/. Embryoï. exp. Morph, Vol. 23, 3, pp. 549-569, 1970
Printed in Great Britain
The role of neurohumours in early embryogenesis
III. Pharmacological analysis of the role of
neurohumours in cleavage divisions
By G. A. BUZNIKOV, 1 A. N. KOST, 2 N. F. K U C H E R O V A , 3
A. L M N D Z H O Y A N , 4 N. N. SUVOROV 5 AND
L. V. B E R D Y S H E V A 1
From the Institute of Developmental Biology, Academy of
Sciences of the USSR, Moscow
In previous papers (Buznikov, Chudakova & Zvezdina, 1964; Buznikov,
Chudakova, Berdysheva & Vyazmina, 1968) we reported that fertilized eggs of
the sea-urchin Strongylocentrotus dwbachiensis synthesized a number of neurohumours, such as serotonin (5-hydroxytryptamine, 5-HT), acetylcholine (ACh),
adrenalin (A), noradrenalin (NA) and dopamine. Synthesis of 5-HT was also
demonstrated in the fertilized eggs of the loach Misgurnus fossilis and some
marine Invertebrata. In experiments with sea-urchin embryos we were able to
trace regular changes in the level of 5-HT, ACh, A and NA, related to the first
cleavage divisions. This early onset of neurohumour synthesis, as well as regular
changes in their level, suggests their direct involvement in the regulation of the
first cleavage divisions.
The functional activity of neurohumours (M) in adult organisms is realized
through their reaction with the active sites of corresponding receptors (R)
according to the following equation :
M+R^MR.
The magnitude of the physiological effect under certain conditions is linearly proportional to the number of complexes MR formed (Turpayev, 1962; Ariëns,
1964). Inhibition of MR complex formation may lead to the decrease or complete disappearance of the physiological effect of the neurohumour.
If neurohumours are indeed directly involved in the regulation of early
1
Authors' address: Institute of Developmental Biology, Academy of Sciences of USSR,
Vavilov Street, 26, Moscow V-133, USSR.
2
Author's address: Moscow State University, Moscow, USSR.
3
Author's address: Institute of Pharmacology and Chemotherapy, Academy of Medical
Sciences, Moscow, USSR.
4
Author's address: Institute of Fine Organic Chemistry, Academy of Sciences of ArmenSSR, Yerevan, ArmenSSR.
5
Author's address: Research Institute of Chemotherapeutics, Moscow, USSR.
549
550
G. A. B U Z N I K O V A N D
OTHERS
embryogenesis, then we must suggest that fertilized eggs possess corresponding
receptors or their functional analogs. Therefore it may be that at least some
neuropharmacological drugs, inhibitors of 'genuine' receptors, would act as
blocking agents of hypothetical receptors in the fertilized eggs. In other words
these drugs would act as antagonists of 5-HT, ACh, A and NA at early developmental stages. This paper describes the search for such antagonists and their use
for the elucidation of the role of neurohumours in early embryogenesis.
MATERIALS AND METHODS
The experiments were conducted using fertilized eggs of several sea-urchin
species: S. dröbachiensis (Barents Sea), S. nudus, S. intermedius (Sea of Japan),
Paracentrotus lividus, Sphaerechlnus granularis and Arbacia lixula (Adriatic Sea).
A few experiments were conducted with the fertilized eggs of the starfish Patiria
pectinifera, lamellibranch mollusc Ostrea gigas (Sea of Japan) and a number of
nudibranch molluscs, Coryphella rufibranchialis, Cuthona nana, Dendronotus
f rondosus and Ancula cristata (Barents Sea). The eggs of Echinodermata and
O. gigas were fertilized artificially; the egg mass of nudibranch molluscs was
obtained from adult organisms kept in an aquarium. Before fertilization the
starfish oocytes were treated with an extract of starfish radiate nerves. This was
necessary for the dissolution of the germinal vesicle (Chaet, 1966; Kanatani &
Ohguri, 1966).
The drugs to be tested were dissolved in sea water. Additional experiments
have shown that the pH of these solutions did not differ significantly from that
of the sea water alone. The eggs were put in these solutions immediately after
fertilization and were kept there until they showed clearly the biological effects
of the drug. In some experiments eggs were exposed to neuropharmacological
drugs for a certain time period and were then washed with sea water. The
efficiency of drugs was estimated from their influence on the cleavage divisions of
fertilized eggs. The results were evaluated by comparison with the control (sea
water without drugs). The percentage of embryos at the 2-blastomere stage was
determined after their fixation with formaldehyde added to a final concentration
of 4 %. Registration by microphotography was employed throughout. More
quantitative characterization of drug efficiency was obtained in experiments
where their influence on the time of formation of the first cleavage furrow was
studied, and in experiments with the incorporation of labelled macromolecular
precursors: [14C]amino acids, [14C]uridine and [14C]thymidine into corresponding macromolecules in vivo. The methods employed in these experiments were
as described in the literature (e.g. Harvey, 1956; Gross & Cousineau, 1964).
Fertilized eggs of S. dröbachiensis and nudibranch molluscs were incubated
at 6-9-5 °C; for Adriatic species the temperature was 14—18 °C, for other
species 20-25 °C. During each individual experiment the temperature was kept
relatively constant.
Neurohumours
in embryogenesis.
Ill
551
Besides commercially available drugs we employed drugs which can be
regarded as potential antagonists of neurohumours but for some reason are not
used in pharmacology. Most of these preparations were first synthesized by the
authors of this paper (A. N. K.,N. F. K., A. L. M.orN. N. S.) or in their laboratories. Several potential antagonists of ACh were kindly supplied by Professor
S. N. Golikov from the Leningrad Institute of Toxicology. Pharmacological
characteristics of the drugs used can be found in the literature (Woolley & Shaw,
1956; Mikhelson, 1957; Barlow, 1957; Mndzhoyan, 1959, 1964; Mashkovsky,
1960,1964; Lazarev, 1961 ; Downing, 1962; Eiduson, Geller, Yuwiler & Eiduson,
1964; Golikov & Razumova, 1964; Zakusov, 1964; Mikhelson & KhromovBorisov, 1964; Suvorov, 1964; Ariëns, 1964; Acheson, 1966). For a more complete list of references and the formulae of preparations used see Buznikov, 1967.
RESULTS
Although there are several observations in the literature concerning the ability
of certain neuropharmacological drugs to affect the processes of early embryogenesis, particularly in the sea-urchin (Rulon, 1941; Villee & Villee, 1952;
Harvey, 1956; Hofmann & Hofmann, 1958; Durante, 1958; Sentein, 1962;
Poulson, 1963; Reddy, Adams & Baird, 1963; Lönning, 1965; Morley, Robson
& Sullivan, 1967) the authors never suggested that the effect of these drugs might
be due to their action on 'prenervous' neurohumoral systems, which were
unknown at that time.
(a) Analogs and antagonists of 5-HT
In search of potential antagonists of 'prenervous' 5-HT we tried about 80
indole derivatives, predominantly indole-alkylamines. About half of them
affected the early development of sea-urchin embryos. When the drugs were given
immediately after fertilization the maximal effect appeared as complete arrest of
cleavage divisions followed by death of the non-cleaving eggs. Lower drug concentrations blocked cleavage divisions completely but did not arrest nuclear
divisions. This resulted in formation of multinucleate cells each having several
dozens of nuclei (Berdysheva & Markova, 1967). When even lower levels of the
drug were used, first cleavage divisions were retarded (Fig. 1). The development of
such embryos usually stopped at the early blastula stage. Often this was accompanied by the formation of unequal blastomeres (Fig. 2B) or 'semiblastulae'
(Fig. 3B): embryos in which one blastomere continued to divide, while the
second was arrested after the first cleavage but contained many nuclei. Finally,
threshold concentrations blocked development at the mid-blastula stage; embryos which were normal at the mid-blastula stage usually showed normal
further development.
It was demonstrated that indole derivatives capable of blocking first cleavage
divisions effectively inhibited the incorporation of [14C]amino acids, [14C]uridine
552
G. A. BUZNIKOV AND OTHERS
14
and [ C]thymidine into corresponding macromolecules of the sea-urchin embryos, that is, they inhibited in vivo the synthesis of protein, RNA and DNA.*
The extent of inhibition of these macromolecular syntheses was very similar.
For a representative experiment of this type see Table 1, where data for the
100
2
80
c
o
IS
I 60
ro
E
o
I 40
c
Q
'w
JO
je
_c
20
0
25
50
75
100
125
Concentration of NK-122 (//g/ml)
150
Fig. 1. The inhibitory action of increasing concentrations of NK-122 on the formation of the first cleavage furrow in fertilized eggs of S. granular is. \ — NK-122;
2 = NK-122 + 5-HT (100/ig/ml). The values are expressed as a percentage of the
control taken as 100 %. At the moment of fixation in this case the first cleavage furrow
is formed in 38-3% of the control, untreated eggs.
substance NK-122 are listed. The degree of inhibition of protein synthesis by
indole derivatives does not change when cleavage divisions are blocked by
colchicine (Fig. 4).
Developmental damage induced by indole derivatives remains reversible for a
certain time period. Under more drastic conditions washing of embryos with sea
water is insufficient to restore development (Fig. 3B); the death of embryos,
however, is delayed. Certain reversibility of drug action was also observed in
experiments with the labelled precursors of macromolecules.
The simplest indole-alkylamine tested—tryptamine (T)—in concentrations
below 100 /ig/ml has practically no effect on the cleavage of sea-urchin eggs.
* (These experiments were conducted in collaboration with Dr G. G. Gause, Jr.)
Neurohumours in embryogenesis. Ill
If
-.
D
k,
553
jfiSfc J l f c - ^ H E
Fig. 2. The action of N, /V-dimethyl-#-3-indolylbutylamine (AK-14, 20/*g/ml) on the
fertilized eggs of S. nudus. (A) Sea water; late blastula. (B) AK-14; from uncleaved
eggs till 32-64-blastomere stage. (C) AK-14+5-HT 50/tg/ml; mid-blastulae; many
embryos are motile. (D) AK-14 4- A 50/tg/ml; weak protective action (all the embryos die at the 32-64-blastomere stage). (E) AK-14+5-HT + A; it is clear that
action of 5-HT and A is not additive, x 42.
Fig. 3. The action of 5-chlortryptamine (5-Cl-T, 500/tg/ml, treatment from 60th to
80th min after fertilization) on the eggs of S. dwbachiensis. (A) sea water; mid-blastulae. (B) 5-Cl-T, washed off with sea water; in most cases no cleavage, in a few cases
abnormal blastula formation. (C) 5-Cl-T, washed off with 5-HT solution in sea
water (100/tg/ml); normal mid-blastulae. x 80.
36
E M B 23
554
G. A. BUZNIKOV AND OTHERS
Table 1. The inhibitory action of NK-122, aprophen and puromyein
on the incorporation of labelled precursors into the TCA-insoluble
fraction of early blastulae ofS. granulans
Inhibition (%)
Treatment
[14C]-lysine
NK-122 (100/tg/ml)
Aprophen (100/tg/ml)
Puromycin (50/ig/ml)
31-8
76-1
65-9
[14C]-uridine [14C]-thymidine
36-5
494
551
34-9
491
57-9
180-min pulse at 14 °C. The puromycin effects are given for comparison.
100 r
100 r
80
O
&
60
è^.
-o
40
4U -
20
10
20
30
.40
Concentration of NK-122 (/*g/m!)
50
0-5
10
1-5
Concentration of aprophen (/<g/ml)
20
Fig. 4. The effect of increasing concentrations of NK-122 (A) and aprophen (B) on
the incorporation of [14C]-lysine into the hot-TCA-insoluble fraction of fertilized
eggs of A. lixula incubated (1) in sea water and (2) in sea water/colchicine solution
(25 /4g/ml). The drugs were added 118 min after fertilization (2-blastomere stage),
[14C]-lysine (0-5/^Ci/ml) 134 min, and TCA 305 min, after fertilization. Colchicinetreated eggs did not cleave.
Active indole-alkylamines differ from T either by the presence of one or several
substituents (methyl, benzyl or haloid) or by the character and position of the
amino alkyl chain. Introduction of a hydroxy group into the indole ring does not
confer the ability to block cleavage divisions. 5-HT, however, affects the time of
onset of the first cleavage furrow (Fig. 1) but this cannot be interpreted as a
Neurohumours
in embryogenesis.
HI
555
delay of development, since the duration of mitotic cycles remains unaltered.
At the blastula stage embryos cultivated from the moment of fertilization in
5-HT do not differ from control ones. Similarly 6-hydroxytryptamine (6-HT)
does not affect sea-urchin development.
If we arrange indole-alkylamines tested in the order of decreasing activity we
obtain the following sequence :
l-benzyl-2,5Haloidtryptamines
,, , ,
„r ,
Methoxydimethylseroto-> NK-122
> Methyl>
• /n A o\
c v. ±u 4, *. •
tryptamines
tryptamines.
Jr
JV
mn (BAS)
5-buthoxytryptamine
The activity of a number of drugs from this series equals the activity of such
inhibitors of cellular division in sea-urchin eggs as colchicine, dinitrophenol or
puromycin.
The activity of indole-alkylamines is decreased when a secondary or tertiary
amino group is substituted for the primary one or when hydroxy, methoxy or
carboxy groups are introduced into the indole ring. The removal of the amino
group or its quaternization results in a complete loss of the activity. This is
illustrated in Table 2.
The sensitivity of sea-urchin embryos to indole derivatives, determined both
by morphological criteria and by the inhibition of protein synthesis, strongly
depends on egg concentration. With respect to this characteristic the drugs can
be divided into two groups. Embryo sensitivity to the drugs of the first group
(NK-122, JV,N-dimethyl-#-3-indolylbutylamine and others) remains relatively
constant over a wide range of egg concentration (from several eggs/ml to as many
as 8000 eggs/ml in A. lixula or 3000 eggs/ml in other sea-urchin species) but if
this maximal concentration is exceeded the sensitivity is decreased. The sensitivity of embryos to the drugs of the second group (haloidtryptamines, methyltryptamines, methoxytryptamines) is high only at low egg concentration (up to
500-600 eggs/ml) and is drastically decreased if the egg concentration is higher.
Developmental aberrations induced by indole derivatives can be partially or
completely prevented or reversed by the addition of 5-HT. Its protective action
can be observed both when 5-HT and the antagonist are given simultaneously or
when 5-HT is given after the removal of the antagonist and washing the embryos
with sea water (Figs. 1, 2, 3). Experimental conditions can be found where the
addition of 5-HT can result in complete normalization of development (Fig. 3).
Under other conditions the protective action is more limited, giving only partial
restoration of the rate of development or the rate of [14C]amino acid incorporation. Sometimes the effect is expressed as a shift of the developmental block to
later stages.
The action of toxic indole derivatives on sea-urchin embryos can be partially
prevented by certain non-toxic indole-alkylamines, containing hydroxy,
carboxy or acetyl groups (melatonin, 6-HT, 5-acetyltryptamine), as well as
by indolyl amino acids or indolyl carboxylic acids (5-hydroxytryptophan,
36-2
556
G. A. BUZNIKOV AND OTHERS
Table 2. The action of some indole derivatives on the fertilized eggs
ofS. dröbachiensis
Drug
Formula
Concentrations (//g/ml)
which arrest development
Before
cleavage
divisions
-CH2—CH,—NHoHCl
Tryptamine
At the
blastula
stage
s^lOO—no effect
NH
5-Chlortryptamine
CI
J
CH2—CH2—NH.-HCI
CH 2 —CH 2 —NH 2 HC1
6-Chlortryptamine
20
20
CI
CH,
CH2—CH2—NH2HC1
5-Methyltryptamine
CH.
5-Methyltryptophan
^N
-CH2—CH—COOH
I
50
10
^100—no effect
NH2
CH2—CH2—CH2—CH2—NH2- HCl
5-3-Indolylbutylamine
50
y
3
CH 2 —CH 2 —CH 2 —CH 2 —N^
HCI
XH,
AW-Dimethyl-£-3-indo Iylbutylamine(AK-14)
^100
y
NK-122
CH 3
I
-CH,—C—NH2HC1
CH,
The drugs were added 5-20 min after fertilization.
20-50
20
Neurohumours
in embryogenesis.
Ill
557
tryptophan, 5-methyTryptophan, 3-indolylacetic acid and others). Both T and 5methoxytryptamine are ineffective. NA and particularly A protect embryos
from the action of toxic indole derivatives (Fig. 2), while choline esters and
histamine are ineffective. However, the magnitude of the protective action of all
these substances except 6-HT is lower than that of 5-HT and is rather variable.
It should be mentioned in this connexion that developmental aberrations
induced by colchicine, various metabolic poisons, actinomycin D, 5-fluorodeoxyuridine and a number of cholino- and adrenolytic drugs are not weakened
or prevented by 5-HT.
m
%
•r
•-
A]
Fig. 5. The action of 7-chlortryptamine (7-C1-T, 50/ig/ml) on the fertilized eggs of
A. crista ta. (A) Sea water; 4-blastomere stage. (B) 7-C1-T; no cleavage. (C) 7-C1T + 5-HT 100/ig/ml; 2-3 blastomeres. x 80.
A number of indole derivatives were tested on fertilized eggs of the starfish
P. pectinifera (10 drugs) and of molluscs (20 drugs). The compounds effective
against fertilized eggs of sea-urchins were also effective against the starfish and
mollusc eggs. Sensitivity of P. pectinifera, O. gigas and A. cristata eggs to active
indole derivatives was approximately the same as that of sea-urchin eggs. For
the developing eggs of C. rufibranchialis, C. nana and D. frondosus it was 5-10
times lower.
5-HT does not affect early development of molluscs and does not protect the
developing eggs of these molluscs from toxic indole derivatives, with the exception of A. cristata. In experiments with A. cristata the protective action of 5-HT
is not less than in experiments with sea-urchin embryos (Fig. 5). The protective
effect of 5-HT can also be observed with P. pectinifera (Fig. 6).
(b) Cholinolytic and cholinomimetic drugs
In experiments with sea-urchin embryos about 80 drugs known as cholinolytics and cholinomimetics were tested. The tested compounds were very different
in both their structure and pharmacological activity. All of them contained
558
G. A. BUZNIKOV AND OTHERS
either a secondary or tertiary amino group or a quaternary ammonium nitrogen atom. About half of the drugs tested affected the development of sea-urchin
embryos. Similar active compounds could be found in all groups of cholinolytics
and cholinomimetics studied, with the exception of curare-like compounds and
anti-cholinesterase drugs. These groups also contained completely inactive
compounds showing no effect in concentrations as high as 500/^g/ml.
The type of action of effective drugs is rather similar to and does not differ much
from the action of indole derivatives. High concentrations of the drugs stop both
cleavage and nuclear divisions; lower concentrations block cleavage, only giving
late inhibition of nuclear divisions; even lower concentrations retard cleavage
divisions and stop development at the early blastula stage; while threshold con-
Fig. 6. The action of 5-C1-T (40/tg/ml) on the fertilized eggs of P. pectinifera. (A)
Sea water; mid-blastulae. (B) 5-C1-T; some of the eggs are not cleaved, some eggs
cleave abnormally. (C) 5-C1-T + 5-HT100 /tg/ml ; in most cases normal mid-blastulae.
x40.
centrations lead to developmental blockade at the mid-blastula stage. Active
compounds, e.g. aprophen, inhibit the incorporation of [14C]amino acids in the
hot-TCA-insoluble fraction of the fertilized eggs (Fig. 4B). The incorporation of
[14C]uridine and [14C]thymidine into the cold-TCA-insoluble fraction is also
inhibited (Table 1). The sensitivity of embryos to active compounds is decreased
with increase of egg concentration and is not changed when the embryos are pretreated with colchicine (Fig. 4 B). Usually, but not always, development is restored
after washing of embryos with sea water.
It should be noted, however, that the action of these drugs has some characteristic features. When given in relatively low concentrations they induce the
formation of thick-walled blastulae with a reduced blastocoel; this is only rarely
observed in experiments with the indole derivatives. On the other hand the
formation of semi-blastulae, frequently observed in embryos treated with indole
compounds, is not observed with the cholinolytics. It may well be that, in contrast to indole derivatives, they affect DNA and RNA synthesis to a lesser extent
Neurohumours
in embryogenes is. Ill
559
than protein synthesis (Table 1). In experiments with A. lixula (but not with
other sea-urchin species) certain cholinolytics such as gangleron and aprophen
showed extremely high activity (Table 3, Fig. 7) which was much higher than the
activity of the most potent indole derivatives.
Additional data about the correlation between structure and activity of cholinolytic and cholinomimetic drugs can be found in other papers (Buznikov,
Table 3. The action of some cholinolytics on the fertilized eggs of sea-urchins
Drug
Aprophen
t
Gangleron
w
C—COO—CH2—CH2—N
CH—CH2—0^
CH 3
r CH3—(CH2)3—o/
L
HCl
V-CH—CH—CH2—N
CH 3
Quateron
Sea-urchin
species
Formula
\ —
CH 3
HCl
^"5
Concentrations
(//g/ml) which
arrest cleavage
divisions
S. dröbachiensis
S. granulans
A. lixula
100
100
01
S. dröbachiensis
S. granulans
A. lixula
50
50
0-1-0-2
C O O — C H — C H — C H 2 — N — C 2 H 5 S. dröbachiensis ^100—no effect
CH 3
CH 3
C2H5
J
The drugs were added 5 min after fertilization.
1966, 1967). Here we only want to stress that the quaternization of nitrogen in
the tested drugs leads to a drastic decrease of their activity. An example is the
transition from gangleron to quateron (Table 3).
ACh and other choline esters in concentrations below 200 /Ag/ml do not affect
the early development of sea-urchins and do not influence the incorporation of
[14C]amino acids by the fertilized eggs. At the same time these compounds
weaken or to some extent neutralize developmental damage induced by active
cholinolytics or cholinomimetics. This protective action can be observed even
if ACh is given to the washed embryos after the removal of the toxic compound, but the effect is not very reproducible. It becomes reproducible only at
very high ACh concentrations many times exceeding the concentration of the
cholinolytic used.
560
G. A. BUZNIKOV AND OTHERS
The results with embryos of A. lixula, possessing very high sensitivity to certain cholinolytics (aprophen, gangleron), were somewhat different. The protective action of ACh and carbachol was quite reproducible and appeared as
more or less complete normalization of development (Fig. 7). It should be noted,
•
•
•
•
•
B
Fig. 7. The action of gangleron (0-1 /*g/ml) on the fertilized eggs of A. lixula. (A)
Gangleron; no cleavage. (B) Gangleron + ACh 200/tg/ml; 4-8-blastomere stage,
as in control (sea water), x 115.
however, that since sensitivity of A. lixula embryos to aprophen and gangleron
is very high, the concentration of choline esters is 1000-2000 times higher than
concentration of blocking drug used. It was found in experiments with the same
species that developmental blockade induced by aprophen or gangleron was not
removed by 5-HT. In other sea-urchin species the protective action of 5-HT and
A against aprophen and gangleron and against some other cholinolytics can be
stronger than the protective action of ACh.
ACh itself does not weaken the action of various mitotic and metabolic
poisons. It does not protect sea-urchin embryos against toxic indole derivatives
or against adrenolytics.
In experiments with the embryos of starfish P. pectinifera 14 cholinolytic and
cholinomimetic drugs were tested. The results were similar to those obtained
with the Strongylocentrotus species. Choline esters do not affect the development
of the starfish; and it is not known whether they can protect against choline
antagonists. It was also found that aprophen and gangleron block the development of O. gigas; ACh does not affect the development of O. gigas and does not
counteract the effects of these two lytics.
Neurohumours
in embryogenesis.
HI
561
(c) Adrenolytic and adrenomimetic drugs
In this section we present the results obtained with two groups of compounds :
adrenomimetic amines (A, NA, isoproterenol (IA), tyramine, amphetamine,
ephedrine) and /^-adrenolytic drugs (dichlorisoproterenol (DC1) and alderlin).
A, NA and IA at 50/tg/ml cause moderate delay of the first cleavage divisions,
leading to a somewhat slower rate of development as compared with the controls. In some experiments A improved the development of poor egg batches or
of embryos developing under unfavourable conditions (low oxygen level,
increased salinity, etc.). Reducing agents such as ascorbic acid (50 /^g/ml) or
mercaptoethanol (16/*g/ml) used as stabilizers of catecholamines had no
influence upon sea-urchin development. Oxidation products of catecholamines,
particularly adrenochrome, were also inactive.
Fig. 8. The action of amphetamine (100/*g/ml) on the fertilized eggs of S. dröbachiensis. (A) Sea water; late blastula. (B) Amphetamine; arrested mid-blastulae.
(C) Amphetamine + A 50/tg/ml; weak protective action. (D) Amphetamine+ NA
50/ig/ml; almost normal and motile mid-blastulae. x42.
Other drugs of the first group—tyramine (200/*g/ml), amphetamine and
ephedrine (50-100 ^g/ml)—caused a marked delay of the first cleavage divisions
and led to the abnormal arrangement of blastomeres. Later this led to the formation of abnormal blastulae, which died before hatching (Fig. 8). Similar
abnormalities were induced by the adrenolytic compound TS-25 (2,5-dimethoxybenzylamine chloride). Sensitivity of developing embryos to all these compounds
decreases markedly with increase of egg concentration.
A and NA reduce or even completely neutralize the effect of both TS-25 and
toxic adrenomimetic amines in fertilized sea-urchin eggs (Fig. 8), but A and NA
are not interchangeable. For example the toxic effect of ephedrine and amphetamine on the embryos of S. dröbachiensis is effectively prevented by NA but only
slightly prevented by A (Fig. 8), while, in the case of TS-25, A is more effective.
In experiments with P. lividus, A is more effective against amphetamine while
562
G. A. BUZNIKOV AND OTHERS
NA is more effective against ephedrine. 5-HT, ACh and histamine usually do not
neutralize the toxic effects of adrenomimetic compounds.
Ephedrine and amphetamine are also active against developing embryos of
P.pectinifera, and the developmental damage is similar to that induced by these
drugs in sea-urchin embryos. Protective action of neurohumours was not tested
in this set of experiments.
In experiments with /?-adrenolytic drugs it was found that DCI effectively
stops cleavage divisions in the sea-urchin, while nuclear divisions are blocked
much later. The effective concentration is 2 yWg/ml for A. lixula and 50-100 /*g/ml
Fig. 9. The action of DCI (100 /*g/ml) on the fertilized eggs of S. dwbachiensis.
(A) Sea water; early blastulae. (B) DCI; no cleavage. (C) DCI + A 50/*g/ml; 4-8blastomere stage, x 62-5.
for other sea-urchin species, the threshold concentration is 0-5 /*g/ml for A. lixula
and 5-10/ig/ml for other sea-urchin species. Alderlin is about 2-5 times more
effective than DCI. In experiments with intact embryos DCI inhibits the incorporation of [14C]amino acids, [14C]uridine and [14C]thymidine into the TCAinsoluble fraction. The inhibitory effect upon development and upon the incorporation of labelled precursors decreases with increase of embryo concentration.
The toxic effects of moderate concentrations of DCI as well as of many other
drugs tested appear to be reversible. Under more drastic treatment conditions,
however, the removal of DCI and washing of embryos with sea water does not
prevent the blockade of development, although embryos die later.
Developmental damage induced by /?-adrenolytic compounds can be diminished
by catecholamines. In experiments with S. dwbachiensis embryos A was the most
efficient protector (Fig. 9); with S. nudus NA was the best. Protective action of
catecholamines can be observed even if they are given after washing off the
inhibitor. Sometimes 5-HT also protects, while choline esters and histamine do
not protect sea-urchin embryos from DCI and alderlin.
DCI also blocks the development of P. pectinifera; effective concentrations
are of the same order of magnitude as for the sea-urchin. The sensitivity of
Neurohumours
in embryogenesis.
Ill
563
O. gigas embryos to DCI is low (the threshold concentration is ^ 100 /tg/ml).
Protective action of neurohumours was not tested.
(d) Phenothiazine derivatives
Seven compounds of this group were tested on developing sea-urchin embryos.
These include promazine, chlorpromazine, dinezine, ethaperazine, fluphenazine,
chloracizine and stelazine. Since the results were published in detail (Buznikov,
1963, 1967) they will be considered here only briefly.
Fig. 10. The action of ethaperazine (30/£g/ml) on the fertilized eggs of S. dröbachiensis. (A) Sea water; early blastulae. (B) Ethaperazine; no cleavage, lysis. (C)
Ethaperazine +5-HT 100/tg/ml; from uncleaved eggs till 2-4-blastomere stage,
lysis. (D) Ethaperazine + NA 50/*g/ml; from uncleaved eggs till 2-4-blastomere
stage, lysis. (E) Ethaperazine + 5-HT 50/*g/ml + NA 50/ig/ml; normal early
blastulae. x 80.
All drugs tested at a final concentration of 10-30 /*g/ml lead to the arrest of
cleavage divisions (Fig. 10); given in concentrations of 1-5/^g/ml they block
development at the mid-blastula stage. The species differences in sensitivity are
564
G. A. B U Z N I K O V A N D
OTHERS
small. Morphological anomalies induced by phenothiazine derivatives are similar
to those induced by toxic indole compounds. Like indole compounds, phenothiazine derivatives can be divided into two groups according to the relationship
between egg concentration and sensitivity. The sensitivity of embryos to promazine and dinezine shows weak dependence on egg concentration; the
sensitivity to other phenothiazine compounds drastically falls with increase in
egg concentration.
In contrast to most other drugs tested, the difference between the minimal
inhibitory concentration of phenothiazine derivatives and the concentration
causing rapid death and lysis of embryos is very small. Sometimes we observed
that after the washing of phenothiazine-treated egg suspension some embryos
continued normal development, while others degenerated and lysed.
The toxic action of phenothiazines on sea-urchin embryos is antagonized by a
number of neurohumours. The spectrum of active neurohumours and/or their
relative effects can differ for different phenothiazine derivatives. Chloracizine is
effectively neutralized by 5-HT, NA and particularly A; chlorpromazine is
neutralized by histamine and particularly by 5-HT and A, etc. It is interesting
that while the action of indole compounds is weakened by 5-HT and 6-HT but
not by 5-methoxytryptamine and T, all these indole compounds protect from
phenothiazine derivatives.
Protective action of different neurohumours against phenothiazine derivatives is additive. Mixture of 5-HT and NA protects S. dröbachiensis embryos
from ethaperazine to a far greater extent than do 5-HT and NA separately
(Fig. 10). This additivity was never observed in experiments with indole derivatives (Fig. 2) or with cholinolytic and adrenolytic drugs. The protective effect of
neurohumours and their mixtures can also be observed when they are given to
embryos after the removal of phenothiazine derivatives and the washing of
embryos with sea water.
DISCUSSION
It was demonstrated in this investigation that a number of neuropharmacological drugs either block or damage the development of fertilized eggs of seaurchins and other animals, while the addition of neurohumours in many cases
prevents or weakens these toxic effects. In this connexion it should be emphasized that :
(a) The protective action of neurohumours is observed in vivo. On the basis of
all pharmacological experience we can reject the possibility of direct interaction
between neurohumours and lytic drugs in vitro. It should be added that if 5-HT
indeed reacts with toxic indole derivatives it would prevent their action not only
on the embryos of Echinodermata but on all organisms studied. However, this
is not the case.
(b) The protective action of neurohumours cannot be explained only by a
decrease of membrane permeability to the lytic compounds. Protective effects
Neurohumours
in embryogenesis.
Ill
565
can be observed even after removal of the lytic drugs by washing, that is, under
conditions where the decrease of membrane permeability cannot be the essential
factor in the protective action.
(c) The protective action of neurohumours has a more or less specific character.
All these facts as well as previous observations demonstrating the synthesis
of 5-HT, A, NA and ACh in developing sea-urchin embryos (Buznikov et al.
1964; Buznikov, 1967; Buznikov et al. 1968) enable us to conclude that many of
the investigated drugs inhibit early development by acting as antagonists of one
or several 'prenervous' neurohumours. Thus, it appears that 5-HT, ACh, A and
NA are necessary for the processes of early development of sea-urchins. The
experiments with A. cristata demonstrate that 5-HT is also necessary for the
early development of nudibranch molluscs.
Neurohumours either separately or in mixtures have very little effect on the
development of embryos. A and NA inhibit the cleavage divisions only in very high
concentrations, while 5-HT causes some delay in the formation of the cleavage
furrow in each mitotic cycle. It may be concluded that the concentrations of
endogenous neurohumours cannot be limiting factors in early embryogenesis.
On the other hand the functions of different neurohumours in developing seaurchin embryos do not appear antagonistic to each other. It is interesting in
this connexion that several effects are common for all groups of the drugs
studied. These include selective inhibition of cleavage (resulting in the formation of multinucleate blastomeres) and inhibition of protein and nucleic acid
synthesis in vivo. The embryos are sensitive to all the effective drugs at any
stage of early development. The sensitivity usually decreases with increase in egg
concentration. In contrast the inhibitory effect of colchicine and puromycin does
not depend on egg concentration, as shown in special control experiments. The
toxic effects of antagonists of 5-HT, ACh or of catecholamines can be weakened
not only by their corresponding neurohumours but also by the other neurohumours. Thus it may well be that the blocking of different neurohumours has
similar consequences and therefore that the functions of different neurohumours
can be similar.
Results of other experiments suggest that the different neurohumours have
different functions in fertilized sea-urchin eggs. For example certain morphological anomalies may be typical for only one group of drugs. The formation of
' semiblastulae ' is typical of the action of indole derivatives, while the reduction
of the blastocoel is characteristic of cholinolytic action. Certain specificity of
induced anomalies follows from the results of cytological analysis (Berdysheva
& Markova, 1967) as well as from isotope experiments (Table 1). Moreover,
toxic effects of indole derivatives are most effectively prevented by 5-HT, while
the effects of adrenolytic compounds are best neutralized by A or NA, etc.
Thus it appears that fertilized sea-urchin eggs contain some serotonin-,
cholino- and adrenoreactive structures. There are no data as to whether they are
genuine receptors, and whether they represent single or different structural
566
G. A. BUZNIKOV AND OTHERS
entities. It is not impossible that the reception of different neurohumours is
accomplished by different active sites of a single macromolecule (Buznikov,
1967). It is interesting in this connexion that phenothiazine derivatives block
some other receptive structures since 5-methoxytryptamine and T protect from
phenothiazine derivatives but do not protect from indole derivatives.
In adult animals the receptive components of neurohumoral systems are
usually found on the outer surface of cell membranes (Eccles, 1964; Martin &
Veale, 1967; Rothstein, 1968). In contrast the reactive structures of fertilized
eggs and early embryos are localized intracellularly. One piece of evidence
supporting this conclusion is the complete or almost complete inactivity of
quaternary analogs of the active tertiary amines (Table 3). It is known from the
literature (Barlow, 1957; Ariëns, 1964) that quaternization of tertiary amines
does not affect their true pharmacological activity but drastically impairs their
ability to pass through the cellular membrane. Poor reproducibility of the protective action of ACh may be due to the intracellular localization of cholinoreceptors. Egg permeability for this quaternary ammonium base is much less
than for effective cholinolytics : secondary or tertiary amines.
The role of neurohumours in early embryogenesis still remains to be elucidated.
It is known that neurohumours participate directly in the regulation of the first
cleavage divisions (Buznikov & Berdysheva, 1966; Chudakova, Berdysheva &
Buznikov, 1966; Buznikov, 1967), but their precise function remains unknown.
Earlier suggestions (Buznikov, Zvezdina & Makeeva, 1966; Buznikov, 1967)
about the role of 'prenervous' neurohumours in the regulation of message
translation still lack firm experimental support.
The relationship between blocking of cell divisions induced by neuropharmacological drugs and the inhibition of protein synthesis also remains unknown.
The inhibition of protein synthesis is sufficient to cause the blocking or suppression of cleavage divisions. The inhibition of protein synthesis as a consequence of blocking cleavage is less probable since the suppression of [14C]amino
acid incorporation by neuropharmacological drugs is observed even in the
presence of colchicine (Fig. 4). These two effects may either be independent of
each other or may be a consequence of inhibition of some primary reaction by
neuropharmacological drugs.
In a number of cases the results of isotope experiments and morphological
observations (Buznikov, 1967; Berdysheva & Markova, 1967) suggest that
neuropharmacological drugs inhibit or damage the nuclear apparatus of the
fertilized egg. It may well be that the structures responsible for the reception of
neurohumours are present not only in the cytoplasm but also in the nucleus. On
the other hand at the present state of our knowledge we cannot exclude the
possibility that inhibition of nucleic acid synthesis is the result of suppression of
some cytoplasmic processes.
In summary, 5-HT, ACh, A and NA in developing sea-urchin embryos (and
5-HT in fertilized nudibranch eggs) act as regulators of some processes of early
Neurohumours in embryogenesis. Ill
567
embryogenesis. The elucidation of the precise role of these substances, their
occurrence among widely different animal species, the changes in their concentration with age is a subject for further investigations.
SUMMARY
1. Many neuropharmacological drugs block the development of sea-urchin
embryos by acting as antagonists of endogenous neurohumours: serotonin
(5-HT), acetylcholine (ACh), adrenaline (A) and noradrenaline (NA) ; the addition of excess of the corresponding neurohumour leads to complete or partial
normalization of development.
2. A number of neuropharmacological drugs inhibit D N A , R N A and protein
synthesis in sea-urchin embryos in vivo.
3. The ability of neuropharmacological drugs to block cleavage divisions was
also demonstrated in fertilized eggs of the starfish Patiria pectinifera and of
several mollusc species. The toxic effects of indole derivatives in embryos of the
nudibranch mollusc Ancula cristata are weakened by the addition of 5-HT.
4. These data indicate that endogenous 5-HT, ACh, A and N A are directly
involved in the processes of early embryogenesis in sea-urchins and that 5-HT is
necessary for early embryogenesis in nudibranch molluscs.
PE3K3ME
Pojib MeflHaTopoB HepBHOH cHCTeMbi B paHHeM 3MÔpHoreHe3e.
.III. qbapMaKOJiorMMecKHH aHajiH3 pojiH Me^HaTopoB BfleneHHax,apo6jieHH5i
1. MHorne
Heopo^apMaKOJiorHHecKHe
npenapaTW
ÔJioKHpyioT pa3BHTHe
MopcKHX ejKeö KaK aHTaroHMCTbi aimoreHHbix HetiporyMopoBicepoTOHHHa (5-HT),
aueTHjixojiHHa (AX), a^peHajmHa (A) HJIH Hopa/npeHajiHHa ( H A ) ; HCKyccTBeHHO
co3,oaHHbw H36biTOK cooTBeTCTByiomero HefiporyMOpa nacTHHHO HJIH nojiHOCTbio
HopMajiH3yeT pa3BHTHe.
2. P a ß HeHpocJ)apMaKOJiorHHecKHx npenapaTOB B o n b u a x in vivo yrueTaiOT
CHHTC3 ÔeJIKOB, P H K H JXHK SMÔpHOHaMH MOpCKHX OKCH.
3. CnocoÔHOCTb Hefipo4)apMaKOJiorH4ecKHX npenapaTOB ÔJioKHpoBaTb pa3BHTHe
oOHapyxeHa TaiŒe B onbiTax Ha onjioAOTBopeHHbix HHuax MopcKofi 3Be3,abi Patiria
pectinifera H p*ma MOJUHOCKOB ; flencTBHe ^epHBaTOB HHAOJia Ha SMÔPHOHOB ro.jio>Ka6epHoro MOJiJiiocKa Ancula cristata ocjiaOJiaeTCH 5-HT.
4. nojiyneHHbie .aaHHbie paccMaTpHBaiOTca Kax npaMoe ,a,OKa3aTejibCTBO HeoóxoAHMOCTH 3H^oreHHbix 5-HT, AX, A H H A fljia npoiieccoB paHHero eM6pHoreHe3a
MopcKHX QyKQpL H Heo6xo/i,HMOCTH 5-HT flJTH npoueccoB paHHero 3MÓpHoreHe3a
rojio)Ka6epHbix MOJTJHOCKOB.
568
G. A. B U Z N I K O V A N D O T H E R S
REFERENCES
ACHESON, G. H. (1966). Second symposium on catecholamines. Pharmac. Rev. 18, 3-803.
ARIENS, E. J. (1964). Molecular Pharmacology, vol. 1. New York: Academic Press.
BARLOW, R. B. (1957). Introduction to Chemical Pharmacology. London: Methuen.
BERDYSHEVA, L. V. & MARKOVA L. N. (1967). Some cytological observations on the action of
antagonists of acetylcholine, catecholamines and serotonin on fertilized sea urchin eggs.
Tsitologiya, 9, 912-21.
BUZNIKOV, G. A. (1963). Tryptamine derivatives applied to the study of the role played by
5-hydroxytryptamine (serotonin) in the embryonic development of Invertebrata. Dokl.
Akad. Nauk SSSR 152, 1270-2.
BUZNIKOV, G. A. (1966). Participation of acetylcholine, adrenaline and noradrenaline in preneural embryogenesis of Echinodermata. Zh. evoluz. Biokhim. Fiziol. 2, 23-30.
BUZNIKOV, G. A. (1967). Low Molecular Weight Regulators in Early Embryogenesis. Moscow:
Nauka.
BUZNIKOV, G. A. & BERDYSHEVA, L. V. (1966). Variations in the functional activity of neurohormones in embryos of Paracentrotus lividus. Dokl. Akad. Nauk SSSR 167, 486-8.
BUZNIKOV,
G. A.,
CHUDAKOVA,
I. V.,
BERDYSHEVA,
L. V. & VYAZMINA, N. M. (1968). The
role of neurohumors in early embryogenesis. II. Acetylcholine and catecholamine content
in developing embryos of sea urchin. / . Embryol. exp. Morph. 20, 119-28.
BUZNIKOV, G. A., CHUDAKOVA, I. V. & ZVEZDINA, N. D. (1964). The role of neurohumours in
early embryogenesis. I. Serotonin content of developing embryos of sea urchin and loach.
/ . Embryol. exp. Morph. 12, 563-73.
BUZNIKOV, G. A., ZVEZDINA, N. D. & MAKEEVA, R. G. (1966). On the possible participation
of serotonin and other neurohormones in the regulation of protein biosynthesis (experiments
carried out on egg-cells of sea urchins). Dokl. Akad. Nauk SSSR 166, 1252-5.
CHAET, A. B. (1966). Neurochemical control of gamete release in starfish. Biol. Bull. mar.
biol. Lab., Woods Hole 130, 43-58.
CHUDAKOVA, I. V., BERDYSHEVA, L. V. & BUZNIKOV, G. A. (1966). Changes in acetylcholine
concentration during the mitotic cycle of fertilized sea urchin eggs. Tsitologiya, 8, 105—7.
DOWNING, D. F. (1962). The chemistry of the psychotomimetic substances. Q. Rev. Biol. 16,
133-62.
DURANTE, M. (1958). Action of Cholinesterase inhibitors on ascidian embryos. Acta Embryol.
Morph, exp. 1, 273-9.
ECCLES, J. C. (1964). The Physiology of Synapses. Berlin: Springer.
EIDUSON, S., GELLER, E., YUWILER, A. & EIDUSON, B. T. (1964). Biochemistry and Behavior.
Princeton : D. van Nostrand.
GOLIKOV, S. N. & RAZUMOVA, M. A. (1964). On the techniques of the study of selective drug
action on the central and peripheral cholinergic systems. Farmak. Toks. 27, 495-8.
GROSS, P. R. & COUSINEAU, G. H. (1964). Macromolecule synthesis and the influence of
actinomycin on early development. Expl Cell Res. 33, 368-95.
HARVEY, E. B. (1956). The American Arbacia and other Sea Urchins. Princeton: Princeton
University Press.
HOFMANN, H. & HOFMANN, E. (1958). Über die pharmakologische Hemmung der Zellteilungsvorgänge durch Derivate des Phenothiazin und ihre Kombinationen mit Narkotika
nach Versuchen am Seeigelei. Pubbl. Staz. zool. Napoli 30, 347-57.
KANATANI, H. & OHGURI, M. (1966). Mechanism of starfish spawning. I. Distribution of
active substance responsible for maturation of oocytes and shedding of gametes. Biol. Bull.
mar. biol. Lab., Woods Hole 131, 104-14.
LAZAREV, N. V. (1961). Handbook on Pharmacology. Leningrad: Medgiz.
LÖNNING, S. (1965). Electron microscopic studies of the block to polyspermy. The influence of
nicotine. Sarsia 18, 17-22.
MARTIN, A. R. & VEALE, J. L. (1967). The nervous system at the cellular level. A. Rev. Physiol.
29, 401-26.
MASHKOVSKY, M. D. (1960). Drugs. Moscow: Publishing House of the Academy of Medical
Sciences, USSR.
Neurohumours in embryogenesis. HI
569
MASHKOVSKY, M. O. (1964). Drugs. Suppl. I. Moscow: Meditsina.
MIKHELSON, M. J. (1957). The Physiological Role of Acetylcholine and Search of New Drugs.
Leningrad: Medgiz.
MIKHELSON, M. J. & KHROMOV-BORISOV, N. V. (1964). The chemical mechanism of physiological acetylcholine action as a basis for the search of new drugs. Zh. vses. khim. Obshch. 9,
418-32.
MNDZHOYAN, A. L. (1959). Gangleron and Results of its Clinical Use. Yerevan: Publ. House
of Acad. Sei. ArmenSSR.
MNDZHOYAN, A. L. (1964). Arpenal and Results of its Clinical Use. Yerevan: Publ. House of
Acad. Sei. ArmenSSR.
MORLEY, P. B., ROBSON, J. M. & SULLIVAN, F. M. (1967). Embryotoxic and teratogenic
action of 5-hydroxytryptamine: mechanism of action in the rat. Br. J. Pharmac. Chemother.
31, 494.
POULSON, E. (1963). Teratogenic effect of 5-hydroxytryptamine in mice. Science, N.Y. 141,
717-18.
REDDY, D. V., ADAMS, F. H. & BAIRD, C. (1963). Teratogenic effects of serotonin. / . Pediat.
63, 394-7.
ROTHSTEIN, A. (1968). Membrane phenomena. A. Rev. Physiol. 30, 15-72.
RULON, O. (1941). The alteration of developmental pattern in the sand dollar by pilocarpine.
Physiol. Zool. 14, 461-9.
SENTEIN, P. (1962). L'analyse du mécanisme des mitoses de segmentation par l'action des
amphétamines. C. r. hebd. Séanc. Acad. Sei., Paris 254, 2224-6.
SUVOROV, N. N. (1964). The current view on the biochemistry of physiologically important
indole derivatives. Zh. vses. khim. Obshch. 9, 395-404.
TURPAYEV, T. M. (1962). The Neurotransmitter role of Acetylcholine and the Nature of Cholinoreceptor. Moscow: Publ. House of Acad. Sei. SSSR.
VILLEE, C. A. & VILLEE, D. T. (1952). Studies on phosphorus metabolism in sea urchin embryos. / . cell. comp. Physiol. 40, 57-71.
WOOLLEY, D. W. & SHAW, E. N. (1956). Antiserotonins in hypertension and the antimetabolite approach to chemotherapy. Science, N.Y. 124, 34.
ZAKUSOV, V. V. (1964). New psychopharmacological drugs (a review). Farmak. Toks. 27,
107-21.
{Manuscript received 12 May 1969, revised 26 September 1969)
37
lî M B 2 3