Combined Pituitary Hormone Deficiency in lhx4-Knockout Zebrafish.

LIM homeobox 4 (LHX4) is a transcription factor crucial for anterior pituitary (AP) development. Patients with LHX4 mutation suffer from combined pituitary hormone deficiency (CPHD), short statures, reproductive and metabolic disorders and lethality in some cases. Lhx4-knockout (KO) mice fail to develop a normal AP and die shortly after birth. Here, we characterize a zebrafish lhx4-KO model to further investigate the importance of LHX4 in pituitary gland development and regulation. At the embryonic and larval stages, these fish express lower levels of tshb mRNA compared with their wildtype siblings. In adult lhx4-KO fish, the expressions of pituitary hormone-encoding transcripts, including growth hormone (gh), thyroid stimulating hormone (tshb), proopiomelanocortin (pomca) and follicle stimulating hormone (fshb), are reduced, the pomca promoter-driven expression in corticotrophs is dampened and luteinizing hormone (lhb)-producing gonadotrophs are severely depleted. In contrast to Lhx4-KO mice, Lhx4-deficient fish survive to adulthood, but with a reduced body size. Importantly, lhx4-KO males reach sexual maturity and are reproductively competent, whereas the females remain infertile with undeveloped ovaries. These phenotypes, which are reminiscent of those observed in CPHD patients, along with the advantages of the zebrafish for developmental genetics research, make this lhx4-KO fish an ideal vertebrate model to study the outcomes of LHX4 mutation.

Glial cells expressing visual cycle genes are vital for photoreceptor survival in the zebrafish pineal gland.

Photoreceptors in the vertebrate eye are dependent on the retinal pigmented epithelium for a variety of functions including retinal re-isomerization and waste disposal. The light-sensitive pineal gland of fish, birds, and amphibians is evolutionarily related to the eye but lacks a pigmented epithelium. Thus, it is unclear how these functions are performed. Here, we ask whether a subpopulation of zebrafish pineal cells, which express glial markers and visual cycle genes, is involved in maintaining photoreceptors. Selective ablation of these cells leads to a loss of pineal photoreceptors. Moreover, these cells internalize exorhodopsin that is secreted by pineal rod-like photoreceptors, and in turn release CD63-positive extracellular vesicles (EVs) that are taken up by pdgfrb-positive phagocytic cells in the forebrain meninges. These results identify a subpopulation of glial cells that is critical for pineal photoreceptor survival and indicate the existence of cells in the forebrain meninges that receive EVs released by these pineal cells and potentially function in waste disposal.

Ectopic over expression of kiss1 may compensate for the loss of kiss2.

Kisspeptin (KISS) is a neuropeptide which plays a central role in the regulation of the hypothalamic-pituitary-gonadal axis, and is essential for sexual maturation and fertility in mammals. Unlike mammals, which possess only one KISS gene, two paralogous genes, kiss1 and kiss2, have been identified in zebrafish and other non-mammalian vertebrates. Previous studies suggest that Kiss2, but not Kiss1, is the reproduction relevant form amongst the two. To better understand the role of each of these isoforms in reproduction, a loss of function approach was applied. Two genetic manipulation techniques-clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN)-were used to generate kiss1 and kiss2 knockout (KO) zebrafish lines, respectively. Examination of these KO lines showed that reproductive capability was not impaired, confirming earlier observations. Further analysis revealed that KO of kiss2 caused a significant increase in expression levels of kiss1, kiss2r and tac3a, while KO of kiss1 had no effect on the expression of any of the examined genes. In situ hybridization analysis revealed that kiss1 mRNA is expressed only in the habenula in wild type brains, while in kiss2 KO fish, kiss1 mRNA-expressing cells were identified also in the ventral telencephalon, the ventral part of the entopeduncular nucleus, and the dorsal and ventral hypothalamus. Interestingly, these regions are known to express kiss2r, and the ventral hypothalamus normally expresses kiss2. These results suggest that a compensatory mechanism, involving ectopic kiss1 expression, takes place in the kiss2 KO fish, which may substitute for Kiss2 activity.

Interference with the Cannabinoid Receptor CB1R Results in Miswiring of GnRH3 and AgRP1 Axons in Zebrafish Embryos.

The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron's axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.

Interactions between the circadian clock and TGF-ß signaling pathway in zebrafish.

BACKGROUND: TGF-ß signaling is a cellular pathway that functions in most cells and has been shown to play a role in multiple processes, such as the immune response, cell differentiation and proliferation. Recent evidence suggests a possible interaction between TGF-ß signaling and the molecular circadian oscillator. The current study aims to characterize this interaction in the zebrafish at the molecular and behavioral levels, taking advantage of the early development of a functional circadian clock and the availability of light-entrainable clock-containing cell lines. RESULTS: Smad3a, a TGF-ß signaling-related gene, exhibited a circadian expression pattern throughout the brain of zebrafish larvae. Both pharmacological inhibition and indirect activation of TGF-ß signaling in zebrafish Pac-2 cells caused a concentration dependent disruption of rhythmic promoter activity of the core clock gene Per1b. Inhibition of TGF-ß signaling in intact zebrafish larvae caused a phase delay in the rhythmic expression of Per1b mRNA. TGF-ß inhibition also reversibly disrupted, phase delayed and increased the period of circadian rhythms of locomotor activity in zebrafish larvae. CONCLUSIONS: The current research provides evidence for an interaction between the TGF-ß signaling pathway and the circadian clock system at the molecular and behavioral levels, and points to the importance of TGF-ß signaling for normal circadian clock function. Future examination of this interaction should contribute to a better understanding of its underlying mechanisms and its influence on a variety of cellular processes including the cell cycle, with possible implications for cancer development and progression.

The not-so-long history of zebrafish research in Israel.

The zebrafish has become a model of choice in fundamental and applied life sciences and is widely used in various fields of biomedical research as a human disease model for cancer, metabolic and neurodegenerative diseases, and regenerative medicine. The transparency of the zebrafish embryo allows real-time visualization of the development and morphogenesis of practically all of its tissues and organs. Zebrafish are amenable to genetic manipulation, for which innovative genetic and molecular techniques are constantly being introduced. These include the study of gene function and regulation using gene knockdown, knockout and knock-in, as well as transgenesis and tissue-specific genetic perturbations. Complementing this genetic toolbox, the zebrafish exhibits measurable behavioral and hormonal responses already at the larval stages, providing a viable vertebrate animal model for high-throughput drug screening and chemical genetics. With the available tools of the genomic era and the abundance of disease-associated human genes yet to be explored, the zebrafish model is becoming the preferred choice in many studies. Its advantages and potential are being increasingly recognized within the Israeli scientific community, and its use as a model system for basic and applied science has expanded in Israel in recent years. Since the first zebrafish-focused laboratory was introduced at Tel Aviv University 16 years ago, seven more zebrafish-centric research groups have been established, along with more than two dozen academic research groups and three bio-medical companies that are now utilizing this model.

The gonadotropin-inhibitory hormone (Lpxrfa) system's regulation of reproduction in the brain-pituitary axis of the zebrafish (Danio rerio).

Gonadotropin-inhibitory hormone (GNIH) was discovered in quail with the ability to reduce gonadotropin expression/secretion in the pituitary. There have been few studies on GNIH orthologs in teleosts (LPXRFamide (Lpxrfa) peptides), which have provided inconsistent results. Therefore, the goal of this study was to determine the roles and modes of action by which Lpxrfa exerts its functions in the brain-pituitary axis of zebrafish (Danio rerio). We localized Lpxrfa soma to the ventral hypothalamus, with fibers extending throughout the brain and to the pituitary. In the preoptic area, Lpxrfa fibers interact with gonadotropin-releasing hormone 3 (Gnrh3) soma. In pituitary explants, zebrafish peptide Lpxrfa-3 downregulated luteinizing hormone beta subunit and common alpha subunit expression. In addition, Lpxrfa-3 reduced gnrh3 expression in brain slices, offering another pathway for Lpxrfa to exert its effects on reproduction. Receptor activation studies, in a heterologous cell-based system, revealed that all three zebrafish Lpxrfa peptides activate Lpxrf-R2 and Lpxrf-R3 via the PKA/cAMP pathway. Receptor activation studies demonstrated that, in addition to activating Lpxrf receptors, zebrafish Lpxrfa-2 and Lpxrfa-3 antagonize Kisspeptin-2 (Kiss2) activation of Kisspeptin receptor-1a (Kiss1ra). The fact that kiss1ra-expressing neurons in the preoptic area are innervated by Lpxrfa-ir fibers suggests an additional pathway for Lpxrfa action. Therefore, our results suggest that Lpxrfa may act as a reproductive inhibitory neuropeptide in the zebrafish that interacts with Gnrh3 neurons in the brain and with gonadotropes in the pituitary, while also potentially utilizing the Kiss2/Kiss1ra pathway.

IGSF10 mutations dysregulate gonadotropin-releasing hormone neuronal migration resulting in delayed puberty.

Early or late pubertal onset affects up to 5% of adolescents and is associated with adverse health and psychosocial outcomes. Self-limited delayed puberty (DP) segregates predominantly in an autosomal dominant pattern, but the underlying genetic background is unknown. Using exome and candidate gene sequencing, we have identified rare mutations in IGSF10 in 6 unrelated families, which resulted in intracellular retention with failure in the secretion of mutant proteins. IGSF10 mRNA was strongly expressed in embryonic nasal mesenchyme, during gonadotropin-releasing hormone (GnRH) neuronal migration to the hypothalamus. IGSF10 knockdown caused a reduced migration of immature GnRH neurons in vitro, and perturbed migration and extension of GnRH neurons in a gnrh3:EGFP zebrafish model. Additionally, loss-of-function mutations in IGSF10 were identified in hypothalamic amenorrhea patients. Our evidence strongly suggests that mutations in IGSF10 cause DP in humans, and points to a common genetic basis for conditions of functional hypogonadotropic hypogonadism (HH). While dysregulation of GnRH neuronal migration is known to cause permanent HH, this is the first time that this has been demonstrated as a causal mechanism in DP‡.

The zebrafish: an emerging animal model for investigating the hypothalamic regulation of reproduction.

Gonadotropin-releasing hormone (GnRH) neurons have a pivotal role in the physiological functions of hypotahlamic-pituitary-gonadal (HPG) axis. The pulsatile releasing of GnRH hormone into the hypophyseal portal circulation at the median eminence represent the first domino in the HPG cascade of events that regulate the development, fertility and aging in all vertebrates. These neurons principally originate in the olfactory placode and migrate during early embryonal stages into the hypothalamus. Alterations in developmental processes or in the releasing of GnRH hormone lead to a rare and complex disorder of the reproductive axis called congenital hypogonadotropic hypogonadism (CHH). Genetic screening of human patients and the use of model systems have led to the identification of several genes involved in the CHH pathogenesis underlying its oligogenic nature. Nevertheless CHH remains, for a large cohort of patients, idiopathic and GnRH neurogenesis processes not fully understood. This is due to intrinsic difficulties that exist in the analysis of earliest embryonic developmental stages and in the methodologies developed to study the CHH-causing genes. In this regard, zebrafish embryos, on account of its external fertilization and development, allow a real-time analysis that could overcome some of the above mentioned limitations. Moreover, the recent availability of several transgenic zebrafish reporter lines makes it an excellent model for the study of the oligogenic mechanisms leading to CHH.

The Dlx5 and Foxg1 transcription factors, linked via miRNA-9 and -200, are required for the development of the olfactory and GnRH system.

During neuronal development and maturation, microRNAs (miRs) play diverse functions ranging from early patterning, proliferation and commitment to differentiation, survival, homeostasis, activity and plasticity of more mature and adult neurons. The role of miRs in the differentiation of olfactory receptor neurons (ORNs) is emerging from the conditional inactivation of Dicer in immature ORN, and the depletion of all mature miRs in this system. Here, we identify specific miRs involved in olfactory development, by focusing on mice null for Dlx5, a homeogene essential for both ORN differentiation and axon guidance and connectivity. Analysis of miR expression in Dlx5(-/-) olfactory epithelium pointed to reduced levels of miR-9, miR-376a and four miRs of the -200 class in the absence of Dlx5. To functionally examine the role of these miRs, we depleted miR-9 and miR-200 class in reporter zebrafish embryos and observed delayed ORN differentiation, altered axonal trajectory/targeting, and altered genesis and position of olfactory-associated GnRH neurons, i.e. a phenotype known as Kallmann syndrome in humans. miR-9 and miR-200-class negatively control Foxg1 mRNA, a fork-head transcription factor essential for development of the olfactory epithelium and of the forebrain, known to maintain progenitors in a stem state. Increased levels of z-foxg1 mRNA resulted in delayed ORN differentiation and altered axon trajectory, in zebrafish embryos. This work describes for the first time the role of specific miR (-9 and -200) in olfactory/GnRH development, and uncovers a Dlx5-Foxg1 regulation whose alteration affects receptor neuron differentiation, axonal targeting, GnRH neuron development, the hallmarks of the Kallmann syndrome.

Novel evolutionary-conserved role for the activity-dependent neuroprotective protein (ADNP) family that is important for erythropoiesis.

BACKGROUND: ADNP is vital for embryonic development. Is this function conserved for the homologous protein ADNP2? RESULTS: Down-regulation/silencing of ADNP or ADNP2 in zebrafish embryos or mouse erythroleukemia cells inhibited erythroid maturation, with ADNP directly associating with the ß-globin locus control region. CONCLUSION: ADNPs are novel molecular regulators of erythropoiesis. SIGNIFICANCE: New regulators of globin synthesis are suggested. Activity-dependent neuroprotective protein (ADNP) and its homologue ADNP2 belong to a homeodomain, the zinc finger-containing protein family. ADNP is essential for mouse embryonic brain formation. ADNP2 is associated with cell survival, but its role in embryogenesis has not been evaluated. Here, we describe the use of the zebrafish model to elucidate the developmental roles of ADNP and ADNP2. Although we expected brain defects, we were astonished to discover that the knockdown zebrafish embryos were actually lacking blood and suffered from defective hemoglobin production. Evolutionary conservation was established using mouse erythroleukemia (MEL) cells, a well studied erythropoiesis model, in which silencing of ADNP or ADNP2 produced similar results as in zebrafish. Exogenous RNA encoding ADNP/ADNP2 rescued the MEL cell undifferentiated state, demonstrating phenotype specificity. Brg1, an ADNP-interacting chromatin-remodeling protein involved in erythropoiesis through regulation of the globin locus, was shown here to interact also with ADNP2. Furthermore, chromatin immunoprecipitation revealed recruitment of ADNP, similar to Brg1, to the mouse ß-globin locus control region in MEL cells. This recruitment was apparently diminished upon dimethyl sulfoxide (DMSO)-induced erythrocyte differentiation compared with the nondifferentiated state. Importantly, exogenous RNA encoding ADNP/ADNP2 significantly increased ß-globin expression in MEL cells in the absence of any other differentiation factors. Taken together, our results reveal an ancestral role for the ADNP protein family in maturation and differentiation of the erythroid lineage, associated with direct regulation of ß-globin expression.

Gucy2f zebrafish knockdown--a model for Gucy2d-related leber congenital amaurosis.

Mutations in retinal-specific guanylate cyclase (Gucy2d) are associated with Leber congenital amaurosis-1 (LCA1). Zebrafish offer unique advantages relative to rodents, including their excellent color vision, precocious retinal development, robust visual testing strategies, low cost, relatively easy transgenesis and shortened experimental times. In this study we will demonstrate the feasibility of using gene-targeting in the zebrafish as a model for the photoreceptor-specific GUCY2D-related LCA1, by reporting the visual phenotype and retinal histology resulting from Gucy2f knockdown. Gucy2f zebrafish LCA-orthologous cDNA was identified and isolated by PCR amplification. Its expression pattern was determined by whole-mount in-situ hybridization and its function was studied by gene knockdown using two different morpholino-modified oligos (MO), one that blocks translation of Gucy2f and one that blocks splicing of Gucy2f. Visual function was assessed with an optomotor assay on 6-days-post-fertilization larvae, and by analyzing changes in retinal histology. Gucy2f knockdown resulted in significantly lower vision as measured by the optomotor response compared with uninjected and control MO-injected zebrafish larvae. Histological changes in the Gucy2f-knockdown larvae included loss and shortening of cone and rod outer segments. A zebrafish model of Gucy2f-related LCA1 displays early visual dysfunction and photoreceptor layer dystrophy. This study serves as proof of concept for the use of zebrafish as a simple, inexpensive model with excellent vision on which further study of LCA-related genes is possible.

Cxcl12a-Cxcr4b signaling is important for proper development of the forebrain GnRH system in zebrafish.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons control pituitary gonadotropin secretion and gametogenesis. In the course of development, these neurons migrate from the olfactory placode to the hypothalamus. The precise molecular mechanism of this neuronal migration is unclear. Here, we investigated whether the chemokine receptor, Cxcr4b, and its cognate ligand, Cxcl12a, are required for proper migration of GnRH3 neurons in zebrafish. Deviated GnRH3 axonal projections and neuronal migration were detected in larvae that carry a homozygote cxcr4b mutation. Similarly, knockdown of Cxcr4b or Cxcl12a led to the appearance of abnormal GnRH3 axonal projections and cell migration, including absence of the characteristic lateral crossing of GnRH3 axons at the anterior commissure and optic chiasm. Double-labeling analysis has shown that cxcr4b and cxcl12a are expressed along the GnRH3 migration pathway (i.e. olfactory placode, terminal nerve and the optic chiasm). The results of this study suggest that the Cxcl12a-Cxcr4b ligand-receptor pair are involved in the migration of GnRH3 neurons in zebrafish, and are therefore crucial for the development of this system.

Targeted gonadotropin-releasing hormone-3 neuron ablation in zebrafish: effects on neurogenesis, neuronal migration, and reproduction.

Hypophysiotropic GnRH neurons are located in the preoptic area and ventral hypothalamus of sexually mature vertebrates. In several species, the embryonic origin of hypophysiotropic GnRH neurons remains unclear. Using the Tg(GnRH3:EGFP) zebrafish line, in which GnRH3 neurons express EGFP, GnRH3 neurons in the olfactory region were specifically and individually ablated during early development using laser pulses. After ablation, the olfactory region maintained the capacity to regenerate GnRH3 neurons. However, this capacity was time-limited. When ablation of GnRH3 cells was conducted at 2 d after fertilization, high regeneration rates were observed, but regeneration capacity significantly decreased when ablation was performed at 4 or 6 d after fertilization. Unilateral GnRH3 neuron ablation results in unilateral soma presence. These unilateral somata are capable of projecting fiber extensions bilaterally. Successful bilateral GnRH3 soma ablation during development resulted in complete lack of olfactory, terminal nerve, preoptic area, and hypothalamic GnRH3 neurons and fibers in 12-wk-old animals. Mature animals lacking GnRH3 neurons exhibited arrested oocyte development and reduced average oocyte diameter. Animals in which GnRH3 neurons were partially ablated exhibited normal oocyte development; however, their fecundity was significantly reduced. These findings demonstrate that the hypophysiotropic GnRH3 populations in zebrafish consist of neurons that originate in the olfactory region during early development. The presence of GnRH3 neurons of olfactory region origin in reproductively mature zebrafish is a prerequisite for normal oocyte development and reproduction.

The zebrafish as a model system for forebrain GnRH neuronal development.

Development and function of the forebrain gonadotropin-releasing hormone (GnRH) neuronal system has long been the focus of study in various vertebrate species. This system is crucial for reproduction and an important model for studying tangential neuronal migration. In addition, the finding that multiple forms of GnRH exist in the CNS as well as in non-CNS tissues, coupled with the fact that GnRH fibers project to many CNS regions, implies that GnRH has a variety of functions in addition to its classic reproductive role. The study of the GnRH system and its functions is, however, limited by available model systems and methodologies. The transgenic (Tg) GnRH3:EGFP zebrafish line, in which GnRH3 neurons express EGFP, allows in vivo study of the GnRH3 system in the context of the entire animal. Coupling the use of this line with the attributes and molecular tools available in zebrafish has expanded our ability to study the forebrain GnRH system. Herein, we discuss the use of the Tg(GnRH3:EGFP) zebrafish line as a model for studying forebrain GnRH neurons, both in developing larvae and in sexually mature animals. We also discuss the potential use of this line to study regulation of GnRH3 system development.

Nasal embryonic LHRH factor plays a role in the developmental migration and projection of gonadotropin-releasing hormone 3 neurons in zebrafish.

The initiation of puberty and the functioning of the reproductive system depend on proper development of the hypophysiotropic gonadotropin-releasing hormone (GnRH) system. One critical step in this process is the embryonic migration of GnRH neurons from the olfactory area to the hypothalamus. Using a transgenic zebrafish model, Tg(gnrh3:EGFP), in which GnRH3 neurons and axons are fluorescently labeled, we investigated whether zebrafish NELF is essential for the development of GnRH3 neurons. The zebrafish nelf cDNA was cloned and characterized. During embryonic development, nelf is expressed in GnRH3 neurons and in target sites of GnRH3 projections and perikarya, before the initiation of their migration. Nelf knockdown resulted in a disruption of the GnRH3 system which included absence or misguiding of GnRH3 axonal outgrowth and incorrect or arrested migration of GnRH3 perikarya. These results suggest that Nelf is an important factor in the developmental migration and projection of GnRH3 neurons in zebrafish.

Ontogeny of the GnRH systems in zebrafish brain: in situ hybridization and promoter-reporter expression analyses in intact animals.

The ontogeny of two gonadotropin-releasing-hormone (GnRH) systems, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), was investigated in zebrafish (Danio rerio). In situ hybridization (ISH) first detected sGnRH mRNA-expressing cells at 1 day post-fertilization (pf) anterior to the developing olfactory organs. Subsequently, cells were seen along the ventral olfactory organs and the olfactory bulbs, reaching the terminal nerve (TN) ganglion at 5-6 days pf. Some cells were detected passing posteriorly through the ventral telencephalon (10-25 days pf), and by 25-30 days pf, sGnRH cells were found in the hypothalamic/preoptic area. Continuous documentation in live zebrafish was achieved by a promoter-reporter expression system. The expression of enhanced green fluorescent protein (EGFP) driven by the sGnRH promoter allowed the earlier detection of cells and projections and the migration of sGnRH neurons. This expression system revealed that long leading processes, presumably axons, preceded the migration of the sGnRH neuron somata. cGnRH-II mRNA expressing cells were initially detected (1 day pf) by ISH analysis at lateral aspects of the midbrain and later on (starting at 5 days pf) at the midline of the midbrain tegmentum. Detection of red fluorescent protein (DsRed) driven by the cGnRH-II promoter confirmed the midbrain expression domain and identified specific hindbrain and forebrain cGnRH-II-cells that were not identified by ISH. The forebrain DsRed-expressing cells seemed to emerge from the same site as the sGnRH-EGFP-expressing cells, as revealed by co-injection of both constructs. These studies indicate that zebrafish TN and hypothalamic sGnRH cell populations share a common embryonic origin and migratory path, and that midbrain cGnRH-II cells originate within the midbrain.

Developmental expression of three forms of gonadotropin-releasing hormone and ontogeny of the hypothalamic-pituitary-gonadal axis in gilthead seabream (Sparus aurata).

To address the complexity of the origin of the GnRH system in perciforms, we investigated the ontogenic expression of three GnRHs in gilthead seabream. Using in situ hybridization, chicken (c) GnRH-II mRNA-expressing cells were detected in the hindbrain at 1.5 days postfertilization (DPF) and in the midbrain at 2 DPF and thereafter; the hindbrain signals became undetectable after 10 DPF. Salmon (s) GnRH mRNA-expressing cells were first seen in the olfactory placode at 3 DPF, started caudal migration at 14 DPF, and reached the preoptic areas at 59 DPF. Seabream (sb) GnRH mRNA-expressing cells were first detected in the terminal nerve ganglion cells (TNgc), ventral part of the ventral telencephalon, nucleus preopticus parvocellularis, and thalamus at 39 DPF, and extended to the nucleus preopticus magnocellularis at 43 DPF, ventrolateral hypothalamus at 51 DPF, and nucleus lateralis tuberis and posterior tuberculum at 59 DPF. Coexpression of sbGnRH and sGnRH transcripts was found in the TNgc. Using real-time fluorescence-based quantitative polymerase chain reaction, transcript levels of cGnRH-II and sGnRH were first detected at 1 and 1.5 DPF, respectively, and increased and remained high thereafter. Transcript levels of sbGnRH remained low after first detection at 1 DPF. Furthermore, these GnRH expression profiles were correlated with the expression profiles of reproduction-related genes in which at least four concomitant increases of GnRH, GnRH receptor, gonadotropin, gonadotropin receptor, and Vasa transcripts were found at 5, 8, 14, and 28 DPF. Our data provide an expanded view of the ontogeny of the GnRH system and reproductive axis in perciforms.

Seasonal variation of the three native gonadotropin-releasing hormone messenger ribonucleic acids levels in the brain of female red seabream.

We studied the seasonal variation of the expression of genes encoding the three native gonadotropin-releasing hormones (GnRHs), namely salmon(s) GnRH, chicken(c) GnRH-II, and seabream(sb) GnRH in red seabream, Pagrus (Chrysophrys) major, in order to better understand the regulatory mechanisms of GnRH gene expression by environmental and endocrine factors. Female red seabream, reared under natural conditions, were collected monthly or bimonthly from October to June, and the levels of the three distinct GnRH messenger ribonucleic acids (mRNAs) in the brains of those fish (n = 4-6) were determined by ribonuclease (RNase) protection analysis. The levels of sbGnRH mRNA correlated well with the observed ovarian histology; the levels of sbGnRH mRNA of immature fish in October and December were low, and increased in February and March in conjunction with active vitellogenesis. The sbGnRH mRNA levels reached a maximum level in April (spawning season), after which they rapidly decreased together with the observed ovarian regression in June. In contrast, the levels of sGnRH mRNA showed no variation, while those of cGnRH-II mRNA were elevated only slightly in March and April. The increase in sbGnRH mRNA levels correlates with the increase in day length, water temperature and serum steroids levels, suggesting that these factors are candidates for regulators of sbGnRH synthesis.

Physiological changes in the spawning gilthead seabream, Sparus aurata, succeeding the removal of males.

The physiological effects triggered in females by the removal of males from a group of spawning fish were examined in the multiple batch spawner, the gilthead seabream, Sparus aurata. One week after the removal of males, a large portion of the oocytes underwent atresia, and sporadic release of low quality eggs continued at low frequency over a period of seven weeks. The transcript levels of the three native gonadotropin releasing hormone (GnRH) forms, salmon (s)GnRH, seabream (sb)GnRH, and chicken (c)GnRH-II, and the two beta GtH subunits were measured. Brain mRNA levels for all three GnRHs and pituitary beta LH mRNA levels significantly declined in the females as a result of removing the males compared to females that were maintained with males. Pituitary beta FSH mRNA levels showed the opposite trend and were significantly higher in females that were separated from males. Circulating levels of LH, testosterone, estradiol, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one, and 17 alpha, 20 beta,21-trihydroxy-4-pregnen-3-one all declined in the group of females without males. These results imply the existence of an endocrine response to socio-sexual stimuli during the reproductive process in the gilthead seabream.