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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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Polybrominated diphenyl ether (PBDE) flame retardants have been implicated as disruptors of the hypothalamic-pituitary-thyroid axis. Animals exposed to PBDEs impact TH-regulated pathways in target tissues. OBJECTIVE: We examined the [...]

Abstract 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.

Abstract Background The aim of this study was to identify differentially expressed ovarian genes during primary and early secondary oocyte growth in coho salmon, a semelparous teleost that exhibits synchronous follicle development. Methods Reciprocal suppression subtractive hybridization (SSH) libraries were generated from ovaries with perinucleolus (P) or cortical alveolus (CA) stage follicles and selected genes were assessed with quantitative PCR (qPCR). An assessment of changes in RNA composition during oocyte growth and its relationship to transcript levels was also conducted. Results SSH revealed several differentially expressed genes during early oogenesis, some which will not likely be utilized until 1–3 years later in salmon. Zona pellucida glycoprotein (zp) genes, vitellogenin receptor (vldlr) isoforms, cathepsin B (ctsba), cyclin E (ccne), a DnaJ transcript (dnaja2), and a ferritin subunit (fth3) were significantly elevated at the P stage, while a C-type lectin, retinol dehydrogenase (rdh1), and a coatomer protein subunit (cope) were upregulated at the CA stage. Putative follicle cell transcripts such as anti-Müllerian hormone (amh), lipoprotein lipase (lpl), apolipoprotein E (apoe), gonadal soma-derived growth factor (gsdf) and follicle-stimulating hormone receptor (fshr) also increased significantly at the CA stage. The analysis of RNA composition during oocyte growth showed that the total RNA yield and proportion of messenger RNA relative to non-polyadenylated RNAs declined as oogenesis progressed. This influenced apparent transcript levels depending on the type of RNA template used and normalization method. Conclusion In coho salmon, which exhibit a dramatic change in oocyte size and RNA composition during oogenesis, use of messenger RNA as template and normalization of qPCR data to a housekeeping gene, ef1a, yielded results that best reflected transcript abundance within the ovarian follicle. Synthesis of zp transcripts and proteins involved in yolk incorporation and processing occurred during primary growth, while increased expression of a CA component and genes related to lipid incorporation occurred concomitant with the appearance of CA, but prior to lipid accumulation. Significant increases in transcripts for fshr, gsdf, and amh at the CA stage suggest a role of FSH and TGFβ peptides in previtellogenic oocyte growth and puberty onset in female salmon.

Studies of microinjection of linear pOnMTGH1 DNA, a genetic construct consisting of the metallothionein-B promoter coupled to the type-1 growth hormone gene, into the blastodisc area of coho salmon eggs whose growth is arrested following fertilization reveal that pOnMTGH1 construct is vital for growth acceleration. The transgenic fishes containing pOnMTGH1 formed a silver body coloration and were markedly larger than non-transgenic fishes. Transgenic fishes also exhibited high winter levels of growth hormone.

Many salmonids have a male heterogametic (XX/XY) sex determination system, and they are supposed to have a conserved master sexdetermining 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 b-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 SdYI183N 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. [ABSTRACT FROM AUTHOR]

While the goal of most conservation hatchery programs is to produce fish that are genetically and phenotypically indistinguishable from the wild stocks they aim to restore, there is considerable evidence that salmon and steelhead reared in hatcheries differ from wild fish in phenotypic traits related to fitness. Some evidence suggests that these phenotypic differences have a genetic basis ( e.g. , domestication selection) but another likely mechanism that remains largely unexplored is that differences between hatchery and wild populations arise as a result of environmentally-induced heritable epigenetic change. As a first step toward understanding the potential contribution of these two possible mechanisms, we describe genetic and epigenetic variation in hatchery and natural-origin adult steelhead, Oncorhynchus mykiss , from the Methow River, WA. Our main objectives were to determine if hatchery and natural-origin fish could be distinguished genetically and whether differences in epigenetic programming (DNA methylation) in somatic and germ cells could be detected between the two groups. Genetic analysis of 72 fish using 936 SNPs generated by Restriction Site Associated DNA Sequencing (RAD-Seq) did not reveal differentiation between hatchery and natural-origin fish at a population level. We performed Reduced Representation Bisulfite Sequencing (RRBS) on a subset of 10 hatchery and 10 natural-origin fish and report the first genome-wide characterization of somatic (red blood cells (RBCs)) and germ line (sperm) derived DNA methylomes in a salmonid, from which we identified considerable tissue-specific methylation. We identified 85 differentially methylated regions (DMRs) in RBCs and 108 DMRs in sperm of steelhead reared for their first year in a hatchery environment compared to those reared in the wild. This work provides support that epigenetic mechanisms may serve as a link between hatchery rearing and adult phenotype in steelhead; furthermore, DMRs identified in germ cells (sperm) highlight the potential for these changes to be passed on to future generations., (Copyright © 2018 Gavery et al.)