牙鲆变态过程中的细胞凋亡

利用整体的原位TUNEL方法检测了牙鲆(Paralichthysolivaceus)变态过程中身体各器官细胞凋亡的分布及变化情况。结果如下:(1)与眼睛移动相关的脑颅骨骼的细胞凋亡右侧眼睛移动开始之后,在额骨、中筛软骨和犁骨软骨中出现细胞凋亡,并保持到眼睛移动结束;(2)中枢神经和感觉器官的细胞凋亡在眼睛移动开始之前,脊髓和脊髓鞘出现细胞凋亡,在眼睛移动开始之后,脊髓和脊髓鞘细胞凋亡停止,而在脑、眼睛和内耳出现细胞凋亡,并一直持续到眼睛移动结束;(3)与游泳、捕食和消化等功能相关的器官的细胞凋亡在眼睛移动开始后,冠状幼鳍的基部出现凋亡;在变态中后期,尾鳍基部出现细胞凋亡;下颌骨、鳃弓以及肝脏在眼睛移动开始之后,出现细胞凋亡,也一直持续到眼睛移动结束。细胞凋亡通过有序地去除多余的细胞来参与器官形态建立和重组,本研究的结果表明,在牙鲆器官功能变化过程中,细胞凋亡在与其相适应的的器官形态重塑中起着重要作用[动物学报52(2):355-361,2006]。     

Isolation of SFRS3 gene and its differential expression during metamorphosis involving eye migration of Japanese flounder Paralichthys olivaceus

Both eyes of flatfishes are located on one side of the body due to asymmetrical eye migration. The molecular mechanisms underlying such asymmetry is poorly understood. As an initial step, we have adopted suppression subtractive hybridization for the identification of upregulated genes during metamorphosis involving eye migration in Japanese flounder, Paralichthys olicaceus. One of the upregulated genes was identified as the splicing factor arginine/serine rich-3 (SFRS3). Sequence analysis of SFRS3 revealed that it encodes a protein of 168 amino acids containing the typical eukaryotic RNA recognition motif (RRM) and an arginine/serine-rich region. The overall amino acid sequences of the Japanese flounder SFRS3 was highly conserved with that of other organisms. The expression of flounder SFRS3 gene increased sharply from the beginning of metamorphosis and reached a high level of expression at stage H of metamorphosis 43 days after hatching. The SFRS3 gene upregulation was mainly limited to the head region, particularly in the rapidly proliferative tissues, the lateral ethmoid and "skin thickness" on blind side, which are thought as two proliferative tissues to push the eye movement. In spite of the upregulated expression of SFRS3 during metamorphosis, its role in metamorphosis involving eye migration requires further studies.     

Identification and differential expression of microRNAs during metamorphosis of the Japanese flounder (Paralichthys olivaceus)

Background

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20–25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA.

Methodology/Principal Findings

Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis.

Conclusions/Significance

We identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder.     

Histological changes in the pituitary-thyroid axis during spontaneous and artificially-induced metamorphosis of larvae of the flounder Paralichthys olivaceus

Summary Histological changes in the pituitary TSH cells and in the thyroid gland of flounder (Paralichthys olivaceus) larvae during spontaneous or artificially induced metamorphosis were studied. Activity of the immunoreactive TSH cells (IrTSH cells) gradually increased during premetamorphosis, reaching the highest level in prometamorphic larvae, and the cells were degranulated in metamorphic climax. The IrTSH cells were most inactive at the post-climax stage. The thyroid gland was morphologically the most active in metamorphic climax when the degranulation occurred in the pituitary IrTSH cells, and appeared inactive at post-climax. A few weeks after metamorphosis, both the IrTSH cells and the thyroid gland appeared to be activated again in the benthic, juvenile flounder. Administration of thyroxine or thiourea revealed negative feedback regulation of the pituitary-thyroid axis in flounder larvae. These results indicate that activation of the pituitary-thyroid axis induces metamorphosis in the flounder.     

Process of pigment cell differentiation in skin on the left and right sides of the Japanese flounder,Paralichthys olivaceus,during metamorphosis

Two groups of larvae of the Japanese flounder,Paralichthys olivaceus, were reared in the laboratory. The survivors of the first group (normal) showed normal pigmentation, and the second group (albinic) exhibited nearly complete pseudoalbinism after metamorphosis. The process of pigment cell differentiation on the left and right sides was observed mainly by transmission electron microscope (TEM) in relation to metamorphosis. In the normal group, chromatoblasts in the left side skin differentiated successfully, but those in the right side skin showed shrinkage and collapse during metamorphosis. Mucus cells are known as typical cells of ocular side skin in flatfish. The ratio of mucus cell density (left side/right side) increased from the onset of metamorphosis. These results suggest, some components of skin changed asymmetrically in process of metamorphosis before differences in fine structures of chromatoblasts were detected between the left and right sides of the normal group. However, in the albinic group, the same process of chromatoblast collapse occurred on the left and right sides, and there was no change in the ratio of mucus cell density during metamorphosis.     

High dietary arachidonic acid levels affect the process of eye migration and head shape in pseudoalbino Senegalese sole Solea senegalensis early juveniles

The effect of high dietary levels of arachidonic acid (ARA) on the eye migration and cranial bone remodelling processes in Senegalese sole Solea senegalensis early juveniles (age: 50 days post hatch) was evaluated by means of geometric morphometric analysis and alizarin red staining of cranial skeletal elements. The incidence of normally pigmented fish fed the control diet was 99·1 ± 0·3% (mean ± s.e .), whereas it was only 18·7 ± 7·5% for those fed high levels of ARA (ARA‐H). The frequency of cranial deformities was significantly higher in fish fed ARA‐H (95·1 ± 1·5%) than in those fed the control diet (1·9 ± 1·9%). Cranial deformities were significantly and negatively correlated with the incidence of normally pigmented animals (r² = ?0·88, P < 0·001, n = 16). Thus, fish displaying pigmentary disorders differed in the position of their eyes with regard to the vertebral column and mouth axes, and by the interocular distance and head height, which were shorter than in fish not displaying pigmentary disorders. In addition to changes in the positioning of both eyes, pseudoalbino fish showed some ARA‐induced osteological differences for some of the skeletal elements from the splanchnocranium (e.g. right premaxillary, dentary, angular, lacrimal, ceratohyal and branchiostegal rays) and neurocranium (e.g. sphenotic, left lateral ethmoid and left frontal) by comparison to normally pigmented specimens. Pseudoalbino fish also had teeth in both lower and upper jaws. This is the first study in Pleuronectiformes that describes impaired metamorphic relocation of the ocular side eye, the right eye in the case of S. senegalensis, whereas the left eye migrated into the ocular side almost normally.     

Growth variability of juvenile soles Solea solea and Solea senegalensis, and comparison with RNA : DNA ratios in the Tagus estuary, Portugal

Growth variability and condition of juvenile soles Solea solea and Solea senegalensis , were assessed through RNA : DNA estimates and compared to absolute growth rates. Higher mean cohort RNA : DNA ratios were observed for cohort I at the beginning of estuarine occurrence for both species (4·42 and 4·87, for S. solea and S. senegalensis respectively). Despite different estuarine colonization habits, no significant differences were observed between RNA : DNA monthly variation for both sole species within the same year ( P  > 0·05 for 2003 and 2004). Juvenile S. senegalensis showed significant differences between RNA : DNA ratios obtained for the two nursery areas ( P  < 0·001). The decrease of seasonal growth rates with fish age was similar to seasonal variation of mean RNA : DNA values. Thus the RNA : DNA pattern of juvenile S. solea and S. senegalensis reflected growth and estuarine colonization patterns.     

Histological and histochemical observations in the stomach of the Senegal sole, Solea senegalensis

An histological and histochemical study was conducted on the stomach of adult Senegal sole, Solea senegalensis specimens. The stomach was made up of four distinct layers: mucosa, lamina propria-submucosa-, muscularis and serosa. Surface epithelial, glandular and rodlet cells were present in the mucosa. Cells of the columnar epithelium contained a basal nucleus. Numerous mitochondria, granular endoplasmic reticulum and Golgi apparatus consisting of several parallel cisternae and vesicles were observed in the cytoplasm of these cells. The lysosomes were small, round and dense. The gastric glands were numerous in the pyloric and fundic regions but absent in the cardiac stomach. These glands were formed by two cell-types: light and dark cells. The light cells were characterised by numerous mitochondria, while dark cells had slightly fewer mitochondria and a tubulo-vesicular system. Rodlet cells similar to those observed in other teleostean fish were present among the epithelial cells. Although the epithelial cells of the mucosa contained a weak presence of neutral and acid mucopolysaccharides/mucosubstances, these substances were abundant in the lamina propria-submucosa. Proteins rich in arginine, lysine, cysteine and cystine were rarely present in the mucosa and lamina propria-submucosa of stomach, while proteins rich in tyrosine were abundant in these layers. Acid phosphatase, and ATP-ase (pH 7.2 and 9.4) activities were detected in the mucosa and lamina propria-submucosa. Alkaline phosphatase activity was not detected.     

Comparative feeding ecology of sympatric Solea solea and S. senegalensis, within the nursery areas of the Tagus estuary, Portugal

Among the stomach contents of 609 individuals of Solea solea and 1104 of S. senegalensis the main food items of S. solea were Corophium spp. and Hediste diversicolor, and of S. senegalensis were Corophium spp., H. diversicolor and Scrobicularia plana. For both species, the importance of larger prey items in the diet, namely H. diversicolor and Crangon crangon, increased with fish size. Feeding activity of S. solea and S. senegalensis increased in spring and summer. Short-term variations were particularly related to the tidal cycle and the two species fed in intertidal areas. Dietary dierences between the two nursery areas reflected prey availability mainly. Although intra- and interspecific length classes overlapped in diet, potential interspecific competition was probably minimized by a dierential habitat use pattern.     

Differential Use of Nursery Areas Within the Tagus Estuary by Sympatric Soles, Solea solea and Solea senegalensis

The spatio-temporal pattern of estuarine use by both Solea solea and Solea senegalensis was investigated between April 1995 and November 1996 by beam trawl surveys. Spatial and seasonal distribution and abundance were different for each species. The highest densities of S. solea were recorded in deeper, warmer, low salinity areas and where the sediment consisted of a high proportion of fine sand and presented a high abundance of amphipods. The 0-group individuals of this species first occurred on the nursery ground in April. Numbers peaked in June and July, and from October to April abundance was low. S. senegalensis had a wider distribution and its abundance was related to food availability. The 0-group individuals of this species occurred in the nursery areas later than S. solea, from June to August.     

Identification of cDNAs encoding two subtypes of vitamin D receptor in flounder, Paralichthys olivaceus

cDNAs encoding two subtypes of the vitamin D receptor (VDR) are cloned from a teleost (flounder, Paralichthys olivaceus). This is the first report of VDR subtypes in fish. Flounder VDR (fVDR) a and b share 86% identity at the amino acid level. With human (h), rat, mouse, quail, and Xenopus VDRs, fVDRa shares 72%, 71%, 71%, 69%, and 71% identity, and fVDRb shares 70%, 69%, 69%, 67%, and 68% identity, respectively. The peptide sequences of the DNA-binding domain (DBD) and hormone-binding domain (HBD) of both subtypes have particularly high homology to those of the tetrapods; e.g. 92% identity for DBP and 74% for HBD between fVDRa and hVDR. In an evolutionary tree constructed with peptide sequences of VDRs and related members of the nuclear receptor superfamily, fVDRa and b are more closely related to each other than to other molecules, and situated in the cluster of VDRs at a position which corresponds well with the evolutional position of fish in the vertebrates. Additional independent genome duplication which is thought to have occurred in ray-finned fish phylogeny may explain the existence of two subtypes of VDR in flounder.     

Photoreceptor development in the pineal organ and the eye of Plecoglossus altivelis and Paralichthys olivaceus (Teleostei)

Summary The ontogenetic apperance of pineal photo-receptors was compared with that of retinal photoreceptors in the ayu Plecoglossus altivelis and the lefteye flounder Paralichthys olivaceus, which hatched 10 days and 3 days after fertilization, respectively. Despite the disparity in incubation time, the outer segments (containing membranous lamellae) of the pineal photoreceptors first appeared from 3 to 4 days after fertilization in both species. In contrast, the outer segments of the retinal photoreceptors first became visible 5 to 6 days after fertilization, although a characteristic retinal stratification and the optic tract leaving the ganglion cell layer were already found 4 days after fertilization in both species. The functional significance of these temporal disparities and/or similarities in photoreceptor development are discussed with special reference to the timing of daily rhythmic activities during the early developmental period of the teleosts.The results were presented at the Annual Meeting of the Japanese Society of Scientific Fisheries on April 2, 1990 (Tokyo)     

Localization of galanin-like immunoreactive structures in the brain of the Senegalese sole, Solea senegalensis

The distribution of galanin-like immunoreactive structures was studied in the brain of the Senegalese sole, Solea senegalensis, using immunohistochemical methods. Periventricular immunoreactive cell bodies were observed in the rostral pole of the preoptic recess, within the pars parvocellularis of the nucleus preopticus parvocellularis. Another galanin-immunoreactive cell population was observed more caudal in the ventromedial hypothalamus, along the medial evaginations of the lateral recess. These cells appear within the cytoarchitectonic limits of the nucleus recessus lateralis pars ventralis. We found an extensive presence of galanin-immunoreactive fibres throughout the entire brain, although the most massive network of fibres was observed in the caudal olfactory bulbs, ventral telencephalon, preoptic area and around diencephalic ventricular recesses. Also, the hypophysis, ventricular mesencephalic area, median reticular formation and viscerosensory rhombencephalon displayed important plexuses of galanin-immunoreactive axons.The widespread distribution of these immunoreactive structures in the brain and pituitary of the Senegalese sole suggests an important role for galanin in neuroendocrine regulation of brain and adenohypophyseal functions.     

Histomorphological and histochemical characteristics of the intestine of the Senegal sole, Solea senegalensis.

The Senegal sole, Solea senegalensis has been exploited extensively in aquaculture from different countries; at present an intensive production of larvae and adults is being achieved with some nutritional problems. Since this species displays very different life styles and feeding habits at different stages of its life history (larvae, metamorphosis, adults), and because digestive mucins are implicated in different physiological processes including: increase of digestive efficiency, promotion of macromolecules-absorption, buffering of intestinal fluid, prevention of proteolytic damage to the epithelium and defence against bacteria, etc., we studied the histomorphological aspects, as well as the histochemical distribution of carbohydrates, (PAS, Alcian Blue), proteins (Bromophenol Blue), lipids (Oil Red O, Black Sudan B) and glycoproteins (Horseradish peroxidase-conjugated lectins) in the intestinal epithelium of adult Solea senegalensis specimens. Our data are compared with those obtained in larvae and adults of this and other fish species. Primary and secondary folds, microvilli of the intestinal enterocytes, as well as mucous or goblet cells were observed with a scanning electron microscope. Enterocytes and mucocytes of the intestine of adult Solea senegalensis were characterized by a rich supply of sugar and oligosaccharides. Carbohydrates (glycogen and mucins), proteins and lipids were present in cytoplasm and microvilli--brush border--of the enterocytes, which contain GalNAc, GlcNAc, Man, Glc and sialic acid-N-acetyl-D-galactosamine glycoconjugates. Intestinal mucous cells were strongly or weakly stained with Alcian Blue (pH 2.5 and 1). PAS reactivity was intense in numerous goblet cells, but some cells were PAS unreactive or weakly stained. Some goblet cells were positive for Bromophenol Blue but numerous cells were unstained; thus many proteins and possibly lipids may be conjugated with sugars. A similar reactivity to WGA and to neuraminidase-WGA was identified in some intestinal goblet, which were Con A unreactive, indicating the absence of Man and/or Glc and NANA glycoconjugates. GalNAc residues were only scarcely present in glycoproteins of some goblet cells.     

Morphological and Molecular Characterization of Dietary-Induced Pseudo-Albinism during Post-Embryonic Development of Solea senegalensis (Kaup, 1858)

The appearance of the pseudo-albino phenotype was investigated in developing Senegalese sole (Solea senegalensis, Kaup 1858) larvae at morphological and molecular levels. In order to induce the development of pseudo-albinos, Senegalese sole larvae were fed Artemia enriched with high levels of arachidonic acid (ARA). The development of their skin pigmentation was compared to that of a control group fed Artemia enriched with a reference commercial product. The relative amount of skin melanophores, xanthophores and iridophores revealed that larval pigmentation developed similarly in both groups. However, results from different relative proportions, allocation patterns, shapes and sizes of skin chromatophores revealed changes in the pigmentation pattern between ARA and control groups from 33 days post hatching onwards. The new populations of chromatophores that should appear at post-metamorphosis were not formed in the ARA group. Further, spatial patterns of distribution between the already present larval xanthophores and melanophores were suggestive of short-range interaction that seemed to be implicated in the degradation of these chromatophores, leading to the appearance of the pseudo-albino phenotype. The expression profile of several key pigmentation-related genes revealed that melanophore development was promoted in pseudo-albinos without a sufficient degree of terminal differentiation, thus preventing melanogenesis. Present results suggest the potential roles of asip1 and slc24a5 genes on the down-regulation of trp1 expression, leading to defects in melanin production. Moreover, gene expression data supports the involvement of pax3, mitf and asip1 genes in the developmental disruption of the new post-metamorphic populations of melanophores, xanthophores and iridophores.     

Ultrastructural and histochemical study on gills and skin of the Senegal sole, Solea senegalensis

This study was undertaken to identify the normal ultrastructural features of gills and skin of the Senegal sole, Solea senegalensis, for a comparative measure to morphological alterations caused by environmental stressors such as reduced water quality and diseases. In the Senegal sole skin, four morphologically distinct layers were identified: cuticle, epidermis, dermis and hypodermis. The epidermis was composed of stratified epithelium containing three cellular layers: the outermost or mucosa layer, the middle or fusiform layer and the stratum germinativum or the basal layer. In the mucosa, two mucous cell types were differentiated: type A cells containing several round vesicles of different electron density and type B cells containing mucosomes of uniform electron density. Senegal sole have five pairs of gill arches, each containing two rows of well‐developed and compactly organized primary filaments and secondary lamellae. Fingerprint‐like microridges were observed on the surface of epithelial cells. The branchial lamellae epithelium consisted of different cell types: pavement, mucous and chloride. Between the chloride cells and the larger pavement cells, accessory cells were observed. Complexes of tight junctions and desmosomes were frequently observed between adjacent chloride and epithelial cells. Neutral mucosubstances and/or glycoconjugates were observed in the epidermis, dermis and hypodermis of S. senegalensis skin. Proteins rich in different amino acids, such as arginine and cysteine, reacted negatively or weakly positive in the epidermis, dermis and hypodermis. In gills, some mucous cells responded weakly positive to periodic acid‐Schiff (PAS) reaction but were strongly stained with Alcian Blue at pH 0.5, 1 and 2.5. When Alcian Blue pH 2.5–PAS reaction was performed, most mucous cells were stained blue (carboxylated mucins) and some mucocytes stained purple, indicating a combination of neutral and acid mucins. Proteins rich in cysteine‐bound sulphydryl (‐SH‐) and cystine disulphide (‐S‐S‐) groups were strongly detected in branchial and epidermal mucous cells, whereas lysine, tyrosine and arginine containing proteins showed very weak staining in both epidermal and branchial mucous cells. Protein reactions were strongly positive in the pillar cells, except for those rich in tryptophan, whereas the branchial cartilaginous tissue did not show an important reaction. The performed lipid reactions were negative in goblet and chloride cells. It is concluded from this study that ultrastructural and cytohistochemical features of the Senegal sole skin and gills may serve as control structures in both natural and aquaculture systems to monitor or detect environmental stress responses at the histological level.     

The evolution and possible role of two Sox8 genes during sex differentiation in Japanese flounder (Paralichthys olivaceus)

Sox8 genes, as members of the Sox family, have been studied widely in mammals. However, regulation of sox8 genes in teleosts has rarely been studied, and functional analysis of these genes in teleosts has rarely been performed. Here, two duplicates of sox8 genes were identified in Japanese flounder, Posox8a and Posox8b. The analysis of expression showed that Posox8a and Posox8b were expressed in Sertoli cells of the testis, indicating that they play important roles in development and functional maintenance of the testis. Positive selection and phylogenetic analysis found that both Posox8a and Posox8b underwent the purification selection during evolutionary and that sox8 was most likely to be the ancestor sox8a. These results suggested that both Posox8a and Posox8b had important biological functions after generation from three rounds of whole‐genome duplication in Japanese flounder. The functional differentiation of Posox8a and Posox8b was verified using cell transfection and dual‐luciferase reporter assays; Posox8a overexpression‐promoted 3β‐hydroxysteroid dehydrogenase expression and Posox8b overexpression‐promoted cytochrome P450 aromatase (cyp19a1; P450arom) expression. Finally, combined with Posox8a and Posox8b expression analysis from 30 to 100 days after hatch, we speculated that Posox8a and Posox8b might participate in the process of sex differentiation and gonadogenesis by regulating sex hormone biosynthesis in the Japanese flounder. Our study is the first to demonstrate the possible mechanism of Posox8a and Posox8b in Japanese flounder sex differentiation and gonadogenesis, laying a solid foundation for functional studies of sox8 genes in teleosts.     

Identification of two differentially expressed forms of progranulin mRNA in the teleost fish Paralichthys olivaceus

Two forms of progranulin mRNA were isolated from kidney and spleen cDNA libraries of the olive flounder, Paralichthys olivaceus. The 3'-untranslated regions (3'-UTRs) of these flounder progranulin (f-pgrn) mRNAs differed in a 20-nucleotide sequence element (5'-AACTGATTACGTTCAACAAC-3') that was present in one mRNA (designated f-pgrn type II) and not in the other (designated f-pgrn type I). Both mRNA sequences contained an open reading frame encoding a 289-amino-acid polypeptide of approximately 33 kDa. Southern blot analysis of the P. olivaceus flounder genome using an f-pgrn cDNA probe and a PCR-based approach identified a single copy of f-pgrn corresponding to the type II mRNA. The expression profiles of the two types of f-pgrn mRNA differed from each other and were tissue- and condition-dependent. The type II mRNA was detected abundantly in studied tissues (gill, kidney, spleen, and intestine) of non-stimulated healthy flounders. The type I mRNA was rarely expressed in any tissues of healthy flounders, but it was continuously increased in the examined tissues of flounders after the intraperitoneal injection of lipopolysaccharide. On the other hand, the expression of type II mRNA was decreased in inverse proportion to the type I mRNA in the LSP-stimulated flounders. These results suggest that type I and type II f-pgrn mRNA are translated into different proteins with different activities in the immune system of flounder.     

Influence of sex, age and season on the feeding habits of the flatfish Solea senegalensis

Synopsis The Senegal sole Solea senegalensis is a commercially valuable fish and it is beginning to be cultured, in an extensive way, in fish farms in southern European countries. Little is known about its feeding habits, unlike other Solea species such as the common sole Solea solea. This paper studies the feeding habits of a wild population of Senegal sole. Differences according to sex, age and season were statistically analyzed. A total of 376 fish, aged between two and six years old, were studied over a single year. Twenty eight different families of prey were identified, belonging, in order of abundance, to crustaceans, polychaetes and molluscs. The Senegal sole trophic profile (preys items with a Simpson's dominance index 2) was: Glyceridae, Nephtydidae, Onuphidae, Apseudidae, Ampeliscidae and Upogebiidae. Feeding habits of Senegal sole were significantly influenced by sex, age and season, with crustaceans being the prey most affected.This paper is dedicated to the memory of Daniel Ray Ogilvie. who died 7 January 1995.     

Distribution of gonadotropin-releasing hormone immunoreactive systems in the brain of the Senegalese sole, Solea senegalensis

The present paper reports the immunohistochemical distribution of the gonadotropin-releasing hormone (GnRH) structures in the brain of the Senegalese sole, Solea senegalensis. In this study, we have used two antibodies against the salmon GnRH and chicken GnRH-II forms and the streptavidin–biotin-peroxidase complex method. Immunoreactive cell bodies are observed at the junction between the olfactory bulbs and the telencephalon (terminal nerve ganglion cells), in the ventral telencephalon, in the preoptic parvocellular nucleus, and in the synencephalic nucleus of the medial longitudinal fasciculus. GnRH-immunoreactive fibres were found extensively throughout the brain, located in the telencephalon, preoptic area, hypothalamus, hypophysis, optic tectum, midbrain and rhombencephalon. The antisera used in this study against the two GnRH forms exhibited cross-reactivity on the same cell masses and did not allow cell populations expressing different GnRH forms to be discriminated clearly. However, anti-salmon GnRH immunostained the GnRH cells and fibres of the forebrain much more intensely, whereas the anti-chicken GnRH antiserum shows a higher immunoreactivity on synencephalic cells of the medial longitudinal fasciculus.