Allelic diversification after transposable element exaptation promoted gsdf as the master sex determining gene of sablefish.

Concepts of evolutionary biology suggest that morphological change may occur by rare punctual but rather large changes, or by more steady and gradual transformations. It can therefore be asked whether genetic changes underlying morphological, physiological, and/or behavioral innovations during evolution occur in a punctual manner, whereby a single mutational event has prominent phenotypic consequences, or if many consecutive alterations in the DNA over longer time periods lead to phenotypic divergence. In the marine teleost, sablefish (Anoplopoma fimbria), complementary genomic and genetic studies led to the identification of a sex locus on the Y Chromosome. Further characterization of this locus resulted in identification of the transforming growth factor, beta receptor 1a (tgfbr1a) gene, gonadal somatic cell derived factor (gsdf), as the main candidate for fulfilling the master sex determining (MSD) function. The presence of different X and Y Chromosome copies of this gene indicated that the male heterogametic (XY) system of sex determination in sablefish arose by allelic diversification. The gsdfY gene has a spatio-temporal expression profile characteristic of a male MSD gene. We provide experimental evidence demonstrating a pivotal role of a transposable element (TE) for the divergent function of gsdfY By insertion within the gsdfY promoter region, this TE generated allelic diversification by bringing cis-regulatory modules that led to transcriptional rewiring and thus creation of a new MSD gene. This points out, for the first time in the scenario of MSD gene evolution by allelic diversification, a single, punctual molecular event in the appearance of a new trigger for male development.

Inhibition of ovarian development and instances of sex reversal in genotypic female sablefish (Anoplopoma fimbria) exposed to elevated water temperature.

This study determined high temperature effects on ovarian development in a marine groundfish species, sablefish (Anoplopoma fimbria), with potential application in sex reversal or sterilization for aquaculture. Monosex female (XX-genotype) sablefish larvae (∼30 mm) were randomly divided into three groups and exposed to control (15.6 °C ±â€¯0.8 °C), moderate (20.4 °C ±â€¯0.5 °C), or high (21.7 °C ±â€¯0.5 °C) temperatures for 19 weeks. Treated fish were then tagged and transferred to ambient seawater (11.2 °C ±â€¯2.3 °C) for one year to determine whether temperature effects on reproductive development were maintained post-treatment. Fish were periodically sampled for gonadal histology, gene expression and plasma 17ß-estradiol (E2) analyses to assess gonadal development. Short-term (4-week) exposure to elevated temperatures had only minor effects, whereas longer exposure (12-19 weeks) markedly inhibited ovarian development. Fish from the moderate and high treatment groups had significantly less developed ovaries relative to controls, and mRNA levels for germ cell (vasa, zpc) and apoptosis-associated genes (p53, casp8) generally indicated gonadal degeneration. The high treatment group also had significantly reduced plasma E2 levels and elevated gonadal amh gene expression. After one year at ambient temperatures, however, ovaries of moderate and high treatment fish exhibited compensatory recovery and were indistinguishable from controls. Two genotypic females possessing immature testes (neomales) were observed in the high treatment group, indicating sex reversal had occurred (6% rate). These results demonstrate that extreme elevated temperatures may inhibit ovarian development or trigger sex reversal. High temperature treatment is likely not an effective sterilization method but may be preferable for sablefish neomale broodstock production.

Initiation of sex change and gonadal gene expression in black sea bass (Centropristis striata) exposed to exemestane, an aromatase inhibitor.

Many teleost fishes exhibit sequential hermaphroditism, where male or female gonads develop first and later undergo sex change. Model sex change species are characterized by social hierarchies and coloration changes, which enable experimental manipulations to better understand these processes. However, other species such as the protogynous black sea bass (Centropristis striata) do not exhibit these characteristics and instead receive research attention due to their importance in fisheries or aquaculture. Black sea bass social structure is unknown, which makes sex change sampling difficult, and few molecular resources are available. The purpose of the present study was to induce sex change using exemestane, an aromatase inhibitor, and assess gonadal gene expression using sex markers (amh, zpc2) and genes involved in steroidogenesis (cyp19a1a, cyp11b), estrogen signaling (esr1, esr2b), and apoptosis or atresia (aen, casp9, fabp11, parg, pdcd4, rif1). Overall, dietary exemestane treatment was effective, and most exposed females exhibited early histological signs of sex change and significantly higher rates of ovarian atresia relative to control females. Genes associated with atresia did not reflect this, however, as expression patterns in sex changing gonads were overall similar to those of ovaries, likely due to a whole ovary dilution effect of the RNA. Still, small but insignificant expression decreases during early sex change were detected for ovary-related genes (aen, casp9, fabp11, zpc2) and anti-apoptotic factors (parg, rif1). Exemestane treatment did not impact spermatogenesis or testicular gene expression, but testes were generally characterized by elevated steroidogenic enzyme and estrogen receptor mRNAs. Further research will be needed to understand these processes in black sea bass, using isolated ovarian follicles and multiple stages of sex change.

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.

Molecular characterization of the gonadal kisspeptin system: Cloning, tissue distribution, gene expression analysis and localization in sablefish (Anoplopoma fimbria).

The kisspeptin system plays pivotal roles in the regulation of vertebrate reproduction. Classically, kisspeptin produced in the brain stimulates brain gonadotropin-releasing hormone signaling, which in turn activates the pituitary-gonad axis. Expression of the kisspeptin system has also been documented in peripheral tissues, including gonads of mammals and fishes. However, the fish gonadal kisspeptin system remained uncharacterized. Herein we report identification and characterization of four kisspeptin system mRNAs (kisspeptin 1 (kiss1), kiss2, and G protein-coupled receptor 54-1 (gpr54-1) and gpr54-2) in sablefish, Anoplopoma fimbria. Sablefish predicted protein sequences were highly similar to those of other marine teleosts, but less so to freshwater teleosts. Tissue distribution analyses revealed that all four kisspeptin-system transcripts were expressed in both brain and gonad. However, kiss2 was the predominant transcript in the gonads and the only transcript detected in ovulated eggs. Ontogenetic analysis of kiss2 expression in juvenile sablefish gonads demonstrated that levels were low during sex differentiation but increased with fish size and gonadal development. Dramatic increases in kiss2 mRNA occurred during primary oocyte growth, while levels remained relatively low in testes. In situ hybridization revealed that kiss2 mRNA was localized to cytoplasm of perinucleolus stage oocytes, suggesting it could play a local role in oogenesis or could be synthesized and stored within oocytes as maternal mRNA. This represents the first study to focus on the gonadal kisspeptin system in fishes and provides important tools for further investigation of both the gonadal and brain kisspeptin systems in sablefish.

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.

Gonadal sex differentiation and effects of dietary methyltestosterone treatment in sablefish (Anoplopoma fimbria).

Methods for sex control are needed to establish monosex aquaculture of sablefish (Anoplopoma fimbria). Here we conducted the first characterization of sex differentiation by histology and hormonal sex reversal experiment in sablefish. Ovarian differentiation was first discernible at ~80 mm fork length (FL) and characterized by development of lamellar structures and onset of meiosis. Testes exhibited a dual-lobe appearance over much of their length and remained non-meiotic until males were ≥520 mm FL (2 years post-fertilization). Juveniles with undifferentiated gonads were provided diets containing 0 (control), 5 or 50 mg 17α-methyltestosterone (MT)/kg for 2 months. Following treatment, controls possessed either ovaries or non-meiotic testes, whereas MT-treated fish exhibited meiotic testes (60% of the fish), intersex gonads (~30%), or gonads that appeared sterile (~10%). A genetic sex marker revealed that all intersex fish were genetic females, although other females appeared to be completely sex reversed (i.e., neomales). One year after treatment, MT-treated fish possessed non-meiotic testes similar to control males or intersex gonads with reduced ovarian features, presumably due to atresia following MT withdrawal. Milt collected from neomales and genetic males 3 years post-treatment permitted sperm motility analyses; however, neomale sperm were virtually immotile. These results demonstrated that sablefish are differentiated gonochorists and that MT treatment from 76 to 196 mm FL induced permanent masculinization of a portion of the genetic females, but acquisition of sperm motility was impaired. Earlier administration of MT may be necessary to sex reverse a higher proportion of genetic females and reduce negative effects on fertility.

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.

Fish reproduction in a warming world: vulnerable points in hormone regulation from sex determination to spawning.

Reproduction in fishes is sensitive to temperature. Elevated temperatures and anomalous 'heat waves' associated with climate change have the potential to impact fish reproductive performance and, in some cases, even induce sex reversals. Here we examine how thermal sensitivity in the hormone pathways regulating reproduction provides a framework for understanding impacts of warmer conditions on fish reproduction. Such effects will differ depending on evolved variation in temperature sensitivity of endocrine pathways regulating reproductive processes of sex determination/differentiation, gametogenesis and spawning, as well as how developmental timing of those processes varies with reproductive ecology. For fish populations unable to shift geographical range, persistence under future climates may require changes in temperature responsiveness of the hormone pathways regulating reproductive processes. How thermal sensitivity in those hormone pathways varies among populations and species, how those pathways generate temperature maxima for reproduction, and how rapidly reproductive thermal tolerances can change via adaptation or transgenerational plasticity will shape which fishes are most at risk for impaired reproduction under rising temperatures. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

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.

Normalizing for biology: accounting for technical and biological variation in levels of reference gene and insulin-like growth factor 1 (igf1) transcripts in fish livers.

Feeding, fasting and re-feeding is a common experimental paradigm for studying growth endocrinology. Herein we demonstrate dynamic changes in the livers of coho salmon under these conditions and how changes in liver composition can influence quantification and interpretation of liver gene expression data. A three-week fast resulted in decreases in hepatosomatic index (liver size), liver glycogen content, and liver DNA concentration. In addition, significant differences were found in liver transcript levels from fed and fasted fish for the reference genes, arp and ef1a, when these were normalized to total RNA. We took the additional step of normalizing reference gene transcript levels to the liver homogenate RNA/DNA ratio to account for differences in RNA yield/cell and the number of cells sampled, normalizing to transcript number per cell rather than transcript number per unit RNA. After this additional step no significant differences in liver transcript levels of reference genes were found. The significance of these results was illustrated by normalizing liver transcript levels of insulin-like growth factor 1 (igf1) to ef1a transcript levels or ef1a transcript levels by RNA/DNA. The different normalization strategies resulted in differing patterns of change in igf1 transcript levels between fed and fasted fish. The novelty of this work rests in a two-step normalization process, attempting to account for both 1) technical errors in reverse transcription and qPCR reactions, and 2) biological variance in liver samples.

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.

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.

Molecular and morphological sex differentiation in sablefish (Anoplopoma fimbria), a marine teleost with XX/XY sex determination.

Phenotypic sex of an organism is determined by molecular changes in the gonads, so-called molecular sex differentiation, which should precede the rise of cellular or anatomical sex-distinguishing features. This study characterized molecular and morphological sex differentiation in sablefish (Anoplopoma fimbria), a marine teleost with established XX/XY genotypic sex determination. Next generation sequencing was conducted on sablefish ovarian and testicular mRNAs to obtain sequences for transcripts associated with vertebrate sex determination and differentiation and early reproductive development. Gene-specific PCRs were developed to determine the distribution and ontogenetic gonadal expression of transcription, growth, steroidogenic and germline factors, as well as gonadotropin and steroid receptors. Molecular changes associated with sex differentiation were first apparent in both XY- and XX-genotype sablefish at ~ 60 mm in body length and prior to histological signs of sex differentiation. The earliest and most robust markers of testicular differentiation were gsdf, amh, dmrt1, cyp11b, star, sox9a, and fshr. Markedly elevated mRNA levels of several steroidogenesis-related genes and ar2 in differentiating testes suggested that androgens play a role in sablefish testicular differentiation. The earliest markers of ovarian differentiation were cyp19a1a, lhcgr, foxl2, nr0b1, and igf3. Other transcripts such as figla, zp3, and pou5f3 were expressed predominantly in XX-genotype fish and significantly increased with the first appearance and subsequent development of primary oocytes. This study provides valuable insight to the developmental sequence of events associated with gonadal sex differentiation in marine teleosts with XX/XY sex determination. It also implicates particular genes in processes of male and female development and establishes robust molecular markers for phenotypic sex in sablefish, useful for ongoing work related to sex control and reproductive sterilization.

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.

Phospholipid and LC-PUFA metabolism in Atlantic salmon (Salmo salar) testes during sexual maturation.

The importance of dietary lipids in male reproduction are not as well understood as in females, in which dietary lipids, such as phospholipids (PL) and associated fatty acids (FA), are important structural components of the eggs and provide energy for their offspring. In mammals, lipids are suggested to be important for spermatogenesis and to structural components of the spermatozoa that could improve fertilization rates. New knowledge of how lipids affect sexual maturation in male Atlantic salmon (Salmo salar), an important global aquaculture species, could provide tools to delay maturation and/or improve reproductive success. Therefore, changes in testicular composition of lipids and gene transcripts associated with spermatogenesis and lipid metabolism were studied in sexually maturing male salmon compared to immature males and females. An increase in total testis content of FA and PL, and a shift to higher PL composition was observed in maturing males, concomitant with increases in mRNA levels for genes involved in spermatogenesis, FA uptake and synthesis, and production of long chain-polyunsaturated fatty acids (LC-PUFA) and PL. A particularly interesting finding was elevated testis expression of acyl-CoA synthetase 4 (acsl4), and acyl-CoA thioesterase 2 (acot2), critical enzymes that regulate intra-mitochondrial levels of 20:4n-6 FA (arachidonic acid), which have been associated with improved cholesterol transport during steroidogenesis. This suggested that FA may have direct effects on sex steroid production in salmon. Furthermore, we observed increased testis expression of genes for endogenous synthesis of 16:0 and elongation/desaturation to 22:6n-3 (docosahexaenoic acid) in sexually maturing males relative to immature fish. Both of these FA are important structural components of the PL, phosphatidylcholine (PC), and were elevated concomitant with increases in the content of phosphatidic acid, an important precursor for PC, in maturing males compared to immature fish. Overall, this study suggests that, similar to mammals, lipids are important to spermatogenesis and serve as structural components during testicular growth and maturation in Atlantic salmon.

Osmoregulation and expression of ion transport proteins and putative claudins in the gill of southern flounder (Paralichthys lethostigma).

The southern flounder is a euryhaline teleost that inhabits ocean, estuarine, and riverine environments. We investigated the osmoregulatory strategy of juvenile flounder by examining the time-course of homeostatic responses, hormone levels, and gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein expression after salinity challenge. Transfer of freshwater (FW)-acclimated flounder to sea water (SW) induced an increase in plasma osmolality and cortisol and a decrease in muscle water content, plasma insulin-like growth factor I (IGF-I) and hepatic IGF-I mRNA, all returning to control levels after 4 days. Gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein levels were elevated in response to SW after 4 days. Transfer of SW-acclimated flounder to FW reduced gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein, increased plasma IGF-I, but did not alter hepatic IGF-I mRNA or plasma cortisol levels. Gill claudin-3 and claudin-4 immunoreactive proteins were elevated in FW versus SW acclimated flounder. The study demonstrates that successful acclimation of southern flounder to SW or FW occurs after an initial crisis period and that the salinity adaptation process is associated with changes in branchial expression of ion transport and putative tight junction claudin proteins known to regulate epithelial permeability in mammalian vertebrates.

Reproductive life history of sablefish (Anoplopoma fimbria) from the U.S. Washington coast.

Sablefish (Anoplopoma fimbria) is a marine groundfish that supports valuable fisheries in the North Pacific Ocean and holds promise for marine aquaculture. Limited information is available, however, about its reproductive biology. This study aimed to characterize the complete reproductive cycle, including seasonal changes in gonadal development (macroscopic and histological), plasma sex steroid levels (17ß-estradiol -E2-, and 11-ketotestosterone -11KT-), gonadosomatic and hepatosomatic indices (GSI, and HSI), and condition factor (K) of female and male sablefish captured off the Washington coast. Adult fish (209 females, 159 males) were caught by longline monthly from August 2012 to August 2013. Early signs of recruitment of ovarian follicles into secondary growth, indicated by oocytes containing small yolk granules and cortical alveoli, were first observed in March. Oogenesis progressed during spring and summer, and fully vitellogenic follicles were first observed in July. Vitellogenic growth was correlated with increases in plasma E2, GSI, HSI and K. Periovulatory females, indicated by fully-grown oocytes with migrating germinal vesicles and hydrated oocytes, were found from November to February. At this stage, plasma E2 and GSI reached maximal levels. In males, proliferating cysts containing spermatocytes were first observed in April. Testicular development proceeded during spring and summer, a period during which all types of male germ cells were found. The first clusters of spermatozoa appeared in July, concomitant with a 5.2-fold increase in GSI. Spermiating males were observed from November to April; at this time, spermatids were absent or greatly reduced, and testis lobules were filled with spermatozoa. The highest levels of plasma 11KT were found in males at this stage. Postspawning ovaries and testes, and basal steroids levels were found in fish captured from February to April. These results suggest that sablefish in coastal Washington initiate their reproductive cycle in March/April and spawn primarily in January/February.

Seasonal physiological dynamics of maturing female southern flounder (Paralichthys lethostigma).

Physiological information is rarely used in descriptions of maturity for managed, wild fish species; however, the use of physiological data holds great promise to provide important detail on the complexities of oocyte development and maturity. Investigating southern flounder (Paralichthys lethostigma)-an overfished commercial and recreational fishery resource-we examined pre-spawn physiological changes in females to provide further detail of the maturation process. Given that adults of this species complete maturation and spawn in unknown offshore locations, information on pre-spawn physiological changes is particularly informative for both size- and age-based patterns of maturity. We evaluated seasonal and ontogenetic changes in hormone concentrations in blood plasma that are commonly associated with sexual maturation, in addition to quantifying and classifying lipid stored in liver tissue. We found a strong positive relationship between body weight and lipid content during all months, as well as evidence for mobilization of lipids among larger females in September and October, presumably for gonadal development. Throughout the sampling period, the lipid content of smaller individuals was dominated by structural lipids (as opposed to storage lipids). In contrast, larger individuals possessed greater amounts of storage lipids. This suggests that larger, putatively maturing individuals were accumulating storage lipids for later production of vitellogenin. Females sampled for blood sex steroids and ovarian histology showed different testosterone and estradiol concentrations between putatively maturing and immature fish, and temporal variation with peaks in October and November. Overall, emerging patterns of liver lipid content and composition and blood steroid concentrations describe a multi-month maturation process that is often managed one dimensionally over short time periods. Insights from this work will improve our understanding of the life history of southern flounder, with the potential for better understanding of the dynamics of offshore spawning migration and informing subsequent species management.

Expression profiles of Fsh-regulated ovarian genes during oogenesis in coho salmon.

The function of follicle-stimulating hormone (Fsh) during oogenesis in fishes is poorly understood. Using coho salmon as a fish model, we recently identified a suite of genes regulated by Fsh in vitro and involved in ovarian processes mostly unexplored in fishes, like cell proliferation, differentiation, survival or extracellular matrix (ECM) remodeling. To better understand the role of these Fsh-regulated genes during oocyte growth in fishes, we characterized their mRNA levels at discrete stages of the ovarian development in coho salmon. While most of the transcripts were expressed at low levels during primary growth (perinucleolus stage), high expression of genes associated with cell proliferation (pim1, pcna, and mcm4) and survival (ddit4l) was found in follicles at this stage. The transition to secondary oocyte growth (cortical alveolus and lipid droplet stage ovarian follicles) was characterized by a marked increase in the expression of genes related to cell survival (clu1, clu2 and ivns1abpa). Expression of genes associated with cell differentiation and growth (wt2l and adh8l), growth factor signaling (inha), steroidogenesis (cyp19a1a) and the ECM (col1a1, col1a2 and dcn) peaked in vitellogenic follicles, showing a strong and positive correlation with transcripts for fshr. Other genes regulated by Fsh and associated with ECM function (ctgf, wapl and fn1) and growth factor signaling (bmp16 and smad5l) peaked in maturing follicles, along with increases in steroidogenesis-related gene transcripts. In conclusion, ovarian genes regulated by Fsh showed marked differences in their expression patterns during oogenesis in coho salmon. Our results suggest that Fsh regulates different ovarian processes at specific stages of development, likely through interaction with other intra- or extra-ovarian factors.