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

Defining Global Gene Expression Changes of the Hypothalamic-Pituitary-Gonadal Axis in Female sGnRH-Antisense Transgenic Common Carp (Cyprinus carpio)

BACKGROUND: The hypothalamic-pituitary-gonadal (HPG) axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH) expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system. METHODOLOGY/PRINCIPAL FINDINGS: In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio) with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH) and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus), 16 and 12 (pituitary), 119 and 93 (ovary), respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified. CONCLUSIONS/SIGNIFICANCE: This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the reproductive system of teleost fish

Gonadal differentiation and effects of temperature on sex determination in southern flounder (Paralichthys lethostigma) PII: S 0 0 4 4 -8 4 8 6 ( 0 2 ) 0 0 4 0 7 -6

Abstract Southern flounder (Paralichthys lethostigma) support valuable North American fisheries and show great promise for aquaculture. Because females grow faster and reach larger adult sizes than males, monosex culture of females is desirable for commercial operations. A detailed understanding of sexual development and its timing is critical to control sex and optimize culture. Structural and cellular sex-distinguishing markers were identified histologically, and then used to describe ovarian development in female and testicular development in male flounder. In presumptive ovaries of southern flounder, development of an ovarian cavity first occurs in fish ranging from 75 to 100 mm total length (TL). This is considerably delayed relative to that observed in the Japanese congener, Paralichthys olivaceus, where an ovarian cavity is seen in fish as small as 40 mm TL. The smallest southern flounder that possessed primary oocytes in the early perinucleolus stage was 115 mm TL. In presumptive testes, the formation of seminiferous tubules first occurs in fish of approximately 100 mm TL. Spermatogonia remained quiescent until most fish were over 100 mm TL. Overall, gonads from southern flounder greater than 120 mm TL commonly possess gonial cells undergoing meiosis, clearly differentiating sex. The effect of temperature on sex determination in southern flounder was addressed in a separate experiment. Juvenile southern flounder were grown at 18, 23, or 28jC for 245 days. High and low temperatures induced phenotypic sex reversal in juvenile southern flounder, producing a higher proportion of males (96% males at high temperature, P < 0.001, 78% males at low temperature, P < 0.01). Raising southern flounder at the midrange temperature held sex ratios close to 1:1. Sex ratios from these trials suggest that southern flounder possess a temperaturesensitive mechanism of sex determination similar to that shown for P. olivaceus, but possibly shifted towards warmer temperatures. These findings indicate that sex differentiation in southern flounder is distinguishable in most fish by 100 -120 mm TL and that sex determination is sensitive to temperature. This information is critical to the development of strategies to maximize the number of faster-growing females for commercial flounder culture.

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

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.publishedVersio

Adaptive Divergence in the Thyroid Hormone Signaling Pathway in the Stickleback Radiation

During adaptive radiations, animals colonize diverse environments, which requires adaptation in multiple phenotypic traits. Because hormones mediate the dynamic regulation of suites of phenotypic traits, evolutionary changes in hormonal signaling pathways might contribute to adaptation to new environments. Here we report changes in the thyroid hormone signaling pathway in stream-resident ecotypes of threespine stickleback fish (Gasterosteus aculeatus), which have repeatedly evolved from ancestral marine ecotypes. Stream-resident fish exhibit a lower plasma concentration of thyroid hormone and a lower metabolic rate, which is likely adaptive for permanent residency in small streams. The thyroid-stimulating hormone-β2 (TSHβ2) gene exhibited significantly lower mRNA expression in pituitary glands of stream-resident sticklebacks relative to marine sticklebacks. Some of the difference in TSHβ2 transcript levels can be explained by cis-regulatory differences at the TSHβ2 gene locus. Consistent with these expression differences, a strong signature of divergent natural selection was found at the TSHβ2 genomic locus. By contrast, there were no differences between the marine and stream-resident ecotypes in mRNA levels or genomic sequence in the paralogous TSHβ1 gene. Our data indicate that evolutionary changes in hormonal signaling have played an important role in the postglacial adaptive radiation of sticklebacks

Toward enhanced MIQE compliance: reference residual normalization of qPCR gene expression data

Normalization of fluorescence-based quantitative real-time PCR (qPCR) data varies across quantitative gene expression studies, despite its integral role in accurate data quantification and interpretation. Identification of suitable reference genes plays an essential role in accurate qPCR normalization, as it ensures that uncorrected gene expression data reflect normalized data. The reference residual normalization (RRN) method presented here is a modified approach to conventional 2^-(ΔΔCt)qPCR normalization that increases mathematical transparency and incorporates statistical assessment of reference gene stability. RRN improves mathematical transparency through the use of sample-specific reference residuals (RRi) that are generated from the mean Ct of one or more reference gene(s) that are unaffected by treatment. To determine stability of putative reference genes, RRN uses ANOVA to assess the effect of treatment on expression and subsequent equivalence-threshold testing to establish the minimum permitted resolution. Step-by-step instructions and comprehensive examples that demonstrate the influence of reference gene stability on target gene normalization and interpretation are provided. Through mathematical transparency and statistical rigor, RRN promotes compliance with Minimum Information for Quantitative Experiments and, in so doing, provides increased confidence in qPCR data analysis and interpretation

Expression profiles of genes associated with tissue or extracellular matrix remodeling during major stages of oogenesis in coho salmon.

<p>Messenger RNA levels were analyzed by qPCR and data were normalized to the geometric mean of four reference genes (<i>eef1a</i>, <i>ctsd</i>, <i>ctsz</i> and <i>actb</i>). PN, perinucleolus stage follicles; CA, cortical alveolus stage follicles; LD, lipid droplet stage follicles; VIT, mid-vitellogenic stage follicles; MAT, postvitellogenic/preovulatory stage follicles. Bars not sharing the same letter are significantly different (P<0.05; n = 4 fish/stage for PN-, CA-, LD-, and VIT-stages, n = 3 fish for MAT-stage, mean ± SEM).</p

Expression profiles of genes associated with growth factor signaling during major stages of oogenesis in coho salmon.

<p>Messenger RNA levels were analyzed by qPCR and data were normalized to the geometric mean of four reference genes (<i>eef1a</i>, <i>ctsd</i>, <i>ctsz</i> and <i>actb</i>). PN, perinucleolus stage follicles; CA, cortical alveolus stage follicles; LD, lipid droplet stage follicles; VIT, mid-vitellogenic stage follicles; MAT, postvitellogenic/preovulatory stage follicles. Bars not sharing the same letter are significantly different (P<0.05; n = 4 fish/stage for PN-, CA-, LD-, and VIT-stages, n = 3 fish for MAT-stage, mean ± SEM).</p

Expression profiles of genes associated with cell differentiation and growth during major stages of oogenesis in coho salmon.

<p>Messenger RNA levels were analyzed by qPCR and data were normalized to the geometric mean of four reference genes (<i>eef1a</i>, <i>ctsd</i>, <i>ctsz</i> and <i>actb</i>). PN, perinucleolus stage follicles; CA, cortical alveolus stage follicles; LD, lipid droplet stage follicles; VIT, mid-vitellogenic stage follicles; MAT, postvitellogenic/preovulatory stage follicles. Bars not sharing the same letter are significantly different (P<0.05; n = 4 fish/stage for PN-, CA-, LD-, and VIT-stages, n = 3 fish for MAT-stage, mean ± SEM).</p