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.

Seasonal variation of pituitary gonadotropin subunit, brain-type aromatase and sex steroid receptor mRNAs, and plasma steroids during gametogenesis in wild sablefish.

Pituitary-hormone signaling plays critical roles in the onset and progression of gametogenesis in vertebrates. This study characterized expression patterns of pituitary gonadotropin beta-subunits (fshb and lhb), brain-type aromatase (cyp19a1b), androgen (ar1, ar2) and estrogen receptors (esr1, esr2a, esr2b), and changes in plasma steroid levels by liquid chromatography/tandem mass spectrometry in wild sablefish (Anoplopoma fimbria, order Scorpaeniformes) during a complete reproductive cycle. Transcripts for fshb increased during early gametogenesis and peaked in late vitellogenic females and late recrudescent males, while expression of lhb reached maximum levels in periovulatory and spermiating fish. Pituitary levels of cyp19a1b and ar1 were strongly correlated with those of lhb in females and males, increasing during gametogenesis and reaching maximum levels prior to spawning. By contrast, expression of ar2, and the three estrogen receptors differed between female and male sablefish. 17ß-estradiol (E2) was the dominant steroid in females during vitellogenesis, while a range of at least 6 steroids (11ß-hydroxyandrostenedione, testosterone [T], E2, 11-ketotestosterone [11KT], 11-deoxycortisol, and 17α,20ß,21-trihydroxyprogesterone) were detected at similar levels in males during testicular development. Prior to spawning, a marked increase in 4-androstenedione, T, 11KT and E2 was found in both periovulatory females and spermiating males. In conclusion, the concomitant changes in plasma androgen levels and pituitary ar1 expression during gametogenesis suggest a specific role for androgens in pituitary hormone regulation of reproduction in sablefish. Further, our data highlight the importance of E2 during final stages of maturation in this species, which may regulate the transcription of pituitary lhb in a paracrine fashion.

A teleost androgen promotes development of primary ovarian follicles in coho salmon and rapidly alters the ovarian transcriptome.

Recent studies using several teleost models have revealed that androgens increase the size of previtellogenic (primary and/or early secondary) ovarian follicles. To explore our hypothesis that androgens drive the development of primary follicles into early secondary follicles, and to determine the mechanisms underlying these androgenic effects, we exposed juvenile coho salmon to near-physiological and relatively sustained levels of the nonaromatizable androgen 11-ketotestosterone (11-KT). This resulted in significant growth of primary ovarian follicles after 10 and 20 days, with follicles after 20 days displaying a morphological phenotype characteristic of early secondary follicles (presence of cortical alveoli). Utilizing the same experimental approach, we then analyzed how 11-KT rapidly altered the ovarian transcriptome after 1 and 3 days of treatment. RNA-Seq analysis revealed that 69 (day 1) and 1,022 (day 3) contiguous sequences (contigs) were differentially expressed relative to controls. The differentially expressed contigs mapped to genes including those encoding proteins involved in gonadotropin, steroid hormone, and growth factor signaling, and in cell and ovarian development, including genes with putative androgen-response elements. Biological functions and canonical pathways identified as potentially altered by 11-KT include those involved in ovarian development, tissue differentiation and remodeling, and lipid metabolism. We conclude that androgens play a major role in stimulating primary ovarian follicle development and the transition into secondary growth.

Regulation of sex steroid production and mRNAs encoding gonadotropin receptors and steroidogenic proteins by gonadotropins, cyclic AMP and insulin-like growth factor-I in ovarian follicles of rainbow trout (Oncorhynchus mykiss) at two stages of vitellogenesis.

At the completion of vitellogenesis, the steroid biosynthetic pathway in teleost ovarian follicles switches from estradiol-17ß (E2) to maturational progestin production, associated with decreased follicle stimulating hormone (Fsh) and increased luteinizing hormone (Lh) signaling. This study compared effects of gonadotropins, human insulin-like growth factor-I (IGF1), and cAMP/protein kinase A signaling (forskolin) on E2 production and levels of mRNAs encoding steroidogenic proteins and gonadotropin receptors using midvitellogenic (MV) and late/postvitellogenic (L/PV) ovarian follicles of rainbow trout. Fsh, Lh and forskolin, but not IGF1, increased testosterone and E2 production in MV and L/PV follicles. Fsh increased steroidogenic acute regulatory protein (star; MV), 3ß-hydroxysteroid dehydrogenase/Δ(5-4) isomerase (hsd3b; MV) and P450 aromatase (cyp19a1a; MV) transcript levels. Lh increased star mRNA levels (MV, L/PV) but reduced cyp19a1a transcripts in L/PV follicles. At both follicle stages, IGF1 reduced levels of hsd3b transcripts. In MV follicles, IGF1 decreased P450 side-chain cleavage enzyme (cyp11a1) transcripts but increased cyp19a1a transcripts. In MV follicles only, forskolin increased star and hsd3b transcripts. Forskolin reduced MV follicle cyp11a1 transcripts and reduced cyp19a1a transcripts in follicles at both stages. Fsh and Lh reduced fshr transcripts in L/PV follicles. Lh also reduced lhcgr transcripts (L/PV). IGF1 had no effect on gonadotropin receptor transcripts. Forskolin reduced MV follicle fshr transcript levels and reduced lhcgr transcripts in L/PV follicles. These results reveal hormone- and stage-specific transcriptional regulation of steroidogenic protein and gonadotropin receptor genes and suggest that the steroidogenic shift at the completion of vitellogenesis involves loss of stimulatory effects of Fsh and Igfs on cyp19a1a expression and inhibition of cyp19a1a transcription by Lh.

Alterations in gene expression during fasting-induced atresia of early secondary ovarian follicles of coho salmon, Oncorhynchus kisutch.

Molecular processes that either regulate ovarian atresia or are consequences of atresia are poorly understood in teleost fishes. We hypothesized that feed restriction that perturbs normal ovarian growth and induces follicular atresia would alter ovarian gene expression patterns. Previtellogenic, two-year old coho salmon (Oncorhynchus kisutch) were subjected to prolonged fasting to induce atresia or maintained on a normal feeding schedule that would promote continued ovarian development. To identify genes that were specifically up- or down-regulated during oocyte growth in healthy, growing fish compared to fasted fish, reciprocal suppression subtractive hybridization (SSH) cDNA libraries were generated using ovaries from fed and fasted animals. Differential expression of genes identified by SSH was confirmed with quantitative PCR. The SSH library representing genes elevated in ovaries of fed fish relative to those of fasted fish contained steroidogenesis-related genes (e.g., hydroxy-delta-5-steroid dehydrogenase), Tgf-beta superfamily members (e.g., anti-Mullerian hormone) and cytoskeletal intermediate filament proteins (e.g., type I keratin s8). Overall, these genes were associated with steroid production, cell proliferation and differentiation, and ovarian epithelialization. The library representing genes elevated in ovaries of fasted fish relative to fed fish contained genes associated with apoptosis (e.g., programmed cell death protein 4), cortical alveoli (e.g., alveolin), the zona pellucida (e.g., zona pellucida protein c), and microtubules (e.g., microtubule associated protein tau). Elevated expression of this suite of genes was likely associated with the initiation of atresia and/or a reduced rate of follicle development in response to fasting. This study revealed ovarian genes involved in normal early secondary oocyte growth and potential early markers of atresia.

Development of approaches to induce puberty in cultured female sablefish (Anoplopoma fimbria).

Efforts to establish sustainable and efficient aquaculture production of sablefish (Anoplopoma fimbria) have been constrained by delayed puberty in cultured females. This study integrates a series of experiments aimed at gaining an understanding of the reproductive physiology of puberty in female sablefish. We detected transcripts for the dopamine D2 receptor (drd2) in brain, pituitary and ovary of sablefish, and prepubertal females exhibited significantly elevated brain and pituitary drd2 expression relative to wild maturing females. Treatments with sustained-release cholesterol pellets containing testosterone (T) and the dopamine D2 receptor antagonist, metoclopramide (Met), stimulated expression of pituitary luteinizing hormone beta subunit (lhb) and follicle-stimulating hormone beta subunit (fshb), respectively, in prepubertal females, whereas a combination of T and gonadotropin-releasing hormone agonist (GnRHa) had a strong synergistic effect on lhb expression (2000-fold higher than control). Although T induced a significant increase in the maximum ovarian follicle volume, none of the treatments tested stimulated onset of vitellogenesis. Using liquid chromatography/tandem mass spectrometry, we demonstrated that Met stimulated production of T by previtellogenic ovarian follicles in vitro, whereas gonadotropin preparations enhanced 17α-hydroxyprogesterone, androstenedione (A4), T and 17ß-estradiol (E2) production. Treatment with T increased production of A4, 11ß-hydroxyandrostenedione, 11ß-hydroxytestosterone, E2, 11-ketotestosterone, and 5α-dihydrotestosterone (DHT). Interestingly, in the presence of high doses of T the previtellogenic ovary preferentially produced A4 and DHT over any other metabolite. Our data suggest the existence of dopamine inhibition of the reproductive axis in female sablefish. Treatments with Met and T elevated gonadotropin mRNAs in prepubertal females but failed to stimulate the transition into vitellogenic growth, suggesting a possible failure in pituitary gonadotropin protein synthesis/release. Previtellogenic ovarian follicles of sablefish are equipped to synthesize steroids, including those required for vitellogenic growth, and DHT, a steroid hormone whose role in reproduction of fishes remains unknown.

In vivo treatment with a non-aromatizable androgen rapidly alters the ovarian transcriptome of previtellogenic secondary growth coho salmon (Onchorhynchus kisutch).

Recent evidence suggests that androgens are a potent driver of growth during late the primary stage of ovarian follicle development in teleosts. We have previously shown that the non-aromatizable androgen, 11-ketotestosterone (11-KT), both advances ovarian follicle growth in vivo and dramatically alters the primary growth ovarian transcriptome in coho salmon. Many of the transcriptomic changes pointed towards 11-KT driving process associated with the transition to a secondary growth phenotype. In the current study, we implanted previtellogenic early secondary growth coho salmon with cholesterol pellets containing 11-KT and performed RNA-Seq on ovarian tissue after 3 days in order to identify alterations to the ovarian transcriptome in early secondary growth. We identified 8,707 contiguous sequences (contigs) that were differentially expressed (DE) between control and 11-KT implanted fish and were able to collapse those to 3,853 gene-level IDs, more than a 3-fold more DE contigs than at the primary growth stage we reported previously. These contigs included genes encoding proteins involved in steroidogenesis, vitellogenin and lipid uptake, follicle stimulating hormone signaling, growth factor signaling, and structural proteins, suggesting androgens continue to promote previtellogenic secondary growth.

Molecular characterization and quantification of sablefish (Anoplopoma fimbria) gonadotropins and their receptors: reproductive dysfunction in female captive broodstock.

Efforts to establish an aquaculture industry for sablefish (Anoplopoma fimbria) are constrained by reproductive dysfunction in wild-caught fish and by lack of reproduction of F1 females. Toward a better understanding of the reproductive dysfunction of captive broodstock, full-length cDNAs encoding the sablefish gonadotropin subunits (fshb, lhb and cga) and their receptors (fshr and lhcgr) were cloned, sequenced and quantitative real-time PCR assays developed. Sablefish gonadotropin subunits display some unique features, such as two additional Cys residues in the N-terminal region of Fshb and a lack of potential N-glycosylation sites in Fshb and Lhb, whereas Fshr and Lhcgr possess conserved structural characteristics described in other vertebrates. Wild females captured in fall completed gametogenesis in captivity the next spawning season, whereas females captured three months earlier, during summer, failed to mature. Interestingly, these wild non-maturing females exhibited similar reproductive features as prepubertal F1 females, including low levels of pituitary gonadotropin and ovarian receptor mRNAs and plasma sex steroids, and ovarian follicles arrested at the perinucleolus stage. In conclusion, this study described the cloning, molecular characterization and development of qPCRs for sablefish gonadotropins and their receptors. Rearing conditions may impair vitellogenic growth of ovarian follicles in sablefish, compromising the reproductive success of broodstock.

Salmonid Pituitary Cells as a Test System for Identifying Endocrine-Disrupting Compounds.

The pituitary gland is a central regulator of reproduction, producing two gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), which regulate gonadal development, sex steroid synthesis, and gamete maturation. The present study sought to optimize an in vitro test system using pituitary cells isolated from previtellogenic female coho salmon and rainbow trout, focusing on fshb and lhb subunit gene expression. Initially, we optimized culture conditions for duration and benefits of culturing with and without addition of endogenous sex steroids (17ß-estradiol [E2] or 11-ketotestosterone) or gonadotropin-releasing hormone (GnRH). The results suggest that culturing with and without E2 was valuable because it could mimic the (+) feedback effects on Lh that are observed from in vivo studies. After optimizing assay conditions, a suite of 12 contaminants and other hormones was evaluated for their effects on fshb and lhb gene expression. Each chemical was tested at four to five different concentrations up to solubility limitations in cell culture media. The results indicate that more chemicals alter lhb synthesis than fshb. The more potent chemicals were estrogens (E2 and 17α-ethynylestradiol) and the aromatizable androgen testosterone, which induced lhb. The estrogen antagonists 4-OH-tamoxifen and prochloraz decreased the E2-stimulated expression of lhb. Among several selective serotonin reuptake inhibitors tested, the sertraline metabolite norsertraline was notable for both increasing fshb synthesis and decreasing the E2 stimulation of lhb. These results indicate that diverse types of chemicals can alter gonadotropin production in fish. Furthermore, we have shown that pituitary cell culture is useful for screening chemicals with potential endocrine-disrupting activity and can support the development of quantitative adverse outcome pathways in fish. Environ Toxicol Chem 2023;42:1730-1742. © 2023 SETAC.

A nonfunctional copy of the salmonid sex-determining gene (sdY) is responsible for the "apparent" XY females in Chinook salmon, Oncorhynchus tshawytscha.

Many salmonids have a male heterogametic (XX/XY) sex determination system, and they are supposed to have a conserved master sex-determining gene (sdY) that interacts at the protein level with Foxl2 leading to the blockage of the synergistic induction of Foxl2 and Nr5a1 of the cyp19a1a promoter. However, this hypothesis of a conserved master sex-determining role of sdY in salmonids is challenged by a few exceptions, one of them being the presence of naturally occurring "apparent" XY Chinook salmon, Oncorhynchus tshawytscha, females. Here, we show that some XY Chinook salmon females have a sdY gene (sdY-N183), with 1 missense mutation leading to a substitution of a conserved isoleucine to an asparagine (I183N). In contrast, Chinook salmon males have both a nonmutated sdY-I183 gene and the missense mutation sdY-N183 gene. The 3-dimensional model of SdY-I183N predicts that the I183N hydrophobic to hydrophilic amino acid change leads to a modification in the SdY ß-sandwich structure. Using in vitro cell transfection assays, we found that SdY-I183N, like the wild-type SdY, is preferentially localized in the cytoplasm. However, compared to wild-type SdY, SdY-I183N is more prone to degradation, its nuclear translocation by Foxl2 is reduced, and SdY-I183N is unable to significantly repress the synergistic Foxl2/Nr5a1 induction of the cyp19a1a promoter. Altogether, our results suggest that the sdY-N183 gene of XY Chinook females is nonfunctional and that SdY-I183N is no longer able to promote testicular differentiation by impairing the synthesis of estrogens in the early differentiating gonads of wild Chinook salmon XY females.

The spatiotemporal expression of multiple coho salmon ovarian connexin genes and their hormonal regulation in vitro during oogenesis.

BACKGROUND: Throughout oogenesis, cell-cell communication via gap junctions (GJs) between oocytes and surrounding follicle cells (theca and granulosa cells), and/or amongst follicle cells is required for successful follicular development. To gain a fundamental understanding of ovarian GJs in teleosts, gene transcripts encoding GJ proteins, connexins (cx), were identified in the coho salmon, Oncorhynchus kisutch, ovary. The spatiotemporal expression of four ovarian cx transcripts was assessed, as well as their potential regulation by follicle-stimulating hormone (FSH), luteinizing hormone (LH) and insulin-like growth factor 1 (IGF1). METHODS: Salmonid ovarian transcriptomes were mined for cx genes. Four gene transcripts designated cx30.9, cx34.3, cx43.2, and cx44.9 were identified. Changes in gene expression across major stages of oogenesis were determined with real-time, quantitative RT-PCR (qPCR) and cx transcripts were localized to specific ovary cell-types by in situ hybridization. Further, salmon ovarian follicles were cultured with various concentrations of FSH, LH and IGF1 and effects of each hormone on cx gene expression were determined by qPCR. RESULTS: Transcripts for cx30.9 and cx44.9 were highly expressed at the perinucleolus (PN)-stage and decreased thereafter. In contrast, transcripts for cx34.3 and cx43.2 were low at the PN-stage and increased during later stages of oogenesis, peaking at the mid vitellogenic (VIT)-stage and maturing (MAT)-stage, respectively. In situ hybridization revealed that transcripts for cx34.3 were only detected in granulosa cells, but other cx transcripts were detected in both oocytes and follicle cells. Transcripts for cx30.9 and cx44.9 were down-regulated by FSH and IGF1 at the lipid droplet (LD)-stage, whereas transcripts for cx34.3 were up-regulated by FSH and IGF1 at the LD-stage, and LH and IGF1 at the late VIT-stage. Transcripts for cx43.2 were down-regulated by IGF1 at the late VIT-stage and showed no response to gonadotropins. CONCLUSION: Our findings demonstrate the presence and hormonal regulation of four different cx transcripts in the salmon ovary. Differences in the spatiotemporal expression profile and hormonal regulation of these cx transcripts likely relate to their different roles during ovarian follicle differentiation and development.

The pituitary-thyroid axis during the parr-smolt transformation of Coho salmon, Oncorhynchus kisutch: quantification of TSH ß mRNA, TSH, and thyroid hormones.

The objective of this investigation was to quantify pituitary thyroid stimulating hormone (TSH) ß mRNA, pituitary and plasma TSH and plasma thyroid hormone levels during the parr-smolt transformation of Coho salmon that occurs in spring from February to May. The status of the pituitary-thyroid axis was assessed using an RNase protection assay for pituitary TSH ß mRNA and radioimmunoassays for salmon pituitary and plasma TSH and thyroid hormones. TSH ß mRNA was highest during late winter (February) (4.9 pg/µg DNA) and gradually declined during spring (2.3 pg/µg DNA). In contrast, pituitary and plasma TSH levels showed a small, but statistically non-significant change during smoltification. Despite minimal change in plasma TSH levels, characteristically large increases in plasma T4 (January-3.3 ng/ml to April-10.2 ng/ml) and significant, but modest increases in plasma T3 (February-2.4 ng/ml to April-5.8 ng/ml) were observed. Regression analysis showed a significant positive relationship between plasma T4 and T3 and negative relationship between plasma T3 and pituitary TSH ß mRNA. However, all other relations were not significant. These data suggest a significant role for peripheral regulation (i.e. T4-T3 conversion, change in tissue sensitivity, hormone degradation rate) as well as evidence of central regulation via negative feedback at the level of the pituitary gland in regulation of thyroid activity in salmon. Furthermore, the increased thyroid sensitivity to TSH (shown previously), in the face of relatively constant plasma TSH levels, may be the major factor responsible for the increased thyroid activity observed during smoltification.

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.

Disruption of the salmon reproductive endocrine axis through prolonged nutritional stress: changes in circulating hormone levels and transcripts for ovarian genes involved in steroidogenesis and apoptosis.

Mechanisms regulating the normal progression of ovarian follicular growth versus onset of atresia in fishes are poorly understood. To gain a better understanding of these processes, we exposed immature female coho salmon (Oncorhynchus kisutch) to prolonged fasting to induce follicular atresia and monitored body growth, development of the ovarian follicles, changes in reproductive hormones, and transcripts for ovarian genes. Prolonged fasting reduced body and ovary weight and increased the appearance of atretic follicles relative to normally fed controls. Endocrine analyses showed that fasting reduced plasma insulin-like growth factor 1 (IGF1), estradiol-17ß (E2), and pituitary, but not plasma, levels of follicle-stimulating hormone (FSH). Transcripts for ovarian fsh receptor (fshr) and steroidogenesis-related genes, such as steroidogenic acute regulatory protein (star), 3ß-hydroxysteroid dehydrogenase (hsd3b), and P450 aromatase (cyp19a1a) were significantly lower in fasted fish. Ovarian expression of apoptosis-related genes, such as Fas-associated death domain (fadd), caspase 8 (casp8), caspase 3 (casp3), and caspase 9 (casp9) were significantly elevated in fasted fish compared to fed fish, indicating that apoptosis is involved in the process of atresia in this species. Interestingly, some genes such as fadd, casp8, casp3, and hsd3b, were differentially expressed prior to increases in the number of atretic follicles and reductions in hormone levels induced by fasting, and may therefore have potential as early indicators of atresia. Together these results suggest that prolonged nutritional stress may disrupt the reproductive system and induce follicular atresia in part via reductions in ovarian IGF and FSH signaling, and downstream effects on steroidogenesis-related genes and E2 production.

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.

Changes in the plasma levels of insulin-like growth factor-I from the onset of spawning migration through upstream migration in chum salmon.

An increase in activity of the pituitary-gonadal axis (PG-axis) and gonadal development are essential for the onset of spawning migration of chum salmon from the Bering Sea. In the Bering Sea, fish with larger body sizes initiated gonadal development and commenced spawning migration to the natal river by the end of summer. We thus hypothesized that insulin-like growth factor-I (IGF-I), a somatotropic signal that interacts with the PG-axis, can be one of such factors responsible for the onset of migration, and examined changes in plasma levels and hepatic expression of IGF-I gene in oceanic and homing chum salmon in 2001-2003. The plasma IGF-I levels and corresponding body sizes in maturing adults, which had developing gonads, were significantly higher than those in immature fish in all years examined. Such increase in the plasma IGF-I levels in maturing fish was observed even in the Gulf of Alaska during February 2006, while coincident increase was not observed in the hepatic amounts of IGF-I mRNA. In autumn, the plasma IGF-I levels in homing adults decreased during upstream migration in the Ishikari River-Ishikari bay water system in Hokkaido, Japan. In conclusion, the plasma IGF-I levels increased with gonadal development when chum salmon migrated from the winter Gulf of Alaska to the summer Bering Sea. Circulating IGF-I may interact with the PG-axis and promote gonadal development that is inseparable from the onset of spawning migration. Circulating IGF-I levels were thereafter lowered in accordance with final maturation during upstream migration in the breeding season.

Pharmacological characterization, localization and quantification of expression of gonadotropin receptors in Atlantic salmon (Salmo salar L.) ovaries.

The gonadotropins Fsh and Lh interact with their receptors (Fshr and Lhr, respectively) in a highly specific manner in mammals with little overlap in biological activities. In fish, the biological activities seem less clearly separated considering, for example, the steroidogenic potency of both Fsh and Lh. Important determinants of the biological activity are the specificity of hormone-receptor interaction and the cellular site of receptor expression. Here, we report the pharmacological characterization of Atlantic salmon Fshr and Lhr, identify receptor-expressing cells in the ovary, and validate receptor mRNA quantification systems. For the pharmacological studies, we used highly purified coho salmon gonadotropins and found that the Fshr preferentially responded to Fsh, but was also activated by approximately 6-fold higher levels of Lh. The Lhr was specific for Lh and did not respond to Fsh. Photoperiod manipulation was used to generate ovarian tissue samples with largely differing stages of maturation. Specific real-time, quantitative (rtq) PCR assays revealed up to 40-fold (fshr) and up to 350-fold (lhr) changes in ovarian expression levels, which correlated well with the differences in ovarian weight, histology, and circulating oestrogen levels recorded in January and June, respectively. Vitellogenic ovaries were used to localise receptor-expressing cells by in situ hybridization. Granulosa cells of small and large vitellogenic follicles were positive for both receptors. Also theca cells of small and large vitellogenic follicles expressed fshr mRNA, while only in large vitellogenic follicles theca cells were (weakly) positive for lhr mRNA. While only ovulatory Lh levels seem high enough to cross-activate the Fshr, expression by both receptors by granulosa and theca cells suggests that homologous ligand receptor interaction will prevail.

Effects of aromatase inhibitors and different doses of testosterone on gonadotropins in one year old male Atlantic salmon (Salmo salar).

The effects of different doses of testosterone (T), the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD) and 4-hydroxy-4-androstene-3,17-dione (4OH), and the combined treatment of T and ATD on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at the onset of puberty in juvenile Atlantic salmon males were investigated. T always increased pituitary LH. Also, ATD increased pituitary LH, though to a lesser extent than T. However, ATD combined with T diminished pituitary LH levels compared to T alone, indicating an aromatase-dependent positive feedback of T on LH in immature males. 4OH, which was less effective than ATD as an aromatase inhibitor, increased LH content. ATD treatment resulted in increased pituitary FSH levels, similar to those of mature controls. Positive effects of ATD on plasma FSH were found, indicating the presence of an aromatase-dependent negative feedback. The 4OH effects on FSH levels were inconsistent. T exerted both positive and negative effects on pituitary FSH and testes growth, depending on dose and season, with the positive effects being more pronounced with the low doses and the negative effects with the high doses. The treatment of T combined with ATD did not affect the positive effect of T alone on pituitary and plasma FSH, indicating the presence of an aromatase-independent positive feedback on FSH. There was a positive correlation between FSH and gonadosomatic index, especially during summer when gonadal development occurs.

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.

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.