Abstract: In teleost fish, bone morphogenetic protein bmp2b is important for the development of skeletons, including the development of secondary sex characters of fins. Sex steroid hormones are also necessary for the development of those characters. In this study, bmp2b expression levels in fins of adult Japanese medaka (Oryzias latipes) after treatment with sex steroid hormones were examined using semi-quantitative RT-PCR. In males, bmp2b expression levels in the posterior part of anal fins were increased at 6 days after 11-ketotestosterone (11-KT) treatments. In contrast, bmp2b expression levels in the posterior part of anal fins were decreased at 6 days after 17β-estradiol (E2) treatments. The bmp2b expression levels in the pelvic fin of males were substantially increased at 3 and 6 days after E2 treatment. No significant difference in the dorsal, anterior part of the anal, pectoral and caudal fins of male was found among time-related courses after 11-KT and E2 treatments. In females, bmp2b expression levels in the dorsal, anterior and posterior part of anal fins were further increased at 3, 12 and 3 days, respectively after 11-KT treatment. Conversely, there was no difference in bmp2b expression levels of pectoral, pelvic and caudal fins of females among time-related courses after treatment with 11-KT and E2. Present results, therefore, suggest that bmp2b regulation in the fin may be involved in 11-KT and E2-mediated regulation depending on the sex-dependent characters of fin morphology in adult Japanese medaka.
INTRODUCTION
Japanese medaka (Oryzias latipes, Teleostei) is one of the best model organisms, which is widely used for experiments in various fields (Lynne, 2008) such as developmental biology (Karim et al., 2009) and endocrine (Zhang et al., 2008). In the genus Oryzias, the morphologies of the dorsal and anal fins are typical secondary sex characters (Hayashi et al., 2007). In adult fish, the dorsal and anal fins of males are usually longer than those of females (Okada and Yamashita, 1944). Especially, in males, papillar processes are present on anal fins (Oka, 1931; Uwa, 1971). The papillar processes are also formed on the pectoral fin of males (Egemi and Ishii, 1956). In the pectoral and pelvic fins, however, there is a difference in the length between males and females. The pectoral and pelvic fins of females are longer than those of males (Iwamatsu et al., 2003). In contrast, the caudal fin does not show any difference in length between males and females (Okada and Yamashita, 1944).
It is well known that, in many teleosts, 11-ketotestosterone is a major androgen that plays physiological roles in reproductive function, sexual differentiation and maintenance of secondary sex characters of males (Borg, 1994; Olsson et al., 2005; Leon et al., 2007). As a primary estrogen in teleosts, 17β-estradiol plays an important role in the maturation of the reproductive organs in females (Nimrod and Benson, 1998; Nilsson et al., 2001; Kagawa et al., 2003).
In all vertebrates, bone morphogenetic proteins are members of transforming growth factor β superfamily and crucial for various developmental processes including bone and cartilage formation (King et al., 1996; Francis-West et al., 1999; Ruggeri et al., 2008). In teleosts, bone morphogenetic protein (bmp) 2b plays an important role in fin formation, differentiation and regeneration (Tiso et al., 2002; Crotwell et al., 2004; Smith et al., 2006). In addition, in mammals androgen and estrogen can increase Bmp mRNA levels, suggesting that those hormones enhance bone formation (Thomas et al., 1998; Ide et al., 1997; Yamamoto et al., 2002; Zhou et al., 2003).
As an objective of this study, we examined the bmp2b mRNA levels how express in the dorsal, anal, pectoral, pelvic and caudal fins of adult Japanese medaka when exposed to 11-ketotestosterone or 17βestradiol.
MATERIALS AND METHODS
Fish: Adult Japanese medaka were purchased from a commercial source in Kanazawa City, Ishikawa, Japan. Their standard length was 24-26 mm. Males and females were kept in separate aquariums with controlling 14:10 h light/dark photoperiod cycle at 26±1°C for 1 week and fed ad libitum with TetraMin (Tokyo, Japan). Sex was determined from the morphology of the secondary sex characters of the dorsal and anal fin according to the criteria of (Okada and Yamashita, 1944). This experiment was conducted from September, 2008 through the end of February, 2009.
Preparation of chemical solutions: In this study, 11-ketotestosterone (11-KT) (Sigma, St. Louis, MO), 17β-estradiol (E2) (Wako, Osaka) and dimethyl sulfoxide (DMSO) solutions (Wako, Osaka) were prepared following the methods of (Iwamatsu, 1999; Leon et al., 2007) and (Gonzalez-Doncel et al., 2008) at suitable concentrations and non-lethal toxicity for Japanese medaka.
For the androgen preparation, 1 mg of 11-KT was dissolved in 0.1 mL of DMSO to make a stock solution (10 mg mL-1). In a final concentration, 0.1 mL of the stock solution was diluted with 1,000 mL of aquarium water to 1 μg mL-1. For the estrogen preparation, 1 mg of E2 was dissolved in 1 mL of DMSO for a stock solution. The stock solution of E2 was diluted with aquarium-water to 0.1 μg mL-1. In each group, three males or four females were immersed in 1,000 mL of aquarium water containing 1 μg mL-1 of 11-KT or 0.1 μg mL-1 of E2 for 3, 6 and 12 days under the above conditions. In the control groups, males or females were treated with only 0.1 mL of DMSO in 1,000 mL of aquarium water without any sex steroid hormones for 12 days. The aquarium-water was changed every three days.
According to the mortality which is shown in Table 1, concentrations of sex steroid hormones seem to be non-lethal and non-acute toxicity for medaka.
| Table 1: | Effects of various durations of DMSO, 11-ketotestosterone (11-KT) and 17β-estradiol (E2) on survival percentages of adult Japanese medaka |
| Fig. 1: | Diagrammatic illustration of the secondary sex characters of fins in (a) male and (b) female medaka. (c) The posterior part of anal fin in male with papillar processes, comprising the 2nd to the 8th fin rays counted from the posterior end, was separated from the anterior part. an, anal fin; ca, caudal fin; d, dorsal fin; pec, pectoral; fin; pel, pelvic fin |
Preparation of fins: Adult males and females were anesthetized with 200 mg L-1 of an ethyl-3-aminobenzoate methanesulphonate (MS-222) solution (Sigma, St. Louis, MO) and placed in a Petri dish. The dorsal, anal, pectoral, pelvic and caudal fins from individual males and females were removed with clean scalpels. In the anal fins, the posterior part with papillar processes, which corresponds to the part from the 2nd to the 8th fin ray counted from the posterior end, was separated from the anterior part according to the criteria of (Iwamatsu et al., 2003) (Fig. 1a, b). Three or four fins were pooled in each tube and a sample of five tubes was collected. Therefore, one experimental group includes fifteen to twenty individuals.
Semi-quantitative RT-PCR: Total RNA from each sample of adult fish was extracted using an isolation reagent Isogen (Wako, Japan) according to the protocol and treated with DNase1 (Takara, Ohtsu) for 30 min at 37°C. Total RNA of 100 ng was reverse-transcribed with AMV reverse transcriptase XL (Takara,Ohtsu) according to the instructions. The solution of 0.5 μL was used for a PCR template.
Primers were all designed on the basis of data of Japanese medaka. Primers for amplification of bmp2b nucleotides were designed as 5’-AAGGGCAAAACAACCCAGCAG-3’ and 5’ GTTCCATCCCACATCGCTGAA-3’ according to the report by Ngamniyom et al. (2009). The β-actin mRNA was amplified in each RT reaction as a loading control and reference. Primers for the amplification of β-actin nucleotides were 5’-AGGGAGAAGATGACC-3’ and 5’-CGCAGGACGCCATACCAA-3’ according to the report by Scholz et al. (2004).
Amplification of cDNA was carried out at cycling conditions of 95°C for 30 sec for the denaturation step and 64°C (bmp2b) or 58°C (β-actin) for 1 min for the annealing step and 72 °C for 1 min for extension step. The cycle numbers were 30 cycles for bmp2b and 20 cycles for β-actin. The PCR products were electrophoresed on 2% agarose gel, immersed in ethidium bromide and visualized on a UV-transilluminator. The amplification rate was quantitated using the software of Scion Image for Windows (Scion, MD, USA). Amplification levels of bmp2b for each fin type were divided by the corresponding amplification level of β-actin to obtain relative expression levels. In this study, no effect of 11-KT and E2 on the levels of β-actin expression in fins were found among time-related courses.
One-way ANOVA with Tukey’s multiple comparison tests was used to examine the differences statistically (Zar, 1984). The data were analyzed using a Statistical Package for the Social Sciences (SPSS) for Windows version 13 (SPSS, Chicago, IL, USA).
RESULTS
Effect of 11-ketotestosterone (11-KT) on the level of bmp2b mRNA expression in fins: In representative gel electrophoresis, alterations of bmp2b mRNA expressions were found in the posterior part of male anal fin at 6 days, anterior part of female anal fin at 12 days and posterior part of female anal fin at 3 days after treatment with 11-KT (Fig. 2b, 3b).
| Fig. 2: | Gel electrophoresis of RT-PCR analysis of bmp2b and β-actin mRNA expression in male and female medaka after treatments of 11-Ketotestosterone. C: Control. (a) male and (b) female |
| Fig. 3: | Gel electrophoresis of RT-PCR analysis of bmp2b and β-actin mRNA expression in male and female medaka after treatments of 17 β-estradiol. C: Control. (a) male and (b) female |
In male medaka, no significant difference in the expression levels of bmp2b mRNA was found in the dorsal, anterior part of anal, pectoral, pelvic and caudal fins by 3, 6 and 12 days between the 11-KT-treated group and the untreated control groups (Fig. 4a). In contrast, the expression levels of bmp2b mRNA were significantly increased in the posterior part of anal fins by 6 days after 11-KT treatments (Fig. 4a).
In female medaka, the expression levels of bmp2b mRNA were significantly higher in the dorsal fins by 3 days after treatment with 11-KT than in the untreated controls (Fig. 4b). In addition, in anal fins, significant increases in the expression levels of bmp2b mRNA were found in the anterior part at 12 days and in the posterior part at 3 days after treatment with 11-KT (Fig. 4b). In contrast, the expression levels of bmp2b mRNA were not significantly changed in the pectoral, pelvic and caudal fins by 3, 6 and 12 days after treatment with 11-KT (Fig. 4b).
In males and females, there was no phenotype change for any of the fin types after treatment of 11-KT.
Effect of 17β-estradiol (E2) on the levels of bmp2b mRNA expression in fins: In representative gel electrophoresis, alterations of bmp2b mRNA expressions were found in the pelvic fins at 3 and 6 days and posterior part of anal fins of males at 3 days after E2 treatments (Fig. 2b, 3b).
In male medaka, no significant difference in the expression levels of bmp2b mRNA was found between the dorsal, anterior part of anal, pectoral and caudal fins by 3, 6 and 12 days after E2 treatment and those from the untreated control groups (Fig. 5a). The expression levels of bmp2b mRNA were significantly decreased in the posterior part of anal fins by 6 days after E2 treatments (Fig. 5a). In contract, the expression levels of bmp2b mRNA were significantly increased in the pelvic fins in response to E2 by 3 and 6 days (Fig. 5a).
| Fig. 4: | Time-related effects of 11-ketotestosterone on bmp2b mRNA expression levels in fins of (a) male and (b) female medaka. Control groups (C) were treated with DMSO. The expression levels in each fin are relative values compared to the expression levels of β-actin mRNA (Mean±SE). Single- and double-asterisk symbols show p<0.05 and p<0.01, respectively (one-way ANOVA followed by Tukey’s multiple comparison test). Each experimental group consisted of five samples of medaka fins |
| Fig. 5: | Time-related effects of 17β-estradiol on bmp2b mRNA expression levels in fins of (a) male and (b) female medaka. Control groups (C) were treated with DMSO. The expression levels in each fin are relative values compared to the expression levels of β-actin mRNA (Mean±SE). Single- and double-asterisk symbols show p<0.05 and p<0.005, respectively (one-way ANOVA followed by Tukey’s multiple comparison test). Each experimental group consisted of five samples of medaka fins |
In female medaka, no significant differences in the expression levels of bmp2b mRNA were found in the dorsal, anal, pectoral, pelvic and caudal fins after E2 treatment (Fig. 5b).
In males and females, no phenotype change was found for any of the fin types after treatment of E2.
DISCUSSION
In the male, bmp2b expression was not different in the dorsal, anterior part of anal, pectoral and pelvic fins among time-related courses after treatment of 11-KT. Kawamoto (1969) reported that, in Japanese medaka, no significant difference was found in the length of the dorsal and anal fin of the adult male after treatment with androstenedione, suggesting that an exogenous androgen might be enough for the normal development of fin characters in an adult fish. Therefore, in an adult male, bmp2b may not require the effect of an additional androgen for the fin growth of dorsal, anterior part of anal and pectoral fins, where the secondary sex characters ultimately develop.
After treatment with 11-KT, however, bmp2b in the posterior part of the anal fins was more highly expressed at 6 days than in the control groups. It is known that, in males, the papillar processes on the anal fin continually develop through the end of the breeding season (May to August) and are increased the number by androgens (Yamamoto, 1975; Shima and Mitani, 2004). This suggests that, in the posterior part of the anal fin, the bmp2b may require an effect of exogenous androgen for inducing the development of papillar processes through the breeding seasons.
In caudal fins, bmp2b did not respond to 11-KT in the various time-related treatments. This result is reasonable because sex dimorphism is not indicated shown by differences in the length of the caudal fin between the male and female.
Effect of androgens is known to extend the length of the dorsal and anal fins of female Japanese medaka as a secondary sex character of the male (Kawamoto, 1969); however, no male phenotype was found in the dorsal, anterior and posterior part of anal fins of adult female after 11-KT treatment, although bmp2b was highly expressed in the dorsal and both parts of anal fins of females at 6 days after 11-KT treatment. Androgen may be required for bmp2b expression to act on those fins that are typical secondary sex character. No significant difference in bmp2b expression was found in the pectoral and pelvic fins after treatment of 11-KT. This suggests that, in adult female fish, an exogenous androgenic hormone may be not required for bmp2b expression in the normal development of at least those fins because estrogen is enough for bmp2b expression to act on them.
The development of dorsal and anal fins is suppressed by the administration of estrogen in the juvenile males of Japanese medaka (Iwamatsu, 1999; Tabata et al., 2001). Therefore, bmp2b expression, which is necessary for fin development, might be low after estrogen treatment. On the other hand, the expression level of bmp2b was similar to that observed in the dorsal, anterior part of anal and pectoral fins of the adult male after treatment with E2. This result suggests that, in the adult male, bmp2b regulation in the dorsal, anterior part of anal and pectoral fins may not be a target for estrogen.
After treatment with E2, low expression of bmp2b in the posterior part of the anal fins was observed at 6 days. Estrogen is also known to suppress the development of papillar processes on the anal fin of males in Japanese medaka (Lin et al., 2004). Therefore, bmp2b expression may be decreased by estrogen for inhibiting or retarding the development of papillar processes involving the posterior part of the anal fin.
In the pelvic fin, the bmp2b expressions were high at 3 and 6 days after treatment with E2. It has been reported that, in Japanese medaka, the length of the pelvic fin of an adult male is elongated by the action of estrogen (Niwa, 1969). Therefore, estrogen may increase the expression level of bmp2 in the pelvic fin to induce the development of the female phenotype in the pelvic fin of the adult male.
In the female, an exogenous estrogen may also be sufficient for the expression of bmp2b in the development of at least the fins because the expression level of bmp2b in the dorsal, both parts of anal, pectoral and pelvic fins was not different after treatment with E2. Similarly to the treatment of 11-KT, no significant difference was detected in the bmp2b expression levels in the caudal fin after treatment with E2.
Recently, Ngamniyom et al. (2009) reported that in Japanese and Thai medaka androgen receptor (AR) and estrogen receptor (ER) levels were higher in dorsal and anal fins than those in pectoral and caudal fins of male and female, respectively. However, no significant difference in Bmp2b level was found among fin types, suggesting steroid hormones was necessary for adequate bmp expression in the normal development of fins. This report of Ngamniyom et al. (2009) related to our results that no response to those hormones was also found in Bmp2b levels of the dorsal and anal fins any time-course experiment, after 11-KT treatments in male and E2 treatments in female. Thus, AR and ER might not require sex steroid hormones for regulating Bmp2b level because AR and ER were excessively expressed for adequate Bmp2b expression in the normal development of at the dorsal and anal fin.
In teleost fish, it is known that the gonadal steroids, including androgen and estrogen, also exert positive or negative feedback and are associated with the regulation of hormone receptors on the metabolism of gonadotrophs (Borg, 1994; Linard et al., 1998; Khan et al., 1999; Larsson et al., 2002). After treatment with sex steroid hormones, the expression level of bmp2b was normal in the posterior part of the anal and pelvic fins of the male at 12 days and in the dorsal and anal fins of the female at 6 days. Therefore, androgen and estrogen regulation on the expression of bmp2b in fins are also involved in positive or negative feedback control, suggesting that the excess expressions of bmp2b may recover to a normal expression by the negative feedback control of androgen and estrogen for the adequate expression of bmp2b in the development of fins. However, it is not clear whether androgen and estrogen regulation on bmp2b are mediated by androgen and estrogen receptors.
The present study demonstrated that bmp2b expressions were affected by 11-KT and E2 depending on the sex and characters of the fin morphology in Japanese medaka. This is the first study in fish to investigate bmp2b regulation in response to sex steroid hormones across five tissues of fin. In addition, this study provides evidence for 11-KT- and E2-mediated regulation on bmp2b expression in the secondary sex characters of fins.