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. 2011 Nov;127(3-5):176-88.
doi: 10.1016/j.jsbmb.2011.03.022. Epub 2011 Apr 14.

Evolutionary Origins of the Estrogen Signaling System: Insights From Amphioxus

Free PMC article

Evolutionary Origins of the Estrogen Signaling System: Insights From Amphioxus

G V Callard et al. J Steroid Biochem Mol Biol. .
Free PMC article


Classically, the estrogen signaling system has two core components: cytochrome P450 aromatase (CYP19), the enzyme complex that catalyzes the rate limiting step in estrogen biosynthesis; and estrogen receptors (ERs), ligand activated transcription factors that interact with the regulatory region of target genes to mediate the biological effects of estrogen. While the importance of estrogens for regulation of reproduction, development and physiology has been well-documented in gnathostome vertebrates, the evolutionary origins of estrogen as a hormone are still unclear. As invertebrates within the phylum Chordata, cephalochordates (e.g., the amphioxus of the genus Branchiostoma) are among the closest invertebrate relatives of the vertebrates and can provide critical insight into the evolution of vertebrate-specific molecules and pathways. To address this question, this paper briefly reviews relevant earlier studies that help to illuminate the history of the aromatase and ER genes, with a particular emphasis on insights from amphioxus and other invertebrates. We then present new analyses of amphioxus aromatase and ER sequence and function, including an in silico model of the amphioxus aromatase protein, and CYP19 gene analysis. CYP19 shares a conserved gene structure with vertebrates (9 coding exons) and moderate sequence conservation (40% amino acid identity with human CYP19). Modeling of the amphioxus aromatase substrate binding site and simulated docking of androstenedione in comparison to the human aromatase shows that the substrate binding site is conserved and predicts that androstenedione could be a substrate for amphioxus CYP19. The amphioxus ER is structurally similar to vertebrate ERs, but differs in sequence and key residues of the ligand binding domain. Consistent with results from other laboratories, amphioxus ER did not bind radiolabeled estradiol, nor did it modulate gene expression on an estrogen-responsive element (ERE) in the presence of estradiol, 4-hydroxytamoxifen, diethylstilbestrol, bisphenol A or genistein. Interestingly, it has been shown that a related gene, the amphioxus "steroid receptor" (SR), can be activated by estrogens and that amphioxus ER can repress this activation. CYP19, ER and SR are all primarily expressed in gonadal tissue, suggesting an ancient paracrine/autocrine signaling role, but it is not yet known how their expression is regulated and, if estrogen is actually synthesized in amphioxus, whether it has a role in mediating any biological effects. Functional studies are clearly needed to link emerging bioinformatics and in vitro molecular biology results with organismal physiology to develop an understanding of the evolution of estrogen signaling. This article is part of a Special Issue entitled 'Marine organisms'.


Figure 1
Figure 1. Phylogenetic trees of (A) aromatase and (B) ER proteins
Trees were constructed to demonstrate the phylogenetic position of our amphioxus aromatase and ER sequences; topologies were consistent with previously published trees [5,7,31] and the evolutionary relationships among taxa [22]. GenBank Accession numbers are given parenthetically. (A) Deduced amino acid sequence of amphioxus aromatase was aligned with vertebrate CYP19 sequences and other representative CYP sequences. The maximum likelihood tree was rooted with the human CYP17 and CYP21 sequences (CYP Clan 2). Accession numbers: Amphioxus CYP19 (ABA47317.1) zebrafish CYP19a1/A (AA65788.1), zebrafish CYP19a2/B (AAK00642.1), killifish CYP19a1/A (AAR97268.1), killifish CYP19a2/B (AAR97269.1), Human CYP19 (NP_112503.1), mouse CYP19 (P28649.1), Human CYP17 (AAA36405; Human CYP21 (NP_000491). Numbers indicate percentage of 100 bootstrap replicates supporting each node. (B) Deduced amino acid sequence of amphioxus ER was aligned with vertebrate ER and ERR sequences, and a Neighbor-Joining tree was constructed. Protostomes (mollusc and annelid) ER sequences were not included in this analysis (see [10] for a thorough analysis of the evolutionary position of these genes). Amphioxus ER (EF 554313.1), teleost ERβb (zebrafish, NP_777287; goldfish, Q9IAL9), teleost ERβa (zebrafish, NP_851297; trout, CAC06714; goldfish, Q9W669; medaka, AAX14000; Salmon AAR92486), mammal ERβ (human, CAA67555; rat, U57439; mouse, AAB51132), Lamprey ER (AAK20929), teleost ERα (Medaka, P50241; Salmon, P50242; Trout, P16058; Goldfish ER, AAL12298; zebrafish, NP_694491; mammal ERα (Mouse, NP_031982; Rat, P06211), ERRs (Amphioxus ERR AAU88062; Human ERRα (NP_004442), ERRβ (O95718), and ERRγ (AAQ93381). Numbers on nodes indicate percentage of 1000 bootstrap replicates supporting each node, and triangles indicate nodes collapsed for simplicity.
Figure 2
Figure 2. Comparison of CYP19 genes in amphioxus, human and zebrafish
The genomic organization of the coding region of the single copy CYP19 gene in the human (A, upper panel; NM 000103.3; [108] is compared to that of amphioxus (HQ115077) and zebrafish CYP19a1(A) and CYP19a2(B) genes (NM 131154.2 and NM131642.1 [109]) (B, lower panel). Exons II – X are labeled in human (panel A) and correspondingly color coded in amphioxus and zebrafish panel B). The translation initiation (*) and the stop (°) codons are indicated. Note that the ovarian promoter/untranslated first exon of the human CYP19 (PII) is contiguous with exon II, whereas the placental (I.1) and brain (I.f) promoters and first exons are located ~93 kb and 33 kb upstream of the ATG in exon II. The untranslated first exon in amphioxus and zebrafish CYP19A1(A) is contiguous with exon II, while the untranslated first exon of zebrafish CYP19A2(B), like that of human I.f is further upstream. Also, the very long exon X (3'-untranslated region) of zebrafish CYP19A2(B) has a ~250-bp region deleted from the mRNA.
Figure 3
Figure 3. Sequence alignment of conserved functional domains of aromatases in amphioxus and representative vertebrates
Boundaries are as described by Simpson et al. [11] for amino acid residues of the human aromatase: I-helix 294–324: aromatic region 376–398: heme-binding domain 426–443). Identical and similar amino acid residues are marked by asterisks and dots, respectively. GenBank accession numbers given in Figure 1 legend or as follows: amphioxus (B. floridae, EF554313.1; B. belcheri, BAF61105.1), Dogfish (ABB53418.1), Killifish (CYP19A1(A): AAR97268, CYP19A2(B): AAR97269), Xenopus (BAA90529), Zebra finch (AAB32404.1), Turtle (AAG09376), Rat (NP_036885.1), Pig (AAB51387)..
Figure 4
Figure 4. Homology model of androstenedione docked within the active site of aromatase in (A) amphioxus and (B) human and plot of non-bonded interactions in (C) amphioxus and (D) human
See sections 2.3 and 3.5 for detailed methods and results describing evolutionarily conserved residues. Also, compare with conserved residues identified by sequence alignment (Fig. 3; section 3.3.2).
Figure 5
Figure 5. Functional comparison of amphioxus ER through binding experiments and cell-based reporter assays
Amphioxus estrogen receptor (BfER) and human estrogen receptor alpha (HsERα) constructs containing a V5 epitope tag were expressed in rabbit reticulocyte (A) and in COS-7 cells (A inset, B). (A) Tritiated estradiol was specifically bound by the human ERα (triangles) as expected, but not by the amphioxus ER (squares). Representative results from one of four independent experiments are shown. (inset) A western blot with a v5 antibody showing expression of BfER and ERα transfected into COS-7 cells. (B) Amphioxus ER (white bars), human ERα (grey bars), or an empty expression vector (pcDNA, black bars) were transfected into COS-7 cells along with a luciferase reporter driven by three estrogen responsive elements (see methods). The y-axis shows the ratio of luminescence by the luciferase reporter to luminescence by a transfection control reporter. Units are normalized such that the value for the DMSO-treated empty expression vector is equal to one.
Figure 6
Figure 6. Tissue-specific expression of (A) CYP19 and (B) ER mRNAs in amphioxus, as determined by semiquantitative RT-PCR analysis
Tissues were collected during the period of reproductive activity. H, head; T, testis; O, ovary; Ta, tail; C, control (no template). Products were separated on 1% agarose gels in 0.5X TBE, stained with ethidium bromide and visualized under ultraviolet light.

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