Limatola N et al. (2019), Nicotine Induces Polyspermy in Sea Urchin Eggs ...

ECB-ART-48685

Cells 2019 Dec 25;91:. doi: 10.3390/cells9010063.

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Nicotine Induces Polyspermy in Sea Urchin Eggs through a Non-Cholinergic Pathway Modulating Actin Dynamics.

Limatola N , Vasilev F , Santella L , Chun JT .

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: While alkaloids often exert unique pharmacological effects on animal cells, exposure of sea urchin eggs to nicotine causes polyspermy at fertilization in a dose-dependent manner. Here, we studied molecular mechanisms underlying the phenomenon. Although nicotine is an agonist of ionotropic acetylcholine receptors, we found that nicotine-induced polyspermy was neither mimicked by acetylcholine and carbachol nor inhibited by specific antagonists of nicotinic acetylcholine receptors. Unlike acetylcholine and carbachol, nicotine uniquely induced drastic rearrangement of egg cortical microfilaments in a dose-dependent way. Such cytoskeletal changes appeared to render the eggs more receptive to sperm, as judged by the significant alleviation of polyspermy by latrunculin-A and mycalolide-B. In addition, our fluorimetric assay provided the first evidence that nicotine directly accelerates polymerization kinetics of G-actin and attenuates depolymerization of preassembled F-actin. Furthermore, nicotine inhibited cofilin-induced disassembly of F-actin. Unexpectedly, our results suggest that effects of nicotine can also be mediated in some non-cholinergic pathways.

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Genes referenced: LOC100887844 LOC576539 LOC590297

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	Figure 1. Nicotine induces polyspermy in a dose-dependent manner. P. lividus eggs were incubated for 5 min in the presence of various concentration of nicotine. About 10 min after fertilization with Hoechst 33342-prestained sperm, the zygotes were examined with a CCD camera to monitor sperm entry and the elevation of the fertilization envelope (FE). (A) Bright field view and the epifluorescence photomicrographs (bottom) showing the control egg and the egg exposed to 6 mM nicotine prior to fertilization. Whereas a single sperm entered the control egg (yellow arrow), numerous sperm were incorporated into the nicotine-exposed eggs. (B) Quantification of the egg-incorporated sperm and the extent of FE elevation. Green bars in the histogram represent the eggs with full-fledged elevation of FE, while brown bars stand for the eggs showing thin modest elevation of the FE. Error bars indicate standard deviation of the sperm counts averaged from multiple eggs specified in Table 1. U-test: # P < 0.05, * P < 0.01, **** P < 0.00001.
	Figure 2. Acetylcholine receptors are not involved in nicotine-induced polyspermy in P. lividus. (A) Prior to fertilization, eggs were pretreated with either acetylcholine (ACh) or carbachol at the same range of concentration (80 to 150 eggs for a given concentration) and for the same incubation time (5 min) as in the nicotine experiments in Figure 1. The number of Hoechst 33342-prestained sperm incorporated into the egg was counted. (B) P. lividus eggs (n = 40 for each condition) were desensitized by excess amount of acetylcholine (ACh) or carbachol (Carb) for 5 min prior to exposure to 3 mM (â) nicotine (5 min). The egg-incorporated sperm were counted 10 min after fertilization. One-way ANOVA: n.s. (differences statistically non-significant). (C) Eggs were pretreated with various inhibitors of ACh receptors 5 min before the addition of nicotine (2 mM) or NSW. Five minutes later, eggs were fertilized (n = 40 for each condition), and the number of egg-incorporated sperm were counted. # Significantly different from the eggs pretreated with 2 mM nicotine without the inhibitor: Tukey HSD test, # P < 0.05.
	Figure 3. Effects of nicotine pretreatment on the Ca2+ signaling in the fertilized eggs of P. lividus. The Ca2+ responses in the fertilized eggs pretreated with various concentrations of nicotine (5 min) were pooled from multiple experiments comprising 5 to 8 eggs for each nicotine dose. (A) The pseudo-colored relative fluorescence images at the top panel represent the sites of instantaneous Ca2+ increases at the key time points in the eggs fertilized with or without nicotine pretreatment (20 mM). The graph shows the trajectory of the intracellular Ca2+ levels based on relative fluorescence unit (RFU) defined in Materials and Methods. Arrows indicate CF (cortical flash). (B) Effects of nicotine on the CF and the peak of the Ca2+ wave. The peak amplitudes of CF and the Ca2+ wave in individual eggs were normalized in reference to the corresponding average values in the control eggs (no nicotine pretreatment) from the same batch of experiment. (C) Effects of nicotine on other aspects of the Ca2+ response at fertilization: counts of Ca2+ wave origins, latent period (i.e., time interval between the CF and the initiation of the Ca2+ wave), and the time required for the Ca2+ wave to travel from the sperm interaction site to the antipode (traverse time). Values significantly different from the control (0 mM nicotine treatment, Tukey HSD): ** P < 0.001, * P < 0.01.
	Figure 4. Effects of nicotine on the structure of the actin cytoskeleton in the subplasmalemmal region of sea urchin eggs. P. lividus eggs microinjected with AlexaFluor 568âphalloidin (10 ÂµM, pipette concentration) were incubated with different doses of nicotine, and the changes of F-actin were monitored on the equatorial plane of the live individual egg by confocal laser scanning microscope. (A) 2 mM nicotine, (B) 5 mM nicotine. The eggs from the same batch without nicotine exposure were used as the control. For each experiment, the moment immediately before nicotine or seawater addition was taken as t = 0. (C) Latrunculin A (LAT-A) attenuates nicotine-induced rearrangement of cortical F-actin. P. lividus eggs microinjected with AlexaFluor 568âphalloidin were incubated in the presence of 3 Î¼M LAT-A or 0.1% DMSO (control) for 20 min. After rinsing in NSW, the eggs were exposed to 10 mM (â)nicotine, and the changes of F-actin were monitored. Each image represents similar results obtained from 4â7 eggs in the given condition.
	Figure 5. Nicotine-induced polyspermy is inhibited by actin drugs in a dose-dependent manner. P. lividus eggs were pretreated with latrunculin A (A) or mycalolide B (B) for 5 min and rinsed twice in NSW prior to the exposure to 6 mM nicotine (5 min). The eggs were then fertilized with Hoechst 33342-prestained sperm, and the number of egg-incorporated sperm was counted 10 min after fertilization. Control eggs without exposure to actin drugs (0 nM) received the same pretreatment with the vehicle of the drugs (0.1% DMSO). Data were pooled from 4 (latrunculin-A) or 3 (mycalolide B) independent experiments. Each treatment in one experiment comprised 20 eggs from the same batch (n = 80 or 60, respectively). U-test: ** P < 0.00001, * P < 0.0001, ## P < 0.025.
	Figure 6. Nicotine renders egg surface hyper-receptive to supernumerary sperm. (A) Comparison of the cell surface topography of the jelly free eggs by scanning electron microscopy after pretreatment of the eggs in the presence or absence of 5 mM nicotine (5 min). (B) Effects of nicotine on the mobilization of cortical actin filaments following fertilization. P. lividus eggs were microinjected with AlexaFluor568-Phalloidin and subsequently incubated in the presence or absence of 20 mM nicotine for 5 min. The eggs were then fertilized in fresh NSW and the distribution of actin filaments were continuously monitored by confocal microscopy. Merged images with the bright field view show lack of fertilization envelope elevation in nicotine-pretreated eggs. The post-fertilization time points were shown below the merged view of the same egg at the given moment. The images labeled 0 min refer to the ones captured immediately before the addition of sperm.
	Figure 7. Nicotine has an intracellular target that is not a nicotinic AChR. (A) P. lividus eggs (n = 40 for each condition) were microinjected with 1 mM (pipette concentration) of ACh or carbachol. Control eggs (0 mM) were microinjected with distilled water. For comparison, eggs were microinjected with 20 mM (â)nicotine (cytosolic concentration). After 5 min incubation, the eggs were fertilized by Hoechst 33342-prestained sperm. The number of egg-incorporated sperm was counted 10 min after insemination. U-test: **** P < 0.00001. (B) P. lividus eggs were microinjected with inhibitors of nicotinic AChR at various doses, i.e., 10 Î¼M or 100 Î¼M (cytosolic concentration): methyllycaconitine (MLA), hexamethonium (HEX) or mecamylamine (MEC). Control eggs (0 Î¼M) were microinjected with distilled water. After 5 min incubation, the eggs were exposed to 6 mM nicotine for 5 min and fertilized. Egg-incorporated sperm were counted 10 min after insemination. In both panels, data were pooled from two independent experiments comprising 20 eggs for each treatment condition. One-way ANOVA test: n.s., non-significant.
	Figure 8. Effects of microinjected ACh, carbachol and nicotine on cortical F-actin of sea urchin eggs. P. lividus eggs were microinjected with Alexa-Phalloidin (10 Î¼M, pipette concentration). After 15 min, the eggs were microinjected again with ACh or carbachol (1 mM, pipette concentration), and the changes of the actin filament in the same living eggs were monitored by confocal microscopy at 10 min intervals. The confocal images immediately before the second microinjection were considered as t = 0 min. For comparison, the second microinjection of some eggs was made with 1 mM (pipette concentration) of (â)nicotine, or with distilled water (negative control). Please note that only in the eggs microinjected with nicotine, the subplasmalemmal F-actin evidently started to get hyperpolymerized by 10â20 min (arrows) or to form thick F-actin bundles (arrowheads). Scale bar = 20 Î¼m. Each image represents similar results obtained from 4â5 eggs in the given condition.
	Figure 9. Unique effects of nicotine on subplasmalemmal actin filaments and the intracellular Ca2+ levels. (A) P. lividus eggs were microinjected with 10 Î¼M Alexa-Phalloidin. After 15 min incubation, the eggs were exposed to 20 mM nicotine (Nic), acetylcholine (ACh), or carbachol (Carb), and the changes of the actin filaments in the same living eggs were monitored by confocal microscopy at 10 min intervals. Note the hyperpolymerization of subplasmalemmal F-actin in the nicotine-incubated eggs that was much pronounced by 20 min (arrows). Each image represents similar results obtained from 8â10 eggs in the given condition. (B) A delayed increase of intracellular Ca2+ in the eggs exposed to 20 mM Nicotine in the 10 mM Ca2+ artificial seawater (ASW), and Ca2+-free seawater (CaFSW). P. lividus eggs microinjected with Calcium Green were exposed to nicotine and other agonists of nAChR. Unlike nicotine, small and large doses of ACh and carbachol commonly fail to induce the Ca2+ changes.
	Figure 10. Direct impact of nicotine on the polymerization and depolymerization dynamics of actin. (A) Actin polymerization kinetics was assessed following the procedures described in Materials and Methods. The trajectories of the actin polymerization kinetics in the presence or absence of 24 Î¼M nicotine were marked by open brown squares and filled green squares, respectively. Each data point in every curve represents mean Â± SD obtained from quadruple samples. Gray triangles represent the virtually unchanging background levels of fluorescence in the wells containing the buffer and nicotine (note that the error bars are small enough to be masked by the symbols). (B) The depolymerization kinetics of pre-polymerized F-actin in the presence (brown squares) or absence (green squares) of 24 Î¼M nicotine. Please note that addition of 1.2 Î¼M cofilin to the reaction mixture steeply accelerated the depolymerization kinetics (open green circles). When 24 Î¼M nicotine was introduced to the F-actin mixture 1 min prior to the cofilin addition, cofilin was appreciably less effective in depolymerizing F-actin (brown open circles).

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Abood, Isolation of a nicotine binding site from rat brain by affinity chromatography. 1983, Pubmed