A. García Ayala

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 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.

An immunocytochemical and ultrastructural study of the endocrine pancreas of pseudemys scripta elegans chelonia

Immunocytochemical and ultrastructural methods have shown four cell types in the endocrine pancreas of the turtle Pseudemys scripta elegans: insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-immunoreactive cells. Each endocrine cell type was distributed differently in the duodenal or splenic regions of the turtle pancreas. Round or fusiform insulin- and glucagon-containing cells could be seen as single scattered cells which were more numerous in the duodenal regions, and the cell groups becoming progressively smaller from splenic to duodenal region. Round or fusiform somatostatin cells with thick processes and spindly pancreatic polypeptide cells with long protrusions were less numerous the nearer they were to the splenic regions; they were isolated in the duodenal zone. Insulin cells were surrounded by somatostatin cells and an outer layer of glucagon cells around the cell groups could be seen. Insulin cells were characterized by their round secretory granules which contained a polygonal, irregular or rod-shaped dense core. They also contained numerous clustered mitochondria, large multivesicular bodies, and cilium. Glucagon cells, joined by desmosomes to adjacent ones, had numerous filamentous mitochondria with longitudinal cristae and round electron-dense secretory granules with closely applied membrane. Somatostatin cells contained two kinds of secretory granules, some of which showed an electron-dense core, while others had moderately electron-dense floccular material. PP cells were characterized by round secretory granules, smaller than those of other cell types, and a large euchromatinic nucleus. Lysosomes, microtubules, bundles of microfilaments, a well-developed Golgi apparatus, and scarce rough endoplasmic reticulum were present in the cytoplasm of all these endocrine cell types.

Pancreatic endocrine cells in sea bass (Dicentrarchus labrax L.): I. Immunocytochemical characterization of glucagon- and PP-related peptides

PP-, PYY-, and glucagon-immunoreactive cells were immunocytochemically identified in the pancreatic islets of Dicentrarchus labrax (sea bass). PYY cells also reacted with anti-PP serum. The specificity control showed that preabsorption of PP antiserum by PYY peptide abolished the immunostaining, while the reaction did not change when the PYY antiserum was preabsorbed by PP. These results suggested the existence of a PP/PYY molecule in the sea bass islets. The islet distribution of PP/PYY-immunoreactive cells differed markedly. Thus, in the principal islet and some intermediate islets few PP/PYY-immunoreactive cells are present (type I islets), whereas in the smaller and some intermediate ones they are numerous (type II islets). Adjacent sections stained by peroxidase-antiperoxidase (PAP) technique and individual sections stained by immunofluorescence double staining showed the coexistence of glucagon and PP/PYY-like immunoreactivities. Both islet types contained cells with PP/PYY coexisting with glucagon peptide, while cells showing solely glucagon immunoreactivity were found in type I islets only.

Identification of mammosomatotropes, growth hormone cells and prolactin cells in the pituitary gland of the gilthead sea bream (Sparus aurata L., Teleostei) using light immunocytochemical methods: an ontogenetic study.

Growth hormone (GH) and prolactin (PRL) immunoreactivities in the adenohypophysis of Sparus aurata specimens from newly hatched until 48-months-old were detected using the peroxidase-antiperoxidase method. GH cells and PRL cells, and cells that were immunoreactive to both GH and PRL antisera, called mammosomatotropes (MS cells), were found. This is the first report on the identification of MS cells in fish, which were found in newly hatched and older larvae and juvenile specimens. GH and PRL cells appeared from two days after hatching. MS cells were first located in the central region of the adenohypophysis and afterwards in the rostral pars distalis. The GH cells were first identified in the dorsal and ventral areas of the middle-posterior part, and the PRL cells in the ventral region of the middle-anterior part. Later, during development, the sequence of appearance of the GH cells was proximal pars distalis, pars intermedia and rostral pars distalis, while for the PRL cells sequence was rostral pars distalis, proximal pars distalis and pars intermedia. This expansion pattern could be due to a GH- and PRL-cell migration although independent cell differentiation may occur in each region. The present results suggest that GH and PRL cells arise from MS cells at the outset of pituitary development, while MS cells procede from PRL cells in old larvae and juveniles.

Pancreatic endocrine cells in sea bass (Dicentrarchus labrax L.) II. Immunocytochemical study of insulin and somatostatin peptides.

Insulin (INS)- and somatostatin (SST)-immunoreactive cells were demonstrated by light immunocytochemistry in the endocrine pancreas of sea bass (Dicentrarchus labrax). INS-immunoreactive cells were identified using bovine/porcine, bonito, and salmon (s) INS antisera; the immunostaining was abolished when each antiserum was preabsorbed with its respective peptide but not with unrelated peptides. These cells also reacted with mammal (m) SST-28 (4-14) antiserum. The immunoreaction did not change when this antiserum was preabsorbed by bovine INS. INS-immunoreactive cells were located in the central part of the endocrine areas of the principal, intermediate, and small islets. Two SST-immunoreactive cell types (D1 and D2) were revealed. D1 cells, immunoreactive to SST 14 (562) and sSST-25 antisera, were located next to the glucagon-immunoreactive cells in the peripheral part of the endocrine areas. D2 cells, immunoreactive to SST-14 (562), SST-14 (566), and mSST-28 (4-14) antisera, were found in apposition to the INS-immunoreactive cells. The specificity controls showed that D1 cells expressed sSST-25-like peptides, while D2 cells might contain SST-14 and/or mSST-28-like peptides. The close topographic association between the different SST-immunoreactive cells and both glucagon- and insulin-immunoreactive cells might indicate the existence of a specific paracrine regulation of each endocrine cell type in the sea bass endocrine pancreas.

Endocrine pancreas of Testudo graeca L. (Chelonia) in summer and winter: an immunocytochemical and ultrastructural study

Insulin-, glucagon-, somatostatin-, and PP-immunoreactive cells were identified immunocytochemically using antisera raised against mammalian hormones in the pancreas of Testudo graeca in both winter and summer. The endocrine cells were present throughout the gland, forming scarce islets except in the splenic region. The insulin cell islets were larger and more numerous in the splenic region than in the duodenal one. Winter glucagon-immunoreactive cells were found mainly in isolation while the summer ones occurred in groups which showed no immunoreactive central area; in both seasons these cells were more numerous in the splenic region than in the duodenal one. Somatostatin-immunoreactive cells were found isolated or grouped together more frequently in the splenic region in the summer specimens. No PP-immunoreactive cells were found in the splenic region, although they were numerous and isolated in the duodenal zone. Four cell types (B, A, D, and PP cells) were ultrastructurally characterized by the shape, size, and electron density of their respective secretory granules. Certain ultrastructural differences were detected in the summer and winter endocrine pancreatic cells. In summer specimens a fifth cellular type was observed. The presence of B, D, and PP cells among the epithelial pancreatic duct cells may confirm the comparatively primitive organization of the T. graeca endocrine pancreas.

Somatostatin 14- and somatostatin 25-like peptides in pancreatic endocrine cells of Sparus aurata (Teleost): A light and electron microscopic immunocytochemical study

An immunocytochemical investigation demonstrates the presence of somatostatin (SST) 14- and salmon somatostatin (sSST) 25-like peptides in two populations of somatostatin (D) cells in the islets of gilthead sea bream (Sparus aurata). Both cell types were identified by their differing immunoreactivities to the somatostatin antisera used. D1 cells in the islet periphery between glucagon cells showed sSST 25-like immunoreactivity and contained large moderate to low electron-dense granules. D2 cells, present only in the central region of the islets between insulin cells, were immunoreactive to the SST 14 antisera and had smaller electron-dense granules. In S. aurata, as in other teleosts, preprosomatostatin I and II are probably synthesized and processed to SST 14- and sSST 25-like peptides, respectively, in different D cell types.

Comparative study on the endocrine cells in the pancreas of Mauremys caspica (chelonia) in summer and winter.

Four endocrine cell types were identified using peroxidase-antiperoxidase (PAP) technique and ultrastructurally characterized in the pancreas of Mauremys caspica in both winter and summer. In winter, insulin-immunoreactive cells were more abundant and the cell groups larger in the splenic than in the duodenal region, whereas in summer, medium or small cell groups were evenly distributed. Glucagon- and somatostatin-immunoreactive cells were found throughout the gland; they were more numerous in the splenic than in the duodenal region. Polypeptide pancreatic (PP)-immunoreactive cells were found only in the duodenal region. Somatostatin-immunoreactive cells were mainly isolated in winter and grouped in summer. Glucagon- and PP-immunoreactive cells had a similar arrangement in both seasons. Somatostatin- and PP-containing cells showed cytoplasmic processes and could be found next to the pancreatic ducts; the latter were also observed near insulin-immunoreactive cells. Some large secretory granules and numerous, isolated and long rough endoplasmic reticulum (RER) cisternae were seen in winter B cells; in summer B cells numerous lysosomes and few, dilated RER cisternae were found. Summer A cells showed well-developed, dilated RER cisternae and numerous vacuoles; secretory granules were more numerous in winter A cells. In winter B cells and summer A cells some nuclear filamentous inclusions were observed. Few RER cisternae were observed in winter D cells and many in summer D cells; secretory granules were found, the shape and electron density of which differed with the season. PP cells were characterized by their small secretory granules, which were less numerous in winter than in summer, being clustered at the cell pole or dispersed in the cytoplasm, respectively; in winter, the well-developed RER cisternae were dilated and irregularly distributed.

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.