Evolution of Steroid-5Α-Reductases and Comparison of Their Function

General and Comparative Endocrinology 166 (2010) 489–497

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General and Comparative Endocrinology

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Review Evolution of steroid-5a-reductases and comparison of their function with 5b-reductase

Valérie S. Langlois a, Dapeng Zhang a, Gerard M. Cooke b, Vance L. Trudeau a,* a Centre for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ont., Canada K1N 6N5 b Toxicology Research Division, Health Canada, 251 Promenade Sir Frederick Banting Driveway Ottawa, Ont., Canada K1A 0K9 article info abstract

Article history: Steroid-5a-reductases (SRD5a) and steroid-5b-reductase (SRD5b) represent a convergence in evolution: Received 25 May 2009 they share similar biological functions, but do not have a common ancestor. In vertebrates, SRD5a and Revised 1 August 2009 SRD5b are involved in C-19 and C-21 steroid biosynthesis, bile acid biosynthesis and erythropoiesis. Accepted 11 August 2009 We compare and contrast the history, evolution, tissue distribution, enzyme characteristics and biological Available online 15 August 2009 functions of SRD5a and SRD5b and suggest possible future directions for research efforts. Both, the unique and overlapping roles that SRD5a and SRD5b play in steroid hormone metabolism, are indicated. Keywords: We also present the phylogeny of the SRD5a. The main SRD5a subfamilies obtained include, not only the 5a-Reductase type 1 well-known SRD5a type 1, type 2 and type 3, but also the synaptic glycoprotein (GPSN2)/trans-2,3-enoly- 5a-Reductase type 2 5a-Reductase type 3 CoA reductase group. Phylogenetic analysis indicated that a eukaryotic ancestor likely underwent 5b-Reductase duplication events to generate these three subfamilies (type 1/2, type 3 and GPSN2 ancestors); both Oxidoreductase SRD5a type 1/2 and GPSN2 subfamilies may have evolved by ancient duplication events at the early stage Aldo–keto reductase of vertebrate and chordate evolution. Steroid biosynthesis Ó 2009 Elsevier Inc. All rights reserved. 5a-DHT 5b-DHT Evolution Function

1. History reverse reactions. Originally, these enzymes were named D4-5a- hydrogenase and D4-5b-hydrogenase for their ability to hydroge- Major research efforts have been devoted to the study of ste- nate the D4 group (Dorfman and Forchielli, 1956). Nowadays, roid-5a-reductase (SRD5a) and the steroid-5b-reductase (SRD5b) SRD5a and SRD5b are currently known under many different in mammalian model organisms because of their implication in appellations (Table 1). Early studies also demonstrated the exis- numerous human diseases such as prostate carcinoma, benign tence of testosterone-reducing activity in liver slices of non-mam- prostate hyperplasia, male pattern baldness, pseudohermaphrod- malian species (Lisboa and Breuer, 1966; Lisboa et al., 1972). ism and hepatic dysfunction (Clayton et al., 1996; Kokontis and Chicken, trout, newt and frog livers catalyzed formation of 5a- Liao, 1999; Peters and Sorkin, 1993; Sinclair, 1998; Wilson et al., and 5b-metabolites after incubation with testosterone suggesting 1993); while little is still known in most non-mammalian species. that both isozymes were also present in those vertebrates (Lisboa The aim of this review is to compare and contrast the history of the and Breuer, 1966). During the same year, Ian Callard’s group re- discovery of SRD5a and SRD5b, their evolution, tissue distribution ported 5b-reduction activity in Rana pipiens ovaries (Callard and and function in vertebrates with a focus on testosterone reduction. Leathem, 1966). Steroid-5-reductases (SRD5a and SRD5b) were first discovered, In the early 1990s, two different types of SRD5a were identified. purified and characterized in rat liver homogenates (Dorfman and Comparative vertebrate studies identified differential sensitivities Forchielli, 1956; Okuda and Okuda, 1984; Samuels et al., 1950). of rat SRD5a and human SRD5a to a 4-azasteroid SRD5a inhibitor. These early experiments demonstrated that these enzymes were It was suggested that these differences were due to the existence of capable of reducing the D4 group of C-19 and C-21 steroids into two SRD5a isozymes (Andersson and Russell, 1990). Later, Jenkins 5a- and 5b-stereoisomers in vertebrates (for example, see Fig. 1). and colleagues (1992) reported that a major tissue difference in pH However, the SRD5a and SRD5b enzymes would not perform the optima existed between SRD5a enzymes from prostate and liver. The liver SRD5a activity was higher in an alkaline pH optimum (now known as the SRD5a type 1, SRD5a1); whereas the prostate * Corresponding author. Fax: +1 613 562 5486. SRD5 activity was favoured under acidic conditions (now known E-mail address: [email protected] (V.L. Trudeau). a

Fig. 1. Biochemical 5a-reduction and 5b-reduction reactions of different steroid biosynthesis pathways in vertebrates and plants.

Table 1 2. Sequence and evolutionary analyses of the SRD5a family List of synonyms of SRD5a and SRD5b steroid enzymes.

SRD5a SRD5b We first attempted to understand the evolutionary and func- EC 1.3.1.22 EC 1.3.1.23 tional relationships between the subtypes of SRD5a and the EC 1.3.99.5 EC 1.3.99.6 SRD5b. Although SRD5a and SRD5b have similar functions in 3-Oxo-5a-steroid-4-dehydrogenase D4-3-Ketosteroid-5b-reductase reducing testosterone, they do not have significant sequence simi- 4 3-Keto-D -steroid-5a-reductase 3-Oxo-5b-steroid-4-dehydrogenase larity (Table 2), they have different amino acid full coding se- 3-Oxosteroid-D-dehydrogenase Aldo–keto reductase-family-1- quences (Table 3) and are not evolutionarily related. SRD5b member-D1 4-Ene-3-ketosteroid-5a- D-(4)-3-Oxosteroid-5b-reductase belongs to an aldo–keto reductase (AKR) superfamily (pfam ID: oxidoreductase PF00248; Charbonneau and The, 2001), whereas SRD5a enzymes 5a-Reductase AKR1D1 form the oxidoreductase superfamily (pfam ID: PF02544; Jenkins 4 D -3-Ketosteroid-5a-reductase 5b-Reductase et al., 1991). Exhaustive phylogenetic analysis of the AKR super- Testosterone 5a-reductase Steroid-5b-reductase-b-polypeptide-1 Steroid-5b-reductase family has been published (Jez et al., 1997b; Jez and Penning, Testosterone-5b-reductase 2001) and SRD5b has been identified and sequenced in vertebrates, 4-Ene-5b-reductase, D(4)5b-reductase for example in human (Faucher et al., 2008) and in rat (Zhu et al., Cytosolic-4-ene-reductase 1996). In contrast, there are three known subtypes of SRD5a: type 1, type 2 and type 3, and another group of SRD5a-like proteins can as the SRD5a type 2, SRD5a2; Jenkins et al., 1992). Furthermore, SRD5a-deficient patients exhibiting male pseudohermaphrodite Table 3 phenotype lacked the SRD5a2 isoform, but exhibited SRD5a1 Amino acid full coding sequences of different vertebrates compared with the Homo sapiens SDR5a and SRD5b sequences. activity (Andersson et al., 1991). This confirmed the presence of two different forms of SRD5a in humans. More recently, with the Species Homo sapiens development of genome-wide gene expression profile analyses, a SRD5a1 SRD5a2 SRD5a3 SRD5b third type of SRD5a (SRD5a3) was identified in hormone-refrac- Homo sapiens 259 254 318 326 tory prostate cancer cells (HRPC, Tamura et al., 2007). The SRD5a3 Rattus norvegicus 255 254 278 326 is able to catalyze the conversion of testosterone into 5a-dihydro- Mus musculus 255 254 330 325 testosterone (5a-DHT) in HRPC cells, in a similar way to SRD5a1 Gallus gallus 239 255 310 448 Xenopus tropicalis 257 239 308 326 (Uemura et al., 2008). Taken together, these results supported Danio rerio 265 252 309 n/a the existence of three different types of SRD5a and only one sub- Average 255 251 310 342 type of SRD5b.

Table 2 Percentage of amino acid identity of different vertebrate sequences compared, for simplicity to only the Homo sapiens SDR5a and SRD5b sequences. Homo sapiens sequences are compared to each other with the compared enzyme in brackets. The online ClustalW2 software was used to calculate percent of homology (http://www.ebi.ac.uk/Tools/ clustalw2/).

Species Homo sapiens SRD5a1 SRD5a2 SRD5a3 SRD5b Homo sapiens 46 (SRD5a2) 25 (SRD5a3) 20 (SRD5a1) 4–7 (SRD5a1,2,3) Rattus norvegicus 60 77 77 79 Mus musculus 63 75 77 82 Gallus gallus 43 67 58 81 Xenopus tropicalis 51 54 56 80 Danio rerio 53 54 44 n/a Average 53 65 61 80 V.S. Langlois et al. / General and Comparative Endocrinology 166 (2010) 489–497 491 be identified by sequence searching using NCBI’s BLAST (http:// ote species and conducted the first phylogenetic analysis of the blast.ncbi.nlm.nih.gov/Blast.cgi), although no previous phyloge- SRD5a family (Fig. 2). The SRD5a family can be classified into three netic analysis has been attempted. These SRD5a-like proteins are main subfamilies: the SRD5a subfamily containing type 1 and type named differently, for example, glycoprotein synaptic 2 (GPSN2) 2 members (SRD5a1/2), the SRD5a type 3 subfamily and the GPSN2 in fish (Danio rerio; GI: 189516842 and 220672879), frogs (Silurana subfamily containing GPSN2 and GPSN2-like. All primary species tropicalis, GI: 60551245; Xenopus laevis, GI: 48231534), rat (Rattus (from plant, amoeba, yeast, to vertebrate) in Eukaryota contain all norvegicus; GI: 19924091) and human (Homo sapiens; GI: three subfamilies, indicating that the duplication event for generat- 24475816); glycoprotein synaptic 2-like (SPSN2-like) in fish (D. re- ing these subfamilies may have occurred in early Eukaryota. In con- rio; GI: 123232867); trans-2,3-enoyl-CoA reductase (TER) in fish trast, type 1 and type 2 SRD5a members (highlighted by green and (D. rerio; GI: 125853747); steroid-5a-reductase 2-like 2 in rat (R. orange lines in Fig. 2) may have evolved by a duplication event from norvegicus; GI: 157819815), steroid-a-reductase family member a chordate SRD5a1/2 ancestor. Another similar ancient duplication in nematoda (Caenorhabditis elegans; GI: 17531783); enoyl reduc- event also generated two types of GPSN2 subfamilies in vertebrates. tase or Tsc13p in yeast (Schizosaccharomyces pombe; GI: The GPSN2 and GPSN2-like families are highlighted by red and blue 19112157); 3-oxo-5a-steroid-4-dehydrogenase/fatty acid elong- lines in Fig. 2. Besides these, other additional lineage- or species- ase/trans-2-enoyl-CoA reductase in plants (Arabidopsis thaliana; specific duplications result in multiple copies of some members GI: 15233250). For the purpose of this review, we will refer to in different kingdoms (represented by bold black lines in Fig. 2), these various proteins as GPSN2 and GPSN2-like. for example, plants (Physcomitrella patens subsp. patens, A. thaliana), To elucidate the evolution of SRD5a, GPSN2 and GPSN2-like, we fungi (Aspergillus oryzae), protists (Entamoeba dispar, Trichomonas further collected SRD5a sequences from many of the main eukary- vaginalis), and Animalia (e.g., C. elegans).

Fig. 2. An unrooted phylogenetic tree of SRD5a family. PSI-BLAST (http://blast.ncbi.nlm.nih.gov/) was used to retrieve SRD5a homologous sequences in Eukaryota. The sequences are presented by its species abbreviation followed by GenBank GI ID. Sequence alignment was generated by Muscle program (Edgar, 2004). Maximum-likelihood analyses were performed with the PhyML program (Guindon and Gascuel, 2003; Guindon et al., 2005). The analyses used a JTT amino acid substitution model with a discrete gamma distribution (four categories) and 100 bootstraps were performed to assess the significance of phylogenetic grouping (Yang et al., 1998). Support values higher than 85% are shown next to their nodes. Colored segments present gene-specific duplication and bolded segments represent species-specific duplication. Abbreviations: Ag, Anopheles gambiae; Ao, Aspergillus oryzae; At, Arabidopsis thaliana; Bm, Brugia malayi; Cc, Coprinopsis cinerea; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Dr, Danio rerio; Dw, Drosophila willistoni; Ed, Entamoeba dispar; Eh, Entamoeba histolytica; Gg, Gallus gallus; Hs, Homo sapiens; Li, Leishmania infantum; Pp, Physcomitrella patens subsp. Patens; Pt, Paramecium tetraurelia; Rn, Rattus norvegicus; Sp, Schizosaccharomyces pombe; Tb, Trypanosoma brucei; Tg, Toxoplasma gondii; Tv, Trichomonas vaginalis; Xl, Xenopus laevis; Xt, Xenopus (Silurana) tropicalis. 492 V.S. Langlois et al. / General and Comparative Endocrinology 166 (2010) 489–497

expressed in the liver at birth followed by scalp and skin with development; whereas SRD5a2 is expressed earlier in the fetal genital skin and also later in skin and scalp (Thigpen et al., 1993). Studies in birds have also indicated the presence of SRD5a and SRD5b in early development (Freking et al., 1998; Grisham et al., 1997); although the types of SRD5a were not determined. In the Western clawed frog (S. tropicalis), mRNAs encoding SRD5a (type 1, type 2 and type 3) and SRD5b were all detected during embryogenesis (Langlois et al., 2008) and also follow multiphasic expression patterns. The presence of these enzymes in early vertebrate life suggests important developmental functions. Tissue distribution surveys have also established the presence of both SRD5a and SRD5b in adult vertebrates. Immunoblotting analysis identified SRD5a in many mammalian tissues such as Fig. 3. Fourth reaction of the fatty acid chain elongation reaction occurring in plants prostate, seminal vesicles, epididymis, skin, liver, fat, sebaceous and animals and requiring the action of GPSN2. gland, brain, scalp and hair follicles (Killinger et al., 1990; Nor- mington and Russell, 1992; Olsen et al., 2005; Thigpen et al., Common Ancestor 1993; Vallarino et al., 2005). Blot hybridization experiments indicate a relatively higher abundance of SRD5a mRNA in male rat liver compared to prostate tissue (Andersson et al., 1989) suggesting Synaptic differential enzyme activity among tissues. SRD5a activity was SRD5α1/2 SRD5α3 glycoproteins also reported in non-mammalian species. In fish and bird studies, SRD5a activity has been mostly reported in gonad, brain and liver tissues (Balthazart, 1991; Lisboa and Breuer, 1966; Pasmanik and SRD5α1 SRD5α2 SRD5α3 GPSN2 GPSN2-like Callard, 1985, 1988; Soma et al., 1999). Moreover, a SRD5a subtype tissue-specificity also occurs in ver- Fig. 4. Schematic of the SRD5a family phylogeny. This schematic is based on the tebrates. The activity of SRD5a1 is mostly high in none androgen proposed SRD5a phylogeny analysis. target tissues (e.g., skin); whereas SRD5a2 is mainly associated with androgen target tissues (e.g., prostate; Normington and Rus- While the functions of the GPSN2 subfamily are not fully under- sell, 1992; Thigpen et al., 1993). In contrast, the recently discov- stood, several reports have shown that GPSN2 members are in- ered SRD5a3 has been recognized as a ubiquitous enzyme in volved in the fourth reaction of fatty acid elongation by reducing mammals. Northern blot and real-time RT-PCR analyses have iden- a fatty chain double bond in fungi (Fig. 3; Han et al., 2002; Kohlw- tified this enzyme in both androgen and non-androgen target hu- ein et al., 2001; Paul et al., 2007), plants (Gable et al., 2004; Song man tissues such as pancreas, brain, prostate cancer cell lines, et al., 2009), and mammals (Moon and Horton, 2003). Although skin, and adipose tissue (Kazutoshi et al., 2008). The presence of the substrate (fatty acid) of GPSN2 members is structurally differ- SRD5a3 has also been reported in whole embryo and larvae bodies ent from that of other two SRD5a subfamilies (testosterone), all of the frog S. tropicalis (Langlois et al., 2008). three subfamilies of SRD5a share a similar biochemical activity A similar broad tissue distribution exists for SRD5b. This en- of reducing a double bond of substrate. This may reflect a divergent zyme has also been detected in vertebrate gonad, brain and liver evolution in activity of the protein families. tissues (Sugimoto et al., 1990). Brain SRD5b activity has been re- The inferred evolutionary history for the SRD5a family is pre- ported in birds, fish, hamster and human (Lisboa et al., 1974; Stei- sented in Fig. 4. We propose that the early eukaryotic ancestor mer and Hutchison, 1981; Terada et al., 1980; Yeung and Chan, underwent duplication events to generate three subfamilies (type 1985). In birds, the brain has been shown to be a major site of 1/2, type 3 and GPSN2). Thereafter, associated with speciation, 5b-androstane biosynthesis; where the main SRD5b metabolite de- three ancestors were duplicated into three subfamilies. At an early tected is 5b-androstanedione (Soma et al., 1999). 5b-Reductase stage of chordate and vertebrate evolution, ancestors of SRD5a activity was also reported in the gonads of human, bird and frog type 1/2 and GPSN2 subfamilies underwent duplication events, (Callard and Leathem, 1966; Charbonneau and The, 2001; Imataka which resulted in type 1 and type 2 within the type 1/2 subfamily et al., 1989). Despite the high occurrence of SRD5a, the liver also and in GPSN2 and GPSN2-like subfamily, in the GPSN2 subfamily. possesses high SRD5b activity in fish (trout, Lisboa and Breuer, 1966), amphibian (frog, Lisboa et al., 1972), bird (chicken, Sugimot- o et al., 1990), and mammals (human, Charbonneau and The, 2001; 3. Developmental profiles and tissue distributions of SRD5a and rat, Dorfman and Forchielli, 1956). SRD5b in vertebrates Species differences in tissue distribution of SRD5a and SRD5b have also been reported in amphibians. Lisboa et al. (1972) ob- Although SRD5a and SRD5b do not share a common evolution, tained higher percentages of liver 5b-reduced metabolites (5b- they do share similarities in biochemical functions. What about androstan-3a,17b-diol and 5b-androstan-3b,17b-diol) in X. laevis biological functions? Enzyme developmental profile and tissue than in Rana temporaria. This dissimilarity was explained by a distribution can give insight into biological functions. Steroid-5- divergence in evolution of hepatic steroid reduction. Similar spe- reductases have been identified during early vertebrate develop- cies differences have also been detected in frog ovaries. While R. ment. Viger and Robaire (1992) reported the pattern of expression pipiens produced mostly 5b-reduced steroids in the ovaries, only of SRD5a mRNA in rat epididymis during postnatal development, 5a-reduced metabolites were identified in R. temporaria ovaries SRD5a mRNA levels increase from several days post-birth and pla- (Ozon et al., 1964). Dorfman and Forchielli (1956) have suggested teau after about 2 months. This increase was explained as a conse- a possible co-inhibition mechanism occurring between both iso- quence of the state of epithelial cell differentiation (Viger and zymes, since SRD5b reduction of radiolabelled steroids is inhibited Robaire, 1992). Immunoblotting experiments also reported SRD5a by SRD5a activity, possibly through testosterone-substrate compe- protein in newborn humans. It was demonstrated that SRD5a1is tition. This complicates the analysis of 5a- and 5b-reduction V.S. Langlois et al. / General and Comparative Endocrinology 166 (2010) 489–497 493 systems since both enzymes are often present in same tissues. New rat (Azzolina et al., 1997; Levy et al., 1995). This partly explains experiments are needed to determine and validate whether sub- the use of the dog over the rat as a model for human prostate strate competition occurs between these two enzymes or if a true SRD5a inhibitor trials. species difference in SRD5a and SRD5b activities is present in SRD5b is a member of the AKR superfamily because of its sim- amphibians. ilarity with 3a-hydroxysteroid dehydrogenase and 17b-hydroxysteroid dehydrogenase (Charbonneau and The, 2001; Jez et al.,

1997a). The AKRs are monomeric (a/b)8-barrel proteins and are 4. Vertebrate enzyme characteristics an ancient superfamily of enzymes since they have been identified from archaebacteria to vertebrates (for review, Jez et al., 1997a). Rat and human SRD5a1 and SRD5a2 contain 5 exons and 4 in- AKRs are NADP(H)-dependant and share a conserved nicotin- trons (Labrie et al., 1992). Russell and Wilson (1994) suggested a amide-cofactor-binding pocket (Jez et al., 1997a). It has been sug- primordial gene duplication event of those two enzymes after they gested that a single amino acid change in the AKR active site may determined that the intron positions were the same in the two iso- modify the catalysis from a carbonyl oxidoreduction, for example zymes. This is confirmed with our SRD5a phylogenetic analysis the hydroxysteroid dehydrogenase function, to a carbon–carbon (Fig. 2). In human, SRD5a1 is located on chromosome 5p15 double-bond reduction, for example the SRD5b function (Jez whereas SRD5a2 is on 2p22 (Jenkins et al., 1991). Humans also car- et al., 1997a). SRD5b catalyzes double-bond reduction by transfer- ry a pseudogene which possesses a nonsense mutation at amino ring a hybrid ion located on the NADPH 4-pro-R position to the 5b- acid 147 rendering it non-functional (Shimazaki, 2002). Charbon- position on its steroid substrate (Berseus and Bjorkhem, 1967) and neau and The (2001) presented the genomic structure of human addition of a proton to the 4a-position (Bjorkhem, 1969). Mouse SRD5b. The SRD5b gene contains 9 exons and 7 large introns. This adrenal tissue characterization determined that SRD5b pH opti- gene is the longest of this family, it possess especially large introns. mum was 6.5 (Collins and Cameron, 1975). Steroid-5b-reductase Future studies have to elucidate which amino acids are essential can catalyze the reduction of the D4 group in testosterone, proges- for the 5b-reduction and to explain the presence of long introns terone, 4-androstenedione, epitestosterone, 17a-hydroxyproges- in the gene. We performed in silico analysis by sequence compari- terone, and 20a-hydroxyprogesterone (Li et al., 1997; sons on human genome and demonstrated the chromosome local- Schumacher et al., 2003). Interestingly, finasteride, the known izations of the other studied genes are SRD5a3 is located at 4q12, SRD5a inhibitor in mammals, has been shown to inhibit SRD5b GPSN2 at 19p13.12, GPSN2-like at 4q13.1 and SRD5b is situated at activity in human embryonic kidney cells (pers. comm., Dr. Van 7q34 (UCSC Genome Bioinformatics; University of California, CA). Luu-The, Université Laval, Québec). Despite a poor protein se- The C-terminal region of hSRD5a is highly conserved among the quence similarity between the SRD5a and SRD5b isozymes, their three types of SRD5a and some residues, such as H232, are likely to proteins must at least share some similarities in their substrate be responsible for the catalytic activity (Wang et al., 1999). It has binding domains to have similar inhibition responses to finaste- been suggested that the N-terminal region is hydrophobic and ride. Inhibition studies and proteomic analyses are required to help could react with aliphatic and aromatic side chains of substrates understand the difference between the three subtypes of SRD5a (Wigley et al., 1994). In addition, some authors suggested that and to elucidate any protein functional similarity between SRD5a the N-terminal region and the H296 residue could have critical and SRD5b (e.g., their substrate binding domains). roles in the SRD5a activity since mutations at both sites abolished SRD5a activity (Uemura et al., 2008). Even though the SRD5a iso- forms share high sequence similarity and are integral membrane 5. Biological functions of vertebrate 5a-reductases and 5b- proteins, some important chemical differences exist among them. reductase SRD5a1 has a broad pH range (6.0–8.5), while SRD5a2 has a sharp pH optimum (5.0–5.5) and SRD5a3 appears to be efficient at pH 6.9 The wide and co-localized distributions of SRD5a and SRD5b (Chang, 2002; Uemura et al., 2008). Under optimal conditions, among vertebrate tissues suggest some similarity or relationship

SRD5a2 has a higher Vm/Km and a 1000-fold greater affinity for ste- in biological function. Androstane biosynthesis is one of the main roid substrates than SRD5a1(Andersson et al., 1991; Chang, 2002; functions of both exerted by SRD5a or SRD5b. A stereospecific Normington and Russell, 1992). No such comparisons exist for hydrogenation of the testosterone double bond at C-4 to C-5 by SRD5a3. The reductases also have different sensitivities to inhibi- either SRD5a or SRD5b yields asymmetric structures that are, tors (for review, Shimazaki, 2002). Both natural and synthetic respectively, 5a-DHT and 5b-DHT. Both reduced steroids are ex- SRD5a inhibitors exist. The green tea (Camellia sinensis) catechin pected to show differential chemical properties and binding affin- is specific for SRD5a1 inhibition (Liao and Hiipakka, 1995); ities due to their stereospecific configuration; 5a-DHT has a ‘‘chair” whereas c-linolenic acid, a natural product found in evening prim- configuration whereas the 5b-DHT has a ‘‘boat” configuration rose (Oenothera biennis) oil and borage (Borago officinalis L.) oil, (Fig. 1). inhibits SRD5a1 and SRD5a2(Huang and Ziboh, 2001; Kokontis The steroid-5a-DHT is known to be the dominant androgen in and Liao, 1999). The 4-aza-3-oxo-1-ene (4-MA) compounds are many vertebrates and its functions are mostly associated with the major class of synthetic SRD5a inhibitors. They include the development and regulation of male secondary characteristics prostate cancer and benign prostatic hyperplasia drugs, finasteride (Russell and Wilson, 1994). Both testosterone and 5a-DHT can and dutasteride. These types of inhibitors require a structure sim- activate the androgen receptor and regulate SRD5a transcription, ilar to 3-oxo-4-ene with a secondary 17b-substituent to success- although 5a-DHT has a stronger affinity for AR (Chang, 2002; Nor- fully bind to the SRD5a-NADPH or SRD5a-NADP+ complexes mington and Russell, 1992). Human SRD5a deficiencies can lead to (Rasmusson et al., 1986; Voigt and Hsia, 1973). It has been shown pseudohermaphroditism, prostate cancer, polycystic ovarian syn- that SRD5a2 is more sensitive to finasteride than SRD5a1, drome and hirsutism (Andersson et al., 1991; Goodarzi et al., although both enzymes respond similarly to dutasteride. No inhi- 2006; Thomas et al., 2009). During human embryogenesis, muta- bition studies have been performed on mammalian SRD5a3(Uem- tions in the SRD5a2 gene result in abnormal external genitalia ura et al., 2008). Furthermore, studies have reported differential and prostate development (Griffin and Wilson, 1989). However, responses to SRD5a inhibitors among vertebrates. The dog, mon- in another study, some SRD5a2 deficient males still exhibited a key and human have similar responses to finasteride (selective to slight virilisation which was explained by peripheral SRD5a1 SRD5a2); whereas both SRD5a subtypes 1 and 2 are inhibited in activity (Thigpen et al., 1993) and possibly SRD5a3 activity. 494 V.S. Langlois et al. / General and Comparative Endocrinology 166 (2010) 489–497

5a-DHT also acts as a potent androgen in amphibians, since expo- An interesting sexual dimorphism has been detected in both sures of Rana clamitans eggs until metamorphosis resulted in a 98% SRD5a and SRD5b expression and activity. Since SRD5a are in- male phenotype (Coady et al., 2004). Furthermore, recent data ob- volved in male secondary characteristic development, it could have tained in our laboratory indicate that chronic exposures to a SRD5a been hypothesized that males have higher SRD5a activity than fe- inhibitor skewed the sex ratio and favoured female phenotypes in male. However, in mammalian and amphibian livers, females ex- S. tropicalis exposed from egg to metamorphic tadpoles (unpub- pressed more SRD5a activity than males (Andersson et al., 1989; lished data). Therefore, SRD5a activity is necessary for 5a-DHT for- Lisboa et al., 1972). In contrast, SRD5b activity is higher in male mation which leads to normal male development and function in rat liver (Mode and Rafter, 1985); whereas female frog liver ex- many species. presses higher SRD5b activity than males (Lisboa et al., 1972). Fur- In marked contrast, the reproductive functions of SRD5b are thermore, sex-reversed male X. laevis treated with estradiol also largely unexplored because of the long held view that 5b-DHT exhibit female SRD5a and SRD5b activity profiles (Lisboa et al., does not have androgenic effects (Kokontis and Liao, 1999). Some 1972). In fish, a differential species expression occur where males experimental evidence supports the view that the 5b-androstanes expressed higher SRD5b levels in goldlined seabream testes (Rhab- are not androgenic and it has been demonstrated that 5b-DHT dosargus sarba; Yeung and Chan, 1985) and lesser in rainbow trout does not compete for the androgen-binding site in mammalian testes (O. mykiss; Baron et al., 2008) compared to ovaries. The rea- prostate (Mainwaring, 1977). This was explained by a steric ef- sons for these species differences are unknown but could relate to fects on the A ring in 5b-androstanes since induction of andro- differences in gonadal maturation or seasonality but such data genic activity needs an electronic effect on the A ring (Liao were not reported in the publications. Nevertheless, the presence et al., 1973). The 5b-androstanes failed to induce a negative feed- of this sexual dimorphism supports the evidence that not only back on plasma luteinizing hormone and follicle stimulating hor- SRD5a but also SRD5b could have functions in androgen availabil- mone (Davies et al., 1980), failed to stimulate cloacal gland ity (e.g., androgen excretion and inactivation). growth (Massa et al., 1980) and failed to induce comb develop- In addition to participation in reproductive functions, both ment in male chicken (Mori et al., 1974). Furthermore, intrahypo- SRD5a and SRD5b are involved in many other biological processes. thalamic 5b-DHT implants did not induce male sexual behavior in Both 5a-androstanes and 5b-androstanes are involved in erythro- birds (Adkins, 1977; Steimer and Hutchison, 1981); while testos- poiesis in mammals (Gordon et al., 1970), birds (Irving et al., terone and 5a-DHT implants induced the erect threat posture in 1976) and amphibians (Garavini and Cristofori, 1984). 5a-Andros- chicks (Groothuis and Ros, 2005). However, there are numerous tanes increase the production of the erythropoietin hormone in the reports to the contrary that indicate important biological func- kidneys; whereas 5b-androstanes stimulate heme production in tions of SRD5b and 5b-DHT. For example, sexual behavior studies the liver (Kokontis and Liao, 1999). The 5b-reduced steroids have indicate important roles of SRD5b, including the regulation of ste- a stronger erythropoietin-inducing activity than the 5a-reduced roid production by testosterone inactivation and/or direct inhibi- metabolites in crested newt (Triturus cristatus camifex; Garavini tion of aromatase (estrogen synthase) by 5b-reduced and Cristofori, 1984). Furthermore, both SRD5a and SRD5b are also metabolites of testosterone in the brain of birds (Balthazart involved in bile biosynthesis, where they catalyze the conversion et al., 1981; Hutchison and Steimer, 1981; Steimer and Hutchison, of 7a,12a-dihydroxy-4-cholesten-3-one into 7a,12a-dihydroxy- 1981). Hutchison and Steimer (1981) suggested that inactivation 5a-cholestan-3-one and 7a,12a-dihydroxy-5b-cholestan-3-one, of testosterone by conversion to 5b-DHT by SRD5b regulates brain respectively. However, only 7a,12a-dihydroxy-5b-cholestan-3- sensitivity to androgens under changing hormonal conditions. one has been shown to be biologically active and is further reduced Long-term castrated doves showed increased levels of 5b-DHT in into bile. The 5a-reduction is suggested to be an inactivation path- the preoptic area (androgen sensitive) compared to intact males way for bile biosynthesis regulation in humans (Kondo et al., (Steimer and Hutchison, 1981) suggesting that 5b-reduction regu- 1994). While speculative, another common function could be pro- lates low androgen levels during non-breeding seasons. Further- posed for SRD5a and SRD5b because they can both regulate bio- more, 5b-DHT has also been shown to exhibit direct biological synthesis by inactivating substrates. For examples, SRD5a can activities. Exposure to 5b-DHT inhibited mammary development inactivate bile biosynthesis and SRD5b can inactivate 5a-DHT in mice (Yanai et al., 1977), and induced vasorelaxation in thoracic biosynthesis. aorta (Montano et al., 2008) and vas deferens (Lafayette et al., 2008) in rats. Even though 5b-DHT does not affect directly male secondary sexual development (compared to 5a-DHT), 5b-DHT 6. Biological functions of 5a- and 5b-reduction in plants can induce other androgen-related functions and 5b-reduction can regulate testosterone availability. Interestingly, plant steroid biosynthesis requires both 5a- and Sex steroid regulation of SRD5b is not fully understood. A recent 5b-reductive enzymes. Brassinosteroids are a group of plant ste- microarray analysis has identified induction of SRD5b mRNA after roid hormones which contain at least 27 carbons (Geuns, 1978) treating immortalized human prostate epithelial cells (androgen- and possess important physiological roles, including cell elonga- responsive and expressing wild-type of AR) with the synthetic tion and photomorphogenesis (Li et al., 1996). De-etiolated 2 androgen R1881 (Bolton et al., 2007). In contrast, an in vivo study (DET2; GI: 15224430) is involved in brassinosteroid biosynthesis has demonstrated that androgens decreased SRD5b mRNA level by catalyzing the campesterol to campestanol conversion (Li in fish gonads (Baron et al., 2008). Hence, treatment of female rain- et al., 1997, 1996). DET2 is a member of the SRD5a1/2 subfamily bow trout (Oncorhynchus mykiss) treated with an exogenous andro- (Fig. 2) and a functional homolog of the mammalian SRD5a1 and gen (11b-hydroxyandrostenedione, 11bOHD4) decreased SRD5b SRD5a2. The A. thaliana DET2 amino acid sequence contains 38– transcription in gonads. In birds, exogenous exposure to testoster- 42% of sequence identity with the mammalian SRD5a1 and one and 17b-estradiol decreased SRD5b activity in the castrated SRD5a2, and all three share similar hydropathy profiles (Li et al., male dove preoptic area of the brain (Steimer and Hutchison, 1997). However, in contrast to human SRD5a1 and SRD5a2 which 1981). Despite the difference in SRD5b responses to androgens, reduces the D4,5 double bond in ring A, it has been suggested that there is evidence that sex steroids do indeed regulate SRD5b tran- DET2 reduces D5,6 in ring B. Li and colleagues (1997) investigated scription in vertebrates. Clearly, a more systematic comparison of this close similarity between these proteins and demonstrated that species differences and tissue responses of SRD5b to hormones when DET2 is expressed in human embryonic kidney cells, it could are required. catalyze the 5a-reduction of human steroid substrates (i.e., testos- V.S. Langlois et al. / General and Comparative Endocrinology 166 (2010) 489–497 495 terone to 5a-DHT). Similarly, human SRD5a1 and SRD5a2 ex- Bagrov, A.Y., Shapiro, J.I., Fedorova, O.V., 2009. Endogenous cardiotonic steroids: pressed in det2 mutant plants can replace DET2 action and partic- physiology, pharmacology, and novel therapeutic targets. Pharmacol. Rev. 61, 9–38. ipate in brassinosteroid biosynthesis by converting campesterol Balthazart, J., 1991. Testosterone metabolism in the avian hypothalamus. J. Steroid into campestanol (Li et al., 1997). However, DET2 is not inhibited Biochem. Mol. Biol. 40, 557–570. by finasteride (Li et al., 1997). Our phylogenetic analysis suggests Balthazart, J., Malacarne, G., Deviche, P., 1981. Stimulatory effects of 5-beta- dihydrotestosterone on the sexual-behavior in the domestic chick. Horm. that many gene duplication events occurred in plants (e.g., A. tha- Behav. 15, 246–258. liana) indicating the existence of more than one type of SRD5a-like Baron, D., Houlgatte, R., Fostier, A., Guiguen, Y., 2008. Expression profiling of protein. Interestingly, some plants also have a 5b-reductase en- candidate genes during ovary-to-testis trans-differentiation in rainbow trout masculinized by androgens. Gen. Comp. Endocrinol. 156, 369–378. zyme, which like vertebrates, can catalyze the stereospecific reduc- Berseus, O., Bjorkhem, I., 1967. Enzymatic conversion of a delta4-3-ketosteroid into 4,5 tion of the D double bond in ring A of several steroids (Gavidia a 3alpha-hydroxy-5beta-steroid – mechanism and stereochemistry of hydrogen et al., 2007). This enzyme is known as progesterone 5b-reductase transfer from NADPH-bile acids and steroids 190. Eur. J. Biochem. 2, 503– 507. and is involved in the biosynthesis of the plant steroid cardenolide Bjorkhem, I., 1969. Stereochemistry of the enzymatic conversion of a delta 4-3- (GI: 45758665; Stuhlemmer and Kreis, 1996). Cardenolide is in- oxosteroid into a 3-oxo-5 beta-steroid. Bile acids and steroids 208. Eur. J. volved in defence mechanisms against predators (Malcolm and Biochem. 7, 413–417. Zalucki, 1996). This toxic cardenolide property is used in the treat- Bolton, E.C., So, A.Y., Chaivorapol, C., Haqq, C.M., Li, H., Yamamoto, K.R., 2007. Cell- and gene-specific regulation of primary target genes by the androgen receptor. ment of heart failure in humans (for review, Bagrov et al., 2009) Genes Dev. 21, 2005–2017. and also in the defence mechanisms of butterflies that eat and Callard, I.P., Leathem, J.H., 1966. Steroid synthesis by amphibian ovarian tissue. Gen. accumulate it to prevent predation (for review, Malcolm and Brow- Comp. Endocrinol. 7, 80–84. Chang, C.-Y., 2002. Androgen and Androgen Receptor. MA Kluwer Academic er, 1989). Gavidia and colleagues (2007) demonstrated that plant Publishers, Boston. progesterone 5b-reductase and mammalian SRD5b do not share Charbonneau, A., The, V.L., 2001. Genomic organization of a human 5 beta- structural homology and suggested an independent 5b-reductive reductase and its pseudogene and substrate selectivity of the expressed enzyme. Biochim. Biophys. Acta Gene Struct. Expr. 1517, 228–235. enzyme evolution within each group. Clayton, P.T., Mills, K.A., Johnson, A.W., Barabino, A., Marazzi, M.G., 1996. Delta(4)- 3-oxosteroid 5 beta-reductase deficiency: failure of ursodeoxycholic acid treatment and response to chenodeoxycholic acid plus cholic acid. Gut 38, 7. Conclusions 623–628. Coady, K., Murphy, M., Villeneuve, D., Hecker, M., Jones, P., Carr, J., Solomon, K., We established the first phylogenetic analysis of all subtypes of Smith, E., Van Der Kraak, G., Kendall, R., Giesy, J., 2004. Effects of atrazine on metamorphosis, growth, and gonadal development in the green frog (Rana SRD5a where we identified major gene duplication events. Steroid- clamitans). J. Toxicol. Environ. Health A 67, 941–957. 5-reductases have highly conserved biochemical functions within Collins, W., Cameron, E.H., 1975. Subcellular localization and properties of mouse the plant and animal kingdoms enhancing the importance of fully adrenal C19-steroid 5beta-reductase. Biochem. J. 147, 165–174. understanding their physiological roles. Even though, SRD5a and Davies, D.T., Massa, R., James, R., 1980. Role of testosterone and of its metabolites in regulating gonadotropin-secretion in the Japanese quail. J. Endocrinol. 84, 211– SRD5b do not possess a common ancestor; they do share similar 222. biochemical and biological functions. This review helps to eluci- Dorfman, R.I., Forchielli, E., 1956. Separation of delta 4-5 alpha-hydrogenases from date the unique and overlapping roles that SRD5a and SRD5b play, rat liver homogenates. J. Biol. Chem. 223, 443–448. Edgar, R.C., 2004. Muscle: multiple sequence alignment with high accuracy and especially in steroid hormone metabolism. Future studies should high throughput. Nucleic Acids Res. 32, 1792–1797. investigate if the enhancement of SRD5b activity and subsequent Faucher, F., Cantin, L., Luu-The, V., Labrie, F., Breton, R., 2008. Crystal structures of alteration of androgen dynamics could help in the treatment of human delta4-3-ketosteroid 5beta-reductase (AKR1D1) reveal the presence of an alternative binding site responsible for substrate inhibition. Biochemistry 47, androgen-related diseases such as prostate cancer and benign 13537–13546. prostate hyperplasia. Furthermore, since both androgenic and Freking, F., Ramachandran, B., Schlinger, B.A., 1998. Regulation of aromatase, 5 anti-androgenic endocrine disrupting chemicals (EDCs) are found alpha- and 5 beta-reductase in primary cell cultures of developing Zebra finch telencephalon. J. Neurobiol. 36, 30–40. in the aquatic environment (Jenkins et al., 2003; Urbatzka et al., Gable, K., Garton, S., Napier, J.A., Dunn, T.M., 2004. Functional characterization of the 2007) and given the physiological roles of both SRD5a and SRD5b Arabidopsis thaliana orthologue of tsc13p, the enoyl reductase of the yeast in androgen dynamics and sexual development among other func- microsomal fatty acid elongating system. J. Exp. Bot. 55, 543–545. Garavini, C., Cristofori, M., 1984. The effect of 5-alpha-dihydrotestosterone and 5- tions, it will also be important to determine if they could be possi- beta-dihydrotestosterone on erythropoiesis of the newt, Triturus cristatus ble targets of EDC action. carnifex (laur). Gen. Comp. Endocrinol. 54, 188–193. Gavidia, I., Tarrio, R., Rodriguez-Trelles, F., Perez-Bermudez, P., Seitz, H.U., 2007. Plant progesterone 5 beta-reductase is not homologous to the animal enzyme. Acknowledgments Molecular evolutionary characterization of p5 betaR from Digitalis purpurea. Phytochemistry 68, 853–864. The authors would like to thank Colin Cameron for help with Geuns, J.M.C., 1978. Steroid-hormones and plant-growth and development. the editing of the manuscript. This research was supported by Phytochemistry 17, 1–14. Goodarzi, M.O., Shah, N.A., Antoine, H.J., Pall, M., Guo, X., Azziz, R., 2006. Variants in the NSERC-DG program (to V.L.T.), the NSERC-PGSD program (to the 5alpha-reductase type 1 and type 2 genes are associated with polycystic V.S.L.), University of Ottawa International Scholarship (to D.Z.) ovary syndrome and the severity of hirsutism in affected women. J. Clin. and Health Canada. Endocrinol. Metab. 91, 4085–4091. Gordon, A.S., Zanjani, E.D., Levere, R.D., Kappas, A., 1970. Stimulation of mammalian erythropoiesis by 5beta-H steroid metabolites. Proc. Natl. Acad. Sci. USA 65, References 919. Griffin, J.E., Wilson, J.D., 1989. The androgen resistance syndromes: 5a-reductase deficiency, testicular feminization and related syndromes. In: Scrivér, C.R., Adkins, E.K., 1977. Effects of diverse androgens on sexual-behavior and morphology Beandet, A.L., Sly, W.S., Valle, D. (Eds.), The Metabolic Basis of Inherited Disease. of castrated male quail. Horm. Behav. 8, 201–207. Springer, New York, pp. 144–199. Andersson, S., Berman, D.M., Jenkins, E.P., Russell, D.W., 1991. Deletion of steroid 5 Grisham, W., Tam, A., Greco, C.M., Schlinger, B.A., Arnold, A.P., 1997. A putative 5 alpha-reductase 2 gene in male pseudohermaphroditism. Nature 354, 159–161. alpha-reductase inhibitor demasculinizes portions of the Zebra finch song Andersson, S., Bishop, R.W., Russell, D.W., 1989. Expression cloning and regulation system. Brain Res. 750, 122–128. of steroid 5 alpha-reductase, an enzyme essential for male sexual Groothuis, T.G.G., Ros, A.F.H., 2005. The hormonal control of begging and early differentiation. J. Biol. Chem. 264, 16249–16255. aggressive behavior: experiments in Black-headed gull chicks. Horm. Behav. 48, Andersson, S., Russell, D.W., 1990. Structural and biochemical properties of cloned 207–215. and expressed human and rat steroid 5 alpha-reductases. Proc. Natl. Acad. Sci. Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate USA 87, 3640–3644. large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704. Azzolina, B., Ellsworth, K., Andersson, S., Geissler, W., Bull, H.G., Harris, G.S., 1997. Guindon, S., Lethiec, F., Duroux, P., Gascuel, O., 2005. Phyml online – a web server Inhibition of rat alpha-reductases by finasteride: evidence for isozyme for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res. differences in the mechanism of inhibition. J. Steroid Biochem. Mol. Biol. 61, 33, W557–W559. 55–64. 496 V.S. Langlois et al. / General and Comparative Endocrinology 166 (2010) 489–497

Han, G., Gable, K., Kohlwein, S.D., Beaudoin, F., Napier, J.A., Dunn, T.M., 2002. The Malcolm, S.B., Brower, L.P., 1989. Evolution and ecological implications of Saccharomyces cerevisiae ybr159w gene encodes the 3-ketoreductase of the cardenolide sequestration in the monarch butterfly. Experientia 45, 284–295. microsomal fatty acid elongase. J. Biol. Chem. 277, 35440–35449. Malcolm, S.B., Zalucki, M.P., 1996. Milkweed latex and cardenolide induction may Huang, Y.-S., Ziboh, V.A., 2001. Gamma-linolenic Acid: Recent Advances in resolve the lethal plant defence paradox. Entomol. Exp. Appl. 80, 193–196. Biotechnology and Clinical Applications. The American Oil Chemistry Society, Massa, R., Davies, D.T., Bottoni, L., 1980. Cloacal gland of the Japanese quail – USA. 259 pp. androgen dependence and metabolism of testosterone. J. Endocrinol. 84, 223– Hutchison, J.B., Steimer, T., 1981. Brain 5-beta-reductase – a correlate of behavioral 230. sensitivity to androgen. Science 213, 244–246. Mode, A., Rafter, I., 1985. The sexually differentiated delta-4-3-ketosteroid 5-beta- Imataka, H., Suzuki, K., Inano, H., Kohmoto, K., Tamaoki, B.I., 1989. Biosynthetic reductase of rat-liver – purification, characterization, and quantitation. J. Biol. pathways of testosterone and estradiol-17-beta in slices of the embryonic ovary Chem. 260, 7137–7141. and testis of the chicken (Gallus domesticus). Gen. Comp. Endocrinol. 73, 69–79. Montano, L.M., Calixto, E., Figueroa, A., Flores-Soto, E., Carbajal, V., Perusquia, M., Irving, R.A., Mainwaring, W.I.P., Spooner, P.M., 1976. Regulation of hemoglobin 2008. Relaxation of androgens on rat thoracic aorta: testosterone concentration synthesis in cultured chick blastoderms by steroids related to 5-beta- dependent agonist/antagonist L-type Ca2+ channel activity, and 5beta- androstane. Biochem. J. 154, 81–93. dihydrotestosterone restricted to L-type Ca2+ channel blockade. Jenkins, E.P., Andersson, S., Imperato-McGinley, J., Wilson, J.D., Russell, D.W., 1992. Endocrinology 149, 2517–2526. Genetic and pharmacological evidence for more than one human steroid 5 Moon, Y.A., Horton, J.D., 2003. Identification of two mammalian reductases involved alpha-reductase. J. Clin. Invest. 89, 293–300. in the two-carbon fatty acyl elongation cascade. J. Biol. Chem. 278, 7335–7343. Jenkins, E.P., Hsieh, C.L., Milatovich, A., Normington, K., Berman, D.M., Francke, U., Mori, M., Suzuki, K., Tamaoki, B.I., 1974. Testosterone-metabolism in rooster comb. Russell, D.W., 1991. Characterization and chromosomal mapping of a human Biochim. Biophys. Acta 337, 118–128. steroid 5 alpha-reductase gene and pseudogene and mapping of the mouse Normington, K., Russell, D.W., 1992. Tissue distribution and kinetic characteristics homologue. Genomics 11, 1102–1112. of rat steroid 5-alpha-reductase isozymes – evidence for distinct physiological Jenkins, R.L., Wilson, E.M., Angus, R.A., Howell, W.M., Kirk, M., 2003. functions. J. Biol. Chem. 267, 19548–19554. Androstenedione and progesterone in the sediment of a river receiving paper Okuda, A., Okuda, K., 1984. Purification and characterization of delta 4-3- mill effluent. Toxicol. Sci. 73, 53–59. ketosteroid 5 beta-reductase. J. Biol. Chem. 259, 7519–7524. Jez, J.M., Bennett, M.J., Schlegel, B.P., Lewis, M., Penning, T.M., 1997a. Comparative Olsen, E.A., Messenger, A.G., Shapiro, J., Bergfeld, W.F., Hordinsky, M.K., Roberts, J.L., anatomy of the aldo–keto reductase superfamily. Biochem. J. 326, 625–636. Stough, D., Washenik, K., Whiting, D.A., 2005. Evaluation and treatment of male Jez, J.M., Flynn, T.G., Penning, T.M., 1997b. A new nomenclature for the aldo–keto and female pattern hair loss. J. Am. Acad. Dermatol. 52, 301–311. reductase superfamily. Biochem. Pharmacol. 54, 639–647. Ozon, R., Breuer, H., Lisboa, B.P., 1964. Étude du métabolisme des hormones Jez, J.M., Penning, T.M., 2001. The aldo–keto reductase (AKR) superfamily: an stéroïdes chez les vertébrés inferieurs.3. Métabolisme in vitro de la testostérone update. Chem. Biol. Interact. 130, 499–525. par l’ovaire et le testicule de la grenouille Rana temporaria. Gen. Comp. Kazutoshi, Y., Labrie, F., Luu-The, V., 2008. Type 3 5-alpha-reductase is an Endocrinol. 4, 577–583. ubiquitous enzyme highly expressed in the brain and strongly inhibited by Pasmanik, M., Callard, G.V., 1985. Aromatase and 5 alpha-reductase in the teleost finasteride and dutasteride. In: 13th International Congress on Hormonal brain, spinal cord, and pituitary gland. Gen. Comp. Endocrinol. 60, 244–251. Steroids and Hormones & Cancer, vol. 53, Québec City, Canada, pp. 107. Pasmanik, M., Callard, G.V., 1988. Changes in brain aromatase and 5 alpha- Killinger, D.W., Perel, E., Daniilescu, D., Kharlip, L., Lindsay, W.R.N., 1990. Influence reductase activities correlate significantly with seasonal reproductive cycles in of adipose-tissue distribution on the biological-activity of androgens. Ann. NY goldfish (Carassius auratus). Endocrinology 122, 1349–1356. Acad. Sci. 595, 199–211. Paul, S., Gable, K., Dunn, T.M., 2007. A six-membrane-spanning topology for yeast Kohlwein, S.D., Eder, S., Oh, C.S., Martin, C.E., Gable, K., Bacikova, D., Dunn, T., 2001. and Arabidopsis tsc13p, the enoyl reductases of the microsomal fatty acid Tsc13p is required for fatty acid elongation and localizes to a novel structure at elongating system. J. Biol. Chem. 282, 19237–19246. the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol. Cell. Biol. 21, Peters, D.H., Sorkin, E.M., 1993. Finasteride – a review of its potential in the 109–125. treatment of benign prostatic hyperplasia. Drugs 46, 177–208. Kokontis, J.M., Liao, S.S., 1999. Molecular action of androgen in the normal and Rasmusson, G.H., Reynolds, G.F., Steinberg, N.G., Walton, E., Patel, G.F., Liang, T.M., neoplastic prostate. Vitam. Horm. Adv. Res. Appl. 55, 219–307. Cascieri, M.A., Cheung, A.H., Brooks, J.R., Berman, C., 1986. Azasteroids – Kondo, K.H., Kai, M.H., Setoguchi, Y., Eggertsen, G., Sjoblom, P., Setoguchi, T., Okuda, structure–activity-relationships for inhibition of 5-alpha-reductase and of K.I., Bjorkhem, I., 1994. Cloning and expression of cDNA of human delta(4)-3- androgen receptor-binding. J. Med. Chem. 29, 2298–2315. oxosteroid 5-beta-reductase and substrate-specificity of the expressed enzyme. Russell, D.W., Wilson, J.D., 1994. Steroid 5 alpha-reductase: two genes/two Eur. J. Biochem. 219, 357–363. enzymes. Annu. Rev. Biochem. 63, 25–61. Labrie, F., Sugimoto, Y., Luu-The, V., Simard, J., Lachance, Y., Bachvarov, D., Leblanc, Samuels, L.T., Sweat, M.L., Levedahl, B.H., Pottner, M., Helmreich, M.L., 1950. G., Durocher, F., Paquet, N., 1992. Endocrinology 131, 1571–1573. Metabolism of testosterone by livers of different species of animals. J. Biol. Lafayette, S.S., Vladimirova, I., Garcez-do-Carmo, L., Monteforte, P.T., Caricati Neto, Chem. 183, 231–239. A., Jurkiewicz, A., 2008. Evidence for the participation of calcium in non- Schumacher, M., Weill-Engerer, S., Liere, P., Robert, F., Franklin, R.J.M., Garcia- genomic relaxations induced by androgenic steroids in rat vas deferens. Br. J. Segura, L.M., Lambert, J.J., Mayo, W., Melcangi, R.C., Parducz, A., Suter, U., Carelli, Pharmacol. 153, 1242–1250. C., Baulieu, E.E., Akwa, Y., 2003. Steroid hormones and neurosteroids in normal Langlois, V.S., Duarte, P., Ing, S., Pauli, B.D., Wade, M.G., Cooke, G., Trudeau, V.L., and pathological aging of the nervous system. Prog. Neurobiol. 71, 2008. Consequences of steroid 5a-reductase and steroid 5b-reductase 3–29. inhibitions during amphibian embryogenesis. In: 13th International Congress Shimazaki, J., 2002. The role of 5alpha-reductase in prostate disease and male on Hormonal Steroids and Hormones & Cancer, Québec, Canada. pattern baldness. In: Chang, C. (Ed.), Androgen and Androgen Receptor: Levy, M.A., Brandt, M., Sheedy, K.M., Holt, D.A., Heaslip, J.I., Trill, J.J., Ryan, P.J., Mechanisms, Functions, and Clinical Applications. Kluwer Academic Morris, R.A., Garrison, L.M., Bergsma, D.J., 1995. Cloning, expression and Publishers, Massachusetts, USA, pp. 155–196. functional-characterization of type-1 and type-2 steroid 5-alpha-reductases Sinclair, R., 1998. Fortnightly review – male pattern androgenetic alopecia. Br. Med. from Cynomolgus monkey – comparisons with human and rat isoenzymes. J. J. 317, 865–869. Steroid Biochem. Mol. Biol. 52, 307–319. Soma, K.K., Bindra, R.K., Gee, J., Wingfield, J.C., Schlinger, B.A., 1999. Androgen- Li, J., Biswas, M.G., Chao, A., Russell, D.W., Chory, J., 1997. Conservation of function metabolizing enzymes show region-specific changes across the breeding season between mammalian and plant steroid 5alpha-reductases. Proc. Natl. Acad. Sci. in the brain of a wild songbird. J. Neurobiol. 41, 176–188. USA 94, 3554–3559. Song, W.Q., Qin, Y.M., Saito, M., Shirai, T., Pujol, F.M., Kastaniotis, A.J., Hiltunen, J.K., Li, J.M., Nagpal, P., Vitart, V., McMorris, T.C., Chory, J., 1996. A role for Zhu, Y.X., 2009. Characterization of two cotton cDNAs encoding trans-2-enoyl- brassinosteroids in light-dependent development of Arabidopsis. Science 272, coa reductase reveals a putative novel NADPH-binding motif. J. Exp. Bot. 60, 398–401. 1839–1848. Liao, S., Hiipakka, R.A., 1995. Selective inhibition of steroid 5 alpha-reductase Steimer, T., Hutchison, J.B., 1981. Metabolic control of the behavioral action of isozymes by tea epicatechin-3-gallate and epigallocatechin-3-gallate. Biochem. androgens in the dove brain – testosterone inactivation by 5-beta-reduction. Biophys. Res. Commun. 214, 833–838. Brain Res. 209, 189–204. Liao, S., Liang, T., Fang, S., Shao, E., Shao, T.C., 1973. Steroid structure and androgenic Stuhlemmer, U., Kreis, W., 1996. Cardenolide formation and activity of pregnane- activity – specificities involved in receptor binding and nuclear retention of modifying enzymes in cell suspension cultures, shoot cultures and leaves of various androgens. J. Biol. Chem. 248, 6154–6162. Digitalis lanata. Plant Physiol. Biochem. 34, 85–91. Lisboa, B.F., Srassner, M., Wulff, C., Hoffmann, U., 1974. 5b-reductase in the human Sugimoto, Y., Yoshida, M., Tamaoki, B.I., 1990. Purification of 5-beta-reductase from foetal brain. Acta Endocrinol. (Copenh) Suppl. 184, 156. hepatic cytosol fraction of chicken. J. Steroid Biochem. Mol. Biol. 37, 717– Lisboa, B.P., Breuer, H., 1966. Studies on the metabolism of steroid hormones in 724. vertebrates. VI. Reductive metabolism of testosterone and related C19-steroids Tamura, K., Furihata, M., Tsunoda, T., Ashida, S., Takata, R., Obara, W., Yoshioka, H., in liver preparations from the trout, triton, frog and fowl. Gen. Comp. Daigo, Y., Nasu, Y., Kumon, H., Konaka, H., Namiki, M., Tozawa, K., Kohri, K., Endocrinol. 6, 114–124. Tanji, N., Yokoyama, M., Shimazui, T., Akaza, H., Mizutani, Y., Miki, T., Fujioka, T., Lisboa, B.P., Breuer, H., Witschi, E., 1972. Studies on the metabolism of steroid Shuin, T., Nakamura, Y., Nakagawa, H., 2007. Molecular features of hormone- hormones in vertebrates. IX. The metabolism of (4–14 C)testosterone in the refractory prostate cancer cells by genome-wide gene expression profiles. liver of the African water frog (Xenopus laevis). Hoppe Seylers Z. Physiol. Chem. Cancer Res. 67, 5117–5125. 353, 1907–1914. Terada, N., Sato, B., Matsumoto, K., 1980. Formation of 5-beta- and 5-alpha-products Mainwaring, W.I.P., 1977. The mechanism of action of androgens. Monogr. as major c19-steroids from progesterone in vitro in immature golden-hamster Endocrinol. 10, 1–178. testes. Endocrinology 106, 1554–1561. V.S. Langlois et al. / General and Comparative Endocrinology 166 (2010) 489–497 497

Thigpen, A.E., Silver, R.I., Guileyardo, J.M., Casey, M.L., McConnell, J.D., Russell, D.W., Wang, M., Bhattacharyya, A.K., Taylor, M.F., Tai, H.H., Collins, D.C., 1999. Site- 1993. Tissue distribution and ontogeny of steroid 5 alpha-reductase isozyme directed mutagenesis studies of the NADPH-binding domain of rat steroid 5 expression. J. Clin. Invest. 92, 903–910. alpha-reductase (isozyme-1) I: analysis of aromatic and hydroxylated amino Thomas, L.N., Douglas, R.C., Rittmaster, R.S., Too, C.K., 2009. Overexpression of acid residues. Steroids 64, 356–362. 5alpha-reductase type 1 increases sensitivity of prostate cancer cells to low Wigley, W.C., Prihoda, J.S., Mowszowicz, I., Mendonca, B.B., New, M.I., Wilson, J.D., concentrations of testosterone. Prostate 69, 595–602. Russell, D.W., 1994. Natural mutagenesis study of the human steroid 5-alpha- Uemura, M., Tamura, K., Chung, S., Honma, S., Okuyama, A., Nakamura, Y., reductase-2 isozyme. Biochemistry 33, 1265–1270. Nakagawa, H., 2008. Novel 5 alpha-steroid reductase (SRD5a3, type-3) is Wilson, J.D., Griffin, J.E., Russell, D.W., 1993. Steroid 5-alpha-reductase-2 deficiency. overexpressed in hormone-refractory prostate cancer. Cancer Sci. 99, 81–86. Endocr. Rev. 14, 577–593. Urbatzka, R., van Cauwenberge, A., Maggioni, S., Vigano, L., Mandich, A., Benfenati, Yanai, R., Mori, T., Nagasawa, H., 1977. Long-term effects of prenatal and neonatal E., Lutz, I., Kloas, W., 2007. Androgenic and antiandrogenic activities in water administration of 5beta-dihydrotestosterone on normal and neoplastic and sediment samples from the River Lambro, Italy, detected by yeast androgen mammary development in mice. Cancer Res. 37, 4456–4459. screen and chemical analyses. Chemosphere 67, 1080–1087. Yang, Z.H., Nielsen, R., Hasegawa, M., 1998. Models of amino acid substitution and Vallarino, M., Mathieu, M., do-Rego, J.L., Bruzzone, F., Chartrel, N., Luu-The, V., applications to mitochondrial protein evolution. Mol. Biol. Evol. 15, 1600– Pelletier, G., Vaudry, H., 2005. Ontogeny of 3{beta}-hydroxysteroid 1611. dehydrogenase and 5{alpha}-reductase in the frog brain. Ann. NY Acad. Sci. Yeung, W.S., Chan, S.T., 1985. The in vitro metabolism of radioactive progesterone 1040, 490–493. and testosterone by the gonads of the protandrous Rhabdosargus sarba at Viger, R.S., Robaire, B., 1992. Expression of 4-ene steroid 5 alpha-reductase various sexual phases. Gen. Comp. Endocrinol. 59, 171–183. messenger ribonucleic acid in the rat epididymis during postnatal Zhu, Y., Fillenwarth, M.J., Crabb, D., Lumeng, L., Lin, R.C., 1996. Identification of the development. Endocrinology 131, 1534–1540. 37-Kd rat liver protein that forms an acetaldehyde adduct in vivo as delta 4-3- Voigt, W., Hsia, S.L., 1973. Further studies on testosterone 5-reductase of human ketosteroid 5 beta-reductase. Hepatology 23, 115–122. skin. Structural features of steroid inhibitors. J. Biol. Chem. 248, 4280–4285.