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GSF-National Research Center for Environment and Health, Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstr. 1, 85764 Neuherberg, Germany
(Requests for offprints should be addressed to J Adamski; Email: adamski{at}gsf.de)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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-reductase giving rise to the most potent androgen dihydrotestosterone and is a substrate for the aromatase yielding estradiol (for a review on steroid hormone formation see also Payne & Hales 2004). The formation of testosterone from androstenedione as well as the respective back-reaction is catalyzed by enzymes of the family of 17ß-hydroxysteroid dehydrogenases (17ß-HSDs). Structurally, nearly all 17ß-HSDs belong to the short-chain dehydrogenase/reductase (SDR) superfamily. In human, mouse and rat, the reduction of androstenedione to testosterone is catalyzed by 17ß-HSD type 3 (Geissler et al. 1994, Tsai-Morris et al. 1999) and type 5 (Dufort et al. 1999, Rheault et al. 1999, Luu-The et al. 2001). In rodents, additionally 17ß-HSD type 1 catalyzes this reaction very efficiently (Akinola et al. 1996, Nokelainen et al. 1996). In mammals, testicular formation of testosterone seems to be mainly governed by 17ß-HSD type 3, which has been shown to be highly and almost exclusively expressed in the testis of human (Geissler et al. 1994), mouse (Mustonen et al. 1997, Sha et al. 1997) and rat (Tsai-Morris et al. 1999). The enzyme is mainly involved in male sexual development resulting in male pseudohermaphroditism upon loss-of-function mutations (Geissler et al. 1994).
In fish, androgens were shown to play important roles in gonadogenesis (Bhandari et al. 2004) and spermatogenesis (Miura et al. 1991, Loir 1999) as well. The latter aspect seems to be closely connected to 11-ketotestosterone, assuming a special role for this modified androgen in fish sexual maturation. Furthermore, the role of such modified together with the so-called classical androgens on mating and nesting behavior of fish has been studied in some selected species (Brantley et al. 1993, Oliveira et al. 2002, Pall et al. 2002). In contrast to mammals, especially the molecular mechanisms underlying androgen function in fish are still widely unknown. The androgen receptors from several different fish species have been cloned and characterized at the molecular level (Ikeuchi et al. 1999, Sperry & Thomas 1999, Takeo & Yamashita 1999, Touhata et al. 1999, Kim et al. 2002, Wilson et al. 2004) and central enzymes of the general pathway leading to the formation of steroid hormones, for example P450 cholesterol side-chain cleavage enzyme (Lai et al. 1998, Hsu et al. 2002) and 17-hydroxylase/17,20-lyase (Sakai et al. 1992, Trant 1995, Kazeto et al. 2000b), have been identified as well. Concerning enzymes of the 17ß-HSD family though, only 17ß-HSD type 1 from eel (Kazeto et al. 2000a) and zebrafish (Mindnich et al. 2004) have been studied in detail and in both species the enzyme does not seem to be involved in androgen metabolism. To get more insight into the molecular regulation of androgen function in fish, we identified and analyzed 17ß-HSD type 3 in zebrafish with respect to its expression pattern and enzymatic activity.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Identification of the zebrafish 17ß-HSD type 3 homolog was carried out by comparison of the human and mouse protein sequences to the zebrafish expressed sequence tag (EST) database at NCBI using the tBLASTn tool (www.ncbi.nlm.nih.gov). EST sequences were selected as putative homologs if either they were already annotated as similar to 17ß-HSD 3 or had an alignment score of >80 bits when their complete putative coding sequence was aligned to the mammalian homologs in the SwissProt database using BLASTx. In this way, two candidate clones were identified and retrieved from the Resource Center, Primary Database (RZPD) of the German Human Genome Project at the Max-Planck-Institute (Berlin, Germany): IMAGp998 N0614301Q3 and IMAGp998 G1614299Q3. Upon sequencing (carried out by SequiServe, Vaterstetten, Germany), the first of these EST clones could be shown to contain the full-length coding sequence while the latter displayed some nucleotide exchanges and lacked the sequence corresponding to exon 2. Only the first clone, IMAGp998N0614301Q3, was considered for further analysis and the annotated sequence was submitted to the GenBank Nucleotide Sequence Database under accession no. AY551081 [GenBank] .
Phylogenetic analysis
The datasets were created by retrieving related sequences from three different sources: a psi-BLAST (Altschul et al. 1997) of the mouse 17ß-HSD 3 protein sequence against the non-redundant protein database at NCBI; a BLink-link sequence of the mouse protein entry in the NCBI database (NP_034605 [GenBank] and NP_032317 [GenBank] ); a translated BLAST sequence (tBLASTn) of the zebrafish 17ß-HSD 3 protein sequence against EST databases of Ciona intestinalis, Caenorhabditis elegans and Drosophila melanogaster. Sequences were aligned by ClustalW (Thompson et al. 1994; www2.ebi.ac.uk/clustalw) and, to avoid redundancy, the alignment was monitored and manually edited in BioEdit (www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogenetic analyses were conducted with MEGA version 2.1 (Kumar et al. 2001 and references therein; www.megasoftware.net) using the neighbor-joining algorithm and 1000 bootstraps. Accession numbers of all selected sequences are as follows: apSPM2 (O57314 [GenBank] ), bn3 KAR (AAO43448 [GenBank] .1), btpSDR (AV601497 [GenBank] and CK836287 [GenBank] ), ceNP505205 (NP_505205 [GenBank] .1), ceNP506448 (NP_506448 [GenBank] .1), ceNP506449 (NP_506449 [GenBank] .1), ciAK116587 (AK116587 [GenBank] .1), ciAL669220 (AL669220 [GenBank] .1), ciBW274894 (BW274894 [GenBank] .1), dmCG6012 (NP_609817 [GenBank] .1), dmRE48687p (AAN71421 [GenBank] .1), ggSDlike (BU137375 [GenBank] ), hsHSD17B12 (NP_057226 [GenBank] .1), hsHSD17B3 (NP_000188 [GenBank] .1), hsSDlike (NP_113651 [GenBank] .3), hv3 KAR (AAB827661.1), mmHSD17B12 (NP_062631 [GenBank] .1), mmHSD17B3 (NP_032317 [GenBank] .1), mmSDlike (CA315224 [GenBank] and BE865064 [GenBank] ), rnHSD17B3 (NP_446459 [GenBank] .1), ssSDlike (BP142073 [GenBank] and BX671798 [GenBank] ), xlHSD17B12 (AAH41194 [GenBank] .1), xlpSDR (BC074162 [GenBank] .1), zfHSD17B12A (AAS58452 [GenBank] ), zfHSD17B12B (AAS58450 [GenBank] ), zfHSD17B3 (AAS58451 [GenBank] ), zfpSDR (AAP74564 [GenBank] ) and zm3 KAR (AAB82767 [GenBank] .1).
RNA preparation and reverse transcriptase (RT)-PCR
Preparation of RNA from zebrafish embryos and adult fish (AB wild-type strain) as well as the reverse transcription into cDNA was performed as described by Mindnich et al.(2004). For RT-PCR, 1 µl cDNA was added to a total reaction volume of 20 µl (1.5 mM MgCl2 and 2.5 U Taq polymerase (New England Biolabs)) and the PCR run on a Robo-Cycler (Stratagene) with one cycle of 3 min at 95 °C and 35 cycles of 30 s at 95 °C, 30 s at 55 °C and 1 min at 72 °C. Zebrafish actin (forward, 5'-CTGGTTGTTGACAACG GATCCG-3'; reverse, 5'-CAGACTCATCGTACTCC TGCTTGC-3') was amplified to monitor cDNA quality. Expression of zebrafish 17ß-HSD 3 was investigated by use of the following primer pair: forward, 5'-AAACAT CGAGGGATTGGATATTGGC-3'; reverse, 5'-TGGC TTCTGATGTCCTGTCATTGC-3'. Each PCR was conducted three to five times including controls without template (water-control) to assure reproducibility.
Cell cultures
All media were supplemented with 10% fetal calf serum (Biochrom), 100 units/ml penicillin G-sodium salt (Invitrogen) and 100 µg/ml streptomycin sulphate (Invitrogen). HeLa cells (ACC57; Deutsche Sammlung von Mickroorganismen und Zellkulturen, Braunschweig, Germany) were maintained in RPMI 1640 medium (Invitrogen) at 37 °C and 5% CO2. HEK-293-Ebna cells (Invitrogen) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 10% fetal bovine serum at 37 ° C and 5% CO2. Zebrafish zf4 cells (Driever & Rangini 1993; LGC Promochem, Germany) were grown in a 1:1 (v/v) mixture of Dulbecco’s (Wessel, Germany) modified Eagle’s medium/Ham’s F12 medium (Invitrogen) at 28 ° C and 5% CO2.
Activity measurements
For activity measurements, the human and zebrafish full-length coding sequences of 17ß-HSD 3 were cloned into the pcDNA3 vector (Invitrogen). Transient transfection of cells in 250 ml cell-culture flasks with 8 µg DNA complexed with 24 µl FuGENE transfection reagent (Roche) in 800 µl medium were carried out as recommended by the supplier. 24 h after transfection, cells were harvested, counted, split into aliquots and the cell pellets frozen at –80 °C until the same lot was subjected to activity measurements with different substrates and cofactors. The aliquots contained 8 x 105 cells in the case of zf4 cells and 3 x 106 cells in case of HEK-293-Ebna cells.
HPLC
One aliquot of frozen cells per measurement was thawed and resuspended in 100 µl reaction buffer (100 mM sodium phosphate, 0.05% BSA and 1 mM EDTA, pH 7.4). 400 µl reaction buffer containing 18.7 nM testosterone (1,2-3H(N); Perkin Elmer) or 13.5 nM androstenedione (1,2,6,7-3H(N); Perkin Elmer) were added. Reactions were started by addition of 50 µl cofactor (5 mg/ml in reaction buffer). After 90 min incubation at 37 °C the reaction was stopped by addition of 100 µl stop solution (0.21 M ascorbic acid in 1% acetic acid in methanol). Samples were extracted on Strata C18-E reverse-phase columns (Phenomenex) and eluted twice with 200 µl methanol. Separation of educt and product in a 20 µl sample was performed through HPLC (Beckman) running isocratic water/acetonitrile (55:45, v/v) on a reverse-phase LUNA 5µ C18 (2) column (Phenomenex) at a flow rate of 1 ml/min. Detection of the tritiated steroids proceeded with an HPLC radioactivity monitor after mixing with scintillation solution (Ready Flow III; Beckman). Integration of the peaks in the HPLC spectra by use of the 32 Karat software (Berthold) yielded the percentage of steroid conversion.
TLC
Per measurement, two aliquots of frozen cells were thawed and resuspended in 100 µl reaction buffer (100 mM sodium phosphate, pH 7.4, 0.05% BSA and 1 mM EDTA). 900 µl reaction buffer and 5 µl of the respective steroid (10 mM) were added and the reaction was started by addition of 50 µl cofactor (5 mg/ml in reaction buffer). Following 4 h of incubation at 37 °C, samples were processed by reverse-phase extraction on Strata C18-E columns as described above. Eluates were vaccum-dried and the steroids resuspended in 30 µl chloroform. Suspensions were spotted on a TLC plate (silica gel 60 F254; Merck) and vials rinsed with 20 µl chloroform, which was also applied to the TLC plate. Steroids were separated in chloroform/methanol (95:5, v/v) and detected by spraying with ethanol/H2SO4 (70:30, v/v) followed by incubating at 140 °C for 4–6 min.
Subcellular localization studies
For localization studies, the human and zebrafish full-length coding sequences of 17ß-HSD 3 were cloned into different LivingColorTM vectors (Clontech) resulting in fluorescent protein-tagged constructs. N-terminal tags were achieved by subcloning into pEGFP-C2; C-terminal tags were constructed by cloning into pEYFP-N1 in the case of the human protein and pECFP-N1 in the case of the zebrafish protein. About 107 cells were plated out in six-well plates containing glass coverslips and transiently transfected with 2 µg DNA/6 µl FuGENE6 as recommended by the supplier (Roche). Medium was changed 6 h after transfection and cells were studied 24 h later. For staining of the endoplasmic reticulum (ER), cells were incubated under growth conditions with fresh medium supplemented with 500 nM ER-Tracker Blue/White (Molecular Probes) for 30 min followed by 5 min incubation at room temperature with fresh medium without dye. Cells were then fixed for fluorescence microscopy by incubation in medium containing 3.7% formaldehyde for 30 min at growth conditions. Cells were washed twice in PBS and once in distilled water and mounted on slides with VectaShield mounting medium (VectorLabs). Samples were analyzed by confocal laser-scanning fluorescence microscopy on a LSM510 Meta microscope with a 63x oil-immersion objective.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In search of zebrafish 17ß-HSD 3 we performed in silico screens on the zebrafish EST database at NCBI with the sequence of the human protein as a probe. Four candidates were retrieved that revealed 40–50% amino acid identity to the human protein. Three of these zebrafish proteins had already been identified in a previous search for 17ß-HSD 3 and were demonstrated not to be the zebrafish homolog (Mindnich et al. 2004). The fourth protein derived from the screen was a new candidate and was thus investigated further. For this sequence, two clones could be ordered directly from RZPD and sequenced, identifying the full-length coding sequence of the putative zebrafish 17ß-HSD 3.
Phylogenetic analysis
Identification of the complete coding sequence of putative zebrafish 17ß-HSD 3 confirmed the close relation to the mammalian homologs by amino acid identities of 45, 44 and 46% to the human, mouse and rat enzymes, respectively. To investigate whether the candidate was indeed the zebrafish 17ß-HSD 3 homolog, a phylogenetic analysis was performed. The resulting dendrogramm (Fig. 1
) revealed a variety of subgroups of different evolutionary origins and a clear separation of 17ß-HSD 3 and 17ß-HSD 12, supported by high bootstrap values. The zebrafish candidate clearly falls into the group of 17ß-HSD 3. Due to this result and further findings (see below), the full-length coding sequence was submitted to the GenBank Nucleotide Sequence Database at NCBI as zebrafish 17 ß-HSD 3 (under accession no. AY551081
[GenBank]
).
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An alignment of the zebrafish protein with the human, mouse and rat homologs (Fig. 2) reveals the functionally important motifs of the SDR family, to which 17ß-HSD type 3 belongs, to be very well conserved. These motifs mediate cofactor binding (GxxxGhG), the catalytic mechanism (GxhhxhSSh and YxASK) and structural integrity (NNAG; Jornvall et al. 1995, Kallberg et al. 2002). Furthermore, residue R80, which in the human 17ß-HSD 3 determines the preference of NADPH over NADH (Geissler et al. 1994, McKeever et al. 2002), is conserved in all four enzymes. Overall amino acid identity is not the same between functionally different parts of the protein. In the preferentially cofactor-binding domain, encompassed by the SDR motifs, about 47% of residues are identical compared with 38% in the part exerting substrate-binding specificity (Fig. 2).
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We tested whether zebrafish 17ß-HSD 3 catalyzes the reduction of androstenedione to testosterone by use of the cofactor NADPH in transiently transfected cells. The zebrafish enzyme indeed catalyzed this reaction with high performance similar to human 17ß-HSD 3 (Fig. 3a). There was a distinct and exclusive preference for NADPH over NADH as cofactor since with the latter no reduction could be detected. HEK-293 and zf4 cells transfected with the vector alone did not show any detectable reductive activity towards androstenedione (data not shown). The reverse reaction was also performed by both enzymes (Fig. 3b) but with an apparently lower efficiency compared with the reduction. Concerning the oxidation, even NAD+ was accepted to some degree, giving rise to 5 and 9% product formation in case of zebrafish and human 17ß-HSD 3, respectively. HEK-293 but not zf4 cells had some weak background activity in the oxidation of testosterone to androstenedione (data not shown), which was considered in data evaluation.
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After measuring a high catalytic activity of zebrafish 17ß-HSD 3 towards the reduction of androstenedione to testosterone we investigated whether other androgenic compounds are accepted as substrates and reduced at C-17. Since these other steroids were not available in tritiated form, the assays were performed with non-radioactive substrates and TLC. The results of these experiments are summarized in Table 1.
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-androstene-3ß,17ß-diol and this reaction was catalyzed by both human and zebrafish 17ß-HSD 3, although the first seemed to be more effective (data not shown). Among the tested 5-reduced androgens, only epiandrosterone was a substrate of both enzymes. Androsterone and androstanedione, though, were reduced at C-17 only by the human but not the zebrafish 17ß-HSD 3. Since it is widely accepted that in fish 11-oxygenated androgens play a more important role than, for example, androstenedione, testosterone and dihydrotestosterone (DHT; Kime 1993, Redding & Patino 1993), we tested whether this type of androgen is a substrate for zebrafish 17ß-HSD 3. Interestingly, 11-ketoandrostenedione as well as 11ß-hydroxyandrostenedione was reduced at C-17, not only by the zebrafish but also by the human enzyme. Zebrafish 17ß-HSD 3 is widely expressed in the adult fish and during embryogenesis
17ß-HSD 3 expression in tissues of male and female adults was investigated by RT-PCR and showed strong sexual dimorphism (Fig. 4a). Transcripts were detected in testis but surprisingly appeared to be present in a much higher degree in ovaries. In male fish the strongest signals were derived from liver. Furthermore, expression was readily detected in male spleen, kidney, heart and intestine whereas in the corresponding female tissues expression was hardly detectable or even absent. The enzyme might also display a sexually dimorphic expression in the skin for only extremely weak signals were obtained from this tissue from males compared with females. 17ß-HSD 3 transcripts could also be detected in muscle, eyes and, to a much lower degree, in the brain of both sexes.
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). The relative amounts, though, might vary between the different developmental stages since observed RT-PCR signals were strongest at the end of somitogenesis (21 somites, 24 h post-fertilization). Zebrafish 17ß-HSD 3 localizes to the ER
We monitored subcellular expression via detection of fluorescent proteins fused to the human and zebrafish enzymes transiently expressed in HeLa and zf4 cells, respectively (Fig. 5). 17ß-HSD 3 tagged at the C-terminus showed a network-like expression centered around and extending from the nucleus which was reminiscent to the structure of the ER (Fig. 5a and c). In case of N-terminally tagged 17ß-HSD 3 (Fig. 5b and d), localization was much more diffuse, reaching further into the cytoplasm but not fully occupying the cytoplasmic area. This localization pattern was distinctly different from that produced by C-terminally tagged proteins and as well from the localization of the fluorescent protein alone, which was equally distributed in the cytoplasm and nucleus (data not shown). Staining of the ER in HeLa cells (Fig. 5 g and h) and zf4 cells (Fig. 5e and f) by ER-Tracker dye revealed patterns similar to those of the C-terminally tagged proteins and produced a complete overlap with the latter expression pattern (Fig. 5i and k). Concerning N-terminally tagged 17ß-HSD 3 though, the staining was rather dissimilar to that of the ER (Fig. 5j and l). Only very small overlaps were detected close to the nucleus in case of the zebrafish enzyme.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The function of testosterone has been investigated in many vertebrates but apart from mammals only little is known about the molecular machinery controlling the formation of this steroid. 17ß-HSD 3 has been suggested to be the major enzyme catalyzing the reduction of androstenedione to form testosterone and resulting in pseudohermaphroditism upon loss-of-function mutations in humans (Geissler et al. 1994). No non-mammalian 17ß-HSD 3 has been characterized so far and we investigated whether the zebrafish homolog might play a similar role to that in mammals.
Screening the EST database at NCBI extracted several putative 17ß-HSD 3 candidates and led to the identification of a single zebrafish homolog that shared about 45% amino acid identity with the mammalian enzymes. Phylogenetic analyses indicate that 17ß-HSD type 3 is closely related to 17ß-HSD type 12 and to 3-ketoacyl reductases. 17ß-HSD type 12 is indeed the human homolog of yeast YBR159p, a 3-ketoacyl-CoA reductase involved in the elongation reaction to generate very-long-chain fatty acids (Moon & Horton 2003). It is as yet unknown whether 17ß-HSD 12 also accepts steroids as substrates. Although its substrate specificity is different, 17ß-HSD 3 still shares some typical aspects with its fatty acid-metabolizing relative. Elongation of very-long-chain fatty acids, for example, takes place at the ER membrane and, concordantly, human 17ß-HSD 12 and the yeast homolog localize to this compartment via an N-terminal localization signal and a subsequent transmembrane helix (Han et al. 2002, Moon & Horton 2003). We found this to be true as well for zebrafish 17 ß-HSD 3, where a stretch of about 50 amino acids preceding the presumed start of the cofactor-binding site may facilitate the direction to and anchoring in the ER membrane. The presence of an N-terminal localization signal might also explain why addition of fluorescent protein to the C-terminus lead to localization at the ER membrane, whereas in the case of the N-terminal tag the putative localization signal seems to have been masked, leading to predominant localization to the cytosol and not the ER. The widespread expression of 17ß-HSD 3, as observed in case of zebrafish, may also be inherited from the ancestor 17ß-HSD 12, which is expressed ubiquitously (Moon & Horton 2003). Although human 17ß-HSD 3 expression is often described as testis-specific, mRNA or protein have also been detected in adipose tissue (Corbould et al. 2002), brain (Stoffel-Wagner et al. 1999, Beyenburg et al. 2000) and bone (Feix et al. 2001). The murine homolog is expressed in a variety of male and female tissues (Sha et al. 1997).
Aside from 17ß-HSD 3, other types of 17ß-HSD are also able to catalyze the reduction of androstenedione to testosterone. In human, 17ß-HSD 5 is widely expressed (Lin et al. 1997, Dufort et al. 1999, Qin & Rosenfield 2000) and accepts similar substrates as 17ß-HSD 3. In mouse, 17ß-HSD 5 is predominantly present in liver (Rheault et al. 1999) but murine 17ß-HSD 1 has been demonstrated to convert androstenedione to testosterone with similar efficiency to 17ß-HSD 3 (Nokelainen et al. 1996) and is expressed in many different organs (Sha et al. 1997). The ability to form testosterone by reduction of androstenedione might be present almost ubiquitously in the vertebrate organism, as was also suggested by measurements of androgenic activity in several tissues of rat (Martel et al. 1992) and monkey (Labrie et al. 1997). On the molecular level though, employment of 17ß-HSD 3 in this context might have evolved differently and be species-specific.
In zebrafish at least, widespread expression of 17ß-HSD 3, for example during embryogenesis and in male adults, may also indirectly serve in the formation of estradiol. Here, 17ß-HSD 1 was found not to be responsible for provision of estradiol, as it could not be detected in the respective tissues (Mindnich et al. 2004). Since aromatase is present during embryogenesis (Kishida & Callard 2001, Trant et al. 2001) and in the male zebrafish (Goto-Kazeto et al. 2004), estradiol could be formed via aromatization of testosterone generated from androstenedione by reductive activity of 17ß-HSD 3.
Enzymatic properties of human and zebrafish 17ß-HSD 3
In our experiments, several structurally different androgens, such as DHEA, epiandrosterone and 11-ketotestosterone, were substrates of human and zebrafish 17ß-HSD 3. But concerning 5-reduced compounds there was a marked difference between these two enzymes since only the human protein was able to reduce androsterone and androstanedione. Comparison of the three-dimensional structure of all measured substrates shows that, in all cases, where the zebrafish 17ß-HSD 3 accepts and converts the steroid, this keto-or hydroxy-group at C-3 is in or above the plane formed by the A-ring, as can be seen in androstenedione, DHEA and epiandrosterone. In androstanedione and androsterone though, the keto- and hydroxy-group are below this plane. Unfortunately, no crystal structure of 17ß-HSD 3 is so far available through which this theory of stereo-specific interaction with the C-3 oxy-group can be investigated further. Taking into account the general architecture of SDRs the most C-terminal part of the enzyme likely conveys recognition and stabilization of this part of the steroid. In this region, zebrafish 17ß-HSD 3 lacks at least four amino acids, including an arginine, conserved in the mammalian homologs (see also Fig. 2). This deviation may result in a structural difference and subsequent loss of stabilization of steroids where the oxy-group at C-3 is oriented below the steroid plane. That certain DHT derivatives are not substrates of zebrafish 17ß-HSD 3 may be because they do not have any important functions in fish compared with mammals. It has been generally accepted that, instead of DHT, 11-ketotestosterone is biologically the most important androgen in teleosts (Kime 1993), which was supported by the findings of Miura et al.(1991) that 11-ketotestosterone by itself induced complete spermatogenesis in the Japanese eel. DHT has been detected in several teleost species (Kime 1993) and teleost androgen receptors may sometimes display an affinity to 5-reduced androgens similar to or even greater than to 11-oxygenated androgens (Ikeuchi et al. 1999, Sperry & Thomas 2000, Braun & Thomas 2004). In the case of zebrafish, there are no data available on the true molecular character of the biologically active androgens and the androgen receptor itself has not been characterized so far. Our finding that at least some 5-reduced androgens are no longer substrates of 17ß-HSD 3 in zebrafish, suggest these androgens to be of minor importance for this organism or, alternatively, that this specific enzymatic function has been taken over by another, as-yet unidentified, 17ß-HSD.
Surprisingly, some androgens that might be considered fish-specific, such as 11ß-hydroxyandrostenedione and 11-ketoandrostenedione, we found to be substrates not only of zebrafish but also of human 17ß-HSD 3. In fish, this may contribute to, but not directly lead to, the formation of 11-ketotestosterone, which is considered to be formed by 11ß-hydroxysteroid dehydrogenase (11ß-HSD) from 11ß-hydroxytestosterone (Kusakabe et al. 2003). 11-Oxygenated androgens are also present in mammals (Oertel & Eik-Nes 1962, Kecskes et al. 1982) due to the activity of 11ß-hydroxylase. Classically, this enzyme is part of the corticoid metabolism leading to the formation of corticosterone and cortisol. 11ß-HSD catalyzes the interconversion of cortisol and cortisone, regulating glucocorticoid activity. 11ß-Hydroxytestosterone and 11-ketotestosterone can inhibit the oxidative and reductive functions of 11ß-HSD, respectively, and thereby may influence the local formation of glucocorticoids (Wang et al. 2002). Both modified androgens are significantly stronger inhibitors than testosterone (Wang et al. 2002), so that production of these steroids by 17ß-HSD 3 may be of physiological importance in the crosstalk between glucocorticoid and androgen metabolism.
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Acknowledgements |
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References |
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Received 5 July 2005
Accepted 8 July 2005
Made available online as an Accepted Preprint 29 July 2005
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