/ . Embryol. exp. Morph. Vol. 60, 83-92 pp. 1980
Printed in Great Britain @ Company of Biologists Limited 1980
83
Essential role of the polyamines in early chick
embryo development
By B. LOWKVIST, 1 O. HEBY 1 AND H. EMANUELSSON 1
From the Department ofZoophysiology, University of Lund
SUMMARY
The polyamines putrescine, spermidine and spermine were analyzed in chick embryos
during the first 2 days of development. A rapid increase in the activity of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine synthesis, was observed
immediately after onset of incubation. Peak activities were found at 15 and 23 h of incubation. The first peak coincides with gastrulation and the second peak with early neurulation
in the embryo. All three polyamines varied in a similar manner as did ODC, with putrescine
and spermidine at about the same level and spermine at a lower level. The ODC activity
was blocked by a-difluoromethylornithine, an enzyme-activated irreversible inhibitor. The
inhibitor was administered to embryos in ovo at 5 h of incubation, i.e. prior to the first
major increase in ODC activity. This block prevented the accumulation of the polyamines
and inhibited development at gastrulation, suggesting a decisive role for polyamines in this
developmental event.
INTRODUCTION
Our present knowledge of the role of the polyamines putrescine, spermidine
and spermine in the development of the vertebrate embryo is very limited and
mainly based on studies of advanced embryonic material (Raina, 1963; Russell
& Snyder, 1968; Caldarera & Moruzzi, 1970; Russell & Lombardini, 1971).
Some investigations suggest that polyamines may be involved in nucleic acid
and/or protein synthesis (Caldarera & Moruzzi, 1970; Russell & Lombardini,
1971). This conclusion, however, is based on circumstantial evidence only.
With the development of specific polyamine synthesis inhibitors, which
deplete cells of their polyamines (for references, see Heby & Janne, 1980), it
has become possible to analyze more directly the physiological function of the
polyamines. This approach has been applied to developing invertebrate embryos
(Emanuelsson & Heby, 1978; Heby & Emanuelsson, 1978; Kusunoki &
Yasumasu, 1978; Brachet et al 1978), and has revealed a requirement of polyamines for cleavage (Kusunoki & Yasumasu, 1978), blastulation (Brachet
et al. 1978) and gastrulation (Emanuelsson & Heby, 1978; Heby & Emanuelsson, 1978). It is important to determine whether these requirements exist in
vertebrates. Therefore, we have analyzed the effects of polyamine depletion in
1
Authors'1 address: Department ofZoophysiology, University of Lund, Helgonaragen 3,
S-223 62 Lund, Sweden.
84
B. LOWKVIST, O. HEBY AND H. EMANUELSSON
A
B
C
Fig. 1. Delineation of the embryo area within the chick blastoderm analyzed for
ODC activity and polyamine concentration. The developmental stages shown
represent the following incubation times: 18 h (A), 30 h (B) and 40 h (C). The arrows
indicate the outline of the area pellucida. The area within the dashed line corresponds
to the material removed and analyzed. At each stage a defined gel puncher (diameter,
3, 5 or 7 mm) was used to remove a constant amount of material and the circular
area was trimmed with a scalpel to the shape delineated (dashed line).
the early chick embryo, which represents an easily accessible and well-known
vertebrate material.
The present study reveals an early activation of polyamine synthesis in the
chick embryo, preceding the one known to occur during organogenesis (Raina,
1963; Russell & Snyder, 1968; Caldarera & Moruzzi, 1970; Russell & Lombardini, 1971). The accumulation of polyamines which begins during cleavage
was prevented by blocking the activity of ornithine decarboxylase (ODC;
L-ornithine carboxy-lyase; EC 4.1.1.17), the initial and rate-limiting enzyme
in polyamine synthesis. Either a-methylornithine, a competitive inhibitor
(Abdel-Monem, Newton & Weeks, 1974; Mamont et al. 1976), or a-difluoromethylornithine, an enzyme-activated irreversible inhibitor (Metcalf et al.
1978; Mamont, Duchesne, Grove & Bey, 1978) was used as the inhibitor of
ODC activity.
MATERIALS AND METHODS
Preparation of embryos
Fertilized hens' eggs (White Leghorn) were incubated at 37-5 ± 0-5 °C.
Embryos were collected at 2-10 h intervals during the first 2 days of development and were prepared in 0-93 % NaCl at 37 °C as follows. The vitelline
membrane with adhering blastoderm was removed from the yolk. The blastoderm was carefully loosened from the membrane and the embryo isolated;
special care was taken to prevent yolk from sticking to the embryo. At all stages
the analyzed embryo represents the embryo area proper, i.e. approximately the
Poly amines in early chick embryos
85
area pellucida of the blastoderm. The exact dimensions of the area analyzed at
various stages are shown in Fig. 1. The developmental stage was determined
according to Hamburger & Hamilton (1951). Depending on age, four to nine
embryos were pooled at each time. The pooled embryos were collected by
centrifugation at 6000 g for 10 min at 4 °C, and were analyzed directly or
stored at — 72 °C. A total of 465 embryos were used to study the ODC activity
and the polyamine concentrations during normal chick embryo development.
Inhibition experiments
The polyamine synthesis inhibitors (DL-a-methylornithine or DL-a-difluoromethylornithine) or control solutions (DL-ornithine or 0-93 % NaCl) were
injected in ovo. Eggs were preincubated for 5 h, and the shell disinfected with
70 % ethanol, and windowed. The vitelline membrane was penetrated with a
bent hypodermic needle and 100 /A of the inhibitor or control solutions were
deposited very slowly 1-2 mm beneath the embryo. The embryos received
5-50/imoles of DL-a-methylornithine (147 embryos), 42/tmoles of DL-adifliioromethylornithine (55 embryos), or 15-70/orioles of DL-ornithine
(35 embryos) in 100/d of 0-93 % NaCl or 100/d of 0-93 % NaCl alone (205
embryos). The window was sealed with adhesive tape and the incubation of the
eggs was continued for various time periods. All solutions were prepared from
distilled and sterilized water, and the experiments were performed under
semi-sterile conditions.
ODC assay
The ODC activity was measured by an in vitro method mainly as described
by Janne & Williams-Ashman (1971). The method is based on capture of
14
CO2 released during the ODC-catalyzed formation of putrescine from DL-114
C-ornithine (18-5 MBq/mmole). The pooled embryos were sonicated at
4°C in 200/fcl of 10 mM Tris/HCl buffer (pH 7-2) containing 0-5 DIM Na a
EDTA, 5-0 mM dithiothreitol, and 0-05 mM pyridoxal 5'-phosphate. A 100 [A
aliquot of the homogenate was analyzed for ODC activity in the presence of a
saturating (1 mM) L-ornithine concentration. The captured 14CO2 was measured
with a Packard TriCarb scintillation spectrometer.
Polyamine analysis
The concentration of the polyamines was determined essentially according to
Seiler (1970). The pooled embryos were sonicated in 250/d of 0-2 M perchloric
acid at 4 °C. After 1 h at 4 °C the homogenate was centrifuged at 6000 g for
10 min and a 200 /A aliquot of the supernatant was dansylated. The dansylated
derivatives were extracted into 500 /A of toluene and 10 [A aliquots were
applied on thin-layer chromatography plates. The plates were developed in
cyclohexane/ethylacetate (1:1). Analysis of the fluorescence intensity was
made with an Aminco-Bowman spectrophotofluorometer.
86
B. LOWKVIST, O. HEBY AND H. EMANUELSSON
Developmental age (h)
Fig. 2. ODC activity in the chick embryo during the first 2 days of development
(solid line). The stages of cleavage (C), gastrulation (G), and neurulation with early
organogenesis (N) are indicated. Mean±s.D. (n = 3; a total of 13-19 embryos at
each time point depending on age). ODC activity in the chick embryo as affected
by a-difluoromethylornithine (42 /^moles/embryo) administered at 5 h of incubation (dashed line). Five embryos were pooled at each time point.
Protein determination
ODC activities and polyamine concentrations are presented on a per mg of
total protein basis. The protein concentrations were determined according to
Lowry, Rosebrough, Farr & Randall (1951), with bovine serum albumin as
standard.
Morphological studies
Embryos treated with inhibitor or control solutions and collected at 26, 30
or 40 h of incubation, were fixed as wholemounts in Clarke's fixative (absolute
ethanol/glacial acetic acid, 3:1) and stained in Gomori's hematoxylin. The
preparations were examined in the light microscope.
Chemicals
DL-a-methylornithine monohydrochloride monohydrate and DL-a-difluoromethylornithine monohydrochloride monohydrate were kindly donated by
Centre de Recherche Merrell International, Strasbourg, France. DL-1- 1 4 Cornithine monohydrochloride was purchased from the Radiochemical Centre,
Amersham, England.
Polyamines in early chick embryos
I
87
Sd
6
.5
-
Sp
10
20
30
40
50
C—IDevelopmental age (h)
Fig. 3. Concentration of putrescine (Pu), spermidine (Sd) and spermine (Sp) in
the chick embryo during the first 2 days of development. The stages of cleavage (C),
gastrulation (G) and neurulation with early organogenesis (N) are indicated.
Mean±s.D. (n = 3-5; a total of 16-30 embryos at each time point depending on
age). Dots without bars are based on 4-5 pooled embryos.
88
B. LOWKVIST, O. HEBY AND H. EMANUELSSON
600
Sd
Pll
9
Sp
£500 1-
f
400 300 ;
/
•i 200 100 -
10
20
0
10
20
Developmental age (h)
0
10
20
Fig. 4. Effect of a-difluoromethylornithine treatment (42 /tmoles/embryo) on the
concentration of putrescine (Pu), spermidine (Sd), and spermine (Sp) in the chick
embryo during the first day of development. a-Difluoromethylornithine was injected
at 5 h of incubation. NaCl-treated chick embryos (solid lines); a-difluoromethylornithine-treated chick embryos (dashed lines). Values are expressed in percentage
of the 5 h polyamine levels. Five embryos were pooled at each time point.
RESULTS
Figure 2 shows the ODC activity during early chick embryo development. Peak
activities were observed at 15 and 23 h of incubation. The first and second peak
activities correspond to seven-fold and nine-fold increases compared to the
5 h controls. Toward the end of the experimental period the ODC activity
again showed a tendency to increase. It is apparent that polyamine synthesis is
activated already during cleavage and attains a first maximum during gastrulation. The second peak occurs during early neurulation and organogenesis.
Following the initial activation of polyamine synthesis during cleavage, the
biosynthetic activity remains elevated during the rest of the experimental
period, which encompasses the completion of the circulatory system in the
embryo.
The concentration of all three polyamines showed a similar pattern as did the
ODC activity (Fig. 3). At peak levels the concentrations of putrescine, spermidine and spermine were roughly 3-5-3-7, 4-6-5-4 and 4-3-4-5 times the 0 h
levels. At the end of the experimental period there was an accumulation of all
three polyamines, amounting to the range of the previous peak levels.
Treatment of the chick embryos with a-difluoromethylornithine from the
5 h stage prevented the surge of ODC activity normally occurring at the time
of gastrulation (Fig. 2). Consistent with this block of the initial step in polyamine synthesis, the increase in the concentrations of all three polyamines was
largely prevented (Fig. 4).
The consequence of polyamine synthesis inhibition for chick embryo morphogenesis is shown in Fig. 5. The average diameter of the 26 h blastoderm
Polyamines in early chick embryos
89
tat.
Fig. 5. Effect of a-difluoromethylornithine treatment (42/tmoles/embryo) on early
chick embryo morphogenesis. (A) 26 h chick embryo treated in ovo with 0-93 %
NaCl (100/tl at 5 h of incubation); (B) 26 h chick embryo treated in ovo with the
polyamine synthesis inhibitor (100/*l of 0-42 M a-difluoromethylornithine in saline).
Notice that embryo development has not progressed beyond the primitive-streak
stage (gastrulation) in embryos treated with a-difluoromethylornithine. Corresponding control embryos have initiated organogenesis and show three distinct
pairs of somites. The observations are based on 35 drag-treated and 20 NaCltreated embryos. Scale bar = 1 mm.
was only 8 mm in the a-difluoromethylornithine-treated specimens compared
to 18 mm in the NaCI-treated controls. Moreover, development did not progress
past the primitive-streak stage (gastrulation) in embryos treated with a-difluoromethylornithine from 5 to 26 h. Even when treatment with the inhibitor was
maintained between 5 and 40 h no further morphogenesis was seen in a study
of three separate embryos (not shown).
Treatment of chick embryos with a-methylornithine, a competitive inhibitor
of ODC, proved to be considerably less effective. At a dose of 40-50 /tmoles
per embryo, a-methylornithine produced only minor changes in the polyamine
levels (not shown). This treatment retarded development in only 10-25 %
of the embryos. Corresponding doses of DL-ornithine produced no adverse
effects on embryo development.
DISCUSSION
In the chick embryo, development begins while the egg is still in the oviduct,
but is interrupted at laying. At the time when development is permitted to
90
B. LOWKVIST, O. HEBY AND H. EMANUELSSON
proceed, by incubation at 37-5 °C, the blastoderm contains about 80000 cells
and represents the late cleavage stage. At this stage one can distinguish within
the blastoderm two distinct regions, an inner area pellucida (the embryo proper)
and an outer area opaca (extra-embryonal material). Analysis of polyamine
metabolism was performed on area pellucida only, thus facilitating observations
of subtle changes that otherwise might have been concealed by the excess of
extra-embryonal material. The present investigation encompasses the early
developmental period up to 2 days of incubation, which includes late cleavage,
gastrulation and early organogenesis. Previous analyses of polyamine metabolism in chick embryos do not include these early events inasmuch as they
were initiated at the 2- or 3-day stage (Raina, 1963; Russell & Snyder, 1968;
Caldarera & Moruzzi, 1970; Russell & Lombardini, 1971).
The rate of polyamine synthesis was found to be markedly increased during
cleavage and reached a first maximum at the culmination of gastrulation. It
remained elevated and attained a second maximum which coincided with early
neurulation and somitogenesis. These two maxima were followed by a significant
decrease. A new activation of polyamine synthesis was not apparent until at the
end of the period investigated in these experiments. This late activation presumably progresses to generate the rise in polyamine synthesis observed at a
comparable stage of development by Raina (1963), Russell & Snyder (1968),
Caldarera & Moruzzi (1970) and Russell & Lombardini (1971).
The pattern of changes in polyamine synthesis (ODC activity) closely resembles those in the concentrations of putrescine, spermidine and spermine.
This fact suggests that the endogenous polyamine levels are determined by
their rates of synthesis. Accordingly, irreversible inhibition of the ODC activity
by a-difluoromethylornithine largely prevented the accumulation of polyamines which normally occurs before gastrulation. The fact that a-methylornithine did not prevent polyamine accumulation is probably due to its
stabilizing action on ODC (McCann, Tardif, Duchesne & Mamont, 1977).
Furthermore, the inevitable dilution in ovo will enhance the reversibility of the
a-methylornithine action.
The possibility that the developmental arrest would be due to side effects of
a-difluoromethylornithine has been previously ruled out. Thus, polyamines
added concomitantly with or after a-difluoromethylornithine have been shown
to counteract and reverse the adverse effects of the inhibitor on vertebrate cells
(Mamont et aJ. 1978). The inhibitor is excreted unchanged, demonstrating that
it is not metabolized in the cell (Seiler, Danzin, Prakash & Koch-Weser, 1978).
The morphological consequences of the inhibition of polyamine synthesis
and accumulation in the early chick embryo were severe and there was no
development beyond the primitive-streak stage (gastrulation), i.e. no neurulation
or formation of somites in the polyamine-depleted embryos. These results are
consistent with our previous observations on an invertebrate system (Emanuelsson & Heby, 1978). Thus, in the polychete Ophryotrocha labronica, inhibition
Polyamines in early chick embryos
91
of pregastrular ODC activity was found to interfere with development at
gastrulation. Taken together, these results lend support to our idea that pregastrular stimulation of polyamine synthesis is a widely existing event necessary
for the successful accomplishment of gastrulation, the developmental process
that delineates the inception of embryonic autonomy.
This investigation was supported by the Swedish Natural Science Research Council and
by gifts from Stiftelsen Lars Hiertas Minne and the Royal Physiographical Society.
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(Received 14 January 1980, revised 23 May 1980)