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

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

Effects of a saponin fraction extracted from Trigonella foenum-graecum L. and two commercially available saponins on sex ratio and gonad histology of Nile tilapa fry, Oreochromis niloticus (L.)

Over three million tonnes (t) of tilapia, mostly Nile tilapia (Oreochromis niloticus, L.), are produced annually making it the second most abundantly produced freshwater fish (FAO, 2010). Tilapia are mouthbreeders that often produce stunted populations under pond conditions; one means of prevention is to produce all-male fish with the additional advantage that males usually grow faster than females. All-male populations can be achieved by supplementing feed with androgens such as 17-α-Methyltestosterone (MT) during days 10–25 post-hatch (Pandian and Sheela, 1995). However, MT is considered to be carcinogenic (Velazquez and Alter, 2004), and Hulak et al. (2008) also showed that effluents of systems in which carp were fed diets containing MT caused masculinization of female fish. Furthermore, in aquaculture the application of hormones to fish destined for human consumption is prohibited in the European Union under directive 96/22/EC, article 5, which also prohibits import of animal products produced with hormones. Kwon et al. (2000) showed that Fadrozole, a non-steroidal compound, caused masculinization in tilapia by inhibiting aromatase, which is the enzyme responsible for the conversion of endogenous androgens to estrogens. Steinbronn et al. (2004) were able to show that a dose of 2000 ppm Quillaja saponins (Sigma S-2149) inhibited reproduction of tilapia after dietary application for 32 days to first-feeding fry, suggesting saponins as a possible alternative to MT. These secondary plant compounds consist of either a steroid or triterpenoid basic structure (aglycone or sapogenin) plus one or more sugar side chains (Francis et al., 2002a). In a previous experiment a saponin fraction from the soapbark tree (Quillaja saponaria M.) inhibited aromatase in vitro (Golan et al., 2008). The fenugreek plant (Trigonella foenum-graecum L), widely cultivated in the Middle East and Asia, also has a high saponin content. The experiment was therefore conducted to test whether saponin fractions from Q. saponaria and from T. foenum-graecum were able to influence the sex ratio and gonad histology of Nile tilapia

Spawning Induction in the Carp: Past Experience and Future Prospects - A Review

Most fish in aquaculture either fail to breed in captivity or their spawning occurs sporadically and late in the season. This is mainly due to the lack of natural cues in captivity, which leads to dysfunction of the endocrine axis regulating oocyte maturation and ovulation. Hypophysation as a remedy for this situation in fish has been employed in aquaculture since the 1930s and is still widely practiced. However, using crude pituitary homogenates in local hatcheries has frequently ended in failures that were attributed to the incon- sistent potency of the injected material and the unknown ovarian stage of the recipient fish. Since the mid 1980s, hypophysation has improved through the introduction of a standardized dry carp pituitary extract in which the luteinizing hormone (LH) content and activity have been calibrated (cal- ibrated carp pituitary extract = CCPE). Induction of spawning, however, is successful mainly in female cohorts in which 65% or more of the oocytes in an ovarian biopsy have migrating germinal vesicles. Further, due to decreasing quantities of industry-processed common carp and the expan- sion of ornamental carp production (koi and goldfish), the growing demand for CCPE could not be met, and an alternative had to be found. A hypo- thalamic approach, introduced into Israeli aquaculture in 1993 (called Dagin), combines a superactive analog of sGnRH (10 μg/kg), with the water-soluble dopamine (D2) receptor antagonist, metoclopramide (20 mg/kg). The progress of oocyte maturation in ovarian biopsies has been studied in parallel with changes in levels of LH, estradiol, and the matura- tion-inducing steroid (MIS; 17α, 20β, dihydroxy-4-pregnene-3-one). The hormone profile indicated that the gradual increases in LH and MIS follow- ing a single administration of Dagin were similar to those in fish treated with priming and resolving doses of CCPE. This would explain why Dagin is effective even when only a single injection is given, saving labor and reduc- ing handling stress. CCPE and Dagin were tested in parallel on common carp in a commercial hatchery. The spawning ratio and embryo viability were similar, although the latency between injection and ovulation was con- siderably longer and more variable in Dagin-treated than in CCPE-treated carp. It is recommended to use CCPE at the beginning and end of the spawning season when the LH content in the pituitary is low, and Dagin in mid-season and in field spawning. Future prospects raise the possibility that by employing molecular tools, a recombinant carp LH will be produced that will have the regular and expected potency of the hypophyseal approach without the risk of spreading pathogens from donor fish to broodstock. Work along this line is currently in progress

Effects of saponin fractions from fenugreek and the soap bark tree in the diet on performance of Nile tilapia, Oreochromis niloticus (L.)

Saponins are generally regarded as anti-nutritional factors in aquaculture diets. However, previous experiments have shown that low dietary levels of saponins derived from Quillaja saponaria Molina do have growth promoting effects on common carp and Nile tilapia. Based on these experiments, we conducted an experiment in which we fed eluated saponin fractions from Q. saponaria and Trigonella foenum-graecum L. (fenugreek) to Nile tilapia in a respirometric system allowing for continuous measurement of oxygen consumption. Saponins were eluated with consecutive methanol/water concentrations (v/v, 40/60, 60/40, 80/20) resulting in three different eluates for each plant. Fractions chosen were the 80% methanol eluate from Q. saponaria (80QS) and all three eluates from T. foenum-graecum (40TS, 60TS and 80TS). Three fish each were fed with low levels (150 mg kg-1 diet) of saponins in the diet and a control diet without saponins. Growth, feed and nutrient utilization, proximate composition, oxygen consumption and metabolic performance were evaluated. The fish grew between 224% (40TS) and 266% (Control) over the eight week period. Feed conversion ratios were between 0.94 (80TS) and 1.15 (40TS) and protein efficiency ratios between 2.54 (80TS) and 2.16 (40TS). Due to low sample sizes, no statistical differences were found between control fish and saponin fed fish. However, numerically one of the tested saponin fractions (40TS) showed inferior performance (Table 1). It is concluded that the tested saponins in the tested concentrations are no potential growth promoter for Nile tilapia. On the contrary, one fraction appears to be a growth inhibitor

Yolk utilization and growth during the early larval life of the Silver Perch, Bidyanus bidyanus (Mitchell, 1838)

The aim of this research was to investigate the yolk sac and oil globule utilization by silver perch (Bidyanus bidyanus) larvae produced from domesticated broodfish. The larvae were kept unfed in the holding tank, sampled, and investigated by image analysis software to determine various characteristics, such as the diameters of ova, water-hardened eggs, yolk-sac, oil globules, and the total length of larvae. The research illustrated that, with the exception of oil globule diameter, all other morphometric parameters were significantly lower (P < 0.05) when compared to the larvae from the wild broodfish. The yolk sac was completely absorbed at 96 h post-hatching (hph) and the oil globule was visible until 240 hph. The larvae exhibited predatory movements and tried to catch rotifer at 4 days post hatching (dph). However, the onset of feeding took place at 5 dph, while 100% of feeding occurred at 6 dph. During the first 96 h (h), larvae grew significantly faster than the next 144 h. Larvae encountered low mortalities (<10%) during the first 96 hph, before increasing significantly in the next 24 h and no unfed larvae survived post 240 h. The results also suggested that the exogenous feed should be available at 96 hph, which is well after the yolk sac is completely depleted. In addition, although most of eggs and larval performance from domesticated broodfish were inferior compared to the wild one, it has larger oil globule that could make longer of its mixed feeding period and therefore could have better in viability

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

Rapid Dopaminergic Modulation of the Fish Hypothalamic Transcriptome and Proteome

Background - Dopamine (DA) is a major neurotransmitter playing an important role in the regulation of vertebrate reproduction. We developed a novel method for the comparison of transcriptomic and proteomic data obtained from in vivo experiments designed to study the neuroendocrine actions of DA. // Methods and Findings - Female goldfish were injected (i.p.) with DA agonists (D1-specific; SKF 38393, or D2-specific; LY 171555) and sacrificed after 5 h. Serum LH levels were reduced by 57% and 75% by SKF 38393 and LY 171555, respectively, indicating that the treatments produced physiologically relevant responses in vivo. Bioinformatic strategies and a ray-finned fish database were established for microarray and iTRAQ proteomic analysis of the hypothalamus, revealing a total of 3088 mRNAs and 42 proteins as being differentially regulated by the treatments. Twenty one proteins and mRNAs corresponding to these proteins appeared on both lists. Many of the mRNAs and proteins affected by the treatments were grouped into the Gene Ontology categorizations of protein complex, signal transduction, response to stimulus, and regulation of cellular processes. There was a 57% and 14% directional agreement between the differentially-regulated mRNAs and proteins for SKF 38393 and LY 171555, respectively. // Conclusions - The results demonstrate the applicability of advanced high-throughput genomic and proteomic analyses in an amendable well-studied teleost model species whose genome has yet to be sequenced. We demonstrate that DA rapidly regulates multiple hypothalamic pathways and processes that are also known to be involved in pathologies of the central nervous system

Preliminary report on the production of transgenic Oreochromis niloticus using tol 2 kit

Pronuclear microinjection of donor plasmid containing the tol 2 transposon that was flanked by a promoter β- actin and green fluorescent protein (GFP) reporter gene were injected into the perinuclear cytoplasm of Oreochromis niloticus fertilized eggs. The aim was to determine if the β-actin promoter will drive the expression of the GFP in specific tissues of the embryo. Few days after hatching, GFP was strongly expressed in the embryos when observed under UV-light microscope. Subsequently, β actin fused with a foreign growth hormone gene in a donor tols 2 transposon along with messenger RNA was co-injected into early stage of fertilized eggs using micro injector. Out of the 30 eggs injected, 15 representing 50% hatchlings were obtained. GH was successfully transposed to the genome of Fo as confirmed from PCR of DNA from fin clip of the founder population. Growth performance in transgenic after 2 months of rearing was 1.06gm (3.7cm) compared with 0.68gm(3.5cm) in nontransgenic individuals. The founder generation is being raised to maturity to determine the level of efficiency in transmission to next generation.Keywords: Transposon; growth enhancement; Oreochromis niloticus; transgenesis; growth hormone gen