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Behavior |
Institute of Marine Biology3
Department of Aquaculture,4 National Taiwan Ocean University, Keelung 20224, Taiwan, Republic of China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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estradiol, pheromones, seasonal reproduction, steroid hormones, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vertebrates, endocrine factors such as sex steroid hormones, androgens (testosterone), and estrogens (estradiol-17ß, E2), play important roles in gametogenesis and reproductive process [9], and aromatase mediates the metabolism of androgens to estrogens [10]. We also hypothesized that the endocrine system is one of the endogenous factors that significantly regulates coral reproduction and spawning. Therefore, sex steroids in coral are the first candidates for investigation.
Several studies have shown that steroids are found in many other invertebrates [9], including mollusks [11], crustaceans [12], and echinoderms [13]. E2 was first detected in coral eggs and seawater during a mass coral spawn in 1992 [14], and estrone and E2 were founded in the tissue of scleractinian coral in 1999 [15]. Progesterone, testosterone, and E2 were detected in a soft coral, Sinularia polydactyla [16]. The annual profiles of steroids were studied in the scleractinian coral, Montipora verrucosa [15], and in S. polydactyla [16], but with no clear profiles. Even so, no increases in E2 were observed in S. polydactyla just prior to spawning [16]. The relationship and roles of E2 and other sex steroids such as testosterone in the reproduction and mass spawning of coral need further study.
Euphyllia ancora is a scleractinian coral that is abundant on the front of a fringing coral reef in Nanwan Bay, in southern Taiwan. E. ancora is a gonochorism, and spawns in late spring in Taiwan via external fertilization. The polyps of E. ancora are extended day and night, and are large enough that they can be collected for endocrine studies. Therefore, E. ancora was selected in order to investigate the presence and the annual profiles of E2 and testosterone in coral tissue and spawning seawater. Aromatase activity in E. ancora was also determined. Using these approaches, we tested the hypothesis that endogenous steroids are important to the mass spawning process in coral.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three female colonies of E. ancora were collected at a depth of 10 m under the sea surface by SCUBA divers from March 1998 to May 1999 in Nanwan Bay of southern Taiwan (21°55'N, 120°59'E). Eight seasonal collections of corals were obtained during these periods. Coral collection was approved by the administration office of the Kenting National Park in Nanwan. Polyps were immediately removed from the coral by scraping and were stored in liquid nitrogen before assay.
Coral was collected daily and the spawning activity was also checked when the predicted spawning date was approaching. The predicted date is the week of the full moon in mid-March (March 15–22) of the lunar calendar. Seawater was also collected daily during this period in order to obtain a sample 1 day before spawning. The entire process of spawning activity could last for 6 hr during the night with a 3-hr peak activity. Therefore, spawning seawater was collected during the peak spawning period after the first sign of spawning activity was observed. The actual spawning occurred on 16 April, 3 May, and 25 April (20 March, 18 March, and 21 March in the lunar calendar) in 1998, 1999, and 2000, respectively.
Seawater (three collections per year) was collected near the surface and near the coral (within 10 cm of a coral colony) in the periods before (1 day), during, and after (1 day) the coral mass spawning in 3 consecutive yr (1998–2000). The coral was not deeper than 10 m from the surface. Seawater (1000 ml per collection) was collected in a bottle, then filtered with a fine plankton net and then stored at -80°C.
Coral Extraction and Alumina Column Chromatography
Coral polyps (1 g) were homogenized with 0.01 M PBS pH 7.0 at 4°C, and then extracted three times with 5-fold (v/v) diethyl ether. The solvent fraction (containing free steroids) and an aqueous fraction (containing conjugated steroids) were obtained. The extracts were purified with an alumina (Sigma, St. Louis, MO) column (2 x 4 cm) to remove the impurities as described previously [12]. The elution process was performed as follows: 30 ml of 100% benzene, 40 ml of 5% ethyl acetate in benzene, 50 ml of 10% ethyl acetate in benzene, 40 ml of 50% ethyl acetate in benzene, 40 ml of 100% ethyl acetate, 40 ml of 5% ethanol in ethyl acetate, and 40 ml of 50% ethanol in water. All fractions were dried with a rotavapor and detected by a validated RIA with a specific antiserum against testosterone or E2. The fractions that contained testosterone or E2 were further purified by HPLC.
High Performance Liquid Chromatography
The immunoreactive fractions from an alumina column were further purified through HPLC using a Mightysil reversed-phase C18 column (4.6 x 250 mm, 5 µm; Kantochemical Co., Inc., Tokyo, Japan). Elution was performed as follows: solvent A, Milli-Q water; solvent B, methyl alcohol; solvent C, acetonitrile; a linear gradient of solvent B (30%–60%) and solvent C (10%–5%) for 60 min, followed by an isocratic elution of 60% solvent B and 40% solvent C for 20 min, and finally with 100% solvent B for 10 min with a flow rate of 1 ml/min. The fractions were dried in a Speed Vac concentrator (Savant), and measured by RIA. The presence of testosterone and E2 in the HPLC coral extract fractions was parallel to that of the fraction of standard steroids. In the sample analysis, the HPLC fractions that contained coral testosterone and E2 (three fractions in each) were pooled for comparison with the retention time of the standard steroid, and the concentrations were then measured by RIA. The recovery of extraction and purification in E2 and testosterone through the alumina column and HPLC for coral tissue was 60%. This recovery was obtained by averaging six representative samples on the basis of adding cold standard steroids (500 µg in each E2 and testosterone) to the coral tissue.
Extraction and Measurement of Sex Steroids in Seawater
A 10-ml seawater sample was extracted twice with 3-fold diethyl ether (v/v). The solvent fraction (with free steroids) and the aqueous fraction (with conjugated steroids) were obtained after extraction. The additional purification (alumina column and reverse-phase HPLC) methods followed those described above in the "Coral Extraction" section. Steroid RIA was then performed.
Hydrolysis of Conjugated Steroids
The aqueous fraction from solvent extraction in coral tissue and seawater was incubated for 2 days at 37°C with 800 µg of ß-glucoronidase (Sigma) in 1 ml of acetate buffer (0.1 M, pH 4.8) or 800 µg of sulfatase (Sigma) in 1 ml of Tris buffer (0.1 M, pH 5.0) to hydrolyze the respective conjugated steroids.
The hydrolyzed solution was then extracted twice with 5-fold diethyl ether (v/v). The organic extracts were dried and measured by RIA. The organic extracts were dried and measured by RIA. The ß-glucoronidase and sulfatase have no cross-reactivity with each other.
Radioimmunoassay
RIA was conducted primarily according to the procedures described in a previous paper [17]. The dried fraction from HPLC was dissolved with 0.01 M phosphate buffer (pH 7.0) and measured by RIA with the specific antisera for testosterone and E2. The concentrations of testosterone and E2 in coral were calculated according to the recoveries. The sensitivities of E2 and testosterone RIA were 1 pg and 12.5 pg, respectively. The samples were measured within a set of assays with 10.2% intraassay variation.
The Presence of Aromatase Activity
Coral tissue was homogenized with potassium phosphate buffer (100 mM KCl, 10 mM KH2PO4, 1 mM EDTA, 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 according to methods described in previous studies [18]. Different concentrations (0–0.8 mM) of substrate (1ß-[3H] androstenedione) were added to obtain a saturation curve. The effects of temperature (20–42°C) on aromatase activity were also conducted. Aromatase activity was expressed in fentomoles of 3H2O/h·mg protein.
Statistical Analysis
The data were expressed as means ± SEM and were not transformed for statistical analysis. The annular profiles of steroids in the coral tissue were analyzed with the Tukey honestly significant difference test after one-way ANOVA (P < 0.05). In one-way ANOVA, the source of variation was as follows: 8 seasonal collections as the treatment with 7 df and within season (3 colonies in each seasonal collection) with 16 df. The Student t-test was applied to the comparison of two sample means in Table 1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fractions from the alumina column were further examined by testosterone and E2 RIA. Only the fifth and sixth fractions showed a parallel curve compared with the respective standard curves for RIA (Figs. 1 and 2). The HPLC fractions in coral extracts contained testosterone or E2 according to the respective RIA data and had the same retention time as the standard steroids in HPLC profiles (Fig. 3).
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Free and Conjugated Forms of Testosterone in Coral Tissue
Free testosterone concentrations ranged from 7.5 ± 2.5 to 43.1 ± 2.3 ng/g tissue in annual profiles (Fig. 4A). The coral collected during the spawning period (April 1998 and May 1999) had significantly low levels of free testosterone compared with those in other seasons (P < 0.05). Significant concentrations of testosterone glucuronide (P < 0.05) were detected in the coral in a season-dependent manner (Fig. 4B). The coral collected during the spawning season had the highest value of testosterone glucuronide (48.8 ± 2.7 ng/g in April 1998 and 36.6 ± 1.2 ng/g in May 1999) (Fig. 4B). No testosterone sulfate was found in the coral extract in any season.
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Free and Conjugated Forms of E2 in Coral Tissue
Significant profiles of free E2 and E2 glucuronide were found in annual samples and both showed statistical significant differences between spawning and nonspawning periods (P < 0.05), from March 1998 to May 1999 (Fig. 5, A and B). The highest concentrations of free E2 and E2 glucuronide were consistently detected just prior to spawning (April 1998 and May 1999). In free E2, coral had 66.0 ± 10.2 to 243.1 ± 49.4 ng of E2/g tissue during the nonspawning season compared with 647.2 ± 22.8 ng of E2/g tissue prior to spawning (Fig. 5A). In its conjugated form (E2 glucuronide), coral had 1.9 ± 0.7 to 4.5 ± 0.6 ng of E2/g tissue during the nonspawning season compared with 241.8 ± 9.5 ng of E2/g tissue prior to spawning (Fig. 5B). No sulfate form of E2 (E2 sulfate) was detected in the coral tissue. During the spawning period, coral had higher levels (P < 0.05) of free E2 (638.3 ± 51.9 and 647.2 ± 22.8 ng/g tissue) than conjugated E2 (241.8 ± 9.5 and 215.8 ± 14.8 ng/g tissue) (Table 1). The concentrations of free and conjugated forms of E2 in coral tissue were higher (P < 0.05) than the concentrations of the respective forms of testosterone (free testosterone, 7.53 ± 2.5 and 13.6 ± 2.2 ng/g; conjugated testosterone, 48.8 ± 2.7, 36.6 ± 1.2 ng/g tissue) (Table 1).
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Steroids in Seawater
Significantly and consistently high concentrations of steroids (free and conjugated forms of testosterone and E2) in seawater were detected only during coral mass spawning but not 1 day before or 1 day after spawning in 1998–2000 (Table 1). E2 and E2 glucuronide were the major steroids in seawater during the spawning period. E2 glucuronide concentrations (14.2–26.0 pg/ml; average 20.3 ± 3.4 pg/ml) had about 3.5-fold higher levels (P < 0.05) than free E2 (4.2–7.1 pg/ml; average 5.8 ± 0.9 pg/ml) in spawning seawater around coral (Table 1). In contrast, only testosterone glucuronide (0.6–1.1 pg/ml; average 0.8 ± 0.2 pg/ml) but not free testosterone (undetectable) was detected in seawater around coral during mass spawning (Table 1). E2 glucuronide (4.3–7.0 pg/ml) and free E2 (1.9–4.1 pg/ml) but not testosterone could also be detected in the seawater collected at the sea surface during the mass spawning period (Table 1). Neither testosterone nor E2 (free or glucuronide) were detected in seawater 1 day before or after spawning (Table 1). No steroid sulfate was detected in seawater (Table 1). The concentrations of free (5.8 ± 0.9 pg/ml) and conjugated (20. 3 ± 3.4 pg/ml) E2 in spawning seawater were higher (P < 0.05) than the concentrations of the respective forms of testosterone (free testosterone was undetectable; conjugated testosterone was 0.8 ± 0.2 pg/ml) (Table 1).
Existence of Aromatase Activity in Coral Tissue
Kinetic characteristics of coral aromatase activity are demonstrated in Figure 6A. The maximum aromatase activity (Vmax) and Michaelis constant (Km) were 820 and 0.08 fmol/h·mg protein, respectively, for 1ß-[3H] androstenedione as a substrate in the coral tissue during the spawning period (Fig. 6A). Aromatase activity was increased when incubated temperatures increased from 20°C to 42°C (Fig. 6B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Significantly higher free E2 concentrations than of E2 glucuronide were found in the coral throughout the year. Higher free testosterone concentrations than testosterone glucuronide were found during the nonspawning period and higher testosterone glucuronide concentrations than free testosterone were detected in the coral during the spawning time. Total E2 (free and conjugated forms) concentrations were much higher than testosterone (free and conjugated forms) in the coral throughout the year, especially just prior to spawning. It seems that biosynthesis of free E2 and E2 glucuronide was significantly elevated in coral approaching the time of spawning. The average ratio of E2 glucuronide to free E2 was 0:03 during nonspawning (March 1998 or April 1999) and it elevated to 0:35 (a 10-fold increase) prior to spawning (April 1998 or May 1999). The average ratio of free E2 concentrations in corals between the prior-to-spawning period (the mass spawning period) and 1 mo before spawning (the nonspawning period, March 1998 or April 1999) was 6:1; in contrast, the ratio of E2 glucuronide between these two periods was increased to 89:1. Similar observation was also found for free testosterone and testosterone glucuronide. The data clearly demonstrate that both E2 glucuronide and testosterone glucuronide concentrations were significantly increased in coral tissue just prior to spawning. No steroid sulfate could be detected. This suggests that the changes in glucuronyl transferase activity may play an important role in the conversion from free steroids to conjugated steroids.
High concentrations of free E2, E2 glucuronide, and testosterone glucuronide but not free testosterone were detected in spawning seawater. Significantly higher concentrations of free E2, E2 glucuronide, and testosterone glucuronide were detected in seawater just around coral than those in seawater from near the sea surface (a depth of 10 m) during mass spawning. It is interesting to find that steroid (E2 and testosterone) glucuronide concentrations were much higher than the respectively free steroid concentrations in seawater. E2 glucuronide was the major compound in seawater compared with the others (free E2 and testosterone, and testosterone glucuronide). High concentrations of E2 glucuronide in spawning seawater were consistent with the greater ratio of steroid glucuronide in the coral tissue just prior to mass spawning. Therefore, we suggest that the increases in free E2 and E2 glucuronide in coral tissues are important for gamete release in seawater. Estrogens may be important for coral reproduction, as has been already demonstrated in other animals.
Higher free E2 concentrations than those of E2 glucuronide were found in the coral tissue. In contrast, higher E2 glucuronide concentrations than those of free E2 were found in spawning seawater. These data suggest that the release of E2 glucuronide in seawater is a specific process and that the physiological significance in coral mass spawning is still unknown. E2 glucuronide may play an important role as a pheromone for the chemical communication among corals during mass spawning. This deserves further investigation in coral. Various pheromones, such as prostaglandin metabolites in goldfish Carassius auratus [19], free and sulfated 17,20ß-dihydroxy-4-pregnen-3-one in goldfish [20, 21], steroid glucuronide in zebrafish Brachydanio rerio [22], and bile acid 7
,12,24-trihydroxy-5-cholan-3-one 24-sulfate in sea lamprey, Petromyzon marinus [23], have also been found in other aquatic animals.
For the first time, we are also able to identify the presence of aromatase activity in coral tissue. The aromatase activity had the typical enzyme kinetics with a maximum activity and enzyme affinity. These data suggest that the biosynthesis of E2 from testosterone could have occurred in coral tissue. The conversion of testosterone to E2 may explain the findings of the decrease in free testosterone concentrations concomitant with the increase in E2 concentrations in coral tissue just prior to mass spawning. Aromatase activity in the coral tissue is temperature-dependent. Aromatase activity did not decrease the temperature of enzymatic reaction, which was 42°C, and which was much higher than survival temperatures in coral. In general, coral could not survive (as evidenced by bleaching) when seawater temperatures were higher than 28°C.
Previous studies have shown that testosterone, but not estrogens, could be synthesized in coral tissue from precursor progesterone [24]. By applying estrone to water, other works have also shown that estrogen could be removed from water by coral and accumulate in coral tissue [25]. These previous data may lead to the conclusion that coral tissues lacked aromatase for the conversion of E2. On the other hand, our study clearly demonstrated that there was no detectable E2 in seawater even just prior to spawning. No E2 was found in seawater 10 h before spawning [18]. The exogenous source of estrogens that accumulate in coral tissue is, therefore, not supported by our data. We clearly demonstrated that a specific aromatase activity was present in the coral tissue. Therefore, we suggest that corals also possess all enzymatic activities necessary for steroid synthesis, including E2. The similarity of steroid synthesis among corals and vertebrates indicates an evolutionary conservation of the critical endocrine system. E2 and E2 glucuronide most likely play important roles in coral reproduction and spawning, respectively. In all previous studies, E2 was suggested to be important in coral spawning [14, 15, 18]. According to our current data, we propose that E2 glucuronide is more important than free E2 as a seawater-mediated chemical signal in the spawning synchrony of corals.
In summary, E2 and testosterone in free and glucuronide forms were consistently detected in E. ancora coral throughout the year. Peak concentrations of free E2, E2 glucuronide, and testosterone glucuronide were measured in the coral prior to spawning. The presence of aromatase in coral tissue may results in the biosynthesis of E2 from testosterone. Higher concentrations of free E2 than E2 glucuronide were found in the coral. In contrast, higher concentrations of E2 glucuronide than free E2 and testosterone glucuronide were found in spawning seawater. It is suggested that E2 glucuronide plays an important role in coral mass spawning.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 886 2 2462 1579; B0044{at}mail.ntou.edu.tw
Received: 17 October 2002.
First decision: 14 November 2002.
Accepted: 21 January 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,20ß-dihydroxy-4-pregnen-3-one functions as a goldfish primer pheromone: preovulatory release is closely associated with male endocrine responses. Gen Comp Endocrinol 1989 75:62-70[CrossRef][Medline]This article has been cited by other articles:
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  T. Mizuta and K. Kubokawa Presence of Sex Steroids and Cytochrome P450 Genes in Amphioxus Endocrinology, August 1, 2007; 148(8): 3554 - 3565. [Abstract] [Full Text] [PDF]
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J. Keay, J. T. Bridgham, and J. W. Thornton The Octopus vulgaris Estrogen Receptor Is a Constitutive Transcriptional Activator: Evolutionary and Functional Implications Endocrinology, August 1, 2006; 147(8): 3861 - 3869. [Abstract] [Full Text] [PDF]
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W.-H. Twan, J.-S. Hwang, Y.-H. Lee, S.-R. Jeng, W.-S. Yueh, Y.-H. Tung, H.-F. Wu, S. Dufour, and C.-F. Chang The Presence and Ancestral Role of Gonadotropin-Releasing Hormone in the Reproduction of Scleractinian Coral, Euphyllia ancora Endocrinology, January 1, 2006; 147(1): 397 - 406. [Abstract] [Full Text] [PDF]
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 A. M. Tarrant Endocrine-like Signaling in Cnidarians: Current Understanding and Implications for Ecophysiology Integr. Comp. Biol., January 1, 2005; 45(1): 201 - 214. [Abstract] [Full Text] [PDF]
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