ClS-adenosyl-L-methionine was used routinely as a measure of spermine
bio synthetic rate.
Determinations of putrescine, spermidine and spermine concentrations
Pools of embryos were homogenized in 4 vol. of 0 1 N-HCl and subjected to
alkaline butanol extraction as previously described (Russell, Medina & Snyder,
19 70).TC A was extracted by ether washes prior to butanol extraction. The butanol
was evaporated to dryness in an evapomix and the residue redissolved in 0-2 ml of
0-1 M-HCI. The amines were separated by high-voltage electrophoresis (80 V/cm
for 1-5 h) in a 0-1 M citric acid buffer, pH 4-3. Concentrations were determined
by staining the chromatography sheet (Whatman 3 MM paper) with a mixture
of 1 g ninhydrin, 100 ml acetone, 5 ml glacial acetic acid, 10 ml H2O and 100 mg
cadmium acetate, drying 60 min at 60 °C, eluting the color, and recording the
absorbancy at 505 nm. Standards were run in the same range as the samples.
Purification of the enzyme
The operations described below were done at 0-5 °C.
Crude extract
Freshly spawned unfertilized eggs of L. pictus were homogenized with 4 vol.
of 0-05 M phosphate buffer, pH 7-2, containing 1-0 mM EDTA, 0-1 mM dithiothreitol, 3 /(M pyridoxal phosphate and 0-5 M sucrose.
Cellular debris was removed from the crude homogenate by centrifugation at
40000 g for 20 min. The resulting supernatant solution was recentrifuged at
100000# (in a Beckman L3-5O ultracentrifuge) for 1 h and then passed through
cheesecloth to remove the lipid layer.
Filtration on Sephadex G-100
A 9 ml portion of the 100000# supernatant solution was applied to a Sephadex G-100 column (45 cm x 4-9 cm2) which had been equilibrated previously
with the homogenizing buffer. The enzyme was eluted with the equilibrating
buffer under a hydrostatic pressure of 20 cm at a flow rate of 25 ml/h and the
eluate was collected in 4 0 ml fractions. The most active fractions were pooled.
248
C.-A. MANEN AND D. H. RUSSELL
DEAE-Cellulose chromatography
The pooled enzyme fraction was dialyzed for 1 h against two changes of 50
vol. of 0-01 M phosphate buffer, pH 7-2, containing 1 mM EDTA, 0-1 mM
dithiothreitol, 3 fm pyridoxal phosphate and 0-5 M sucrose. Then 30 ml of the
dialyzed preparation were applied to a DEAE-cellulose column (15 cm x 4-9
cm2) which had been equilibrated previously with the same buffer as used in the
dialysis. After the column had been washed with 90 ml of the equilibrating
buffer, the active enzyme was eluted with 300 ml of a linear gradient of 0-1 M-KCI
in the equilibrating buffer. Fractions (4 ml) were collected, and the most active
fractions were pooled. At this stage enzyme activity was stable on storage at
— 20 °C for more than 6 months.
Labelling
Fertilized eggs were centrifuged gently (270 g for 1 min) and resuspended in
250 ml of sea water with 2 mCi of [14C]putrescine (0-1 mmoles). After 30 min
the eggs were washed twice with x 50 vol. and cold-pulsed for 10 min with 1 mM
putrescine. They were then washed twice and resuspended in 300 ml of sea
water.
The polyamines were extracted as described above with the exception that the
ninhydrin-stained spots were cut out and eluted with 5 ml of methanol for 30
min, after which 10 ml of toluene omnifluor was added and the radioactivity
counted on a Beckman LS-150 liquid scintillation counter. Corrections were
made for the quench due to the ninhydrin color.
RESULTS
Ornithine decarboxylase activity {putrescine formation)
Earlier we reported a marked elevation in ornithine decarboxylase activity in
L.pictus eggs within 1 h of fertilization (Manen & Russell, 1973). When enzymic
activity was measured at earlier times, it was found that ornithine decarboxylase
activity had increased over fourfold within \ h and within 1 h the activity had
dropped to twofold above the level present in unfertilized eggs (Fig. 2). Ornithine
decarboxylase activity continued to decline and was below the control level at
3 h post-fertilization.
Ornithine decarboxylase activity in fertilized eggs of S. purpuratus exhibits
two marked cycles within the first 4 h (Fig. 3), one which has a maximum at \ h,
similar to that of L. pictus, and another which is maximal at 2 h, at which time
ornithine decarboxylase activity is 14-fold greater than the level found in unfertilized eggs.
Cyclical changes in polyamine synthesis
249
1800
1
2
3
Time after fertilization (h)
4
Fig. 2. Ornithine decarboxylase activity in early cleavage stages of L. pictus.
Activity was determined by measuring the evolution of 14CO2 from ["QL-ornithine
(Russell & Snyder, 1968; Russell & Snyder, 1969). Each point represents the mean ±
S.E. for four separate determinations.
Putrescine-stimulated S-adenosyl-L-methionine decarboxylase (spermidine
formation)
As previously reported, we found that putrescine-stimulated S-adenosyl-Lmethionine decarboxylase exhibits considerable activity in unfertilized eggs of
L. pictus (Manen & Russell, 1973). After fertilization the activity drops markedly
with a low point \ h after fertilization, and then cycles with maxima at \\ and
3 h respectively (Fig. 4). This is in contrast to the maxima at \ and 2 h found for
ornithine decarboxylase activity in L. pictus. The cycles are precisely 1 h out of
synchrony.
The same cyclical patterns were detected for putrescine-stimulated S-adenosylL-methionine decarboxylase of S. purpuratus (Fig. 5). However, the initial levels
250
C.-A. MANEN AND D. H. RUSSELL
1
2
3
Time after fertilization (h)
4
Fig. 3. Ornithine decarboxylase activity in early cleavage stages of S. purpuratus.
Activity was determined by measuring the evolution of 14CO2 from [14C]L-ornithine
(Russell & Snyder, 1968; Russell & Snyder, 1969). Each point represents the mean ±
S.E. for four separate determinations.
of this enzyme are lower in S.purpuratus and there is not the post-fertilization
drop in activity.
Spermidine-stimulated S-adenosyl-L-methionine decarboxylase (spermine
formation)
Since the level of spermidine-stimulated S-adenosyl-L-methionine decarboxylase is lower than that of putrescine-stimulated S-adenosyl-L-methionine
decarboxylase by a factor of 2, the cycles are not as striking but they are still
evident (Figs. 4, 5). There is low activity at \ and 2\ h respectively, and maximal
activities at \\ and 3 h in both species of sea urchins studied.
Variations in the polyamine pooh during early cleavage stages
After fertilization of S. purpuratus eggs, preliminary experiments indicate that
all three amines exhibit maximal concentrations 2\ h post-fertilization, followed
by marked declines by telophase of the second division.
A marked increase in the polyamines between 2 and 3 h after fertilization,
followed by a dramatic drop in concentrations, was measured in L. pictus also
(Fig. 6).
Cyclical changes in polyamine synthesis
251
6400
— Putrcscinc-stimulatcd
— Spcrmidinc-stimulated
5600
4800
4000
X
o on
3 sE
•e
3E
§d
3200
2400
1600
0
1
2
3
4
Time after fertilization (h)
Fig. 4. Putrescine- and spermidine-stimulated S-adenosyl-L-methionine decarboxylase activity in early cleavage stages of L. pictus. Activity was determined by
measuring the 14CO2 released from 14COOH-S-adenosyl-L-methionine in the
presence of the appropriate amine (see Materials and Methods). Each point
represents the mean ± S.E. for four separate determinations.
Therefore, not only do the polyamine biosynthetic enzymes exhibit cyclical
patterns, but also the products themselves. There must be either active secretion
of polyamines at discrete times or active degradation.
Distribution of radiolabel after incubation of embryos with [uC]putrescine
After the incubation of fertilized eggs of L. pictus with [14C]putrescine for \ h,
the eggs were washed with sea water containing cold putrescine, then resuspended in sea water alone. Samples were removed at \ h intervals and assayed
for radiolabeled amines. Radiolabel is detectable very rapidly, not only in
endogenous putrescine but also in spermidine and spermine (Fig. 7). The label
is present mainly in putrescine and spermidine. This agrees with the greatly
increased accumulations there during early after fertilization. These data
indicate further that the polyamine biosynthetic pathway is similar in sea urchins
to that already established for other major groups.
252
C.-A. MANEN AND D. H. RUSSELL
1600
\
r
Putrcscinc-stimulatcd
Spcrmidine-stimulatcd
1400 -
200
0
1
2
3
4
5
Time after fertilization (h)
6
Fig. 5. Putrescine- and spermidine-stimulated S-adenosyl-L-methionine decarboxylase activity in early cleavage stages of S. purpuratus. Activity was determined by
measuring the 14CO2 released, from "COOH-S-adenosyl-L-methionine in the
presence of the appropriate amine (see Materials and Methods). Each point
represents the mean ± S.E. for four separate determinations.
Properties of S-adenosyl-L-methionine decarboxylase from sea urchins
Through the use of the double-reciprocal plot, the apparent Kni for putrescine
was determined. Saturating levels of -S-adenosyl-L-methionine were used and
partially purified S-adenosyl-L-methione decarboxylase preparations from L.
pictus were utilized as the enzyme source in these assays. The Km for putrescine
under these conditions, 3 x 10~5 M, is an order of magnitude lower than the Km
for putrescine of this enzyme in rat liver (Table 1). The calculated Km for
spermidine obtained from enzyme preparations from L. pictus was 7 x 10~4 M.
This is similar to the Km calculated for the enzyme partially purified from S.
purpuratus (5 x 10~4 M). In both cases the values are lower than those obtained
from enzyme preparations of rat liver. The lower amounts of putrescine and
spermidine necessary to optimize their conversion to spermidine and spermine
respectively may account for both the lower amounts of putrescine present
in sea urchins and the higher levels of spermine that accumulate.
253
Cyclical changes in polyamine synthesis
180
160
E 140
o
JT 120
o
E
Putrescine
c
E 100
Spermidine
80
1«
D.
40 -
20 -
Spermine
-,
J>
1 2
3
4
Time after fertilization (h)
1 2
3
4
Time after fertilization (h)
Fig. 7
Fig. 6
Fig. 6. Polyamine pools in early cleavage stages oiL.pictus. Pools of embryos were
assayed for putrescine, spermidine and spermine by extraction of amines into
alkaline-1-butanol and separation by high-voltage electrophoresis (Russell, Medina
& Snyder, 1970). Each point represents the mean for two or more duplicate
determinations.
Fig. 7. 14C content of putrescine, spermidine and spermine in early cleavage stages
oiL.pictus. Fertilized eggs were incubated with [14C]putrescine for \ h at the start
of the experiment (see Materials and Methods). Each point represents the mean
for two separate determinations.
Table 1. S-adenosyl-h-methionine decarboxylase: comparison of calculated Kn
values for putrescine and spermidine from rat liver and sea urchin
Putrescine
Rat liver*
L. p ictus
4
3 x 10- M
3 x 10-5 M
Spermidine
2 x 10-3
M
7X10-4M
* From Feldman, Levy & Russell (1972).
254
C.-A. MANEN AND D. H. RUSSELL
DISCUSSION
During early cleavage stages in sea urchins there are numerous reports of
cyclical variations of metabolic parameters, with these variations occurring at a
definite time in relation to cell division (Nagano & Mano, 1968; Lovtrup &
Iverson, 1969; Mano, 1970). For instance, protein synthesis in sea urchins in
early cleavage is elevated during prometaphase-metaphase and is depressed
during anaphase-telophase of the next mitotic division (Mano, 1970). Studies
conducted on the cell cycle in a variety of cells capable of being synchronized in
some manner indicate that the above-mentioned cell cycle stage specificity of
synthesis is a general phenomenon (Friedman, Bellantone & Canellakis, 1972;
Mitchell & Rusch, 1972). Preliminary data from our laboratory of the relationship between initiation of polyamine biosynthetic activity as related to the cell
cycle indicate that in mammalian tissue culture cells ornithine decarboxylase
activity increases early in Gx phase and putrescine- and spermidine-stimulated
iS-adenosyl-L-methionine decarboxylase activities are enhanced during S phase
(Heby & Russell, in preparation).
Ornithine decarboxylase activity increases rapidly after fertilization, reaching
a maximum \ h after fertilization. A rise in ornithine decarboxylase activity
therefore appears to slightly precede and extends through the first period of
DNA synthesis. This period reportedly initiates in S. purpuratus at 20 min postfertilization and lasts approximately 15 min (Hinegardner, Rao & Feldman,
1964). There may be slight variations in these times attributable to the temperature of the sea water in which the embryos are grown. There is not a Gx phase
in the cell cycle of this sea urchin (Nemer, 1962; Ficq, Aiello & Scarano, 1963).
The second S phase begins in telophase prior to the first cell division and
extends into interphase of the next cell cycle. The second rise in ornithine
decarboxylase activity again appears to coincide with the second S phase.
Putrescine- and spermidine-stimulated S-adenosyl-L-methioninc decarboxylase activities do not appear to increase significantly until after the first S phase their activities appear to be specific for prophase-metaphase. This correlates
with general protein synthesis (Mano, 1970).
Another aspect of this study that might be of great importance is the cyclical
nature of the levels of the polyamines themselves. This applies to all three amines
that are present in sea urchins, i.e. putrescine, spermidine, and spermine. In S.
purpuratus there is an increase in all three amines 2\ h after fertilization although
this experiment was performed only once and further gametes could not be
obtained. L. pictus also exhibits maximal polyamine concentrations 2\ h after
fertilization. However, the spermine concentration is always much greater than
the concentrations of either putrescine or spermidine (Fig. 6). The rapid declines
in the concentrations of these compounds must mean (1) that there are enzymes
present for their selective degradation, or (2) these compounds are secreted into
the surrounding medium at this time. It would be of value to determine how
Cyclical changes in polyamine synthesis
255
these rapid changes occur, as routes of polyamine degradation are almost unknown in mammalian systems. Perhaps an understanding of this mechanism in
the sea urchin would facilitate the elucidation of such mechanisms in higher
organisms.
C.-A. Manen is a University Fellow, University of Maine. This manuscript is a portion of
the research completed in partial fulfillment of the requirement for the degree of Doctor of
Philosophy in Zoology.
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14
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OGATA,
{Received 1 January 1973, revised 10 March 1973)