Evaluation of endocrine and transcriptomic markers of male maturation in winter-run Steelhead Trout (Oncorhynchus mykiss).

Steelhead Trout (Oncorhynchus mykiss) display a varied life-history, including precocious male maturation at age-1 or age-2. In wild fish, precocious male maturation represents an important component of a diverse life-history portfolio. In hatchery programs, however, it is undesirable if rearing practices increase rates of early male maturation and reduce numbers of anadromous male adults. Our study aimed to develop endocrine and molecular markers for identifying males at early stages of maturation in the spring (prior to smolt release) and evaluated the potential use of these markers for quantifying early male maturation rates at a hatchery scale. In a laboratory study, Skookumchuck winter-run Steelhead Trout were reared at a high growth rate in order to increase the occurrence of precocious male maturation. Fish were lethally sub-sampled in February, prior to the time of smolt release; in May, at the time of smolt release; and in September, when 1+ age maturing males that would spawn the following spring were clearly identifiable based solely on gonadosomatic index (GSI). In February and May samples, we measured GSI, plasma 11-ketotestosterone (11KT), mRNAs for pituitary follicle stimulating hormone (fshb) and luteinizing hormone (lhb) beta subunits, and analyzed stage of spermatogenesis by testis histology. Additionally, in May, we measured testis anti-Müllerian hormone (amh) and insulin-like growth factor 3 (igf3) mRNA. Our primary goal was to evaluate the aforementioned maturation indices for their efficacy in forecasting the proportion of fish initiating early male maturation in the spring (approximately 1 year prior to spermiation), compared to the proportion that actually matured. Combining measures of GSI, plasma 11KT, and pituitary fshb and lhb mRNA expression provided a useful, but conservative, estimate of the proportion of males initiating maturation in the spring (21%) compared to the proportion that were ultimately destined to mature (37%) the following spring. These results suggest that maturation may be less synchronous than previously appreciated and some males may have initiated maturation after our census in May.

Temporal Dynamics of DNA Methylation Patterns in Response to Rearing Juvenile Steelhead (Oncorhynchus mykiss) in a Hatchery versus Simulated Stream Environment.

Genetic selection is often implicated as the underlying cause of heritable phenotypic differences between hatchery and wild populations of steelhead trout (Oncorhynchus mykiss) that also differ in lifetime fitness. Developmental plasticity, which can also affect fitness, may be mediated by epigenetic mechanisms such as DNA methylation. Our previous study identified significant differences in DNA methylation between adult hatchery- and natural-origin steelhead from the same population that could not be distinguished by DNA sequence variation. In the current study, we tested whether hatchery-rearing conditions can influence patterns of DNA methylation in steelhead with known genetic backgrounds, and assessed the stability of these changes over time. Eyed-embryos from 22 families of Methow River steelhead were split across traditional hatchery tanks or a simulated stream-rearing environment for 8 months, followed by a second year in a common hatchery tank environment. Family assignments were made using a genetic parentage analysis to account for relatedness among individuals. DNA methylation patterns were examined in the liver, a relatively homogeneous organ that regulates metabolic processes and somatic growth, of juveniles at two time points: after eight months of rearing in either a tank or stream environment and after a subsequent year of rearing in a common tank environment. Further, we analyzed DNA methylation in the sperm of mature 2-year-old males from the earlier described treatments to assess the potential of environmentally-induced changes to be passed to offspring. Hepatic DNA methylation changes in response to hatchery versus stream-rearing in yearling fish were substantial, but few persisted after a second year in the tank environment. However, the early rearing environment appeared to affect how fish responded to developmental and environmental signals during the second year since novel DNA methylation differences were identified in the livers of hatchery versus stream-reared fish after a year of common tank rearing. Furthermore, we found profound differences in DNA methylation due to age, irrespective of rearing treatment. This could be due to smoltification associated changes in liver physiology after the second year of rearing. Although few rearing-treatment effects were observed in the sperm methylome, strong family effects were observed. These data suggest limited potential for intergenerational changes, but highlight the importance of understanding the effects of kinship among studied individuals in order to properly analyze and interpret DNA methylation data in natural populations. Our work is the first to study family effects and temporal dynamics of DNA methylation patterns in response to hatchery-rearing.

Development of a multiplex gene expression assay for components of the endocrine growth axis in coho salmon.

This study explores the efficacy of the Quantigene plex (QGP) technology for measuring a panel of endocrine growth-related transcripts in coho salmon, Oncorhynchus kisutch. The QGP technology permits the simultaneous quantification of multiple targeted mRNAs within a single tissue homogenate using sequence-specific probes and requires no reverse transcription (RT) or amplification as is required for RT-quantitative PCR (RT-qPCR). Using liver homogenates from coho salmon under fed and fasted conditions, we compared the detectable fold differences of steady-state mRNA levels between the QGP and probe-based RT-qPCR assays for insulin-like growth factors (igf1 and igf2), insulin-like growth factor binding proteins (igfbp1b, igfbp2a, and igfbp2b), somatolactin receptor (slr), and growth hormone receptors (ghr1 and ghr2). Significant, positive correlations for all genes between the two assays were found. In addition, the relatively low variance of results from the QGP assay suggests that this is a suitable method for a comprehensive analysis of endocrine growth-related transcripts and could potentially be used to develop assays for other gene networks in teleosts.

Follicle-stimulating hormone regulation of ovarian transcripts for steroidogenesis-related proteins and cell survival, growth and differentiation factors in vitro during early secondary oocyte growth in coho salmon.

Little is known about follicle-stimulating hormone (FSH) function during oocyte growth in fishes. The goal of this study was to gain a fundamental understanding of FSH action on ovarian follicles during early secondary oocyte growth by examining changes in ovarian gene expression and steroidogenesis in response to FSH. Coho salmon (Oncorhynchus kisutch) mid to late cortical alveolus stage follicles were incubated with or without salmon FSH in time-course and concentration-response experiments. Steroid levels were determined in the culture medium by immunoassay and levels of target ovarian mRNAs were determined by quantitative RT-PCR. Medium estradiol-17ß (E2) levels increased in response to FSH and plateaued by 36h, while testosterone levels increased similarly but were lower and more variable than E2. Gonadotropin receptor transcripts were differentially regulated, with fshr and lhcgr being down- and up- regulated, respectively. Transcripts encoding proteins involved in steroidogenesis, such as star and hsd3b were significantly upregulated by FSH, whereas aromatase (cyp19a1a) mRNA was unaffected by FSH and declined over time in culture. A recently identified teleost gene, bmp16, was suppressed by FSH and an anti-apoptotic factor, clusterin 1 (clu1), was upregulated by FSH. Lesser FSH effects were observed on igf2, cyp11a1 and cyp17a1, which were stimulated, and igf1ra, inhbb, amh and apoe, which were suppressed. As evident by the significant increases in steroid production and transcripts for specific steroidogenesis-related proteins, FSH influences steroidogenesis during early secondary growth and may be a critical signal for puberty onset. Effects of FSH on ovarian anti-apoptotic and growth factor genes suggest roles for FSH in cell survival, growth and differentiation in teleosts.

Regulation of pituitary GnRH receptor and gonadotropin subunits by IGF1 and GnRH in prepubertal male coho salmon.

Insulin-like growth factor 1 (IGF1) is a key somatotropic hormone that may convey growth status to the reproductive endocrine system. This study examined effects of IGF1 alone or in combination with gonadotropin-releasing hormone (GnRH) on pituitary transcripts for GnRH receptor (GnRHR) variants, follicle-stimulating hormone (FSH), luteinizing hormone (LH), growth hormone (GH), and IGF, as well as secretion of FSH in vitro. Three experiments were conducted with dispersed pituitary cells of prepubertal male coho salmon (Oncorhynchus kisutch) to determine the time course of the response to IGF1, IGF1 concentration response, and GnRH concentration response. IGF1 consistently elevated pituitary transcripts for gnrhr1 and the four gonadotropin subunits (fshb, lhb, cga1, and cga2) by day 10 of culture, while suppressing gh and igf2. Short-term treatment with GnRH (24h) induced minor increases in transcripts for fshb, cga1, and cga2, but suppressed lhb and strongly inhibited gnrhr1 expression. IGF1 significantly increased GnRH-stimulated FSH protein release by the pituitary cells, although not as robustly as previously observed in more reproductively advanced salmon. Our results demonstrate that IGF1 increases steady-state mRNA levels of gnrhr1 and four gonadotropin subunits, and may act alone or with GnRH to increase pituitary FSH release in male coho salmon, over 1year before puberty. These findings suggest that IGF1 may prime pituitary gonadotrope cells of prepubertal salmon to respond to GnRH by stimulating synthesis of GnRHR and FSH during puberty onset.

Thyroid hormone regulation of mRNAs encoding thyrotropin beta-subunit, glycoprotein alpha-subunit, and thyroid hormone receptors alpha and beta in brain, pituitary gland, liver, and gonads of an adult teleost, Pimephales promelas.

Thyroid hormones (THs) regulate growth, morphological development, and migratory behaviors in teleost fish, yet little is known about the transcriptional dynamics of gene targets for THs in these taxa. Here, we characterized TH regulation of mRNAs encoding thyrotropin subunits and thyroid hormone receptors (TRs) in an adult teleost fish model, the fathead minnow (Pimephales promelas). Breeding pairs of adult minnows were fed diets containing 3,5,3'-triiodo-L-thyronine (T(3)) or the goitrogen methimazole for 10 days. In males and females, dietary intake of exogenous T(3) elevated circulating total T(3), while methimazole depressed plasma levels of total thyroxine (T(4)). In both sexes, this methimazole-induced reduction in T(4) led to elevated mRNA abundance for thyrotropin beta-subunit (tshbeta) in the pituitary gland. Fish treated with T(3) had elevated transcript levels for TR isoforms alpha and beta (tralpha and trbeta) in the liver and brain, but reduced levels of brain mRNA for the immediate-early gene basic transcription factor-binding protein (bteb). In the ovary and testis, exogenous T(3) elevated gene transcripts for tshbeta, glycoprotein hormone alpha-subunit (gphalpha), and trbeta, while not affecting tralpha levels. Taken together, these results demonstrate negative feedback of T(4) on pituitary tshbeta, identify tralpha and trbeta as T(3)-autoinduced genes in the brain and liver, and provide new evidence that tshbeta, gphalpha, and trbeta are THs regulated in the gonad of teleosts. Adult teleost models are increasingly used to evaluate the endocrine-disrupting effects of chemical contaminants, and our results provide a systemic assessment of TH-responsive genes during that life stage.

Activity of the pituitary-gonadal axis is increased prior to the onset of spawning migration of chum salmon.

The activity of the pituitary-gonadal axis (PG axis) in pre-migratory and homing chum salmon was examined because endocrine mechanisms underlying the onset of spawning migration remain unknown. Pre-migratory fish were caught in the central Bering Sea in June, July and September 2001, 2002 and 2003, and in the Gulf of Alaska in February 2006. They were classified into immature and maturing adults on the basis of gonadal development. The maturing adults commenced spawning migration to coastal areas by the end of summer, because almost all fish in the Bering Sea were immature in September. In the pituitaries of maturing adults, the copy numbers of FSHbeta mRNA and the FSH content were 2.5- to 100-fold those of the immature fish. Similarly, the amounts of LHbeta mRNA and LH content in the maturing adults were 100- to 1000-fold those of immature fish. The plasma levels of testosterone, 11-ketotestosterone and estradiol were higher than 10 nmol l(-1) in maturing adults, but lower than 1.0 nmol l(-1) in immature fish. The increase in the activity of the PG-axis components had already initiated in the maturing adults while they were still in the Gulf of Alaska in winter. In the homing adults, the pituitary contents and the plasma levels of gonadotropins and plasma sex steroid hormones peaked during upstream migration from the coast to the natal hatchery. The present results thus indicate that the seasonal increase in the activity of the PG axis is an important endocrine event that is inseparable from initiation of spawning migration of chum salmon.

Dietary exposure to 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) alters thyroid status and thyroid hormone-regulated gene transcription in the pituitary and brain.

BACKGROUND: Polybrominated diphenyl ether (PBDE) flame retardants have been implicated as disruptors of the hypothalamic-pituitary-thyroid axis. Animals exposed to PBDEs may show reduced plasma thyroid hormone (TH), but it is not known whether PBDEs impact TH-regulated pathways in target tissues. OBJECTIVE: We examined the effects of dietary exposure to 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47)-commonly the highest concentrated PBDE in human tissues-on plasma TH levels and on gene transcripts for glycoprotein hormone alpha-subunit (GPHalpha) and thyrotropin beta-subunit (TSHbeta) in the pituitary gland, the auto-induced TH receptors alpha and beta in the brain and liver, and the TH-responsive transcription factor basic transcription element-binding protein (BTEB) in the brain. METHODS: Breeding pairs of adult fathead minnows (Pimephales promelas) were given dietary PBDE-47 at two doses (2.4 microg/pair/day or 12.3 microg/pair/day) for 21 days. RESULTS: Minnows exposed to PBDE-47 had depressed plasma thyroxine (T(4)), but not 3,5,3'-triiodothyronine (T(3)). This decline in T(4) was accompanied by elevated mRNA levels for TStHbeta (low dose only) in the pituitary. PBDE-47 intake elevated transcript for TH receptor alpha in the brain of females and decreased mRNA for TH receptor beta in the brain of both sexes, without altering these transcripts in the liver. In males, PBDE-47 exposure also reduced brain transcripts for BTEB. CONCLUSIONS: Our results indicate that dietary exposure to PBDE-47 alters TH signaling at multiple levels of the hypothalamic-pituitary-thyroid axis and provide evidence that TH-responsive pathways in the brain may be particularly sensitive to disruption by PBDE flame retardants.

Molecular cloning and sequence analysis of multiple cDNA variants for thyroid-stimulating hormone beta subunit (TSHbeta) in the fathead minnow (Pimephales promelas).

We cloned and sequenced full-length cDNAs encoding the beta subunit of thyroid-stimulating hormone (TSHbeta) from the pituitary of fathead minnow (Pimephales promelas) using 5'- and 3'-rapid amplification of cDNA ends (RACE). Three cDNA variants for TSHbeta with lengths of 1184-, 1093-, and 818-bp were identified. The cDNA variant of 1184-bp included 453-bp of open-reading frame and 610-bp of 3' UTR followed by a poly(A)site. This cDNA encodes 150 amino acids including a 19 residue signal peptide and a mature TSHbeta protein of 131 residues with sequence identities of 97-53% to other fishes and 42-39% to mammals. The 1093-bp cDNA variant was identical to the 1184-bp variant in the open-reading frame, but contained a deletion of 40-bp in the 3' UTR. The 818-bp cDNA variant, however, contained 498-bp of open-reading frame followed by 227-bp of 3' UTR and a poly(A)site. The deduced amino acid sequence for this cDNA variant showed 99.2% homology with the 1184- and 1093-bp variants of TSHbeta, but a single deletion of 332-bp nucleotides spanning the predicted stop codon and 3' UTR resulted in a deduced amino acid sequence with 15 additional residues on the C terminus. The presence of this 818-bp cDNA variant in the pituitary was further confirmed by PCR using primers developed to the 5' and 3' UTR. PCR and Southern blot analyses of genomic DNA suggested only one gene for TSHbeta. Sequencing of this gene revealed a hairpin loop structure of approximately 300-bp located in the 3' UTR and corresponding to the region of the 332-bp deletion in the 818-bp transcript.