| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Mechanisms of Hormone Action |
Department of Aquaculture,3 National Taiwan Ocean University, Keelung 202, Taiwan, Republic of China
National Museum of Marine Biology and Aquarium,4 Pintung 944, Taiwan, Republic of China
Laboratory of Reproductive Biology,5 National Institute for Basic Biology, Okazaki 444, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ERß) and an androgen receptor (AR) were monitored. An AR cDNA was cloned and characterized. ER, ERß, and AR were differentially transcribed in bisexual testicular and ovarian tissue according to reverse transcription polymerase chain reaction (RT-PCR) and Southern analysis. A real-time quantification PCR analysis was further developed for the measurement of AR, ER, and ERß transcripts. ER and AR transcripts were significantly more plentiful in bisexual testis than in bisexual ovary in 1+- and 2+-yr-old fish. ER, ERß, and AR transcripts decreased in the functional testis of 3-yr-old fish. Similar levels of ERß and AR were detected in the ovary of sex-changed females and in functional testis of 3-yr-old males. Significantly decreased AR transcripts were found in testicular tissue of bisexual and functional male and female gonads in 3-yr-old fish as compared with 1- and 2-yr-old fish. In contrast, increased ER transcripts were detected in the bisexual ovary and sex-changed ovary of 3-yr-old fish as compared with the bisexual ovary of 1- and 2-yr-old fish. The data suggest a differential sensitivity in the bisexual testicular and ovarian tissue of black porgy.
androgen receptor, developmental biology, estradiol receptor, mechanisms of hormone action, steroid hormone receptors
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Oral administration of 17ß-estradiol (E2; 4–6 mg/kg feed) induced the increase of gonadal aromatase activity and resulted in sex change from male to female [3, 6–8]. However, differential plasma levels of E2 and 11-ketotestosterone (11-KT) but not testosterone (T) were previously found in sex-changing black porgy [2, 4, 6, 9]. Aromatase inhibitors completely blocked the natural sex change [9]. In contrast, low doses of E2 (0.25 or 1.0 mg/kg feed) stimulated testicular development and spermiation in 1-yr-old fish [8]. 11-KT but not T was higher in males than in sex-changed females during the spawning season [6–8]. Therefore, the involvement of sex steroids in the natural and controlled sex change is suggested in black porgy [2, 4, 9]. Androgen receptor (AR) and estrogen receptor (ER) have been considered the critical mediators for the action of sex steroids on male and female differentiation and development in both fish and mammals. At least two types of ER (ER and ERß) have been found in many animals, including black porgy [10]. ER and ERß may play different roles in gene regulation [11] and in mouse ovarian development [12]. Therefore, the importance of E2 and androgens on the development of testis and ovary leads us to suggest that AR and ER may be an important part of the mechanism of sex change in hermaphroditic fish.
The bisexual gonad (ovarian plus testicular tissue) of black porgy provides a unique model to further investigate the mechanism of sex change. We hypothesize that bisexual testicular tissue is more responsive than bisexual ovarian tissue to the stimulation of endogenous hormones. The responsiveness of gonadal tissue has been hypothesized to be associated with the transcription of AR and ER in these tissues. In this study, we monitored the transcript levels of AR and ER in the gonad of protandrous black porgy at different stages of development.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Black porgy of different ages (1–3 yr) and seasons were obtained from ponds (Table 1). All experimental fish were acclimated to ponds at the culture station (National Taiwan Ocean University, Keelung, Taiwan) with a seawater and natural light system. The water temperatures ranged from 19°C (winter season) to 27°C (summer season). The fish were fed ad libitum with a commercial feed once daily (Fwu Sou Feed Co., Taichung, Taiwan). Bisexual testicular and ovarian tissues (defined as the testicular and ovarian tissue in the bisexual gonad, respectively) were carefully collected from each fish for the analysis of ER, ERß, and AR transcripts. The fish were handled in an appropriate way to conform to animal experimentation procedure guidelines.
|
Cloning of AR
Total RNA from testis was extracted by homogenization in Trizol reagent (Gibco BRL, Grand Island, NY). Reverse transcription (RT) was performed using Superscript II (Gibco BRL) with oligo (dT)12–18 primers under the following conditions: 42°C for 50 min, 37°C for 15 min, and 70°C for 15 min. The degenerated primers for AR cloning were designed as follows: primer 1 (nucleotides [nt] 1232–1255), 5'-ACATGTWCCCMATGGAGTTCT-3'; primer 2 (nt 2291–2316), 5'-CTASTYGTGRAAMARGATTGGTTT-3'. The polymerase chain reaction (PCR) assay was carried out for 32 cycles: 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min. An approximately 1115-base pair (bp) product was amplified. The primer pair consisting of degenerated primer 3 (nt 361–384), 5'-GCYGTBTCYGTGTCDCTGGGMYTG-3', and specific primer 4 (nt 1232–1255), 5'-AACAGATCATGCACATCCTCTGA-3', and another pair consisting of degenerated primer 5 (nt-8–11), 5'-TCTGGGASATGAGCCAAAC-3', and specific primer 6 (nt 410–428), 5'-GGGAGAGCAGCGTCCATG-3', were designed to obtain full-length AR cDNA (2316 bp with 771 deduced amino acids for the open reading frame). PCR products were gel purified and cloned into then pGEM-T vector (Promega, Madison, WI) and then further amplified and purified from plasmid preparations. Sequencing was performed using a dye terminator cycle sequencing kit (Perkin Elmer, Foster City, CA).
ER and ERß cDNA
The ER and ERß cDNA sequences are available in GenBank under the accession numbers AY074779 for ER and AY074780 for ERß [10].
RT-PCR and Southern Blot Analysis of ER, ERß, and AR Transcripts in Ovarian and Testicular Tissue of Bisexual Gonad
One microgram of total RNA extracted from various stages of the representative fish was reverse transcribed to first-strand cDNA using Superscript II with the oligo (dT)12–18 primers. The first-strand cDNA was ready for the PCR and Southern blot analysis. No PCR product was found in these RNA samples by adding respectively specific ER, ERß, and AR primers for PCR, which indicates that the RNA extract was not contaminated with genomic DNA. Specific primers were designed for AR (nt 420–438, 5'-ATGGACGCTGCTCTCCC-3'; nt 1119–1137, 5'-GCTGGAGTTGGGATATGG-3'), ER (nt 697–708, 5'-CAGGCTTGCCGTCTTAGG-3'; nt 1598–1618, 5'-CATGCCTTTGTTGCTCATGTG-3'), ERß (nt 739–757, 5'-ACAGGGCAGAACCAACGG-3'; nt 1265–1283, 5'-GACTCTGCAGCTCCTCGC-3'), and ß-actin (nt 46–67, 5'-CTACAACGAGCTGAGAGTTGC-3'; nt 414–434, 5'-CACGTAGGAGAGCTTCTCCTT-3'). ß-Actin was used as an internal control. PCR conditions were as follows: 94°C for 1 min, 62.5°C for 1 min, and 72°C for 1.5 min for 30 cycles. This number of PCR cycles was preliminarily tested and was in the range of the linear curve for the relationship between the number of cycles and the amount of PCR product. Fragments of about 707 bp, 921 bp, 544 bp, and 388 bp were specifically amplified using AR, ER, ERß, and ß-actin, respectively, as probes. The probe was labeled with P32-dCTP with a random primer labeling kit (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, England) for the Southern hybridization according to previous studies [10].
Quantification of ER, ERß, and AR Transcripts by Real-Time PCR Analysis
Absolute quantification with real-time PCR analysis was modified from the methods and principles previously reported [13]. Specific primers were designed for the real-time PCR for ER (nt 500–516, 5'-GGATCCGCTGGGTTTGA-3'; nt 553–574, 5'-ATGGTACCCAGAGGCATAATCG-3'), ERß (nt 203–221, 5'-CAATCCGCCGAGCATTTCA-3'; nt 258–276, 5'-CATGGCTGGGCCAAAATAA-3'), and AR (nt 806–824, 5'-GAGACCGTGGCGGCTCTA-3'; nt 849–871, 5'-TGCTCTCCATGACTTCCATGAA-3'). Templates were then obtained for ER (695 bp; primers: nt 1–22, 5'-GCCCTGTGGACCAGTACAGA-3'; nt 672–695, 5'-AGCTCTTCCTCCGATTCCTGTCAA-3'), ERß (514 bp; primers: nt 167–186, 5'-CATACCTTTCTACAGTCCAA-3'; nt 662–680, 5'-CATCCCACACTTGGTCATG-3'), and AR (895 bp; primers: nt 361–384, 5'-GCTGTTTCTGTGTCGCTGGGATTG-3'; nt 1232–1255, 5'-AACAGATCATGCACATCCTCTGA-3'). Linear plasmid DNA (containing respective inserts of ER, ERß and AR in the PGEM-T vector; Promega) was obtained by cutting with SalI (New England BioLabs, Beverly, MA). In vitro transcription was conducted with T7 polymerase (Promega) to obtain standard RNA. Different concentrations of RNA (1, 0.1, 0.01, 0.001, and 0.0001 µg) were prepared, and the respective cDNAs were synthesized using SuperScript II RT (Gibco BRL) for the standard curve. One microgram of sample total RNA was also prepared, and a cDNA was synthesized to analyze the receptor mRNA expression levels. Gene quantification of standards, samples, and controls was simultaneously conducted by a real-time PCR (GeneAmp 5700 Sequence Detection System; Applied Biosystems, Foster City, CA) with SYBR green I as a double-strand DNA minor-groove binding dye. Single and specific PCR product was identified in the respective dissociation curve assay, and the threshold cycle (CT; the calculated fractional cycle number at which the PCR fluorescence product is detectable above a threshold, based on the variability of baseline data in cycles 6–15) was measured. The respective standard curve of log of transcript concentrations versus CT was obtained. The CT value is inversely proportional to the log of the initial mRNA copy number. The correlation of the standard curve was in the range of -0.990 to -0.999 for ER, ERß, and AR. The values detected from different amounts of RNA (1, 0.1, 0.01, and 0.001 µg) from the representative samples were parallel with the respective standard curve.
Data Analysis
All data are expressed as mean ± SEM. The values were subjected to a one-way ANOVA to test significance (P < 0.05) followed by a Duncan multiple-range test [14].
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
One type of black porgy AR was cloned (GenBank accession AY219702). The sequence of cDNA and the deduced amino acids (AA) are shown in Figure 1. The C domain (AA 409–495) had two zinc finger motifs with eight cysteines located at AA 415, 418, 432, 435, 451, 457, 467, and 470. The C domain also had a proximal box (AA 433-GSCKV-437) for binding specifically to the response element and a distal box (AA 452-ASKND-456) involved in the recognition of spacing between half-sites of the response element and in dimerization. The homology of the deduced AA sequence ranged from 52% to 89% compared with other teleosts and was 61% compared with the human sequence.
|
ER and AR in the Bisexual Gonad Analyzed by RT-PCR and Southern Blot
ER and ERß were differentially expressed in ovarian and testicular tissue of the bisexual gonad of representative 1-, 1+-, 2+-, and 3-yr-old black porgy (Fig. 2). High levels of ER transcripts were found in the functional testis in 1- and 2-yr-old fish, in testicular tissue of bisexual gonad in 2+-yr-old fish, and in vitellogenic ovary in 3-yr-old fish compared with levels in the respective bisexual ovarian tissue (Fig. 2). No difference in ERß transcripts was found between testicular and ovarian tissue of 1+- and 2+-yr-old fish (Fig. 2). Differences between ovarian and testicular tissue were greater for ER transcripts than for ERß transcripts (Fig. 2).
|
AR transcripts were significantly more plentiful in testicular tissue than in ovarian tissue in 1+- and 2+-yr-old fish (Fig. 3). AR transcripts in testicular tissue of 3-yr-old fish were virtually undetectable, and levels were similar to those in the respective ovarian tissues (Fig. 3).
|
ER Transcripts in Gonadal Tissue as Determined by Real-Time PCR
Bisexual testicular tissue had higher levels of ER transcripts (50- and 40-fold) than the respective ovarian tissue in 1+- and 2+-yr-old fish (Fig. 4A). No difference in ER transcripts was found between bisexual testicular and ovarian tissue in 3-yr-old fish (Fig. 4A). Bisexual testicular tissue had higher levels of ER transcripts in 1+- and 2+-yr-old fish than in 3-yr-old fish (Fig. 4B). No difference in ER transcripts was found among functional testis in 1-, 2-, and 3-yr-old fish and bisexual testis in 3-yr-old fish (Fig. 4B). Female ovary and bisexual ovary in 3-yr-old fish had significantly higher levels of ER transcripts than did bisexual ovarian tissue in 1+- and 2+-yr-old fish (Fig. 4C; note difference in scale compared with Fig. 4, A and B).
|
ERß Transcripts in Gonadal Tissue
No difference in ERß transcripts was found between bisexual testicular and ovarian tissue in 1+-, 2+-, and 3-yr-old fish (Fig. 5A). There was also no difference in ERß transcripts among bisexual testis of fish of different ages (Fig. 5B), but transcript levels in functional testis were lower in 3-yr-old fish than in 1- and 2-yr-old fish (Fig. 5B). Bisexual ovary and female ovary also had similar levels of ERß (Fig. 5C). Similar but lower levels of ERß transcript were detected in bisexual ovary of 2+- and 3-yr-old fish and in female ovary of 3-yr-old fish than in bisexual ovary of 1+-yr-old fish (Fig. 5C).
|
AR Transcripts in Gonadal Tissue
Bisexual testis had higher levels of AR transcripts than did bisexual ovary in 1+- and 2+-yr-old fish (6.6-fold and 13.5-fold higher, respectively) (Fig. 6A). Bisexual testis in 3-yr-old fish had significantly lower AR transcript levels than did testis in 1+- and 2+-yr-old fish (Fig. 6B). Functional testis had significantly lower AR transcript levels in 3-yr-old males than in 1- and 2-yr-old fish (Fig. 6B). Female ovary (3-yr-old fish) had lower AR transcript levels than did bisexual ovary (1+- to 3-yr-old fish) (Fig. 6C; note difference in scale compared with Fig. 6, A and B).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and AR transcripts than did ovarian tissue. These data support the hypothesis that testicular tissue is more sensitive (responsive) than ovarian tissue in the bisexual gonad of 1+- and 2+-yr-old fish (before sex change) to the stimulation of endocrine hormones. Therefore, under certain hormone stimulation regimens, bisexual testicular tissue will develop and bisexual ovary will regress, and the fish will remain male.
In previous studies, levels of plasma E2, T, and 11-KT were low (
100 pg/ml) in male black porgy during the nonspawning season [2, 3, 6, 8, 15], and E2 was significantly increased in the natural sex-changing females (300–800 pg/ml) [2, 4, 6]. Plasma levels of T (200–700 pg/ml) and 11-KT (300–1000 pg/ml) also increased in 1- to 3-yr-old functional males, but the increased amplitude of androgens was much smaller in 3-yr-old males than in younger males [2, 3, 7–9, 15]. Various concentrations of E2 may act to stimulate spermatogenesis (low E2) or to stimulate sex change (high E2) in black porgy [3, 6–8]. Based on results from previous studies on the involvement of sex steroids in sex change and the current experiments, high ER in testicular tissue may indicate that E2 also has significant effects on the stimulation of spermatogenesis. E2 (in high doses) decreased plasma 11-KT and T levels in black porgy [7, 15] and may suppress ER and AR transcripts in testicular tissue and stimulate ER transcripts in ovarian tissue, resulting in sex change. Similar biphasic actions of E2 have been found in other species. E2 induces spermatogonial stem cell renewal in an eel (Anguilla japonica) [16]. However, E2 inhibits 11-KT production in Atlantic croaker (Micriopogonias undulates) in vitro [17]. Androgens also may be mediated by AR to stimulate the development of testicular tissue; thus, the fish continues to function as a male, as previously demonstrated by oral administration of T in black porgy [18]. The association between levels of sex steroids and ER/AR transcripts needs to be further characterized. It is not clear whether steroid receptors detected in this study represent the expression of steroid receptor protein.
Our data on ER are consistent with those of other studies in protandrous gilthead seabream, Sparus aurata [19]. ER transcripts in the testicular tissue were more plentiful than those in ovarian tissue and then decreased in the process of sex change in that fish [19]. Higher levels of ER transcripts in the bisexual ovary were found in 3-yr-old black porgy than in 1+- and 2+-yr-old fish, although primary oocytes appeared in ovarian tissue of those fish. Therefore, ER transcripts were ovarian stage-dependent in black porgy, as also shown for gonochoristic rainbow trout (Oncorhynchus mykiss) [20]. ERß exhibited little change compared with ER in black porgy. Furthermore, similar but decreased ERß transcript levels were detected in the ovary of sex-changed females and in the functional testis of 3-yr-old male fish. Consequently, the importance of ERß in the development of testis and ovary in black porgy is not clear. In gonochoristic rainbow trout, ER transcripts are not different in testis and ovary at various stages [21, 22]. The importance of ER in ovarian development has been demonstrated with ER- and ERß-knockout mice [12] and rats [23].
In contrast to the decrease in ERß in the sex-changed fish, ER transcript levels in female ovary were much higher in sex-changed fish than in 1+- and 2+-yr-old fish. ERß transcript levels were not different in the bisexual ovary (2+- and 3-yr-old fish) and female ovary (3-yr-old fish). These data suggest that ER but not ERß is associated with sex change in black porgy. However, in gonochoristic rainbow trout, ER genes were detected early but with no sex difference in both male and female gonad before or during sex differentiation [21].
Higher AR transcript levels were expressed in bisexual testicular tissue and functional testis compared with respectively ovarian tissue in 1+- and 2+-yr-old fish. Bisexual testis had similar or even higher AR transcript levels than did the functional testis. These data suggest that bisexual testis remains at the active stage before the sex change to female. Significantly decreased AR and ER transcript levels were found in the bisexual testis of 3-yr-old fish. These data suggest that bisexual testicular tissue becomes less sensitive when fish reach 3 yr of age (approaching the year of sex change). The decrease in AR and ER in functional testis of 3-yr-old fish also may be related to the low production of sperm and 11-KT in 3-yr-old male fish compared with 1- and 2-yr-old functional males [7, 9, 24].
Low AR transcript levels were found in bisexual ovary and female gonad. Primary oocytes and vitellogenic oocytes are the major germ cells in the bisexual ovary (before sex change) and sex-changed female gonad, respectively, of black porgy [4, 5]. The finding of lower AR transcript levels in vitellogenic oocytes than in bisexual ovarian tissue (with primary oocytes) suggests that decreased AR transcript levels are associtated with the process of sex change. In contrast, similar ER or ERß transcript levels were found for bisexual ovary (with primary oocytes) and female ovary (with vitellogenic oocytes) in 3-yr-old fish. These data suggest that other factors (such as decreased AR in this study and increased aromatase [9] in addition to ER) also are involved in the sex change mechanism in 3-yr-old black porgy. Our current findings on the relative abundance of AR transcripts (bisexual testicular tissue 
functional testis > ovarian tissue) in black porgy differ from those obtained in gonochoristic species such as rat and pig [24, 25]. AR transcript levels are increased in maturing testis in rat [25] and also in the developing ovary in pig [24].
One type of AR was cloned from testis of black porgy in contrast to two types in tilapia (GenBank accessions AB 045211 and AB 045212), eel (A. japonica) [26, 27], rainbow trout [28], and a cichlid fish (Astatotilapia burtoni) (GenBank accessions AF 121257.1, AY 082342) and one type in red seabream (Chrysophrys major) [29], goldfish (Carassius auratus) (GenBank accession AY 090897), and human [30]. The homology of black porgy AR AA sequence was 52–60% for AR and ARß in eel (A. japonica) [26, 27] and rainbow trout [28] and 89% for red seabream AR [29]. 11-KT is also probably more potent than T in affecting the action of black porgy AR, as demonstrated in eel (A. japonica) AR and ARß [27]. The possible role of other ERs on sex change cannot be completely excluded because a third type of ER (ER
) was found in Atlantic croaker [31].
Black porgy, which have a bisexual gonad, provide another model to study the endocrine mechanism of sex change. The present data on the levels of ER, ERß, and AR transcripts suggest that ovarian tissue is much less responsive than testicular tissue to hormone stimulation, which determines whether the bisexual gonad remains a functional testis or becomes an ovary. Alternatively, testis- or ovary-specific expression of steroidogenic enzymes may result in a local increase of steroids acting in a paracrine or autocrine manner, in the absence of any competition. Increased ER transcript levels appear more important than ERß levels to stage and sex change of gonadal tissue. These findings provide the foundation for a new hypothesis, the differential sensitivity of bisexual testicular/ovarian tissue, to help explain the complicated process of sex change, using black porgy as a model.
|
FOOTNOTES |
|---|
2 Correspondence: Ching-Fong Chang, Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan. FAX: 886 2 2462 1579; b0044{at}mail.ntou.edu.tw
Received: 2 January 2003.
First decision: 22 January 2003.
Accepted: 24 March 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ERß at AP1 sites. Science 1997 277:1439 (ER) and ß (ERß) on mouse reproductive phenotypes. Development 2000 127:4277-4291[Abstract] messenger RNA and it expression in the ovary during the reproductive cycle. J Mol Endocrinol 2000 25:243-251[Abstract] and ß. Science 1999 286:2328-2331This article has been cited by other articles:
|
|
 |
|
  M. S. Hossain, A. Larsson, N. Scherbak, P.-E. Olsson, and L. Orban Zebrafish Androgen Receptor: Isolation, Molecular, and Biochemical Characterization Biol Reprod, February 1, 2008; 78(2): 361 - 369. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-L. Du, Y.-H. Lee, W.-S. Yueh, and C.-F. Chang Seasonal Profiles of Brain and Pituitary Gonadotropin-Releasing Hormone and Plasma Luteinizing Hormone in Relation to Sex Change of Protandrous Black Porgy, Acanthopagrus schlegeli Biol Reprod, April 1, 2005; 72(4): 922 - 931. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-H. Lee, G.-C. Wu, J.-L. Du, and C.-F. Chang Estradiol-17{beta} Induced a Reversible Sex Change in the Fingerlings of Protandrous Black Porgy, Acanthopagrus schlegeli Bleeker: The Possible Roles of Luteinizing Hormone in Sex Change Biol Reprod, October 1, 2004; 71(4): 1270 - 1278. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
T1, 1-yr-old functional testis; 
O1+ and T1+, bisexual ovarian (O1+) and testicular (T1+) tissue in 1+-yr-old fish; 
