Gonadotropin-releasing hormone and gonadotropin in goldfish and masu salmon

Reproductive activities in vertebrates are regulated by an endocrine system, consisting of the brain-pituitary-gonad axis. In teleosts, gonadotropin-releasing hormone (GnRH) in the brain stimulates gonadotropin (GTH) release in the pituitary gland, but because of lack of the portal vessel, it is not known when and how much GnRH is released for the regulation of GTH release. There are multiple molecular types of GnRH in teleosts and several distinct populations of GnRH neurons in the brain. However, we do not know which types and populations of GnRH neurons regulate reproductive activities. Here we summarize our recent studies on GnRH and GTH in masu salmon Oncorhynchus masou and goldfish Carassius auratus. Immunocytochemistry showed the location and molecular types of GnRH neurons. Salmon (sGnRH) and chicken-II GnRH (cGnRH-II) neuronal fibers were widely distributed in the brain of both masu salmon and goldfish. Only sGnRH fibers were observed in the pituitary of masu salmon, whereas both sGnRH and cGnRH-II fibers were observed in the goldfish pituitary, indicating that species specific GnRH profiles are involved in the regulation of pituitary function in teleosts. A series of experiments in masu salmon and goldfish suggest that among GnRH neuron populations GnRH neurons in the ventral telencephalon and the hypothalamus regulate GTH release, and that GnRH of the terminal nerve origin is not essential to gonadal maturation and ovulation. The biological function of other GnRH neurons remains unkown. Two GTHs appear to be characteristic of teleost; however, regulation of reproduction by these GTHs is a question that remains to be elucidated. In salmonid species, it is proposed that GTH I stimulates early gonadal development, whereas GTH II acts in later stages. When GTH expression was examined in goldfish, both GTH I and II mRNA levels in the pituitary gland showed increases in accordance with gonadal development, unlike the sequential expression of GTH subunits in salmonids. The expression of these GTH subunit mRNAs were affected by water temperature, starvation, and steroid hormones in goldfish, but in what manner these two GTHs regulate gonadal development remains to be clarified.     

Intracellular mechanisms mediating gonadotropin and growth hormone release in the goldfish,Carassius auratus

Evidence for the involvement of Ca²⁺, protein kinase C, cAMP, and arachidonic acid metabolism in mediating gonadotropin (GTH) and growth hormone (GH) release in the goldfish is reviewed. Models for the signal transduction pathways mediating GTH-releasing hormone (GnRH) and dopamine actions on GTH and GH secretion are postulated. A novel hypothesis that two GnRHs which bind to the same receptor type activate different transduction cascade in two different cell types (GTH vs. GH) as well as within the same cell type (GTH) is presented.

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Résumé Cette revue présente les données expérimentales démontrant l'implication de Ca⁺⁺, de la protéine kinase C et du métabolismes de l'acide arachidonique dans les mécanismes régulant la sécrétion des hormones gonadotrope (GTH) et de croissance (GH). Des modèles de signaux de transduction de l'action de la gonadolibérine (GnRH) et de la dopamine sur la sécrétion de GTH et de GH sont proposés. Les deux GnRHs existant chez le poisson rouge pourraient se lier au même type de récepteur et activer différentes voies de transduction dans deux différents types cellulaires (GTH vs. GH) ou dans un seul type (GTH).

     

Characterization of growth hormone binding sites in the goldfish,Carassius auratus: effects of hypophysectomy and hormone injection

A recombinant carp growth hormone (rcGH) was used to develop for a GH radioreceptor binding assay in the goldfish (Carassius auratus). Specific binding of¹²⁵I-rcGH to goldfish liver membranes was a pH, time, temperature, and membrane protein dependent process. Scatchard and LIGAND analysis indicated a single class of high affinity and low capacity binding site, with an association constant (Ka) of 1.9×10¹⁰ M^–1 and a maximum binding capacity (B_max) of 9 fmol mg^–1 protein. Liver tissue displayed the highest¹²⁵I-rcGH binding of all the tissues examined. Displacement of¹²⁵I-rcGH with various unlabeled teleost and mammalian GHs and prolactins revealed that the goldfish hepatic binding site was highly specific for teleost GH. Intraperitoneal administration of 0.1, 1.0, and 10 g rcGH g^–1 body weight to hypophysectomized goldfish resulted in a 27, 52, and 68% decrease in total binding sites, respectively. Injection of a high dose of rat prolactin (rPRL) (5 g rPRL g^–1 body weight) also resulted in a 32% decrease in total binding sites. These results suggest that endogenous GH may have a role in the regulation of its own receptors in the goldfish.     

Hypothalamic peptides influencing growth hormone secretion in the goldfish,Carassius auratus

In vivo andin vitro techniques were used to examine the influence of various vertebrate peptides on growth hormone (GH) secretion in the goldfish. Tetradecapeptide somatostatin (SRIF-14) was found to inhibit GH secretionin vitro from perifused pituitary fragments, whereas similar concentrations of a salmonid SRIF peptide (sSRIF-25) did not affect GH secretion from the goldfish pituitary fragments. This indicates that SRIF receptors on the goldfish pituitary are very specific for SRIF-14-like peptides. Salmon gonadotropin (GTH)-releasing hormone (sGnRH) was found to elevate serum GH levels in male goldfish. The dopamine antagonist pimozide alone or injected in combination with sGnRH did not influence serum GH levels, although injection of pimozide alone significantly elevated serum GTH levels, in addition to potentiating the effects of sGnRH on GTH secretion. sGnRH stimulated GH secretion from goldfish pituitary fragmentsin vitro, indicating that sGnRH acts directly at the level of the pituitary to stimulate GH secretion in the goldfish. These results suggest that GnRH may also function as a GH-releasing factor in the goldfish, although the release-inhibitory factors for GH and GTH secretion do appear to be separate and distinct. Two human GH-releasing hormone (hGHRH) peptides were found to be ineffective in altering GH secretionin vitro from the perifused pituitary fragments. Consequently, a role for a mammalian GHRH-like peptide in the hypothalamic regulation of GH secretion in the goldfish remains questionable.     

China rose (Hibiscus rosasinensis) petals: a potent natural carotenoid source for goldfish (Carassius auratus L.)

Goldfish (Carassius auratus) are in demand in world markets due to their attractive golden colour. Carotenoids are the primary source of colour in the skin of fish. To optimize the colour in captivity, fish must obtain an adequate level of carotenoids in their feed. With this objective, four natural colour enhancers were tested. A common batch of feed was divided into five equal portions and colour ingredients, spirulina (D‐S), china rose petals (D‐C), marigold petals (D‐M) and Lactobacil, a commercial probiotic (D‐L), were added at 5 mg kg⁻¹ to four portions of feed; one portion (D‐O) was kept as a control without any additive. A feeding trial was conducted for 8 weeks. Each 70 L aquarium was stocked with 10 fish (average weight 1.6 g) and feed was given at 5% of the body weight. Growth rate, survival, biochemical composition and pigmentation in the skin of fish were measured. Histological studies of gonads were also conducted. Growth of fish in different treatments was significantly different. There was no difference in the proximate composition of the fish at the start of the experiment but after 8 weeks of feeding, fish fed the diet supplemented with china rose petals had a lower moisture content (70.48%) and higher protein (17.7%) and lipid (5.25%) levels than the group fed the control diet. Pigmentation was the highest (4.01 μg g⁻¹) in D‐C, followed by D‐M (3.16 μg g⁻¹), D‐S (2.92 μg g⁻¹) and D‐L (2.84 μg g⁻¹) and the lowest (0.24 μg g⁻¹) in D‐O. Gonad development of fish fed with the D‐C diet was better compared with the gonads of control (D‐O) fish, followed by D‐M‐, D‐L‐ and D‐S‐fed fish. Gonads of fish, fed D‐C, showed well‐marked changes in the testis where a large number of seminiferous tubules bound together by means of a thin layer of connective tissue were observed. These tubules were highly convoluted and were separated from each other by thin connective tissue stroma. The intra space contained connective tissue, blood capillaries and interstitial cells. The spermatogonia could be seen as a large spherical cell containing a large central nucleus with a distinct nucleolus. The study shows that the china rose (Hibiscus rosasinensis) petal is a potent natural carotenoid source for goldfish to enhance its colour and also accelerate gonadal development.     

A comparison of artificial and natural foods and their combinations in the rearing of goldfish, Carassius auratus (L.)

Intensive grow‐out of goldfish, Carassius auratus (L.), larvae and juveniles in closed systems requires the control of environmental conditions and feeding. This study investigates the use of different types of live food and combinations of live food and dry food in a series of four rearing experiments. Juvenile goldfish can be weaned from Artemia onto live food at about 24 days after the onset of feeding without causing a reduction in growth and survival. The replacement of Artemia by Daphnia at day 10 appears feasible, as growth and survival were not significantly affected. Fish fed decapsulated Artemia cysts grew better than fish fed live Artemia. Within the first 14 days, goldfish juveniles should be fed at least 155 cysts per fish per day to achieve fast growth and to minimize size variation.     

Hematological response in fish: pronephric and splenic involvements in the goldfish,Carassius auratus L.

Pronephric and splenic involvements in erythropoiesis and in stress-induced hematological response by goldfish,Carassius auratus, were examined under conditions of minimal stress and following transient (3h) temperature-induced elevation of O₂ demand and transient (3h) exposure to hypoxia. Although hemoglobin content and red cell numbers were little affected, the ontogenic composition of the circulating erythrocyte population was significantly altered by both types of respiratory challenge and also by the stresses associated with capture, air exposure during transfer and intraperitoneal administration of label. Juvenile cell numbers increased sharply while mature erythrocyte abundances declined. Consistent with this, [³H] methylthymidine-labeled cell numbers rose in both spleen and pronephros following imposition of stress. Splenic erythropoietic activity was modest relative to that of the pronephros. Our observations point to a readily triggered response involving: [1] release of cells reservoired in the spleen and pronephros, [2] increased erythropoiesis and [3] karyorrhexis. We suggest that while the magnitude of response may depend on the quality and magnitude of the imposed stress, its central features are essentially constant.     

Dopamine functions as a growth hormone-releasing factor in the goldfish,Carassius auratus

In vivo and in vitro approaches have been used to examine the role of dopamine (DA) as a growth hormone (GH)-releasing factor in the goldfish. DA stimulated GH release from perifused pituitary fragments of goldfish in a dose-dependent manner. The GH-releasing effect of DA was seasonal, being the highest in sexually regressed fish, intermediate in recrudescent fish, and the lowest in sexually mature (prespawning) fish. The GH response to DA was blocked by the D1 antagonist (+)SCH23390, confirming the involvement of D1 receptors in DA-stimulated GH release. In studies using static incubation of pituitary cells, somatostatin, a known physiological GH-release inhibitor in the goldfish, abolished the GH response to DA. Intraperitoneal injection of apomorphine, a non-selective DA agonist, also increased the plasma GH levels and enhanced the linear body growth of goldfish. These results strongly suggest that DA, by acting through DA D1 receptors, functions as a GH-releasing factor in the goldfish.     

Isolation of a cyprinid herpesvirus 2 from goldfish, Carassius auratus (L.), in the UK

Haematopoietic necrosis virus [cyprinid herpesvirus 2 (CyHV-2)] was isolated during disease outbreaks in goldfish, Carassius auratus, at an ornamental fish retail site in southern England in 2004. Signs of disease included lethargy and inappetence and were first seen after water temperatures increased from 14-15 to 19-21 degrees C. External gross pathology included pale patches on the gills and skin and internally the spleen was enlarged, often with distinctive white nodules. The most prominent histopathological changes observed were necrotic lesions in the spleen and kidney and focal patches of necrosis in the gill lamellae. Necrotic cells often contained nuclei with marginated chromatin and pale intranuclear inclusions. Ultrastructural examination of the spleen tissue revealed typical herpesvirus-like particles measuring 100 nm in diameter. The virus was isolated from extracts of gill tissue in KF-1 cells at 20 degrees C and oligonucleotide primer sets were designed based on conserved gene sequences and used to amplify viral DNA by polymerase chain reaction (PCR). The PCR assays were then used to detect the virus in DNA extracted from tissues sampled during earlier disease investigations at the retail site owner's holding facility in 2002 and 2003 and stored at -70 degrees C since then. Polymerase gene-specific PCR amplification products obtained from tissue samples and from the virus isolated in cell culture shared 100% nucleotide sequence identity with the published sequence for CyHV-2.     

Histomorphometric effects of calcitonin on pharyngeal bone in fed and starved goldfish Carassius auratus

ABSTRACT: The effects of salmon calcitonin (sCT) on osteoblasts and osteoclasts were histologically and histomorphometrically investigated in the pharyngeal bone of fed and starved goldfish Carassius auratus . The thickness of the osteoid and the height of osteoblasts were also measured. Fish were given sCT intraperitoneally at a dose of 10 ng/g bodyweight four times every other day under either fed or starved conditions. The sCT treatment induced the retraction of osteoclasts and their disengagement from the bone surface. Salmon calcitonin had no effect on the histomorphometric parameters of bone formation activity, but suppressed the parameter (OcP/EP) that showed osteoclast activity. Salmon calcitonin did not affect the thickness of the osteoid, but increased the height of osteoblasts in starved fish. Histological and histomorphometric results demonstrated that osteoclast activity was suppressed when sCT was given to starved estrogenized goldfish five times for 10 days. Overall, results suggest that calcitonin is involved in bone assimilation by suppressing osteoclast activity and by enhancing osteoblast activity in the pharyngeal bone of goldfish.     

Variable versus constant temperature acclimation regimes: Effects on hemoglobin isomorph profile in goldfish,Carassius auratus

Goldfish,Carassius auratus, were acclimated for 2 to 3.5 weeks to three temperature regimes: [1] temporally-constant (10, 20 and 30°C), [2] diurnally-cycling (20 ± 10°C) and [3] randomly-fluctuating (± 2°C at approximately 2h intervals between extremes of 10 and 30°C). No significant differences in hematocrit were evident. Hemoglobin levels in fish at constant 30°C and under randomly fluctuating temperature were significantly elevated. Of the three hemoglobin isomorphs observed, the two minor components (G₁, G₃) tended to decrease in relative abundance with increase in constant temperature, but increased under varying temperature regimes. The converse was true of the principal hemoglobin, G₂. Extent of isomorph variation was correlated with extent of temperature variability. These observations confirm that temperature variability significantly effects thermoacclimatory response. The functional significance of changes in isomorph abundances during the acclimatory process is considered.     

Effect of short‐term treatment with potassium permanganate on stress markers and blood biochemistry in goldfish Carassius auratus

Potassium permanganate (PM) is a therapeutic agent in aquaculture with strong oxidant properties. In this study, the effects of potassium permanganate treatment (PMT) on stress response and blood biochemistry were investigated in goldfish Carassius auratus. Two triplicate groups of fish were exposed to either 10 mg L^?1 PM or clean water, over 30 min. Blood samples were collected at 0 and 0.5 h after exposure as well as 3 and 24 h. PMT caused significant increase in serum cortisol and glucose levels, 0.5 h post exposure, which stayed elevated until 24 h. Sodium decreased after exposure and did not return to initial levels. Chloride decreased at 0.5 and 3 h, but returned to initial levels at 24 h. Calcium decreased at 0.5 h and returned to initial at 3 h, but significantly increased at 24 h. Total protein, albumin and globulin levels increased at 3 h and stayed elevated until 24 h. Albumin:globulin ratio (A:G) levels decreased at 24 h post exposure. Results indicated that short‐term PMT caused stress response, hydromineral imbalance and possibly change in blood protein profile in goldfish, which lasted until 24 h post treatment.     

First detection of Cyprinid Herpesvirus 2 (CyHV‐2) in goldfish (Carassius auratus) in France

Massive mortalities of Carassius auratus (L.) occurred in a farm in France during summer 2014. Fish presented anorexia, loss of scales and large amounts of mucus on the gills. Necrosis of the distal tip of the filament and the lamellae, combined with fusion of the lamellae, was observed, as well as necrosis in the hematopoietic organs and in the digestive tract. The histological examination led to hypothesize the implication of a virus in the mortality. The presence of cyprinid herpesvirus 2 (CyHV‐2) in dead fish was demonstrated by amplification and sequencing of portions of the DNA polymerase and helicase genes, both sequences exhibiting 100% identity with CyHV‐2 from Japan. In an attempt to find genetic markers of variation, two regions containing tandem repeats in the Japanese genome were amplified from a virus‐positive sample from the present outbreak. A first region (mB) was fully identical to the Japanese isolate. However, the second region (mA) exhibited a range of deletions and substitutions compared to CyHV‐2 from Japan. This is the first report of CyHV‐2 in France in association with mortality of goldfish and the first identification of a molecular marker for its tracing.     

久效磷对金鱼精巢超显微结构和特征性酶活性的影响

用0.01、0.10、1.00 mg.L-1久效磷暴露金鱼21 d,测定了精巢乳酸脱氢酶(LDH)、酸性磷酸酶(ACP)、碱性磷酸酶(ALP)的活性,并观察了精巢显微和超微结构的变化。结果表明:久效磷暴露21 d,精巢小叶基膜断裂,Leydig氏细胞水肿,小叶间质扩大,支持细胞核膜溶解,胞质内脂滴和髓磷脂象数量增多。1.00 mg.L-1久效磷造成了生殖腺指数的显著下降,对精巢发育有一定程度的延迟作用。随着久效磷暴露浓度的升高,LDH,ACP,ALP的活性逐渐下降,0.10、1.00 mg.L-1久效磷暴露组精巢LDH,ACP的活性下降显著,而各暴露组的精巢ALP活性呈现不显著下降趋势。推测久效磷可能通过损伤精巢超显微结构和降低LDH,ACP和ALP活性,干扰精巢能量代谢,造成对雄性金鱼的生殖毒性。     

金鱼望天眼性状受环境和遗传影响的初步研究

试验旨在探索研究不同的水深和容器的透光性是否影响望天眼金鱼最终成功翻眼率,并通过杂交试验,探讨金鱼望天眼性状是否稳定遗传。试验首先选取红望天眼金鱼两雌一雄和一雄偏龙睛五花金鱼,进行人工混合繁殖,随机选取后代分四组,分别在透明玻璃缸水深15 cm(A)、透明玻璃缸水深30 cm(B)、不透光塑缸水深15 cm(C)和不透光塑缸水深30 cm(D)环境中养殖290 d,统计最终金鱼成功翻眼率,进行双因素方差分析,同时进行望天眼金鱼与翻眼不成功的介于望天和龙睛中间体的金鱼进行正反交和各自自交,统计后代最终的眼睛性状。结果显示:水深对望天眼金鱼成功翻眼率影响极显著,容器透光性对其影响不显著;亲本翻眼程度越大,其后代望天比例越高,表明望天眼性状受遗传影响较大,并且一定程度上受母体遗传效应影响。     

The effect of 2-phenoxyethanol and transport packing density on the post-transport survival rate and metabolic activity in the goldfish, Carassius auratus

To test the hypothesis that the anaesthetic 2-phenoxyethanol would reduce the metabolic rate and allow for higher transport packing densities, goldfish (3.93 +/- 5 g) were transported for 48 h at 25, 50 and 75 fish per 500 ml combined with anaesthetic concentrations of 0, 0.25 and 0.35 ml l-1. The anaesthetic did not affect the survival rate or the oxygen and ammonia concentrations. Thus, its use could not be recommended for the transport of goldfish. It is suggested that optimum packing densities be based on a minimum post-transport oxygen value of 4 mg l-1 for goldfish