RESEARCH ARTICLE
Extracellular Ca21 Influx Is Crucial for the Early Embryonic Development of the Sea Urchin Echinometra lucunter JOCELMO CA´SSIO DE ARAU´JO LEITE AND LUIS FERNANDO MARQUES-SANTOS Departamento de Biologia Molecular, Centro de Cieˆncias Exatas e da Natureza, Universidade Federal da Paraı´ba, Joa˜o Pessoa, Paraı´ba, Brazil
ABSTRACT
J. Exp. Zool. (Mol. Dev. Evol.) 318:123--133, 2012
The involvement of Ca21 in the activation of eggs and in the first steps of the embryonic development of several species is a well-known phenomenon. An association between Ca21 sources with the fate of the blastopore during embryonic development has been investigated by several authors. Ca21 influx mediated by voltage-gated channels and Ca21 mobilization from intracellular stores are the major sources of Ca21 to egg activation and succeeding cell divisions. Studies on sea urchins embryonic development show that intracellular Ca21 stores are responsible for egg activation and early embryogenesis. In the present work we investigated the involvement of extracellular Ca21 in the first stages of the embryonic development of the sea urchin Echinometra lucunter. Divalent cation chelators EDTA and EGTA strongly blocked the early embryonic development. Adding to this, we demonstrated the involvement of voltage-gated Ca21 channels in E. lucunter embryogenesis since Ca21 channel blockers powerfully inhibited the early embryonic development. Our data also revealed that Ca21 influx is crucial for embryonic development during only the first 40 min postfertilization. However, intracellular Ca21 remains mandatory to embryonic development 40 min postfertilization, seen that both the intracellular Ca21 chelator BAPTA-AM and calmodulin antagonists trifluoperazine and chlorpromazine inhibited the first stages of development when added to embryos culture 50 min postfertilization. Our work highlights the crucial role of extracellular Ca21 influx through voltage-gated Ca21 channels for the early embryonic development of the sea urchin E. lucunter and characterizes an exception in the phylum Echinodermata. J. Exp. Zool. (Mol. Dev. Evol.) 318:123--133, 2012. & 2011Wiley Periodicals, Inc. How to cite this article: Leite JCA, Marques-Santos LF. 2011. Extracellular ca21 influx is crucial for the early embryonic development of the sea urchin Echinometra lucunter. J. Exp. Zool. (Mol. Dev. Evol.) 318:123--133.
The concept of Ca21 as a conductor of intracellular signals was introduced in 1883 by the British scientist Sydney Ringer, who studied the contraction of heart muscle cells isolated from rats (Miller, 2004). Since then, Ca21 has emerged as a ubiquitous second messenger (Rasmussen and Rasmussen, ’90; Borodinsky and Spitzer, 2006). Physiological functions mediated by Ca21 are regulated by changes in cytosolic Ca21 concentration ([Ca21]c). [Ca21]c is normally kept at very low levels (107 M) compared to the extracellular medium (103 M). Under the most diverse stimuli, [Ca21]c transiently increased to levels that vary according to cell type: 101 M in striated skeletal muscle cells; 103 M in
gametes; and 105 M in immune system cells (Nakamura and Yasumasu, ’74; Iino, 2010). The increase in [Ca21]c may be due to Ca21 influx from the extracellular milieu or the mobilization of Correspondence to: Luis Fernando Marques-Santos, Laborato´rio de Biologia do Desenvolvimento, Departamento de Biologia Molecular, Universidade Federal da Paraı´ba, Campus I, Cidade Universita´ria, Joa˜o Pessoa, Paraı´ba, CEP 58051-900, Brazil. E-mail:
[email protected] Received 4 June 2011; Revised 14 August 2011; Accepted 4 October 2011 Published online 14 November 2011 in Wiley Online Library (wileyonline library.com). DOI: 10.1002/jezmde.21450
& 2011 WILEY PERIODICALS, INC.
124 2 this ion from intracellular stores, as well as the sum of these two mechanisms. Organelles such as mitochondria, endosomes, lysosomes, calciosomes, secretory vesicles, Golgi apparatus, and nucleus can store large amounts of Ca21 (Patel and Docampo, 2010). However, the major Ca21 intracellular store is the endoplasmic reticulum, which can accumulate about 102 M of this ion (Montero et al., ’95). On the other hand, the influx is the main source for a rapid increase in [Ca21]c. Extracellular Ca21 influx activates several physiological responses such as skeletal muscle contraction, excitation–contraction coupling in heart muscle, vesicle fusion and the release of neurotransmitters, among others. Ca21 influx is conducted through various channels on the plasma membrane, which includes Ca21 receptor-operated Ca21 channels, second messenger-operated Ca21 channels, store-operated Ca21 channels and voltage-gated Ca21 channels (Cav). Ca21 entry through these channels is driven by the electrochemical gradient of the ion without energetic expense (Berridge et al., 2003). The evidence that ions are essential for the formation of new organisms extends from the pioneering works of the Germanborn scientist Jacques Loeb (’13), who investigated the influence of inorganic ions in the activation of sea urchins eggs (Loeb, ’13). The first report of the intracellular Ca21 increase in the activation of animal female gametes was done by Daniel Mazia studying the early embryonic development of the sea urchin Arbacia punctulata (Mazia, ’37). Several cellular events that occur after the fusion of gametes are dependent of an increase in [Ca21]c, including exocytosis of cortical granules, migration and fusion of the pro-nuclei, cytosol alkalinization and reactivation of the metabolism of the egg (Whitaker, 2008). Ca21 is also responsible for cell-cycle resumption in sea urchin embryonic cells, regulating DNA replication, chromatin condensation, nuclear envelope breakdown, formation of the mitotic spindle, separation of sister chromatids, segregation of the chromosomes and cytokinesis (Poenie et al., ’86; Whitaker and Patel, ’90; Groigno and Whitaker, ’98; Philipova et al., 2005). The source of Ca21 during egg activation has been the subject of debate since the last century. Lionel Jaffe (Jaffe, ’83) proposed an association between Ca21 source with the fate of blastopore during embryonic development. The author hypothesized that Ca21 influx mediated by the voltage-gated channels on the plasma membrane is responsible for the increase in [Ca21]c and egg activation in protostomes (animals where embryonic blastopore gives rise to the mouth). On the other hand, Ca21 mobilization from intracellular stores, mainly the endoplasmic reticulum, would be responsible for the same events in deuterostomes (animals where embryonic blastopore becomes the anus). According to Jaffe, in this group of animals, extracellular Ca21 influx is not essential for egg activation and the first cell divisions. However, later reports showed exceptions to this hypothesis. Studies performed in the mussel Mytilus edulis (Deguchi et al., ’96), in the marine shrimp Sicyonia ingentis J. Exp. Zool. (Mol. Dev. Evol.)
LEITE AND MARQUES-SANTOS (Lindsay et al., ’92), and in the worm Cerebratulus lacteus (Stricker and Smythe, 2006), protostome animals, revealed mechanisms of egg activation independent of extracellular Ca21. Similarly, researches on the deuterostome Phallusia mammillata showed a high dependence on Ca21 influx by voltage-gated channels to fertilization and early embryo development (Goudeau and Goudeau, ’93). Thus far, studies on the deuterostome sea urchins are in agreement with the hypothesis proposed by Jaffe. The first evidence of the independence of extracellular Ca21 influx for the activation of eggs in sea urchins comes from the works of Steinhardt and Epel (’74), which showed that Ca21 ionophore A23187 was able to induce the activation of eggs of Lytechinus pictus and Strongylocentrotus purpuratus in a Ca21-free medium. Takahashi and Sugiyama (’73) had already demonstrated that extracellular Ca21 was not required to egg activation of five sea urchin species—Pseudocentrotus depressus, Hemicentrotus pulcherrimus, Toxopneustes pileolus, Tripneustes gratilla and Echinometra mathaei. The relevance of Ca21 mobilization from intracellular stores to egg activation was demonstrated in studies performed in Lytechinus variegates, L. pictus and S. purpuratus, where exogenous Ca21 was not required for fertilization and subsequent cleavage and pharmacological blockage of extracellular Ca21 influx did not inhibit Ca21 wave (Azarnia and Chambers, ’76; Steinhardt et al., ’77; Schmidt et al., ’82; Shen and Buck, ’93). However, some authors suggest that extracellular Ca21 influx is important in the first seconds postfertilization through spermatozoon Ca21 channel (Cre´ton and Jaffe, ’95). In the present work we studied the involvement of extracellular Ca21 in the first stages of development of the sea urchin Echinometra lucunter (Linnaeus, 1758). We show that extracellular Ca21 influx through voltage-gated Ca21 channels is crucial for the early embryonic development of the sea urchin E. lucunter in a time-dependent fashion. These data characterize an exception in the phylum Echinodermata.
MATERIALS AND METHODS Animals Capture and Maintenance E. lucunter (Linnaeus, 1758) adult sea urchins were collected at Cabo Branco Beach, Joa˜o Pessoa, Paraı´ba, Brazil (71090 S, 341470 O). Animals were transported to the laboratory in plastic containers filled with local seawater. Animals were extensively washed with filtered seawater (FSW) to remove biological contaminants and disposed in a glass tank with 4 l of FSW per animal, under constant oxygen supply. Animal capture was authorized by ICMBios (Instituto Chico Mendes de Conservac- a˜o da Biodiversidade/Authorization code number: 11545-1). Drugs Ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid (EGTA), verapamil
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CA21 INFLUX IN E. lucunter EMBRYONIC DEVELOPMENT (VP), nifedipine (NF), diltiazem (DT), ouabain (OUA), valinomycin (VAL), nigericin (NIG), reversin 205 (REV), MK571, Fluo-3-AM, pluronic acid, ionomycin, trifluoperazine (TFP) and chlorpromazine (CPZ) were purchased from Sigma (St. Louis, MO). Stock solutions of verapamil, diltiazem, ouabain, trifluoperazine and chlorpromazine were prepared in distilled water and stored at 41C. Reversin 205, MK571, BAPTA-AM, Fluo-3-AM and pluronic acid stock solutions were prepared in DMSO and stored at 201C. Nifedipine, ionomycin, valinomycin and nigericin stock solutions were prepared in ethanol and stored at 201C. Vehicles (distilled water, EtOH or DMSO) had no effect on E. lucunter embryonic development. Collection of Gametes and Eggs Fertilization Spawning was induced by an intracoelomic injection of 0.5 M KCl (2–3 ml) and gametes obtaining limited to 15 min after injection. Sperm were collected with a glass Pasteur pipette and kept at 41C until use (not more than 7 days). Eggs were collected directly in FSW and after decantation they were washed twice in 500 ml of fresh FSW. Egg suspension was adjusted to 1 104 cells/ml in artificial sea water (ASW: 425.0 mM NaCl; 9.0 mM KCl; 9.3 mM CaCl2.2H2O; 19.9 mM MgCl2.6H2O; 25.5 mM MgSO4.7H2O; 2.1 mM NaHCO3). Dry sperm were diluted to 1:5,000 in ASW for the fertilization procedure. The fertilization envelope elevation was monitored under phase contrast microscopy. Embryonic Development Assay Embryos (1 104 embryos/ml) were incubated or not with compounds and cultured in sterile 24-multiwell plates at 26721C protected from light. At 90 min (2-cell embryos/first cleavage stage), 120 min (2–4 cell embryos/second cleavage stage) or 240 min (morula stage) postfertilization, embryos aliquots were fixed with 2% formaldehyde and stored at 41C. Analyzes of the embryonic developmental stages were performed with a light microscope at 400 magnification. A total of 100 embryos were evaluated for each sample. All experiments were performed in triplicate and repeated at least three times. Intracellular Staining of [Ca21 ] Eggs were previously treated with MK571 or reversin 205 (10 mM, 10 min) and incubated with 10 mM Fluo-3-AM calcium indicator (20% pluronic acid) for 50 min before fertilization procedure. Fertilization was performed on a glass slide with coverslip under capillarity method. At required time intervals, 20 eggs (fertilized or not) were analyzed under fluorescence microscopy (Olympus BX41 equipped with mercury arc lamp—excitation at 488 nm). Cell images were acquired with SONY Cyber-Shot H3 (Carl Zeiss lens), 1/4 second exposure time, F 3.5 diaphragm aperture and ISO 800. The Ca21 ionophore ionomycin (10 mM) was used as positive control. All images were captured with 40 objective. Fluorescence intensity was analyzed with the software IMAGE J
(U.S. National Institutes of Health, Bethesda) and values expressed as mean of fluorescence intensity (MFI). Experiments were repeated at least three times and data represent mean7 standard error of the mean. Effect of Valinomycin, Ouabain and Nigericin in the Inhibitory Effect of Verapamil on the Embryonic Development Two protocols were used to investigate the effects of valinomycin, nigericin and ouabain on verapamil inhibitory effect. In the first protocol (postverapamil treatment), 1 104 embryos/ml were incubated with verapamil (100 mM), 10 min postfertilization and treated with valinomycin (10 mM), ouabain (25 mM) or nigericin (250 nM) 5 min later. In the second protocol (preverapamil treatment), embryos were incubated with valinomycin (10 mM), ouabain (25 mM) or nigericin (250 nM) 5 min postfertilization and treated with verapamil (100 mM) 5 min later. Embryonic development was monitored as described above. Time-Dependent Ca21 Influx Assays Embryos were treated with verapamil (100 mM) or EGTA (342 mM) at different time intervals postfertilization and embryonic development monitored as described above. Fertilization envelope elevation was monitored under phase contrast microscopy to ensure that fertilization had occurred. All experiments were performed in triplicate and repeated at least three times. Effect of Reversin 205 on the Verapamil Inhibitory Effect Embryos were incubated with reversin 205 (5 mM) 5 min after fertilization and treated or not with verapamil (100 mM) at 20, 30, 40, 60 and 80 min postfertilization. The progression to 2–4 cell embryos stage was monitored as described above. All experiments were performed in triplicate and repeated at least three times. Investigation of the Involvement of Extracellular Ca21 on E. lucunter Embryonic Development 50 min Postfertilization E. lucunter embryos were treated with the intracellular calcium chelator BAPTA-AM (10 mM) and the calmodulin antagonists trifluoperazine (75 mM) or chlorpromazine (35 mM) 50 min postfertilization. The progression to 2–4 cell and morula stage were monitored as describe above. All experiments were performed in triplicate and repeated at least three times.
RESULTS Divalent Cation Chelators Block the Progression to the First and Second Cleavage To investigate the role of extracellular Ca21 in the early events of E. lucunter embryogenesis, fertilized eggs were incubated in ASW with different concentrations of two divalent cation chelators. EDTA and EGTA incubation blocked the first two cell division in a concentration-dependent fashion (Fig. 1). However, EGTA was found to be more effective than EDTA in the blockage of the J. Exp. Zool. (Mol. Dev. Evol.)
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Figure 1. Divalent cation chelators block the progression to the first and second cleavage. Embryos (10 min postfertilization) were incubated with different EDTA () or EGTA ( & ) concentrations. (A) 2-cell embryos (%) represent the percentage of embryos that completed the first cell division. (B) 2–4 cell embryos (%) represent the percentage of embryos that completed the second cell division. Data represent mean7standard error of the mean of four independent experiments performed in triplicates. Po0.001 compared to control (one-way ANOVA followed by Tukey’s test). progression to both cleavage stages. EC50 value of EGTA was 1.90 lesser than EC50 value of EDTA on the progression to the first cleavage (341.9 mM against 649 mM) and 1.76 lesser on the progression to the second cleavage (324.3 mM against 572.1 mM). Ca21 Channel Blockers Inhibit E. lucunter Embryonic Development Since the influx of extracellular Ca21 occurs mainly through the voltage-gated Ca21 channels we studied the effect of three calcium channel blockers on the embryonic development of E. lucunter until morula stage. All compounds were able to block E. lucunter embryonic development in a concentration-dependent manner. Figure 2A shows that nifedipine (NF) was more effective than verapamil (VP) or diltiazem (DT) on the blockage of the first cell division (2-cell embryo stage). This effect was more evident at the highest concentrations where NF inhibited about J. Exp. Zool. (Mol. Dev. Evol.)
Figure 2. Ca21 channel blockers inhibit E. lucunter embryonic development. Embryos (10 min postfertilization) were treated with different nifedipine (), verapamil ( & ) or diltiazem (m) concentrations. (A) 2-cell embryos (%) represent the percentage of embryos that completed the first cell division. (B) 2–4 cell embryos (%) represent the percentage of embryos that completed the second cell division. (C) Morula embryos (%) represent the percentage of embryos that reached the morula stage. Data represent mean7standard error of the mean of four independent experiments performed in triplicates. Po0.001 compared to control (one-way ANOVA followed by Tukey’s test).
CA21 INFLUX IN E. lucunter EMBRYONIC DEVELOPMENT
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70% (300 mM) to 85% (400 mM) of cell division. VP and DT at the higher concentrations (400 mM) inhibited only 45 and 40% of the progression to 2-cell stage, respectively. However, nonspecific Ca21 channel blockers VP and DT were more effective than NF on the blockage of the progression to 2–4 cell and morula stages (Fig. 2B and C). VP and DT showed lower EC50 values (113.5 and 87.9 mM, respectively) than NF (219.5 mM) on the blockage of the progression to the second cleavage stage. Progression to the morula stage was strongly inhibited by the unspecific Ca21 channel blockers, where a complete blockage was obtained with 150 mM (VP) and 250 mM (DT) (Fig. 2C). VP and DT also exhibited lower EC50 values than the L-type calcium channel blocker NF on the blockage to morula stage—71.6 mM (VP) and 78.0 mM (DT) against 239.1 mM (NF). Fluo-3-AM Fluorescence Did Not Increase in E. lucunter Activated Eggs To address the distribution pattern of intracellular Ca21 in E. lucunter activated eggs, we used the membrane permeant fluorescent Ca21 probe Fluo-3-AM. No increase in fluorescence signal was observed when eggs were activated with spermatozoa, even when eggs were preincubated with MK571 or reversin 205, two ABC transporter inhibitors. Fluo-3-AM MFI was 43.1272.31 in activated eggs and 39.9871.98 in unfertilized eggs. Additionally, only a slight cortical fluorescence was observed when eggs were treated with the Ca21 ionophore ionomycin in the presence of 10 mM extracellular Ca21 (MFI 5 45.8973.11). Valinomycin and Ouabain but not Nigericin Prevent the Inhibitory Effect of Verapamil on the Embryonic Development Valinomycin (VAL) is a K1 ionophore that has been widely used as a hyperpolarization agent. Its effect on plasma membrane can induce the opening of some types of Cav as well as influence on its pharmacological regulation. To investigate the effect of plasma membrane hyperpolarization on the inhibitory effect of Ca21 channel blockers on the first stages of the embryonic development, E. lucunter fertilized eggs were treated with VAL before or after VP (100 mM). VAL (10 mM) remarkably circumvented the inhibitory effect of VP on embryonic development when it was added to fertilized eggs before VP. Under this protocol 93.271.6% of eggs reached the 2–4 cell embryos stage against 53.373.2% treated only with VP (Fig. 3). Interestingly, VAL was not able to circumvent VP inhibitory effect when it was added 5 min after the Ca21 channel blocker. Under the same protocols, the electro-neutral carrier nigericin (NIG, 250 nM) did not circumvent the inhibitory effect of VP on embryonic development when added before or after VP (Fig. 3). However, similar reversion results were obtained with the Na1/K1-ATPase inhibitor ouabain (OUA). OUA (25 mM) improved embryonic development when it was added to zygotes before VP (91.572.1% against 53.373.2%, 2–4 cell embryos stage). Adding to this, OUA did not circumvent the inhibitory effect of
Figure 3. Valinomycin (VAL) and ouabain (OUA) but not nigericin (NIG) prevent the inhibitory effect of verapamil on the embryonic development. Embryos were treated with VAL, NIG or OUA before (pre VP) or after (post VP) verapamil addition. 2–4 cell embryos (%) represent the percentage of embryos that completed the second cell division compared to the control group. Data represent mean7standard error of the mean of four independent experiments performed in triplicates. Po0.001 compared to control (one-way ANOVA followed by Tukey’s test). VP when it was added to fertilized eggs after the Ca21 channel blocker (Fig. 3). Time-Dependent Ca21 Influx To verify whether there was an association between the dependence of extracellular Ca21 influx and the time course of the E. lucunter early embryonic development, we investigated the involvement of Cav during the first 80 min of the embryogenesis. VP (100 mM) strongly blocked the progression to 2–4 cell and morula stages mainly when it was added in the first minutes postfertilization (Fig. 4A and B). The inhibitory effect was more pronounced on the progression to morula stage, where the percentage of inhibition ranged from 100% (30 sec postfertilization) to 7071.4% (40 min postfertilization). The Ca21 channel blocker had no effect on embryonic development when added 50 min postfertilization. Extracellular Ca21 Influx Is Crucial for E. lucunter Embryonic Development During the First 40 min Postfertilization Like other Ca21 channel blockers, VP is an ABCB1 protein (also known as P-glycoprotein) substrate. ABCB1 protein is fully active 20 min after fertilization in E. lucunter embryonic cells (De Souza et al., 2010). To investigate whether the loss of the inhibitory effect of VP on the embryonic development, 50 min postfertilization, was due to activity of ABCB1 protein, embryos were incubated with reversin 205 (REV, 5 mM) and treated or not with VP (100 mM) 20–50 min postfertilization. Embryonic development was analyzed in the 2–4 cell stage. Our data show J. Exp. Zool. (Mol. Dev. Evol.)
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Figure 4. Verapamil inhibitory effect on E. lucunter embryonic development is time-dependent. Embryos were treated with verapamil (100 mM) at different times postfertilization. (A) 2–4 cell embryos (%) represent the percentage of embryos that completed the second cell division compared to the control group. (B) Morula embryos (%) represent the percentage of embryos that reached the morula stage compared to the control group. Data represent mean7standard error of the mean of four independent experiments performed in triplicates. Po0.001 compared to control (one-way ANOVA followed by Tukey’s test). that inhibition of ABCB1 protein activity did not confer an inhibitory effect to VP 50 min postfertilization considering that the same percentage of embryos reached that stage of development regardless REV incubation (Fig. 5A). Adding to this, REV per si did not block the progression to 2–4 cell stage. To confirm whether extracellular Ca21 influx was not necessary to early embryogenesis 50 postfertilization, E. lucunter embryos were incubated with EGTA (340 mM) at different time intervals postfertilization and the progression to the second cleavage stage was analyzed. The data revealed a clear time-dependent profile on the inhibitory effect induced by the Ca21 chelator (Fig. 5B). EGTA strongly inhibited embryonic development when it was added in the first 10 min after spermatozoa addition (100.0 to 51.475.8%, J. Exp. Zool. (Mol. Dev. Evol.)
LEITE AND MARQUES-SANTOS
Figure 5. Extracellular Ca21 influx is crucial for E. lucunter embryonic development during the first 40 minutes post-fertilization. (A) Embryos were treated with verapamil (100 mM) at different times postfertilization in the presence (REV1VP) or absence (VP) of reversin 205 (10 mM). Reversin 205 was added 5 min postfertilization. REV—group treated only with reversin (B) Embryos were treated with EGTA (340 mM) at different times postfertilization. 2–4 cell embryos (%) represent the percentage of embryos that completed the second cell division compared to the control group. Data represent mean7standard error of the mean of three independent experiments performed in triplicates. Po0.001 compared to control (one-way ANOVA followed by Tukey’s test). 30 sec and 10 min after fertilization, respectively). The blockage of development was attenuated when EGTA was added 20 min postfertilization (32.874.6%). EGTA had no effect on embryonic development when added 50 min postfertilization.
Intracellular Ca21 Is Required to E. lucunter Embryonic Development 50 min Postfertilization In order that our data show that extracellular Ca21 influx is not required to E. lucunter embryonic development 50 min
CA21 INFLUX IN E. lucunter EMBRYONIC DEVELOPMENT post-fertilization, we examined the requirement of intracellular Ca21 to the first stages of development. The well-known membrane permeable Ca21 chelator BAPTA-AM (10 mM) strongly inhibited the progression to 2–4 cell and morula stages even when added 50 min postfertilization (Fig. 6). Ca21 -Calmodulin Complex Is Involved in E. lucunter Embryonic Development To access the involvement of calmodulin in the first stages of the embryonic development of E. lucunter, embryos were treated with two calmodulin inhibitors: trifluoperazine and chlorpromazine. Both phenothiazines blocked the progression to 2–4 cell and morula stages when added to culture 50 min postfertilization. Trifluoperazine (75 mM) inhibited to 8071.8% the progression to 2–4 cell stage and 86.471.1% the progression to morula stage. Chlorpromazine (35 mM) had similar effects on the progression to 2–4 cell stage (72.371.2%) and morula stage (79.572.2%) (Fig. 7).
DISCUSSION Since the pioneer works of Jacques Loeb in late Eighteen Century, science has been studying the involvement of ions in fertilization and embryonic development of several species. Studies in sea urchins have contributed to the understanding of the role of ions, particularly Ca21, in embryogenesis. Over the past four decades, a series of works have made the involvement of Ca21 in the early stages of embryonic development an amazing and challenging issue. In the present work we show pharmacological evidences that demonstrate the dependence of extracellular Ca21 influx
Figure 6. Intracellular Ca21 is required to E. lucunter embryonic development 50 min postfertilization. Embryos were incubated with BAPTA-AM (10 mM) 50 min postfertilization and the progression to 2–4 cell embryos (white bar) or morula stage (black bar) analyzed. 2–4 cell embryos (%) represent the percentage of embryos that completed the second cell division compared to the control group and morula embryos (%) represent the percentage of embryos that reached the morula stage compared to the control group. Data represent mean7standard error of the mean of three independent experiments performed in triplicates. Po0.001 compared to control (one-way ANOVA followed by Tukey’s test).
129 7 through the Cav for the early embryonic development of the sea urchin E. lucunter. To our knowledge, there is no report on the involvement of Ca21 in the fertilization and embryonic development of E. lucunter. Our data showed that divalent cation chelators EDTA or EGTA inhibited the progression to 2-cell and 2–4 cell stages in a concentration-dependent manner (Fig. 1). However, EGTA was about two times more effective than EDTA, suggesting that extracellular Ca21 chelation and not other divalent ions chelation is involved in the blockage induced by the buffers. These data are in agreement with the fact that EGTA has a much higher affinity for Ca21 than EDTA (Williams, ’70). The employ of Ca21 chelators has been extensively used to study the involvement of extracellular Ca21 in several cellular processes. Schmidt et al. (’82), assessing fertilization with acrosome-reacted sperm in EGTA-buffered media, demonstrated that exogenous Ca21 is not required for fertilization and subsequent cleavage of Strongylocentrotus purpuratus and Lytechinus pictus eggs. On the other hand, Cre´ton and Jaffe (’95) had shown that the calcium buffer BAPTA inhibited Lytechinus variegatus egg activation and its subsequent cleavage. However, this effect was obtained only when the chelating agent was added in the first 20 sec postfertilization. Similar results were previously obtained by Presley and Baker (’70) using EDTA in the study of egg activation of the sea urchins Psammechinus miliar and Echinus esculentus. The inhibitory effect of chelating agents in sea urchin egg activation has been attributed to the blockage of the cortical flash, a fast increase in [Ca21]c in the periphery of the egg induced by influx of Ca21 through the spermatozoon (conduit model, see Cre´ton and Jaffe, ’95). Since EGTA and EDTA were added to zygote 10 min postfertilization our data rule out the conduit model for E. lucunter egg activation, suggesting that Ca21 influx occurs through fertilized egg membrane. Considering the relevance of exogenous Ca21 in E. lucunter egg activation suggested by the chelating agent assays, we decided to investigate the involvement of Cav in the early embryonic development of E. lucunter. The phenylalkylamine calcium channel blocker verapamil and the benzothiazepine calcium channel blocker diltiazem were more effective than the dihydropyridine calcium channel blocker nifedipine, suggesting the involvement of other Cav beyond Cav1 channels (L-type) in the early embryonic development of E. lucunter. Shen and Buck (’93) demonstrated that the L-type Cav blocker nifedipine inhibited cortical flash but had no effect on calcium wave and L. pictus egg activation. The unspecific Cav blocker lanthanum inhibited the first cleavages of L. variegatus activated eggs, but this effect was observed only when the compound was added to eggs until the first 20 sec postfertilization. This feature led authors to attribute lanthanum inhibitory effect to the blockage of calcium entry through spermatozoon instead of egg calcium channels (Cre´ton and Jaffe, ’95). According to our experimental design, the impairment of calcium entry must be attributed to the J. Exp. Zool. (Mol. Dev. Evol.)
130 8 effect of calcium channel blocker on egg calcium channels as addition of the calcium channel blockers was done 10 min postfertilization and fertilization envelope had already been elevated (data not shown). We did not observe an increase in Fluo-3-AM fluorescence in E. lucunter activate eggs, even when eggs were exposed to the Ca21 ionophore ionomycin. These results can be attributed to the activity of ABCB1 or ABCC1 protein in E. lucunter eggs since Fluo-3-AM is a strong ABCB1 protein substrate (Orlicky et al., 2004). Hence, further investigations to address the pattern of intracellular Ca21 increase are needed. Additional assays were performed to confirm the involvement of Cav in the early embryogenesis of E. lucunter. First, we investigate the effect of the potassium ionophore valinomycin in the inhibitory effect of verapamil on the embryonic development. Valinomycin is known to alter plasma membrane potential and regulate voltage-sensitive ionic channels. This compound has been used as a pharmacological tool to induce Ca21 influx in some cell types (Boitano and Omoto, ’91; Colden-Stanfield and Scanlon, 2000). Our results showed that verapamil was not able to block embryonic development when E. lucunter embryos were pretreated with valinomycin (Fig. 3). Some works have demonstrated that Ca21 influx induced by valinomycin is mediated by Cav (Nazarenko et al., 2003; Guzman-Grenfell and GonzalezMartinez, 2004) and that membrane hyperpolarization is able to remove inactivation of Ca21 channels (Krasznai et al., 2000). Interestingly, when valinomycin was added after verapamil it did not circumvent the inhibitory effect of the calcium channel blocker. The same pattern of response was obtained when embryos were pretreated with ouabain, a classical Na1/K1-ATPse inhibitor which leads to the activation of the reverse mode of the Na1/Ca21 exchanger (Bonting and Becker, ’64; Namekata et al., 2009). Similar results were obtained in rat vascular smooth muscle where ouabain blocked the inhibition of the contraction induced by verapamil (Ashida et al., ’91). Furthermore, Ca21 ionophores ionomycin and A23187 (nanomolar to micromolar range) were also not able to circumvent verapamil inhibitory effect on E. lucunter embryonic development when added after the calcium channel blocker (data not shown). This set of data suggests that the onset of Ca21 influx mediated by Cav is crucial for E. lucunter early embryogenesis and development is fated to proceed once extracellular Ca21 entry is established. Considering that valinomycin induces alterations in intracellular K1 concentration (Dise and Goodman, ’85), and to certify that the blockage of verapamil inhibitory effect induced by the potassium ionophore was due to a change in membrane potential, we evaluated the effect of the electroneutral K1/H1 exchange ionophore nigericin (Daniele et al., ’78) on the inhibitory effect of the calcium channel blocker on E. lucunter early embryonic development. Nigericin did not block verapamil inhibitory effect on E. lucunter embryogenesis, which suggests that membrane hyperpolarization and subsequent Ca21 influx must J. Exp. Zool. (Mol. Dev. Evol.)
LEITE AND MARQUES-SANTOS be responsible for blocking the inhibitory effect of verapamil and not a change in K1 intracellular concentration. Nigericin is able to induce a pH reduction that has been associated with the inhibition of DNA synthesis and cell cycle (Margolis et al., ’87). However, it is important to note that nigericin per si did not inhibit E. lucunter embryonic development under the concentration tested. Several Ca21-dependent events occur few minutes after gametes fusion, such as activation of NAD kinase and increase in NADP1 and NADPH concentration, O2 consumption and intracellular pH (Gilbert, 2010). These events have been described in several sea urchin species where the source of Ca21 responsible for an increase in [Ca21]c has been associated with Ca21 mobilization from intracellular stores (Whitaker, 2008). Nonetheless, our data show that E. lucunter embryonic development display an unusual model into the phylum Echinodermata given that verapamil exhibited a clear time-dependent inhibition that extends for, at least, the first 40 min postfertilization (Fig. 4). In all times monitored, fertilization envelope elevation was observed under optical microscopy, which ensures that gamete fusion and zygote formation was accomplished (data not shown). A similar profile of Ca21 influx was observed in the sea urchin Anthocidaris crassispina, which exhibits a sustained 45Ca uptake during the first hour postfertilization that is inhibited by diltiazem or verapamil (Fujino et al., ’85). However, A. crassispina embryonic development is not affected by calcium channel blockers. In resume, our findings reveal a timedependent profile of Ca21 influx on the early embryogenesis of E. lucunter that differs from other sea urchin species where Ca21 influx seems to be crucial during the first 20–30 sec postfertilization, regardless of the type of cell involved in Ca21 transport— spermatozoon or egg (Paul and Johnston, ’78; Cre´ton and Jaffe, ’95). Recently, our group characterized ABCB1 (P-glycoprotein) and ABCC1 (multidrug resistance protein) protein activity in gametes and embryonic cells of E. lucunter (De Souza et al., 2010). ABC transporters constitute a large family of transmembrane proteins involved in the transport of several compounds (Bellamy, ’96). A high activity of ABCB1 and ABCC1 proteins is reached 20 min postfertilization in E. lucunter embryonic cells. Verapamil is a well-known ABC transporter modulator as well as an ABCB1 substrate (Tsuruo et al., ’81). Therefore, the loss of verapamil inhibitory effect (50 min postfertilization) could be attributed to ABCB1 activity. Nevertheless, previous incubation with reversin 205—an ABCB1 inhibitor—did not alter the verapamil time-dependent inhibitory pattern (Fig. 5). Adding to this, when embryos were treated with EGTA at different time intervals postfertilization, the chelating agent exhibited the same inhibitory pattern observed with verapamil under the same treatment protocol. These findings confirm that extracellular Ca21 influx is essential for embryonic development during the first 50 min postfertilization.
131 9
CA21 INFLUX IN E. lucunter EMBRYONIC DEVELOPMENT
Figure 7. Ca21-Calmodulin complex is involved in E. lucunter embryonic development. Embryos were treated with trifluoperazine (75 mM) or chlorpromazine (35 mM) 50 min postfertilization and the progression to 2–4 cell embryos (white bar) or morula stage (black bar) analyzed. 2–4 cell embryos (%) represent the percentage of embryos that completed the second cell division compared to the control group and morula embryos (%) represent the percentage of embryos that reached the morula stage compared to the control group. Data represent mean7standard error of the mean of three independent experiments performed in triplicates. Po0.001 compared to control (one-way ANOVA followed by Tukey’s test). A question that rose from these data is: is intracellular Ca21 needed for the late events that regulate the first cell divisions? In order to answer this question, E. lucunter embryos were incubated with the intracellular Ca21 chelator BAPTA-AM 50 min postfertilization. BAPTA-AM strongly blocked E. lucunter embryogenesis (Fig. 6), showing that intracellular Ca21 remains necessary for the progression of the first stages of the embryonic development even after extracellular Ca21 is no longer available. Moreover, we show that calmodulin is involved in this process since two calmodulin antagonists (trifluoperazine and chlorpromazine) inhibited the progression to 2–4 cell embryo and morula stage (Fig. 7). Our data suggest that intracellular mechanisms can assume [Ca21]c regulation 50 min postfertilization. It has been described that the configuration of endoplasmic reticulum in microdomains is associated with the capability of intracellular Ca21 mobilization in the responsiveness to fertilization stimulus induced by spermatozoon (Stricker and Smythe, 2003). The time lapse between gametes fusion and the first hour postfertilization may be necessary to reorganization of endoplasmic reticulum, which enables this organelle to assume the control of the regulation of [Ca21]c. Further investigations are necessary to address this issue. Additional studies are also needed to investigate a possible down-regulation of Cav during the first hour postfertilization. In addition to the findings obtained with the pharmacological assays, a physiological evidence that drew our attention was the fact that E. lucunter activated eggs exhibit a shorter distance between the plasma membrane of the fertilized egg and the
fertilization envelope, which comprehends the region named perivitelline space. In E. lucunter, this distance measures only 3.5 mm (data not shown) against 12.5 mm in S. purpuratus (Vogel et al., ’99). Elevation of the fertilization envelope depends on the cortical granules exocytosis, an event that is Ca21-dependent (Vogel et al., ’91). Therefore, we suggest that the difference between E. lucunter perivitelline space and other sea urchin perivitelline space may be due to the potential of the distinct source of calcium during egg activation in inducing the increase in [Ca21]c. Since Steinhardt et al. (’77) had elegantly demonstrated that L. pictus eggs can be activated by Ca21 ionophore in a Ca21-free sea water, many works have been conducted in sea urchin species to study the role of Ca21 in the early development. An association between blastopore formation and Ca21 source during fertilization has been investigated by several authors (see Jaffe, ’83; Stricker, ’99). A collection of data shows that extracellular Ca21 influx is predominant in protostomes while mobilization from the intracellular Ca21 stores is the main source of Ca21 in deuterostomes. Our data show a clear extracellular Ca21 influx dependence to E. lucunter embryonic development that resembles protostome fertilization process. Other deuterostome organisms also show this behavior, such as Phallusia mammillata, which exhibits a high dependence on Ca21 influx by voltage-gated channels to the early embryo development (Goudeau and Goudeau, ’93). The goal of our work is to demonstrate that the sea urchin E. lucunter strongly depends on Ca21 influx by voltage-gated channels, what makes this organism an exception into the phylum Echinodermata. Jacques Loeb said that ‘‘all the spermatozoon needs to carry into the egg for the process of fertilization are ions’’. In E. lucunter, it seems that male gametes have the key to open the gates of female gametes to ‘‘the ion.’’
ACKNOWLEDGMENT We thank Dr. George Emmanuel Cavalcanti de Miranda (Departamento de Sistema´tica e Ecologia, Universidade Federal da Paraı´ba) for the filtered sea water Dr. Jose´ Pinto de Siqueira Ju´nior (Departamento de Biologia Molecular, Universidade Federal da Paraı´ba) who has kindly supplied chlorpromazine. We also thank Elis Torrezan for careful English revision of the manuscript. Jocelmo Cassio de Arau´jo Leite is the recipient of graduate fellowship (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico—CNPq).
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