Characterizing the SREB G protein-coupled receptor family in fish: Brain gene expression and genomic differences in upstream transcription factor binding sites.

The SREB (Super-conserved Receptors Expressed in Brain) family of orphan G protein-coupled receptors is highly conserved in vertebrates and consists of three members: SREB1 (orphan designation GPR27), SREB2 (GPR85), and SREB3 (GPR173). SREBs are associated with processes ranging from neuronal plasticity to reproductive control. Relatively little is known about similarities across the entire family, or how mammalian gene expression patterns compare to non-mammalian vertebrates. In fish, this system may be particularly complex, as some species have gained a fourth member (SREB3B) while others have lost genes. To better understand the system, the present study aimed to: 1) use qPCR to characterize sreb and related gene expression patterns in the brains of three fish species with different systems, and 2) identify possible differences in transcriptional regulation among the receptors, using upstream transcription factor binding sites across 70 ray-finned fish genomes. Overall, regional patterns of sreb expression were abundant in forebrain-related areas. However, some species-specific patterns were detected, such as abundant expression of receptors in zebrafish (Danio rerio) hypothalamic-containing sections, and divergence between sreb3a and sreb3b in pufferfish (Dichotomyctere nigroviridis). In addition, a gene possibly related to the system (dkk3a) was spatially correlated with the receptors in all three species. Genomic regions upstream of sreb2 and sreb3b, but largely not sreb1 or sreb3a, contained many highly conserved transcription factor binding sites. These results provide novel information about expression differences and transcriptional regulation across fish that may inform future research to better understand these receptors.

Phoenixin-14 alters transcriptome and steroid profiles in female green-spotted puffer (Dichotomyctere nigroviridis).

Phoenixin (PNX) is a highly conserved, novel hormone with diverse functions, including hypothalamic control of reproduction, appetite modulation, and regulation of energy metabolism and inflammation. While some functions appear conserved across vertebrates, additional research is required to fully characterize these complex pleiotropic effects. For instance, very little is known about transcriptome level changes associated with PNX exposure, including responses in the hypothalamic-pituitary-gonadal (HPG) axis, which is critical in vertebrate reproduction. In addition, the PNX system may be especially complex in fish, where an additional receptor is likely present in some species. The purpose of this study was to assess hypothalamic and ovarian transcriptomes after PNX-14 administration in female vitellogenic green-spotted puffer (Dichotomyctere nigroviridis). Steroid-related changes were also assessed in the liver and blood plasma. Hypothalamic responses included pro-inflammatory signals such as interleukin 1ß, possibly related to gut-brain axis functions, as well as suppression of cell proliferation. Ovarian responses were more widely downregulated across all identified pathways, which may reflect progression to a less transcriptionally active state in oocytes. Both organs shared regulation in transforming growth factor-ß and extracellular matrix remodeling (periostin) pathways. Reproductive processes were in general downregulated, but both inhibiting (bone morphogenetic protein 15 and follistatin) and promoting (17-hydroxyprogesterone) factors for oocyte maturation were identified. Select genes involved in reproduction (vitellogenins, estrogen receptors) in the liver were unresponsive to PNX-14 and higher doses may be needed to induce reproductive effects in D. nigroviridis. These results reinforce the complexity of PNX actions in diverse tissues and highlight important roles for this hormone in regulating the immune response, energy metabolism, and cell growth.

Characterization of the G protein-coupled receptor family SREB across fish evolution.

The SREB (Super-conserved Receptors Expressed in Brain) family of G protein-coupled receptors is highly conserved across vertebrates and consists of three members: SREB1 (orphan receptor GPR27), SREB2 (GPR85), and SREB3 (GPR173). Ligands for these receptors are largely unknown or only recently identified, and functions for all three are still beginning to be understood, including roles in glucose homeostasis, neurogenesis, and hypothalamic control of reproduction. In addition to the brain, all three are expressed in gonads, but relatively few studies have focused on this, especially in non-mammalian models or in an integrated approach across the entire receptor family. The purpose of this study was to more fully characterize sreb genes in fish, using comparative genomics and gonadal expression analyses in five diverse ray-finned (Actinopterygii) species across evolution. Several unique characteristics were identified in fish, including: (1) a novel, fourth euteleost-specific gene (sreb3b or gpr173b) that likely emerged from a copy of sreb3 in a separate event after the teleost whole genome duplication, (2) sreb3a gene loss in Order Cyprinodontiformes, and (3) expression differences between a gar species and teleosts. Overall, gonadal patterns suggested an important role for all sreb genes in teleost testicular development, while gar were characterized by greater ovarian expression that may reflect similar roles to mammals. The novel sreb3b gene was also characterized by several unique features, including divergent but highly conserved amino acid positions, and elevated brain expression in puffer (Dichotomyctere nigroviridis) that more closely matched sreb2, not sreb3a. These results demonstrate that SREBs may differ among vertebrates in genomic structure and function, and more research is needed to better understand these roles in fish.

Metal stress-related gene expression patterns in two marine invertebrates, Hediste diversicolor (Annelida, Polychaeta) and Littorina littorea (Mollusca, Gastropoda), at a former mining site.

Abandoned mines often contaminate sediments with dissolved heavy metals and are known to impact many taxa. Physiological responses needed to avoid oxidative stress from metal toxicity include the upregulation of detoxification and metal-binding proteins such as glutathione-s-transferases and metallothioneins, which have been studied in diverse species. Fewer studies, however, have focused on gene expression changes to better understand these molecular mechanisms, especially across multiple species at a single contaminated site. To this end, the purpose of this study was to characterize metal stress-related gene expression in two species from different phyla, Hediste diversicolor (Annelida, Polychaeta) and Littorina littorea (Mollusca, Gastropoda), at a former mine site (Callahan Mine, Maine, USA). Both species and sediments were collected from a mine-affected tidal estuary (Goose Pond) and a nearby reference site. Elevated sediment metal levels were confirmed at Goose Pond. H. diversicolor individuals weighed significantly less at Goose Pond, while L. littorea weighed similarly at both sites. Transcript levels were stable in H. diversicolor but weakly upregulated in L. littorea, which likely reflect the importance of other physiological strategies for metal sequestration, or variable metal exposure at the individual level, respectively. In addition, patterns in glutathione-s-transferase expression differed across isoforms in H. diversicolor, while L. littorea exhibited divergent expression patterns in foot muscle and hepatopancreas. Overall, these results reinforce that diverse species likely undergo different physiological responses to metal toxicity, and more research is needed to investigate these mechanisms.

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.

The effects of stress, cortisol administration and cortisol inhibition on black sea bass (Centropristis striata) sex differentiation.

Sex differentiation in many lower vertebrates (e.g. reptiles, amphibians, and fishes) can be influenced by environmental factors experienced during sensitive developmental periods. Environmental stressors, acting through cortisol, masculinize some teleost fishes during development by limiting gonadal cytochrome P450 aromatase (cyp19a1a), the enzyme that irreversibly converts testosterone to 17ß-estradiol. In this study, we examined the influence of cortisol, cortisol inhibitors and a repeated, acute stressor (net-chasing) on sex differentiation in black sea bass (BSB; Centropristis striata), a protogynous hermaphroditic teleost. Wild-caught, sexually-undifferentiated, BSB juveniles (~90 mm) were collected from Rhode Island waters, raised in recirculating systems and fed diets supplemented with cortisol, a cortisol receptor antagonist (mifepristone), a cortisol synthesis inhibitor (metyrapone), or net-chased twice a week for two min until gonads were differentiated (77-89 days). Long term cortisol administration partially masculinized all female fish, but repeated net-chasing did not alter sex differentiation relative to the control group. Blocking cortisol receptor binding delayed sex differentiation in some individuals, but overall led to increased masculinization compared to control fish. The proportion of treatment fish that developed as males suggests a functionally, diandric protogynous reproductive strategy in this species. We also identified a glucocorticoid response element in the gonadal aromatase (cyp19a1a) promoter, indicating a possible relationship between cortisol and cyp19a1a gene expression.

Fine-scale population genetic structure of sugar kelp, Saccharina latissima (Laminariales, Phaeophyceae), in eastern Maine, USA.

There is an interest to develop sugar kelp (Saccharina latissima) cultivation in the rural, eastern Maine region of the USA. Future farming efforts would benefit from an understanding of the genetic diversity and population structure of kelp, to inform management and conservation, and to identify genetic resources. The purpose of the present study was to characterize the fine-scale population genetic structure of kelp in eastern Maine, using twelve microsatellite loci. A total of 188 samples were genotyped from five sampling locations. Overall, kelp exhibited relatively low genetic diversity and small but significant differentiation among populations (FST = 0.0157). The greatest genetic difference was detected between two geographically close populations in Penobscot and Frenchman Bays, which is likely due to patterns in the Eastern Maine Coastal Current that may limit meiospore recruitment. The population structure could not be fully explained by an isolation-by-distance model. Fine-scale structuring was also detected among populations along the more continuous, eastern Maine coastline. These differences highlight that sugar kelp populations are finely structured across small spatial scales, and that future management and farming efforts should aim to maintain genetic diversity and assess the culture potential of local populations.

Genome Sequences of Four Subcluster L2 Mycobacterium Phages, Finemlucis, Miley16, Wilder, and Zakai.

Four subcluster L2 mycobacteriophages, Finemlucis, Miley16, Wilder, and Zakai, that infect Mycobacterium smegmatis mc2155 were isolated. The four phages are closely related to each other and code for 12 to 14 tRNAs and 130 to 132 putative protein-coding genes, including tyrosine integrases, cro, immunity repressors, and excise genes involved in the establishment of lysogeny.

The effects of temperature on ovarian aromatase (cyp19a1a) expression and sex differentiation in summer flounder (Paralichthys dentatus).

Female summer flounder grow considerably faster and larger than males, and a tremendous increase in performance can therefore be realized through production of monosex female populations. Rearing temperature has been shown to affect sex differentiation in other teleost species by influencing expression of genes encoding transcription factors or enzymes involved in endocrine function. Cyp19a1a is a well-studied gene that had been shown to play a role in ovarian development, and exhibits sexually dimorphic expression in other species. In the present study, summer flounder (37 days post-hatch; DPH) were raised at 13, 16 or 19 °C. Fish from all three treatments were sampled throughout development and analyzed in qPCR to determine cyp19a1a gene expression levels. Sex ratios of additional fish grown to ≥150 mm at each temperature treatment were determined. Low female production was achieved overall (26.9, 17.6 and 0% at 13, 16 and 19 °C, respectively). Cyp19a1a expression was significantly lower at 52 DPH (~15 mm total length) at the male-producing temperature (19 °C) and increased to similar levels as other treatments at 66 DPH. Expression levels later in juvenile development (66-191 DPH) largely decreased with fish size. The period of sex differentiation in summer flounder remains unknown, but cyp19a1a expression patterns suggest that it may occur earlier in development than that of congenerics. Further research is necessary to understand the sex-determining mechanisms in this species before sexually dimorphic growth can be used to achieve economic advantages in commercial production.

Identification and expression of GnRH2 and GnRH3 in the black sea bass (Centropristis striata), a hermaphroditic teleost.

We cloned two cDNAs for two gonadotropin-releasing hormones, GnRH2 (chicken GnRH-II) and GnRH3 (salmon GnRH), respectively, from the black sea bass (Centropristis striata). Black sea bass are protogynous hermaphroditic teleosts that change from females to males between 2 and 5 years of age. Similar to other GnRH precursors, the precursors of black sea bass GnRH2 and GnRH23 consisted of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide. Our analyses failed to identify GnRH1. GnRH3 precursor transcript was more widely distributed in a variety of tissues compared with GnRH2. Further examination of GnRH expression and gonadal histology was done in black sea bass from three different size groups: small (11.4-44.1 g), medium (179.4-352.2 g) and large (393.8-607.3 g). Interestingly, GnRH3 expression occurred only in the pituitaries of males in the small and medium groups compared with expression of GnRH2. Future functional studies of the sea bass GnRHs will be valuable in elucidating the potential underlying neuroendocrine mechanisms of black sea bass reproduction and may ultimately contribute to management advances in this commercially important fish.

Brain aromatase (cyp19a1b) and gonadotropin releasing hormone (gnrh2 and gnrh3) expression during reproductive development and sex change in black sea bass (Centropristis striata).

Teleost fish exhibit diverse reproductive strategies, and some species are capable of changing sex. The influence of many endocrine factors, such as gonadal steroids and neuropeptides, has been studied in relation to sex change, but comparatively less research has focused on gene expression changes within the brain in temperate grouper species with non-haremic social structures. The purpose of the present study was to investigate gonadotropin releasing hormone (GnRH) and brain aromatase (cyp19a1b) gene expression patterns during reproductive development and sex change in protogynous (female to male) black sea bass (Centropristis striata). Partial cDNA fragments for cyp19a1b and eef1a (a reference gene) were identified, and included with known gnrh2 and gnrh3 sequences in real time quantitative PCR. Elevated cyp19a1b expression was evident in the olfactory bulbs, telencephalon, optic tectum, and hypothalamus/midbrain region during vitellogenic growth, which may indicate changes in the brain related to neurogenesis or sexual behavior. In contrast, gnrh2 and gnrh3 expression levels were largely similar among gonadal states, and all three genes exhibited stable expression during sex change. Although sex change in black sea bass is not associated with dramatic changes in GnRH or cyp19a1b gene expression among brain regions, these genes may mediate processes at other levels, such as within individual hypothalamic nuclei, or through changes in neuron size, that warrant further research.

Characterizing ovarian gene expression during oocyte growth in Atlantic cod (Gadus morhua).

Vertebrate oocytes undergo dramatic changes during development as they accumulate many RNA transcripts, glycoproteins, and yolk proteins, necessary to ensure proper fertilization and embryogenesis. Oogenesis in teleosts often requires several years for completion, but very little is known about the early developmental stages. Recently, two-stage gene expression comparisons were made during oocyte growth in coho salmon (Oncorhynchus kisutch) and Atlantic cod (Gadus morhua), but more broad-scale, comprehensive assessments have not been conducted. The objectives of the present study were to characterize the gene expression patterns throughout oocyte growth in cod and compare them to changes previously identified in coho salmon. A quantitative PCR survey was conducted using 50 genes at six ovarian stages, ranging from the onset of primary growth (oocyte differentiation) to late vitellogenesis. Most expression patterns could be grouped into three major clusters, consisting of oocyte-derived (cluster 1) and likely follicle cell (clusters 2 and 3) genes. Oocyte genes were elevated during primary growth, while many follicle cell transcripts were abundant during oocyte differentiation and vitellogenesis. Few expression changes identified in coho salmon were evident in cod, which is likely due to differences in reproductive strategies. These results demonstrate that dynamic changes in gene expression occur during oocyte growth in teleost fish.

Identification of ovarian gene expression patterns during vitellogenesis in Atlantic cod (Gadus morhua).

Follicular maturational competence and ovulatory competence in teleost fish refer to the ability of the ovarian follicle to undergo final oocyte maturation and ovulation, respectively, in response to gonadotropin stimulation and other external cues. Some gene products related to competence acquisition are likely synthesized during vitellogenic growth, as these follicles gain in vivo responsiveness to exogenous gonadotropin stimulation and can be induced to undergo maturation and ovulation. In Atlantic cod (Gadus morhua), gonadotropin responsiveness has been shown to be oocyte size-dependent, and only ovaries containing late-stage vitellogenic follicles can be induced to ovulate. The purpose of the present study was to compare gene expression patterns between mid (unresponsive) and late (responsive) vitellogenic ovaries to identify genes involved in gonadotropin responsiveness and the acquisition of maturational and ovulatory competencies. Representational difference analysis was conducted in two reciprocal comparisons using intact ovarian fragments and follicle wall-enriched tissues, and genes of interest were used in real time quantitative PCR to confirm differential expression. Few differences were detected in intact ovarian fragments, but type IV ice-structuring protein and gephyrin were upregulated later in development and may be involved in lipid and sulfur metabolism, respectively. Candidate gene assays for luteinizing hormone receptor and aromatase also exhibited significant upregulation during vitellogenesis. Many genes were differentially expressed in follicle wall-enriched tissues, including endocrine maturational regulators and smooth muscle genes. Overall, maturational and ovulatory competencies during vitellogenesis in Atlantic cod are associated with up- and downregulation of many genes involved in lipid metabolism, endocrine regulation, and ovulatory preparation.