Abigail Elizur

Immunohistochemical localization of three different prepro-GnRHs in the brain and pituitary of the European sea bass (Dicentrarchus labrax) using antibodies to the corresponding GnRH-associated peptides

The distribution of the cells expressing three prepro‐gonadotrophin‐releasing hormones (GnRH), corresponding to salmon GnRH (sGnRH), seabream GnRH (sbGnRH), and chicken GnRH‐II (cGnRH‐II) forms, was studied in the brain and pituitary of the sea bass (Dicentrarchus labrax) by using immunohistochemistry. To circumvent the cross‐reactivity problems of antibodies raised to GnRH decapeptides, we used specific antibodies generated against the different sea bass GnRH‐associated peptides (GAP): salmon GAP (sGAP), seabream GAP (sbGAP), and chicken‐II GAP (cIIGAP). The salmon GAP immunostaining was mostly detected in terminal nerve neurons but also in ventral telencephalic and preoptic perikarya. Salmon GAP‐immunoreactive (ir) fibers were observed mainly in the forebrain, although sGAP‐ir projections were also evident in the optic tectum, mesencephalic tegmentum, and ventral rhombencephalon. The pituitary only receives a few sGAP‐ir fibers. The seabream GAP‐ir cells were mainly detected in the preoptic area. Nevertheless, sbGAP‐ir neurons were also found in olfactory bulbs, ventral telencephalon, and ventrolateral hypothalamus. The sbGAP‐ir fibers were only observed in the ventral forebrain, innervating strongly the pituitary gland. Finally, chicken‐II GAP immunoreactivity was only detected in large synencephalic cells, which are the origin of a profuse innervation reaching the telencephalon, preoptic area, hypothalamus, thalamus, pretectum, posterior tuberculum, mesencephalic tectum and tegmentum, cerebellum, and rhombencephalon. However, no cIIGAP‐ir fibers were detected in the hypophysis. These results corroborate the overlapping of sGAP‐ and sbGAP‐expressing cells in the forebrain of the sea bass, and provide, for the first time, unambiguous information on the distribution of projections of the three different GnRH forms expressed in the brain of a single species. J. Comp. Neurol. 446:95–113, 2002.

Differential expression of three different prepro‐GnRH (gonadotrophin‐releasing hormone) messengers in the brain of the european sea bass (Dicentrarchus labrax)

The expression sites of three prepro‐gonadotrophin‐releasing hormones (GnRHs), corresponding to seabream GnRH (sbGnRH: Ser8‐mGnRH, mammalian GnRH), salmon GnRH (sGnRH: Trp7Leu8‐mGnRH), and chicken GnRH‐II (cGnRH‐II: His5Trp7Tyr8‐mGnRH) forms were studied in the brain of a perciform fish, the European sea bass (Dicentrarchus labrax) by means of in situ hybridization. The riboprobes used in this study correspond to the three GnRH‐associated peptide (GAP)‐coding regions of the prepro‐GnRH cDNAs cloned from the same species (salmon GAP: sGAP; seabream GAP: sbGAP; chicken GAP‐II: cIIGAP), which show little oligonucleotide sequence identity (sGAP versus sbGAP: 42%; cIIGAP versus sbGAP: 36%; sGAP versus cIIGAP: 41%). Adjacent paraffin sections (6 mm) throughout the entire brain were treated in parallel with each of the three anti‐sense probes and the corresponding sense probes, demonstrating the high specificity of the hybridization signal. The results showed that both sGAP and sbGAP mRNAs had a broader expression in the olfactory bulbs, ventral telencephalon, and preoptic region, whereas cIIGAP mRNA expression was confined to large cells of the nucleus of the medial longitudinal fascicle. In the olfactory bulbs, both the signal intensity and the number of positive cells were higher with the sGAP probe, whereas sbGAP mRNA‐expressing cells were more numerous and intensely stained in the preoptic region. Additional isolated sbGAP‐positive cells were detected in the ventrolateral hypothalamus. These results demonstrate a clear overlapping of sGAP‐ and sbGAP‐expressing cells in the forebrain of the European sea bass, in contrast to previous reports in other perciforms showing a clear segregation of these two cell populations. J. Comp. Neurol. 429:144–155, 2001.

Differential Effects of Gonadotropin-Releasing Hormone, Dopamine and Somatostatin and Their Second Messengers on the mRNA Levels of Gonadotropin IIβ Subunit and Growth Hormone in the Teleost Fish, Tilapia

In cultured pituitary cells of tilapia, gonadotropin-releasing hormone (GnRH; 10 nM 4-24 h), elevation of cyclic AMP (by 10 microM forskolin or 0.2 mM 3-isobutyl-1-methylxanthine: IBMX 0.5-36 h) or activation of protein kinase C (PKC; by 12.5 nM tetradecanoyl phorbol-13-acetate: TPA, 0.5-24 h) all increased gonadotropin (GtH) II beta steady state mRNA levels by three to four-fold. The involvement of PKA and PKC in the GnRH stimulatory effect on both GtH release and GtH II beta mRNA levels was corroborated by use of the PKA and PKC inhibitors, H89 and GF109203X, respectively (100 nM) which attenuated the GnRH effect. Incubation with actinomycin D (8 microM, 4-21 h) after preexposure for 24 h to either forskolin (10 microM) or TPA (12.5 nM), revealed that rates of transcript degradation were slower in forskolin-treated cells (T 1/2 = 14.1 h) than in control or TPA-treated cells (T 1/2 = 8.47 or 8.38 h), suggesting a stabilizing effect on the mRNA. Dopamine (DA; 10 microM, 4-36 h) had no apparent effect on steady state mRNA levels of GtH II beta, but reduced GtH release by as much as 75%. Steady state levels of growth hormone (GH) mRNA were not affected by exposure to GnRH (10 nM, 4-24 h), although GH release was more than doubled. Similarly, activation of PKC (by TPA 12.5 nM, 1.5-36 h), which was shown to be essential for the GnRH-stimulatory effect on GH release, did not alter levels of the GH transcript, but increased GH release by more than fivefold. DA (10 microM, 4-24 h) moderately increased GH transcript levels (160%) with similar kinetics but lower potency than direct elevation of cAMP (by 10 microM forskolin or 0.2 mM IBMX, 0.5-36 h) which increased transcript levels by more than fourfold. The involvement of PKA in the DA effect was confirmed when the PKA inhibitor H89 (100 nM, 15 min prior to DA exposure) attenuated the DA effect on GH mRNA levels. Exposure of cells to actinomycin D (8 microM, 2-16 h) after treatment with forskolin (10 microM, 24 h) led to a slower rate of transcript degradation than in control cells (T 1/2 = 6.5 h vs. T 1/2 = 4.36 h), suggesting that cAMP also elicits a stabilizing effect on GH mRNA. Somatostatin (100 nM, 0.5-36 h) had no clear effect on GH transcript levels, but reduced GH release by as much as 90%. These results suggest that activation of either cAMP-PKA or PKC pathways can, possibly by different mechanisms, stimulate mRNA levels of the GtH II beta gene, but that only the cAMP-PKA pathway stimulates GH mRNA levels. It would appear therefore that GnRH, although stimulating GH release, does not regulate GH transcription in this fish.

Long photoperiod delayed spawning and increased somatic growth in gilthead seabream (Sparus aurata)

The effects of extended photoperiods, mimicking the longest day of the year, were studied in 1- and 2-year seabream. The photoperiod regimes started in late July, 36 and 39 days after the summer solstice and continued for 11 months, well beyond the natural reproductive season of December–March. Regime 1 (long day, 15.5L:8.5D), which used natural and fluorescent light, reduced the incidence of maturity in both year classes and females did not spawn although some gonadal development was observed. Among all 1-year sampled fish of regime 1, a maximum of 5% became spermiating males (March) and 5% reached the yolk granule stage of vitellogenesis (VO3; 250–400 μm diameter) by May. Among 2-year sampled fish of regime 1, 45% became spermiating males and 25% were females, which reached the advanced vitellogenesis stage (VO4; 400–600 μm) by April. Regime 2 (skeleton photoperiod), consisting of natural light and a 1.5-h pulse of fluorescent light during the period 14–15.5 h after sunrise, postponed gonadal development and spawning for up to 3 months. In this regime, a maximum of 80% of 1-year sampled fish were spermiating males in February and a maximum of 10% were VO3 stage females in March. In the sampled 2-year fish, the maximum levels were 50% spermiating males in February and 25% VO3 stage females in March. Control fish, which were exposed to the natural photoperiod (29°34′N), spawned during their natural season. The maximum levels for 1-year sampled control fish were 95% spermiating males and no females in December, while among 2-year sampled fish, maxima of 75% males in February and 45% VO4 stage females in November. Final average weights of photoperiod treated fish (1-year=430 g—regime 1, 400 g—regime 2; 2-year=582 g—regime 1, 518 g—regime 2) were significantly greater (p regime 2 (skeleton photoperiod)>control.

Regulation of Gonadotropin-Releasing Hormone (GnRH)-Receptor Gene Expression in Tilapia: Effect of GnRH and Dopamine

Abstract The present work was designed to study certain aspects of the endocrine regulation of gonadotropin-releasing hormone receptor (GnRH-R) in the pituitary of the teleost fish tilapia. A GnRH-R was cloned from the pituitary of hybrid tilapia (taGnRH-R) and was identified as a typical seven-transmembrane receptor. Northern blot analysis revealed a single GnRH-R transcript in the pituitary of approximately 2.3 kilobases. The taGnRH-R mRNA levels were significantly higher in females than in males. Injection of the salmon GnRH analog (sGnRHa; 5–50 μg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 μg/kg and at 36 h. At the higher dose of sGnRHa (50 μg/kg), taGnRH-R transcript appeared to be down-regulated. Exposure of tilapia pituitary cells in culture to graded doses (0.1–100 nM) of seabream (sbGnRH = GnRH I), chicken II (cGnRH II), or salmon GnRH (sGnRH = GnRH III) resulted in a significant increase in taGnRH-R mRNA levels. The highest levels of both LH release and taGnRH-R mRNA levels were recorded after exposure to cGnRH II and the lowest after exposure to sbGnRH. The dopamine-agonist quinpirole suppressed LH release and mRNA levels of taGnRH-R, indicating an inhibitory effect on GnRH-R synthesis. Collectively, these data provide evidence that GnRH in tilapia can up- regulate, whereas dopamine down-regulates, taGnRH-R mRNA levels.

Domestication of the white grouper, Epinephelus aeneus 1. Growth and reproduction☆

Abstract The growth and reproductive biology of the white grouper, Epinephelus aeneus, were studied in captive fish to determine its potential for aquaculture. About 250 fish were captured by fishermen along the Mediterranean coast and maintained in 16 m3 concrete tanks supplied with sea water in a flow-through system. The captive fish fed readily on dry pellets supplemented with chopped frozen fish and gained an average of 3.3 g/day during the initial growth phase (0.5-1.5 kg), and 11.3 g/day during the secondary growth phase (1.5-3.0 kg). Using ovarian biopsies, the sexual development of 17 females was closely monitored for 3 years, and for 1 year in an additional 47 females. In adult females held in captivity, the oocytes reached the final stages of vitellogenesis, however, final oocyte maturation, ovulation and spawning did not occur. Sustained release of [ d -Ala6,Pro9NEt]-GnRH from implanted devices was highly effective in inducing ovulation, but did not result in natural spawning. Repeated implantations resulted in 2–3 ovulations per reproductive season, which lasted from April through September. The ovulated females were manually stripped and the eggs were artificially fertilized, resulting in millions of fertilized eggs and larvae. The average fecundity per female was 242 343 eggs (kg BW)−1 yr−1. In some of the young females, early vitellogenesis did not lead to the final stages of vitellogenesis. Instead, the vitellogenic oocytes underwent rapid atresia. Monitoring individual fish demonstrated that E. aeneus is a protogynous hermaphrodite, changing sex from female to male, confirming reports by other authors. Sex inversion occurred both spontaneously and after implantation with 17α-methyltestosterone. The rapid growth rates and the potential for induced spawning in captivity make the white grouper an excellent candidate for mariculture.

Spatial analysis of biomineralization associated gene expression from the mantle organ of the pearl oyster Pinctada maxima

BackgroundBiomineralization is a process encompassing all mineral containing tissues produced within an organism. One of the most dynamic examples of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this architecture. However general understanding of how this process is achieved remains ambiguous. The mantle is a conserved organ involved in shell formation throughout molluscs. Specifically the mantle is thought to be responsible for secreting the protein component of the shell. This study employs molecular approaches to determine the spatial expression of genes within the mantle tissue to further the elucidation of the shell biomineralization.ResultsA microarray platform was custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from mantle tissue. This microarray was used to analyze the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and analyzed for differential gene expression with PmaxArray 1.0. Over 2000 ESTs were determined to be differentially expressed among the tissue sections, identifying five major expression regions. In situ hybridization validated and further localized the expression for a subset of these ESTs. Comparative sequence similarity analysis of these ESTs revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell related genes.ConclusionsThis investigation has mapped the spatial distribution for over 2000 ESTs present on PmaxArray 1.0 with reference to specific locations of the mantle. Expression profile clusters have indicated at least five unique functioning zones in the mantle. Three of these zones are likely involved in shell related activities including formation of nacre, periostracum and calcitic prismatic microstructure. A number of novel and known transcripts have been identified from these clusters. The development of PmaxArray 1.0, and the spatial map of its ESTs expression in the mantle has begun characterizing the molecular mechanisms linking the organics and inorganics of the molluscan shell.

The KiSS1/GPR54 system in fish.

The KiSS1/GPR54 system has now been identified in non-mammalian vertebrates. Transcripts encoding for KiSS1 and its receptor, GPR54, have been isolated from a number of fish species. The expression of their genes was characterized in the context of temporal and spatial expression and in response to endocrine manipulations. GPR54 sequence is conserved between mammals and fish, with a second receptor sequence identified in zebrafish. The KiSS1 gene sequence is highly divergent between mammals and fish, yet the human kisspeptin is capable of activating the fish GPR54. As in mammals, the fish KiSS1/GPR54 system appears to be partially regulated by gonadal steroids. The data available for fish are fragmented, yet indicate that the KiSS1/GPR54 system is functionally conserved in non-mammalian vertebrates and supports the notion that it has a role in pubertal development and reproduction in piscine systems.

Developmental expression of three different prepro-GnRH (gonadotrophin-releasing hormone) messengers in the brain of the European sea bass (Dicentrarchus labrax).

In this study, we have analyzed the ontogenic expression of three gonadotrophin-releasing hormones (GnRH) systems expressed in the brain of a perciform fish, the European sea bass, using in situ hybridization. The riboprobes used correspond to the GnRH-associated peptide (GAP) coding regions of the three prepro-GnRH cDNAs cloned from the same species: prepro-salmon GnRH, prepro-seabream GnRH and prepro-chicken GnRH II. On day 4 after hatching, the first prepro-chicken GnRH-II mRNA-expressing cells appeared in the germinal zone of the third ventricle. They increased in number and size from 10 to 21 days, reaching at day 30 their adult final position, within the synencephalic area, at the transitional zone between the diencephalon and the mesencephalon. First prepro-salmon GnRH mRNA-expressing cells became evident on day 7 arising from the olfactory placode and migrating towards the olfactory nerve. On day 10, this cell group reached the olfactory bulb, being evident in the ventral telencephalon and preoptic area from days 15 and 45, respectively. Weakly labeled prepro-seabream GnRH mRNA-expressing cells were first detected at 30 days in the olfactory area and ventral telencephalon. On day 45, prepro-seabream GnRH mRNA-expressing cells were also present in the preoptic region reaching the ventrolateral hypothalamus on day 60. The results obtained in sea bass indicate that sGnRH and sbGnRH cells have a common origin in an olfactory primordium suggesting that both forms might arise from a duplication of a single ancestral gene, while cGnRH-II cells develop from a synencephalic primordium.

Developmental Expression of Three Forms of Gonadotropin-Releasing Hormone and Ontogeny of the Hypothalamic-Pituitary-Gonadal Axis in Gilthead Seabream (Sparus aurata)

Abstract 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.