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Department of Medicine University of California, San Diego La Jolla, California 92093-0693
Address all correspondence and requests for reprints to: Michael E. Baker, Department of Medicine, 0693, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0693.
In the 1950s, Jensen’s laboratory began investigating the mechanism of action of estradiol, which lead to the identification of the estrogen receptor (ER) as a mediator of estrogen action (1). The 1970s witnessed the characterization of the other adrenal and sex steroid receptors: the progesterone receptor (PR) (2), the glucocorticoid receptor (GR) (3), the mineralocorticoid receptor (MR) (4), and the androgen receptor (AR) (5).
An important next step, determining the sequences of these steroid receptors, came more slowly because it was difficult to get sufficient purified protein for sequencing. This obstacle was resolved in the mid-1980s, when molecular techniques led to the cloning of the genes for adrenal and sex steroid receptors (2, 6, 7), which permitted an evolutionary analysis of their relationships with each other. Unexpectedly, steroid receptors were found to be related to the thyroid hormone receptor, retinoic acid receptor, and vitamin D receptor, all of which are now classified as belonging to the nuclear receptor family of transcription factors (8, 9, 10). Sequence analyses placed adrenal and sex steroid receptors on a branch separate from other nuclear receptors (9, 10, 11, 12). The AR, PR, GR, and MR cluster together, with the ER on a separate branch.
In the 1990s, further advances in cloning technology ushered in a Golden Age for evolutionary biology. It became possible to think about cloning entire genomes of bacteria, unicellular eukaryotes, and even multicellular animals and plants, which it was hoped would yield important medical and agricultural benefits, as well as providing information for understanding the evolution of life on earth. With an inventory of genes from bacteria, yeast, plants, flies, fish, amphibians, and mammals, among others, it might be possible to understand the transition from prokaryotes to eukaryotes, the origins of plants, multicellular animals, and vertebrates and the evolution of various signaling pathways, including that of steroids. Indeed, as gene sequences from diverse organisms became available, and newspapers increasingly contained articles on evolution, such as the relationship of humans to other primates, and the origins of eyes, Dobzhansky’s statement in 1973 that "Nothing in biology makes sense except in the light of evolution" (13) captured the imagination of biologists and the public at large.
As various genomes were sequenced, there were surprises. Steroid receptors and other nuclear receptors were not found in either yeast or plants, despite evidence that the ER (14) and GR (15) could function in yeast, and the GR could function in plants (16). These studies indicated that the transcriptional machinery for responding to nuclear receptors is present in yeast and plants, but nuclear receptors evolved later in multicellular animals. Another surprise was the absence of adrenal and sex steroid receptors in the fruit fly and roundworm, although both invertebrates have nuclear receptors. And, although distant ancestors of the ER have been found in octopus, snails, and other mollusks, these ERs are active in the absence of steroids and do not bind estradiol or other steroids (17, 18, 19, 20).
When did adrenal and sex steroids begin to regulate gene transcription in animals (11, 21)? Steroid receptors are found in vertebrates, including lamprey and hagfish (22, 23), which are jawless fish at the base of the vertebrate line (Fig. 1
). Is this when adrenal and sex steroids first began to regulate gene transcription, or was it earlier? Recently, the sea urchin genome, a basal deuterostome (Fig. 1), was sequenced and found to contain 33 nuclear receptors (24). None, however, were orthologs of a steroid receptor.
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). Amphioxus has a similar body plan as vertebrates, with the experimental advantage of a simpler anatomy and physiology than vertebrates. As a result, amphioxus is an important model organism for studying the evolution of development in vertebrates (25, 26), which led to selection of the amphioxus genome for sequencing. When the amphioxus genome is sequenced, it should answer many questions about when proteins involved in steroid hormone action evolved.
In this issue, Mizuta and Kubokawa (27) begin to answer some of these questions by providing sequences of three cytochrome P450 genes, CYP11A, CYP17, and CYP19, which they cloned from B. belcheri, a pacific amphioxus. These three proteins catalyze key steps in the synthesis of progesterone, testosterone, and estradiol from cholesterol (28, 29). CYP11A catalyzes the conversion of cholesterol to pregnenolone. CYP17 catalyzes the conversion of pregnenolone to 17
-hydroxypregnenolone, which CYP17 converts to dehydroepiandrosterone (DHEA), a precursor of androgens, estrogens and neurosteroids. CYP19 catalyzes the conversion of androstenedione and testosterone to estrone and estradiol, respectively. Thus, amphioxus has the CYP450s that are essential for the synthesis of three reproductive steroids: progesterone, testosterone, and estradiol. Mizuta and Kubokawa also cloned a 17ß-hydroxysteroid dehydrogenase, which could metabolize androstenedione and estrone to testosterone and estradiol, respectively. However, the gene for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), which converts pregnenolone to progesterone, and DHEA to androstenedione, was not cloned. Nor did they clone CYP21, which catalyzes the synthesis of glucocorticoids from progesterone and 17-hydroxyprogesterone.
Mizuta and Kubakawa (27) present other evidence for the presence of 3ß-HSD in amphioxus. They used a RIA to measure progesterone, testosterone, and estradiol from extracts of gonads from mature male and female amphioxus collected during the breeding season in July, for comparison with similar steroid extracts from amphioxus collected during the nonbreeding season in March. Ovaries contained substantially more estradiol and progesterone in July than in March. Interestingly, progesterone levels were over 2-fold higher in testes than in ovaries during the breeding season. Testosterone levels in testis were about 4% of the progesterone levels. The presence of estradiol and testosterone in amphioxus gonads indicates that DHEA was metabolized to androstenedione by an enzyme with 3ß-HSD activity, and that amphioxus contains an enzyme with 17ß-reductase activity. The same 3ß-HSD also metabolized pregnenolone to progesterone. In summary, Mizuta and Kubakawa provide strong evidence that the enzyme pathway for the synthesis of three steroids with reproductive activity in vertebrates is present in amphioxus. This contrasts with the tunicate, Ciona intestinalis, another protochordate that is close to vertebrates (Fig. 1), which lacks steroid receptors and CYP450s for steroid synthesis (30). Thus, amphioxus is the most ancient animal that is known to have CYP450 enzymes for synthesis of vertebrate sex steroids.
Mizuta and Kubakawa (27) open the door for wide range of studies to decipher the physiological roles of sex steroids in amphioxus and their mechanism of action. Exposure of amphioxus at different stages of development to progesterone, estradiol, or testosterone, as well as exposure to inhibitors of the three cloned CYP450s will provide important information on sex steroid action in amphioxus. This can yield new insights into the functions of sex steroids early in vertebrate evolution; some of these functions are likely to be conserved in mammals.
Of course, the next step is to clone the amphoxius steroid receptor gene(s). Indeed, an important question is whether amphioxus contains only one steroid receptor, which evolved into the full complement of adrenal and sex steroid receptors through a series of gene duplications (9, 11, 21, 22). If there is only one steroid receptor gene, is it an ER, as is implied from studies with lamprey (22)? Does this receptor only respond to estradiol or does it also respond to progesterone or other steroids (31, 32)? Or does progesterone in amphioxus activate a nuclear PR or a membrane PR (33) or both? Another puzzle is whether there is an amphioxus CYP21 that catalyzes glucocorticoid synthesis, or did this enzyme (and glucocorticoids) evolve later?
So there is much more to discover about the steroid response in amphioxus and the role of steroids in the evolution of vertebrates. Exciting discoveries, some with important clinical applications, will come from understanding the origins of steroid physiology in vertebrates. And as Mizuta and Kubakawa have demonstrated (27), we do not have to wait for a genome consortium to provide the relevant sequences.
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Acknowledgments |
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Footnotes |
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Abbreviations: AR, Androgen receptor; DHEA, dehydroepiandrosterone; ER, estrogen receptor; GR, glucocorticoid receptor; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; MR, mineralocorticoid receptor; PR, progesterone receptor.
Received April 26, 2007.
Accepted for publication May 8, 2007.
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