Gonadotropin regulation of activin betaA and activin type IIA receptor expression in the ovarian follicle cells of the zebrafish, Danio rerio

We have previously demonstrated that both activin and its receptors are expressed in the zebrafish ovary, suggesting paracrine roles for activin in the ovarian functions. Activin significantly stimulated zebrafish oocyte maturation in vitro, and this effect could be blocked by follistatin, an activin-binding protein. Interestingly, follistatin also blocked the stimulatory effect of gonadotropin (hCG) on the oocyte maturation. Taken together, these results have led to a hypothesis that the ovarian activin system may play a role in mediating the actions of gonadotropin in the ovary. To test this hypothesis, the present study was undertaken to investigate if gonadotropin has any effect on the expression of activin betaA subunit and activin type IIA (ActRIIA) receptor in the zebrafish ovary. A primary culture of zebrafish ovarian follicle cells was established in the present study, and the cultured cells expressed both activin betaA and ActRIIA receptor when assayed with RT-PCR. The primary culture consisted of three major types of cells, presumably the fibroblasts, the thecal cells and the granulosa cells, according to the morphological features, histochemical staining for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and RT-PCR for aromatase. Using a semi-quantitative RT-PCR with beta-actin as the internal control, we demonstrated that hCG significantly stimulated mRNA expression of both activin betaA and ActRIIA receptor in the cultured follicle cells in a time- and dose-dependent manner. Treatment of the cells with hCG quickly increased the steady-state mRNA levels of activin betaA and ActRIIA receptor, and the effect peaked at 2 h of treatment. The stimulatory effect of gonadotropin diminished with longer treatment and no effect was observed at 8 h of treatment. The effect of hCG also exhibited strong dose dependence when assayed at 2 h of treatment. The levels of activin betaA and ActRIIA receptor mRNA elevated with increasing dose of hCG; however, the effect significantly decreased at dosage higher than 15 IU/ml. Consistent with the stimulatory effect of gonadotropin on the expression of activin betaA and ActRIIA receptor, IBMX, forskolin and 8-Br-cAMP all significantly increased the mRNA levels of activin betaA and ActRIIA receptor. These results suggest that gonadotropin activates the activin system in the zebrafish ovary by increasing the expression of both activin and its receptors.     

Gonadotropin regulation of follistatin expression in the cultured ovarian follicle cells of zebrafish, Danio rerio

Follistatin is a single-chain glycoprotein initially identified in the mammalian ovary. As a specific binding protein of activin, it effectively modifies the paracrine/autocrine roles of activin in a variety of tissues including the ovary. In the zebrafish, we have demonstrated that the human chorionic gonadotropin (hCG)-induced oocyte maturation and oocyte maturational competence can be blocked by follistatin, suggesting a role for ovarian activin in the signaling pathway of gonadotropin in the zebrafish ovary. The up-regulation of zebrafish ovarian activin betaA subunit by gonadotropin further supports this hypothesis. Since follistatin has extremely high affinity for activin, its expression level in various tissues is critical in fine-tuning local activin activities. In the present study, we investigated the regulation of follistatin expression by gonadotropin in a primary culture of zebrafish ovarian follicle cells using semi-quantitative RT-PCR. Both hCG and goldfish pituitary extract strongly increased the expression of follistatin in the cultured follicle cells in clear time- and dose-dependant manners. The effect of hCG (15IU/ml) reached the maximal level at 2h of treatment and longer treatment (4-8h) led to decreased response. The up-regulation of follistatin expression by hCG could be mimicked by all the drugs that increase the intracellular cAMP level including 8-Br-cAMP, db-cAMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX). The hCG (15IU/ml)- and forskolin (10microM)-induced follistatin expression could be blocked by H89 (10microM), a specific protein kinase A (PKA) inhibitor. These results strongly suggest that the regulation of follistatin expression in the zebrafish ovary by gonadotropin is primarily mediated by cAMP-PKA signaling pathway. The up-regulation of follistatin mRNA by gonadotropin in cultured zebrafish ovarian follicle cells, together with our previous studies on gonadotropin regulation of activin beta subunits, suggests that the ovarian activin-follistatin system is tightly controlled by gonadotropin in fish ovary.     

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

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20β-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17β, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.     

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

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20beta-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17beta, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.     

Cloning of transforming growth factor-beta 1 (TGF-beta 1) and its type II receptor from zebrafish ovary and role of TGF-beta 1 in oocyte maturation

TGF-beta is a multifunctional factor involved in regulating a variety of cellular activities. In mammals, TGF-beta is known to regulate reproduction, including ovarian functions. The role of TGF-beta in lower vertebrates, such as fish, is poorly understood. To examine the role of TGF-beta in fish reproduction, cDNAs encoding TGF-beta 1 and the type II TGF-beta receptor (T beta RII) were cloned from the zebrafish ovary using PCR- based strategies. The mature peptide region of the zebrafish TGF-beta 1 shows 70-85% identity with TGF-beta 1 from other species. The zebrafish T beta RII cDNA sequence is the first to be reported from a fish species, and it shows a high level of conservation at the kinase domain. Using RT-PCR, we have detected mRNA expression of TGF-beta 1, T beta RII, as well as its downstream signaling molecules Smad2, 3, and 4 in ovarian follicles at different stages of development. In addition, we have examined the effect of TGF-beta 1 on oocyte maturation. TGF-beta 1 significantly inhibited both gonadotropin- and 17 alpha, 20 beta-dihydroxyprogesterone-induced oocyte maturation in a dose- and time-dependent manner. These findings demonstrate, for the first time, that TGF-beta 1 plays a role in regulating oocyte maturation in fish and suggest that a TGF-beta/Smad signaling pathway is present in the zebrafish ovary.     

Cloning,regulation of messenger ribonucleic acid expression,and function of a new isoform of pituitary adenylate cyclase-activating polypeptide in the zebrafish ovary

Increasing evidence suggests that pituitary adenylate cyclase-activating polypeptide (PACAP) acts as a local factor in the ovary of mammals. In nonmammalian vertebrates, although the expression of PACAP has also been demonstrated in the ovary, the information on its functions and regulation is limited. In the present study, we identified a new type of PACAP, zebrafish (zf)PACAP38-2, from the zebrafish ovary. The precursor of GHRH-zfPACAP38-2 consists of 175 amino acids with only 64% homology with another type of zebrafish PACAP, zfPACAP38-1. RT-PCR analysis detected two messengers of zfPACAP38-2 in the zebrafish ovary. The short product was more abundant, and it encodes zfPACAP38-2 only, whereas the long form codes for both zfPACAP38-2 and GHRH. Using a primary culture of zebrafish follicle cells, we demonstrated that gonadotropin (human chorionic gonadotropin and goldfish pituitary extract) significantly stimulated zfPACAP38-2 expression within 2 h; however, the effect decreased to the control level after 8 h of treatment. The stimulation of zfPACAP38-2 expression by gonadotropin could be mimicked by cAMP analogs and forskolin but suppressed by H89 (10 mum), suggesting the involvement of the cAMP-protein kinase A signaling pathway. We also examined the expression of PACAP receptor VPAC2-R in the zebrafish ovary. Unlike zfPACAP38-2, which showed a trend of increase during follicle development, the expression of VPAC2-R mRNA in the follicles showed no significant stage-dependent variation, and its expression in the follicle cells did not respond to gonadotropin treatment. Our studies further demonstrated that synthetic zfPACAP38-2 stimulated oocyte maturation and increased the expression of follistatin in zebrafish ovarian follicle cells. These results suggest that zfPACAP38-2 is a potential ovarian factor that mediates gonadotropin actions in paracrine/autocrine manners, and its functional roles are likely, to some extent, related to the ovarian activin/follistatin system.     

A shift in steroidogenesis occurring in ovarian follicles prior to oocyte maturation

Gonadotropins (GTHs; FSH and LH) require two major steroidal mediators, estradiol-17 beta (E(2)) and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP) to act as critical hormones to execute oocyte growth and maturation, respectively. A two-cell type model has been proposed, where the theca cells provide the precursor steroids, and the granulosa cells produce the two steroidal mediators under the direct influence of FSH and LH. A distinct shift in steroidogenesis, i.e. from E(2) to 17 alpha,20 beta-DP as well as the steroidogenic enzyme genes from ovarian cytochrome P450 aromatase (oP450arom) to 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD), occurs in the granulosa layers of ovarian follicles prior to oocyte maturation. The triggering of the steroidogenic shift by GTHs in granulosa cells occurs through the subjugation of Ad4BP/SF-1 expression in respect of oP450arom, followed by an over-expression of 20 beta-HSD probably through the CREB.     

Isoleucine-15 of rainbow trout carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase is critical for coenzyme (NADPH) binding

Carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase (CR/20beta-HSD) is an enzyme that converts 17alpha-hydroxyprogesterone to 17alpha, 20beta-dihydroxy-4-pregnen-3-one (the maturation-inducing hormone of salmonid fish). We have previously isolated two types of CR/20beta-HSD cDNAs from ovarian follicle of rainbow trout (Oncorhynchus mykiss). Recombinant proteins produced by expression in Escherichia coli in vitro showed that one (type A) had CR and 20beta-HSD activity but that the other (type B) did not. Among the three distinct residues between the protein products encoded by the two cDNAs, two residues (positions 15 and 27) are located in the N-terminal Rossmann fold, the coenzyme binding site. To investigate the structure/function relationships of CR/20beta-HSDs, we generated mutants by site-directed mutagenesis at the following positions: MutA/I15T, MutB/T15I, and MutB/Q27K. Enzyme activity of wild-type A was abolished by substitution of Ile-15 by Thr (MutA/I15T). Conversely, enzyme activity was acquired by the replacement of Thr-15 with Ile in type B (MutB/T15I). MutB/T15I mutant showed properties similar to the wild-type A in every aspect tested. Mutation MutB/Q27K had only partial enzyme activity, indicating that Ile-15 plays an important role in enzyme binding of cofactor NADPH.     

20beta-Hydroxysteroid dehydrogenase of the Japanese eel ovary: its cellular localization and changes in the enzymatic activity during sexual maturation

In many species of teleost, 20beta-hydroxysteroid dehydrogenase (20beta-HSD) is a key steroidogenic enzyme in the production of the oocyte maturation-inducing steroid (MIS), 17alpha, 20beta-dihydroxy-4-pregnen-3-one. In this study, 20beta-HSD in the ovary of the Japanese eel was biochemically characterized using a cell-free system. 20beta-HSD activity was located mainly in the membrane-bound fractions of the mitochondria and microsome, with lower levels detected in the cytosolic fraction. The enzymatic activity of membrane-bound 20beta-HSD was strikingly enhanced by treatment of eels with gonadotropin-rich salmon pituitary homogenate. The activity of eel ovarian mitochondrial 20beta-HSD in the presence of different solubilizing detergents was then assessed. Mitochondrial fractions solubilized by sodium deoxycholate and octhylthioglucoside retained approximately 30% of 20beta-HSD activity when compared to those of nontreated mitochondria. These results suggest that Japanese eel ovarian 20beta-HSD is composed of membrane-bound and soluble activities, and that the membrane-bound component is stimulated by gonadotropin.     

The role of the insulin-like growth factor (IGF) system in zebrafish (Danio rerio) ovarian development

The presence of an ovarian IGF system in teleosts suggests a distinct role in reproductive physiology. This study investigates the role of the ovarian IGF system in oocyte maturation, the acquisition of maturational competence and steroidogenesis in the zebrafish (Danio rerio). Recombinant human IGF-I and IGF-II stimulated germinal vesicle breakdown (GVBD) in early vitellogenic (EV; 0.35-0.44 mm), midvitellogenic (MV; 0.45-0.56 mm) and full grown (FG; 0.57-0.65 mm) follicles incubated in vitro. By comparison, the maturation inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) only induced GVBD in MV and FG follicles. Collectively these studies suggest that IGF is involved in oocyte maturation and that follicles become responsive to IGFs at an earlier stage compared to 17,20β-P. IGF-I also increased the responsiveness of the follicle to 17,20β-P, suggesting a role in promoting maturational competence. IGF-I alone and in combination with human chorionic gonadotropin (hCG) stimulated the production of 17,20β-P by ovarian follicles incubated in vitro. However, IGF-I had no effect on the production of 17β-estradiol (E₂) or the expression of genes involved in steroidogenesis (20β-hydroxysteroid dehydrogenase; 20β-HSD and P450c17-II). These results provide evidence that the IGF system plays an important role in the promotion of oocyte maturation and ovarian development in the zebrafish.     

Androgen metabolism via 17beta-hydroxysteroid dehydrogenase type 3 in mammalian and non-mammalian vertebrates: comparison of the human and the zebrafish enzyme

Formation and inactivation of testosterone is performed by various members of the 17beta-hydroxysteroid dehydrogenase (17beta-HSD) family. The main player in testosterone formation is considered to be 17beta-HSD type 3, which catalyzes the reduction of androstenedione to testosterone with high efficiency and is almost exclusively expressed in testis. So far, only the mammalian homologs have been characterized but nothing is known about the role of 17beta-HSD type 3 in other vertebrates. In this study, we describe the identification and characterization of the zebrafish homolog. We found zebrafish 17beta-HSD type 3 to be expressed in embryogenesis from sphere to 84 h post-fertilization. Expression was also detected in various tissues of both male and female adults, but displayed sexual dimorphism. Interestingly, expression was not highest in male testis but in male liver. In female adults, strongest expression was observed in ovaries. At the subcellular level, both human and zebrafish 17beta-HSD type 3 localize to the endoplasmic reticulum. The zebrafish enzyme in vitro effectively catalyzed the conversion of androstenedione to testosterone by use of NADPH as cofactor. Among further tested androgens epiandrosterone and dehydroepiandrosterone were accepted as substrates and reduced at C-17 by the human and the zebrafish enzyme. Androsterone and androstanedione though, were only substrates of human 17beta-HSD type 3, not the zebrafish enzyme. Furthermore, we found that both enzymes can reduce 11-ketoandrostenedione as well as 11beta-hydroxyandrostenedione at C-17 to the respective testosterone forms. Our results suggest that 17beta-HSD type 3 might play slightly different roles in zebrafish compared with human although testosterone itself is likely to have similar functions in both organisms.     

Characterization and regulation of the insulin-like growth factor (IGF) system in the zebrafish (Danio rerio) ovary

Locally produced peptide hormones play an important role in the paracrine/autocrine regulation of ovarian development. The insulin-like growth factor (IGF) system is one family of local factors that has not been well studied in the ovary of fish. This study characterized the zebrafish (Danio rerio) ovarian IGF system, its spatial and temporal expression and regulation by gonadotropins and steroids. Three ligands (igf2a, igf2b, igf3) and two receptors (igf1ra and igf1rb) were demonstrated in the ovary using RT-qPCR. Though it was examined, igf1 expression was not detected in the zebrafish ovary. Igf3 expression significantly decreased in the hours prior to ovulation and was confined to the follicle cells. Igf2a, igf2b and the two receptors were detected in both the follicle cells and the oocyte and were constitutively expressed in ovarian tissue across the daily ovulatory cycle. In vitro addition of human chorionic gonadotropin (hCG; 20 IU/ml) stimulated a significant increase in igf3 expression in both midvitellogenic (MV; 0.45-0.56 mm) and full grown (FG; 0.57-0.65 mm) follicles while igf2b expression increased only in FG follicles. Treatment of follicles in vitro with 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P; 10 ng/ml) significantly decreased igf3 and igf2b expression in both MV and FG follicles. 17β-Estradiol (E₂; 25 ng/ml) had no effect on the expression of igf3 in MV or FG follicles. Igf1rb expression did not change after treatment with hCG, 17,20β-P or E₂. Collectively, these results demonstrate the presence of an ovarian IGF system in zebrafish that is differentially regulated by gonadotropin and steroids.     

Involvement of cyclic adenosine 3',5'-monophosphate in the differential regulation of activin betaA and betaB expression by gonadotropin in the zebrafish ovarian follicle cells

Activin is a dimeric protein consisting of two similar but distinct beta-subunits, betaA and betaB. In our previous studies, both activin A (betaAbetaA) and activin B (betaBbetaB) have been demonstrated to stimulate oocyte maturation and promote oocyte maturational competence in the zebrafish. Follistatin, a specific activin-binding protein, can block both activin- and gonadotropin-induced final oocyte maturation in vitro, suggesting that activin is likely a downstream mediator of gonadotropin actions in the zebrafish ovary. In the present study, a full-length cDNA encoding zebrafish ovarian activin betaA was cloned and sequenced. The precursor of zebrafish activin betaA consists of 395 amino acids and its mature region exhibits about 78% homology with that of mammals. Using an in vitro primary culture of the ovarian follicle cells and semiquantitative RT-PCR assays, we examined the regulation of activin betaA and betaB expression by human chorionic gonadotropin (hCG) and its intracellular signal transduction mechanisms. hCG (15 IU/ml) increased the mRNA level of activin betaA-subunit; however, it significantly down-regulated the steady-state expression level of activin betaB in a time- and dose-dependent manner. The differential regulation of the two beta-subunits by hCG could be mimicked by 3-isobutyl-1-methylxanthine, forskolin, and dibutyryl-cAMP, suggesting involvement of the intracellular cAMP pathway. Interestingly, H89 (a specific inhibitor of protein kinase A, PKA) could effectively block hCG- and forskolin-stimulated activin betaA expression at 10 micro M, but it was unable to reverse the inhibitory effects of hCG and forskolin on betaB expression. This suggests that the hCG-stimulated activin betaA expression is dependent on the activation of the cAMP-PKA pathway, whereas the inhibitory effect of hCG on activin betaB expression is likely mediated by PKA-independent pathway(s).     

Identification of 17,20beta,21-trihydroxy-4-pregnen-3-one as an oocyte maturation-inducing steroid in black porgy, Acanthopagrus schlegeli

The identity of the maturation-inducing steroid (MIS) in black porgy, Acanthopagrus schlegeli, a marine protandrous teleost, is unknown. Previous studies demonstrated that two teleost MISs, the progestins 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) and 17,20beta-dihydroxy-4-pregnen-3-one (DHP) can induce maturation of black porgy oocytes in vitro. The purpose of the present study was to identify the major progestin produced during oocyte maturation (OM) in black porgy and investigate whether its secretion increases during this process. Females were injected twice with a LHRH analog to induce OM. Ovarian follicles undergoing OM were incubated in vitro with tritiated [3H]pregnenolone precursor and the tritiated products were extracted, purified, and identified by HPLC, TLC, acetylation, and recrystallization. Significant amounts of tritiated products were biosynthesized from [3H]pregnenolone that co-migrated with 20beta-S but not with DHP on HPLC and TLC. Similar TLC profiles were obtained with the tritiated products isolated from the HPLC/TLC 20beta-S fraction and standard 20beta-S after the acetylation reaction. The identity of the tritiated products as 20beta-S was confirmed by recrystallization. 20beta-S had a slightly higher potency than DHP in the inducing in vitro final oocyte maturation. Plasma 20beta-S concentrations increased significantly during the oocyte maturation after injection with a LHRH analog. The present data suggest that 20beta-S is the MIS in black porgy.     

Differential regulation of gonadotropin receptors by bone morphogenetic proteins in the zebrafish ovary

Follicle-stimulating hormone receptor (fshr) and luteinizing hormone/choriogonadotropin receptor (lhcgr) exhibit differential temporal expression patterns during zebrafish folliculogenesis with fshr being dominant during vitellogenic growth and lhcgr increasing its expression dramatically before maturation. The dynamic and distinct expression patterns of fshr and lhcgr suggest that they are under tight regulatory control. However, the underlying mechanisms for the differential expression of the two receptors remain unknown. We have recently demonstrated that members of bone morphogenetic protein (BMP) family are largely expressed in the oocyte, while their receptors are exclusively localized on the follicle cells, suggesting a potential paracrine signaling from the oocyte to the follicle cells by BMPs. In this study, we investigated the effects of zebrafish BMP2b (zfBmp2b) and BMP4 (zfBmp4) on the expression of fshr and lhcgr using a novel co-culture approach. The recombinant zfBmp2b or zfBmp4-producing CHO cells were co-cultured with the zebrafish follicle cells followed by real-time qPCR analysis of fshr and lhcgr expression. Our results showed that zfBmp2b and zfBmp4 both down-regulated fshr, while up-regulated lhcgr expression at 24 h of co-culturing. This finding, together with the high expression level of BMP receptors in the follicle cells prior to oocyte maturation, strongly suggests a potential role for BMPs in the differential expression of fshr and lhcgr, especially in the full-grown follicles before maturation. As BMPs are largely expressed in the oocyte, this also implies an important role for the oocyte in orchestrating the differentiation and function of the follicle cells.     

Discovery of a novel enzyme mediating glucocorticoid catabolism in fish: 20beta-hydroxysteroid dehydrogenase type 2

Hydroxysteroid dehydrogenases (HSDs) are involved in metabolism and pre-receptor regulation of steroid hormones. While 17beta-HSDs and 11beta-HSDs are extensively studied in mammals, only few orthologs are characterized in fish. We discovered a novel zebrafish HSD candidate closely related to 17beta-HSD types 3 and 12, which has orthologs in other species. The enzyme catalyzes the conversion of cortisone to 20beta-hydroxycortisone identified by LC-MS/MS. We named the new enzyme 20beta-HSD type 2. All 20beta-HSD type 2 orthologs localize in the endoplasmic reticulum. Zebrafish 20beta-HSD type 2 is expressed during embryonic development showing the same expression pattern as 11beta-HSD type 2 known to oxidize cortisol to cortisone. In adult tissues 20beta-HSD type 2 shows a ubiquitous expression pattern with some minor sex-specific differences. In contrast to other enzymes metabolizing C21-steroids and being mostly involved in reproduction we propose that novel type 2 20beta-HSDs in teleost fish are important enzymes in cortisol catabolism.