Molecular cloning, characterization and expression of thyroid-stimulating hormone receptor in channel catfish.

Thyroid-stimulating hormone receptors (TSHRs) are primarily expressed in the thyroid of vertebrates, however recently, transcripts encoding TSHR have been found abundantly in the gonads in a variety of fish species. The purpose of this study is to characterize the channel catfish TSHR and to examine whether the transcript are translated into protein in the gonad or store the transcript as maternal RNA for later use. The cDNA encoding the TSHR was isolated from the channel catfish thyroid but the transcript was determined to be expressed in a number of tissues, including the gonads. In fact, the ovarian expression of TSHR changed significantly during the reproductive season and peaked after the vitellogenic growth phase. Furthermore, the TSHR transcript was also detected in unfertilized eggs but not in fertilized egg of catfish. LM-PAT analysis demonstrated that catfish TSHR transcripts were fully polyadenylated in thyroidal follicles, gonads and unfertilized eggs suggesting that they were translated into protein opposed to being "stored mRNA". Western blot analysis using polyclonal antibodies against the catfish TSHR verified this assumption by visualizing immunoreactive protein in the thyroid, testis, and the post-vitellogenic ovary in abundance. A functional assay clearly showed that the recombinant catfish TSHR was specifically activated by bovine TSH but not by recombinant catfish follicle-stimulating hormone (FSH) and luteinizing hormone (LH). As in other species, the heterologous gonadotropin, hCG, partially activated the receptor. These results suggested that TSHR plays important roles for gametogenesis rather than embryogenesis.

Leydig cells express follicle-stimulating hormone receptors in African catfish.

This report aimed to establish, using African catfish, Clarias gariepinus, as model species, a basis for understanding a well-known, although not yet clarified, feature of male fish reproductive physiology: the strong steroidogenic activity of FSHs. Assays with gonadotropin receptor-expressing cell lines showed that FSH activated its cognate receptor (FSHR) with an at least 1000-fold lower EC50 than when challenging the LH receptor (LHR), whereas LH stimulated both receptors with similar EC50s. In androgen release bioassays, FSH elicited a significant response at lower concentrations than those required to cross-activate of the LHR, indicating that FSH stimulated steroid release via FSHR-dependent mechanisms. LHR/FSHR-mediated stimulation of androgen release was completely abolished by H-89, a specific protein kinase A inhibitor, pointing to the cAMP/protein kinase A pathway as the main route for both LH- and FSH-stimulated steroid release. Localization studies showed that intratubular Sertoli cells express FSHR mRNA, whereas, as reported for the first time in a vertebrate, catfish Leydig cells express both LHR and FSHR mRNA. Testicular FSHR and LHR mRNA expression increased gradually during pubertal development. FSHR, but not LHR, transcript levels continued to rise between completion of the first wave of spermatogenesis at about 7 months and full maturity at about 12 months of age, which was associated with a previously recorded approximately 3-fold increase in the steroid production capacity per unit testis weight. Taken together, our data strongly suggest that the steroidogenic potency of FSH can be explained by its direct trophic action on FSHR-expressing Leydig cells.

Japanese eel follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): production of biologically active recombinant Fsh and Lh by Drosophila S2 cells and their differential actions on the reproductive biology.

Two gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), control gonadal steroidogenesis and gametogenesis in vertebrates, including teleost fish. Here, we report on the production of biologically active recombinant Fsh (rec-Fsh) and Lh (rec-Lh) in Japanese eel using Drosophila S2 cells. The three subunits composing Gths, i.e., glycoprotein hormone, alpha polypeptide (Cga), follicle-stimulating hormone, beta polypeptide (Fshb), and luteinizing hormone, beta polypeptide (Lhb), were at first independently produced and were proven to be glycosylated and secreted as the mature peptides. Each beta subunit, along with its Cga, was simultaneously coexpressed to produce heterodimeric rec-Fsh and rec-Lh that were subsequently highly purified. The biological activity of rec-Gths was demonstrated in various in vitro assays. The rec-Gths differentially activated their receptors, which resulted in an increase in 11-ketotestosterone (11KT) secretion, a differential alteration of gene expression of steroidogenic enzymes in immature testis, and the induction of the complete process of spermatogenesis in vitro. The data strongly suggest that Fsh and Lh differentially play important roles in the reproductive physiology of the Japanese eel. By contrast, these rec-Gths exhibited little activity in the gonad when administered in vivo. This difference between in vitro and in vivo bioactivity is probably due to the qualitative nature of glycosylation in S2 cells, which resulted in degradation of the recombinant protein in vivo. These differences in the carbohydrate moieties need to be elucidated and ameliorated.

Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions.

Due to the lack of purified, native gonadotropins (GtH) for almost all species of fish, we designed a system for the production of recombinant bioactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) using the channel catfish (Ictalurus punctatus) as a model animal. The strategy was to produce the three subunits composing FSH and LH, i.e. the common alpha-subunit (alpha-glycoprotein hormone (alpha-GP)), beta-FSH, and beta-LH subunit, individually in stable recombinant insect cells (S2) with C-terminal His-tag. This expression system was also used to co-express the alpha-subunit without the His-tag with each of the His-tagged beta-subunits. The recombinant S2 cells were capable of secreting FSH and LH heterodimers and alpha-GP in abundance; however, expression of the individual beta-subunits was much less successful. The recombinant GtHs were partially purified from the cell medium by immobilized metal affinity chromatography to ~15% purity with a yield of 7 and 4 mg per liter of medium for FSH and LH respectively. These recombinant GtHs activated their receptors in vitro, enhanced estrogen secretion, up-regulated several steroidogenic enzyme genes in channel catfish ovarian follicles, and increased androgen secretion from African catfish testis. Interestingly, the FSH and LH dose-response curves for each of these biological activities clearly demonstrate differences in their cellular action and physiological roles. This expression system may be an important development for the production of species-specific GtHs so that FSH- and LH-specific mechanisms of actions within the reproductive endocrine processes can finally be examined with homologous, albeit recombinant, hormones.

Effects of dibutylphthalate and ethynylestradiol on liver peroxisomes, reproduction, and development of zebrafish (Danio rerio).

The aim of the present work was to study the effects of the peroxisome proliferator dibutylphthalate (DBP) and the xenoestrogen 17alpha-ethynylestradiol (EE2) on liver peroxisomes, reproduction, and development of zebrafish (Danio rerio). In experiment 1, newly fertilized zebrafish eggs were exposed for five weeks, covering the entire period of sexual determination, to nominal concentrations of 25 and 100 microg/L of DBP and 5 microg/L of EE2. In experiment 2, adult female zebrafish were exposed for 15 d to 100 and 500 microg/L of DBP and 5 microg/L of EE2, and afterward, they were paired with untreated males to study the effects in the resultant offspring. Ethynylestradiol provoked marked mortality (approximately 50%) and delayed development of larvae as well as sterility of adult females, possibly related to alterations in aromatase gene expression. Ethynylestradiol up-regulated vitellogenin expression in the early life stages and increased vitellogenin synthesis and accumulation in adult females. Ethynylestradiol caused liver peroxisome proliferation in early life stages but not in adult females. Dibutylphthalate caused teratogenic effects in early life stages and mortality of the larvae obtained from exposed females. Dibutylphthalate provoked liver peroxisome proliferation and up-regulation of cytochrome P450A1 in early life stages at the end of the exposure and in adult females. Dibutylphthalate also up-regulated the expression of aromatase genes. In conclusion, the xenoestrogen EE2 caused liver peroxisome proliferation in early life stages of zebrafish, but the peroxisome proliferator DBP did not behave as a typical xenoestrogen. Overall, changes in gene expression were more marked during early life stages than in adult female zebrafish.

Short-term exposure to low concentrations of the synthetic androgen methyltestosterone affects vitellogenin and steroid levels in adult male zebrafish (Danio rerio).

Short-term effects of methyltestosterone (MT) on the endocrine system of adult male zebrafish (Danio rerio) were examined. Males were exposed to 0, 4.5, 6.6, 8.5, 19.8, 35.9, 62.3 ng MT/l and ethinylestradiol (EE2) (26.4 ng/l) for 7 days. Several physiological endpoints that may be affected by endocrine disrupters were analysed, specifically vitellogenin (VTG) concentration, estradiol (E2), testosterone (T), and 11-ketotestosterone (KT) content, brain aromatase activity and gene expression of CYP19A1 and CYP19A2 in the testis. Exposure to the lowest MT concentration (4.5 ng MT/l), and the EE2 increased the concentration of VTG significantly compared to solvent control group. Exposure to higher concentrations of MT did not increase VTG levels. Endogenous KT and T levels decreased significantly in a concentration-dependent manner in response to the MT exposure and the lowest effective concentrations were 6.4 and 8.5 ng MT/l, respectively. The levels of KT and T were also significantly suppressed by EE2 when compared to the solvent control group. Significant decreases in endogenous E2 levels were found in some MT groups but it was not possible to distinguish a simple concentration-response relationship. No effects of MT or EE2 on the brain aromatase activity or on testicular gene expression of CYP19A1 and CYP19A2 were detected. The results show that androgens such as MT can act as endocrine disrupters even at very low concentrations.

Molecular characterization of three forms of putative membrane-bound progestin receptors and their tissue-distribution in channel catfish, Ictalurus punctatus.

Membrane-bound progestin receptors (mPR) were recently cloned and characterized as a new class of steroid receptors that transduce cell-signals through alteration of MAP kinase- and cAMP-dependent pathways. To further develop our understanding of this new class of steroid receptors, we characterized the cDNAs and genes of thealpha, beta and gamma forms of the channel catfish mPRs (IpmPR). The predicted alpha and beta proteins have 49% sequence identity, whereas they only have 30% and 27% identities, respectively, with the gamma form. Furthermore, IpmPRalpha and IpmPRbeta genes have similar structures featuring intronless coding regions, while IpmPRgamma gene is composed of 8 exons and 7 introns. The 5'-flanking region of each IpmPR gene differs, but each contains putative transcriptional regulatory elements of factors known to influence reproductive physiology and endocrine disruption, for example, responsive elements for cAMP and steroids and the recognition sites for steroidogenic factor-1 and for the aryl hydrocarbon receptor. The IpmPRalpha gene was detected in all the tissues tested with relatively greater expression in brain, pituitary, muscle and testis. The expression of IpmPRbeta was much lower than that of IpmPRalpha and the transcript was predominantly observed in brain, pituitary, ovary and testis. In contrast, the IpmPRgamma transcript was mainly detected in gill, ventral aorta, intestine, and trunk kidney. In conclusion, all the structural features of the IpmPRs strongly suggest that the closely related alpha and beta forms are distantly related to the gamma form. Additionally, regulatory features of the 5'-flanking regions and the differences in tissue-specific expression of each IpmPR gene suggest that they are involved in different endocrine functions in catfish.

Membrane-bound progestin receptors in channel catfish and zebrafish ovary: changes in gene expression associated with the reproductive cycles and hormonal reagents.

Membrane-bound progestin receptors (mPRs) are potential intermediaries in meiotic maturation of fish oocytes and other physiological processes. In this study, gene expression of the mPRs in the ovary of catfish and zebrafish during the reproductive cycle and the hormonal regulation of the expression were investigated. The transcript abundance of catfish mPRalpha gradually increased in conjunction with ovarian growth and then decreased prior to spawning period whereas the ovarian gene expression of mPRbeta varied little throughout the reproductive cycle. In contrast, mPRgamma gene expression peaked early in the mid-vitellogenic stage. The transcript abundance of zebrafish mPRalpha and beta was low in ovarian follicles at early stages of oogenesis and gradually increased after the onset of vitellogenic growth and, thereafter, the gene expression did not vary. Gonadotropic treatment did not modulate the ovarian expression of mPRalpha and beta genes in either catfish or zebrafish. On the other hand, exposure to 17,20beta-dihydroxy-4-pregenen-3-one (the maturation-inducing steroid in this species) resulted in the down-regulation of mPRalpha in catfish ovary whereas gene expression was significantly induced by estradiol-17beta. Taken together, these findings suggest that gonadotropin-induced final oocyte maturation may not require an induction of mPR(s) expression or that the gonadotropin stimulates mPR protein production at the post-transcriptional level, presuming these receptors are indispensable for oocyte maturation.

Localization and expression of aromatase mRNA in adult zebrafish.

Estradiol plays a key role in the control of many behavioral and physiological aspects of reproduction therefore the expression of cytochrome P450 aromatase (CYP19), the enzyme responsible for the conversion of androgens to estrogens, is of vital interest. The zebrafish, and many other teleosts, have two aromatase genes (CYP19A1 and CYP19A2) that are expressed predominantly in the ovary and brain, respectively, however, the physiological impact of extra-gonadal aromatase has been poorly described. In this study, in situ hybridizations of whole-mount and paraffin sections of adult zebrafish brains, pituitaries, and ovarian follicles showed that CYP19A2 was strongly expressed in the olfactory bulb (OB), ventral telencephalon (TEL), preoptic area (POA), and ventral/caudal hypothalamic zone (HT) of the brain, and in the anterior and posterior lobes of the pituitary. The regional distribution of the CYP19A2 mRNA did not vary with sex however transcript abundance varied within (male "high expressers" had much higher expression in the OB, TEL, and HT than in "low expressers") and between sexes (higher in OB, TEL, and HT of males than in females). In situ hybridizations of CYP19A1 failed to develop a signal in the brain or pituitary but were detectable by RT-PCR. CYP19A1 was highly expressed in Stage III B follicles (>500 nm) with significantly lower levels in the Stage IV follicles (>680 nm), Stage III A follicles (>350 nm), and Stage I and II follicles (350 microm) which were embedded in connective tissues. The differential expression of the aromatase genes, particularly CYP19A2 in the brain, suggests that the two aromatase genes play different roles in the reproductive behavior and/or physiology of bony fish.

Effects of endocrine disrupting chemicals on the expression of CYP19 genes in zebrafish (Danio rerio) juveniles.

Cytochrome P450 aromatase (CYP19) is the key steroidogenic enzyme responsible for conversion of androgens to estrogens which play a critical role in developmental sex differentiation and adult reproductive cycles in vertebrates. To evaluate the potential roles of endocrine disrupting chemicals (EDC) on reproductive physiology of fish, the influence of multiple classes of EDC on the transcript abundance of two CYP19 isoforms, CYP19A1 and A2, were investigated in zebrafish juveniles. The pharmaceutical, clofibrate, and the pesticide, atrazine, did not influence the expression of either CYP19 gene. Estrogenic compounds, nonylphenol (NP) and a pharmaceutical estrogen, ethinylestradiol (EE), strongly enhanced the expression of CYP19A2 gene in dose-dependent manner. Exposure to benzo[a]pyrene (BaP) significantly increased CYP19A2 transcript abundance. Furthermore, BaP when co-treated with EE partially suppressed EE-induced upregulation of CYP19A2. In contrast, the expression of CYP19A1 was basically resistant to EDC treatment although EE at high concentration (1-100 nM) downregulated its expression. These findings suggest that multiple classes of EDC may potentially perturb developmental and reproductive physiology in fish through differential transcriptional modulation of the CYP19 genes with the most evident disruption in neural tissue.

Hypophyseal gene expression profiles of FSH-beta, LH-beta, and glycoprotein hormone-alpha subunits in Ictalurus punctatus throughout a reproductive cycle.

A determination of the seasonal changes in the expression of the genes encoding the subunits of gonadotropic hormones is an important first step in the understanding of the molecular control of the onset of puberty and the reproductive cycle in fish. In this study, the abundance of transcripts encoding the glycoprotein hormone alpha (GpH-alpha), follicle-stimulating hormone beta (FSH-beta), and luteinizing hormone beta (LH-beta) subunits in pituitaries of female channel catfish were systematically tracked throughout an annual reproductive cycle. All three genes showed a concurrent elevation coinciding with the onset of ovarian recrudescence but then each showed a second elevation at different times of the ovarian cycle. In addition to the initial peak at recrudescence, the expression of FSH-beta and GpH-alpha gene peaked again during mid- and late-vitellogenic growth, respectively. The LH-beta gene expression remained low during the phases of regression and vitellogenic growth but was moderately elevated (7-fold) at the onset of ovarian recrudescence and dramatically elevated (36-fold) just prior to spawning (June-July) when the FSH-beta levels were at their lowest. The expression patterns of FSH-beta and LH-beta are remarkably similar to the ovarian expression of their respective receptors.

Ovarian steroidogenesis in white sturgeon (Acipenser transmontanus) during oocyte maturation and induced ovulation.

Ovarian follicles and plasma were collected from two female white sturgeon, Acipenser transmontanus, injected with sturgeon pituitary homogenate followed 12h later with GnRHa to induce ovulation. The oocytes of one female underwent germinal vesicle breakdown (GVBD) but ovulation did not occur in response to hormonal stimulation (Female 1), while the oocytes of the other female underwent GVBD and ovulation (Female 2). Follicles collected 12h after the first injection to induce ovulation were incubated with radioinert pregnenolone (P5) or tritiated-P5 ([3H]P5) plus radioinert P5. Steroids were extracted from media and intact follicles, and the extracts were fractionated by high performance liquid chromatography (HPLC). Fractions from the incubation with radioinert precursor were used in a bioassay to determine the potency of the steroid products to induce GVBD. Plasma levels of testosterone (T), estradiol, and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) were measured by radioimmunoassay during induced ovulation, and plasma collected at the time of ovulation (actual or expected) was analyzed by HPLC. A peak in plasma 17,20beta-P was detected at the time of the second injection to induce ovulation in Female 2 (the time at which follicles were collected for incubation with [3H]P5). The HPLC analysis revealed several progestins in the plasma of Female 2 at ovulation that were not present in Female 1 at the time of expected ovulation. A variety of C19 and C21 steroids were produced in vitro by ovarian follicles from both females. The "suggestive" identities of the major metabolites were 11-deoxycortisol, androstenedione, 17-hydroxyprogesterone (17OHP), and 17,20beta-P in Female 1 and cortisol, 17,20beta, 21-trihydroxyprogesterone (20beta-S), 11-deoxycortisol, T, 17OHP, and 17,20beta-P in Female 2. Several of the steroids were active in a GVBD bioassay, but the fractions that contained the steroid coeluting the authentic 11-deoxycortisol on the HPLC and 17,20beta-P (positively identified by gas chromatography-mass spectrometry) were found to be the most potent. The results from this study combined with the results of Webb et al. (2001b) suggest the potential roles of 11-deoxycortisol, 17,20beta-P, 20beta-S, and P4 as maturation-inducing steroids in sturgeon.