The Presence and Ancestral Role of Gonadotropin-Releasing Hormone in the Reproduction of Scleractinian Coral, Euphyllia ancora
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《内分泌学杂志》
Institute of Marine Biology (W.-H.T., J.-S.H.) and Department of Aquaculture (W.-H.T., Y.-H.T., H.-F.W., C.-F.C.), National Taiwan Ocean University, Keelung 202, Taiwan
National Museum of Marine Biology and Aquarium (W.-H.T., Y.-H.L.), Pingtung 944, Taiwan
Department of Aquaculture, National Kaohsiung Marine University (S.-R.J., W.-S.Y.), Kaohsiung 811, Taiwan
Unite Mixte de Recherche, 5178 Centre National de la Recherche Scientifique, Biologie des Organismes Marins et Ecosystemes, Departement des Milieux et Peuplements Aquatiques, Museum National d’Histoire Naturelle (S.D.), 75231 Paris, France
Abstract
The objectives of this study were to investigate the presence of immunoreactive GnRH (irGnRH) in scleractinian coral, Euphyllia ancora, study its seasonal variation, and evaluate its biological activity. irGnRH was detected and quantified in coral polyps. The biological activity of coral irGnRH was tested on pituitary cells from black porgy by evaluating its ability to stimulate LH release. Coral extracts (10–9–10–5 M irGnRH) as well as mammalian (m) GnRH agonist (10–10–10–6 M) had a similar dose-dependent effect on LH release. Furthermore, GnRH receptor antagonist dose-dependently inhibited the stimulation of LH release in response to coral extracts (10–5 M irGnRH) and mGnRH agonist (10–6 M). Peak levels of irGnRH (10-fold increase) were observed during the spawning period in a 3-yr investigation. Significantly higher aromatase activity and estradiol (E2) levels were also detected during the period of spawning compared with the nonreproductive season. In in vivo experiments, mGnRH agonist time- and dose-dependently stimulated aromatase activity as well as the concentrations of testosterone and E2 in free and glucuronided forms in coral. In conclusion, our data indicate that irGnRH does exist in coral, with its ability to stimulate LH release in fish. Seasonal variations of coral irGnRH, with a dramatic increase during the spawning period, concomitant to that in aromatase and E2, as well as the ability of mGnRH agonist to stimulate coral aromatase, steroidogenesis, and steroid glucuronization suggest that irGnRH plays an important role in the control of oocyte growth and mass spawning in corals.
Introduction
MOST BROADCAST SCLERACTINIAN corals synchronously release their gametes during a brief annual spawning period (1). The spawning period follows the lunar phase (around mid-March in Taiwan). The previous studies on scleractinians suggest that several environmental factors may play an important role in determining the timing of reproduction and spawning (2, 3, 4, 5, 6, 7). The environmental factors include temperature, lunar periodicity, illumination, tidal surge, physical shock, and presence/abundance of food (2, 3, 4, 5, 6, 7, 8, 9), but few studies aimed to elucidate how the endogenous factors may regulate and synchronize gametogenesis and mass spawning in corals. Recently, we found that significantly higher estradiol (E2) than glucuronided E2 levels were detected in the scleractinian coral Euphyllia ancora during the spawning season (10). In contrast, significantly higher glucuronided E2 than free E2 levels were detected in the spawning seawater (10). A role of E2, and more specifically of its glucuronide as secreted in seawater, has been suggested in the communication between polyps and coral colonies and induction of mass spawning (10). Aromatase activity, an important enzyme related to oocyte development in vertebrates, was also clearly detected in this species (10). These findings provide the foundation for additional investigation of the endocrine factors related to the control of reproduction in corals.
Hypothalamus-pituitary-gonadal axis is the main pathway in the regulation of reproduction in vertebrates. The hypothalamic neuropeptide, GnRH, a decapeptide, was discovered to regulate pituitary gonadotropins (LH and FSH), and consequently sex steroids and reproduction in vertebrates (11, 12). In addition to vertebrates, several forms of GnRH have been found in protochordate (tunicate, Chelyosoma productum) (13, 14) and mollusca (15, 16, 17). GnRH with a 12-amino acid peptide was recently characterized in the octopus (18). To date, 16 known forms of GnRH in vertebrates with known amino acid sequence have been characterized (19). Immunological studies have also suggested the presence of GnRHs (from as many as 10 species) in other low invertebrates, such as Porifera, Cnidarians, and Nematodes (19, 20). Therefore, an ancient origin of this neurohormone before the emergence of the vertebrates has been suggested.
In vertebrates, in addition to its regulatory function on pituitary LH and FSH and gonadal maturation, GnRH may also play a variety of roles. Indeed, GnRH was shown to act as a neurotransmitter and neuromodulator in the nervous system (15, 21, 22) and as a local hormone in the gonad (23, 24, 25, 26). For instance, in teleosts, GnRH acts as a brain neuromediator on sexual behavior and as a paracrine gonadal neurohormone on oogenesis (27, 28, 29). GnRH also increased sex steroid levels in protochordate (30, 31). In invertebrates, GnRH induced gamete release in the pond snail (32) and the mollusk (33). This suggests an ancient role of GnRH on reproductive function, before the emergence of vertebrates, possibly exerted directly at the gonadal level, before the appearance of the pituitary. Based on these evolutionary perspectives, the present study aimed at investigating whether GnRH could be a major endogenous mechanism regulating coral reproductive function.
E. ancora is a scleractinian coral abundant on front of fringing coral reefs in Nanwan Bay, southern Taiwan. E. ancora is a gonochoristic species, and spawns in the late spring in Taiwan via external fertilization. The polyps of E. ancora are extended day and night, and they are large enough to obtain tissue samples for endocrine studies. Therefore, E. ancora was selected for the present study. We investigated the presence and seasonal profiles of immunoreactive GnRH (irGnRH) in coral polyps sampled in situ throughout 3 yr. Comparisons were made with aromatase activity and sex steroid levels in coral tissue. The biological activity of coral irGnRH was assessed by its ability to stimulate LH release from teleost pituitary cells in vitro. Finally, the possible physiological roles of irGnRH in the regulation of aromatase and sex steroids were also determined by in vivo experiments on corals. By these approaches, we tested the hypothesis that the GnRH-steroid axis may play a key role in the induction of reproduction (steroid production/oocyte growth) and mass spawning (production of glucuronided steroids) in corals.
Materials and Methods
Coral collection
Samples from three female colonies of coral, E. ancora, were collected from a depth of a 7-meter sea surface by scuba diving from March 1998 to April 2000 in Nanwan Bay of southern Taiwan (21°55'N, 120°59'E). Eleven seasonal collections of corals were obtained during these periods for E2 and GnRH RIA. The corals were collected on April 25, 2000 (spawning period), and used for the identification of coral irGnRH by Sep-Pak column-HPLC-RIA. Monthly collection was also obtained from January to June 2004 for seasonal aromatase activity (total of seven collections; two collections in May). Coral collection was approved by the administration office of the Kenting National Park in Nanwan. Polyp was immediately removed from coral by scraping and was stored in liquid nitrogen before assay.
Coral was collected daily, and spawning activity was checked when the predicted spawning date was approaching. Actual spawning occurred on April 16, May 3, April 25, and May 13 in the western calendar (March 20, March 18, March 21, and March 22 in the lunar calendar) in 1998, 1999, 2000, and 2004, respectively.
Extraction of peptides and preparation of crude extracts
Coral polyps were homogenized with 0.1 N HCl in 0.01 M phosphate buffer saline (PBS; pH 7.0) at 4 C and then centrifuged at 15,000 x g for 30 min. The supernatant was neutralized to pH 6.0 with NaOH in 0.01 M PBS and then purified by a Sep-Pak C18 cartridge column (Shandon Corp., Cheshire, UK) pretreated with 100% methanol and then with 0.12% trifluoroacetic acid (TFA). The elution program was as follows: 10 ml 0.12% TFA and 12 ml 40% acetonitrile. The eluate from 40% acetonitrile was dried in a Speed Vac concentrator (Savant Instruments, Holbrook, NY). The dried eluates (crude coral extracts) were further purified by HPLC before RIA for the identification of coral irGnRH or were directly measured by GnRH RIA in the seasonal samples.
HPLC
The dried materials obtained from the Sep-Pak column were dissolved in 0.12% TFA and further purified through HPLC using a Mightysil reverse phase C18 column (4.6 x 250 mm, 5 μm; Kanto Chemical Co., Tokyo, Japan) and a Jupiter reverse phase C18 column (4.6 x 250 mm; Phnomenex, Torrance, CA). Elution was performed as follows: solvent A, 0.1% TFA; solvent B, acetonitrile in 0.1% TFA; a linear gradient of solvent B 9.8–50.3% for 50 min, 50.3–75% for 5 min, 75% for 5 min with a flow rate of 1 ml/min. The fractions were dried in a Speed Vac concentrator (Savant Instruments) and measured by RIA. The methods of extraction, crude purification, and HPLC separation for coral irGnRH were modified from previous studies (34). Standard seabream (sb) GnRH, salmon (s) GnRH, chicken (c) GnRH-I, cGnRH-II, mammalian (m) GnRH, and lamprey (l) GnRH I were purchased from Bachem AG (Bubendorf, Switzerland).
In vitro effects of coral extract and GnRH agonist on LH release by primary culture of fish pituitary cells
Preparation of dispersed fish pituitary cells and static culture.
Black porgy fish (Acanthopagrus schlegeli) pituitary cell culture was developed. For each culture experiment, fifteen 2+-yr-old black porgy fish were decapitated, and pituitary cell culture was performed using enzymatic and mechanic procedures according to the previous studies (35). Pituitary cells were resuspended in culture medium (CM): medium 199 with Earle’s salts, sodium bicarbonate, 100 U penicillin/ml, 100 μg streptomycin/ml, and 250 ng fungizone/ml (Invitrogen Life Technologies, Inc., Gaithersburg, MD). Cells were cultured in 96-well tissue culture plates (Falcon, BD Biosciences, Franklin Park, NJ) at a density of 62,000 cells/125 μl CM/well with 1% fetal bovine serum (Invitrogen Life Technologies) in a tissue culture incubator (NU4500, CO2 water-jacketed incubator; NUAIRE, Plymouth, MN) at 24 C under 5% CO2 and saturated humidity. Measurements of LH release were performed on the third day of culture (d 0). Before addition of treatments, cells were washed three times and incubated in CM (0.25 ml/well) in the presence of 1.0% fetal bovine serum. Six wells were used for each treatment. Aliquots of culture medium were removed after 1, 2, and 4 h of culture in the respective four treatments. LH concentrations in the CM were measured by RIA.
Treatment with GnRH agonist and GnRH receptor antagonist in dispersed fish pituitary cells.
In the first experiment, a mGnRH agonist (a synthetic mGnRH agonist, [D-Ala6]-HRH ethylamide; Sigma-Aldrich Corp., St. Louis, MO) was tested over 4 h at various concentrations (10–6, 10–8, and 10–10 M). This mammalian agonist has been previously shown to be active on LH release in other vertebrates, including various teleosts, namely, the black porgy (35). In the second experiment, dispersed pituitary cells were treated with medium alone (blank) or with mGnRH agonist (10–8 and 10–6 M) alone or in the presence of various concentrations of GnRH receptor antagonist (10–9, 10–7, and 10–5 M). The mGnRH receptor antagonist (GnRH receptor antagonist, [D-Phe2,D-Ala6]LHRH) was purchased from Sigma-Aldrich Corp. This mGnRH receptor antagonist has been previously shown to be able to block the GnRH stimulatory effect on LH release in vivo or in vitro in other vertebrates, including the black porgy (35).
Treatment with coral extracts and GnRH receptor antagonist in dispersed fish pituitary cells.
irGnRH concentrations in coral extracts (collected from the spawning period) purified through a Sep-Pak column were quantified, and irGnRH concentrations (10–3 M) were prepared as a stock. In the third experiment, coral extracts containing various doses of irGnRH (10–5, 10–7, and 10–9 M) were tested in the dispersed pituitary cells over 4 h.
In the fourth experiment, cells were treated with medium alone (controls) or with coral extracts (10–5 M irGnRH) alone or in the presence of various concentrations of GnRH receptor antagonist (10–9, 10–7, and 10–5 M).
Effects of GnRH agonist on the coral aromatase activity and steroidogenesis in vivo
Twelve pieces (8 x 8 cm/piece) of corals were cultured in three tanks (four pieces per tank with 5 liters seawater at 25 C) in a set of experiment. The whole set of the experiment was repeated three times. Corals were respectively obtained during the nonspawning period (March 7, May 3, and November 7 of 2002). mGnRH agonist was added to seawater on d 0, 2, and 4 at final concentrations of 0 (blank), 0.5, and 1.5 μM, respectively. Corals were collected from each tank on d 0, 2, 4, and 6. Aromatase activity, testosterone (T), and E2 levels in free and glucuronided forms in corals were measured. To combine three sets of experimental data, the value of aromatase activity, T or E2 on d 0 was considered as 100%, and the data from d 2, 4, and 6 were calculated according to the value on d 0.
LH assay
Concentrations of LH in the culture medium were measured using a homologous RIA employing purified black porgy LH as a standard with antiblack porgy LH serum as an antibody according to previous study (36).
GnRH assay
Coral extracts from a Sep-Pak column (in the seasonal samples) or further purified by HPLC (for the identification of coral irGnRH) were dissolved in 0.01 M PBS with gelatin for GnRH RIA according to previous methods (37). Coral extracts did not cross-react with antisera strictly specific to sbGnRH (Fig. 1A), sGnRH, cGnRH-II, or mGnRH (data not shown). Therefore, an RIA for coral irGnRH was developed using an sGnRH antiserum with a broad spectrum of cross-reactivity. This sGnRH antiserum was induced in a rabbit using sGnRH linked to BSA. sGnRH was iodinated with chloramine T and separated in a Sephadex G-25 column. The RIA employed sGnRH (Sigma-Aldrich Corp.) as standard and [125I]sGnRH as labeled peptide. The antiserum cross-reacted with various forms GnRH, such as sGnRH, mGnRH, sbGnRH, cGnRH-I, cGnRH-II, and lGnRH I (Fig. 1B), but did not have any cross-reactivity with other neuropeptides, such as TRH, isotocin, vasotocin, or somatostatin.
Sex steroid RIA
For studies on seasonal profiles (free E2) and effects of GnRH agonist on sex steroids (free and glucuronided forms) in coral, sex steroid (T and E2) RIAs were conducted according to previous studies in coral (10). Coral polyps were homogenized with 0.01 M PBS with gelatin (pH 7.0) at 4 C. Coral homogenate was extracted three times with 5-fold (vol/vol) diethyl ether, and finally an ether phase (first round ether extracts contained free steroids) and a liquid phase were obtained. The liquid phase was further extracted once with diethyl ether (fourth ether extracts), and the remaining liquid phase (contained glucuronided steroids) was hydrolyzed with -glucuronidase (Sigma-Aldrich Corp.) to hydrolyze the conjugated steroids (10). The hydrolyzed solution was extracted three more times with diethyl ether (second round ether extracts contained glucuronided steroids). The extracts (first and second round ether extracts and fourth ether extracts) were purified in an alumina column and reverse phase column HPLC before steroid RIA (10). Results were expressed as nanograms of steroid per gram of tissue (wet weight). The levels of sex steroids in the fourth ether extracts were undetectable. T and E2 in the first and second round ether extracts were free and glucuronided sex steroids, respectively.
Measurement of aromatase activity
Coral tissue was homogenized with a potassium phosphate buffer [100 mM KCl, 10 mM KH2PO4, 1 mM EDTA, and 10 mM dithiothreitol (pH 7.4)] and centrifuged at 1000 x g for 10 min at 4 C. Aromatase activity was measured in the supernatant with 1-[3H]androstenedione as a substrate according to previous studies in coral (10). Aromatase activity was expressed as femtomoles of 3H2O per hour per milligram of protein.
Statistical analysis
The data were expressed as the mean ± SEM. The data were analyzed with Tukey’s honestly significant difference test after one-way ANOVA.
Results
Characterization of irGnRH in coral extracts by a Sep-Pak column and RIA
Fractions from a Sep-Pak column were assayed by GnRH RIA. Coral extract could not react with antisera strictly specific against sbGnRH (Fig. 1A), sGnRH, or cGnRH-II (data not shown). In contrast, coral extract reacted with a broad specific GnRH antiserum, showing a parallel curve comparable to the standard curve of RIA (Fig. 1B). RIA of HPLC fractions of coral extract, obtained after separation by one of two types of columns (Mightysil and Jupiter reverse phase C18 columns), showed one peak of immunoreactivity, suggesting that coral extract contained one form of irGnRH (Fig. 2, A and B). Furthermore, the irGnRH content of the peak was proportional to the amount of coral extract (Fig. 2, A and B). Coral irGnRH had a different retention time than mGnRH, sbGnRH, cGnRH-II, sGnRH, and lGnRH I in HPLC profiles (Fig. 2C), suggesting the occurrence of a different molecular form of GnRH in corals compared with the known GnRH standards.
Seasonal profiles of irGnRH in corals
Significant seasonal variations in irGnRH content in coral polyps were found in annual samples over a 3-yr period of collection in situ from the coral reefs. The highest concentrations of irGnRH (10-fold increase) were consistently detected during the spawning period (April 1998, May 1999, and April 2000; P < 0.01) compared with the nonspawning season (Fig. 3). Corals had about 171 ± 11 to 308 ± 44 pg irGnRH/g tissue during the nonspawning season compared with 2637 ± 354 (April 1998) to 3208 ± 209 pg irGnRH/g tissue (May 1999) during the spawning period (Fig. 3).
Seasonal profiles of E2 in corals
Significant changes in E2 (6- to 8-fold increase) in the coral polyps were detected during the spawning period compared with the nonspawning season (P < 0.05; Fig. 4).
Seasonal profiles of aromatase activity in corals
Aromatase activity was consistently detected in coral from January to June (Fig. 5). Significantly higher aromatase activity (9-fold) was detected during the period of spawning (May 13, 2004) compared with the nonspawning season (P < 0.01; Fig. 5). Aromatase activity on the day of spawning was even higher than in the corals collected 10 d before spawning (Fig. 5).
Biological activity of coral GnRH in vitro on LH release by fish pituitary cells
Effects of mGnRH agonist and GnRH receptor antagonist.
mGnRH agonist (10–10–10–6 M) significantly (P < 0.05) and dose-dependently stimulated the release of LH after 1, 2, and 4 h of incubation compared with controls (Fig. 6A). A GnRH receptor antagonist (10–9–10–5 M) significantly inhibited the stimulation of LH release in response to mGnRH agonist (10–6 M; Fig. 6B).
Effects of coral irGnRH and GnRH receptor antagonist.
The irGnRH content of coral extracts was quantified by RIA to prepare various doses (10–9–10–5 M) for in vitro treatment. Coral extracts significantly (P < 0.05) and dose-dependently stimulated the release of LH after 1, 2, and 4 h of incubation compared with controls (Fig. 7A). GnRH receptor antagonist (10–9–10–5 M) significantly (P < 0.05) and dose-dependently inhibited the stimulation of LH release in response to coral extracts (10–5 M irGnRH; Fig. 7B).
Biological activity of mGnRH agonist on coral steroidogenesis in vivo
Stimulation of aromatase activity in coral tissue by mGnRH agonist.
Addition of mGnRH agonist to the seawater aquaria in which coral samples were maintained in culture induced a time- and dose-dependent increase in aromatase activity in coral extracts. The highest stimulation in aromatase activity was a 5-fold increase (after 6 d of treatment with the highest dose of mGnRH agonist; P < 0.05; Fig. 8A).
Stimulation of T and E2 (free and glucuronided forms) in coral tissue by mGnRH agonist.
Addition of mGnRH agonist to the seawater aquaria also induced a time- and dose-dependent increase in T concentrations (up to 4-fold increase after 6 d of treatment; P < 0.05; Fig. 8B) and in E2 concentrations (up to 5-fold increase after 6 d of treatment; P < 0.05) in coral extracts (Fig. 8C). The high dose of mGnRH agonist also stimulated the production of glucuronided T and E2 concentrations in coral (P < 0.05; Fig. 9).
Discussion
Our data combining HPLC purifications and GnRH RIA clearly demonstrated significant concentrations of irGnRH in the tissue extracts of coral polyps, E. ancora. As suggested by the retention time on HPLC, coral irGnRH may only have one molecular form, which would be different from the already known GnRHs, such as mGnRH, sGnRH, sbGnRH, cGnRH-II, and lGnRH I.
The biological activity of coral irGnRH was assessed using a teleost (black porgy) (35) pituitary cell culture system. In vitro, mGnRH agonist stimulated the release of LH in a time- and dose-dependent fashion after 1, 2, and 4 h of incubation. The stimulatory effect of mGnRH agonist was inhibited in the presence of a specific GnRH receptor antagonist. By using this in vitro system, we found that coral irGnRH could also stimulate LH release from fish pituitary cells in a time- and dose-dependent manner. This demonstrates that coral irGnRH exhibit GnRH-like biological activity. Furthermore, specific GnRH receptor antagonist was able to dose-dependently inhibit the action of coral irGnRH on LH release. This indicates that coral irGnRH induced LH release by interacting with specific GnRH receptor from fish pituitary cells. These data suggest that the structure-function of coral irGnRH is conserved enough compared with that of mGnRH, enabling them possibly to act through a similar GnRH receptor pathway as in teleost pituitary cells, leading to the stimulation of LH release.
Samples collected over a 3-yr period from three colonies of corals in situ on the reefs in Nanwan Bay of southern Taiwan enabled us to provide the first evidence of dramatic seasonal variations in irGnRH content in coral. The significant variations in annual irGnRH concentrations were consistently found over a 3-yr period, especially in relation to the mass spawning time. Peak concentrations of irGnRH were detected in coral tissue during the spawning period. This is the first report demonstrating the presence of irGnRH in coral and the abrupt elevation of irGnRH (10-fold increase) during the spawning period compared with other seasons.
We had previously demonstrated a seasonal increase in coral E2 content in free and glucuronided forms at the time of spawning (10). In the present study we found concomitant increases in irGnRH and E2 concentrations. We also found a close relationship between irGnRH concentrations and aromatase activity in coral approaching the reproductive season and spawning period.
The parallel increases in irGnRH concentrations, aromatase activity, and E2 concentrations at the time of spawning suggested an important role of irGnRH in the control of steroidogenesis and reproduction in coral. We therefore aimed at proving this function of GnRH by conducting in vivo experiments in coral. However, from an ecological point of view, to preserve coral populations, we have been reluctant to collect too many coral samples to identify the amino acid sequence of coral irGnRH and to directly study the in vivo action of extracted coral irGnRH. Therefore, a mGnRH agonist was used instead of extracted coral irGnRH in the experimental studies of the regulation of coral aromatase activity and sex steroid production. Our current studies will aim at characterizing the sequences of coral irGnRH and GnRH receptor by developing molecular approaches.
The mGnRH agonist significantly stimulated aromatase activity and T and E2 concentrations with a time- and dose-dependent effect in coral polyps in vivo. This demonstrated that mGnRH agonist may directly act on the gonad and stimulate aromatase activity as well as other steroidogenic enzymes. The stimulation of coral aromatase activity by mGnRH agonist in in vivo experiments as well as the parallel seasonal profiles of coral irGnRH and aromatase in situ in the coral reefs suggest that irGnRH may be important, through aromatase, in the regulation of oocyte growth in coral, as indicated in vertebrates (38, 39). Indeed, aromatase has been known to play important roles in the biosynthesis of E2, ovarian differentiation and development, and oocyte growth in vertebrates (38, 39). Our recent experiments also demonstrated that adding mGnRH agonist to seawater in the coral aquaria (0.05 nM) at a 3-d intervals for 15 d could induce oocyte growth in coral (oocyte diameter, 312 ± 4 vs. 377 ± 7 μm in control vs. GnRH-treated, respectively; our unpublished observations). Additional studies should investigate whether this effect of GnRH on oocyte growth is mediated by E2.
This is the first report showing that mGnRH agonist stimulated the production of glucuronided E2 and T in coral polyps during in vivo experiments. This indicates that the mGnRH agonist may directly act on the gonad and stimulate steroid glucuronization in addition to aromatase and other steroidogenic enzymes. These data are consistent with our previous findings in situ in the coral reefs that the concentrations of glucuronided E2 and T were elevated in coral polyps during the spawning period (10). Therefore, irGnRH may control steroid glucuronization in corals. We previously proposed that glucuronided E2 may play important roles in the induction and synchronization of coral mass spawning (10). Taken together, the results obtained in the present and previous studies (10) suggest that irGnRH may play an important role in the induction of coral mass spawning via its positive effect on steroid synthesis and glucuronization.
The biological activity of a mammalian GnRH agonist in coral indicates that mammalian agonist is able to activate a putative coral GnRH receptor signaling pathway in coral. Reciprocally, as discussed above, we also demonstrated that coral irGnRH could activate GnRH receptor and signaling pathway in the pituitary of a teleost, leading to the release of LH. This finding also supports the strong evolutionary conservation of the structure-function of GnRHs and GnRH receptors in the animal kingdom. Therefore, it is possible that GnRH axis is phylogenically ancient and conserved among the eukaryote phyla.
The present study, combining in situ seasonal profiles and in vivo experiments, provides the first evidence for a key role of irGnRH in the control of reproduction in corals. In vertebrates, in addition to its presence in the brain and main neuroendocrine action on the pituitary gonadotrophs, GnRH has been found in the gonad of mammals [human (23) and rat (24)] as well as of teleosts [goldfish (25) and rainbow trout (26)]. GnRH could directly stimulate oocyte maturation and steroidogenesis in teleosts (25). GnRH could also stimulate an increase in sex steroids (30, 31) and spawning in tunicate, Ciona intestinalis (27). Our data, obtained in coral, which represents a very ancient group of metazoan, strongly suggest that the reproductive axis (at least for the GnRH-gonadal sex steroid pathway) would have been developed early in evolution, from low invertebrates. The direct action of irGnRH on gonadal activity might have represented the ancestral role of GnRH in the control of reproduction, long before the appearance of the pituitary as a relay, with the emergence of the vertebrates.
Our findings provide the foundation to understanding the mechanism of reproduction and mass spawning in corals on the basis of the current and previous studies (10). We propose that coral irGnRH, as a neuroendocrine factor, may be responsible for the integration of signals from environmental cues and regulate oocyte growth and mass spawning in coral via its effects on gonadal steroidogenesis and steroid glucuronization, respectively.
Acknowledgments
Specific antiserum against respective sGnRH and cGnRH-II was donated by Dr. K. Okuzawa (National Research Institute of Aquaculture, Tamaki, Japan) and Dr. K. Aida (University of Tokyo, Tokyo, Japan). We are grateful to Dr. Sherly Tomy and Rory Arrowsmith for English editing.
Footnotes
First Published Online September 29, 2005
Abbreviations: c, Chicken; CM, culture medium; E2, estradiol; irGnRH, immunoreactive GnRH; l, lamprey; m, mammalian; s, salmon; sb, seabream; T, testosterone; TFA, trifluoroacetic acid.
Accepted for publication September 22, 2005.
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National Museum of Marine Biology and Aquarium (W.-H.T., Y.-H.L.), Pingtung 944, Taiwan
Department of Aquaculture, National Kaohsiung Marine University (S.-R.J., W.-S.Y.), Kaohsiung 811, Taiwan
Unite Mixte de Recherche, 5178 Centre National de la Recherche Scientifique, Biologie des Organismes Marins et Ecosystemes, Departement des Milieux et Peuplements Aquatiques, Museum National d’Histoire Naturelle (S.D.), 75231 Paris, France
Abstract
The objectives of this study were to investigate the presence of immunoreactive GnRH (irGnRH) in scleractinian coral, Euphyllia ancora, study its seasonal variation, and evaluate its biological activity. irGnRH was detected and quantified in coral polyps. The biological activity of coral irGnRH was tested on pituitary cells from black porgy by evaluating its ability to stimulate LH release. Coral extracts (10–9–10–5 M irGnRH) as well as mammalian (m) GnRH agonist (10–10–10–6 M) had a similar dose-dependent effect on LH release. Furthermore, GnRH receptor antagonist dose-dependently inhibited the stimulation of LH release in response to coral extracts (10–5 M irGnRH) and mGnRH agonist (10–6 M). Peak levels of irGnRH (10-fold increase) were observed during the spawning period in a 3-yr investigation. Significantly higher aromatase activity and estradiol (E2) levels were also detected during the period of spawning compared with the nonreproductive season. In in vivo experiments, mGnRH agonist time- and dose-dependently stimulated aromatase activity as well as the concentrations of testosterone and E2 in free and glucuronided forms in coral. In conclusion, our data indicate that irGnRH does exist in coral, with its ability to stimulate LH release in fish. Seasonal variations of coral irGnRH, with a dramatic increase during the spawning period, concomitant to that in aromatase and E2, as well as the ability of mGnRH agonist to stimulate coral aromatase, steroidogenesis, and steroid glucuronization suggest that irGnRH plays an important role in the control of oocyte growth and mass spawning in corals.
Introduction
MOST BROADCAST SCLERACTINIAN corals synchronously release their gametes during a brief annual spawning period (1). The spawning period follows the lunar phase (around mid-March in Taiwan). The previous studies on scleractinians suggest that several environmental factors may play an important role in determining the timing of reproduction and spawning (2, 3, 4, 5, 6, 7). The environmental factors include temperature, lunar periodicity, illumination, tidal surge, physical shock, and presence/abundance of food (2, 3, 4, 5, 6, 7, 8, 9), but few studies aimed to elucidate how the endogenous factors may regulate and synchronize gametogenesis and mass spawning in corals. Recently, we found that significantly higher estradiol (E2) than glucuronided E2 levels were detected in the scleractinian coral Euphyllia ancora during the spawning season (10). In contrast, significantly higher glucuronided E2 than free E2 levels were detected in the spawning seawater (10). A role of E2, and more specifically of its glucuronide as secreted in seawater, has been suggested in the communication between polyps and coral colonies and induction of mass spawning (10). Aromatase activity, an important enzyme related to oocyte development in vertebrates, was also clearly detected in this species (10). These findings provide the foundation for additional investigation of the endocrine factors related to the control of reproduction in corals.
Hypothalamus-pituitary-gonadal axis is the main pathway in the regulation of reproduction in vertebrates. The hypothalamic neuropeptide, GnRH, a decapeptide, was discovered to regulate pituitary gonadotropins (LH and FSH), and consequently sex steroids and reproduction in vertebrates (11, 12). In addition to vertebrates, several forms of GnRH have been found in protochordate (tunicate, Chelyosoma productum) (13, 14) and mollusca (15, 16, 17). GnRH with a 12-amino acid peptide was recently characterized in the octopus (18). To date, 16 known forms of GnRH in vertebrates with known amino acid sequence have been characterized (19). Immunological studies have also suggested the presence of GnRHs (from as many as 10 species) in other low invertebrates, such as Porifera, Cnidarians, and Nematodes (19, 20). Therefore, an ancient origin of this neurohormone before the emergence of the vertebrates has been suggested.
In vertebrates, in addition to its regulatory function on pituitary LH and FSH and gonadal maturation, GnRH may also play a variety of roles. Indeed, GnRH was shown to act as a neurotransmitter and neuromodulator in the nervous system (15, 21, 22) and as a local hormone in the gonad (23, 24, 25, 26). For instance, in teleosts, GnRH acts as a brain neuromediator on sexual behavior and as a paracrine gonadal neurohormone on oogenesis (27, 28, 29). GnRH also increased sex steroid levels in protochordate (30, 31). In invertebrates, GnRH induced gamete release in the pond snail (32) and the mollusk (33). This suggests an ancient role of GnRH on reproductive function, before the emergence of vertebrates, possibly exerted directly at the gonadal level, before the appearance of the pituitary. Based on these evolutionary perspectives, the present study aimed at investigating whether GnRH could be a major endogenous mechanism regulating coral reproductive function.
E. ancora is a scleractinian coral abundant on front of fringing coral reefs in Nanwan Bay, southern Taiwan. E. ancora is a gonochoristic species, and spawns in the late spring in Taiwan via external fertilization. The polyps of E. ancora are extended day and night, and they are large enough to obtain tissue samples for endocrine studies. Therefore, E. ancora was selected for the present study. We investigated the presence and seasonal profiles of immunoreactive GnRH (irGnRH) in coral polyps sampled in situ throughout 3 yr. Comparisons were made with aromatase activity and sex steroid levels in coral tissue. The biological activity of coral irGnRH was assessed by its ability to stimulate LH release from teleost pituitary cells in vitro. Finally, the possible physiological roles of irGnRH in the regulation of aromatase and sex steroids were also determined by in vivo experiments on corals. By these approaches, we tested the hypothesis that the GnRH-steroid axis may play a key role in the induction of reproduction (steroid production/oocyte growth) and mass spawning (production of glucuronided steroids) in corals.
Materials and Methods
Coral collection
Samples from three female colonies of coral, E. ancora, were collected from a depth of a 7-meter sea surface by scuba diving from March 1998 to April 2000 in Nanwan Bay of southern Taiwan (21°55'N, 120°59'E). Eleven seasonal collections of corals were obtained during these periods for E2 and GnRH RIA. The corals were collected on April 25, 2000 (spawning period), and used for the identification of coral irGnRH by Sep-Pak column-HPLC-RIA. Monthly collection was also obtained from January to June 2004 for seasonal aromatase activity (total of seven collections; two collections in May). Coral collection was approved by the administration office of the Kenting National Park in Nanwan. Polyp was immediately removed from coral by scraping and was stored in liquid nitrogen before assay.
Coral was collected daily, and spawning activity was checked when the predicted spawning date was approaching. Actual spawning occurred on April 16, May 3, April 25, and May 13 in the western calendar (March 20, March 18, March 21, and March 22 in the lunar calendar) in 1998, 1999, 2000, and 2004, respectively.
Extraction of peptides and preparation of crude extracts
Coral polyps were homogenized with 0.1 N HCl in 0.01 M phosphate buffer saline (PBS; pH 7.0) at 4 C and then centrifuged at 15,000 x g for 30 min. The supernatant was neutralized to pH 6.0 with NaOH in 0.01 M PBS and then purified by a Sep-Pak C18 cartridge column (Shandon Corp., Cheshire, UK) pretreated with 100% methanol and then with 0.12% trifluoroacetic acid (TFA). The elution program was as follows: 10 ml 0.12% TFA and 12 ml 40% acetonitrile. The eluate from 40% acetonitrile was dried in a Speed Vac concentrator (Savant Instruments, Holbrook, NY). The dried eluates (crude coral extracts) were further purified by HPLC before RIA for the identification of coral irGnRH or were directly measured by GnRH RIA in the seasonal samples.
HPLC
The dried materials obtained from the Sep-Pak column were dissolved in 0.12% TFA and further purified through HPLC using a Mightysil reverse phase C18 column (4.6 x 250 mm, 5 μm; Kanto Chemical Co., Tokyo, Japan) and a Jupiter reverse phase C18 column (4.6 x 250 mm; Phnomenex, Torrance, CA). Elution was performed as follows: solvent A, 0.1% TFA; solvent B, acetonitrile in 0.1% TFA; a linear gradient of solvent B 9.8–50.3% for 50 min, 50.3–75% for 5 min, 75% for 5 min with a flow rate of 1 ml/min. The fractions were dried in a Speed Vac concentrator (Savant Instruments) and measured by RIA. The methods of extraction, crude purification, and HPLC separation for coral irGnRH were modified from previous studies (34). Standard seabream (sb) GnRH, salmon (s) GnRH, chicken (c) GnRH-I, cGnRH-II, mammalian (m) GnRH, and lamprey (l) GnRH I were purchased from Bachem AG (Bubendorf, Switzerland).
In vitro effects of coral extract and GnRH agonist on LH release by primary culture of fish pituitary cells
Preparation of dispersed fish pituitary cells and static culture.
Black porgy fish (Acanthopagrus schlegeli) pituitary cell culture was developed. For each culture experiment, fifteen 2+-yr-old black porgy fish were decapitated, and pituitary cell culture was performed using enzymatic and mechanic procedures according to the previous studies (35). Pituitary cells were resuspended in culture medium (CM): medium 199 with Earle’s salts, sodium bicarbonate, 100 U penicillin/ml, 100 μg streptomycin/ml, and 250 ng fungizone/ml (Invitrogen Life Technologies, Inc., Gaithersburg, MD). Cells were cultured in 96-well tissue culture plates (Falcon, BD Biosciences, Franklin Park, NJ) at a density of 62,000 cells/125 μl CM/well with 1% fetal bovine serum (Invitrogen Life Technologies) in a tissue culture incubator (NU4500, CO2 water-jacketed incubator; NUAIRE, Plymouth, MN) at 24 C under 5% CO2 and saturated humidity. Measurements of LH release were performed on the third day of culture (d 0). Before addition of treatments, cells were washed three times and incubated in CM (0.25 ml/well) in the presence of 1.0% fetal bovine serum. Six wells were used for each treatment. Aliquots of culture medium were removed after 1, 2, and 4 h of culture in the respective four treatments. LH concentrations in the CM were measured by RIA.
Treatment with GnRH agonist and GnRH receptor antagonist in dispersed fish pituitary cells.
In the first experiment, a mGnRH agonist (a synthetic mGnRH agonist, [D-Ala6]-HRH ethylamide; Sigma-Aldrich Corp., St. Louis, MO) was tested over 4 h at various concentrations (10–6, 10–8, and 10–10 M). This mammalian agonist has been previously shown to be active on LH release in other vertebrates, including various teleosts, namely, the black porgy (35). In the second experiment, dispersed pituitary cells were treated with medium alone (blank) or with mGnRH agonist (10–8 and 10–6 M) alone or in the presence of various concentrations of GnRH receptor antagonist (10–9, 10–7, and 10–5 M). The mGnRH receptor antagonist (GnRH receptor antagonist, [D-Phe2,D-Ala6]LHRH) was purchased from Sigma-Aldrich Corp. This mGnRH receptor antagonist has been previously shown to be able to block the GnRH stimulatory effect on LH release in vivo or in vitro in other vertebrates, including the black porgy (35).
Treatment with coral extracts and GnRH receptor antagonist in dispersed fish pituitary cells.
irGnRH concentrations in coral extracts (collected from the spawning period) purified through a Sep-Pak column were quantified, and irGnRH concentrations (10–3 M) were prepared as a stock. In the third experiment, coral extracts containing various doses of irGnRH (10–5, 10–7, and 10–9 M) were tested in the dispersed pituitary cells over 4 h.
In the fourth experiment, cells were treated with medium alone (controls) or with coral extracts (10–5 M irGnRH) alone or in the presence of various concentrations of GnRH receptor antagonist (10–9, 10–7, and 10–5 M).
Effects of GnRH agonist on the coral aromatase activity and steroidogenesis in vivo
Twelve pieces (8 x 8 cm/piece) of corals were cultured in three tanks (four pieces per tank with 5 liters seawater at 25 C) in a set of experiment. The whole set of the experiment was repeated three times. Corals were respectively obtained during the nonspawning period (March 7, May 3, and November 7 of 2002). mGnRH agonist was added to seawater on d 0, 2, and 4 at final concentrations of 0 (blank), 0.5, and 1.5 μM, respectively. Corals were collected from each tank on d 0, 2, 4, and 6. Aromatase activity, testosterone (T), and E2 levels in free and glucuronided forms in corals were measured. To combine three sets of experimental data, the value of aromatase activity, T or E2 on d 0 was considered as 100%, and the data from d 2, 4, and 6 were calculated according to the value on d 0.
LH assay
Concentrations of LH in the culture medium were measured using a homologous RIA employing purified black porgy LH as a standard with antiblack porgy LH serum as an antibody according to previous study (36).
GnRH assay
Coral extracts from a Sep-Pak column (in the seasonal samples) or further purified by HPLC (for the identification of coral irGnRH) were dissolved in 0.01 M PBS with gelatin for GnRH RIA according to previous methods (37). Coral extracts did not cross-react with antisera strictly specific to sbGnRH (Fig. 1A), sGnRH, cGnRH-II, or mGnRH (data not shown). Therefore, an RIA for coral irGnRH was developed using an sGnRH antiserum with a broad spectrum of cross-reactivity. This sGnRH antiserum was induced in a rabbit using sGnRH linked to BSA. sGnRH was iodinated with chloramine T and separated in a Sephadex G-25 column. The RIA employed sGnRH (Sigma-Aldrich Corp.) as standard and [125I]sGnRH as labeled peptide. The antiserum cross-reacted with various forms GnRH, such as sGnRH, mGnRH, sbGnRH, cGnRH-I, cGnRH-II, and lGnRH I (Fig. 1B), but did not have any cross-reactivity with other neuropeptides, such as TRH, isotocin, vasotocin, or somatostatin.
Sex steroid RIA
For studies on seasonal profiles (free E2) and effects of GnRH agonist on sex steroids (free and glucuronided forms) in coral, sex steroid (T and E2) RIAs were conducted according to previous studies in coral (10). Coral polyps were homogenized with 0.01 M PBS with gelatin (pH 7.0) at 4 C. Coral homogenate was extracted three times with 5-fold (vol/vol) diethyl ether, and finally an ether phase (first round ether extracts contained free steroids) and a liquid phase were obtained. The liquid phase was further extracted once with diethyl ether (fourth ether extracts), and the remaining liquid phase (contained glucuronided steroids) was hydrolyzed with -glucuronidase (Sigma-Aldrich Corp.) to hydrolyze the conjugated steroids (10). The hydrolyzed solution was extracted three more times with diethyl ether (second round ether extracts contained glucuronided steroids). The extracts (first and second round ether extracts and fourth ether extracts) were purified in an alumina column and reverse phase column HPLC before steroid RIA (10). Results were expressed as nanograms of steroid per gram of tissue (wet weight). The levels of sex steroids in the fourth ether extracts were undetectable. T and E2 in the first and second round ether extracts were free and glucuronided sex steroids, respectively.
Measurement of aromatase activity
Coral tissue was homogenized with a potassium phosphate buffer [100 mM KCl, 10 mM KH2PO4, 1 mM EDTA, and 10 mM dithiothreitol (pH 7.4)] and centrifuged at 1000 x g for 10 min at 4 C. Aromatase activity was measured in the supernatant with 1-[3H]androstenedione as a substrate according to previous studies in coral (10). Aromatase activity was expressed as femtomoles of 3H2O per hour per milligram of protein.
Statistical analysis
The data were expressed as the mean ± SEM. The data were analyzed with Tukey’s honestly significant difference test after one-way ANOVA.
Results
Characterization of irGnRH in coral extracts by a Sep-Pak column and RIA
Fractions from a Sep-Pak column were assayed by GnRH RIA. Coral extract could not react with antisera strictly specific against sbGnRH (Fig. 1A), sGnRH, or cGnRH-II (data not shown). In contrast, coral extract reacted with a broad specific GnRH antiserum, showing a parallel curve comparable to the standard curve of RIA (Fig. 1B). RIA of HPLC fractions of coral extract, obtained after separation by one of two types of columns (Mightysil and Jupiter reverse phase C18 columns), showed one peak of immunoreactivity, suggesting that coral extract contained one form of irGnRH (Fig. 2, A and B). Furthermore, the irGnRH content of the peak was proportional to the amount of coral extract (Fig. 2, A and B). Coral irGnRH had a different retention time than mGnRH, sbGnRH, cGnRH-II, sGnRH, and lGnRH I in HPLC profiles (Fig. 2C), suggesting the occurrence of a different molecular form of GnRH in corals compared with the known GnRH standards.
Seasonal profiles of irGnRH in corals
Significant seasonal variations in irGnRH content in coral polyps were found in annual samples over a 3-yr period of collection in situ from the coral reefs. The highest concentrations of irGnRH (10-fold increase) were consistently detected during the spawning period (April 1998, May 1999, and April 2000; P < 0.01) compared with the nonspawning season (Fig. 3). Corals had about 171 ± 11 to 308 ± 44 pg irGnRH/g tissue during the nonspawning season compared with 2637 ± 354 (April 1998) to 3208 ± 209 pg irGnRH/g tissue (May 1999) during the spawning period (Fig. 3).
Seasonal profiles of E2 in corals
Significant changes in E2 (6- to 8-fold increase) in the coral polyps were detected during the spawning period compared with the nonspawning season (P < 0.05; Fig. 4).
Seasonal profiles of aromatase activity in corals
Aromatase activity was consistently detected in coral from January to June (Fig. 5). Significantly higher aromatase activity (9-fold) was detected during the period of spawning (May 13, 2004) compared with the nonspawning season (P < 0.01; Fig. 5). Aromatase activity on the day of spawning was even higher than in the corals collected 10 d before spawning (Fig. 5).
Biological activity of coral GnRH in vitro on LH release by fish pituitary cells
Effects of mGnRH agonist and GnRH receptor antagonist.
mGnRH agonist (10–10–10–6 M) significantly (P < 0.05) and dose-dependently stimulated the release of LH after 1, 2, and 4 h of incubation compared with controls (Fig. 6A). A GnRH receptor antagonist (10–9–10–5 M) significantly inhibited the stimulation of LH release in response to mGnRH agonist (10–6 M; Fig. 6B).
Effects of coral irGnRH and GnRH receptor antagonist.
The irGnRH content of coral extracts was quantified by RIA to prepare various doses (10–9–10–5 M) for in vitro treatment. Coral extracts significantly (P < 0.05) and dose-dependently stimulated the release of LH after 1, 2, and 4 h of incubation compared with controls (Fig. 7A). GnRH receptor antagonist (10–9–10–5 M) significantly (P < 0.05) and dose-dependently inhibited the stimulation of LH release in response to coral extracts (10–5 M irGnRH; Fig. 7B).
Biological activity of mGnRH agonist on coral steroidogenesis in vivo
Stimulation of aromatase activity in coral tissue by mGnRH agonist.
Addition of mGnRH agonist to the seawater aquaria in which coral samples were maintained in culture induced a time- and dose-dependent increase in aromatase activity in coral extracts. The highest stimulation in aromatase activity was a 5-fold increase (after 6 d of treatment with the highest dose of mGnRH agonist; P < 0.05; Fig. 8A).
Stimulation of T and E2 (free and glucuronided forms) in coral tissue by mGnRH agonist.
Addition of mGnRH agonist to the seawater aquaria also induced a time- and dose-dependent increase in T concentrations (up to 4-fold increase after 6 d of treatment; P < 0.05; Fig. 8B) and in E2 concentrations (up to 5-fold increase after 6 d of treatment; P < 0.05) in coral extracts (Fig. 8C). The high dose of mGnRH agonist also stimulated the production of glucuronided T and E2 concentrations in coral (P < 0.05; Fig. 9).
Discussion
Our data combining HPLC purifications and GnRH RIA clearly demonstrated significant concentrations of irGnRH in the tissue extracts of coral polyps, E. ancora. As suggested by the retention time on HPLC, coral irGnRH may only have one molecular form, which would be different from the already known GnRHs, such as mGnRH, sGnRH, sbGnRH, cGnRH-II, and lGnRH I.
The biological activity of coral irGnRH was assessed using a teleost (black porgy) (35) pituitary cell culture system. In vitro, mGnRH agonist stimulated the release of LH in a time- and dose-dependent fashion after 1, 2, and 4 h of incubation. The stimulatory effect of mGnRH agonist was inhibited in the presence of a specific GnRH receptor antagonist. By using this in vitro system, we found that coral irGnRH could also stimulate LH release from fish pituitary cells in a time- and dose-dependent manner. This demonstrates that coral irGnRH exhibit GnRH-like biological activity. Furthermore, specific GnRH receptor antagonist was able to dose-dependently inhibit the action of coral irGnRH on LH release. This indicates that coral irGnRH induced LH release by interacting with specific GnRH receptor from fish pituitary cells. These data suggest that the structure-function of coral irGnRH is conserved enough compared with that of mGnRH, enabling them possibly to act through a similar GnRH receptor pathway as in teleost pituitary cells, leading to the stimulation of LH release.
Samples collected over a 3-yr period from three colonies of corals in situ on the reefs in Nanwan Bay of southern Taiwan enabled us to provide the first evidence of dramatic seasonal variations in irGnRH content in coral. The significant variations in annual irGnRH concentrations were consistently found over a 3-yr period, especially in relation to the mass spawning time. Peak concentrations of irGnRH were detected in coral tissue during the spawning period. This is the first report demonstrating the presence of irGnRH in coral and the abrupt elevation of irGnRH (10-fold increase) during the spawning period compared with other seasons.
We had previously demonstrated a seasonal increase in coral E2 content in free and glucuronided forms at the time of spawning (10). In the present study we found concomitant increases in irGnRH and E2 concentrations. We also found a close relationship between irGnRH concentrations and aromatase activity in coral approaching the reproductive season and spawning period.
The parallel increases in irGnRH concentrations, aromatase activity, and E2 concentrations at the time of spawning suggested an important role of irGnRH in the control of steroidogenesis and reproduction in coral. We therefore aimed at proving this function of GnRH by conducting in vivo experiments in coral. However, from an ecological point of view, to preserve coral populations, we have been reluctant to collect too many coral samples to identify the amino acid sequence of coral irGnRH and to directly study the in vivo action of extracted coral irGnRH. Therefore, a mGnRH agonist was used instead of extracted coral irGnRH in the experimental studies of the regulation of coral aromatase activity and sex steroid production. Our current studies will aim at characterizing the sequences of coral irGnRH and GnRH receptor by developing molecular approaches.
The mGnRH agonist significantly stimulated aromatase activity and T and E2 concentrations with a time- and dose-dependent effect in coral polyps in vivo. This demonstrated that mGnRH agonist may directly act on the gonad and stimulate aromatase activity as well as other steroidogenic enzymes. The stimulation of coral aromatase activity by mGnRH agonist in in vivo experiments as well as the parallel seasonal profiles of coral irGnRH and aromatase in situ in the coral reefs suggest that irGnRH may be important, through aromatase, in the regulation of oocyte growth in coral, as indicated in vertebrates (38, 39). Indeed, aromatase has been known to play important roles in the biosynthesis of E2, ovarian differentiation and development, and oocyte growth in vertebrates (38, 39). Our recent experiments also demonstrated that adding mGnRH agonist to seawater in the coral aquaria (0.05 nM) at a 3-d intervals for 15 d could induce oocyte growth in coral (oocyte diameter, 312 ± 4 vs. 377 ± 7 μm in control vs. GnRH-treated, respectively; our unpublished observations). Additional studies should investigate whether this effect of GnRH on oocyte growth is mediated by E2.
This is the first report showing that mGnRH agonist stimulated the production of glucuronided E2 and T in coral polyps during in vivo experiments. This indicates that the mGnRH agonist may directly act on the gonad and stimulate steroid glucuronization in addition to aromatase and other steroidogenic enzymes. These data are consistent with our previous findings in situ in the coral reefs that the concentrations of glucuronided E2 and T were elevated in coral polyps during the spawning period (10). Therefore, irGnRH may control steroid glucuronization in corals. We previously proposed that glucuronided E2 may play important roles in the induction and synchronization of coral mass spawning (10). Taken together, the results obtained in the present and previous studies (10) suggest that irGnRH may play an important role in the induction of coral mass spawning via its positive effect on steroid synthesis and glucuronization.
The biological activity of a mammalian GnRH agonist in coral indicates that mammalian agonist is able to activate a putative coral GnRH receptor signaling pathway in coral. Reciprocally, as discussed above, we also demonstrated that coral irGnRH could activate GnRH receptor and signaling pathway in the pituitary of a teleost, leading to the release of LH. This finding also supports the strong evolutionary conservation of the structure-function of GnRHs and GnRH receptors in the animal kingdom. Therefore, it is possible that GnRH axis is phylogenically ancient and conserved among the eukaryote phyla.
The present study, combining in situ seasonal profiles and in vivo experiments, provides the first evidence for a key role of irGnRH in the control of reproduction in corals. In vertebrates, in addition to its presence in the brain and main neuroendocrine action on the pituitary gonadotrophs, GnRH has been found in the gonad of mammals [human (23) and rat (24)] as well as of teleosts [goldfish (25) and rainbow trout (26)]. GnRH could directly stimulate oocyte maturation and steroidogenesis in teleosts (25). GnRH could also stimulate an increase in sex steroids (30, 31) and spawning in tunicate, Ciona intestinalis (27). Our data, obtained in coral, which represents a very ancient group of metazoan, strongly suggest that the reproductive axis (at least for the GnRH-gonadal sex steroid pathway) would have been developed early in evolution, from low invertebrates. The direct action of irGnRH on gonadal activity might have represented the ancestral role of GnRH in the control of reproduction, long before the appearance of the pituitary as a relay, with the emergence of the vertebrates.
Our findings provide the foundation to understanding the mechanism of reproduction and mass spawning in corals on the basis of the current and previous studies (10). We propose that coral irGnRH, as a neuroendocrine factor, may be responsible for the integration of signals from environmental cues and regulate oocyte growth and mass spawning in coral via its effects on gonadal steroidogenesis and steroid glucuronization, respectively.
Acknowledgments
Specific antiserum against respective sGnRH and cGnRH-II was donated by Dr. K. Okuzawa (National Research Institute of Aquaculture, Tamaki, Japan) and Dr. K. Aida (University of Tokyo, Tokyo, Japan). We are grateful to Dr. Sherly Tomy and Rory Arrowsmith for English editing.
Footnotes
First Published Online September 29, 2005
Abbreviations: c, Chicken; CM, culture medium; E2, estradiol; irGnRH, immunoreactive GnRH; l, lamprey; m, mammalian; s, salmon; sb, seabream; T, testosterone; TFA, trifluoroacetic acid.
Accepted for publication September 22, 2005.
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