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. 2007 Oct 4;149(1):161–173. doi: 10.1210/en.2007-0938

Molecular Cloning and Characterization of Estrogen, Androgen, and Progesterone Nuclear Receptors from a Freshwater Turtle (Pseudemys nelsoni)

Yoshinao Katsu 1, Rie Ichikawa 1, Toshitaka Ikeuchi 1, Satomi Kohno 1, Louis J Guillette Jr 1, Taisen Iguchi 1
PMCID: PMC2734501  PMID: 17916628

Abstract

Steroid hormones are essential for the normal function of many organ systems in vertebrates. Reproductive activity in females and males, such as the differentiation, growth, and maintenance of the reproductive system, requires signaling by the sex steroids. Although extensively studied in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens, androgens, and progestins) action are poorly understood in reptiles. Here we evaluate hormone receptor ligand interactions in a freshwater turtle, the red-belly slider (Pseudemys nelsoni), after the isolation of cDNAs encoding an estrogen receptor alpha (ERα), an androgen receptor (AR), and a progesterone receptor (PR). The full-length red-belly slider turtle (t)ERα, tAR, and tPR cDNAs were obtained using 5′ and 3′ rapid amplification cDNA ends. The deduced amino acid sequences showed high identity to the chicken orthologs (tERα, 90%; tAR, 71%; tPR, 71%). Using transient transfection assays of mammalian cells, tERα protein displayed estrogen-dependent activation of transcription from an estrogen-responsive element-containing promoter. The other receptor proteins, tAR and tPR, also displayed androgen- or progestin-dependent activation of transcription from androgen- and progestin-responsive murine mammary tumor virus promoters. We further examined the transactivation of tERα, tAR and tPR by ligands using a modified GAL4-transactivation system. We found that the GAL4-transactivation system was not suitable for the measurement of tAR and tPR transactivations. This is the first report of the full coding regions of a reptilian AR and PR and the examination of their transactivation by steroid hormones.

STEROID HORMONES PLAY important roles in the reproductive biology of vertebrates including reptiles. Many of the currently reported actions of steroid hormones, including estrogens, androgens, and progestins, are mediated by specific receptors that are localized in the nucleus of target cells. Nuclear steroid hormone receptors form a superfamily of transcription factors that include progestogens, androgens, glucocorticoids, mineralocorticoids, the vitamin D, and the retinoic acid receptors (1). Three distinct types of estrogen receptor (ER) have been isolated, to date, in vertebrates. Teleost fish express ERα, ERβ, and ERγ forms, but the teleost ERγ form appears to be closely related to the teleost ERβ, suggesting that it reflects the gene duplication event that occurred within the teleosts (2). Thornton (3) proposed that the ancestral condition for the jawed vertebrates (Gnathostomata) is the presence of two forms of ER, corresponding to ERα and ERβ. Both forms of ER have been found in fish, amphibians, reptiles, birds, and mammals. To date, cDNAs encoding full-length ERα have been cloned from relatively few reptiles and amphibians (4,5,6,7,8) compared with other vertebrate classes, yet these receptors appear to hold a basal location in the evolution of vertebrate steroid receptors.

Estradiol-17β (E2) is the principle estrogen in circulation and appears essential for normal ovarian development in many vertebrates, including reptiles (9,10,11). Embryonic exposure to inhibitors of aromatase, the enzyme responsible for the conversion of testosterone to E2, causes genetic females to become phenotypic males in chicken and turtle (12,13,14) and disrupts normal ovarian development in the alligator (15). A number of studies strongly suggest that endogenous E2 acts as a natural inducer of ovarian differentiation in nonmammalian vertebrates (11,16,17), and in many freshwater turtles and crocodilians, environmental sex determination appears to involve an estrogenic signal for normal ovarian development (11). However, the molecular mechanisms of estrogen action on ovarian differentiation in nonmammalian vertebrates, especially reptiles, remain poorly studied. In turtles, the presence of an ER has been reported using steroid-binding assays (18,19,20,21), and Bergeron et al. (22) reported the cloning of a partial sequence of cDNA encoding an ER from the red-eared slider turtle, Trachemys scripta.

Androgens play essential roles in vertebrate spermatogenesis, the growth and differentiation of the male reproductive duct system, and the development of many male secondary sex characteristics, but their specific regulatory actions in reptiles have not been fully clarified. Elevated blood androgen concentrations have been observed in males and females of a number of reptiles, including the turtles Sternotherus odoratus, Chelonia mydass, Chrysemys picta, and Caretta caretta (23,24,25,26). Selcer et al. (27) reported that an androgen receptor (AR) was present in the oviduct of the turtle T. scripta using immunohistochemical and immunoblot analyses. However, the molecular cloning of a turtle AR, or any reptilian AR has not been reported, and the binding and transactivation of a reptilian AR and its ligand is still required.

Progesterone has been implicated in the reproductive biology of all reptiles studied to date, because elevated plasma concentrations are observed after ovulation and during pregnancy in viviparous forms. In 1979, Dube and Tremblay (28) established the presence of a progesterone (P4) receptor (PR) in turtles using competitive binding with [3H]R5020. Evidence for turtle PR proteins has been obtained using heterologous antibody and steroid-binding assays (29). As with the chicken (30), two isoforms of the PR are expressed in the turtle liver (31). The partial sequence of a turtle, C. picta, PR and the change in its gene expression seasonally or after exposure to steroids has been reported (29,31,32). They observed that only the hepatic expression of the larger PR mRNA transcript (4.5 kb) varied with the annual reproductive cycle (31), whereas exposure to exogenous E2 induced mRNA for both isoforms and P4 decreased their expression (32,33). A partial sequence for the alligator PR has also been reported (4), but the full coding region of a reptilian PR has not been reported to date. Like estrogens and androgens, P4 is found in the circulation of reptiles, especially during pregnancy or gravidity, and is derived primarily during this period from the corpus luteum or the endocrine placenta of some viviparous reptiles (34,35,36,37).

To further understand the molecular biology of the reptilian endocrine system as well as provide additional data on the evolution of the vertebrate steroid hormone receptors, we isolated cDNA clones encoding Pseudemys nelsoni homologs of ER, AR, and PR. We analyzed their phylogenic relationship with other known vertebrate receptors. Furthermore, the transactivation functions of ERα, AR, and PR were determined by expressing these receptors in transiently transfected culture cells using a general reporter gene assay and a modified GAL4 system.

Materials and Methods

Animals

An adult female turtle, red-belly slider (P. nelsoni) was obtained from Orange Lake, Alachua County, Florida. Additional embryonic tissues were obtained from P. nelsoni eggs obtained as part of larger research projects. Animals were overdosed with sodium pentobarbital and tissues obtained by sterile necropsy.

Chemical reagents

We obtained chemicals from Sigma-Aldrich Corp. (St. Louis, MO): 17β-trenbolone (Tre), 17α-methyltestosterone (MT), testosterone (T), 5α-dihydrotestosterone (DHT), E2, P4, corticosterone (Cor), diethylstilbestrol (DES), 17α-ethynylestradiol (EE2), estrone (E1), estriol (E3), 17α-hydroxypregnenolone (17OH-Preg), 17α-hydroxyprogesterone (17OH-Prog), pregnenolone (Preg), and 4-androstene-3,17-dione (AD). All chemicals were dissolved in dimethylsulfoxide (DMSO). The concentration of DMSO in the culture medium did not exceed 0.1%.

Molecular cloning of steroid hormone receptors

For ERα, two conserved amino acid regions in the DNA-binding domain (CAVCNDY) and the ligand-binding domain (MKCKNVV) of ERα were selected, and degenerate oligonucleotides were used as primers for PCR. As a template for PCR, the first-strand cDNA was synthesized from 2 μg total RNA isolated from the turtle liver. After amplification, an additional primer set, MCPATNQ and KCVEGMV, was used for second PCR.

Two conserved amino acid regions, GCHYGV and AGMVKP, of the PR were selected, and degenerate oligonucleotides were used as primers for PCR. First-strand cDNA was synthesized from 2 μg total RNA isolated from the liver of turtle after amplification, and an additional primer set, EEFLCM and EFPEMM, was used for second PCR.

We selected two conserved amino acid regions, AEGKQKY and KVKPIYFH, of the AR and produced degenerate oligonucleotides that were used as primers for PCR. As with the other receptors, the first-strand cDNA was synthesized from 2 μg total RNA isolated from the liver of the turtle. After amplification, a primer set, DCTIDKF and PEMMAEII, was used for second PCR.

The amplified DNA fragments were subcloned with TA-cloning plasmid pGEM-T Easy (Promega, Madison, WI), sequenced using a BigDye Terminator Cycle Sequencing kit (PE Biosystems, Foster City, CA) with T7 and SP6 primers, and analyzed on the ABI PRISM 377 automatic sequencer (PE Biosystems). The 5′- and 3′-ends of the ERα, PR, and AR cDNAs were amplified by rapid amplification of cDNA ends (RACE) using a SMART RACE cDNA amplification kit (BD Biosciences Clontech, Palo Alto, CA).

Construction of plasmid vectors

pcDNA3.1(+)-tERα (turtle ERα), pcDNA3.1(+)-tPR, and pcDNA3.1(+)-tAR were constructed by PCR amplification of the entire protein-coding region with KOD DNA polymerase (TOYOBO Biochemicals, Osaka, Japan). The PCR products were gel-purified and ligated into pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA). An estrogen-regulated reporter vector that had four estrogen-responsive elements (ERE) (GGTCAnnnTGACC), named pGL3-Basic-4xERE-tk-Luc, was constructed as described previously (5). An androgen- and progestin-regulated reporter vector named pGV2-MMTV was also constructed as described previously (38).

pBIND-tERs also were constructed by PCR amplification using 1) the full coding region (amino acids 1–587), 2) a deletion mutant-1 (amino acids 179–587), 3) a deletion mutant-2 (amino acids 245–587), and 3) a deletion mutant-3 (amino acids 344–587). All PCR products were ligated into the pBIND vector (Promega). pBIND-tAR-D and pBIND-tPR-D were constructed by PCR amplification of the C to F domains of each receptor, using the receptor terminology of Krust et al. (39).

Transactivation assays

HEK293 (for tERα) or HepG2 (for tAR and tPR) cells were cultured and seeded in 24-well plates at 5 × 104 cells per well in phenol-red-free DMEM for HEK293 or phenol-red-free MEM for HepG2 (Sigma) and supplemented with 10% charcoal/dextran-treated fetal bovine serum (Hyclone, South Logan, UT). After 24 h, the cells were transfected with 400 ng pGV2-MMTV for AR and PR or pGL3-Basic-4xERE tk-Luc for ER, 100 ng pRL-TK (as an internal control to normalize variation in transfection efficiency; contains the Renilla reniformis luciferase gene with the herpes simplex virus thymidine kinase promoter; Promega), and 200 ng pcDNA3.1-tERα, pcDNA3.1-tAR, or pcDNA3.1-tPR using Fugene 6 transfection reagent (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions. After 20 h of incubation, steroid hormones were introduced to the media. After an additional 24 h, the cells were collected, and the luciferase activity of the cells was measured by a chemiluminescence assay employing the Dual-Luciferase Reporter Assay System (Promega). Luminescence was measured using a Turner Designs Luminometer TD-20/20 (Promega). Promoter activity was calculated as firefly (Photinus pyralis) luciferase activity/sea pansy (R. reniformis) luciferase activity. All transfections were done at least three times, employing triplicate sample points in each experiment.

The analyses of the transcriptional activations of ERα, AR, and PR using the GAL4 system were performed as described previously (5). CHO-K1 cells were transfected with 100 ng pG5-luc and 100 ng pBIND-tERα, -tAR, or -tPR (containing the R. reniformis luciferase gene as a control for normal variation in transfection efficiency) using the Fugene 6 transfection reagent (Roche).

Database and sequence analysis

All sequences generated were searched for similarity using Blastn and Blastp on the web servers of the National Center of Biotechnology Information. Multiple sequence alignments and construction of unrooted phylogenetic trees were performed using the CLUSTALW and TREEVIEW programs (40).

Phylogenetic analysis

Phylogenetic trees were constructed by the PHYLIP program package (41). The predicted amino acid sequences were aligned using Clustal X (42) and were edited with minor manual rearrangements on MEGA program (43). Any gaps in aligned sequences were removed within the same receptor, but there were still some gaps in comparison among the full length of three receptors. In addition, there was no gap in comparison of hinge region and ligand-binding domain. The branch length (expected amino acid substitutions per site) was calculated by the maximal likelihood method with the JTT model using Protdist program in PHYLIP. The reliability of branching was estimated by the bootstrap resampling (1000 times) and the majority-rule consensus tree method using Seqboot, Protdist, and Consense programs in PHYLIP.

GenBank accession nos. for the predicted amino acid sequences of ERα are P81559 for Xenopus laevis, Q9YHT3 for Anolis carolinensis, BAB79437 for Aspidoscelis uniparens, BAD08348 for Alligator mississippiensis, BAB79436 for Caiman crocodiles, BAE45626 for Crocodylus niloticus, ABF20062 for Centropus grillii, Q91250 for Taeniopygia guttata, P06212 for Gallus gallus, and AF442965 for Coturnix japonica. In addition, the accession nos. for AR are AAC97386 for X. laevis, BAE95686 for A. mississippiensis, NP_001070156 for T. guttata, AAA17402 for Serinus canaria, BAE80463 for G. gallus, and BAD38679 for C. japonica. Moreover, the accession nos. for PR are AAH68635 for X. laevis, AAB35740 for A. uniparens, BAD08350 for A. mississippiensis, AAB81722 for Crocodylus siamensis, and P07812 for G. gallus.

Statistical methods

Results are presented as mean ± sem. Comparisons between two groups were performed using t test, and all multigroup comparisons were performed using ANOVA followed by Bonferroni. Software used was GraphPad Prism (version 4.0c; GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered statistically significant.

Results

Cloning, characterization, and transactivation of turtle ERα

Using standard PCR techniques described above, partial DNA fragments were amplified from turtle liver RNA. A DNA fragment was obtained, and sequence analysis showed that the fragment had similarity to ERα (data not shown). Using RACE, we were able to clone a full-length turtle ERα cDNA (GenBank accession no. AB301060, designated P. nelsoni ERα, tERα). The cDNA for tERα is composed of a predicted 587 amino acids with a calculated molecular mass of 66.4 kDa (Fig. 1A). Phylogenetic analysis indicates that tERα is more closely related to the tERα genes obtained from crocodilians and birds, than to the available sequences from other reptiles, such as lizards (Figs. 2A). In fact, comparison of sequences demonstrates strong sequence conservation among crocodilians and turtles with divergence in the squamates (lizards) (Fig. 2A). Using the nomenclature of Krust et al. (39), the tERα sequence can be divided into four domains based on its sequence homology to other steroid hormone receptors (Fig. 1B). If our turtle sequence is compared with four other available ERα sequences (human, chicken, American alligator, Xenopus), tERα shared 92–65% identity in the A/B domain (Fig. 1B). In contrast, the similarity to medaka (fish) ERα is significantly lower (37%). In general, the A/B domains of the fish ERs are different from other vertebrates. When tERα was compared with other domains of common vertebrates, we found a 100–96% homology in the C domain (DNA-binding domain), 92–37% homology in the D domain, 93–57% homology in the E/F domain (the ligand-binding domain) (Fig. 1B). Thus, domains C (DNA-binding domain) and E/F (ligand-binding domain) are highly conserved among all vertebrate ERs studied to date. Amino acid sequences of tERα and alligator ERα show an overall homology of 92%. The overall homologies of tERα with chicken, human, or Xenopus ERαs were 90, 78, and 77%, respectively.

Figure 1.

Figure 1

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The turtle ERα. A, The deduced amino acid sequence of tERα. Numbers on the side represent the position of amino acid residues in sequence. B, Comparison of tERα protein with ERs of several species (H, human; C, chicken; A, American alligator; X, X. leavis; M, medaka; GenBank accession nos. are: human ERα, P03372; chicken ERα, P06212; Xenopus ERα, P81559; medaka ERα, D28954; and American alligator ERα, BAD08348). The functional A/B to F domains are schematically represented with the numbers of amino acid residues indicated.

Figure 2.

Figure 2

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Phylogenetic trees of predicted amino acid sequences based on the hinge and ligand-binding regions of ERα (A), AR (B), or PR (C). The trees were constructed using the maximal likelihood method with the JTT model and the bootstrap resampling (1000 times). Branch lengths reflect estimated numbers of substitutions along each branch. The scale bar indicates 0.05 expected amino acid substitutions per site. The width of branches indicates reliability (bootstrap value) of tree branching.

In transactivation of ERs in mammalian cells, the ERs recognize a response element named ERE consisting of a palindromic repeat of the GGTCAnnnTGACC site (44) and induce a transcript of its downstream region (45). We examined the transcriptional activity of tERα using a reporter gene assay. E2 was effective in inducing luciferase activity. The significance of induction compared with vehicle treatment (P < 0.001) was found at 10−11 m E2. DHT also induced the transcriptional activity of tERα in a dose-dependent manner. However, the significance of induction was found at 10−6 m. No induction was found in the presence of Cor or P4 (Fig. 3A). We also observed that other estrogens, natural estrogens E1 and E3 and synthetic estrogens EE2 and DES, stimulated luciferase activity in a dose-dependent manner (Fig. 3B). EC50 was 7.113 × 10−12 m for E2, 3.665 × 10−10 m for E1, 1.761 × 10−10 m for E3, 3.206 × 10−12 m for EE2, and 8.366 × 10−12 m for DES. Relative potency was calculated and indicated that tERα was more sensitive to EE2 as compared with other estrogens. The relative potency of EC50 of EE2 was 2-fold higher than that of E2 (Table 1).

Figure 3.

Figure 3

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Transcriptional activities of tERα. (A) Transcriptional activities of tERα for various steroids. HEK293 cells were transiently transfected with the ERE-containing vector together with a tERα expression vector. Cells were incubated with increasing concentrations of E2 (10−16 to 10−7 m), DHT (10−15 to 10−5 m), P4 (10−15 to 10−5 m), and Cor (10−15 to 10−5 m). Data are expressed as a ratio of steroid to vehicle (DMSO). Each column represents the mean of triplicate determinations, and vertical bars represent the mean ± se. B, Dose-response profile of tERα activation by estrogens. HEK293 cells were transiently transfected with the ERE-containing vector together with a tERα expression vector. Cells were incubated with increasing concentrations of E1/E3 (10−15 to 10−5 m), and EE2/DES (10−16 to 10−7 m), Each point represents the mean of triplicate determinations, and vertical bars represent the mean ± se.

Table 1.

Gene transcriptional activities of estrogens mediated by tERα

tERα EC50 (m) 95% CIa (m) RPb (%)
E2 7.113 × 10−12 4.984 × 10−12 to 1.015 × 10−11 100
E1 3.665 × 10−10 1.827 × 10−10 to 7.353 × 10−10 1.9
E3 1.761 × 10−10 9.537 × 10−11 to 3.251 × 10−10 4.1
EE2 3.206 × 10−12 1.822 × 10−12 to 5.642 × 10−12 222.5
DES 8.366 × 10−12 5.357 × 10−12 to 1.307 × 10−11 85.3
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a

95% CI, 95% confidence intervals of EC50. 

b

RP, Relative potency = (EC50 E2/ EC50 chemical X) × 100. 

Cloning, characterization, and transactivation of a turtle AR

Using standard PCR techniques, we determined the full coding sequence of a turtle AR cDNA. The AR sequence contained an open reading frame encoding 790 amino acid residues [molecular mass, 86,109 Da, GenBank accession no. AB3011061, designated P. nelsoni AR (tAR)] (Fig. 4A). Other than this sequence, no full-length sequences of AR are available for reptiles, and our initial phylogenetic analysis supports that the AR of a bird is closely related to that of the turtle (Figs. 2B). Recently, the full sequence of chicken AR was reported. Its cDNA contains 703 amino acid residues, and we observed that tAR has an overall homology of 71% with chicken AR (46). Using the sequence information for the ligand-binding region alone (see Discussion), this conclusion is further supported, indicating that the turtle AR is more closely related to that of the crocodilians and birds than the squamates (Fig. 2B). Using neighbor-joining methods, we observed that tAR had an overall homology of 62.7% with chicken AR. The putative DNA-binding domain (C domain) and ligand-binding domain (E/F domain) of tAR showed high homology with those of other ARs (C domain, 100–87%; E/F domain, 97–68%) (Fig. 4B). The overall homology percentages of tAR were 55.1% (human AR), 48.7% (Xenopus AR), 29.8% (eel AR1), 8.0% (turtle ERα), and 24.4% (turtle PR; see below).

Figure 4.

Figure 4

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Nucleotide sequence and the deduced amino acid sequence (A) and structure comparison (B) with other species of tAR. A, The numbers on the right refer to the position of the nucleotides and the amino acids. The functional A/B to E/F domains of tAR is schematically represented with the numbers of amino acid residues. Percent homology of the domain relative to the turtle AR is depicted. E, Eel; H, human; T, turtle; X, Xenopus. GenBank accession nos. are: human AR, P10275; Xenopus AR, AAC97386; eel AR1, AB023960; and eel AR2, AB025361.

We examined the transcriptional activity of tAR using a reporter gene assay. DHT was effective in inducing luciferase activity. The significance of induction compared with vehicle treatment (P < 0.001) was found at 10−9 m DHT. E2 and P4 also induced the transcriptional activity of turtle AR in a dose-dependent manner. However, the significance of induction was found at 10−6 m for E2 (P < 0.001) and 10−6 m for P4 (P < 0.05). No induction was found in the presence of Cor (Fig. 5A). Next, we examined the dose-dependent activation by androgens T, MT, Tre, and AD and found that all four androgens stimulated luciferase activity through tAR in a dose-dependent manner (Fig. 5B). EC50 was 2.729 × 10−10 m for DHT, 2.412 × 10−10 m for MT, 2.097 × 10−9 m for T, 1.681 × 10−10 m for Tre and 2.434 × 10−8 m for AD. Relative potency was calculated and indicated that tAR was more sensitive to Tre as compared with other androgens. The relative potency of EC50 of Tre was 1.5-fold higher than that of DHT (Table 2).

Figure 5.

Figure 5

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Transcriptional activities of tAR. A, Transcriptional activities of tAR for various steroids. HepG2 cells were transiently transfected with the MMTV-luciferase vector together with a tAR expression vector. Cells were incubated with increasing concentrations of E2/P4/Cor (10−15 to 10−5 m) or DHT (10−17 to 10−7 m). Data are expressed as a ratio of steroid to vehicle (DMSO). Each column represents the mean of triplicate determinations, and vertical bars represent the mean ± se. B, Dose-response profile of tAR activation by androgens MT, Tre, AD, and T. Each point represents the mean of triplicate determinations, and vertical bars present the mean ± se.

Table 2.

Gene transcriptional activities of chemical mediated by tAR

tAR EC50 (m) 95% CIa (m) RPb (%)
DHT 2.729 × 10−10 1.371 × 10−10 to 5.433 × 10−10 100
MT 2.412 × 10−10 1.155 × 10−10 to 5.036 × 10−10 113.1
T 2.097 × 10−9 1.018 × 10−9 to 4.320 × 10−9 13.0
Tre 1.681 × 10−10 7.383 × 10−11 to 3.827 × 10−10 162.3
AD 2.434 × 10−8 1.601 × 10−8 to 3.700 × 10−8 1.1
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a

95% CI, 95% confidence intervals of EC50. 

b

RP, Relative potency = (EC50 DHT/ EC50 chemical X) × 100. 

Cloning, characterization, and transactivation of a turtle PR

The PR sequence contains a long open reading frame encoding 823 amino acid residues [molecular mass, 90,175 Da, GenBank accession no. AB3011062, designated P. nelsoni PR (tPR)] (Fig. 6A). Like tERα and tAR, the tPR is closely related in sequence to that of birds and crocodilians (Fig. 2C). The putative DNA-binding domain (C domain) and ligand-binding domain (E/F domain) of tPR showed high homology with those of other vertebrates (C domain, 98–83%; E/F domain, 94–70%) (Fig. 6B). The overall homology percentages of tPR were 48.3% (human PR), 58.7% (chicken PR), 45.7% (Xenopus PR), 34.0% (eel PR1), and 7.8% (turtle ERα).

Figure 6.

Figure 6

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Nucleotide sequence and the deduced amino acid sequence (A) and structure comparison (B) with other species of tPR. A, The numbers on the right refer to the position of the nucleotides and the amino acids. The functional A/B to E/F domains of tPR is schematically represented with the numbers of amino acid residues. Percent homology of the domain relative to the tPR is depicted. C, Chicken; E, Eel; H, human; T, turtle; X, Xenopus. GenBank accession nos. are: human PR, P06401; chicken PR, P07812; Xenopus PR, AAH68635; eel PR1, AB032075; and eel PR2, AB028024.

We examined the transcriptional activity of tPR using a reporter gene assay. All steroids, E2, DHT, P4 and Cor, induced the luciferase activity via tPR. The significance of induction compared with vehicle treatment (P < 0.001) was found at 10−9 m P4, 10−7 m Cor, 10−6 m DHT, and 10−5 m E2. EC50 of P4 was 3.781 × 10−10 m and 95% confidence intervals of EC50 was 2.618 × 10 to 5.462 × 10−10 m. Next, we examined the dose-dependent activation by 17OH-Preg, 17OH-Prog, and Preg and found that 17OH-Prog and Preg, but not 17OH-Preg, stimulated luciferase activity through tPR in a dose-dependent manner (Fig. 7B). The significance of induction was found at 10−7 m for 17OH-Prog (P < 0.001) and 10−8 m for Preg (P < 0.001).

Figure 7.

Figure 7

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Transcriptional activities of tPR. A, Transcriptional activities of tPR for various steroids. HepG2 cells were transiently transfected with the MMTV-luciferase vector together with a tPR expression vector. Cells were incubated with increasing concentrations of E2/DHT/Cor (10−15 to 10−5 m) or P4 (10−16 to 10−6 m). Data are expressed as a ratio of steroid to vehicle (DMSO). Each column represents the mean of triplicate determinations, and vertical bars represent the mean ± se. B, Dose-response profile of tPR activation by 17OH-Preg, 17OH-Prog, and Preg. Each point represents the mean of triplicate determinations, and vertical bars present the mean ± se.

Transactivation of ERα, AR, and PR using GAL4-assay system

Previously, we developed and reported an assay system for the measurement of transactivation of the giant salamander ER (5). This system is a modification of the mammalian two-hybrid assay and provides a simple method with high activity to examine the effect of steroids on transactivation of a receptor. We established this GAL4-assay system for tERα. The GAL4-DNA-binding domain construct was fused to the full coding region of tERα and was introduced into CHO-K1 cells. This system was then challenged by the addition of E2 to the culture medium. We found that E2 stimulated reporter gene activity in a dose-dependent manner (Fig. 8A). The significance of induction compared with vehicle treatment (P < 0.001) was found at 10−9 m E2. We also examined the domain requirements for the activation of the GAL4-assay system. Because the tERα sequence can be divided into four distinct domains based on its sequence homology relative to other steroid hormone receptors, we constructed a deletion series from the N-terminal domain: 1) the full-length (F) receptor, 2) deletion of the A/B domain (C), 3) deletion of the A/B and C domains (D), and deletion of the A/B, C, and D domains (E) (Fig. 8B). GAL4-tERα constructs were introduced into CHO-K1 cells, and then E2 was added to the culture media. We observed that E2 stimulated reporter gene activity of constructs F, C, and D in a dose-dependent manner, whereas E2-induced transcriptional activity of construct E, representing a deletion of the A/B, C, and D domains, was not observed (Fig. 8C). We found that only the ligand-binding domain (E/F domains) and hinge region (D domain) were required for induction of GAL4-ERα activity by E2. Furthermore, construct C, containing the C, D, and E/F domain of tERα, induced the greatest luciferase activity in response to E2 when compared with constructs F and D (Fig. 8C). Three natural estrogens (E1, E2, and E3) also induced transcriptional activity of the full GAL4-tERα construct in a dose-dependent manner (data not shown).

Figure 8.

Figure 8

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Transcriptional activities of tERα using GAL4 system. A, CHO-K1 cells were transiently transfected with the pBIND or pBIND-tERα together with pG5-luc vector. Cells were incubated with increasing concentrations of E2 (10−12 to 10−7 m). Each point represents the mean of the triplicate determinations, and vertical bars present the mean ± se. B, Schematic representation of tERα deletion mutants used in this study. C, Dose-response profile of tERα activation by E2. CHO-K1 cells were transiently transfected with the pG5-luc vector together with N-terminal deletion constructs of tERα expression vector. Cells were incubated with increasing concentrations of E2 (10−13 to 10−7 m). Each point represents the mean of the triplicate determinations, and vertical bars present the mean ± se.

Finally, we also used the GAL4 assay approach to examine transactivation by the tAR and tPR. The full-length tAR and tPR were fused to GAL4-DNA-binding domain in the pBIND vector. GAL4-tAR or -tPR constructs were introduced into CHO-K1 cells, and then DHT for tAR or P4 for tPR were added to the culture media. We observed that P4 stimulated reporter gene activity of GAL4-tPR in a dose-dependent manner, whereas DHT-induced transcriptional activity of GAL4-tAR was not observed (Fig. 9A). We found that luciferase activity of GAL4-tPR stimulated by P4 rose only 6-fold (Fig. 9A), whereas using a conventional murine mammary tumor virus (MMTV)-luciferase reporter assay system, we observed that P4-induced luciferase activity was over 80-fold (Fig. 7B). We changed the culture cells from CHO-K1 to HepG2, which are suitable for the conventional MMTV-luciferase reporter assay system, but could not detect elevated luciferase activity of the GAL4-tAR and GAL4-tPR constructs. Furthermore, we used deletion constructs that contain C, D, and E/F domains, as described above for tERα. Again, we could not detect reporter activity by steroid hormones of the GAL4-tAR and GAL4-tPR modified constructs.

Figure 9.

Figure 9

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Transcriptional activities of tAR and tPR. A, Dose-response profile of tAR and tPR activation. CHO-K1 cells were transiently transfected with the pG5-luc vector together with a tPR expression vector (tPR, •) or a tAR expression vector (tAR, ○). Cells were incubated with increasing concentrations of P4 for tPR or DHT for tAR (10−13 to 10−7 m). Each point represents the mean of the triplicate determinations, and vertical bars present the mean ± se. B, Transcriptional activity of tAR was determined in CHO-K1 cells transiently transfected with reporter MMTV-luc construct. After transfection, cells were incubated with (black bar) or without (white bar) 10−8 m DHT. Each point represents the mean of the triplicate determinations, and vertical bars present the mean ± se.

These results suggest that the GAL4-DNA-binding domain fused to tAR and tPR proteins may not interact with the ligand in an appropriate orientation or may not bind to coactivator(s) owing to unusual conformation changes. Although we saw very low transactivation with P4 in this system using a GAL4-tPR construct, we observed no activity with the GAL4-tAR construct in this system (Fig. 9A). Doesburg et al. (47) reported that the amino-terminal, transactivation domain and ligand-binding domain are necessary for the ligand-dependent transactivation of mammalian AR. GAL4-fusion AR may interrupt the interaction of transactivation domain and ligand-binding domain owing to unusual conformation change. The N-terminal region of tAR (tAR-N; 1–337) and C-terminal region of tAR (tAR-C; 407–790) were cotransfected into CHO-K1 cells with the pGV2-MMTV reporter construct. Separate expression of tAR-N or tAR-C did not result in androgen-induced luciferase activity, whereas coexpression of tAR-N and tAR-C resulted in androgen-induced luciferase activity. The significance of induction compared with vehicle treatment (P < 0.001) was found in coexpression of tAR-N and tAR-C (Fig. 9B). These results indicate that, like other mammalian AR studied to date, both the N-terminal transactivation domain and the ligand-binding domain are necessary for hormone-dependent tAR activation.

Discussion

Steroid hormone receptors are implicated in a wide array of reproductive activities in vertebrates, such as gonadal differentiation, maturation of the female and male reproductive tracts, and reproductive behaviors (48,49,50). In vertebrates, estrogens appear to induce both genomic and nongenomic cellular actions via nuclear and possibly G-coupled membrane receptors (49,51). To date, the characterization of reptilian nuclear steroid hormone receptors is rare. First, Young et al. (52) isolated partial DNA fragments of ER, PR, and AR from the unisexual whiptail lizard, Cnemidophorus uniparents, and examined the expression level of these receptors in oviduct. In 2000, the partial sequence of green anole, A. carolinensis, ERα was reported, and ligand interactions were examined (53). In 2001, the first full sequence for the reptilian nuclear ER was reported in caiman (C. crocodiles) and whiptail lizard (C. uniparens), and hormone-dependent transactivation was characterized (7). Since that time, additional sequences have been reported for two crocodilians (4,6). We report here the full sequence for ERα in a freshwater turtle, the red-belly slider (P. nelsoni). Partial sequences of reptilian AR and PR have also been previously reported (4,52,54,55), but full coding sequences for AR and PR in reptiles remained unknown until our current study. Although reptiles represent a basal lineage in the amniote vertebrates, relatively little is known concerning the molecular action of steroids in this group. A growing literature exists on sequence and evolutionary phylogeny for various vertebrate steroid receptors; however, few studies examine reptilian receptors and fewer still have examined hormone-induced transcriptional activity of these receptors with various known steroids so that cross-species comparisons can be made.

We cloned and sequenced a turtle ER that has strong sequence similarity to ERα genes reported from crocodilians and birds. Reptiles, like other amniote vertebrates, have two ERs belonging to the nuclear receptor family (4). Recent phylogenetic studies examining various mitochondrial and nuclear genes support the hypothesis that turtles are more closely related to Archosaurs, the birds, crocodilians, and extinct dinosaurs, than other reptiles. The Chelonia (turtles) are currently thought to have shared a common ancestor with crocodilians, unique to that of squamates. We observed that the DNA-binding region of ERα, the C region in the terminology of Krust et al. (39), was highly conserved, having 100% sequence similarity with the ERα cloned from humans, chicken, and alligator. These data suggest that the EREs in these diverse species are also likely to be highly conserved. Our data clearly support the concept that turtles represent a sister group to the crocodilians and, likely, the birds.

Although fewer data are available to produce a similar comparison for the AR and PR, our data again support the highly conserved sequence similarities among species of amniotes in the DNA- and ligand-binding regions of these receptors. Like ERα, the AR and PR of the turtle are closely related phylogenetically to similar receptors found in birds or crocodilians. Given the diverse roles of various progestogens among vertebrates, it is not surprising to see more variation in sequence similarities in the DNA and ligand-binding regions.

As with other vertebrates, turtles exhibit pronounced cycles in plasma concentrations of sex steroids associated with sexual maturation and reproductive cyclicity. Furthermore, pioneering work on environmental sex determination has been performed by two groups using freshwater turtles as a model system, and these studies have identified steroid hormones, principally E2, as major regulatory agents (10,11,56,57), as reported in other vertebrates. Unlike reports from mammals, estrogens play an important role in the development of the ovary in reptiles, especially those with environmental sex determination such as the American alligator (58) and freshwater turtles (11,56). Abnormalities in the development of the reproductive system, including complete sex reversal, have been reported after environmental or laboratory exposure to environmental estrogens in reptiles (59,60,61,62,63). Exposure to environmental estrogens also altered steroidogenesis and morphology of the ovary (63). It has previously been shown that a wide array of environmental contaminants have a potential to bind ERs from an array of vertebrates, including reptiles (64,65). E2 also displays a well-defined seasonal cycle in the plasma of reptiles, including turtles, and is associated with reproductive cyclicity, including reproductive tract proliferation and hepatic vitellogenesis (31,66). In turtle populations exposed to environmental contaminants, it has been reported that contaminant-exposed females had significantly reduced plasma E2 and vitellogenin concentrations when compared with less exposed reference populations (67). Additionally, exposed females displayed a normal hepatic vitellogenic response to exogenous E2 but exhibited significantly less responsiveness to exogenous gonadotropin, as measured by changes in plasma E2 concentration (68). More work needs to be done to characterize the expression of ERα and ERβ in turtle tissues and their relative roles in development and functioning of the reproductive system as well as the potential of this system to be disrupted by environmental contaminants with potential to act as ER ligands or antagonists.

Androgens are important steroid hormones for the expression of the male phenotype in all vertebrate species studied to date. They have characteristic roles during male sexual differentiation, development and maintenance of secondary male characteristics, and the initiation and maintenance of spermatogenesis (69). Differentiation of the masculine phenotype is directed by secretion of steroid hormones, usually androgens, such as T and DHT in most vertebrates studied to date (70,71). Many actions are initiated through binding and activation of the nuclear AR, a ligand-dependent transcription factor that belongs to the steroid nuclear receptor superfamily (72). Selcer et al. (27) reported the presence of AR in the oviduct of a freshwater turtle, T. scripta. They proposed that androgens could act in the female reproductive tract, as suggested previously for several mammalian species (73,74). Like the ER, AR is a target for an array of endocrine-disrupting chemicals, leading to a number of developmental defects of the male reproductive system, such as hypospadias, cryptorchidism, and altered semen quality (75). AR is also expressed in females and could act on the female reproductive system. Future work needs to further characterize the potential functions of androgens in female reptiles to determine whether contaminants with androgenic or antiandrogenic potential represent a reproductive health risk in females as they do in males.

P4, besides being a precursor for sex and stress steroids, also plays a role as a coordinator of all aspects of female reproductive activity in vertebrates, especially during pregnancy and gravidity. P4 is synthesized in the ovarian follicle before ovulation in reptiles and is secreted in large concentrations from the postovulatory follicle, the reptilian corpus luteum (35,36). Reptilian chorioallantoic placentae also have been suggested as a source of P4 during pregnancy in viviparous lizards (76). The physiological effects of P4 are mediated, in part, by interaction of the hormone with specific intracellular PRs that are members of the nuclear receptor superfamily of transcription factors. PRs are generally thought to be composed of three proteins, termed PR-A, PR-B, and PR-C, that are expressed from a single gene in rodents and humans as a result of transcription from alternative promoters (77,78,79). Two forms have been reported, to date, in the chicken with translation initiation at two alternative AUG initiation codons (30). In fish, two types of PR have been reported that were obtained from different genes (80). The observation of two different genes for PR in fish could be due, in part, to a gene duplication that occurred in teleost fishes and suggests that alternate isoforms could also be present that have not been isolated to date. We have cloned one cDNA for turtle PR. Although we did not obtain multiple chelonian PRs, previous studies have reported two or three mRNAs for PRs in the freshwater turtle C. picta (31). However, no full-length sequences for these isoforms of chelonian PR have been reported. The isoform we obtained is believed to be a splice variant of PR that is similar to the long-sequence PR-A reported for other species. This is supported by the sequence alignments provided in the phylogenetic analyses. The PR knockout mouse has demonstrated that P4 is essential for pregnancy-associated mammary gland ductal side branching as well as alveologenesis and that these morphological changes are dependent on P4-induced mammary epithelial proliferation (81,82). Furthermore, it is now well established that P4 plays a central role in the establishment and termination of uterine myometrial quiescence for pregnancy (77). The role of P4 has been studied in reptiles, and P4 appears to mediate (inhibitory) vitellogenesis and uterine contractions in turtles (83) as well as other reptiles (34,36).

In addition to the nuclear steroid receptors, the identities of the membrane receptors mediating the majority of rapid, cell surface-initiated, nongenomic steroid actions described to date are unclear in reptiles. Three novel seven-transmembrane G protein-coupled proteins, representing three distinct classes of steroid membrane receptors, a membrane PR and a membrane ER, GPR30, and a membrane AR have been identified in several vertebrate species (84,85). Recently, we have isolated the cDNAs for membrane PR and GPR30 from the American alligator and turtle (Katsu et al., in preparation). We are currently comparing and contrasting interactions of the nuclear and membrane types of steroid hormone receptors with the endogenous steroid hormones as well as environmental contaminants with known steroid actions.

Our current work was performed, in part, to develop in vitro assay techniques to study the basic endocrinology of the reptilians and other nonmammalian vertebrates as well as to determine whether these species were uniquely susceptible to the endocrine-disruptive effects of various environmental contaminants capable of acting as ER, AR, and PR agonists or antagonists. For example, recent studies have demonstrated that ERα from the Japanese medaka (Oryzias latipes) was more responsive to a number of environmental estrogens, such as nonylphenol, when compared with the response of mammalian ERα (86). Because the red-belly slider, P. nelsoni, lives in freshwater ecosystems, and numerous other species have been reported to respond to an array of environmental endocrine-disrupting contaminants (87), it is important to examine the ligand and species specificity of ERs, AR, and PRs in various aquatic species. Previously, a synthetic androgen, Tre, was shown to induce gonopodium differentiation from the anal fin of adult female and juvenile mosquitofish (88) and to act as a potent androgen in the fathead minnow (89). We have examined the transactivity of turtle AR and confirmed that Tre has strong androgenic activity at the level of receptor transactivation. In fact, Tre has more activity compared with the native steroid DHT (1.5-fold). Thus, future studies are needed to examine the basic regulation of ER, AR, and PR, in detail, as well as potential effects of environmental chemicals on the sex determination and growth of larvae or neonates in a wide array of species.

In summary, we cloned and sequenced turtle ERα, AR, and PR-A and characterized these receptors from a freshwater turtle, the red-belly slider (P. nelsoni). This is the first report of the full-sequence information and characterization of in vitro transcriptional activity by ligands of reptilian AR and PR. These data provide a useful approach for future studies examining the basic endocrinology of nonmammalian steroid hormone receptors. Reporter gene assays, using turtle ERα, AR, and PR genes, could provide an in vitro level screening tool for estrogenic or androgenic substances that could be followed by in vivo studies examining ovotestis formation, testis-ova induction, or sex conversion. Furthermore, these data help provide basic molecular data useful in examining the role of ER, AR, and PR in gonadal development and reproductive biology of reptiles. The almost global distribution of turtle makes this species an interesting biological model for assessing endocrine disruptors in a wide range of aquatic environments.

Acknowledgments

We thank colleagues in our laboratories, especially Ms. M. Hinago for technical assistance.

Footnotes

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology; grants from the Ministry of Environment, Japan (Y.K. and Ta.I); and grants to L.J.G. (UF Opportunity Fund, Homeland Foundation, NIEHS R21 ES014053-01A1).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 4, 2007

Abbreviations: AD, 4-Androstene-3,17-dione; AR, androgen receptor; Cor, corticosterone; DES, diethylstilbestrol; DHT, 5α-dihydrotestosterone; DMSO, dimethylsulfoxide; E1, estrone; E2, 17β-estradiol; E3, estriol; EE2, 17α-ethynylestradiol; ER, estrogen receptor; ERE, estrogen response element; MMTV, murine mammary tumor virus; MT, 17α-methyltestosterone; P4, progesterone; 17OH-Preg, 17α-hydroxypregnenolone; 17OH-Prog, 17α-hydroxyprogesterone; PR, progesterone receptor; Preg, pregnenolone; RACE, rapid amplification of cDNA ends; T, testosterone; Tre, 17β-trenbolone.

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