In previous papers (Buznikov, Chudakova & Zvezdina, 1964; Buznikov, Chudakova, Berdysheva & Vyazmina, 1968) we reported that fertilized eggs of the sea-urchin Strongylocentrotus dröbachiensis 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:
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 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.

The experiments were conducted using fertilized eggs of several sea-urchin species: S. dröbachiensis (Barents Sea), S. nudus, S. intermedias (Sea of Japan), Paracentrotas lividus, Sphaerechinas granalaris 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, Dendronotas frondosas 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.

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 & Khromov-Borisov, 1964; Suvorov, 1964; Ariëns, 1964; Acheson, 1966). For a more complete list of references and the formulae of preparations used see Buznikov, 1967.

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.

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. 1 = NK-122; 2 = NK-122-I-5-HT (100μg/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.

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. 1 = NK-122; 2 = NK-122-I-5-HT (100μg/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.

Fig. 2.

The action of N, N-dimethyl-δ-3-indolylbutylamine (AK-14, 20 μg/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 μg/ml; mid-blastulae; many embryos are motile. (D) AK-144-A 50μg/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, × 42.

Fig. 2.

The action of N, N-dimethyl-δ-3-indolylbutylamine (AK-14, 20 μg/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 μg/ml; mid-blastulae; many embryos are motile. (D) AK-144-A 50μg/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, × 42.

Fig. 3.

The action of 5-chlortryptamine (5-CI-T, 500 μg/ml, treatment from 60th to 80th min after fertilization) on the eggs of S. dröbachiensis. (A) sea water; mid-blastulae. (B) 5-CI-T, washed off with sea water; in most cases no cleavage, in a few cases abnormal blastula formation. (C) 5-CI-T, washed off with 5-HT solution in sea water (100μg/ml); normal mid-blastulae, × 80.

Fig. 3.

The action of 5-chlortryptamine (5-CI-T, 500 μg/ml, treatment from 60th to 80th min after fertilization) on the eggs of S. dröbachiensis. (A) sea water; mid-blastulae. (B) 5-CI-T, washed off with sea water; in most cases no cleavage, in a few cases abnormal blastula formation. (C) 5-CI-T, washed off with 5-HT solution in sea water (100μg/ml); normal mid-blastulae, × 80.

It was demonstrated that indole derivatives capable of blocking first cleavage divisions effectively inhibited the incorporation of [14C]amino acids, [14C]uridine and [14C]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 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).

Table 1.

The inhibitory action of NK-122, aprophen and puromycin on the incorporation of labelled precursors into the TCA-insoluble fraction of early blastulae of S. granularis

The inhibitory action of NK-122, aprophen and puromycin on the incorporation of labelled precursors into the TCA-insoluble fraction of early blastulae of S. granularis
The inhibitory action of NK-122, aprophen and puromycin on the incorporation of labelled precursors into the TCA-insoluble fraction of early blastulae of S. granularis
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 μg/ml). The drugs were added 118 min after fertilization (2-blastomere stage), [14C]-lysine (0·5 μCi/ml) 134 min, and TCA 305 min, after fertilization. Colchicinetreated eggs did not cleave.

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 μg/ml). The drugs were added 118 min after fertilization (2-blastomere stage), [14C]-lysine (0·5 μCi/ml) 134 min, and TCA 305 min, after fertilization. Colchicinetreated eggs did not cleave.

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 μg/ml has practically no effect on the cleavage of sea-urchin eggs.

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 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 :

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.

Table 2.

The action of some indole derivatives on the fertilized eggs of S. drobachiensis

The action of some indole derivatives on the fertilized eggs of S. drobachiensis
The action of some indole derivatives on the fertilized eggs of S. drobachiensis

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, N,N-dimethyl-δ-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,tryptophan, 5-methyltryptophan, 3-indolylacetic acid and others). BothT and 5-methoxytryptamine 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-fluoro-deoxyuridine and a number of cholino- and adrenolytic drugs are not weakened or prevented by 5-HT.

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. frondosas 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).

Fig. 5.

The action of 7-chlortryptamine (7-C1-T, 50μg/ml) on the fertilized eggs of A. cristata. (A) Sea water; 4-blastomere stage. (B) 7-C1-T; no cleavage. (C) 7-C1-T + 5-HT 100μg/ml; 2–3 blastomeres, × 80.

Fig. 5.

The action of 7-chlortryptamine (7-C1-T, 50μg/ml) on the fertilized eggs of A. cristata. (A) Sea water; 4-blastomere stage. (B) 7-C1-T; no cleavage. (C) 7-C1-T + 5-HT 100μg/ml; 2–3 blastomeres, × 80.

Fig. 6.

The action of 5-C1-T (40μg/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-HT 100 μg/ml ; in most cases normal mid-blastulae. ×40.

Fig. 6.

The action of 5-C1-T (40μg/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-HT 100 μg/ml ; in most cases normal mid-blastulae. ×40.

(b) Cholinolytic and cholinomimetic drags

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 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 μg/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-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. 4B). 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 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.

Table 3.

The action of some cholinolytics on the fertilized eggs of sea-urchins

The action of some cholinolytics on the fertilized eggs of sea-urchins
The action of some cholinolytics on the fertilized eggs of sea-urchins
Fig. 7.

The action of gangleron (0–1 μg/ml) on the fertilized eggs of A. lixula. (A) Gangleron; no cleavage. (B) Gangleron + ACh 200 μg/ml; 4–8-blastomere stage, as in control (sea water), × 115.

Fig. 7.

The action of gangleron (0–1 μg/ml) on the fertilized eggs of A. lixula. (A) Gangleron; no cleavage. (B) Gangleron + ACh 200 μg/ml; 4–8-blastomere stage, as in control (sea water), × 115.

Additional data about the correlation between structure and activity of cholinolytic and cholinomimetic drugs can be found in other papers (Buznikov, 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 μg/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.

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, 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.

(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 β-adrenolytic drugs (dichlorisoproterenol (DCI) and alderlin).

A, NA and IA at 50 μg/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 μg/ml) or mercaptoethanol (16μg/ml) used as stabilizers of catecholamines had no influence upon sea-urchin development. Oxidation products of catecholamines, particularly adrenochrome, were also inactive.

Other drugs of the first group—tyramine (200μg/ml), amphetamine and ephedrine (50–100 μg/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-dimethoxy-benzylamine chloride). Sensitivity of developing embryos to all these compounds decreases markedly with increase of egg concentration.

Fig. 8.

The action of amphetamine (100μg/ml) on the fertilized eggs of S. drô-bachiensis. (A) Sea water; late blastula. (B) Amphetamine; arrested mid-blastulae. (C) Amphetamine + A 50μg/ml; weak protective action. (D) Amphetamine + NA 50μg/ml; almost normal and motile mid-blastulae. ×42.

Fig. 8.

The action of amphetamine (100μg/ml) on the fertilized eggs of S. drô-bachiensis. (A) Sea water; late blastula. (B) Amphetamine; arrested mid-blastulae. (C) Amphetamine + A 50μg/ml; weak protective action. (D) Amphetamine + NA 50μg/ml; almost normal and motile mid-blastulae. ×42.

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. drobachiensis 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 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 β-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 μg/ml for A. lixula and 50–100 μg/ml for other sea-urchin species, the threshold concentration is 0·5 μg/’ml for A. lixula and 5–10 μg/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 TCA-insoluble 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 dimin ished by catecholamines. In experiments with S’, dröbachiensis 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.

Fig. 9.

The action of DCI (100 μg/ml) on the fertilized eggs of S. dröbachiensis. (A) Sea water; early blastulae. (B) DCI; no cleavage. (C) DCI + A 50μg/ml; 4–8-blastomere stage, × 62·5.

Fig. 9.

The action of DCI (100 μg/ml) on the fertilized eggs of S. dröbachiensis. (A) Sea water; early blastulae. (B) DCI; no cleavage. (C) DCI + A 50μg/ml; 4–8-blastomere stage, × 62·5.

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 O. gigas embryos to DCI is low (the threshold concentration is ⩾ 100 μg/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.

All drugs tested at a final concentration of 10–30 μg/ml lead to the arrest of cleavage divisions (Fig. 10); given in concentrations of l–5μg/ml they block development at the mid-blastula stage. The species differences in sensitivity are 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.

Fig. 10.

The action of ethaperazine (30μg/ml) on the fertilized eggs of S. drb-bachiensis. (A) Sea water; early blastulae. (B) Ethaperazine; no cleavage, lysis. (C) Ethaperazine + 5-HT 100μg/ml; from uncleaved eggs till 2–4-blastomere stage, lysis. (D) Ethaperazine + NA 50μg/ml; from uncleaved eggs till 2–4-blastomere stage, lysis. (E) Ethaperazine + 5-HT 50μg/ml + NA 50μg/ml; normal early blastulae, × 80.

Fig. 10.

The action of ethaperazine (30μg/ml) on the fertilized eggs of S. drb-bachiensis. (A) Sea water; early blastulae. (B) Ethaperazine; no cleavage, lysis. (C) Ethaperazine + 5-HT 100μg/ml; from uncleaved eggs till 2–4-blastomere stage, lysis. (D) Ethaperazine + NA 50μg/ml; from uncleaved eggs till 2–4-blastomere stage, lysis. (E) Ethaperazine + 5-HT 50μg/ml + NA 50μg/ml; normal early blastulae, × 80.

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. drobachiensis 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.

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 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 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 chohnolytics: 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 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.

  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 DNA, RNA 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 NA are directly involved in the processes of early embryogenesis in sea-urchins and that 5-HT is necessary for early embryogenesis in nudibranch molluscs.

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*

(These experiments were conducted in collaboration with Dr G. G. Gause, Jr.)