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Atlantic sea lamprey contains two corticoid receptors (CRs), CR1 and CR2, that are identical except for a four amino acid insert (Thr-Arg-Gln-Gly) in the CR1 DNA-binding domain (DBD). Steroids are stronger transcriptional activators of CR2 than of CR1 suggesting that the insert reduces the transcriptional response of lamprey CR1 to steroids. The DBD in elephant shark mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), which are descended from a CR, lack these four amino acids, suggesting that a CR2 is their common ancestor. To determine if, similar to lamprey CR1, the presence of this insert in elephant shark MR and GR decreases transcriptional activation by corticosteroids, we inserted these four CR1-specific residues into the DBD of elephant shark MR and GR. Compared to steroid activation of wild-type elephant shark MR and GR, cortisol, corticosterone, aldosterone, 11-deoxycorticosterone and 11-deoxycortisol had lower transcriptional activation of these mutant MR and GR receptors, indicating that the absence of this four-residue segment in the DBD in wild-type elephant shark MR and GR increases transcriptional activation by corticosteroids.

Atlantic sea lamprey contains two corticoid receptors (CRs), CR1 and CR2, that are identical except for a four amino acid insert (Thr-Arg-Gln-Gly) in the CR1 DNA-binding domain (DBD). Steroids are stronger transcriptional activators of CR2 than of CR1 suggesting that the insert reduces the transcriptional response of lamprey CR1 to steroids. The DBD in elephant shark mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), which are descended from a CR, lack these four amino acids, suggesting that a CR2 is their common ancestor. To determine if, similar to lamprey CR1, the presence of this insert in elephant shark MR and GR decreases transcriptional activation by corticosteroids, we inserted these four CR1-specific residues into the DBD of elephant shark MR and GR. Compared to steroid activation of wild-type elephant shark MR and GR, cortisol, corticosterone, aldosterone, 11-deoxycorticosterone and 11-deoxycortisol had lower transcriptional activation of these mutant MR and GR receptors, indicating that the absence of this four-residue segment in the DBD in wild-type elephant shark MR and GR increases transcriptional activation by corticosteroids., 18 pages, 3 figures. arXiv admin note: text overlap with arXiv:2210.04111

Lampreys are jawless fish that evolved about 550 million years ago at the base of the vertebrate line. Modern lampreys contain a corticoid receptor (CR), the common ancestor of the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), which first appear in cartilaginous fish, such as sharks. Until recently, 344 amino acids at the amino terminus of adult lamprey CR were not present in the lamprey CR sequence in GenBank. A search of the recently sequenced lamprey germline genome identified two CR sequences, CR1 and CR2, containing the 344 previously un-identified amino acids at the amino terminus. CR1 also contains a novel four amino acid insertion in the DNA-binding domain (DBD). We studied corticosteroid activation of CR1 and CR2 and found their strongest response was to 11-deoxycorticosterone and 11-deoxycortisol, the two circulating corticosteroids in lamprey. Based on steroid specificity, both CRs are close to elephant shark MR and distant from elephant shark GR. HEK293 cells transfected with full-length CR1 or CR2 and the MMTV promoter have about 3-fold higher steroid-mediated activation compared to HEK293 cells transfected with these CRs and the TAT3 promoter. Deletion of the amino-terminal domain (NTD) of lamprey CR1 and CR2 to form truncated CRs decreased transcriptional activation by about 70% in HEK293 cells transfected with MMTV, but increased transcription by about 6-fold in cells transfected with TAT3, indicating that the promoter has an important effect on NTD regulation of CR transcription by corticosteroids., 27 pages, 6 figures

[Image: see text] We investigated progestin and corticosteroid activation of the progesterone receptor (PR) from elephant shark, a cartilaginous fish belonging to the oldest group of jawed vertebrates. Comparison with the human PR provides insights into the evolution of steroid activation of the human PR. At 1 nM steroid, the elephant shark PR is activated by progesterone, 17-hydroxy-progesterone, 20β-hydroxy-progesterone, 11-deoxycorticosterone (21-hydroxyprogesterone), and 11-deoxycortisol. The human PR, in comparison, is activated at 1 nM steroid, only by progesterone and 11-deoxycorticosterone, indicating increased progestin and corticosteroid specificity during the evolution of the human PR. RU486, an important clinical antagonist of the human PR, did not inhibit progesterone activation of the elephant shark PR. Cys-528 in the elephant shark PR corresponds to Gly-722 in the human PR, which is essential for RU486 inhibition of the human PR. Confirming the importance of Cys-528 in the elephant shark PR, RU486 inhibited progesterone activation of the Cys528Gly mutant PR. To investigate the physiological relevance of Gly-722 in the human PR and Cys-528 in the elephant shark PR, we studied steroid activation of the Gly722Cys human PR and Cys528Gly elephant shark PR. Compared to the wild-type human PR, there was an increase in the activation of human Gly722Cys PR by11-deoxycortisol and a decrease in activation by corticosterone, which may have been important in selection for the mutation corresponding to the human glycine-722 PR that first evolved in the platypus PR, a basal mammal.

A distinct mineralocorticoid receptor (MR) ortholog first appears in cartilaginous fishes, such as sharks, skates, rays and chimaeras. Although aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, is a transcriptional activator of skate MR and elephant shark MR, aldosterone is not synthesized by cartilaginous fishes. Aldosterone, first appears in lungfish, which are lobe-finned fish that are forerunners of terrestrial vertebrates. Aldosterone activation of the MR regulates internal homeostasis of water, sodium and potassium, which was critical in the conquest of land by vertebrates. We studied transcriptional activation of the slender African lungfish (Protopterus dolloi) MR by aldosterone, other corticosteroids and progesterone and find that aldosterone, 11-deoxycorticosterone, 11-deoxycortisol and progesterone have half-maximal responses (EC50s) below 1 nM and are potential physiological mineralocorticoids. In contrast, EC50s for corticosterone and cortisol were 23 nM and 66 nM, respectively. Unexpectedly, truncated lungfish MR, consisting of the DNA-binding domain, hinge domain and steroid-binding domain, had a stronger response to aldosterone, other corticosteroids and progesterone than did full-length lungfish MR, indicating that an allosteric action of the N-terminal domain represses steroid activation of lungfish MR. This contrasts to human MR in which the N-terminal domain contains an activation function. BLAST searches of GenBank did not retrieve a GR ortholog, leading us to test dexamethasone and triamcinolone for activation of lungfish MR. At 10 nM, both synthetic glucocorticoids are about 4-fold stronger than 10 nM aldosterone in activating full-length lungfish MR, leading us to propose that lungfish MR also functions as a GR.

Naked mole-rats (Heterocephalus glaber) inhabit subterranean burrows in savannas and are, thus, unable to access free water. To identify their mechanism of osmoregulation in xeric environments, we molecularly cloned and analyzed the nuclear receptor subfamily 3 group C member 2 (NR3C2) gene encoding the mineralocorticoid receptor (MR), required for hormone-dependent regulation of genes contributing to body fluid homeostasis. Most vertebrates harbor a single MR homolog. In contrast, we discovered that MR is duplicated in naked mole-rats. The amino acid sequence of naked mole-rat MR1 is 90% identical to its mouse ortholog, and MR1 is abundantly expressed in the kidney and the nervous system. MR2 encodes a truncated protein lacking DNA- and ligand-binding domains of MR1 and is expressed in diverse tissues. Although MR2 did not directly transactivate gene expression, it increased corticosteroid-dependent transcriptional activity of MR1. Our results suggest that MR2 might function as a novel regulator of MR1 activity to fine-tune MR signaling in naked mole-rats.

We investigated progestin and corticosteroid activation of the progesterone receptor (PR) from elephant shark (Callorhinchus milii), a cartilaginous fish belonging to the oldest group of jawed vertebrates. Comparison with human PR experiments provides insights into the evolution of steroid activation of human PR. At 1 nM steroid, elephant shark PR is activated by progesterone, 17-hydroxy-progesterone, 20b-hydroxy-progesterone, 11-deoxycorticosterone (21-hydroxyprogesterone) and 11-deoxycortisol. At 1 nM steroid, human PR is activated only by progesterone and11-deoxycorticosterone indicating increased specificity for progestins and corticosteroids during the evolution of human PR. RU486, an important clinical antagonist of human PR, did not inhibit progesterone activation of elephant shark PR. Cys-528 in elephant shark PR corresponds to Gly-722 in human PR, which is essential for RU486 inhibition of human PR. Confirming the importance of this site on elephant shark PR, RU486 inhibited progesterone activation of the Cys528Gly mutant PR. There also was a decline in activation of elephant shark Cys528Gly PR by 11-deoxycortisol, 17-hydroxy-progesterone and 20b-hydroxy-progesterone and an increase in activation of human Gly722Cys PR by 11-deoxycortisol and decreased activation by corticosterone. One or more of these changes may have selected for the mutation corresponding to human glycine-722 PR that first evolved in platypus PR, a basal mammal.

Orthologs of human glucocorticoid receptor (GR) and human mineralocorticoid receptor (MR) first appear in cartilaginous fishes. Subsequently, the MR and GR diverged to respond to different steroids: the MR to aldosterone and the GR to cortisol and corticosterone. We report that cortisol, corticosterone and aldosterone activate full-length elephant shark GR, and progesterone, which activates elephant shark MR, does not activate elephant shark GR. However, progesterone inhibits steroid binding to elephant shark GR, but not to human GR. Together, this indicates partial functional divergence of elephant shark GR from the MR. Deletion of the N-terminal domain (NTD) from elephant shark GR (truncated GR) reduced the response to corticosteroids, while truncated and full-length elephant shark MR had similar responses to corticosteroids. Swapping of NTDs of elephant shark GR and MR yielded an elephant shark MR chimera with full-length GR-like increased activation by corticosteroids and progesterone compared to full-length elephant shark MR. Elephant shark MR NTD fused to GR DBD+LBD had similar activation as full-length MR, indicating that the MR NTD lacked GR-like NTD activity. We propose that NTD activation of human GR evolved early in GR divergence from the MR.

Sex steroid hormones including estrogens and androgens play fundamental roles in regulating reproductive activities and they act through estrogen and androgen receptors (ESR and AR). These steroid receptors have evolved from a common ancestor in association with several gene duplications. In most vertebrates, this has resulted in two ESR subtypes (ESR1 and ESR2) and one AR, whereas in teleost fish there are at least three ESRs (ESR1, ESR2a and ESR2b) and two ARs (ARα and ARβ) due to a lineage-specific whole genome duplication. Functional distinctions have been suggested among these receptors, but to date their roles have only been characterized in a limited number of species. Sexual differentiation and the development of reproductive organs are indispensable for all animal species and in vertebrates these events depend on the action of sex steroid hormones. Here we review the recent progress in understanding of the functions of the ESRs and ARs in the development and expression of sexually dimorphic characteristics associated with steroid hormone signaling in vertebrates, with representative fish, amphibians, reptiles, birds and mammals.

Deepwater Horizon spilled over 200 million gallons of oil into the waters of the Gulf of Mexico in 2010. In an effort to contain the spill, chemical dispersants were applied to minimize the amount of oil reaching coastal shorelines. However, the biological impacts of chemically-dispersed oil are not well characterized, and there is a particular lack of knowledge concerning sublethal long-term effects of exposure. This study examined potential estrogenic effects of CWAF, Corexit 9500-enhanced water-accommodated fraction of oil, by examining its effect on estrogen receptors and sex determination in the American alligator, Alligator mississippiensis. The alligator exhibits temperature-dependent sex determination which is modulated by estrogen signals, and exposure to 17β-estradiol (E2) and estrogenic compounds in ovo during the thermosensitive period of embryonic development can induce ovarian development at a male-producing temperature (MPT). CWAF induced transactivation up to 50% of the maximum induction by E2 via alligator estrogen receptors in vitro. To determine potential endocrine-disrupting effects of exposure directly on the gonad, gonad-adrenal-mesonephric (GAM) organ complexes were isolated from embryos one day prior to the thermosensitive period and exposed to E2, CWAF, or medium alone in vitro for 8–16 days at MPT. Both CWAF and E2 exposure induced a significant increase in female ratios. CWAF exposure suppressed GAM mRNA abundances of anti-Mullerian hormone (AMH), sex determining region Y-box 9, and aromatase, whereas E2 exposure suppressed AMH and increased Forkhead box protein L2 mRNA abundances in GAM. These results indicate that the observed endocrine-disrupting effects of CWAF are not solely estrogenically mediated, and further investigations are required.

Aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, first appears in lungfish, which are lobe-finned fish that are forerunners of terrestrial vertebrates. Aldosterone activation of the MR regulates internal homeostasis of water, sodium and potassium, which was critical in the conquest of land by vertebrates. We studied transcriptional activation of the slender African lungfish MR by aldosterone, other corticosteroids and progesterone and find that aldosterone, 11-deoxycorticosterone, 11-deoxycortisol and progesterone have half-maximal responses (EC50s) below 1 nM and are potential physiological mineralocorticoids. In contrast, EC50s for corticosterone and cortisol were 23 nM and 66 nM, respectively. Unexpectedly, truncated lungfish MR, consisting of the DNA-binding, hinge and steroid-binding domains, had a stronger response to corticosteroids and progesterone than full-length lungfish MR, indicating that the N-terminal domain represses steroid activation of lungfish MR, unlike human MR in which the N-terminal domain contains an activation function. BLAST searches of GenBank did not retrieve a GR ortholog, leading us to test dexamethasone and triamcinolone for activation of lungfish MR. At 10 nM, both synthetic glucocorticoids are about 4-fold stronger than 10 nM aldosterone in activating full-length lungfish MR, leading us to propose that lungfish MR also functions as a GR., 27 pages, 6158 words, 6 figures, 1 table

The mineralocorticoid receptor (MR) is descended from a corticoid receptor (CR), which has descendants in lamprey and hagfish, cyclostomes (jawless fish), a taxon that evolved at the base of the vertebrate line. A distinct MR and GR first appear in cartilaginous fishes (Chondrichthyes), such as sharks, skates, rays and chimeras. Skate MR has a strong response to corticosteroids that are mineralocorticoids and glucocorticoids in humans. The half-maximal responses (EC50s) for skate MR for the mineralocorticoids aldosterone and 11-deoxycorticosterone are 0.07 nM and 0.03 nM, respectively. EC50s for the glucocorticoids cortisol and corticosterone are 1 nM and 0.09 nM, respectively. The physiological mineralocorticoid in ray-finned fish, which do not synthesize aldosterone, is not fully understood because several 3-ketosteroids, including cortisol, 11-deoxycortisol, corticosterone, 11-deoxycorticosterone and progesterone are transcriptional activators of fish MR. Further divergence of the MR and GR in terrestrial vertebrates, which synthesize aldosterone, led to emergence of aldosterone as a selective ligand for the MR. Here, we combine sequence analysis of the CR and vertebrate MRs and GRs, analysis of crystal structures of human MR and GR and data on transcriptional activation by 3-ketosteroids of wild-type and mutant MRs and GRs to investigate the evolution of selectivity for 3-ketosteroids by the MR in terrestrial vertebrates and ray-finned fish, as well as the basis for binding of some glucocorticoids by human MR and other vertebrate MRs.

The progesterone receptor (PR) mediates progesterone regulation of female reproductive physiology, as well as gene transcription in non-reproductive tissues, such as brain, bone, lung and vasculature, in both women and men. An unusual property of progesterone is its high affinity for the mineralocorticoid receptor (MR), which regulates electrolyte transport in the kidney in humans and other terrestrial vertebrates. In humans, rats, alligators and frogs, progesterone antagonizes activation of the MR by aldosterone, the physiological mineralocorticoid in terrestrial vertebrates. In contrast, in elephant shark, ray-finned fishes and chickens, progesterone activates the MR. Interestingly, cartilaginous fishes and ray-finned fishes do not synthesize aldosterone, raising the question of which steroid(s) activate the MR in cartilaginous fishes and ray-finned fishes. The simpler synthesis of progesterone, compared to cortisol and other corticosteroids, makes progesterone a candidate physiological activator of the MR in elephant sharks and ray-finned fishes. Elephant shark and ray-finned fish MRs are expressed in diverse tissues, including heart, brain and lung, as well as, ovary and testis, two reproductive tissues that are targets for progesterone, which together suggests a multi-faceted physiological role for progesterone activation of the MR in elephant shark and ray-finned fish. The functional consequences of progesterone as an antagonist of some terrestrial vertebrate MRs and as an agonist of fish and chicken MRs are not fully understood. Indeed, little is known of physiological activities of progesterone via any vertebrate MR., 17 pages, 5 figures

During the Deepwater Horizon oil spill, vast quantities of a chemical dispersant Corexit 9500 were applied in remediation efforts. In addition to the acute toxicity, it is essential to evaluate Corexit further with a broader scope of long-term sublethal endocrine endpoints. The American alligator (Alligator mississippiensis) is an excellent organism for such an endeavor. It exhibits temperature-dependent sex determination, in which egg incubation temperatures during a thermosensitive period (TSP) in embryonic development determine the sex of embryos. Estrogen signals play a critical role in this process. For example, a single exposure to exogenous estrogen during the TSP overrides the effects of temperature and leads to skewed sex ratios. At a concentration of 100 ppm, Corexit significantly induced transcriptional activity of both alligator nuclear estrogen receptors 1 and 2 in vitro in reporter gene assays. To investigate the estrogenic effects of Corexit on gonadal development, alligator eggs were exposed to Corexit at environmentally relevant concentrations (0.25, 2.5 and 25 ppm) before the TSP in ovo. Exposure to Corexit at 0.25 and 25 ppm significantly delayed hatching and growth. Corexit exposure at any treatment level did not affect sex ratios or testicular mRNA abundance as measured at 1-week post-hatching, suggesting that the combination of Corexit components did not synergize enough to induce ovarian development in ovo. These results point to a need for further investigations on individual and combined components of Corexit to understand better their long-term effects on the development and reproductive health of alligators and other coastal aquatic wildlife.

One or more distinct forms of the nuclear estrogen receptor (ER) have been isolated from many vertebrates to date. To better understand the molecular evolution of ERs, we cloned and characterized er cDNAs from the inshore hagfish, Eptatretus burgeri, a modern representative of the most primitive vertebrates, the agnathans. Two er cDNAs, er1 and er2, were isolated from the liver of a reproductive female hagfish. A phylogenetic analysis placed hagfish ER1 into a position prior to the divergence of vertebrate ERs. Conversely, hagfish ER2 was placed at the base of the vertebrate ERβ clade. The tissue distribution patterns of both ER subtype mRNAs appeared to be different, suggesting that each subtype has different physiological roles associated with estrogen actions. An estrogen responsive-luciferase reporter assay using mammalian HEK293 cells was used to functionally characterize these hagfish ERs. Both ER proteins displayed estrogen-dependent activation of transcription. These results clearly demonstrate that the hagfish has two functional ER subtypes.

Steroid hormones are a key regulator of reproductive biology in vertebrates, and are largely regulated via nuclear receptor families. Estrogen signaling is regulated by two estrogen receptor (ER) subtypes alpha and beta in the nucleus. In order to understand the role of estrogen in vertebrates, these ER from various species have been isolated and were functionally analyzed using luciferase reporter gene assays. Interestingly, species difference in estrogen sensitivity has been noted in the past, and it was reported that snake ER displayed highest estrogen sensitivity. Here, we isolated additional ER from three lizards: chameleon (Bradypodion pumilum), skink (Plestiodon finitimus), and gecko (Gekko japonicus). We have performed functional characterization of these ERs using reporter gene assay system, and found high estrogen sensitivity in all three species. Furthermore, comparison with results from other tetrapod ER revealed a seemingly uniform gradual pattern of ligand sensitivity evolution. In silico 3D homology modeling of the ligand-binding domain revealed structural variation at three sites, helix 2, and juncture between helices 8 and 9, and caudal region of helix 10/11. Docking simulations indicated that predicted ligand-receptor interaction also correlated with the reporter assay results, and overall squamates displayed highest stabilized interactions. The assay system and homology modeling system provides tool for in-depth comparative analysis of estrogen function, and provides insight toward the evolution of ER among vertebrates.

The response to a panel of steroids by the mineralocorticoid receptor (MR) from Amur sturgeon and tropical gar, two basal ray-finned fish, expressed in HEK293 cells was investigated. Half-maximal responses (EC50s) for transcriptional activation of sturgeon MR by 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, cortisol and aldosterone, and progesterone were between 13 pM and 150 pM. For gar MR, EC50s were between 8 pM and 55 pM. Such low EC50s support physiological regulation by these steroids of the MR in sturgeon and gar. Companion studies with human MR and zebrafish MR found higher EC50s compared to EC50s for sturgeon and gar MR, with EC50s for zebrafish MR closer to gar and sturgeon MR than was human MR. For zebrafish MR, EC50s were between 75 pM and 740 pM; for human MR, EC50s were between 65 pM and 2 nM. In addition to progesterone, spironolactone and 19nor-progesterone were agonists for all three fish MRs, in contrast to their antagonist activity for human MR, which is hypothesized to involve serine-810 in human MR because all three steroids are agonists for a mutant human Ser810Leu-MR. Paradoxically, sturgeon, gar and zebrafish MRs contain a serine corresponding to serine-810 in human MR. Our data suggests alternative mechanism(s) for progesterone, spironolactone and 19nor-progesterone as MR agonists in these three ray-finned fishes and the need for caution in applying data for progesterone signaling in zebrafish to human physiology.

Estrogens regulate many physiological responses in vertebrates by binding to the estrogen receptor (ER), a ligand-activated transcription factor. To understand the evolution of vertebrate ERs and to investigate how estrogen acts in a jawless vertebrate, we used degenerate primer sets and PCR to isolate DNA fragments encoding two distinct ER subtypes, Esr1a and Esr1b from the Japanese lamprey, Lethenteron japonicum. Phylogenetic analysis indicates that these two ERs are the result of lineage-specific gene duplication within the jawless fishes, different from the previous duplication event of Esr1 (ERα) and Esr2 (ERβ) within the jawed vertebrates. Reporter gene assays show that lamprey Esr1a displays both constitutive and estrogen-dependent activation of gene transcription. Domain swapping experiments indicate that constitutive activity resides in the A/B domain of lamprey Esr1a. Unexpectedly, lamprey Esr1b does not bind estradiol and is not stimulated by other estrogens, androgens or corticosteroids. A 3D model of lamprey Esr1b suggests that although estradiol fits into the steroid binding site, some stabilizing contacts between the ligand and side chains that are found in human Esr1 and Esr2 are missing in lamprey Esr1b.

The mineralocorticoid receptor (MR) is a nuclear receptor and part of a large and diverse family of transcription factors that also includes receptors for glucocorticoids, progesterone, androgens, and estrogens. The corticosteroid aldosterone is the physiological activator of the MR in humans and other terrestrial vertebrates; however, its activator is not known in cartilaginous fish, the oldest group of extant jawed vertebrates. Here, we analyzed the ability of corticosteroids and progesterone to activate the full-length MR from the elephant shark (Callorhinchus milii). On the basis of their measured activities, aldosterone, cortisol, 11-deoxycorticosterone, corticosterone, 11-deoxcortisol, progesterone, and 19-norprogesterone are potential physiological mineralocorticoids. However, aldosterone, the physiological mineralocorticoid in humans and other terrestrial vertebrates, is not found in cartilaginous or ray-finned fish. Although progesterone activates MRs in ray-finned fish, progesterone does not activate MRs in humans, amphibians, or alligator, suggesting that during the transition to terrestrial vertebrates, progesterone lost the ability to activate the MR. Both elephant shark MR and human MR are expressed in the brain, heart, ovary, testis, and other nonepithelial tissues, suggesting that MR expression in diverse tissues evolved in the common ancestor of jawed vertebrates. Our data suggest that 19-norprogesterone- and progesterone-activated MR may have unappreciated functions in reproductive physiology.

The mineralocorticoid receptor (MR) and its kin, the glucocorticoid receptor (GR) evolved from an ancestral corticoid receptor (CR) in a cyclostome (jawless fish) through gene duplication and divergence. Distinct MR and GR orthologs first appear in cartilaginous fishes, such as sharks, skates, rays and chimaeras. Although aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, is not synthesized by cyclostomes or cartilaginous fishes, cyclostome CR and cartilaginous fish MR and GR are activated by aldosterone. Aldosterone first appears in lungfish, lobe-finned fish that are forerunners of terrestrial vertebrates. Further sequence divergence of the MR and GR in terrestrial vertebrates led to emergence of aldosterone as a selective ligand for the MR. Interestingly, ray-finned fish do not synthesize aldosterone, leaving the identity of their physiological mineralocorticoid(s) unresolved. Several steroids: cortisol, 11-deoxycortisol, corticosterone, 11-deoxycorticosterone and progesterone activate fish MR and are potential mineralocorticoids in ray-finned fish. Here we review the evolution of the MR in cartilaginous fish, terrestrial vertebrates and ray-finned fish, and discuss new insights into progesterone activation of the MR in ray-finned fish.

The mineralocorticoid receptor (MR) and its kin, the glucocorticoid receptor (GR) evolved from an ancestral corticoid receptor (CR) in a cyclostome (jawless fish) through gene duplication and divergence. Distinct MR and GR orthologs first appear in cartilaginous fishes, such as sharks, skates, rays and chimaeras. Although aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, is not synthesized by cyclostomes or cartilaginous fishes, cyclostome CR and cartilaginous fish MR and GR are activated by aldosterone. Aldosterone first appears in lungfish, lobe-finned fish that are forerunners of terrestrial vertebrates. Further sequence divergence of the MR and GR in terrestrial vertebrates led to emergence of aldosterone as a selective ligand for the MR. Interestingly, ray-finned fish do not synthesize aldosterone, leaving the identity of their physiological mineralocorticoid(s) unresolved. Several steroids: cortisol, 11-deoxycortisol, corticosterone, 11-deoxycorticosterone and progesterone activate fish MR and are potential mineralocorticoids in ray-finned fish. Here we review the evolution of the MR in cartilaginous fish, terrestrial vertebrates and ray-finned fish, and discuss new insights into progesterone activation of the MR in ray-finned fish.

Although multiple steroid ligands of the glucocorticoid, mineralocorticoid, and progestin families bind to and regulate the activity of mineralocorticoid receptors (MRs), the responses to these ligands differ across species. To understand how the different domains of MRs contribute to the ligand-induced activation or inhibition of MR activity, we studied the response to eight steroids (aldosterone, 11-deoxycorticosterone, 11-deoxycortisol, cortisol, corticosterone, progesterone, 19-norprogesterone, and spironolactone) of human, chicken, alligator, frog, and zebrafish full-length MRs and truncated MRs, which lacked the N-terminal domain (NTD) and DNA binding domain (DBD). Compared to full-length MRs, some truncated MRs were not activated by the steroids, and others required higher steroid concentrations for activation. Progesterone, 19-norprogesterone, and spironolactone did not activate full-length or truncated human, alligator, or frog MRs. However, at 10 nM, these steroids activated full-length chicken and zebrafish MRs, whereas at 100 nM, these steroids had little activity for truncated chicken MRs, but they retained activity for truncated zebrafish MRs. This suggests that regulation of the activation of the chicken MR by progestin resides in the NTD-DBD and that of the zebrafish MR resides in the hinge-LBD. Zebrafish and chicken MRs contain a serine corresponding to Ser810 in human MR, which is required for the antagonist activity of progesterone for human MR, suggesting a previously uncharacterized mechanism of regulation of progestin activation of chicken and zebrafish MRs. These findings suggest that progesterone may be a physiological activator of chicken and zebrafish MRs.

Androgens are essential for the development, reproduction, and health throughout the life span of vertebrates, particularly during the initiation and maintenance of male sexual characteristics. Androgen signaling is mediated by the androgen receptor (AR), a member of the steroid nuclear receptor superfamily. Mounting evidence suggests that environmental factors, such as exogenous hormones or contaminants that mimic hormones, can disrupt endocrine signaling and function. The American alligator (Alligator mississippiensis), a unique model for ecological research in that it exhibits environment-dependent sex determination, is oviparous and long lived. Alligators from a contaminated environment exhibit low reproductive success and morphological disorders of the testis and phallus in neonates and juveniles, both associated with androgen signaling; thus, the alterations are hypothesized to be related to disrupted androgen signaling. However, this line of research has been limited because of a lack of information on the alligator AR gene. Here, we isolated A mississippiensis AR homologs (AmAR) and evaluated receptor-hormone/chemical interactions using a transactivation assay. We showed that AmAR responded to all natural androgens and their effects were inhibited by cotreatment with antiandrogens, such as flutamide, p,p'-dichlorodiphenyldichloroethylene, and vinclozolin. Intriguingly, we found a spliced form of the AR from alligator cDNA, which lacks seven amino acids within the ligand-binding domain that shows no response to androgens. Finally, we have initial data on a possible dominant-negative function of the spliced form of the AR against androgen-induced AmAR.

Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p'-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research.

Cancer is as unique as the person fighting it. With the exception of a few biomarker-driven therapies, patients go through rounds of trial-and-error approaches to find the best treatment. Using patient-derived cell lines, we show that zebrafish larvae xenotransplants constitute a fast and highly sensitive in vivo model for differential therapy response, with resolution to reveal intratumor functional cancer heterogeneity. We screened international colorectal cancer therapeutic guidelines and determined distinct functional tumor behaviors (proliferation, metastasis, and angiogenesis) and differential sensitivities to standard therapy. We observed a general higher sensitivity to FOLFIRI [5-fluorouracil(FU)+irinotecan+folinic acid] than to FOLFOX (5-FU+oxaliplatin+folinic acid), not only between isogenic tumors but also within the same tumor. We directly compared zebrafish xenografts with mouse xenografts and show that relative sensitivities obtained in zebrafish are maintained in the rodent model. Our data also illustrate how

Two cDNAs of RXR were isolated, for the first time, from the ivory shell, Babylonia japonica, and the transcriptional activities were tested in vitro to compare with other gastropod (Thais clavigera and Nucella lapillus) RXR isoforms. The transcriptional activities of all of these RXR isoforms were significantly induced by mammalian RXR agonist, 9-cis retinoic acid (9cRA). The transcriptional activity of T. clavigera RXR-1 was also examined by using 9cRA and 16 organotin compounds, and significant ligand-dependent transactivations were observed by 9cRA and 5 organotins (tributyltin (TBT), tetrabutyltin (TeBT), tripropyltin (TPrT), tricyclohexyltin (TcHT) and triphenyltin (TPhT)). These 5 organotins also induced significant transcriptional activities in N. lapillus and B. japonica RXR isoforms. These 4 organotins, except for TeBT, have been reported to promote the development of imposex after a month of a single injection each, using female T. clavigera. To investigate the function of gastropod RXR isoforms, the effects of mammalian specific pan-agonist, PA024, and pan-antagonist, HX531, were examined, and significant induction of transcriptional activity by PA024 was demonstrated in these gastropod RXR isoforms. The additions of HX531 significantly suppressed the transcriptional activities of these gastropod RXR isoforms by 9cRA and 5 organotins. Using the mammalian two retinoic acid response elements, the transcriptional activities by 2 agonists, 9cRA and PA024, were different among the RXR isoforms of each gastropod species. With retinoid X response element (RXRE), transcriptional activities of TcRXR-1, BjRXR-1, and NlRXRa were significantly higher than those of TcRXR-2, BjRXR-2, and NlRXRb. Transcriptional activities of TcRXR-2, BjRXR-2, and NlRXRb, however, were significantly higher than those of TcRXR-1, BjRXR-1, and NlRXRa with thyroid hormone response element, TREpal. Thus, induction of imposex in prosobranch gastropods is strongly suggested to be triggered by 9cRA and certain organotins, such as TBT and TPhT through the activation of RXRs. These gastropod RXRs might control the different gene transcription by forming homo- or heterodimer complex with their own isoforms. These findings will contribute to our understanding of the fundamentals of the endocrine system in molluscs, particularly on RXR signaling pathway.

Contaminants of emerging concern (CECs) are ubiquitous in aquatic environments with well-established endocrine-disrupting effects. A data matrix of 559 water samples was queried to identify two commonly occurring CECs mixtures in Great Lakes tributaries. One mixture consisted of eight agricultural CECs (AG), while another contained 11 urban CECs (UB). The known estrogenic compounds bisphenol A, estrone and nonylphenol were present in both mixtures. According to the EPA Tox21 in ToxCast database, AG and UB mixture at an environmentally relevant concentration were estimated to account for 6.5% and 3.4% estrogenicity of the model endocrine disruptor estradiol-17β, respectively. Two isoforms of the estrogen receptor (Esr1 and -2, former Erα and Erβ) cloned from fathead minnow, bluegill sunfish, American alligator and human, responded differently to AG and UB mixtures. Human and bluegill Esr1 were the most sensitive to AG and UB mixtures, respectively. Fathead minnow Esr1 and Esr2b were the least sensitive to 10× AG and UB in estrogen dose equivalents, respectively. Even at environmentally documented concentrations, UB significantly activated bluegill Esr1. Moreover, 100× concentrated UB hyperstimulated fathead minnow Esr1 beyond the maximum induction of estradiol-17β. These results indicate that efficacious receptors and species differ in their response to CEC mixtures. Furthermore, estrogenicity may be present in some CECs not previously considered estrogenic, or, alternatively, estrogenicity of a mixture may be enhanced through chemical interactions. Our study highlights the need for further studies of CECs utilizing a variety of receptors cloned from diverse species.

The organotin compounds have a high affinity for the retinoid X receptor (RXR), which is a transcriptional factor activated by retinoids that induce imposex in gastropods. However, the molecular mechanisms underlying the regulation of RXR and its related genes in gastropods remain unclear. We isolated a retinoic acid receptor (RAR)-like cDNA (TcRAR) in the rock shell, Thais clavigera, and examined the transcriptional activity of the TcRAR protein by using all-trans retinoic acid (ATRA). However, we did not observe any ligand-dependent transactivation by this protein. We also examined the transcriptional activity of the TcRAR-ligand binding domain fused with the GAL4-DNA binding domain by using retinoic acids, retinol, and organotins and again saw no noteworthy transcriptional induction by these chemicals. Use of a mammalian two-hybrid assay to assess the interaction of the TcRAR protein with the TcRXR isoforms suggested that TcRAR might form a heterodimer with the RXR isoforms. The transcriptional activity of domain-swapped TcRAR chimeric proteins (the A/B domain of TcRAR combined with the D-F domain of human RARα) was also examined and found to be ATRA-dependent. These results suggest that TcRAR is not activated by retinoic acids, but can form a heterodimer with TcRXR isoforms. These data contribute to our understanding of the mechanism by which RXR functions in gastropods.

Steroid hormones are essential for health in vertebrates. Corticosteroids, for example, have a regulatory role in many physiological functions, such as osmoregulation, respiration, immune responses, stress responses, reproduction, growth, and metabolism. Although extensively studied in mammals and some non-mammalian species, the molecular mechanisms of corticosteroid hormone (glucocorticoids and mineralocorticoids) action are poorly understood in reptiles. Here, we have evaluated hormone receptor-ligand interactions in the American alligator (Alligator mississippiensis), following the isolation of cDNAs encoding a glucocorticoid receptor (GR) and a mineralocorticoid receptor (MR). The full-length alligator GR (aGR) and aMR cDNAs were obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequences exhibited high identity to the chicken orthologs (aGR: 83%; aMR: 90%). Using transient transfection assays of mammalian cells, both aGR and aMR proteins displayed corticosteroid-dependent activation of transcription from keto-steroid hormone responsive, murine mammary tumor virus promoters. We further compared the ligand-specifity of human, chicken, Xenopus, and zebrafish GR and MR. We found that the alligator and chicken GR/MR have very similar amino acid sequences, and this translates to very similar ligand specificity. This is the first report of the full-coding regions of a reptilian GR and MR, and the examination of their transactivation by steroid hormones.

Thyroid hormones are essential for the regulation of a wide range of biological processes associated with normal development and metabolism in vertebrates. For the screening of chemicals with a potential thyroid hormone and anti-thyroid hormone activities, we have established transient transactivation assay systems using thyroid hormone receptors (TRα and TRβ) from three frog species (Xenopus laevis, Silurana tropicalis and Rana rugosa), a fish (Oryzias latipes), an alligator (Alligator mississippiensis) and a human (Homo sapiens). In all species examined, similar transcriptional activities were found for triiodothyronine (T3 : 10(-11) M in TRα and 10(-10) M in TRβ) and thyroxine (T4 : 10(-9) M in TRα and 10(-8) M in TRβ). Analogs of thyroid hormone (3,5,3',-triiodothyroacetic acid and 3,3',5,5'-tetraiodothyroacetic acid) exhibited weaker activity, requiring 10-fold higher concentrations for induction of activity when compared with T3 and T4 . These results provide support for the usefulness of in vitro screening assay systems as part of an approach to test chemicals for potential thyroid hormone receptor activity. In addition, we observed that T3 -stimulated transcriptional activity of the O. latipes TRα was inhibited by 10(-5) M tetrabromobisphenol A (TBBPA). In contrast, TR antagonist activities on TRα were not encountered in other species, even with TBBPA concentrations at 10(-5) M. In vitro transactivation assay systems using TRs from various species can be used for the screening of chemicals with thyroid-receptor agonist and antagonist activities. They also can be used for studies that examine evolutionary differences among species in the potency of TR activation.

Proliferation and differentiation of cells in female reproductive organs, the oviduct, uterus and vagina, are regulated by endogenous estrogen. In utero exposure to a synthetic estrogen, diethylstilbestrol (DES), induces vaginal clear-cell adenocarcinoma in humans. In mice, perinatal exposure to DES results in abnormalities such as polyovular follicles, uterine circular muscle disorganization and persistent vaginal epithelial cell proliferation. We reported the persistent gene expression change such as interleukin-1 (IL-1) related genes, insulin-like growth factor-I (IGF-I) and its downstream signaling in the mouse vagina exposed neonatally to DES. In this study, we found persistent up-regulation of Wnt4 and persistent down-regulation of Wnt11 in the vagina of mice exposed neonatally to DES and estrogen receptor α specific ligand. Also Wnt4 expression in vagina is correlated to the stratification of epithelial cells with the superficial keratinization of vagina, but not epithelial cell stratification only.

The occurrence of oocytes in the testis (testis-ova) of several fish species is often associated with exposure of estrogenic chemicals. However, induction mechanisms of the testis-ova remain to be elucidated. To develop marker genes for detecting testis-ova in the testis, adult male medaka were exposed to nominal concentration of 100 ng L(-1) of 17α-ethinylestradiol (EE2) for 3-5 weeks, and 800 ng estradiol benzoate (EB) for 3 weeks (experiment I), and a measured concentration of 20 ng L(-1) EE2 for 1-6 weeks (experiment II). Histological analysis was performed for the testis, and microarray analyses were performed for the testis, liver and brain. Microarray analysis in the estrogen-exposed medaka liver showed vitellogenin and choriogenin as estrogen responsive genes. Testis-ova were induced in the testis after 4 weeks of exposure to 100 ng L(-1) EE2, 3 weeks of exposure to 800 ng EB, and 6 weeks of exposure to 20 ng L(-1) EE2. Microarray analysis of estrogen-exposed testes revealed up-regulation of genes related to zona pellucida (ZP) and the oocytes marker gene, 42Sp50. Using quantitative RT-PCR we confirmed that Zpc5 gene can be used as a marker for the detection of testis-ova in male medaka.

Environmental contaminant exposure can influence gonadal steroid signaling milieus; however, little research has investigated the vulnerability of non-steroidal signaling pathways in the gonads. Here we use American alligators (Alligator mississippiensis) hatched from field-collected eggs to analyze gonadal mRNA transcript levels of the activin–inhibin–follistatin gene expression network and growth differentiation factor 9. The eggs were collected from Lake Woodruff National Wildlife Refuge, a site with minimal anthropogenic influence, and Lake Apopka, a highly contaminated lake adjacent to a former EPA Superfund site. The hatchling alligators were raised for 13 months under controlled conditions, thus limiting differences to embryonic origins. Our data reveal sexually dimorphic mRNA expression in 13-month-old alligator gonads similar to patterns established in vertebrates with genetic sex determination. In addition, we observed a relationship between lake of origin and mRNA expression of activin/inhibin subunits α and βB, follistatin, and growth differentiation factor 9. Our study suggests that embryonic exposure to environmental contaminants can affect future non-steroidal signaling patterns in the gonads of a long-lived species.

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in vertebrates and promote the growth and differentiation of the female reproductive system. Importantly, environmental estrogens can influence the reproductive system and have been shown to disrupt gametogenesis in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor ligand interactions in the carp, Cyprinus carpio, a species used widely for both field- and laboratory-based studies, we cloned all three carp estrogen receptors (ER; ERα ,E Rβ1 and ERβ2) and applied an estrogen-responsive (ERE)- luciferase reporterassay systemto characterize theinteractions of thesereceptors with steroidalandsynthetic estrogens. DNA fragments encoding all three ERs in carp, ERα ,E Rβ1 and ERβ2, were obtained from the ovary using degenerate primer sets and PCR techniques, and full-length carp ER (cER) cDNAs were then obtained using RACE (rapid amplification of the cDNA end) techniques. Amino acid sequences of cERs showed overall homology of 46% (α vs β1), 49% (α vs β2) and 53% (β 1v sβ2). In the transient transfection ERE-luciferase reporter assay system (using mammalian cells) the cER proteins displayed estrogen- dependent activation of transcription and cERβ2 showed a higher sensitivity to the natural steroid oestrogen, 17β-estradiol, than cERα. The assay system developed is a powerful assay for toxicology and provides a tool for future studies examining the receptor-environmental chemical interactions and estrogen-disrupting mechanisms in carp. The data presented also expand our knowledge of estrogen receptor evolution. Copyright © 2011 John Wiley & Sons, Ltd.

The organotin compounds tributyltin (TBT) and triphenyltin (TPT) belong to a diverse group of widely distributed environmental pollutants that induce imposex in gastropods. These organotins have high affinity for retinoid X receptor (RXR), which is a transcription factor activated by retinoids, such as 9-cis retinoic acid (9cRA), in vertebrates. However, the molecular mechanisms underlying the regulation of RXR by retinoids and organotins have not been clarified in gastropods. We isolated two isoforms of RXR cDNAs, RXR isoform 1 (TcRXR-1) and RXR isoform 2 (TcRXR-2), in the rock shell Thais clavigera. The deduced amino acid sequences of TcRXR-1 and TcRXR-2 are highly homologous with those of other gastropods. These TcRXR isoforms displayed 9cRA-dependent activation of transcription in a reporter gene assay using COS-1 cells. The transcriptional activity of TcRXR-2, the encoded protein of which has five additional amino acids in the T-box of the C domain, was significantly lower than that of TcRXR-1. Decreases of the transcriptional activity by TcRXR-1 were observed when more than equal amount of TcRXR-2 fused expression vector was existed in a co-transfection assay. Immunoblot analysis showed several shifted bands for TcRXR isoforms resulting from phosphorylation. Mutation of potential phosphorylation sites from serine to alanine in the A/B domain of TcRXR-1 showed that, in the S89A/S103A mutant, there was a band shift and significantly higher transcriptional activity than in the controls when stimulated with 9cRA. Our findings could contribute to a better understanding of the role of interactions between RXR and retinoids and organotins, not only in the induction mechanism of imposex in gastropods but also in the endocrinology of mollusks.

The ability of targeted and nontargeted metabolomics to discover chronic ecotoxicological effects is largely unexplored. Fenitrothion, an organophosphate pesticide, is categorized as a "red list" pollutant, being particularly hazardous to aquatic life. It acts primarily as a cholinesterase inhibitor, but evidence suggests it can also act as an androgen receptor antagonist. Whole-organism fenitrothion-induced toxicity is well-established, but information regarding target and off-target molecular toxicities is limited. Here we study the molecular responses of male roach ( Rutilus rutilus ) exposed to fenitrothion, including environmentally realistic concentrations, for 28 days. Acetylcholine was assessed in brain; steroid metabolism was measured in testes and plasma; and NMR and mass spectrometry-based metabolomics were conducted on testes and liver to discover off-target toxicity. O-demethylation was confirmed as a major route of pesticide degradation. Fenitrothion significantly depleted acetylcholine, confirming its primary mode of action, and 11-ketotestosterone in plasma and cortisone in testes, showing disruption of steroid metabolism. Metabolomics revealed significant perturbations to the hepatic phosphagen system and previously undocumented effects on phenylalanine metabolism in liver and testes. On the basis of several unexpected molecular responses that were opposite to the anticipated acute toxicity, we propose that chronic pesticide exposure induces an adapting phenotype in roach, which may have considerable implications for interpreting molecular biomarker responses in field-sampled fish.

In fish, estradiol-17β (E2) regulates various reproductive processes by acting through estrogen receptors (ERs). Here, we cloned three ER subtypes from medaka and examined their developmental expression in the gonads and liver of genetically females and males from embryos to adults. During embryogenesis, marked increases in the expression of ERβ2, but not either ERα or ERβ1, were found in genetically female embryos during sex differentiation. E2 treatment induced marked up-regulation of ERβ2 expression in genetically male embryos. In adult ovaries, ERα levels were high in follicles (granulosa cells) during oocyte growth. In the testis, ERβ1 expression exhibited a distinct peak at 10 days post hatching (dph). In the liver, very high levels of ERβ2 were found in both females and males throughout the sampling period with significantly higher levels in females during 30-50 dph. These findings suggest each action of E2 to be mediated by different types of ERs.

Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERα and ERβ) from the Japanese firebelly newt (Cynops pyrrhogaster), Tokyo salamander (Hynobius tokyoensis), axolotl (Ambystoma mexicanum), and Raucous toad (Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5′ and 3′ rapid amplification of cDNA ends. The predicted amino acid sequences of these amphibian ERs showed a high degree of amino acid sequence identity (over 70%) to each other. We analyzed the relationships of these amphibian ER sequences to other vertebrate ER sequences by constructing a phylogenetic tree. We verified that these were bona fide estrogen receptors using receptor dependent reporter gene assays. We analyzed the effects of natural estrogens, ethinylestradiol, and DDT and its metabolites on the transactivation of the four amphibian species listed above, and Xenopus tropicalis ERs and found that there were species-specific differences in the sensitivity of these ERs to hormones and environmental chemicals. These findings will expand our knowledge of endocrine-disrupting events in amphibians.

Development of the reproductive organs can be strongly affected by the hormonal environment. In the mouse, exposure to estrogens and androgens during the critical developmental period induces estrogen-independent cell proliferation and differentiation in the adult vaginal epithelium, which often results in cancerous lesions later in life. In the present study, we assessed the contributions of estrogen receptor 1 (alpha) (ESR1) to the developmental effects of the nonaromatizable androgen 5alpha-dihydrotestosterone (DHT) on female mouse vagina and external genitalia. The vagina of Esr1(-/-) mice treated neonatally with DHT showed atrophic epithelium, whereas the vaginal epithelium of Esr1(+/+) mice was stratified and keratinized even after ovariectomy. In addition, neonatal treatment with DHT led to persistent phosphorylation of ESR1 in the vaginae of 60-day-old ovariectomized mice. We infer from these data that ESR1 is obligatory for the induction and maintenance of persistent vaginal epithelial changes induced by neonatal administration of DHT. Neonatal DHT treatment also induced hypospadias in both Esr1(-/-) and Esr1(+/+) mice. In contrast, DHT-induced formation of an os penis-like large bone in the clitoris was found in Esr1(-/-) mice but not in Esr1(+/-) or Esr1(+/+) mice. These results shed light on mechanisms of the induction of developmental effects elicited by sex steroid hormones on the developing animals.

Estrogens are necessary for ovarian differentiation during critical developmental windows in most vertebrates and promote the growth and differentiation of the adult female reproductive system. Estrogen actions are largely mediated through the estrogen receptors (ERs), which are ligand-activated transcription factors. To understand the molecular evolution of sex steroid hormone receptors, we isolated cDNAs encoding two steroid receptors from Japanese amphioxus, Branchiostoma belcheri: an ER ortholog and a ketosteroid receptor (SR) ortholog. Reporter gene assays revealed that the SR ortholog has molecular functions similar to those of the vertebrate ER. Surprisingly, the ER ortholog is an estrogen-insensitive repressor of SR-mediated transcription. Furthermore, we found that the SR ortholog can bind to both estrogen-responsive elements (EREs) and androgen-responsive elements (AREs) and mediates transcriptional activation by estrogens through both types of elements. Our findings suggest that the ancestral SR, but not ER, could bind estrone and induce the ERE- and ARE-dependent transactivation and that it gained the ability to be regulated by ketosteroid and recognize ARE specifically before jawless vertebrates split. These results highlight the importance of comparative experimental approaches for the evolutionary study of endocrine systems.