Evolutionary origins of the estrogen signaling system: Insights from amphioxus

https://doi.org/10.1016/j.jsbmb.2011.03.022Get rights and content

Abstract

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’.

Introduction

Based primarily on evidence from humans and laboratory mammals, it is well established that estrogens play a critical regulatory role in many different life processes beginning in early stages of embryogenesis. The term “estrogen” derives from its first perceived function as a female reproductive hormone, specifically associated with the period of sexual receptivity in female mammals (estrus = Latin oestrus meaning frenzy or gadfly). Although early investigators used the urine of pregnant women to isolate estrone, the first steroid found to have hormonal activity, subsequent studies soon reported the presence of estrogens and the biosynthesis of estradiol, estrone and estriol from small acyclic precursors in both males and females of a wide range of vertebrates from fish to mammals [1]. It is now generally accepted that estrogen not only is required for the normal growth, development and functioning of the reproductive system but also has a critical role in diverse other tissue types and organ systems, including brain, bone, skin, fat, cardiovascular and metabolic. Excesses or deficiencies of estrogen are associated with various pathological states, such as breast and prostate cancer and osteoporosis. Environmental chemicals that are estrogen-like in their bioactivity have been implicated in developmental abnormalities and endocrine-disrupting effects in humans and animals. Not surprisingly, factors and mechanisms regulating estrogen production and signal transduction continue to be a matter of intense research interest (reviewed by [2], [3]).

Classically, the estrogen signaling system has two core components: cytochrome P450 aromatase, 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 this viewpoint continues to serve as a valuable template for basic and clinical studies, advances in molecular endocrinology reveal that the complexity and diversity of estrogen physiology is accomplished by multiple signaling modes (endocrine, paracrine, autocrine/intracrine), as defined by the nature, proximity and topographical relationship of aromatase and ER expressing cells; two or more genetically distinct ER subtypes and multiple ER splice variants; diverse other classes of membrane- and nuclear-localized receptors; and an array of different cellular signal transduction pathways (genomic, nuclear-mediated; non-genomic/membrane-mediated) (see Section 1.2.1).

Fundamental questions remain regarding the evolution of the estrogen mediated signaling system. What are the evolutionary origins and molecular nature of the core components (aromatase and ER)? Which receptor signal transduction pathway is most ancient? Is the original messenger molecule the endogenously synthesized estrogen we know in vertebrates (estradiol, estrone)? Or did estrogen-like environmental molecules have the earliest signaling role? The basic anatomy, physiology and biochemistry of estrogen signaling have been extensively studied in representatives of all major groups of jawed vertebrates, signifying an ancient and evolutionarily conserved regulatory role. More recently, the structures and phylogenetic distribution of genes encoding aromatase (Fig. 1A [4], [5]) and ER (Fig. 1B [6], [7], [8], [9], [10]) have been documented, reinforcing the earlier work, but mechanistic details of estrogen-mediated signaling in organisms that predate the gnathostomes is not entirely clear. One approach to addressing the question is to study the closest invertebrate relatives of vertebrates and to determine precursors of vertebrate-specific molecules and pathways in these organisms. In addition to vertebrates, the phylum Chordata includes two invertebrate groups: urochordates (e.g., the ascidian Ciona intestinalis) and cephalochordates (e.g., the amphioxus of the genus Branchiostoma). In this paper, we briefly review the evolutionary history of the aromatase and ER genes, with a particular emphasis on insights from amphioxus and other invertebrates, and then present new analyses of aromatase and ER in amphioxus.

The critical enzyme for estrogen synthesis is aromatase, a member of the cytochrome P450 (CYP) superfamily of monooxygenase enzymes [11]. The membrane-associated aromatase complex catalyzes the transformation of androgens (androstenedione and testosterone) to estrogens (estradiol and estrone) and is the product of a single CYP19A1 gene in humans. Although most highly expressed in estrogen secreting glandular tissues, such as placenta and gonads, aromatase is expressed in a wide array of other tissue types: brain, fat, bone, pituitary in humans; brain, pituitary, retina in teleost fish. Of these, certain cell/tissue types are competent to transform acyclic precursors stepwise through cholesterol all the way to estrogen (ovary), whereas others are competent in the final aromatization step but are lacking one or more of the earlier enzymes in the steroidogenic pathway. Human placenta, for example, lacks C17,20 lyase (CYP17) and relies on androgen precursors supplied by the fetal adrenal for estrogen production.

The aromatase protein is monomeric and is anchored within the endoplasmic reticulum by a membrane-spanning region of the amino terminus [12], [13]. The crystal structure of the human aromatase protein has recently been determined [14]. The 503-residue polypeptide chain folds into 12 major α-helices and 10 β-strands and forms a heme group and adjacent steroid binding site near the geometric center of the protein [15]. This overall folding pattern is similar to other membrane-bound P450s, and several regions show strong sequence conservation including helices H–K, the aromatic region and especially the heme-binding region. Of the conserved helices, the “I-helix” is particularly important because it contains several hydrophobic residues that help to form the catalytic cleft and incorporates a key bend at Pro308 that provides additional space to accommodate a steroid substrate [15], [16].

Aromatase activity (for review [17]) and the CYP19 gene(s) have been well-documented in all major classes of gnathostome (jawed) vertebrates. The CYP19 gene has undergone independent duplications in several lineages, most notably the teleost fish [18], [19] and suiform mammals [20], [21]. Whereas the teleostean gene duplicates are thought to reflect a whole genome duplication event [22], the three CYP19 genes of pigs are the result of much more recent tandem duplication events. Duplicate aromatases retain the ability to synthesize estrogens but also exhibit functional differences. Within the teleost fish, duplicated CYP19 genes differ dramatically in their tissue expression patterns [19], [23] as well as in their relative affinity for different androgen and inhibitor substrates [24], [25] and inducibility by estrogens and xenoestrogens [18], [23], [26], [27]. Similarly, in suiform mammals, duplicated aromatase genes differ in expression patterns, substrate affinity and product formation [20], [21]. While humans possess only a single CYP19 gene, expression is regulated by 11 promoters and alternative first exons, which are used in a tissue specific manner [28], [29]. Along with the diverse roles played by estrogens, this complexity of aromatase regulation indicates the importance and richness of the estrogen signaling pathway.

Phylogenetic analyses of the CYP superfamily have not revealed close relationships of CYP19 with any other family members [4], [30]; thus, it is not currently possible to trace the origin of aromatase activity from ancestral CYPs that served other metabolic functions. CYP19 orthologs have recently been identified within amphioxus [4], [5]. However, CYP19 has not been identified within the sequenced genomes of urochordates, echinoderms, or protostomes, nor have they been identified outside of the bilaterian animals [31], [32]. Although we cannot rule out the possibility that a recognizable ancestral CYP19-like gene or CYP19 itself was secondarily lost in these groups, the cephalochordate lineage represents the earliest known occurrence of CYP19 to date. In addition to CYP19, amphioxus contains orthologs of other enzymes in the steroidogenic sequence leading to estrogen biosynthesis: CYP17, and 17β-hydroxysteroid dehydrogenase [5], [33]. In addition, amphioxus contains CYP11-like genes that, along with some uncharacterized cnidarian and placozoan CYPs, are positioned as an outgroup to the vertebrate CYP11 clade [5], [31]. CYP11A catalyzes cleavage of the side chain from the sterol D-ring; side chain cleavage by CYP11A (or a functional equivalent) is necessary for de novo synthesis of steroids. Because the catalytic activities of the amphioxus CYP11-like genes have not been determined and side-chain cleavage has not been documented, it remains unclear whether amphioxus can synthesize steroids from sterol precursors.

Measurements of steroidogenic activity using radiolabeled precursors and steroid-like immunoreactivity in amphioxus are consistent with the molecular studies described above. Aromatase activity in amphioxus was first demonstrated through the conversion of tritiated 19-hydroxyandrostenedione to estrone and estradiol by homogenates of body segments containing gonads [34]. Interestingly, activity was not detected in homogenates of brain or tail segments. Mizuta et al. [35] similarly measured estrogen synthesis by amphioxus ovarian homogenates and documented a suite of steroidogenic conversions. Estrogen synthesis primarily occurred in mature ovarian tissues prior to spawning. Estradiol-like, as well as progesterone- and testosterone-like molecules, have been quantified in amphioxus gonads using radioimmunoassay [5]. Similar to the patterns in aromatase activity, immunoactive estrogen was present in both ovaries and testes, but not in non-gonadal extracts, and concentrations in the ovary were greatest prior to spawning [5].

In vertebrates, the classical mechanism of estrogen signaling occurs through specific binding of estradiol to ERs, which are encoded by Esr genes. Within the nuclear receptor superfamily, the ERs form a family with two other receptor groups: the estrogen-related receptors (ERRs), and other vertebrate-type steroid receptors (SRs, which include androgen receptors, progesterone receptors, and corticoid receptors). The human genome contains two ERs, ERα (NR3A1, Esr1 [36]) and ERβ (NR3A2, Esr2 [37]), due to a duplication of the Esr gene early in the vertebrate lineage [38]. Unique among the vertebrates, however, teleost fish have one ERα but two ERβs (ERβa and ERβb).

Like other nuclear receptors, ERs have a modular structure divided into key functional domains (A–F) [39]. At the amino terminus, the A/B domains contain the ligand-independent AF-1 activation function [40]. The DNA-binding domain (DBD, C domain) is the most highly conserved region and contains two zinc fingers that enable binding of the ER to specific estrogen responsive elements (EREs) on the DNA. The hinge region (D-domain) has a more variable sequence, contains a nuclear localization signal, and enables synergism between the activation functions (AF-1 and AF-2) for full transcriptional activity [41]. At the amino terminus, the ligand binding domain (E/F) LBD is highly conserved, and serves to bind ligands, enable dimerization, recruit co-factors and stimulate transcription through the ligand-dependent AF-2 region.

In the absence of ligand, ERs generally occur in complexes with chaperones, such as Hsp90 [42]. Upon binding of estradiol or another agonist, ERs dissociate from the chaperones, form homo- or heterodimers [43], recruit cofactors, bind to DNA and modulate transcription of target genes. Utilization of multiple promoters and alternative splicing creates additional complexity in ER signaling. Eight promoters have been identified for human ERα and two for ERβ, which function in tissue-specific expression [44], [45], [46], [47]. Alternate splicing generates an exceptional number of ER isoforms lacking one or more functionally important domains; these variants differ in their expression patterns and functional properties [47]. For example, a human ERβ isoform (ERβcx) truncated at the C-terminus has been reported to heterodimerize with wild-type ERβ and function as a dominant negative [47], [48], [49].

In addition to modulating the activity of nuclear receptors, steroids can also stimulate rapid cellular responses which are mediated through membrane-bound receptors [50], [51]. With respect to estrogen signaling, rapid effects have been attributed to interactions with classical nuclear ERs that are localized within the cell membrane [52], [53], [54] as well as with GPR30, a G-protein coupled receptor [55]. To date, membrane-bound ERs have only been rigorously characterized in mammals and fish [56], [57]. Estrogens have been shown to exert similar rapid effects on cell signaling in molluscs [58]; however, the genes encoding membrane-bound ERs have not yet been identified in invertebrates, and it has not yet been demonstrated that estradiol is the endogenous activator of this receptor.

ERs have been identified and shown to be activated by steroidal estrogens in all classes of vertebrates, including the agnathan sea lamprey [6]. Among invertebrates, homologs to the ERs have been identified in amphioxus [7], [33] as well as in molluscs [9], [59] and annelids [10]. Previous phylogenetic analyses conducted using a variety of methods (parsimony, likelihood, Bayesian) have shown that chordate ERs (vertebrate and amphioxus) form a clade [7], [10] and that the protostome ERs (mollusc and annelid) comprise a sister group [9], [10]. In addition, Keay and Thornton [10] found that this bilaterian ER clade was supported as a sister group to the SRs. In their study, the position of the protostomes ERs was only moderately supported, but much of the observed uncertainty could be attributed to the effects of a long branch associated with the amphioxus SR.

As demonstrated by reporter assays in mammalian cell lines, ERs from amphioxus [6], [8], [60] and from molluscs [9], [59] are not activated by steroidal estrogens. In contrast, ERs from two annelid species bind estrogens with high affinity and activate transcription in response to low concentrations (EC50 < 10 nM estradiol) of estrogens [10], although it remains to be determined whether steroidal estrogens are physiological ligands for these annelid receptors. Based on phylogenetic patterns and reconstructions of predicted ancestral receptors, it has been hypothesized that the ancestral ER originated early in the bilaterian lineage and was activated by estrogens ([10], [61], but see also [6], [31]). One interpretation is that ER activation by estrogens was a property that was lost within the lineage leading to the cephalochordates and that the ER gene per se was lost from echinoderms, urochordates and several protostome lineages.

Within the large nuclear receptor superfamily (48 genes in human, 33 in amphioxus [33]), the ERs form a family (NR3A) with two other receptor groups: the estrogen-related receptors (ERRs, NR3B), and other steroid receptors (SRs, NR3C, which include androgen receptors, progesterone receptors, and corticoid receptors). Amphioxus has one representative gene in each of these three groups [7], [33]. As mentioned above, cell-based reporter assays indicate the amphioxus ER ortholog does not stimulate transcription of ERE-driven reporters or interact with the coactivator SRC-1 in response to estradiol. Somewhat surprisingly (but as hypothesized by Paris et al. [6]), reporter assays indicate that the amphioxus SR stimulates transcription through EREs and AREs (androgen-responsive elements) in response to estradiol and estrone [8], [60]. Amphioxus ER and SR share overlapping affinities for DNA binding sites, and reporter assays indicate that ER can competitively repress estradiol-induced signaling by SR [8] as well as by human ERα and ERβ [6]. Binding of ligands to amphioxus ER was not directly measured in these studies, but limited proteolysis assays suggested that the amphioxus ER is unlikely to bind estradiol or several other ligands for vertebrate ERs [6]. Cell-based reporter assays have been used to screen a variety of ligands (e.g., 3β-androstenediol, resveratrol, enterolactone, diethylstilbestrol [6]) for their ability to modulate signaling by amphioxus ER, but no functional ligands have been identified. Interestingly, although limited proteolysis assays suggested that the plasticizer bisphenol A can bind amphioxus ER, this ligand did not affect transactivation [6].

Bridgham et al. [8] noted that 11 of the 18 residues that line the ligand-binding pocket of human ERα are altered in amphioxus ER, but only 4 of 18 in amphioxus SR. Through comparison with the human ERα crystal structure, they identified two key substitutions likely to disrupt hydrogen bonding and packing interactions that would normally stabilize the ligand within the binding pocket in a trancriptionally active conformation. They then conducted site-directed mutagenesis, and experimentally demonstrated that the two substitutions (corresponding to amino acids 394 and 404 in the LBD of human ERα) are indeed sufficient to confer repressive activity on the SR.

As part of a long term program of research in this laboratory that focuses on the origin and evolution of estrogen signaling in vertebrates, we sought to obtain insights by studying aromatase and ER in amphioxus. Here we confirm and extend studies cited above, and present new information on CYP19 gene organization, including an in silico model of the aromatase protein.

Section snippets

Animals, treatments, and nucleic acid extraction

Amphioxus (Branchiostoma floridae) were purchased from Gulf Specimen Marine Lab (Panacea, FL). Animals were obtained in May, when adults were reproductively active and readily sexed by visualizing the gonads through the transparent body wall. Immediately upon receipt, animals were chilled to 4 °C on ice, sexed, and divided into cephalic (anterior to the gonads), caudal (posterior to the gonads), and central (gonad-containing) regions under a dissecting microscope as previously described [34].

Isolation of aromatase cDNA and sequence analysis

The assembled amphioxus CYP19 cDNA consensus sequence (GenBank accession number HQ010363) consisted of a single translation initiation site, a 1581 bp open reading frame (ORF) that encoded a predicted protein sequence of 527 aa, and 5′ and 3′ UTR of 5 and 1194 bp, respectively. The 3′-UTR terminated in a polyA tail. Compared with the in silico sequence initially reported by Nelson [65], our cloned sequence showed 13 overall residue substitutions and a 5 amino acid insertion at the boundary of

Conclusions and future perspectives

The basic requirements of a functional chemical signaling system are (a) a messenger molecule; (b) a cellular receptor for recognition and signal transduction; and (c) a biological response. Results presented here reinforce the view that the cephalochordate amphioxus has the ability to synthesize estrogen, and also has the core molecular elements of a classical vertebrate ER-mediated signal transduction pathway. While modeling and docking studies predict that amphioxus aromatase will bind

Acknowledgements

Supported by grants from the NIEHS P42 ES07381 (GVC, SV) and EPA (STAR-RD831301) (GVC), a Ruth L Kirschstein National Research Service Award (AT, F32 ES013092-01), an NIH traineeship (SS, SG), a NATO Fellowship (AN) and the Boston University Undergraduate Research Program (LC). The human ERαand 3xERE-TATA-luc reporter plasmids were generously provided by Dr. Donald McDonnell.

References (110)

  • D. Nelson

    Metazoan cytochrome P450 evolution

    Comp. Biochem. Physiol. C: Toxicol. Pharmacol.

    (1998)
  • G. Callard et al.

    In vitro conversion of androgens to estrogen in amphioxus gonadal tissues

    Gen. Comp. Endocrinol.

    (1984)
  • V. Kumar et al.

    Functional domains of the human estrogen receptor

    Cell

    (1987)
  • L. Tora et al.

    The human estrogen receptor has two independent non acidic transcriptional activation functions

    Cell

    (1989)
  • M. Kos et al.

    Upstream open reading frames regulate the translation of the multiple mRNA variants of the estrogen receptor alpha

    J. Biol. Chem.

    (2002)
  • I. Poola et al.

    Identification of twenty alternatively spliced estrogen receptor alpha mRNAs in breast cancer cell lines and tumors using splice targeted primer approach

    J. Steroid Biochem. Mol. Biol.

    (2000)
  • I. Poola et al.

    Expression of alternatively spliced estrogen receptor alpha mRNAs is increased in breast cancer tissues

    J. Steroid Biochem. Mol. Biol.

    (2001)
  • S. Hirata et al.

    Isoform/variant mRNAs for sex steroid hormone receptors in humans

    Trends Endocrinol. Metab.

    (2003)
  • I. Poola et al.

    Identification of ten exon deleted ERbeta mRNAs in human ovary, breast, uterus and bond tissues: alternative splicing pattern of estrogen receptor beta mRNA is distinct from that of estrogen receptor alpha

    FEBS Lett.

    (2002)
  • A. Pedram et al.

    A conserved mechanism for steroid receptor translocation to the plasma membrane

    J. Biol. Chem.

    (2007)
  • P. Thomas et al.

    Conserved estrogen binding and signaling functions of the G protein-coupled estrogen receptor 1 (GPER) in mammals and fish

    Steroids

    (2010)
  • Y. Pang et al.

    Involvement of estradiol-17beta and its membrane receptor, G protein coupled receptor 30 (GPR30) in regulation of oocyte maturation in zebrafish, Danio rario

    Gen. Comp. Endocrinol.

    (2009)
  • L. Canesi et al.

    Rapid effects of 17β-estradiol on cell signaling and function of Mytilus hemocytes

    Gen. Comp. Endocrinol.

    (2004)
  • T. Matsumoto et al.

    Oyster estrogen receptor: cDNA cloning and immunolocalization

    Gen. Comp. Endocrinol.

    (2007)
  • S. Greytak et al.

    Estrogen responses in killifish (Fundulus heteroclitus) from polluted and unpolluted environments are site- and gene-specific

    Aquat. Toxicol.

    (2010)
  • S. Greytak et al.

    Isolation and characterization of two cytochrome P450 aromatase forms in killifish (Fundulus heteroclitus): differential expression in fish from polluted and unpolluted environments

    Aquat. Toxicol.

    (2005)
  • S. Greytak et al.

    Cloning of three estrogen receptors (ER) from killifish (Fundulus heteroclitus): differences in populations from polluted and reference environments

    Gen. Comp. Endocrinol.

    (2007)
  • I. McDonald et al.

    Satisfying hydrogen bonding potential in proteins

    J. Mol. Biol.

    (1994)
  • M. Baker et al.

    Motif analysis of amphioxus, lamprey and invertebrate estrogen receptors: toward a better understanding of estrogen receptor evolution

    Biochem. Biophys. Res. Commun.

    (2008)
  • Y. Kazeto et al.

    The 5′-flanking regions of CYP19A1 and CYP19A2 in zebrafish

    Biochem. Biophys. Res. Commun.

    (2001)
  • A. Kamat et al.

    Mechanism in tissue-specific regulation of estrogen biosynthesis in humans

    Trends Endocrinol. Metab.

    (2002)
  • G. Callard et al.

    Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish

    J. Steroid Biochem. Mol. Biol.

    (2001)
  • D. Chen et al.

    Regulation of breast cancer-associated aromatase promoters

    Cancer Lett.

    (2009)
  • J. Elliston et al.

    Comparative analysis of estrogen receptors covalently labeled with an estrogen and an antiestrogen in several estrogen target cells as studied by limited proteolysis

    J. Steroid Biochem.

    (1988)
  • S. Chen et al.

    Positive and negative transcriptional regulation of aromatase expression in human breast cancer tissue

    J. Steroid Biochem. Mol. Biol.

    (2005)
  • L. Engel

    The biosynthesis of estrogen

  • R. Santen et al.

    History of aromatase: saga of an important biological mediator and therapeutic target

    Endocr. Rev.

    (2009)
  • N. Heldring et al.

    Estrogen receptors: how do they signal and what are their targets

    Physiol. Rev.

    (2007)
  • L. Castro et al.

    The genomic environment around the Aromatase gene: evolutionary insights

    BMC Evol. Biol.

    (2005)
  • T. Mizuta et al.

    Presence of sex steroids and cytochrome P450 genes in amphioxus

    Endocrinology

    (2007)
  • M. Paris et al.

    An amphioxus orthologue of the estrogen receptor that does not bind estradiol: insights into the estrogen receptor evolution

    BMC Evol. Biol.

    (2008)
  • M. Schubert et al.

    Nuclear receptor signaling in amphioxus

    Dev. Genes Evol.

    (2008)
  • J. Bridgham et al.

    Evolution of a new function by degenerative mutation in cephalochordate steroid receptors

    PLoS Genet.

    (2008)
  • J. Keay et al.

    The Octopus vulgaris estrogen receptor is a constitutive transcriptional activator: evolutionary and functional implications

    Endocrinology

    (2006)
  • J. Keay et al.

    Hormone-activated estrogen receptors in annelid invertebrates: implications for evolution and endocrine disruption

    Endocrinology

    (2009)
  • E. Simpson et al.

    Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis

    Endocr. Rev.

    (1994)
  • B. Amarneh et al.

    Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-direct mutagenesis

    Mol. Endocrinol.

    (1993)
  • D. Ghosh et al.

    Structural basis for androgen specificity and oestrogen synthesis in human aromatase

    Nature

    (2009)
  • M. Kishida et al.

    Distinct cytochrome P450 aromatase isoforms in zebrafish (Danio rerio) brain and ovary are differentially programmed and estrogen regulated during early development

    Endocrinology

    (2001)
  • A. Conley et al.

    Adaptive evolution of mammalian aromatases: lessons from Suiformes

    J. Exp. Zool. Part A: Ecol. Genet. Physiol.

    (2009)
  • Cited by (55)

    • Hypothalamic aging and hormones

      2021, Vitamins and Hormones
    • A critical evaluation of some of the recent so-called ‘evidence’ for the involvement of vertebrate-type sex steroids in the reproduction of mollusks

      2020, Molecular and Cellular Endocrinology
      Citation Excerpt :

      Although, in all these papers, the authors claimed that immunostaining was evidence of the presence of aromatase, one must take into account, firstly, that an aromatase gene has yet to be identified in the genome of any invertebrate (let alone any mollusks) and, secondly, that immunostaining (especially using polyclonal antibodies against mammalian proteins) is a highly unreliable procedure for identifying or localizing specific proteins in tissues (especially when applied to invertebrates). In conclusion, there is little or no evidence that CYP19A itself, or a homologue of CYP19A, is present in any animals other than vertebrates and their immediate ancestors (Fig. 2) (Callard et al., 2011; Di Cristo and Koene, 2017; Markov et al., 2009, 2017). If CYP19A is not present in mollusks, then what could be the explanation for the trace production of estrogens by mollusks (as in the in vivo study carried out by Hallmann et al., 2019)?

    View all citing articles on Scopus
    View full text