INTRODUCTION

Serotonin (5HT) and dopamine are well-known classic prenervous transmitters performing a number of regulatory functions during the embryogenesis. The presence of functionally active serotonergic and dopaminergic systems at the early stages of embryonic development long before the formation of nervous cells was shown in a wide range of species [1]. Sea urchins are the classic subjects for studies of prenervous neurotransmitter functions, their early embryos were shown to be highly sensitive to neuropharmaca [2]. Several mechanisms of their effects related with signal transduction pathways involving metabotropic and ionotropic receptors and impacts on the processes of cleavage divisions, blastomere interactions, rigidity of cytoskeleton, and ciliary motility were described [1, 3]. Early embryos of sea urchin are sensitive to agonists and antagonists of serotonin, dopamine, and adrenergic receptors, and these effects are specific, as they are suppressed or prevented by these transmitters or their agonists [4]. However, the specificity of receptor-mediated effects in this particular case should be evaluated with regard of significant differences between transmitter receptors of sea urchins and mammals. Earlier we have shown that the gene homologous to mammalian D2-receptor, as well as several genes annotated as 5HT receptors’ homologues are expressed at all stages of the development of sea urchin Paracentrotus lividus, from oocyte to pluteus [4]. Some components of signal transduction pathways in early sea urchin embryos were studied; it was shown in particular that these mechanisms involve adenylate cyclase system and phosphatidyl inositol pathway, including changes in intracellular levels of free calcium ions [5, 6]. The data show that various cytoskeleton elements are the final target of these signal cascades [3, 7]. The present paper is devoted to the influence of antagonists of 5HT and dopamine receptors on the state of actin and tubuline cytoskeleton in the model of blockade of the first cleavage division of sea urchin embryo [8] allowing quantitative evaluation of the effect of embryotoxic drugs.

MATERIALS AND METHODS

The study was carried out on the embryos of sea urchin Paracentrotus lividus (Lamarck, 1816), collected in Trašte Bay (Adriatic Sea), at the Institute of Marine Biology (Dobrota, Montenegro). Handling of animals, obtaining of gametes and performing artificial fertilization was carried out according to standard protocols [9]. Elevation of vitelline membrane was recorded 5 min after fertilization, only batches with more than 95% fertilized embryos were used for experiments. Experiments using model of the blockade of the first cleavage divisions were carried out according to the protocol described previously [8]. Haloperidol hydrochloride (0931 Tocris Bioscience, USA) and cyproheptadine hydrochloride (C6022 Sigma Aldrich, USA) were administered into the medium in required concentration 10 min after fertilization. Photorecords were made 40 min after fertilization using inverted light microscope Opton (Carl Zeiss, Germany) and Dcm130 Microscope Camera Minisee (Scopetek, China). Percent of full cleavages was counted in images using not less than 100 embryos (% Clv). Eight experiments were carried out to study concentration dependence of the effect. Experimental data approximation and IC50 determination were performed by GraphPad Prism software (GraphPad Software, Inc., USA) using the model of four parameter dose–effect plot (4PL):

$$Y = \frac{{{\text{Bottom}} + ({\text{Top}} - {\text{Bottom}})}}{{1 + {{{10}}^{{(\log {\text{IC5}}0 - X){\text{HillSlope}}}}}}}\,\,\,\,[10].$$

Embryos exposed to ligands at the minimal concentration, at which cleavages were blocked, were fixed for 16 h in 4% paraformaldehyde at 4°C and then were stored in PBS with 0.05% sodium azide for further immunocytochemical analysis. Fibrillar actin was stained by phalloidine conjugated with Alexa Fluor 546 (A12 380, Invitrogen, USA) and microtubules were detected by immunostaining using mouse monoclonal tubulin antibodies (T6793, Sigma–Aldrich, USA) and goat anti-mouse Ig conjugated with Alexa Fluor 488 (ab150 113, Abcam, UK). DNA was stained by Hoechst 33 342 (40046, Biotium, USA). The obtained specimens were mounted into Fluoroshield medium (ab104 135, Abcam, UK) and studied using confocal laser scanning microscope Olympus FV10i (Lab of confocal microscopy, Center of Collective Use of the Moscow Lomonosov State University). Medial optical sections obtained at equal parameters of illumination intensity and detector sensitivity were analyzed for the state of cytoskeleton using software pack Fiji [11]. Quantitative evaluation of the fibrillar actin distribution along radial axis of blastomeres was performed all around the cell using plugin Clock Scan [12]. Each graph was plotted on the basis of averaged values obtained for 10 embryos. Statistical processing of all data was performed using GraphPad Prism program (GraphPad Software, Inc., USA).

RESULTS AND DISCUSSION

Experiments using the model of first cleavage division blockage were carried out to ascertain the dose dependence of haloperidol and cyproheptadine effects shown previously [4]. Figure 1 presents the dose-dependence curves for the antagonists in the concentration range of 10–100 μM, obtained using the model of four-parameter “dose–response” curve [13]. Statistically significant effect of D2-like receptor antagonist haloperidol was detected at 25 μM and averaged 27.1% cleaving embryos. According to this model, IC50 for haloperidol is 21.14 μM. Minimal concentration of the 5HT2 receptors antagonist cyproheptadine evoking statistically significant effect is 75 μM (mean level of cleaving embryos is 32.9%) and IC50 is 59.99 μM. The data on the minimal effective concentrations of antagonists under study are in a good agreement with previously obtained results [4] and were further used in the experiments. It is worth mentioning that the embryo sensitivity to dopamine receptor antagonist haloperidol was nearly threefold higher than to 5HT-antagonist cyproheptadine.

Fig. 1.
figure 1

Concentration dependence of the effects of haloperidol (a) and cyproheptadine (b) on the model of the cleavage division blockage in sea urchin P. lividus. % Clv, percent of embryos that completed the first cleavage division. Mean ± SEM, *p < 0.05 (Dunn’s multiple comparisons test). IC50 is shown by dotted line.

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To study the mechanisms of the cytostatic effect of transmitter receptor antagonists, additional experiments were performed on the effects of minimal blocking concentrations of haloperidol and cyproheptadine on the cleaving embryos and subsequent immunocytochemical examination of the cytoskeleton state. Haloperidol (25 μM) causes blockage of cleavage divisions (Fig. 2b) accompanied with an increase of the depth of cortical actin cytoskeleton and formation of the cytoplasmic granules of polymerized actin (Figs. 2k, 2n). Quantitative evaluation of actin distribution along radial axis of embryo (Fig. 3a) shows that haloperidol induces a significant increase in the amount of fibrillary actin both in cytocortex (Fig. 3c) and in the cytoplasm (Fig. 3d). This observation is in a good agreement with literature data on the transmitters’ influence on the cytoskeleton of sea urchin embryos, which indicate that catecholamines and serotonin antagonists decrease the rigidity of cortical layer of sea urchin zygotes, whereas serotonin and catecholamine antagonists produce opposite effects [3, 7]. These facts demonstrate a negative influence of D2-like receptor on the actin polymerization and mechanical parameters of cytocortex that play an important role in the process of cleavage division. Immunohistochemical staining of microtubules in the embryos incubated in haloperidol reveal serious anomalies of tubuline cytoskeleton organization: absence of peripheral microtubules, asymmetrical mitotic spindle (Fig. 2h), and anomalies of chromosome separation leading to the formation of micronuclei (Fig. 2e). The obtained data testify in favor of a possible role of D2-like receptor in maintaining of regular organization of mitotic spindle in the cleaving sea urchin embryos.

Fig. 2.
figure 2

Influence of haloperidol and cyproheptadine on the cytoskeleton of cleaving embryos of sea urchin P. lividus. Optic microscopy (a–c), phase contrast microscopy with inverted fluorescent DNA staining (d–f), immunofluorescence of tubuline cytoskeleton (g–i), fluorescent labelling of F-actin (j–o). Control (a, d, g, j, m), haloperidol 25 μM (b, e, h, k, n), cyproheptadine 75 μM (c, f, i, l, o).

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Fig. 3.
figure 3

Quantitative analysis of haloperidol (25 μM) and cyproheptadine (75 μM) effects on the cytoskeleton of cleaving embryos of P. lividus. Changes in the fibrillar actin distribution along radial axis of embryos incubated in the presence of cyproheptadine (a) and haloperidol (b). Quantitative evaluation of cytocortical actin polymerization degree in cytocortex (c) and cytoplasm (d). Mean ± SEM, *p < 0.05 (Mann–Whitney test).

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Cyproheptadine (75 μM) blocked the first cleavage division in sea urchin zygotes (Fig. 2c). Granules and chaotically oriented short actin filaments occur in the cytoplasm (Figs. 2l, 2o). Analysis of fibrillary actin distribution along radial embryo axis (Fig. 3b) show a significant increase of actin polymerization both in the cytoplasm (Fig. 3d) and in the cortical layer (Fig. 3c). Total content of immunohistochemically detected tubuline in intact and cyproheptadine-treated embryos does not differ. At the same time, tubuline cytoskeleton of embryos incubated with cyproheptadine was totally depolymerized (Fig. 2i). Besides, there were no signs of karyokinesis (Fig. 2f), suggesting a critical impact of cyproheptadine on the earliest phases of the first cell cycle.

Thus, the obtained data indicate that cytostatic effect of antagonists of dopamine and serotonin receptors in cleaving sea urchin embryos is coupled with their influence on the cytoskeleton elements. Both antagonists increase polymerization of actin cytoskeleton in the cytoplasm and in the cortical layer. Furthermore, both antagonists affect the tubuline cytoskeleton; haloperidol mainly evokes disturbances of mitotic spindle spatial organization, whereas cyproheptadine induces total depolymerization of tubuline and blockage of the mitotic processes.

Analysis of the concentration dependence of cytostatic effect of the antagonist have shown that sea urchin embryos are threefold more sensitive to D2-receptor antagonist haloperidol then to the 5HT2-receptor antagonist cyproheptadine. Earlier it was shown that the cytostatic effect of transmitter receptor ligands weakens in presence of transmitter themselves and that the efficiency of dopamine, serotonine, and adrenaline, as well as their lipophilic analogues, is comparable [4]. In the present work the effects of the transmitters on the cleavage divisions and the state of cytoskeleton were not revealed. At the same time, cyproheptadine and haloperidol influence the state of microfilaments in a similar way, which suggests possible crosstalk mechanisms of serotonergic and dopaminergic signal pathways in the regulation of early stages of the development. One of the most probable key link of these processes is metabotropic receptor that is homolog of D2 receptor whose expression was shown in the early stages of P. lividus development starting from the zygote stage [4]. Difference in the effects of haloperidol and cyproheptadine on the state of tubuline cytoskeleton is probably coupled to peculiar details of signal transduction mechanisms triggered by D2-like and 5-HT2-like receptors. In the case of D2‑like receptors antagonist activates cAMP signalling cascade. It is known that haloperidol is able to disorganize tubuline cytoskeleton by affecting the activity of PKA and Akt kinases [14] and effector proteins Tau [15], Aurora A [16], and KSP/Eg5 [17]. Cyproheptadine blocked PKC signaling cascade that also plays important role in the stabilization of both actin and tubuline cytoskeleton [18]. It seems probable that cytostatic action of dopamine and serotonin antagonists on the cleaving sea urchin embryos are based on similar and/or interconnected molecular mechanisms that require further investigations.