M. P. García Hernández

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.

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.

Ontogeny of some endocrine cells of the digestive tract in sea bass (Dicentrarchus labrax) : an immunocytochemical study

Serotonin- and ten peptide-immunoreactive (IR) cell types were identified in the digestive tract of sea bass (Dicentrarchus labrax L.) larvae of four morphofunctional phases ranging in age from hatching to 61 days. The sequence of appearance and location of endocrine cells during ontogenetic development of the larvae was determined. The differentiation of endocrine cells followed a distal-proximal gradient in the gut which paralleled the morphofunctional differentiation. Serotonin-IR cells were identified in the last portion of the digestive tract from phase I onwards and in the gastric region from phase III, before these regions were morphofunctionally differentiated; met-enkephalin-IR cells were identified from phase II onwards in both the differentiated rectum and the undifferentiated intestine; cholecystokinin (CCK)- and synthetic human gastrin-34-IR cells were located only in the intestine and first found in the undifferentiated intestine of phase II; human gastrin-17-, peptide YY (PYY)- and neuropeptide Y (NPY)-IR cells appeared in the intestine from phase II and in stomach in phase IV, when it showed gastric glands; pancreatic polypeptide (PP)- and glucagon-IR cells were observed in both intestine and stomach, but insulin- and somatostatin-IR cells only in stomach, from phase III, during which the intestine but not the stomach was differentiated. PP- and PYY-, PP- and glucagon-, and PYY- and glucagon-like immunoreactivities coexisted from their first appearance in some cells of the gut.

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax)

SummaryThe development of the endocrine pancreas of the teleost sea bass (Dicentrarchus labrax, L.) was examined from hatching to 61 days, using the peroxidase-antiperoxidase technique for light microscopy. Mammalian and bonito insulin (mI and bI)-, salmo somatostatin-25 (SST-25)-, somatostatin-14 (SST-14a and b)-, glucagon-, bovine pancreatic polypeptide (PP)-, peptide tyrosine-tyrosine (PYY)- and salmo neuropeptide Y (NPY)-like immunoreactivity was demonstrated. Four ontogenetic stages were established according to the organization and immunostaining of the endocrine cells. One cell strand or primordial cord showing mI/bI- and SST-25/SST-14a-like immunoreactivity was first found at hatching in the dorsal epithelium of the anterior zone of the midgut (stage 1). One primitive islet, comprising outer SST-25/SST-14a- and inner mI/bI- and SST-14a/ SST-14b-immunoreactive cells, was found in 2- to 5-day-old larvae (stage 2). One single islet, in which glucagon-immunoreactive cells appear in the periphery, was found in larvae from 9 to 20 days after hatching (stage 3). One big islet containing, in addition, PP-immunoreactive cells in the outer region and slender cell processes which showed PYY-like immunoreactivity, was found from 25 to 61 days after hatching. During this period, primordial islets, composed of SST-25- and bI-immunoreactive cells, and clustered or isolated pancreatic endocrine cells, close to the pancreatic duct, as well as small and intermediate islets (secondary islets), in which glucagon, PP, PYY and NPY seem to be co-localized, were progressively found (stage 4). The origin of the endocrine pancreas of sea bass, and the ontogenetic and phylogenetic significance, are discussed.

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

Abstract 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.

Electron-microscopic immunocytochemical study of the endocrine pancreas of sea bass (Dicentrarchus labrax)

Insulin (B)-, somatostatin 25 (SST-25) (D1)-, somatostatin 14 (SST-14) (D2)-, glucagon (A)-, and glucagon PP/PYY/NPY (PP-like)-immunoreactive cells in islets of sea bass (Dicentrarchus labrax) were characterized according to their ultrastructure and immunogold labeling. Cells labeled with antisera to bonito and salmon insulin had numerous secretory granules with a small halo and round core, and a few with wide halo and round or crystalloid core. Gold particles were found throughout the granule in tissue labeled with the former but only in the core in tissue labeled with the latter. D1 cells had large granules with a medium electron-dense content and some with a darker core. D2 had smaller medium or high electron-dense secretory granules than D1 cells, located mainly in cell periphery. Glucagon-immunoreactive cells contained some granules with a polygonal core that was heavily labeled and other granules with a round core with no or hardly any labeling. Glucagon and PP-like immunoreactivity were co-localized in secretory granules, in which the gold particles showed no different distribution with the various antisera used. PYY-immunoreactive granules were also found in nerve endings. All the pancreatic endocrine cell types showing involutive characteristics are found.

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. II. The big and secondary islets

The big and secondary islets of sea bass larvae were characterized ultrastructurally from, 25 to 60 days after hatching. From the 25th day, big islets consisted of inner type II and III, external type I and peripheral type IV cells. From the 55th day, type V cells appeared in limited peripheral areas. Secondary islets, first found in 32-day-old larvae, were made up of inner type II and III, external type I, and peripheral either type IV and V cells (type I islets), or only type V cells (type II islets). Type I cells contained secretory granules with a fine granular, low-medium electron-dense material, whereas the secretory granules of type II cells were smaller and had a high electron-dense core with diffused limits; needle and rod-like crystalloid contents were occasionally found. Type III secretory granules posessed a homogeneous, high or medium electron-dense material with or without a clear halo. Type IV cells had secretory granules with a polygonal dense core embedded in a granular matrix and granules containing a high or medium electron-dense material. Type V cells had secretory granules with a fine granular, high or medium electron-dense content. These cell-types correlated with cells previously identified immuno-cytochemically, as regards to their distribution in the islets, and related to those characterized ultrastructurally in adult specimens. Thus, types I, II, III, IV and V correspond to D1, B, D2, A and PP cells, respectively. From the 32nd day onwards, endocrine cells of all the different types were found grouped, type V cells also being observed in isolation close to pancreatic ducts and/or blood vessels. Small groups consisting of type I and II cells were found in 40-day-old larvae. A mitotic centroacinar ductular cell containing some secretory granules similar to those of type I cells, was seen adjacent to a type I cell. As the larvae grew older, the endoplasmic reticulum developed, the number of free ribosomes decreased, and the number and size of the secretory granules increased. Dark type I, II, III, IV and V cells were found in the islets and cell clusters from the 55th day onwards.

Development of a homologous radioimmunoassay for Mediterranean yellowtail (Seriola dumerilii, Risso 1810) LH

Abstract Purified Mediterranean (M.) yellowtail luteinizing hormone-like gonadotropin (MyLH) and its β-subunit (MyLHβ) served to develop a radioimmunoassay (RIA) for MyLH. The rabbit antisera against MyLH and MyLHβ used for this purpose were tested on pituitary sections by immunocytochemistry. Anti-MyLHβ specifically detected a single type of cells, which were located at the periphery of the proximal pars distalis (PPD) and surrounding the pars intermedia (PI). Anti-MyLH, however, also recognized two other cell types, thyrotropin β-immunoreactive (ir) cells and putative follicle-stimulating hormone-like (MyFSH)-producing cells. Labeling of the two latter cell types was prevented by preabsorption of anti-MyLH with M. yellowtail pituitary glycoprotein α-subunit. The standard curve for the RIA was generated using purified MyLH, 125 I-labeled MyLHβ and anti-MyLHβ at a dilution of 1:70,000, which resulted in the binding of 30% of the tracer added. The standard curve ranged from 0.25 to 50 ng/ml. The midrange of the assay (ED50) was obtained with 5.48–7.87 ng LH/ml. The variation between assays was less than 15%. An average cross-reactivity of FSH in the LH RIA of 8.4% was found. Serial dilutions of M. yellowtail pituitary extracts displaced radiolabelled MyLHβ parallel to the MyLH standard. Application of the LH RIA to blood samples and pituitary cell culture medium provided physiological validation of the assay. Significant increases in LH levels were recorded after salmon GnRH treatments in vivo and in vitro. Serum LH levels from wild fish sampled at the spawning season were significantly higher than those from captive fish sampled in the same period.

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.

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. I. The primordial cord and the primitive, single and primordial islets

The primordial cord and the primitive, single and primordial islets present in the 3 earliest stages of the developing endocrine pancreas of sea bass were studied ultrastructurally. The primordial cord consisted of type I and II cells and was included in the gut. Besides these cell types, X cells were seen in the primitive islet. The single islet was made up of type I, II, III and IV cells. A correlation between these endocrine cell-types and cells previously identified immunocytochemically, was established. Type I, II, III and IV cells, correlated respectively with SST-25-, insulin-, SST-14- and glucagon-immunoreactive cells, and could be related to the D1, B, D2 and A cells, respectively, of older larvae and adult sea bass. Each cell type shows characteristic secretory granules from its first appearance. A progressive development of the organelles and an increase in the number and size of the secretory granules, whose ultrastructure also varied, was observed in the endocrine cells of the primordial cord and the succeeding islets. In 25-day-old larvae at the beginning of the fourth developmental stage, the primordial islet, the first ventral islet found, was close to a pancreatic duct and blood vessel, and consisted of type I and II cells whose ultrastructure was similar to that of the type I and II cells in the primordial cord. These data suggest a ductular origin for the pancreatic endocrine cells in the ventral pancreas. It is suggested that although endocrine cells undergo mitosis, their increase in number during the earliest development stages is principally due to the differentiation of surrounding cells.