J. Embryo!, exp. Morph. Vol. 29, 2, pp. 33l~345, 1973
331
Printed in Great Britain
Spermine is major polyamine
in sea urchins: studies of polyamines and their
synthesis in developing sea urchins
By CAROL-ANN MANEN 1 AND DIANE H. RUSSELL 1
From the University of Maine, Department of Zoology and
the National Institutes of Health, National Cancer Institute,
Baltimore Cancer Research Center
SUMMARY
Polyamine synthesis and accumulation was studied in several species of developing sea
urchins. Most striking is the presence in the gametes of a large amount of spermine, with low
amounts of putrescine and spermidine. This contrasts with the pattern of polyamines present
in both micro-organisms and mammals. Micro-organisms contain mainly putrescine and
spermidine and adult mammalian tissues usually contain equimolar concentrations of
spermidine and spermine.
During development (i.e. to gastrulation) there is a large increase in the spermidine
concentration with relatively little change in either putrescine or spermine levels. After
gastrulation, both spermidine and spermine concentrations are elevated. These new accumulations parallel the new synthesis of rRNA that occurs after gastrulation.
Enzyme activity patterns parallel the changes detected in the concentrations of the
polyamines with the exception of ornithine decarboxylase activity. This exception may be
due to the rapid turnover of putrescine, the precursor for the synthesis of spermidine and
spermine.
INTRODUCTION
Polyamines have been looked for and found at almost every level of the plant
and animal kingdom (Tabor & Tabor, 1964; Cohen & Raina, 1967; Cohen,
1971). In bacteria, the polyamines present are predominantly spermidine and its
precursor, putrescine, whereas in animal tissues spermidine and spermine are
found in highest concentrations (Tabor & Tabor, 1964). Certain bacteria and
tissue cell lines exhibit an absolute growth requirement for polyamines (Herbst
&Snell, 1949; Ham, 1964).
In animal tissues several lines of evidence have indicated a relationship of
polyamines to rapid tissue growth: (1) polyamine biosynthesis is one of the
earliest detectable events in growth systems, i.e. regenerating rat liver (Russell &
Snyder, 1968; Janne & Raina, 1968), embryonic development (Russell, 1970,
1971; Russell & McVicker, 1972), cardiac hypertrophy (Russell, Shiverick,
1
Authors' address: Laboratory of Pharmacology, National Institutes of Health, National
Cancer Institute, Baltimore Cancer Research Center, Baltimore, Maryland 21211, U.S.A.
332
C.-A. MANEN AND D. H. RUSSELL
Hamrell & Alpert, 1971; Feldman & Russell, 1972), and neoplastic growth
(Russell & Levy, 1971); (2) polyamine biosynthesis and accumulation are
enhanced by hormonal stimulation; i.e. growth hormone in rat liver (Russell
& Snyder, 1969, Janne & Raina, 1969; Russell, Snyder & Medina, 1970),
testosterone in rat prostate (Pegg & Williams-Ashman, 1968), and estradiol in
rat uterus (Cohen, O'Malley & Stastny, 1970; Russell & Taylor, 1971); (3) high
polyamine accumulations in mammalian systems appear to precede slightly the
ability to accumulate higher levels of rRNA and thus the ability of a tissue to
maintain higher rates of protein synthesis (Cohen, 1971; Dykstra & Herbst,
1965; Russell & Lombardini, 1971; Caldarera, Barbiroli & Moruzzi, 1965). To
date the literature in mammalian tissues indicates that the most probable role
for spermidine is the enhancement of the rate of rRNA synthesis.
Of interest, also, is the polyamine biosynthetic pathway. The enzymes
involved in polyamine synthesis in bacteria appear very different from those
found in mammalian systems. In bacteria, spermidine is synthesized by two
enzymes: S-adenosyl-L-methionine is first decarboxylated by S-adenosyl-Lmethionine decarboxylase and the decarboxylated -S-adenosyl-L-methionine
then serves as a propylamine donor for putrescine (Tabor & Tabor, 1964). A
propylamine transferase then completes spermidine synthesis by transferring a
propylamine moiety from decarboxylated S-adenosyl-L-methionine to putrescine
(Tabor & Tabor, 1964). In mammals, the evidence indicates that decarboxylated
S-adenosyl-L-methionine is never a free intermediate but there is controversy
as to whether these are two separable enzymes which catalyze: (1) decarboxylation of S-adenosyl-L-methionine, and (2) transfer of the propylamine group
from decarboxylated S-adenosyl-L-methionine to putrescine, to form spermidine.
We have not been able to separate these two functions in enzyme preparations
from rat liver which exhibit over a 400-fold purification (Feldman, Levy & Russell,
1972), although there is a report of separation of these two functions in enzyme
preparations from the rat ventral prostate (Janne & Williams-Ashman, 1971).
In this paper, we report that crude enzyme preparations (20000g supernatant
solution) from sea urchins do not exhibit any separation of decarboxylation and
propylamine transfer activity. In sea urchins putrescine and spermidine stimulate
enzymic activity, effects similar to those of yeast and mammalian enzymic
systems (Pegg & Williams-Ashman, 1969; Coppoc, Kallio & Williams-Ashman,
1971; Feldman et al. 1972). Further studies are being conducted to determine
if further purification will allow for the separation of these activities. Spermine
formation may be catalysed by the same enzyme or enzyme complex that
synthesizes spermidine (Feldman etal. 1972; Russell &Potyraj, 1972). The sea
urchin species that were studied herein present a unique system to study spermine
formation as they contain high amounts of spermine relative to spermidine and
putrescine. In fact, unfertilized eggs contain about 10 molecules of spermine to
every 1 molecule of spermidine.
One of the few areas where general information about polyamine distribution
Polyamines
in sea urchins
333
and biosynthesis are lacking is the invertebrate phyla. In this paper we report
studies of polyamine biosynthesis and accumulation in several species of sea
urchins.
MATERIALS AND METHODS
Materials
Ripe Lytechinus variegatus were collected at Mote Marine Laboratory,
Sarasota, Florida. Ripe L. pictus and Strongylocentrotus purpuratus were
obtained from Pacific Bio-Marine. S. droehbachiensis was collected at Limon
beach, Limon, Maine. Spawning was induced by injection of 0-55 M-KC1. Eggs
were collected and washed in artificial sea water, except in the case of L.
variegatus, whose embryos were cultured in sterilized sea water. Embryos were
grown in 4 1 beakers under natural light conditions at 20 °C. After gastrulation,
the embryos were transferred to clean sea water containing approximately 5 ml
of a Duniella culture. Only those cultures which showed at least 90 % normal
development were used. [l-4-14C]Putrescine dihydrochloride (17-72 mCi/mM),
[14C]spermine tetrahydrochloride (14-4 mCi/mM), [14C]spermidine trihydrochloride (10-7 mCi/mM), 5-adenosyl-L-| carboxy-14C]methionine (7-3 mCi/mM) and
DL-ornithine-l-14C (3-31 mCi/mM) were obtained from New England Nuclear.
Putrescine dihydrochloride and spermidine trihydrochloride were obtained from
Calbiochem and routinely purified by paper chromatography before use in
enzyme assays.
Determination of putrescine, spermidine and spermine concentrations
Pools of embryos were homogenized in 4 vol. of 0-1 N HC1 and subjected to
alkaline butanol extraction as previously described (Russell, Medina & Snyder,
1970). 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-NaOH buffer, pH 4-3.
Concentrations were determined by staining the chromatography sheet (Whatman 3MM 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 60min at 60 °C,
eluting the color and recording the absorbancy at 505 nm. Standards were run
in the same range as the samples. 14C-Amines were used to determine the
recovery rates and appropriate calculations made.
Preparation of enzyme solutions
The material was homogenized in 4 vol of 0-05 M sodium-potassium phosphate
buffer, pH 7-2, containing 0-1 mM dithiothreitol. These enzymes are inhibited
by Tris buffer, as are the mammalian enzymes. The homogenate was centrifuged
at 20000g for 20min, the pellet discarded, and the supernatant used in the
assays. Since the supernatant after centrifugation at 100000 g for 90min gave
the identical enzyme activities, the 20000 g supernatant was used routinely.
22
E M B 29
334
C.-A. MANEN AND D. H. RUSSELL
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,
1969). Although the substrate concentration (0-1 mM as L-ornithine used
routinely) was non-saturating, the same changes were noted when excess
ornithine (2 nnvi) was used as substrate in some experiments.
Assay for putrescine-stimulated adenosyl-L-methionine decarboxylase
Enzyme activity was determined by measuring the liberation of 14CO2 from
*S-adenosyl-L-[carboxy-14C]methionine as previously described (Pegg & WilliamsAshman, 1969). Incubation mixtures consisted of 0-2-2-0 mM S-adenosyl-L[carboxy-14C]methionine, 50 mM sodium-potassium phosphate buffer (pH
7-2), 50 ^M pyridoxal phosphate, 2-5 HIM putrescine, and 01—015 ml (3-5 mg
protein) of enzyme solution in 0-2 ml. This reaction required putrescine for
maximal activity (Table 1). When the formation of spermidine from unlabeled
S-adenosyl-L-methionine was estimated, 0-33/«nole of [l,4-14C]putrescine and
4-0 /tmoles of unlabeled putrescine were added to an assay containing the same
concentration of reactants in 2-0 ml. The reaction was stopped by the injection
of 2-0 ml of 5 % (w/v) trichloroacetic acid containing 0-4 /jmole of spermidine
and spermine. After removal of the protein precipitate by centrifugation the
supernatant was washed three times with 5 vol of ether. The ether was discarded
and the solution put directly on 5 cm x 0-25 cm2 Dowex 50 (H + ) column.
The labeled putrescine was removed with 200 ml of 0-8 M-HCI. The labeled
spermidine was removed with 20 ml of 6 M-HCI and evaporated to dryness
under reduced pressure and then dissolved in 0-5 ml of 0-01 M-HCI. Of this
solution 0-1 ml was spotted on Whatman 3MM chromatography paper and
subjected to electrophoresis in 0-1 M citric acid, pH4-3. The chromatograms
were developed with ninhydrin and the appropriate spots were cut out, eluted,
and their radioactivity determined, as previously described (Russell, Medina &
Snyder, 1970). There was a stoichiometric relationship between amount of
[14C]spermidine formed under these conditions and the evolution of 14CO2 when
*S-adenosyl-L-[carboxy-14C]methionine was used.
Assay for spermidine-stimulated S-adenosyl-L-methiottine decarboxylase activity
This assay is identical to that for putrescine-dependent-5-adenosyl-L-methionine decarboxylase except 5 mM spermidine was added instead of 2-5 mM putrescine. This reaction required the addition of spermidine for maximal activity
(Table 1). When the formation of spermine from labelled S-adenosyl-L-methionine was estimated, 0-33/tmole of [l,4-14C]spermidine and 4-0/mioles of
unlabeled spermidine were added to an assay containing the same concentration
Polyamines in sea urchins
335
Table 1. Stimulation of S-adenosyl-L-methionine decarboxylase activity
by putrescine and spermidine
Putrescine (nivi)
0
2-5
50
100
200
Spermidine (mM)
0
0125
0-25
0-5
10
5*-adenosyl-L-methionine
decarboxylase activity
expressed in pmoles
14
Co2/30 min/mg protein
Fold-increase
5
220
534
1870
4070
—
44
107
374
813
5
118
330
716
2340
—
26
73
159
521
Frozen unfertilized eggs of L. pictus were assayed as described in Methods with the
exception that a saturating concentration of 5-adenosyl-L-methionine was used. The values
are expressed as the mean of three or more determinations.
of reactants in 2-0 ml. This reaction was carried out as described above. There
was a stoichiometric relationship between the amount of [14C]spermine formed
under these conditions and the evolution of 14CO2 when S-adenosyl-L-[carboxy14
C]methionine was used.
RESULTS
Polyamine content of developing embryos of sea urchins
Lytechinuspictus. Both the sperm and the eggs of L. pictus contain low amounts
of putrescine and spermidine and rather large amounts of spermine (Table 2).
However, eggs contain two to three times more of each polyamine than do
sperm. Upon fertilization, the earliest increase appears to be in the spermidine
pool, which increases linearly to the last point assayed, a 5-day pluteus larva.
Throughout development, there is much more spermine present than spermidine.
At gastrulation there is about one molecule of spermidine to three molecules
of spermine and at the early pluteus stage, there is approximately one molecule
of spermidine to two molecules of spermine. In unfertilized eggs and in sperm
there is approximately one molecule of spermidine to 10 molecules of spermine.
Adult tissues of L. pictus have large amounts of spermine and much smaller
amounts of putrescine and spermidine (Table 3). For instance, the testis contains
nearly twenty times as much spermine as spermidine. The pattern is similar
in the ovary. The gut, in contrast, contains considerable putrescine, and the
ratio of spermidine to spermine is about 4.
Lytechinus variegatus. When the endogenous concentrations of polyamines
336
C.-A. MANEN AND D. H. RUSSELL
Table 2. Polyamine content of developing embryos o/Lytechinus pictus
nmoles/mg protein
Stage
Sperm (4)
Eggs (4)
2-4 cell (4)
Morula (1)
Blastula (8)
Gastrula(lO)
Pluteus-1 day (7)
Pluteus-2 day (2)
Pluteus-4 day (1)
Piuteus-5 day (1)
Putrescine
Spermidine
Spermine
0-39 ±018
1-13 + 0-25
0-85 + 0-50
0-75
0-17 ±0-04
1-37 ±0-39
2-18 ±0-77
0-68 ±0-68
2-87
1-91
0-20 ± 0 0 7
0-83 ± 0 1 0
1-11 ±0-37
1-57
0-52 + 0-12
1-91 ±0-41
308 ±0-54
4-32±l-56
7-12
7-83
3-56±0-36
605 ±0-98
5-12+1 00
5-68
6-90 ±1-24
6-89 ±0-76
7-71 ±0-56
15-01 ±3-36
24-75
11-50
(n) = Number of separate pools assayed.
Pools of embryos were assayed for endogenous levels of putrescine, spermidine, and
spermine, by extraction of amines into alkaline 1-butanol and separation by high voltage
electrophoresis as described previously (Russell, Medina & Snyder, 1970). Data are presented
as the mean±s.E. for duplicate determinations of the number of separate pools shown in
parentheses.
Table 3. Polyamine content of adult tissues o/Lytechinus pictus
nmoles/mg protein
Stage
Putrescine
Spermidine
Spermine
Ovary (7)
Testis (6)
Gut (7)
0-63 ± 0 1 0
0-51 ± 0 1 2
2-53 ±0-79
0-82 ±0-20
l-54±0-35
l-43±016
12-15 ±1-32
1908 ±1-70
904 ±0-96
(n) = Number of separate pools assayed.
Pools of embryos were assayed for endogenous levels of putrescine, spermidine, and
spermine, by extraction of amines into alkaline 1-butanol and separation by high voltage
electrophoresis as described previously. Data are presented as the mean±s.E. for duplicate
determinations of the number of separate pools shown in parentheses.
were assayed in L. variegatus, it was found that sperm contained rather large
amounts of putrescine, spermidine and spermine, while eggs contained no
detectable putrescine, a small amount of spermidine and a rather large amount
of spermine (Table 4). After fertilization, both spermidine and spermine concentrations increase linearly. At the last point assayed, a 2-day pluteus, spermidine
concentration was four times that found in the blastula stage and spermine
content was about two times that found in the blastula stage. Therefore, during
early embryogenesis there does appear to be a dramatic accumulation of
spermidine and spermine. However, the amount of spermidine present during
development is much lower than the corresponding amount at the same stage
of L. pictus (Table 2). The only exception is the blastula where the concentrations
are similar.
Polyamines in sea urchins
337
Table 4. Polyamine content of developing embryos o/Lytechinus variegatus
nmoles/mg protein
Stage
Sperm (!)
Eggs (2)
Blastula(l)
Gastrula (3)
Pluteus-1 day (2)
Pluteus-2 day (2)
r
Putrescine
2-10
<0-25
1-75
0-30 ±0-30
<0-25
<0-25
Spermidine
Spermine
10
0-25 ±0-25
0-45
0-51 +0-21
0-80 ±0-30
205 ± 005
5-90
3-05 ±0-75
4-80
6-67 ±0-78
6-50 ±2-90
8-35 ±0-85
(/;) = number of separate pools assayed.
Pools of embryos were assayed for endogenous levels of putrescine, spermidine, and
spermine, by extraction of amines into alkaline 1-butanol and separation by high voltage
electrophoresis. Data are presented as the mean±s.E. for duplicate determinations of the
number of separate pools shown in parentheses.
Table 5. Polyamine content of developing embryos of
Strongylocentrotusdroehbachiensis
nmoles/mg protein
Stage
Sperm (3)
Eggs (5)
Morula (2)
Blastula (3)
Pluteus-8 day (2)
Putrescine
Spermidine
Spermine
<0-25
<0-25
<0-25
<0-25
<0-25
1 09 ±012
0-94 ±014
18-20 ±1-57
9-88 ±0-71
11-20 ±0-30
11-93 ±3-24
28-90 ±1-39
1-49 ±023
2-30+1-28
404 ±200
(/;) = number of separate pools assayed.
Pools of embryos were assayed for endogenous levels of putrescine, spermidine and
spermine, by extraction of amines into alkaline 1-butanol and separation by high voltage
electrophoresis. Data are presented by the mean±s.E. for duplicate determinations of the
number of separate pools shown in parentheses.
Strongylocentrotus droehbachiensis. Neither the eggs nor the sperm of this
species contained a detectable amount of putrescine (Table 5). Both the sperm
and the eggs contain about equal concentrations of spermidine but the sperm
contains twice the amount of spermine as do eggs. Again, both spermidine and
spermine appear to increase rapidly after fertilization, although we do not have
determinations for either the gastrula or early pluteus larva.
Strongylocentrotus purpuratus. The pattern of polyamine accumulation in S.
purpuratus is very similar to those of both L. variegatus (Table 4) and L. pictus
(Table 2). However, the gametes contain much lower amounts of spermidine
(Table 6). Again a dramatic increase in the spermidine content is detectable at
gastrulation.
338
C.-A. MANEN AND D. H. RUSSELL
Table 6. Polyamine content of developing embryos of
Strongylocentrotus purpuratus
nmoles/mg protein
Stage
Sperm (5)
Eggs (5)
2-4 cell (1)
Blastula (3)
Gastrula (3)
Putrescine
Spermidine
Spermine
061 ±005
015 ±003
0-77
0-58 + 002
0-68 ±0-25
O-38±O-12
016 ±002
0-63
0-67 ±015
4-84 ±1-06
5-20 ±0-30
703
6-47 ±0-82
5-63 ±1-33
115 + 015
(/?) = number of separate pools assayed.
Pools of embryos were assayed for endogenous levels of putrescine, spermidine and
spermine, by extraction of amines into alkaline 1-butanol and separation by high voltage
electrophoresis. Data are presented as the mean ± S.E. for duplicate determinations of the
number of separate pools shown in parentheses.
Table 7. Polyamine content of adult tissues of
Strongylocentrotus purpuratus
nmoles/mg protein
Tissue
Ovary (5)
Testis (5)
Gut (5)
Putrescine
Spermidine
Spermine
0-37 ±004
0 41+006
0-17 ±0-07
0 40 ±011
0-64 ±0-10
0-21 ±007
12-63 + 1-30
17-29± 1-21
5-39 ±0-40
(«) = number of separate pools assayed.
Pools of embryos were assayed for endogenous levels of putrescine, spermidine, and
spermine, by extraction of amines into alkaline 1-butanol and separation by high voltage
electrophoresis. Data are presented as the mean ± S.E. for duplicate determinations of the
number of separate pools shown in parentheses.
Adult tissues have very high spermine concentrations and low concentrations
of both putrescine and spermidine (Table 7). These tissues all have over 20-fold
more spermine than spermidine.
Polyamine biosynthesis in developing sea urchins
Ornithine decarboxylase activity {putrescine formation). Polyamine biosynthesis was studied in two species, L. pictus and S. purpuratus.
Ornithine decarboxylase shows a marked elevation in activity early after
fertilization of L. pictus eggs (Fig. 1), exhibits further elevation by gastrulation,
and increases to high levels in plutei larvae.
In S. purpuratus, ornithine decarboxylase activity is markedly elevated shortly
after fertilization (Fig. 2). After the blastula stage ornithine decarboxylase
339
Polyamines in sea urchins
2400 -
•> 'd 2000
'•2
g
"t, = 1600 -
•o P 1200
Fertilization
Blastula
Gastrula
Prism
Developmental stage of Z.. pictus
Pluteus
2-day Pluteus
Fig. 1. Ornithine decarboxylase activity was determined by measuring the evolution
of 14 CO 2 from ornithine-l- 14 C (Russell & Snyder, 1968, 1969). Each point
represents the mean±s.E. of at least five determinations on at least five separate
pools of embryos.
activity rises markedly and by the early gastrula stage is 50-fold greater than
control levels (unfertilized eggs).
Putrescine-stimulated S-adenosyl-L-methionine decarboxylase. We measured
putrescine-stimulated S-adenosyl-L-methionine decarboxylase activity in L.
pictus, and found a marked increase in enzyme activity after fertilization
(Fig. 3). Thereafter, enzyme activity actually decreased with another marked
elevation in the early pluteus.
It is interesting to note the very high activity of putrescine-stimulated Sadenosyl-L-methionine decarboxylase in unfertilized eggs. The reason for this is
unclear. However, it correlates with the higher concentration of spermidine
found in L. pictus eggs.
This enzyme exhibited a much different pattern of activity in S. purpuratus
(Fig. 4). The level in unfertilized eggs is very low (approximately 800 pmoles/30
min/mg protein) as opposed to L. pictus (5000 pmoles/30 min/mg protein).
Again, this correlates well with the low amount of spermidine found in S.
purpuratus eggs. After fertilization, the enzyme activity increases rapidly and
reaches levels 5-fold above controls (unfertilized eggs) in the early gastrula.
Spermidine-stimulatea S-adenosyl-L-methionine decarboxylase. In L. pictus
(Fig. 5), the pattern of spermidine-stimulated S-adenosyl-L-methionine decarboxylase was very similar to that found with the putrescine-stimulated enzyme,
340
C.-A. MANEN AND D. H. RUSSELL
Fertilization
Blaslula
Gastrula
Prism
Developmental stage of S. pitrpuratu.s
Fig. 2. Ornithine decarboxylase activity was assayed by measuring the evolution
of 14CO2 from ornithine-l-14C (Russell & Snyder, 1968, 1969). Each point
represents the mean ± S.E. of at least five determinations on at least five separate
pools of embryos.
1= 7000
73
Fertilization
Blastula
Gastrula
Prism
Pluteus
2-day Plutcus
Developmental stage of L. piclus
Fig. 3. Putrescine-stimulated S-adenosyl-L-methionine decarboxylase was assayed
by detecting the 14CO2 released from "COOH-S-adenosyl-L-methionine when
putrescine was present (see Materials and Methods). Each point represents the
mean ± S.E. of at leastfivedeterminations on at least five separate pools of embryos.
Polyamines in sea urchins
Fertilization
Blastula
Gastrula
Prism
Developmental stage of S. purpuratus
Fig. 4. Putrescine-stimulated S-adenosyl-L-methionine decarboxylase was assayed
by detecting the 14CO2 released from "COOH-S-adenosyl-L-methionine when
putrescine was present (see Materials and Methods). Each point represents the
mean ± S.E. of at least five determinations on at least five separate pools of embryos.
Fertilization
Blastula
Gastrula
Prism
Pluteus 2-day Pluieus
Developmental stage of L. pic tits
Fig. 5. Spermidine-stimulated 5-adenosyl-L-methionine decarboxylase was assayed
by detecting the 14CO2 released from "COOH-S-adenosyl-L-methionine when
spermidine was present (see Materials and Methods). Each point represents the
mean ± S.E. of at least five determinations on at leastfiveseparate pools of embryos.
341
342
C.-A. MANEN AND D. H. RUSSELL
Fertilization
Blastula
Gastrula
Prism
Developmental stage of S. purpurutus
Fig. 6. Spermidine-stimulated S-adenosyl-L-methionine decarboxylase was assayed
by detecting the 14CO2 released from 14COOH-5-adenosyl-L-methionine when
spermidine was present (see Materials and Methods). Each point represents the
mean ± S.E. of at least five determinations on at least five separate pools of embryos.
with the exception that the enzyme preferred putrescine to spermidine with a
ratio of about 2. In S. purpuratus (Fig. 6), spermidine-stimulated S-adenosylL-methionine decarboxylase reached its highest activity at gastrulation, when
it was two-fold that found in the unfertilized egg.
DISCUSSION
This paper demonstrates that embryos of certain invertebrates, similar to
embryos of amphibians, birds and mammals, exhibit marked polyamine biosynthesis during development. However, there are certain differences worth
noting. For example, sea urchins contain very large amounts of spermine, and
low levels of both putrescine and spermidine.
The route of polyamine biosynthesis appears to be the same in sea urchins
as in other systems (Tabor & Tabor, 1964; Pegg & Williams-Ashman, 1969).
That is, if eggs are incubated with labeled putrescine and then fertilized, the
label can be detected at various times in spermidine and spermine (unpublished
observations). Further, S-adenosyl-L-methionine decarboxylase appears to be
putrescine and/or spermidine dependent, which resembles the characteristics of
the mammalian enzyme system (Pegg & Williams-Ashman, 1969; Feldman et ah
1972). In most cases, an accumulation of spermidine follows fertilization and
increases through gastrulation. In fact, in developing embryos of L. pictus, the
ratio of spermine to spermidine changes from approximately 10 in unfertilized
eggs to about three at gastrula stage. The change is due to the increased amount
of spermidine at the gastrula stage and is not due to a change in the spermine
pool. After gastrulation, both the pools of spermidine and of spermine increase
exponentially, at least in L. pictus.
Polyamines in sea urchins
343
It is interesting to note the very high activity of putrescine-stimulated-Sadenosyl-L-methionine decarboxylase and spermidine stimulated S-adenosylL-methionine decarboxylase in unfertilized eggs of L. pictus. This would explain
the very high ratio of spermine to spermidine contained in the unfertilized eggs
of the species. This enzyme system(s) must be expressed in catalysing the conversion of the precursor, putrescine, all the way to spermine. This would
indicate that spermine synthesis could best be studied in the unfertilized eggs
of L. pictus. There are very few other examples in the invertebrate or vertebrate
kingdom in which the rate of spermine synthesis is substantial enough to
actually consider studying the enzyme(s) involved.
The relationships between polyamine biosynthesis and accumulation, and of
RNA synthesis in sea urchins, are not at all clear at this time. However, major
bursts of polyamine synthesis occur well in advance of any increases in rRNA
synthesis. At present, it is the general consensus that there is a constant rate
of rRNA synthesis/cell prior to gastrulation (Emerson & Humphreys, 1971).
At blastulation there is synthesis of a large amount of high molecular weight
DNA-like RNA which could be the precursor of rRNA. This reaction is more
dramatic at gastrulation and thereafter (Guidice & Mutolo, 1967; Slater &
Spiegelman, 1970). There are, however, active spermidine and spermine accumulations when the plutei larvae begin to feed and these increases correlate with
the increased synthesis and accumulation of ribosomal RNA that occur at this
time (Emerson & Humphreys, 1970).
The differences in polyamine patterns exhibited by various species of sea
urchins are indeed interesting and worthy of some discussion. Environmental
factors are not likely to affect the temporal pattern of changes occurring prior
to gastrulation as these changes are thought to be governed by the maternal
genome. However, after gastrulation such environmental factors as food supply,
water temperature, day length, etc., may well prescribe the growth rate, and
may be reflected in the ultimate ratio of spermidine/spermine during growth
periods. In mammals, a ratio of spermidine/spermine greater than one is found
during growth periods. In sea urchins, the ratio during growth phases may be as
high as 0-7. This must be interpreted in light of the ratio in the unfertilized egg,
which may be as low as 0-1. Therefore the patterns of polyamine metabolism
exhibited by those species of sea urchins studied may well be closely linked to
their ecology.
C.-A.M. is a University Fellow, University of Maine. This manuscript is a portion of
research completed in partial fulfilment of the requirements for the degree of Doctor of
Philosophy in Zoology.
344
C.-A. MANEN AND D. H. RUSSELL
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{Received 6 June 1972, revised 20 August 1972)