Role of 5α-dihydrotestosterone in testicular development of gilthead seabream following finasteride administration.

In teleosts, spermatogenesis is regulated by pituitary gonadotropins and sex steroids. 5α-dihydrotestosterone (DHT), derived from testosterone (T) through the action of 5α-reductase, has recently been suggested to play a physiologically important role in some fish species. In this study, gilthead seabream, Sparus aurata L., males received an implant of 1µgT/g body mass (bm) or vehicle alone and, 7days later, 1mg finasteride (FIN, an inhibitor of 5α-reductase)/kg bm or vehicle. Serum levels of T, 11-ketotestosterone (11KT), DHT and 17ß-estradiol (E2), and the mRNA levels of the main enzymes involved in their synthesis, were analysed. T promoted a transient increase in the serum levels of T, 11KT and E2 but a decrease in those of DHT at day 15 following T injection, in accordance with the up-regulation of mRNA levels of the enzymes involved in T transformation to 11KT (coding genes: cyp11b1 and hsd11b) and the down-regulation of mRNA levels of the enzyme responsible for T transformation to DHT (coding gene: srd5a). Interestingly, a similar effect was observed when FIN was injected. However, when fish were injected with T and FIN successively (T+FIN), control levels were not recovered at the end of the experimental period (28days). DHT seems to regulate E2 serum levels via the down-regulation of mRNA levels of aromatase (coding gene: cyp19a1a), which is needed for the transformation of T into E2. The testis histology, together with the proliferative rates recorded upon T, FIN or T+FIN treatment, suggests that DHT is involved in the onset of the meiotic phase of spermatogenesis.

Tamoxifen disrupts the reproductive process in gilthead seabream males and modulates the effects promoted by 17α-ethynylestradiol.

17α-Ethynylestradiol (EE2), which is used in oral contraceptives and hormone replacement therapy, is a well documented estrogenic endocrine disruptor and an aquatic contaminant. In the present study, adult male specimens of the marine hermaphrodite teleost gilthead (Sparus aurata L.) were fed a diet containing tamoxifen (Tmx), an estrogen receptor ligand used in cancer therapy, alone or combined with EE2, for 25 days and then fed a commercial diet for a further 25 days (recovery period). The effects of short (5days) and long (25 days) treatments on several reproductive and gonad immune parameters and the reversibility of the disruptive effects after the recovery period were examined. Our data showed that Tmx acted as an estrogenic endocrine disruptor as revealed by the increase in the hepatic transcription of the vitellogenin gene in males, the serum levels of 17ß-estradiol and the gonad expression levels of the estrogen receptor α and G protein-coupled estrogen receptor genes, and the recruitment of leukocytes into the gonad, a well known estrogenic-dependent process in gilthead seabream males. On the other hand, Tmx also increased sperm concentration and motility as well as the serum levels of androgens and the expression levels of genes that codify for androgenic enzymes, while decreasing the expression levels of the gene that code for gonadal aromatase. When applied simultaneously, Tmx and EE2 could act in synergy or counteract, each other, depending on the parameter measured. The disruptive effect of EE2 and/or Tmx was not reversible after a 25 day recovery period.

FSH-, LH-, and TSH-expressing cells during development of Sparus aurata L. (Teleostei). An immunocytochemical study.

Follicle-stimulating hormone-like gonadotropin (FSH), luteinizing hormone-like gonadotropin (LH), and thyrotropin (TSH) cells were detected in adult and developing pituitary gland of gilthead seabream. Antisera obtained against the alpha- and beta-subunits of FSH (anti-My FSH) and the beta-subunit of LH (anti-My LHbeta), respectively, of the teleost Mediterranean yellowtail, and an antiserum against the beta-subunit of human TSH (anti-h TSHbeta), were applied to identify and follow these cells during ontogeny using light microscopy. FSH cells were immunoreactive to anti-My FSH serum, LH cells were immunoreactive to anti-My LHbeta and anti-My FSH sera, and TSH cells were immunoreactive to anti-h TSHbeta and anti-My FSH sera. In adult specimens, FSH and LH cells were located in both the proximal pars distalis (ppd) and the pars intermedia (pi) in strands or compact groups and as isolated cells. FSH cells were less numerous than LH cells. Some FSH and LH cells had a vacuolated appearance. TSH cells were mainly arranged as a mass in the anterior ppd, although some isolated cells could also be observed. FSH, LH, and TSH cells appeared at different times during development. FSH cells were observed for the first time in 22-day-old larvae and LH cells in juvenile specimens when the gonad was still undifferentiated. No vacuolated FSH and LH cells were present in larvae or juveniles. TSH cells were observed for the first time at hatching. As the fish developed, FSH, LH, and TSH cells progressively increased in number and showed the same distribution as in adult specimens.

Development of the digestive tract of sea bass (Dicentrarchus labrax L). Light and electron microscopic studies.

The developing gut of sea bass was studied by light and electron microscopy, four phases being established. Phase I, from hatching to the opening of the mouth, was a lecitotrophic period, in which the gut appeared as a straight undifferentiated tube lined by a simple epithelium that became stratified in the most caudal region. The epithelial cells increased in length towards the caudal zone, as did the number and height of the apical microvilli and the magnitude of the lamellar structures in their basal region. Cilia were more numerous in the caudal region than in the rest of the gut. Signs of lipid but not of protein absorption were found in the epithelial cells at this phase. Phase II, from the opening of the mouth to the complete resorption of the yolk sac, was a lecitoexotrophic period in which an esophagus, a gastric region, an intestine and a rectum, the last two separated by a valve, were present. During this phase the differentiation of the gut started at the esophagus and the rectum. In the esophagus, the epithelium became stratified and goblet cells containing acid mucosubstances, including sulphomucins, appeared. In the epithelial cells of the rectum, supranuclear vacuoles and an incipient endocytotic apparatus that seemed to be involved in the absorption and digestion of proteins were found. In both regions the mucosa was folded. Phase III, from the complete resorption of the yolk sac to the appearance of the first gastric glands, initiated the exclusively exotrophic period. During this phase the intestine formed the mucosa folds, while the first pyloric caeca and the epithelial cells acquired the ultrastructural features of mature absorptive cells with many lipid inclusions. Goblet cells containing neutral mucosubstances appeared and increased in number in both the intestine and the rectum. Neutral mucosubstances were also present in the cells lining the gastric region. During phase IV, from the appearance of the first gastric glands onwards, the intestinal absorptive surface increased with the formation of new pyloric caeca and two intestinal loops. The stomach acquired its definitive anatomy and histology with the development of the caecal and pyloric regions alongside differentiated gastric glands. The glandular cells had the ultrastructural features of the cells that secrete both pepsinogen and hydrochloride acid in the adult teleost stomach.

Immunocytochemical and ultrastructural characterization of somatolactin cells from the gilthead sea bream (Sparus aurata L., Teleostei): an ontogenic study (from newly hatched to adults).

For the first time, somatolactin (SL) cells have been ultrastructurally identified and characterized during the ontogeny of gilthead sea bream, Sparus aurata, using specimens ranging in age from hatching to 15 months. The SL cells were identified by an immunogold method using anti-cod SL serum. The SL-immunoreactivity was mostly located on the secretory granules of the cells, although some vesicles of variable size and shape with a medium electron-dense content, and some irregular secretory granules and polymorphic or very irregular masses that can arise from the fusion of several secretory granules, also presented immunogold labeling. In adults, the SL cells were mainly found in the pars intermedia, where they were organized in discontinuous cell cords lying against the neurohypophysis or surrounding the neurohypophyseal branches. Some SL cells, however, appeared isolated or in small groups in the pars intermedia, in the proximal pars distalis and, rarely, in the rostral pars distalis. The SL cells were variable in shape, with processes directed towards the neurohypophysis or blood vessels, or intermingling among other adenohypophyseal cells. The secretory granules were mostly round, although some were oval, bilobate or pear-shaped, with a homogeneous, very electron-dense content and a narrow, dense or clear, halo. Different SL cell populations can be distinguished according to secretory granule size. Our findings indicate that SL is stored in the secretory granules and released by exocytosis. SL cells showing involutive features were only found in adults. SL cells can be ultrastructurally identified in one-day-old larvae although similar characteristics to those found in adults can be positively identified only after 4 days. Secretory granules increased in number, size and heterogeneity during development. Synaptic-like structures between axon terminals of the neurohypophysis and the SL cells were found in larvae from one-day-old onwards. In juveniles of 118 days of age, two different populations of secretory granules (immunogold-labeled and non-immunogold-labeled) can be found in the same or different SL cells, findings that suggest the existence of two different molecular forms of SL at this age. There was a clear increase in the complexity of the pituitary gland and in the heterogeneity of the SL cells during development, the latter observation probably reflecting different functional cell stages or production of SL molecules.

Identification of the pancreatic endocrine cells of Pseudemys scripta elegans by immunogold labeling.

The endocrine pancreatic cells of Pseudemys scripta elegans were investigated immunocytochemically by light and electron microscopy. Insulin-, somatostatin (SST)-1, SST-28 (1-12)-, salmon (s)SST-25-, glucagon-, pancreatic polypeptide (PP)-, peptide tyrosine tyrosine (PYY)-, and neuropeptide tyrosine (NPY)-like immunoreactivities were observed. Insulin cells were immunogold labeled with bonito insulin antiserum and secretory granules were characterized by a wide halo and a dense core of varying shape. Consecutive PAP-immunostained sections showed that SST-28 (1-12), SST-14, and sSST-25 immunoreactivities occurred in the same cells. However, preabsorption tests demonstrated that anti-sSST-25 serum detected the invariant SST-14 molecule. The SST-28 (1-12)/SST-14-immunogold-labeled cells mainly had round or ovoid medium electron-dense granules. Glucagon-IR cells were characterized by round secretory granules with an electron-dense core, with or without a narrow clear halo. There were PP, PYY, and NPY (NPY-like) immunoreactivities in a population of glucagon-IR cells in the pancreatic duodenal region (glucagon/NPY cells). Most of the secretory granules of these glucagon/NPY-like cells had an electron-dense content and were round, although there were also pyriform or ovoid secretory granules which were smaller than those of glucagon-IR cells. Preabsorption tests proved that the NPY-like peptides detected in the endocrine pancreas of P. scripta elegans were more similar to NPY or PYY than to PP.

Glucagon- and NPY-related peptide-immunoreactive cells in the gut of sea bass (Dicentrarchus labrax L.): a light and electron microscopic study.

Glucagon and peptide of the neuropeptide Y (NPY) family immunoreactivities were studied in the gut of sea bass (Dicentrarchus labrax) using antisera against bovine/porcine glucagon, porcine glucagon, glicentin (10-30), bovine pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), salmon PYY (sPYY), and NPY. Glucagon-, glicentin-, PYY-, and NPY-immunoreactive (ir) cells were detected in the stomach, and glucagon-, PP-, PYY-, sPYY-, and NPY-ir cells in the intestine. PP, PYY, and NPY immunoreactivities coexisted in intestinal endocrine cells (NPY-like peptide containing cells), in some of which there was also glucagon immunoreactivity. Preabsorption tests indicated that different products of the glucagon gene(s) are probably expressed in the stomach and intestine of sea bass and that the peptides belonging to the NPY family in the endocrine cells of the intestine are more similar to NPY than to other peptides of this family. Glucagon-ir cells in the stomach, and glucagon/NPY-like containing cells in the intestine, were characterized by conventional and immunogold electron-microscopic techniques. The glucagon cells had secretory granules with a clotted content, the gold particles being observed in both the core and the halo. Glucagon/NPY-like cells showed two types of secretory granules differing in size, both of which were immunogold labeled with anti-NPY and anti-sPYY; the smaller granules were weakly immunogold labeled with anti-glucagon.

Gonadotropic and thyrotropic cells from the Mediterranean yellowtail (Seriola dumerilii; Risso, 1810): immunocytochemical and ultrastructural characterization.

BACKGROUND: Gonadotropins GTH I and GTH II from the pituitary of Mediterranean (M.) yellowtail (Seriola dumerilii) were isolated and characterized, and antisera to the whole GTH II molecule (anti-My alpha,betaGTH II) and to its beta-subunit (anti-My betaGTH II) were obtained. At the light microscopic level, anti-My alpha,betaGTH II reacted with My betaGTH II-immunoreactive cells (GTH II cells), thyroid-stimulating hormone (TSH) cells, and a third cell population, which could have been GTH I cells. The aim of this study was the ultrastructural characterization of GTH and TSH cells in M. yellowtail using the immunogold method in order to provide a basis for future research into reproduction of this species. METHODS: Pituitaries from mature male and female specimens reared in captivity were dissected out and processed for electron microscopy. The immunogold method was carried out by using anti-My alpha,betaGTH II, anti-My alpha,betaGTH II preabsorbed with the alpha subunit of the M. yellowtail GTH (My alphaGTH-subunit), anti-My betaGTH II, anti-human (h) alpha,betaTSH, and anti-h betaTSH sera to reveal gonadotropic and thyrotropic cells. RESULTS: M. yellowtail gonadotropic cells were very heterogeneous with regard to their size, shape, and ultrastructural features. Cells were found with numerous, round, variably electron-dense, secretory granules and globules; others were found with their cytoplasm occupied mostly by dilated cisternae of rough endoplasmic reticulum (RER) and scarce secretory granules; and other intermediate cell forms were found that showed varying proportions of secretory granules and dilated RER. The secretory granules and globules were immunogold labeled with anti-My alpha,betaGTH II, and the reaction was weaker in the latter. A similar immunogold-labeling pattern was found with anti-My betaGTH II and with anti-My alpha,betaGTH II preabsorbed with the My alphaGTH-subunit, although some cells that showed the same ultrastructural features described above were not immunogold labeled and could have been GTH I cells. Thyrotropic cells had small, round, secretory granules of medium or high electron density that were immunogold labeled with anti-My alpha,betaGTH II, anti-h alpha,betaTSH, and anti-h betaTSH sera, but not with anti-My betaGTH II or anti-My alpha,betaGTH II serum preabsorbed with the My alphaGTH-subunit. All of the cell forms described for gonadotropes and thyrotropes were also found in a state of involution. CONCLUSIONS: Gonadotropes that are of a single morphological type but that vary in ultrastructure are present in the pituitary of captive M. yellowtail. GTH II- and putative GTH I-producing cells were distinguishable from one another and from TSH cells by their different reactions to anti-My alpha,betaGTH II, anti-My betaGTH II, and anti-My alpha,betaGTH II preabsorbed with the My alphaGTH-subunit.

Ontogeny of immunoreactive somatolactin cells in the pituitary of gilthead sea bream (Sparus aurata L., Teleostei).

This is the first report on the identification of somatolactin (SL) cells during the early developmental stages of the teleost fish Sparus aurata. The SL cells were followed from newly hatched until 46 months. SL cells were immunocytochemically identified at light microscopical level with anti-cod SL in the developing pituitary using the peroxidase-antiperoxidase method. SL cells first appeared in newly hatched specimens, in which the pituitary gland lacked the neurohypophysis. They were scarce and located from the middle to the posterior region of the adenohypophysis. As the fish developed, the cells progressively increased in number and surrounded the developing neurohypophysis, which could be distinguised from 12-day-old larvae onwards in the caudal region of the gland. From 51 days onwards, SL cells were found in a discontinuous layer surrounding the neurohypophysis branches that entered the pars intermedia as clustered or isolated cells among non-SL-immunoreactive cells of the pars intermedia, and in the proximal pars distalis. The somatolactin-immunoreactive cells are periodic acid-Schiff-positive only in the adult stages. These data confirm, previous findings concerning the presence of two molecular forms of SL, glycosylated and nonglycosylated, in this species.

Immunocytochemical and ultrastructural characterization of melanotropin and adrenocorticotropin cells from the Mediterranean yellowtail (Seriola dumerilii, Risso 1810).

BACKGROUND: Melanotropin (MSH) and adrenocorticotropin (ACTH) are pituitary hormones derived from a common precursor: the proopiomelanocortin (POMC), which is processed differently in the melanotropic and corticotropic cells of several vertebrates. While ACTH is a major final product in corticotropes, it is further processed into alpha-MSH and corticotropin-like intermediate lobe peptide (CLIP) in melanotropes. Cells which are immunoreactive to ACTH (ACTH cells) and to both alpha-MSH and ACTH (MSH cells) have been described in a number of teleosts, including the Mediterranean yellowtail, by light microscopic immunocytochemistry. However, these cells have been ultrastructurally characterized only in a few species. In this paper, we use electron microscopy to identify and characterize the cells producing MSH and ACTH in M. yellowtail (Seriola dumerilii). METHODS: Pituitaries from adult specimens were dissected and processed for conventional and immunocytochemical electron microscopy. An immunogold technique was performed using anti-synthetic alpha-MSH and anti-human (h) ACTH (1-24) sera. RESULTS: MSH cells had round secretory granules with a granular content of varying electron density and compactness, which were immunogold-labeled with anti-alpha-MSH. Homogeneous and electron-dense secretory granules found in the Golgi area of these cells reacted with both anti-alpha-MSH and anti-hACTH (1-24). ACTH cells had round secretory granules with a homogeneous and medium or high electron-dense core and narrow clear halo, which were grouped in the cell area near the neurohypophysis (NH). Some granules showed an osmiophilic semicore in the medium electron-dense content, which has not been described in other teleost pituitary cells. Immunogold-labeling over the secretory granules only was obtained with all the antisera used. Some ACTH cells showed involutive features. CONCLUSIONS: MSH and ACTH are respective final products of the POMC in two ultrastructurally different cells of the pituitary of M. yellowtail, MSH and ACTH cells. The immature granules in the Golgi area of MSH cells seem to be the site of proteolitic cleavage of ACTH into alpha-MSH and CLIP.

Isolation and characterization of two distinct gonadotropins from the pituitary gland of Mediterranean yellowtail, Seriola dumerilii (Risso, 1810).

Two gonadotropins, GTH I and GTH II, were isolated and chemically characterized from the pituitary of Mediterranean yellowtail. They were extracted with 35% ethanol-10% ammonium acetate, separated by ion-exchange chromatography on a DE-52 column, and purified by reversed-phase high-performance liquid chromatography on Asahipak C4P-50 and subsequently by gel filtration chromatography on Superdex 75. The molecular weights were estimated at 47 kDa for GTH I and 29 kDa for GTH II by SDS-PAGE and at 49 kDa for GTH I and 42 kDa for GTH II by gel filtration. GTH II was completely dissociated, while GTH I was partially dissociated into alpha- and beta-subunits by treatment with 0.1% trifluoroacetic acid. The complete amino acid sequences of GTH alpha-, GTH I beta-, and GTH II beta-subunits were determined. The GTH alpha-subunit consisted of 91 amino acid residues. The GTH I beta and GTH II beta consisted of 105 and 115 amino acid residues, respectively, and had a 28% sequence identity to each other. They had the highest sequence identity with the respective gonadotropin subunits of bonito, tuna, and striped bass: 81-83% for GTH alpha, 67-71% for GTH I beta, and 91-93% for GTH II beta. The sequence identity of the GTH alpha-subunit with those of other teleosts and human and bovine LH and FSH was 57-67%. The GTH I beta-subunit showed a low sequence identity with other known fish GTH I beta s (36-51%) and was more similar to human and bovine FSH beta s (34% identity) than to human and bovine LH beta s (29% identity). The sequence identity of the GTH II beta-subunit with those of other teleosts was higher (60-73%), being more similar to LH beta s (43% identity) than FSH beta s (38% identity). Thus, two distinct gonadotropins, GTH I and GTH II, homologous to mammalian FSH and LH, respectively, are synthetized by M. yellowtail pituitary glands.

Immunocytochemical and ultrastructural characterization of prolactin, growth hormone, and somatolactin cells from the Mediterranean yellowtail (Seriola dumerilii, Risso 1810).

BACKGROUND: Prolactin (PRL), growth hormone (GH), and somatolactin (SL) are structurally related pituitary hormones that belong to a peptide family. Whereas growth hormone and prolactin are present in the hypophysis of all vertebrates, somatolactin, a recently discovered hormone, has been found only in fish. It has been demonstrated immunocytochemically in a few teleost species; ultrastructurally, cells producing this hormone have been characterized only in one species of salmon. In this paper, we identify and characterize ultrastructurally the cells producing these three hormones in Mediterranean yellowtail (Seriola dumerilii). METHODS: Pituitaries from adult specimens were dissected out and processed for electron microscopy. The immunogold technique was performed in some ultrathin sections using fish primary antibodies. RESULTS: PRL cells had round, peripherally distributed, very electron-dense, homogeneous secretory granules of variable size. GH cells had dense, round secretory granules with a conspicuous scalloped membrane, which were grouped in the cell area near the neurohypophysis. SL cells had round, polymorphic, or very irregularly shaped secretory granules, the last seeming to arise from the fusion of various secretory granules. The population of secretory granules varied greatly from one cell to another. In all cases, immunogold labeling was seen exclusively in the secretory granules. Exocytosis was observed in all cell types. Some of the PRL, GH, and SL cells showed involutive features. CONCLUSIONS: PRL, GH, and SL, although structurally and functionally related, are secreted by ultrastructurally different cells in the pituitary of M. yellowtail.

The adenohypophysis of Mediterranean yellowtail, Seriola dumerilii (Risso, 1810): an immunocytochemical study.

The adenohypophysis (ADH) of the Mediterranean yellowtail was studied using the peroxidase-antiperoxidase technique. Human corticotropin (ACTH) (1-24)-immunoreactive (ir) cells were found bordering the neurohypophysis (NH) and salmon prolactin (PRL)-ir cells were arranged in thick cords, both in the rostral pars distalis (RPD). Gonadotropin (GTH)-, thyrotropin (TSH)- and growth hormone (GH)-ir cells were observed in the proximal pars distalis (PPD). Anti-chum salmon GTH I and anti-chum salmon GTH II immunostained the same cells in the outermost part of the ADH at the level of the PPD and the PI. In addition to these cells, some cells grouped in the inner areas of the posterior PPD were revealed by catfish alpha, beta-GTH antiserum. Human beta-TSH-ir cells formed small groups and discontinuous strands in the PPD often in contact with the NH. Tilapia GH-ir cells formed cords mainly surrounding the NH in the central PPD, while cod somatolactin- and alpha MSH-ir cells mainly surrounded the NH branches in the PI.

Early organization of the pituitary gland in Sparus aurata L. (Teleostei). An ultrastructural study.

The cell organization of the pituitary gland and the relationship between neurohypophysis and adenohypophysis in early developmental stages of the gilthead sea bream, Sparus aurata, were studied by electron microscopy. In newly hatched larvae, the pituitary gland was embedded in the ventral floor of the diencephalon and separated from the hypothalamus by a continuous basal lamina. Elongated mesenchymal cells next to the ventral surface were observed. At this stage, there was no neurohypophysis and the adenohypophysis consisted of undifferentiated endocrine cells with small scarce secretory granules and a few stellate cells, with no distinctive zonation. An incipient neurohypophysis was present in 1-day-old larvae. The first evagination of the neurohypophysis into the adenohypophysis were observed in 2-day-old larvae and developed progressively with age, being deeper in the caudal zone. Two regions in the adenohypophysis, one anterior--the presumptive pars distalis--and one posterior--the presumptive pars intermedia--were found in 2-day-old larvae. Three regions (rostral and proximal pars distalis and pars intermedia) were clearly distinguishable in 4-day-old larvae. The ultrastructural features of the pituitary endocrine cells varied during gland differentiation, with the secretory granules gradually increasing in number and size, accompanying organelle development. Nevertheless, even in the oldest larvae studied (65 days), undifferentiated cells similar to those in the earliest stages were observed. The first blood vessels appeared in the neurohypophysis around 16 days after hatching. During early development, the pituitary gland progressively emerged from the ventral floor of the brain. By 16 days, the principal pattern of the pituitary gland architecture appeared to be established.

Occurrence of somatostatin and insulin immunoreactivities in the stomach of sea bass (Dicentrarchus labrax L.): light and electron microscopic studies.

Somatostatin (SST)- and insulin (INS)-immunoreactive (ir) cells were identified in the gut of sea bass (Dicentrarchus labrax) by immunofluorescence double staining and peroxidase-antiperoxidase (PAP) techniques for light microscopy and by immunogold method for electron microscopy using antisera to mammalian and fish peptides. SST-14 and SST-25 immunoreactivities coexisted in cells mainly located among the mucous neck cells of the gastric glands. Preabsorption controls showed that some SST-25- and, possibly, some SST-14-like peptides appeared in these cells. Immunoreactivity to fish INS, but not to mammalian INS (mINS) or insulin-like growth factor I (mIGF-I), was observed in all the SST-ir cells. The preabsorption controls suggest a cross-reaction of the fish INS antisera with SST-containing or type I cells. These cells displayed ovoid or round secretory granules with fibrous, medium electron-dense or homogeneous osmiophilic materials. Some gastric cells (type II) with round secretory granules of variable electron density, which were gold immunolabeled with bonito INS but not with mINS, mIGF-I, or SST antisera, were also found. INS-related peptide in type II cells of the sea bass stomach is suggested.

Ontogeny of the endocrine cells of the intestine and rectum of sea bass (Dicentrarchus labrax L.): an ultrastructural study.

Several endocrine cell types were ultrastructurally characterized during the differentiation of the intestine and rectum of sea bass (Dicentrarchus labrax L.) larvae. Only one cell type (type I) was found in the posterior region of the undifferentiated gut of 5-day-old larvae (phase I). Types V and VI were found in both the intestine and rectum, types II, III and IV in the intestine, and types VII and VIII in the rectum of 9- and 12-day-old larvae (phase II), the rectum alone showing signs of functional differentiation. In phase III larvae, in which both the intestine and rectum were differentiated, types IX, X, XI, XII, XIII, XIV and XV were found in the intestine, only types X, XI and XII being seen in the rectum. Besides these, a new cell type, XVI, was observed in the intestine of 55- and 60-day-old larvae (phase IV), in which the digestive tract was completely differentiated. The endocrine cells appearing in phases I and II showed very scarce secretory granules and the ultrastructural features of undifferentiated cells. Some endocrine cell types in the earliest developmental stages were related to some of those found later. A maturational process of the endocrine cell types paralleled the differentiation of the intestine and rectum, with an apparent increase in the number of secretory granules accompanying organelle development.

Ontogeny of the endocrine cells of the stomach of sea bass (Dicentrarchus labrax L.): an ultrastructural study.

The endocrine cells present in the developing stomach of sea bass larvae have been characterized ultrastructurally. Only one endocrine cell type (type I) was found in the presumptive stomach of 9- and 12-day-old larvae, one (type II) and five (types III, IV, V, VI and VII) in the aglandular stomach of 32-, and of 39- to 46-day-old larvae, respectively, and five (types III, VIII, IX, X and XI) in the differentiated stomach of 55- and 60-day-old larvae. A maturation process was established for some of these cells. Types I, II and III and types IV and X were thought to be different maturational stages of the same endocrine cell type.