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Endocrinology Vol. 145, No. 11 5221-5230
Copyright © 2004 by The Endocrine Society

Molecular and Functional Characterization of Sex Hormone Binding Globulin in Zebrafish

Solange Miguel-Queralt, Michelle Knowlton, George V. Avvakumov, Rana Al-Nouno, Greg M. Kelly and Geoffrey L. Hammond

Department of Obstetrics and Gynaecology (S.M.-Q., G.L.H.), University of British Columbia, and B.C. Research Institute for Children’s and Women’s Health, Vancouver, Canada; and Canadian Institutes of Health Research Group in Fetal and Neonatal Health and Development (G.V.A., R.A.-N., G.L.H.), Departments of Biology and Pediatrics (M.K., G.M.K.), and Child Health Research Institute (G.M.K.), University of Western Ontario, London, Ontario, Canada

Address all correspondence and requests for reprints to: Geoffrey L. Hammond, Ph.D., British Columbia Research Institute for Children’s and Women’s Health, 950 West 28th Avenue, Vancouver, British Columbia, Canada V5Z 4H4. E-mail: ghammond{at}cw.bc.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
SHBG (sex hormone binding globulin) transports androgens and estrogens in the blood of vertebrates including fish. Orthologs of SHBG in fish are poorly defined, and we have now obtained a zebrafish SHBG cDNA and characterized the zebrafish SHBG gene and protein through molecular biological, biochemical, and informatics approaches. Amino-terminal analysis of zebrafish SHBG indicated that its deduced precursor sequence includes a 25-residue secretion polypeptide and exhibits 22–27% homology with mammalian SHBG sequences and 41% with a deduced fugufish SHBG sequence. The 356-residue mature zebrafish SHBG (39,243 Da) sequence comprises a tandem repeat of laminin G-like domains typical of SHBG sequences; contains three N-glycosylation sites; and exists as a 105,000 ± 8700 Da homodimer. Zebrafish SHBG exhibits a high affinity and specificity for sex steroids. An RT-PCR indicated that SHBG mRNA first appears in zebrafish larva, and SHBG mRNA was localized within the liver and gut at this stage of development by whole-mount in situ hybridization. In adult fish, SHBG mRNA was found in liver, testis, and gut. In the liver, immunoreactive SHBG was present in hepatocytes and concentrated in intrahepatic bile duct cells, whereas in the testis it was confined to cells surrounding the seminiferous tubule cysts. In the intestine, immunoreactive SHBG was present in the stroma and epithelial cells of the villous projections and the surrounding muscle. The production and presence of SHBG in the gut of developing and adult zebrafish suggests a novel role for this protein in regulating sex steroid action at this site.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SHBG (SEX HORMONE BINDING globulin) IS THE MAJOR TRANSPORT protein for sex steroids in the blood of a wide variety of mammals and other vertebrate species, including fish (1). Plasma SHBG is produced in the liver and regulates the bioavailability and metabolic clearance of its steroid ligands in the blood (2), but mammalian SHBG genes are expressed in tissues other than the liver, and this suggests that SHBG may act differently outside the blood system (3). For instance, the testis produces an SHBG homolog, known as the testicular androgen-binding protein, which is secreted into the seminiferous tubular lumen and regulates androgen availability in the male reproductive tract (4). The human SHBG gene is also expressed in the germ cells of the testis, and an SHBG isoform with unknown function accumulates in the sperm acrosome (5). During fetal development in mammals, there is a transient burst of SHBG gene expression in the liver that coincides with critical stages of reproductive organogenesis (6, 7), and there is some indication that it is expressed in the fetal gut (7). Changes in the tissue-specific expression of SHBG are therefore likely to have a significant impact on the actions of sex steroids during development, and our knowledge of this critical component of sex steroid action in fish is lacking.

Although SHBG can be detected in the blood of fish, little is known about the structure of fish SHBGs or the site(s) of SHBG expression in this vertebrate class. Studies of the SHBG steroid-binding characteristics in several fish species (8, 9, 10, 11, 12, 13, 14, 15) have shown that its affinity for endogenous sex steroids (testosterone and estradiol), and xenobiotics (13, 16, 17, 18, 19) varies between species. There is also some evidence that plasma SHBG levels fluctuate in fish during the reproductive cycle (20, 21, 22), but it is not known where SHBG is produced in fish or how expression of the SHBG gene is regulated. Information about SHBG gene expression during early fetal development in mammals is also limited (7), and zebrafish represent a powerful model system for studying the temporal and spatial expression of specific genes during development (23). We have therefore cloned a zebrafish SHBG cDNA, and this has allowed us to characterize a SHBG molecule in this nonmammalian vertebrate species at both the biochemical and molecular levels. We have also defined the sites of SHBG gene expression in zebrafish throughout development and studied the cellular distribution of SHBG in adult zebrafish tissues by immunohistochemistry.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Zebrafish (Danio rerio) were housed in dechlorinated tap water at 28 C and kept on a 12-h light, 12-h dark cycle. Embryos were collected 15 min after the beginning of a light cycle, rinsed in Instant Ocean (Aquarium Systems Inc., Mentor, OH) and incubated at 29.5 C. Animals were maintained under the guidelines of the Canadian Council of Animal Care.

Steroids
[3H]Testosterone (55 Ci/mmol), [3H]estradiol (41 Ci/mmol), and [3H]5{alpha}-dihydrotestosterone (40 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). Unlabeled steroids were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada) or Steraloids Inc. (Wilton, NH) and used as supplied.

Cloning and DNA sequence analysis
Total RNA isolated from adult zebrafish using TRIzol Reagent (Invitrogen Canada Inc., Burlington, Ontario, Canada) was used as template in a RT-PCR to amplify an approximately 789-bp fragment (forward primer, 5'-TCTGTGCAGGAGAGCAGCAGGTG; reverse primer, 5'-CGGTTGATCCATCTTTCAGGCGG). These two primers were designed based on two expressed sequence tag (EST) sequences (fb66h07.y1 and fb67f10.y1) in the zebrafish EST database (www.genetics.wustl.edu/fish_lab/frank/cgi-bin/fish). The full-length zebrafish SHBG cDNA sequence was then obtained by 5' rapid amplification of cDNA ends (RACE) and a 3' RACE using the SMART RACE cDNA amplification kit (BD Biosciences Canada, Mississauga, Ontario, Canada) protocol. Major PCR products were cloned into a pCR-Blunt II-Topo vector (Invitrogen) and sequenced using a DNA sequencing kit or an ALF DNA sequencer (Amersham Pharmacia Biotech Inc., Baie d’Urfé, Quebec, Canada). The resulting DNA sequences were compared by BLAST analysis of mammalian SHBG sequences in the NCBI GenBank database (http://www.ncbi.nlm.nih.gov) and the zebrafish genome database (http://www.sanger.ac.uk/Projects/D_rerio).

Expression of zebrafish SHBG in Chinese hamster ovary cells
The coding region of the zebrafish SHBG cDNA was inserted into pRc/cytomegalovirus eucaryotic expression vector (Invitrogen). The resulting plasmid was used for transfection of Chinese hamster ovary (CHO) cells using LipofectAMINE reagent (Invitrogen) according to the protocol suggested by the manufacturer. After selection in the presence of Geneticin (Invitrogen), stably transformed cells were grown to near confluence, washed twice with PBS to remove fetal bovine serum, and then cultured in HyQ PF-CHO LS medium (HyClone, Logan, UT) for 3–5 d.

Analytical gel filtration chromatography
Gel filtration chromatography of concentrated (Ultrafree 15 with a 30-kDa cut-off limit, Millipore, Billerica, MA) cell culture medium containing zebrafish SHBG was performed using a fast protein liquid chromatography (FPLC) system (Amersham Pharmacia Biotech). Samples (150 µl) were loaded onto a 1 x 30 cm Superdex 75 column equilibrated with 20 mM Tris-HCl (pH 7.0), 250 mM NaCl, 0.05% NaN3, and calibrated using gel filtration marker proteins (Amersham Pharmacia Biotech). Elution was performed with the same buffer, and 250-µl fractions were collected and analyzed for steroid binding activity (24).

Zebrafish SHBG purification, characterization, and antibody production
Culture medium (30 dishes, each containing 30 ml HyQ PF-CHO LS) from CHO cells expressing zebrafish SHBG cDNA was concentrated by ultrafiltration through a PM-30 membrane (Millipore) and dialyzed overnight at 4 C in Slide-A-Lyzers (Sigma-Aldrich) against 20 mM Tris-HCl (pH 8.0) and 0.05% NaN3. The dialyzed solution was loaded onto an FPLC column packed with 8 ml anion-exchange resin SOURCE 15Q (Amersham Pharmacia Biotech) and equilibrated with 20 mM Tris-HCl (pH 8.0) and 0.05% NaN3. Elution was performed using a linear (0–0.25 M) gradient of NaCl in the same buffer. Fractions containing SHBG were identified using a steroid-binding capacity assay (24), combined, and used for steroid-ligand affinity chromatography (25) using an elution buffer containing 250 mg testosterone/liter. The SHBG-containing fractions were combined, concentrated, and subjected to gel filtration on a Sepharose 6 FPLC column, and fractions containing SHBG were identified by a steroid-binding capacity assay (24), as described below. Final purification of zebrafish SHBG was achieved by preparative PAGE under nondenaturing conditions using a model 491 Prep Cell (Bio-Rad Laboratories, Hercules, CA), and fractions containing pure zebrafish SHBG were identified by SDS-PAGE, and staining with Coomassie Brilliant Blue. Zebrafish SHBG was deglycosylated using N-glycosidase F following the protocol supplied by the enzyme manufacturer (Roche Diagnostics, Basel, Switzerland) and analyzed in the same way. An amino-terminal protein sequence analysis of pure zebrafish SHBG was performed using an ABI 492 Procise cLC sequencer in the UVic-Genome BC Proteomics Centre (http://www.proteincenter.com). Pure zebrafish SHBG (500 µg) was also used to raise antisera in a rabbit using a standard immunization protocol approved by the Animal Care Committee of the University of Western Ontario.

Steroid-binding assays
The steroid-binding capacity of zebrafish SHBG isolated from CHO cell cultures or diluted (1:200) zebrafish plasma was determined by saturation analysis using [3H]testosterone or [3H]estradiol as labeled ligands and dextran-coated charcoal (DCC) to separate bound and free steroid (24). The apparent dissociation rates of zebrafish SHBG-bound steroid ligands ([3H]testosterone, [3H]estradiol, and [3H]5{alpha}-dihydrotestosterone) were assessed by exposure of the tritium-labeled steroid-protein complex to DCC for increasing time intervals at 0 C. Equilibrium dissociation constants of zebrafish SHBG ligand interactions were measured by Scatchard analysis, and relative binding affinities for different steroids were determined using [3H]testosterone as labeled ligand and varying concentrations of unlabeled steroids as competitors (24).

RT-PCR
To detect SHBG mRNA in developing zebrafish and adult zebrafish tissues, RT-PCR assays were performed using zebrafish SHBG-specific forward (5'-GTGCTTTCACTGCGTGATGGC) and reverse (5'-TCCCAGGGGGTGCTGAG) primers that amplify a 332-bp region within coding sequence for zebrafish SHBG. Total RNA extracts from embryo, larva, juvenile, or adult tissues were reverse transcribed into single-stranded DNA using oligo(dT) primer and SuperScript II, and the single-stranded cDNA product was used in a standard PCR using Taq polymerase (Invitrogen). After amplification, an aliquot of the PCR mixture was analyzed by agarose gel electrophoresis and staining with ethidium bromide to confirm the presence of a single amplicon of the expected size. The amplicon was sequenced to confirm its identity. Amplification of a ribosomal RNA sequence was performed under the same conditions as an RT-PCR control.

Whole-mount in situ hybridization
Based on the RT-PCR screen for the onset of SHBG expression in developing zebrafish (see above), d 4 and 5 larvae were collected and prepared for whole-mount in situ hybridization (26). These were incubated in hybridization solution containing sense or antisense digoxigenin probes generated using the Maxiscript kit (Ambion Inc., Austin, TX). Briefly, larvae were washed extensively to remove unbound probe; blocked in 2 mg/ml BSA, 5% goat serum, 5% dimethyl sulfoxide, and PBS containing 0.01% Tween 20; and then incubated overnight at 4 C in blocking solution containing anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche). After an extensive wash in NTMT [100 mM NaCl, 100 mM Tris HCl (pH 9.5), 50 mM MgCl2, and 0.1% Tween 20], larvae were incubated in BM Purple (Roche). After the color reaction, larvae were fixed in PBS containing 4% paraformaldehyde, transferred to methanol, and then cleared in benzylbenzoate/benzyl alcohol before examination using differential interference contrast microscopy. Photomicrographs were taken using a Wild MPS52 camera (Leica, Québec, Canada) mounted on a DMRBE microscope (Leica), and images were captured with a digital camera.

Western blot analysis
Diluted zebrafish serum and pure native and deglycosylated recombinant zebrafish SHBG were heat denatured in loading buffer and subjected to discontinuous SDS-PAGE with 4 and 10% polyacrylamide in the stacking and resolving gels, respectively. Proteins in the gel were transferred (27) to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech). The membranes were blocked using a 5% skim milk solution and then incubated 1 h at room temperature with an antizebrafish SHBG antiserum diluted (1:800) in PBS containing 0.01% Tween 20 and 5% skim milk powder. The blots were then washed several times with PBS containing 0.01% Tween 20 to remove excess antiserum, and specific antibody-antigen complexes were identified using secondary antibodies (horseradish peroxidase-conjugated goat antirabbit IgG) and chemiluminescent peroxidase substrate reagents (Amersham Biosciences) by exposing the nitrocellulose membrane to x-ray film.

Immunohistochemistry
Adult zebrafish were fixed in PBS containing 4% paraformaldehyde at 4 C for 24 h and subsequently dehydrated with a series of ethanol solutions and embedded in paraffin. Paraffin sections were dewaxed and treated with proteinase K (20 µg/ml) in PBS. The sections were then treated with 0.03% hydrogen peroxide solution for 7 min and blocked (room temperature for 1–2 h) with BSA (5 mg/ml) in PBS. The primary antibody for zebrafish SHBG was first diluted (1:7500) in either culture medium harvested from wild-type CHO cells or medium from CHO cells that express sufficient quantities zebrafish SHBG to block the anti-SHBG antibodies. This was done to confirm the specificity of the anti-zebrafish SHBG antibodies. After an overnight incubation at 4 C with either the unblocked or blocked anti-zebrafish SHBG antisera, immunoreactive zebrafish SHBG was detected using the EnVision + System, horseradish peroxidase (diaminobenzidine) from Dako (Carpinteria, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Molecular cloning of the zebrafish SHBG gene
Screening the zebrafish EST database revealed two EST sequences that displayed significant coding sequence homologies to human SHBG and rat androgen-binding protein (see above). Based on the sequences of these ESTs, zebrafish SHBG-specific oligonucleotide primers were used in a RT-PCR to obtain a 789-bp zebrafish SHBG cDNA. Additional oligonucleotide primers were then used to obtain a more complete 1.4-kb zebrafish cDNA sequence by using 5' and 3' RACE methods (Fig. 1Go). This sequence contained an open reading frame for the 381-amino acid-residue zebrafish SHBG precursor polypeptide. To define the amino terminus of the mature SHBG sequence, recombinant zebrafish SHBG produced in CHO cells was subjected to amino-terminal sequence analysis. The resulting amino-terminal sequence (DQISGR) indicates that a 25-residue leader sequence must be removed from the zebrafish SHBG precursor to yield a 39,243-Da polypeptide of 356 amino acids, which includes two laminin G-like (LG) domains and three N-linked glycosylation sites (Fig. 1Go).



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  		FIG. 1. Full-length cDNA and deduced amino acid sequences of the zebrafish SHBG. The amino acids corresponding to the signal peptide and the putative polyadenylation signal are underlined. The stop codon is marked with asterisks. The nucleotide number is shown on the left side, and amino acid residue number is on the right in relation to the amino terminus of the mature polypeptide. Empty circles indicate the N-glycosylation sites. The two LG domains are marked on the right side.

 
We used the zebrafish SHBG cDNA sequence to screen the fugufish genome database, and this allowed us to deduce the coding sequence for the fugufish SHBG precursor polypeptide (Fig. 2Go). Comparison of the zebrafish and fugufish SHBG sequences reveal 41% sequence identity between them, with the amino-terminal LG domain being the most highly conserved (50% sequence identity) region. In addition, the locations of N-linked glycosylation sites were perfectly conserved between the two fish SHBG sequences (Fig. 1Go).



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  		FIG. 2. Amino acid sequence comparison between zebrafish SHBG (zfSHBG), fugufish SHBG (ffSHBG), and human SHBG (hSHBG). The three N-glycosylations sites present in zebrafish and fugufish SHBGs are indicated with empty circles. The cysteine residues that form intramolecular disulfide bridges within the two LG domains and the conserved residues important for the steroid-binding site are shown in bold. Sequence alignment was obtained using the Clustal method. Gaps were introduced to maximize sequence homologies. The amino acids corresponding to the signal peptides are underlined. Residues that are identical or conserved are marked by asterisks and dots, respectively.

 
To identify other structurally and/or functionally important regions of the fish SHBG sequences, we compared them with the human SHBG primary structure (Fig. 2Go). This further illustrated the relatively high level of amino acid sequence identity within their amino-terminal LG domains as well as the cysteine residues that are conserved structural elements of the carboxy-terminal regions of LG-domain modules (28). Remarkably, the N-linked glycosylation sites in the SHBGs of humans and other mammalian species are confined to the caboxy-terminal LG domain. Whereas at least two of the three N-linked glycosylation sites in zebrafish and fugufish SHBGs are clearly located within their amino-terminal LG domains, the third N-linked glycosylation site in these fish species is located between the two LG domains (Fig. 2Go). The most highly conserved region of all three SHBG sequences spans a portion of the amino-terminal LG domain that is known to comprise the steroid-binding site (29), and within this region several key structural elements of the steroid-binding site are invariably conserved (Fig. 2Go). These include a serine residue (Ser42 in human SHBG) and an aspartic acid residue (Asp65 in human SHBG) that hydrogen bond with the functional groups at C3 and C17 of sex steroids that bind SHBG with high affinity (29, 30).

Comparison of the zebrafish cDNA sequence against the zebrafish genome database (http://www.sanger.ac.uk/Projects/D_rerio) has also allowed us to define the structural organization of the zebrafish SHBG gene (Fig. 3Go). This indicated that the transcription unit encoding zebrafish SHBG spans 12,945 bp, and contains eight exons of 81–189 bp and intron sequences of 78–5,108 bp in length. The sequences of intron-exon boundaries conform to consensus sequences for 5'-donor and 3'-acceptor splice sites and follow the GT/AG rule (Fig. 3Go).



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  		FIG. 3. Genomic organization of the zebrafish SHBG gene. A, Diagram of the zebrafish SHBG gene structure. The exons are numbered and are shown in relation to their sizes as black boxes. The region encoding zebrafish SHBG is distributed over eight exons and spans 13 kb of genomic DNA. B, Zebrafish SHBG exon and intron sizes and junction sequences. The intron-exon junctions revealed no deviation in 5'-donor and 3'-acceptor sequences; all splice junctions followed the GT/AG rules. Consensus splice sites are in bold. Exon and intron sequences are shown in upper and lowercase letters, respectively.

 
Biochemical analysis of zebrafish SHBG
To circumvent the difficulty in obtaining sufficient amounts of zebrafish blood for detailed analysis of SHBG in this species, the zebrafish SHBG cDNA was inserted into the pRc/cytomegalovirus eucaryotic expression vector and expressed in CHO cells. Stably transfected cells were cloned by limiting dilution, and cell culture medium from clones expressing high levels of SHBG was collected and used for biochemical analysis as well as purification of the protein.

Gel filtration analysis of CHO medium containing zebrafish SHBG indicates that the hydrodynamic properties of zebrafish SHBG correspond to a molecular mass of 105,000 ± 8700 Da (Fig. 4AGo), and this is consistent with the presence of a homodimer of 39.2-kDa subunits that comprise up to three N-linked oligosaccharide chains. This was confirmed by SDS-PAGE analysis of purified zebrafish SHBG, which revealed the apparent molecular mass sizes of the intact (~52 kDa) and deglycosylated (~40 kDa) subunits (Fig. 4BGo).



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  		FIG. 4. A, Gel filtration of zebrafish SHBG. Specific binding of [3H]testosterone was determined in the fractions eluted from a Superdex 75 column. Arrows indicate positions of the peaks of marker proteins, BSA (67 kDa), and catalase (232 kDa). B, Western blot of zebrafish serum (dilution 1:400) and pure zebrafish SHBG before (–) and after (+) treatment of N-glycosidase F to remove N-linked oligosaccharides. The positions of protein size markers are shown on the left.

 
The apparent dissociation rates of zebrafish SHBG-bound [3H]testosterone, [3H]estradiol, and [3H]5{alpha}-dihydrotestosterone (DHT) were assessed by exposure to DCC for increasing time intervals at 0 C (Fig. 5AGo). [3H]Testosterone was found to dissociate most slowly from the zebrafish SHBG steroid-binding site. The preferential binding of testosterone to zebrafish SHBG was confirmed by Scatchard analysis (Fig. 5Go, B and C), which clearly demonstrated that zebrafish SHBG binds testosterone (dissociation-rate constant = 1.8 nM) with slightly higher affinity than estradiol (affinity constant = 2.2 nM). We therefore selected [3H]testosterone as the preferred labeled ligand for a comprehensive analysis of the steroid-binding specificity of zebrafish SHBG.



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  		FIG. 5. A, Dissociation of steroids from recombinant zebrafish SHBG. Dissociation was measured by monitoring the effect of the duration of DCC exposure time on the removal of [3H]DHT, [3H]estradiol, and [3H]testosterone from the zebrafish SHBG steroid-binding site. The DCC was added at 0 C to assay tubes, and the mixtures were incubated at 0 C for the times indicated before centrifugation. B, Scatchard analysis of the binding of [3H]testosterone to zebrafish SHBG. C, Scatchard analysis of the binding of [3H]estradiol to zebrafish SHBG.

 
Zebrafish SHBG binds selectively several physiologically important androgens and estrogens and has a modest affinity for progesterone (Table 1Go). These data indicate that androstenedione has approximately the same affinity for zebrafish SHBG as testosterone and confirm the lower affinities for estradiol and DHT when compared with testosterone. The biologically active piscine androgen, 11-ketotestosterone, showed only moderate affinity to the zebrafish SHBG (about 10-fold less than testosterone and 3- to 4-fold lower than estradiol), whereas the major piscine androgen metabolites (11-hydoxyandrostenedione and 11-hydroxytestosterone) bind with even lower affinities than 11-ketotestosterone. Unlike SHBG in several mammalian species (1, 31), zebrafish SHBG does not bind 2-methoxyestradiol (Table 1Go).


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  		TABLE 1. Relative binding affinities (RBAs) of different steroids for zebrafish SHBG

 
Plasma SHBG levels were also determined in male (n = 5) and female (n = 9) zebrafish using a saturation ligand-binding assay in which [3H]estradiol was used as labeled ligand (24). The results indicate that the mean ± SD plasma levels of SHBG in male (191 ± 54 nM) and female (178 ± 46 nM) zebrafish are not different.

Spatial and temporal expression of zebrafish SHBG mRNA during zebrafish development
We analyzed the spatial and temporal expression of the zebrafish SHBG gene during zebrafish development using a combination of RT-PCR and whole-mount in situ hybridization approaches. The temporal expression profile of zebrafish SHBG mRNA was first examined by a RT-PCR assay. Zebrafish SHBG cDNA amplicons of the expected size (332 bp) were not detected in embryos (data not shown) but were first detected in d 5 and d 6 larval fish (Fig. 6AGo). Zebrafish SHBG RT-PCR-generated amplicons were also detected in juvenile fish (data not shown). Whole-mount in situ hybridization was used to explore the tissue distribution of SHBG mRNA in d 5 zebrafish larva, and the antisense riboprobe revealed that SHBG transcripts are confined to the liver and gut (Fig. 6BGo). When the tissue-specific expression of the zebrafish SHBG gene was examined in adult tissues, RT-PCR amplicons were readily detected using RNA extracts from liver and gut but not from brain, heart, and ovary, whereas low levels of RT-PCR amplicons were produced from testes RNA extracts (Fig. 6CGo).



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  		FIG. 6. Temporal and spatial accumulation of SHBG mRNA in zebrafish. A, Zebrafish SHBG transcripts where first detected by RT-PCR in a d 5 larval fish and were present in all developmental stages thereafter including adult fish. B, In situ hybridization with an antisense SHBG riboprobe localized SHBG mRNA to the liver (L) and gut (G). C, This localized pattern of expression was confirmed by RT-PCR, using first-strand cDNA generated from RNA isolated from adult, brain, gut, heart, liver, ovary, and testes. Relatively low amounts of SHBG amplicon were obtained using testis RNA, whereas larger quantities were routinely obtained using gut and the liver RNA as template in the RT-PCRs. The performance of RT-PCR was monitored by using primers to the constitutively expressed L37A ribosomal RNA (A and C, bottom panels). To control for genomic contamination, RNA subjected to RT-PCR in either the presence (+) or absence (–) of reverse transcriptase (A). In C, the RT-PCR was performed using RNA (+) or water containing no RNA (–).

 
Localization of SHBG in adult zebrafish tissues
The antiserum against purified zebrafish SHBG was initially evaluated by examining a serum sample in a Western blotting experiment, and this indicated that it recognizes only the fully glycosylated protein and a minor protein band that corresponds in size to unglycosylated zebrafish SHBG (Fig. 4BGo). Blocking this antiserum with recombinant zebrafish SHBG was therefore also used to confirm its specificity during immunohistochemistry (Fig. 7Go, B, D, and F). In the liver (Fig. 7AGo), low levels of immunoreactive SHBG (brown staining) could be detected within hepatocytes, but more intense staining was present within the luminal aspects of cells lining intrahepatic bile ducts. In the testis (Fig. 7CGo), the SHBG-positive staining (brown) surrounded the seminiferous tubule cysts. In the adult zebrafish intestine (Fig. 7EGo), immunoreactive SHBG was present throughout the surrounding muscles as well as in the stroma of the intestinal villous fold and the epithelial cells. In the epithelial cells, the staining for SHBG exhibited a punctuate appearance under higher-power magnification, suggesting its presence in granules.



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  		FIG. 7. Immunohistochemical localization of SHBG in adult male zebrafish tissues. Serial sections were probed with rabbit antizebrafish SHBG antisera (A, C, and E) or the same dilution of the antisera blocked with recombinant zebrafish SHBG (B, D, and F). Controls using preimmune rabbit serum were also performed, and these were uniformly negative. A shows the liver and part of the intestine, with an arrow pointing to the intense immunoreactivity (brown staining) within the luminal aspect of bile ducts. C shows the testis with spermatogonia (sg) and sperm (sp) identified. E shows the intestine. All images were acquired at x40 magnification.

 
Other sites in which specific immunoreactive SHBG was concentrated in zebrafish tissues included the muscle, particularly the fin muscle, and what appears to be mucous cells of the skin (data not shown). Although SHBG mRNA cannot be detected in skeletal muscle by RT-PCR, low levels are present in skin (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Genome and EST databases for model organisms, such as zebrafish and fugufish, represent an invaluable resource, but they are incompletely annotated. Partial zebrafish and fugufish SHBG coding sequences are available within public databases, and we combined this information with conventional molecular biological approaches to obtain the complete open reading frames for the SHBG precursor polypeptides in both these fish species. Amino-terminal sequence analysis of recombinant zebrafish SHBG produced in CHO cells also allowed us to define the amino terminus of the mature SHBG polypeptide and deduce the position at which cleavage of the hydrophobic leader sequence for secretion occurs. The overall coding sequence identity between fish and mammalian SHBG genes is relatively poor, and this has undoubtedly hampered previous attempts to clone piscine SHBG cDNAs using conventional approaches. However, close inspection of the zebrafish, fugufish, and human SHBG sequences indicate that these are structurally related proteins, which comprise two LG domains, each with conserved cysteine residues that participate in intramolecular disulfide bridges. It is also evident that key residues within the human SHBG steroid-binding site, which participate in hydrogen bonding with functional groups of androgens and estrogens, are perfectly conserved in both zebrafish and fugufish SHBG sequences.

Like SHBGs in mammalian species, fish SHBG is a glycoprotein. The N-glycosylation of plasma proteins is generally assumed to influence their biological half-lives, but it has also been suggested that a highly conserved consensus site for N-glycosylation in the carboxy-terminal LG domain of mammalian SHBGs has some other functional role (32). It was therefore surprising that this site is not conserved in fish SHBG sequences and that none of the three consensus sites for N-glycosylation sites in fish SHBGs are located within the carboxy-terminal LG domain as they are in mammalian SHBG sequences. Moreover, all three N-glycosylation sites in zebrafish SHBG are conserved within the amino-terminal LG domains of SHBG sequences of fugufish and several other fish species (Miguel-Queralt, S., G. V. Avvakumov, M. Blázquez, F. Piferrer, and G. L. Hammond, unpublished data). These phylogenetic differences in the location of N-linked carbohydrate chains within SHBG sequences may provide new clues as to the functional roles of specific oligosaccharide chains associated with this protein.

The cloning of a near full-length zebrafish SHBG cDNA allowed us to identify the genomic organization of the zebrafish SHBG gene by analysis of sequences within the zebrafish genome database. This indicated that there is a perfect conservation with respect to the intron boundaries of the eight exons within the transcription units encoding SHBG in humans and zebrafish. However, the introns in the zebrafish SHBG gene are much larger than in the human (33) or rat (34) SHBG genes. In line with the compacted nature of the fugufish genome (35), the fugufish SHBG appears to span only 2.2 kb, and this is about 50% smaller than the transcription units encoding mammalian SHBGs (33, 34). We examined sequences within the zebrafish and fugufish genomes within 1 kb immediately 5' from the translation initiation codon for the SHBG precursor polypeptide but have been unable to identify any regions corresponding to those of the human and rat SHBG proximal promoters that contain conserved regulatory sequences known to influence the tissue-specific expression of the gene (36, 37).

Expression of zebrafish SHBG in CHO cells allowed us to produce sufficient quantities of the protein for detailed analyses of its biochemical characteristics and steroid-binding properties. It is evident that zebrafish SHBG exists as a homodimer, and it is likely that both subunits contain a functional steroid binding site, as they do in the human SHBG homodimer (38). The steroid-binding specificity of SHBG in piscine species varies to a much greater extent than in mammals, with SHBG in some fish binding androgens with greater affinity than estrogens (9, 10, 12, 20), whereas in others the opposite is true (8, 9, 13, 20). Two forms of the androgen receptor with differences in their relative affinities for androgens have been described in some fishes (39, 40, 41), and 11-ketotestosterone is quantitatively the most important androgen in the blood of many fish species during the breeding season (42). It is therefore interesting that zebrafish SHBG, like other fish SHBGs studied to date, has a lower affinity for this androgen than for testosterone.

Our measurements of SHBG concentrations in the plasma of adult zebrafish are consistent with those reported in several other fish species including goldfish (10, 12), flounder (9), and spotted seatrout (11), with no sexual dimorphism in levels, as previously noted in goldfish (12). It should be noted, however, that these are among the highest plasma SHBG levels reported in fish species, with some species having plasma SHBG levels lower than 50 nM (9). Because plasma SHBG levels have been reported to increase by only about 50% in some fish during the breeding season (12, 20, 21, 22), large increases in plasma 11-ketotestosterone at this time would result in the displacement of testosterone and androgen precursors, such as androstenedione, from the SHBG steroid-binding site and enhance their delivery to androgen sensitive target tissues. Furthermore, in those fish species in which estradiol is the preferred ligand of SHBG, large increases in plasma 11-ketotestosterone in females as well as males would also enhance the bioavailability of this active estrogen. This highlights the need for a greater understanding of how changes in SHBG gene expression may impact on the actions of both androgens and estrogens in the context of their specific target tissues during reproduction in different fish species.

Our studies of zebrafish during development indicate that the liver and gut are major sites of SHBG gene expression. As in mammalian fetuses (6, 7), the liver appears to produce SHBG soon after its formation in zebrafish larvae (43), and this most likely results in the secretion of SHBG into the developing blood circulation in which it presumably influences the way sex steroids influence gonadal development at this stage of development (44, 45). The amount of immunoreactive SHBG in zebrafish hepatocytes is modest despite the fact that the liver is clearly the major organ responsible for the production of plasma SHBG. This can be attributed to the rapid secretion of protein from hepatocytes, which would be in accordance with similar observations in mammals (46). We observed, however, more intense SHBG immunoreactivity within the luminal aspect of cells lining intrahepatic bile ducts in adult zebrafish, suggesting that SHBG in this location may influence the bilary excretion of steroids in fish (19).

Like liver, the adult zebrafish testis contains both SHBG mRNA and immunoreactive protein, which is confined to the outer margins of the seminiferous tubules. Although this corresponds to the location in which Sertoli cells are found in the fish testis, the SHBG immunoreactivity is not confined to a specific cell type and likely provides a different function in terms of regulating androgen access to developing sperm than it does in mammals such as rat (4). This was not unexpected because there is considerable variation in the morphology of the testis and the site of SHBG gene expression even in mammalian species (5).

The presence of SHBG mRNA in the gut of zebrafish larva was also not unexpected because human SHBG transcripts have been identified in the intestines of fetal mice that express a human SHBG transgene (7). However, it is remarkable that SHBG mRNA is readily detectable in the adult zebrafish gut, and immunoreactive SHBG is concentrated in granules within intestinal epithelial cells. Although the source of SHBG in these granules is not clear, it is possible that SHBG is either produced and released by these intestinal cells or reflects their uptake of SHBG released within the bile. Irrespective of its source, these observations suggest that SHBG in this location plays a novel role in regulating the uptake and recycling of steroids from the gut.


   Acknowledgments
 
The authors thank Dr. Francina Munell and Dr. Francesc Piferrer for helpful comments and suggestions and Tracie Galbraith for assistance with the preparation of the manuscript.


   Footnotes
 
This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada. M.K. is supported by an Ontario Graduate Scholarship Studentship.

The nucleotide sequence reported in this article has been submitted to the GenBank database: accession no. AY640176.

Abbreviations: CHO, Chinese hamster ovary; DCC, dextran-coated charcoal; DHT, dihydrotestosterone; EST, expressed sequence tag; FPLC, fast protein liquid chromatography; LG, laminin G-like; RACE, rapid amplification of cDNA ends; SHBG, sex hormone binding globulin.

Received May 27, 2004.

Accepted for publication July 21, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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