/ . Embryol. exp. Morph. Vol. 20, 1, pp. 119-28, August 1968
With 1 plate
Printed in Great Britain
119
The role of neurohumors in early embryogenesis
II. Acetylclioline and catecholamine content in developing
embryos of sea urchin
By G. A. BUZNIKOV,1 I. V. CHUDAKOVA,1
L. V. BERDYSHEVA1 & N. M. VYAZMINA1
{From the Institute of Developmental Biology, Academy of Sciences
of the USSR, Moscow)
In a previous paper (Buznikov, Chudakova & Zvezdina, 1964) it has been
reported that serotonin (5-hydroxytryptamine, 5-HT) may be involved in early
embryogenesis in various groups of animals. This conclusion was confirmed by
Baker's recent publication (Baker, 1965) concerning 5-HT synthesis in Xenopus
laevis embryos.
Some other low molecular weight substances, neurohumors or related compounds, are known to be synthesized in fertilized eggs as well. Acetylcholine
(ACh) synthesis in sea-urchin eggs and embryos was demonstrated by Numanoi
(1953, 1955, 1959, 1961). It seems possible that ACh can be synthesized in
fertilized insect eggs as well (Morley & Schachter, 1963; Schachter, 1964).
The synthesis of another neurohumor, dopamine (DA), in early insect embryos
seems to be indisputable (Furneaux & McFarlane, 1965). However, in most
cases changes in the level of neurohumors with age have not been studied.
In the present paper data concerning the change of concentration with age of
ACh and catecholamines (adrenaline (A) and noradrenaline (NA)) in early seaurchin embryos will be presented. We thought it would be of interest to investigate the suggestion that serotonin is not the only neurohumor synthesized in
embryos of the sea urchin Strongylocentrotus drobachiensis. In addition, we
compare the changes in the levels of various neurohumors with age.
MATERIALS AND METHODS
The experiments were carried out on developing embryos of the sea urchin
S. drobachiensis. Most of the experiments were carried out at the Murmansk
Marine Biological Institute (on the Barents Sea). After artificial fertilization,
sea urchin eggs were incubated at 7 °C. During the time when we were taking
1
Authors' address: Institute of Developmental Biology, Academy of Sciences of USSR,
Vavilov Street, 26, Moscow V-133, USSR.
120
G. A. BUZNIKOV AND OTHERS
samples some eggs were fixed with Carnoy's fluid to obtain total preparations
(according to Melander & Wingstrand, 1953) for subsequent determination of
mitotic phases. The results were expressed in /ig of active substance (ACh, A or
NA) per 106 embryos.
(a) Estimation of acetylcholine
A dense egg suspension was washed twice with 0-04 N - H C I and then homogenized. Prior to homogenization 1 ml of 0 0 4 N - H C I and 5-6 ml acetone were
added to 1 ml of suspension. The homogenate was incubated for 1 hr at 20-22 °C.
The precipitate was removed by centrifugation. The supernatant was evaporated
on a water-bath, the dry residue extracted with Locke solution diluted 2-4 times
with double-distilled water. In some cases, the Loewi-Hellauer technique was
employed for ACh extraction, which allows exclusion of evaporation (Loewi &
Hellauer, 1938). It was found that the loss of active substance was quite small
and reproducible and need not be taken into account. The extracts obtained
were tested on leech dorsal muscle (Minz, 1955). Prior to the tests the muscle
was treated with Locke solution diluted 2-4 times containing eserine or prostigmine (H0~ 6 g/ml). Before the experiments 'dose-response' curves for ACh
were plotted. The amplitude of muscle contraction produced by ACh served as
a measure of the effect. In some cases to accelerate the relaxation, the muscle
was washed with 5-HT solution (10~8 g/ml) as described by Poloni (1955) and
Schain (1961). To test the specificity of the effects observed, nicotine was used,
which is known to block cholinoreceptors of leech dorsal muscle (Minz, 1955).
The threshold ACh concentration in our experiments was 5 x lO"10 to 1 x 10"9"
g/ml. Some experiments were carried out on the Straub frog heart preparation.
In this case only qualitative estimation of ACh could be made.
(b) Estimation of catecholamines
A dense suspension of the eggs was washed twice with 0-04 N-HCI, then
homogenized in acidified ethanol (9 ml of 96 %ethanoland 1 ml of 0-25 N-HCl/ml
of suspension). The homogenates were sealed in glass ampules and transported
to Moscow. Prior to the analyses the homogenates were centrifuged and the
supernatants evaporated on a water-bath or lyophilized. The dry residues were
extracted with a 5 % solution of trichloroacetic acid, A and NA contents in
extracts were determined by the highly specific colorimetric method developed
by Manukhin (Manukhin, 1961; Manukhin & Vyazmina, 1967). The threshold
values of A and NA which can be detected by this method were 0-001 and 001 /*g
respectively, accuracy ± 10 %. In some cases, simultaneously with analyses by
Manukhin's method, the effect of the extracts on Straub frog heart preparations
treated with atropine was tested. In some instances catecholamines were also
identified by paper chromatography after preliminary purification of extracts on
ion-exchange resin Dowex-50 (Manukhin, Pustovoytova & Vyazmina, unpublished data).
Acetylcholine in embryos
121
The time from taking samples to the beginning of analyses was 3-7 days: for
36 h the samples were kept at a room temperature and for the rest of the time
in a refrigerator. Catecholamines are stable in acid solutions and any loss can
be neglected.
RESULTS
(a) Acetylcholine
ACh was found to be present in mature unfertilized eggs of S. drobachiensis
(Text-fig. 1). In the first minutes after fertilization the ACh concentration drops
to zero. Then it rises quickly and reaches a maximum by 90 min after fertilization.
This period coincides with the period of approach and contact of female and
male pronuclei and with the rise of 5-HT concentration (Buznikov, Chudakova &
Zvezdina, 1964). By the end of the second hour of development ACh practically
disappears from the embryos.
0
1 2 3
4
5 6 7 8 9
10
20 21
30
Age (h)
37 38 39 40 41
48
62 63
75
Text-fig. 1. The changes of ACh content in developing embryos of the sea urchin
S. drobachiensis. (1-4) First, second, third and fourth cleavage divisions respectively;
(5) the motile blastula stage (hatching).
The first two cleavage divisions are accompanied by two almost equal
(7-8 /*g/106 embryos) increases in ACh concentration. In the interval between
them we did not find any detectable amounts of ACh in the samples. The formation of blastulae is characterized by two more rises of the ACh content, the
second one coinciding with the onset of embryonal motility and with hatching.
One analysis made during gastrulation revealed a rather low ACh level (0-8 /tg/106
embryos). At the stage of prism the ACh concentration increases again.
122
G. A. BUZNIKOV AND OTHERS
There are convincing reasons to propose that age changes of ACh level are
much more complicated than would appear from the curve in Text-fig. 1, which
is plotted from comparatively few experimental points. The thorough analysis
of changes of ACh concentration with age which occur during the first mitotic
cycles after fertilization confirms this suggestion (Text-fig. 2). Each point is the
mean of 3-4 determinations. It has been found that in each of the first cell
cycles there are not one but two peaks of ACh concentration. The first peak
1 r
Anaphase Telophase Interphase Prophase Metaphase Anaphase Telophase
Text-fig. 2. The changes of ACh content in fertilized eggs of S. drobachiensis
during the first mitotic cycle.
begins at the metaphase and reaches a maximum at anaphase. The second, more
pronounced rise is less prolonged, and coincides with the late telophase, i.e. with
the formation of the cleavage furrow. A two-peaked curve of this kind is typical
at least of the first two mitotic cycles. The identification of ACh-like substance
as ACh is supported by the following data:
1. Leech dorsal muscle, highly sensitive to ACh, does not contract under the
influence of the majority of endogenous physiologically active substances which
have been studied in this respect (Minz, 1955; Konzett & Riedler, 1963), but it
does contract under the influence of the ACh-like substance.
2. Nicotine inhibits the sensitivity of leech dorsal muscle both to ACh and the
ACh-like substance of sea-urchin eggs.
3. 5-HT abolishes the muscle contraction which is produced both by ACh and
and the ACh-like substance.
4. Serum cholinesterase preparations (activity about 1 unit/ml) have been
found to destroy specifically the active substance in extracts of sea urchin
embryos. Extracts were treated with serum cholinesterase preparations for
40-60 min. at 12-14 °C. After such a treatment extracts lost their ACh-like effect
/. Embryol. exp. Morph., Vol. 20, Part 1
PLATE 1
The effect of extracts of mature unfertilized eggs of S. drobachiensis on the isolated frog heart.
Fig. A. The effect produced by the extract fraction, purified from ATP, on the heart previously
treated with an adrenolytic. f , Time of introduction, j , time of removal, a, d, the extract
fractions; b, atropine (cholinolytic); 5 x 10~5 g/ml; c, ACh, 1 x lO"7 g/ml.
Fig. B. The effect of an extract fraction, purified from ATP, on a heart which was not previously
treated. A two-phase effect is observed, typical for solutions containing both ACh and A.
G. A. BUZN1KOV, I. V. CHUDAKOVA, L. V. BERDYSHEVA & N. M. VYAZMINA
facing p. 122
Acetylcholine in embryos
123
both on leech dorsal muscle and on frog heart preparation. If the duration of
incubation was less than 40-60 min, the extracts did not lose their potency. This
suggests that cholinesterase acts on the active substance of extracts and not the
pharmacological receptors of the test-objects. The effect of serum cholinesterase
preparations on the ACh-like substance was abolished by eserine (1-5 x 10~5 g/ml).
5. The effect of the ACh-like substance, like that of ACh, on a frog heart has
been found to be prevented by atropine (Plate 1).
6. Numanoi (1955) showed by means of paper chromatography that ACh and
not other choline esters is present in sea urchin eggs.
The technique of extraction used in the present experiments allows us to
determine total ACh. Thus we studied the true ACh synthesis in fertilized eggs
but not the release of this substance from pre-existing inactive complexes.
We did not study the mechanism of ACh synthesis in fertilized sea-urchin
eggs. As to the mechanisms of ACh loss during the first cell cycles, it does not
seem to be connected with the presence of acetylcholinesterase (AChE). Using
histochemical methods (Pearse, 1960) and a titrimetric micromethod, we found
AChE activity in fertilized eggs only from the stage of motile blastula onwards.
Similar results were reported previously by Augustinsson & Gustafson (1949).
(b) Catecholamines
Both NA and A are present in mature unfertilized eggs of S. drobachiensis.
The concentration of these substances varies from close to zero to a few tenths
of a /*g/106 eggs. In the first 20-40 min after fertilization catecholamines are
absent. Later they reappear, the variations in their concentrations being very
sharp (from 0 to tens of /*g/106 eggs). Unfortunately, we have not been able to
detect any regularity underlying these variations.
Much clearer results are obtained when examining the changes of NA and A
contents during the second mitotic cycle. Four series of such analyses were
carried out.
Despite significant individual variations, the changes during mitotic cycle
were regular. They were more or less uniform in all the experiments, with two
clearly pronounced rises of concentration at interphase and prophase-prometaphase (Text-fig. 3). There may also be a third rise at metaphase-anaphase,
simultaneously with the first rise in ACh concentration (cf. Text-fig. 2). Between
these rises the A concentration drops, sometimes to zero.
The NA content changes less regularly (Table 1). Increases in NA concentration are observed at interphase and at anaphase-telophase.
The results obtained in experiments with isolated frog heart preparations,
confirmed the presence of A in unfertilized sea-urchin eggs (Plate 1) and embryos. It has been shown that embryonic extracts affect the isolated atropinized
frog heart in a similar way to A. This effect is completely prevented by adrenolytics. We also succeeded in identifying A and NA in extracts of fertilized sea
urchin eggs by paper chromatography.
124
G. A. BUZNIKOV AND OTHERS
As judged by preliminary data, dihydroxyphenylalanine (DOPA) and dopamine (DA) are also present in fertilized sea urchin eggs. DOPA and DA may
cause elevated values of A and NA when determined by Manukhin's method.
Even so it can be concluded that regular changes in catecholamine level (A and
NA per se or together with DA and DOPA) do occur during the first cell cycles.
/-p
7-1
P-Pm
Pm-M
M
A-T
Text-fig. 3. Changes of A content in fertilized eggs of S. drobachiensis during the
second mitotic cycle. T, telophase; I, interphase; P, prophase; Pm, prometaphase;
M, metaphase; A, anaphase. The predominant mitotic phase is underlined. The
arrows show the onset of the first and second cleavage furrow formation.
Table 1. Changes of NA content in fertilized eggs of Strongylocentrotus
drobachiensis during the second mitotic cycle
NA content G«g/106 eggs)
Egg sets
— 0-54 0-79
I
0-45 0
2-54
II
— 0-55 1-25
III
Mitotic phases T
T-l I
—
0
0
/-P
0
—
0
032 2-30
0-53
011 0
—
P
V-Pm Pm-M
0
—
—
M
— 1-64 —
0 102 0
0
M-A A A-T T
Abbreviations as in Text-fig. 3.
The method of extraction used in the present experiments is such that total
catecholamine content is estimated. Thus in this case, as that of ACh, we
observe total synthesis of neurohumors in fertilized eggs rather than the release
of neurohumors from inactive pre-existing complexes.
Acetylcholine in embryos
125
We have not studied the pathways of synthesis and inactivation of catecholamines in early embryos. However, inactivation of catecholamines is unlikely to
be due to monoamine oxidase action. At present it is not known whether
catecholamine inactivation is caused by other enzymes or by their elimination
into the surrounding water.
One of the catecholamines, NA, has been found in fertilized eggs of the loach
Misgurnus fossilis. Colorimetric and chromatographic methods have been used
in these experiments; changes of NA level with age have not been studied.
DISCUSSION
Fertilized eggs and early sea-urchin embryos have been shown to synthesize
a number of neurohumors: serotonin, acetylcholine, adrenaline, noradrenaline
and, possibly, dopamine; loach embryos synthesize serotonin and noradrenaline.
This is the main conclusion drawn from the results obtained in our previous and
present experiments.
Regular changes of concentrations during the first cell cycles are likely to be
characteristic of all neurohumors studied. It should be noted that the curves
presented in the paper can reflect both the rate of the synthesis and that of the
removal of neurohumors. This makes their analysis difficult (Text-figs. 2, 3).
For example, we do not know whether the rises of the ACh level at the anaphase
and telophase (Text-fig. 2) are caused by an increase in synthesis or a slowing of
removal of this substance. The first possibility seems to be more probable.
However, irrespective of the cause of the sharp rises in the level of the neurohumors, the fact that these rises do occur suggests that these substances play
a part in the regulation of the first cell division cycles.
One cannot exclude the possibility that the real ACh content in sea-urchin
embryos is higher than measured owing to the presence of significant amounts
of ATP in the samples. As ATP level in sea urchin embryos remains constant
throughout the period of cleavage divisions (Chambers & Mende, 1953; Hultin,
1957; Nilsson, 1961; Taguchi, 1962; Epel, 1963; Zotin, Milman & Faustov,
1965) this difference between real and experimental ACh content should be the
same in all the samples. Therefore this difference could not have a fundamental
influence on the character of the age curves.
Further confirmation that neurohumors do play a regulatory role in this case
is given by the concentrations of these substances. Concentrations of neurohumors in sea-urchin embryos are much lower than those of usual metabolites
and similar to the concentrations of neurohumors in adults, where they do play
a regulatory role.
The relationship between increases of neurohumor concentrations and events
during cell division is confirmed by the data obtained on infusoria Tetrahymena
pyriformis (Sullivan & Sullivan, 1964). Experiments on synchronized cultures of
T. pyriformis have shown that the ACh content rises rhythmically during cell
126
G. A. BUZNIKOV AND OTHERS
divisions. Our experiments on the action of phenothiazine derivatives on fertilized eggs of different sea-urchin species give confirmatory evidence for direct
participation of neurohumors in the regulation of cell division (Buznikov, 1966,
1967; Buznikov & Berdysheva, 1966). It should be noted that the character of the
intercellular regulatory functions of these substances is still not quite understood.
SUMMARY
1. Acetylcholine (ACh) is found in unfertilized eggs of sea urchin S. drobachiensis. Immediately after fertilization the level of this substance drops sharply,
but in the course of cleavage divisions and at the stages of early blastula, late
blastula and prism the concentration shows transient sharp increases.
2. In the course of the second mitotic cycle, two increases of ACh content
are observed in fertilized eggs of S. drobachiensis. The first rise occurs at
metaphase-anaphase and the second, more pronounced, during the formation
of the cleavage furrow.
3. Adrenaline (A) and noradrenaline (NA) are also found in unfertilized eggs
of S. drobachiensis. After fertilization, sharp changes in the content of these
substances occur which we have not followed in detail.
4. Increases in A concentration are observed at interphase and prophaseprometaphase. The content of NA increases at interphase. It is suggested that
such changes in the level of ACh, A and NA take place during both of the first
cleavage divisions and that these substances play a part in the regulation of cell
division.
PE3I0ME
1. AijeTHJixojiHH (AX) o6Hapy?KeH B spejitix HeonjioaoTBopeHHtix nftueKJieTKax S. drobachiensis.
Ilocjie onjioflOTBopeHHH ypoBem> aToro BemecTBa
pe3K0 CHHHfaeTCH. IToflteMLi KomjeHTpaiTHH A X OTMeneHLi Ha 90-ii
nocjie onjioflOTBopemiH, a TaK?Ke Ha CTaflimx ji;po6jieHHH, paHHeii
noflBHffiHOH: gjiacTyjiH H npn3MH.
2. AflpeHajiHH (A) H HopaflpeHajniH (HA) TaKHte o6HapyjKeHLi
S. drobachiensis.
Ilocjie omioflOTBopemiH Ha6jiioflaioTCH pe3Kne
KOHUeHTpaUHH 8THX BemeCTB,flGTaJIBHOHe H3yieHHHe.
3 . BO BpeMH BTOpOrO MHTOTHHeCKOrO UHKJia B OnJIOflOTBOpeHHMX HHIjeS. drobachiensis
Ha6jnoflaeTCH ^Ba nojri»eMa KOHi^eHTpaunH A X .
H3 no.n'BeMOB npoHcxoflHT BO BpeMH MeTa<J)a3Li aHa(J)a3H, a BTopoft,
6OJIBIUHH no aScojnoTHOfi BejiHHHHe — BO BpeMH o6pa3OBaroiH 6opo3Ati «po6KOHneHTpanira A TaKJKe Ha6jno,o;aiOTCH ^Ba?KABi — BO
H npo(J)a3ti-npoMeTa$a3Bi, anofl^eM KOHLteHTpai];HH H A — BO
HHTep(|)a3Bi. npe^nojiaraeTCH, HTO TaKne HSMeHeHHH ypoBHeii A X , A H H A
BO BpeMH Kawfloro H 3 nepBHx ,n;ejieHHii ji;po6jieHHH.
Acetylcholine in embryos
127
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