The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like compounds are widely encountered toxic substances suspected of interfering with the endocrine systems of humans and wildlife, and of contributing to the loss of fertility. In this study, we determined the changes in testicular gene expression caused by in utero exposure to TCDD along with the intra-testicular testosterone levels, epididymal sperm reserves, daily sperm production (DSP) and testis histology. To this purpose, female pregnant Sprague-Dawley rats orally received TCDD (10, 100 or 200 ng/kg body weight) or vehicle at embryonic day 15, and the offspring was killed throughout development. Hepatic Cyp1a1 gene expression was measured in the offspring to confirm the exposure to TCDD. The gross histology of the testes and intra-testicular testosterone levels were normal among the studied groups. Sperm reserves were altered in 67-day-old rats of the TCDD-200 group, but not in 145-day-old animals or in the other TCDD-exposed groups. Nonetheless, fertility was not altered in males of the TCDD-200 group, and the F2 males generated had normal sperm reserves and DSP. Microarray analysis permitted the identification of eight differentially expressed genes in the 4-week-old testes of the TCDD-200 compared with that of the control group (cut-off value +/- 1.40), including the down-regulated chemokine Ccl5/Rantes. Inhibition of Ccl5/Rantes gene expression was observed throughout development in the TCDD-200 group, and at 67 and 145 days in the TCDD-100 group (animals of younger ages were not examined). Ccl5/Rantes gene expression was mostly confined in Leydig cells. F2 males generated from males of the TCDD-200 group had normal levels of Ccl5/Rantes in testis and Cyp1a1 in liver, which might indicate that Ccl5/Rantes is a marker of TCDD exposure in testis such as Cyp1a1 in liver. In conclusion, we demonstrated a decrease in Ccl5/Rantes RNA levels and a transitory decline in sperm reserves in the testes of rats of TCDD-dosed dams.

Ontogenesis of thyrotropin-releasing hormone in the human fetal pancreas. A combined radioimmunological and immunocytochemical study.

The ontogenesis of pancreatic thyrotropin-releasing hormone (TRH) in the human fetal gland was studied by radioimmunoassay or immunocytochemistry. The highest TRH concentrations (1,508.5 +/- 382.3 pg/mg wet wt) were detected between 6 and 8 wk of gestation. From 9 to 12 wk, TRH declined to 365.2 +/- 127.4 pg/mg wet wt and remained low thereafter (96.1 +/- 28.9 pg/mg wet wt). The immunocytochemical procedure was performed on semithin and thin sections from 12- to 19-wk-old human fetuses. At the light microscope level, TRH was found interspersed among the islet cell clusters (12 wk), and later (16 wk) inside the typical islets of Langerhans. Consecutive semithin sections treated by TRH and insulin antisera showed the same immunoreactive cells. Electron microscopy showed TRH in B cell secretory granules. These results are consistent with an eventual implication of TRH in the endocrine regulation of metabolism or in the fetal development of pancreas.

Histidyl-proline diketopiperazine (His-Pro DKP) immunoreactivity is present in the glucagon-containing cells of the human fetal pancreas.

Histidyl-proline diketopiperazine (His-Pro DKP) cells in the pancreas of human fetuses aged between 12 and 19 wk were localized by the indirect antibody-enzyme method on semithin sections. To study their fine structure, two techniques were used: a superimposition technique consisting of comparison of the same cells in semithin and electron microscopic preparations, and an immunocytochemical technique on ultrathin sections using the unlabeled antibody peroxidase-antiperoxidase method. Our results show that (a) the same cells are positive for both His-Pro DKP and glucagon/glicentin, (b) His-Pro DKP immunoreactive cells possess extremely electron-opaque secretory granules, implying that these cells correspond to the A cells, and (c) His-Pro DKP immunoreactivity is found over the secretory granules. We hypothesize that the two peptides His-Pro DKP and thyrotropin-releasing hormone (TRH) have independent origins, since TRH is found in the B cells.

Processing of thyrotropin-releasing hormone prohormone (pro-TRH) in the adult rat pancreas: identification and localization of pro-TRH-related peptides in beta-cells of pancreatic islets.

Rat TRH prohormone (pro-TRH) contains five separate copies of the TRH progenitor sequence, Gln-His-Pro-Gly. All five sequences are flanked by paired basic amino acid cleavage sites and linked together by connecting sequences. RIAs to synthetic TRH and prepro-TRH-(178-199) were used to investigate pro-TRH processing in the endocrine pancreas of adult rats. HPLC analysis of adult rat pancreatic extracts showed the presence of a major immunoreactive peptide eluting at the position of prepro-TRH-(178-199). An additional peak coeluting with [less than Glu172]prepro-TRH-(172-199) (less than Glu = pyroglutamyl) revealed the presence of a C-terminally extended form of TRH. Quantification of TRH in pancreatic extracts indicated the presence of 22 mol TRH/mol prepro-TRH-(178-199) and 17 mol TRH/mol [less than Glu172]prepro-TRH-(172-199). Treatment of rats with streptozotocin markedly reduced the pancreatic content of both immunoreactive TRH (-84%) and immunoreactive prepro-TRH-(178-199) (-62%). Light microscopic immunocytochemistry showed that prepro-TRH-(178-199)-like immunoreactivity was exclusively located within insulin-containing cells of the pancreatic islets. At the electron microscopic level, prepro-TRH-(178-199) immunoreactivity appeared to be concentrated in secretory granules. The present study demonstrates that processing of pro-TRH generates both non-TRH- and TRH-related peptides in the adult rat pancreas. Our data also indicate that beta-cells of the endocrine pancreas are the major source of TRH- and pro-TRH-derived peptides.

Persistence of peptidylglycine alpha-amidating monooxygenase activity and elevated thyrotropin-releasing hormone concentrations in fetal rat islets in culture.

Two experimental systems were used to investigate the relationship between TRH content and peptidylglycine alpha-amidating monooxygenase (PAMase) activity of the neonatal rat pancreatic islets: freshly isolated islets from rats aged 1-14 days, and fetal islets maintained in culture for 3 weeks. TRH was present in freshly isolated islets and in newly formed fetal islets in culture. However, while the TRH concentration in freshly isolated islets measured by RIA followed the same ontogenic pattern as that in the total pancreas, peaking during the first week of life (78 pg/micrograms DNA on day 4) and decreasing thereafter to reach 4 pg/micrograms DNA on day 14, the TRH content of fetal islets in culture did not decrease with time (65 pg/micrograms DNA on day 1 and 80 pg/micrograms DNA after 3 weeks in culture). Similarly, using immunocytochemistry, immunoreactive cells were only seen at day 2 in freshly isolated islets. In contrast, in fetal islets, TRH cells were present throughout the culture period. In both experimental systems, TRH was localized in beta-cells. PAMase activity paralleled TRH concentration, peaking during the first week of life in freshly isolated islets and remaining unchanged in the fetal islets in culture. PAMase activity is, therefore, present in the endocrine pancreas. The results suggest that PAMase activity is a rate-limiting step in TRH biosynthesis in this tissue.

Sorbin in the porcine gastrointestinal tract and pancreas: an immunocytochemical analysis.

Sorbin is a 153-amino acid peptide that was initially discovered in the porcine duodenum. We have reported previously that this peptide regulates intestinal electrolyte transport and have described accumulation sites in the rat digestive tract. In the present study, we investigated the anatomical distribution and the site(s) of sorbin production in the porcine digestive tract using immunocytochemistry. The use of polyclonal antisera, which by cross-reaction studies were shown to be specific for different regions of the molecule, revealed a diversified distribution. Sorbin predominated in endocrine cells preferentially localized in the pyloric glands, duodenal crypts of Lieberkühn, and pancreatic islets; in the gastrointestinal tract, sorbin coexisted with Met-enkephalin or with substance P in a small fraction of serotonin-storing [enterochromaffin (ED)] cells, i.e. EC2 cells and EC1 cells, respectively; in the pancreas, sorbin coexisted with insulin in the beta-cells, also considered as serotonin-storing cells in the pig, and with EC cells in the exocrine pancreas. An enteric neuronal system containing sorbin was also reported. Our results demonstrate that sorbin is a component of the serotonin-storing cell type in the porcine gastrointestinal tract and pancreas, and suggest potential directions to investigate the functions of this new regulatory peptide.

Expression and regulation of transforming growth factor-beta 1 messenger ribonucleic acid and protein in cultured porcine Leydig and Sertoli cells.

In the present work, the expression and secretion of transforming growth factor-beta 1 (TGF beta 1) by immature pig Leydig and Sertoli cells were investigated. Both cell types express two TGF beta 1 mRNA transcripts of 2.5 and 3.5 kilobases, and the levels were 2.6-fold higher in Leydig than in Sertoli cells. In the latter cells, mRNA levels were enhanced when cultured cells were stimulated by epidermal growth factor and phorbol ester (4-beta-phorbol 12-myristate 13-acetate) and significantly decreased by FSH and testosterone. Using a polyclonal antibody raised against a synthetic peptide that corresponded to the carboxyl-terminal region of TGF beta 1 and recognized this peptide, but not TGF beta 2 or TGF beta 3, specific immunostaining of both Leydig and Sertoli cells was demonstrated in situ, after cell isolation, and during culture. The immunostaining was more marked in Leydig cells than in Sertoli cells. Western blot analysis of Leydig or Sertoli cell-conditioned medium demonstrated a band of 25 kilodaltons, which was shifted to 12.5 kilodaltons under reducing conditions. Using the mink lung epithelial cell bioassay for TGF beta 1, we could demonstrate the presence of TGF beta 1-like activity in Leydig and Sertoli cell-conditioned media after acid treatment, but not before activation. The inhibitory effects of both pure TGF beta 1 and acidified conditioned medium were almost completely blunted by the TGF beta 1 antibody. The amounts of TGF beta 1 secreted by Sertoli and Leydig cells were not significantly different and varied between 400-800 pg/48 h.10(6) cells. These studies demonstrate for the first time that both pig Leydig and Sertoli cells express TGF beta 1 mRNA, and the TGF beta 1-like activity secreted by these cells corresponds to TGF beta 1. As TGF beta 1 has been demonstrated to have strong effects on testicular cells, in particular on Leydig cell functions, it is suggested that local secreted TGF beta 1 may play a role in the autocrine/paracrine regulation of testicular functions.

ACTH and angiotensin II regulation of insulin-like growth factor-I and its binding proteins in cultured bovine adrenal cells.

Insulin-like growth factor-I (IGF-I) is required for the maintenance of differentiated functions of bovine adrenal fasciculata cells in culture. We have investigated, by immunocytochemistry, the presence of IGF-I in cells cultured in the absence or presence of ACTH and angiotensin II (AII), as well as the secretion of IGF-I and its binding proteins (IGFBPs). In control cultures, very few cells were specifically stained with the anti-IGF-I serum. Following 2 days of treatment with AII (1 microM) or ACTH (10 nM) the number of stained cells increased by 5- and 14-fold respectively. In all cases the staining was specific, since it was abolished when non-immune rabbit serum replaced the anti-IGF serum or when the anti-IGF-I serum was preincubated with saturating concentrations of the peptide. Under the same experimental conditions the secretion of IGF-I into the medium, evaluated by a specific radioimmunoassay, was increased two- and sevenfold by AII and ACTH respectively. Using the method of Western ligand blotting, the major form of IGFBP secreted by control adrenal cells was found to be a 38-42 kDa doublet protein. Two minor forms with apparent molecular weights of 28-31 kDa and 24 kDa have also been identified. Following acid-ethanol extraction of the conditioned medium, all the IGFBPs were recovered in the pellet, whereas most of the IGF-I was in the supernatant. ACTH and, to a lesser extent, AII pretreatment increased the 38-42 kDa IGFBP by several fold, decreased the 28-31 kDa IGFBP and had no effect on the 24 kDa IGFBP. In conclusion, these results demonstrate (i) that bovine adrenal cells contain IGF-I-like immunoreactive material, (ii) that the stimulatory effects of ACTH and AII on IGF-I secretion by bovine adrenal cells are due mainly to an increase in the number of IGF-I-producing cells and (iii) that ACTH and AII modulate the secretion of IGFBP by adrenal cells. Although the roles of IGFBPs have not been defined in adrenal cells, they are capable of modulating the biological action of IGFs in other cell cultures. Regulation of both IGF-I and its binding proteins by the two specific hormones ACTH and AII suggests important roles for these binding proteins in modulating the action of IGF-I in bovine adrenal cell function.

Anatomical distribution of somatostatin immunoreactivity in the infant brainstem.

The distribution of somatostatin-immunoreactive structures in the infant brainstem was investigated using the peroxidase-antiperoxidase technique. A wide distribution of somatostatin-immunoreactive cell bodies and fibers was observed throughout the brainstem. Numerous somatostatin-immunoreactive cell bodies and fibers were present in several areas of the brainstem including the substantia grisea centralis and the reticular formation. Some immunoreactive cell bodies were seen in cranial nerve nuclei such as the nucleus praepositus, the nucleus nervi hypoglossi and the vestibular nuclei. Immunoreactive fibers were seen in the nucleus cuneatus, the locus coeruleus, the nucleus tractus solitarius, the nucleus ambiguus, the nucleus tractus spinalis nervi trigemini and the dorsal horn of the spinal cord. These data were in agreement with previous works on the human adult. However, a high density of somatostatin-immunoreactive cell bodies and fibers in the interpeduncular nucleus and in the nucleus centralis superior, and a dense network of somatostatin-immunoreactive fibers in the dorsal part of the nucleus inferior olivarius, were also observed. The role of somatostatin in some brainstem nuclei and its probable implication in some specific neuropathological diseases of the infant brainstem is discussed.

Overexpression of a dominant-negative type II TGFbeta receptor tagged with green fluorescent protein inhibits the effects of TGFbeta on cell growth and gene expression of mouse adrenal tumor cell line Y-1 and enhances cell tumorigenicity.

Transforming growth factor beta (TGFbeta) has been reported to be a potent growth inhibitor of epithelial cells. The purpose of the present work was to study in vitro and in vivo the effects of overexpression of a dominant-negative type II TGFbeta receptor on the proliferation and differentiation of Y-1 cells. Stable transfections were performed with a mutant TbetaRII (TbetaRII-KR) fused with the Enhanced Fluorescent Green Protein (EGFP). The expression of this fusion protein and its overexpression were demonstrated by northern blot and immunoblot with EGFP and TbetaRII probes and antibodies respectively. The membrane localization of this fusion protein was confirmed by confocal microscopy. The functionality of this fusion protein was demonstrated by its blocking effects on TGFbeta action on DNA synthesis and on Y-1 expression of steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Moreover, in nude mice the tumorigenicity of cells stably transfected with the fusion protein was higher than that of cells stably transfected with EGFP alone. Taken together, the present results show that TbetaRII-KR/EGFP blocks the effects of TGFbeta1 on Y-1 cells and acts as a potent dominant-negative receptor preventing TGFbeta signaling.

Immunocytochemical evidence for a substance related to the bovine pancreatic polypeptide-peptide YY group of peptides in the human fetal gastrointestinal tract.

The time of the first appearance and distribution of substance(s) reacting with the bovine pancreatic polypeptide (BPP) antiserum No. 146-6, i.e., BPP-like immunoreactivity, were studied in the gastrointestinal tract of 5-24-week-old human fetuses using an indirect immunoperoxidase method. The first immunostaining was identified at the 12th week of gestation in the oxyntic and colonic mucosa, and at the 10th week in the ileum. Serial sections alternately labelled with BPP and glicentin (GLI-1) antisera show several patterns. In the enteric and oxyntic mucosa, there is a cell population reacting only with the GLI-1 antiserum intermixed with cells containing both BPP-like and GLI-1-like immunoreactivities. In the oxyntic mucosa, however, certain cells might store BPP-like material only. Specificity tests illustrate cross-reactivity occurring in immunocytochemical studies of extrapancreatic BPP. The ability of synthetic BPP or a chemically related peptide, peptide YY to abolish the BPP antiserum immunoreaction, as well as previous radioimmunoassay data, raise the question of the presence of authentic BPP in GLI-1-containing cells.

Ontogeny of immunoreactive glicentin in the human gastrointestinal tract and endocrine pancreas.

The gestational time of appearance and distribution of immunoreactive glicentin was compared to that of immunoreactive glucagon in the gastrointestinal tract and endocrine pancreas of human fetuses, aged between 5 and 24 weeks, by an indirect immunoperoxidase method. With the glicentin antiserum No. R 64, the first immunoreactive cells were detected at the 10th week of gestation in the oxyntic mucosa and proximal small intestine, at the 8th week in the ileum and at the 12th week in the colon. In the endocrine pancreas, the first immunoreactive cells were observed as early as 8 weeks within the walls of the primitive pancreatic ductules. At a more advanced stage of development (12 weeks), they were found interspersed among the islet cell clusters and still later (16 weeks) inside the recognizable islets of Langerhans. With the glucagon antiserum No. GB 5667, no immunoreactive cells were demonstrated in the gastrointestinal tract whatever the age of the fetuses. In the endocrine pancreas, the first immunoreactive cells were observed at the 8th week of gestation in the pancreatic parenchyma. The distribution of glucagon-containing cells in the pancreas was similar to that of glicentin immunoreactivity throughout ontogenesis. In the pancreatic islets of one 18-week-old human fetus, the study of consecutive semithin sections treated by both antisera showed that the same cells were labelled. The significance of these findings concerning the role of glicentin as a glucagon precursor is discussed.

Immunocytochemical evidence for the presence of S-100 protein in insulin-containing cells of cultured fetal rat islets.

S-100 protein was long considered to be specific to glial and Schwann cells, but was subsequently proved to be present in various organs. In particular, S-100 proteinimmunoreactivity was demonstrated in the parathyroid gland, adenohypophysis and endocrine pancreas. In the present study cultured fetal rat islets were investigated in view of the possible presence of S-100 protein immunoreactivity in their cells. In the initial 5-day period, continuity between islets and ducts could be demonstrated, and the islets appeared to bud from the ducts. During this time, S-100 protein-immunoreactive cells were found in either the budding islets or ducts. The colocalization of S-100 protein and insulin was demonstrated immunocytochemically. In contrast, the newly formed islets from endocrine monolayers did not display S-100 protein immunoreactivity. After this initial period, numerous free-floating islets were observed, but only some of them contained S-100 protein immunoreactivity. S-100 protein-immunoreactive cells had the same distribution as those storing insulin, again suggesting the coexistence of the two peptides. The results suggest that S-100 protein might be involved in the regulation of islet function.

Immunocytochemical location of thyrotropin-releasing hormone (TRH) in the B-cells of adult hypothyroid rat pancreas.

Thyrotropin-releasing hormone (TRH) is present in small quantities in the rat adult pancreas. As hypothyroidism increases dramatically the pancreatic content of this peptide, this model was used to localize TRH in the gland by immunocytochemistry. Immunocytochemical staining of semithin (0.5-1.0 micron) and thin (golden) sections was performed as well as antibody and method controls to check the specificity of the immunoperoxidase staining. At the light microscope level, a very faint TRH-like immunoreactivity was apparent in the pancreas of normal untreated animals. In hypothyroid rats, a strong TRH immunostaining was observed in the central portion of the islets of Langerhans. On the contrary, in previously hypothyroid rats made euthyroid, no TRH-like immunoreactivity was found. Serial sections alternately labelled with TRH and insulin antisera revealed the simultaneous occurrence of both immunoreactivities. In addition, the TRH immunoreactive cells were distinct from glucagon- or somatostatin-containing cells. At the electron microscope level, immunoreactive TRH was found over the secretory granules of insulin-containing cells. Hypothyroid animals offer therefore a suitable model for the study of TRH in the pancreas.

Anatomical distribution of LHRH-immunoreactive neurons in the human infant hypothalamus and extrahypothalamic regions.

The morphological features and distribution of luteinizing hormone-releasing hormone (LHRH)-immunoreactive cell bodies and fibers of the hypothalamic and the neighboring mesencephalic regions were studied in the normal newborn infant by immunohistochemistry. Within the hypothalamus, numerous LHRH-immunoreactive like (IL) cell bodies were found mainly in the ventral portion of the infundibular nucleus close to the median eminence and at a lower extent in the medial preoptic area. In addition, sparse immunoreactive cell bodies were displayed in the paraventricular and medial mammillary nuclei. The mesencephalon also exhibited rare immunoreactive cell bodies in the periaqueductal gray. LHRH-IL fibers, predominantly varicose, formed a continuum from the septo-preoptico level to the mesencephalon. In the hypothalamus, the median eminence exhibited the highest LHRH innervation. LHRH-IL fibers are also observed in the lamina terminalis, the medial preoptic area, the suprachiasmatic, the supraoptic, the peri- and the paraventricular nuclei. In the last two nuclei, some fibers projected to the dorsomedial and ventromedial nuclei whereas others were in close relation with the ependyma. The mesencephalon displayed low LHRH-IL fibers, present essentially in the raphe and interpeduncular nuclei and around the ependyma. When compared with data obtained in other mammals, the present findings agree well with the general distribution and morphological features of LHRH-IL neuronal structures reported elsewhere.

Immunohistochemical distribution of somatostatin in the infant hypothalamus.

Somatostatin (SS)-containing neurons were mapped in the normal infant hypothalamus with immunohistochemistry, using the peroxidase anti-peroxidase technique. Neurons displaying SS immunoreactivity show a widespread distribution throughout the hypothalamic region. Principal SS-immunoreactive like (SS-IL) perikarya are located in the paraventricular, infundibular and posterior nuclei and in the preoptic region. High SS innervation is also found in the ventromedial and in the lateral mammillary nuclei, and in the median eminence. In general this distribution of SS-IL agrees well with that reported for rat. Compared to the immunohistochemical distribution of SS in human adult hypothalamus, this mapping in the infant hypothalamus is grossly similar. However some differences may be underlined: the presence of a moderately dense group of SS-IL perikarya in the tuberal and posterior nuclei, and a dense innervation of the ventromedial nucleus and in the median eminence. This first detailed distribution of SS immunoreactivity in infant hypothalamus can provide basic knowledge for further studies of infant neuropathology.

Anatomical distribution of substance P-immunoreactive neurons in human brainstem during the first postnatal year.

The localization of substance P (SP)-immunoreactive structures in the infant brainstem was investigated by immunohistochemistry using the peroxidase antiperoxidase technique. SP-immunoreactive structures are widely distributed throughout the brainstem region. SP-immunoreactive cell bodies are prominent in the superior colliculis, the central grey, the nucleus tractus solitarius and the reticular formation. A high density of SP-immunoreactive fibers is found in the nucleus tractus solitarius, the trigeminal nucleus and the dorsal horn of the spinal cord. Large SP-immunoreactive fibers are seen in the substantia nigra. In the present study, we also investigated the development of substance P-immunoreactive fibers in the infant brainstem during the first postnatal year. We note a qualitative increase in the density of SP-immunoreactivity in some brainstem regions such as colliculus superior and substantia nigra with respect to age.

[Demonstration of the secretion of IGF-I and of its binding proteins by bovine adrenal fasciculata cells in culture. Immunocytochemistry, radioimmunoassay and ligand blotting]. / Mise en évidence de la sécrétion d'IGF-I et de ses protéines de liaison par les cellules fasciculées du cortex surrénalien bovin en culture. Immunocytochimie, dosage radioimmunologique et "ligand blotting".

Insulin-like growth factor I (IGF-I) is required for the maintenance of differentiated functions of bovine adrenal fasciculata cells in culture. We have investigated, by immunocytochemistry, the presence of IGF-I in cells cultured in the absence or presence of corticotropin (ACTH) and angiotensin II (A-II), as well as the secretion of IFG-I and its binding proteins (IGF-BP). In control cultures, very few cells were specifically stained with the anti-IGF-I serum. Following 2 days treatment with A-II (10(-6)M) or ACTH (10(-8)M) the number of stained cells increased 5 and 14 fold, respectively. In all cases the staining was specific since it was abolished when non-immune rabbit serum replaced the anti-IGF serum or when the anti-IGF-I serum was preincubated with saturating concentrations of the peptide. Under the same experimental conditions the secretion of IGF-I in the medium, evaluated by a specific radioimmunoassay, was increased 2- and 7-fold by A-II and ACTH, respectively. Using the method of western ligand blot, we found that the major form of IGF-BP secreted by control adrenal cells is a 38-42 kDa doublet protein. Two minor forms with apparent mol wt of 28-31 kDa and 24 kDa have also been identified. Following acid-ethanol extraction of the conditioned medium all the IGF-BP were recovered in the pellet, whereas most of the IGF-I was in the supernatant. ACTH and, to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Light and electron microscopic immunocytochemical evidence that delta sleep-inducing peptide and gonadotropin-releasing hormone are coexpressed in the same nerve structures in the guinea pig median eminence.

The relationships between delta sleep-inducing peptide (DSIP) and GnRH immunoreactivity within the guinea pig median eminence are investigated by light and electron microscopic immunocytochemistry. Indirect immunofluorescence and elution-restaining experiments show that at the light microscopic level the distribution patterns of DSIP and GnRH immunoreactivity are indistinguishable. This suggested the possible coexistence of both immunoreactivities within the same fibers and neurosecretory endings. At the electron microscopic level, a preembedding double-immunolabeling technique using both indirect immunoperoxidase and immunogold methods, clearly indicate that DSIP and GnRH immunoreactivity are frequently colocalized within single secretory granules. In addition DSIP/GnRH immunoreactive nerve endings were also observed often in close proximity to tanycyte elements. Taken together, the present results provide for the first time ultrastructural evidence for the presence of DSIP immunoreactivity and demonstrate that DSIP and GnRH immunoreactivities may be coexpressed within the same neuronal elements in the median eminence.

Coexistence of thyrotropin-releasing hormone and insulin in cultured fetal rat islets: a light and electron microscopic immunocytochemical study during islet neoformation.

Pancreatic thyrotropin-releasing hormone (TRH) is without doubt localized in the insulin-containing beta-cells. A previous study reported cellular continuity between beta-cells and ducts in cultured fetal rat islets, but it is not known whether these insulin-containing beta-cells form a cell type that is different from the TRH cells producing insulin. On the other hand, the subcellular coexistence of both peptides as yet remains unresolved. To overcome these problems the present study was conducted, using light microscopic immunocytochemistry, to verify the cellular distribution of TRH in cultured fetal rat islets with particular regard to the interrelationship between beta-cells and ducts, and using electron microscopic double labeling cytochemistry, to study the subcellular distribution of TRH and insulin. Our data show that both TRH and insulin are expressed in the same cells during islet cell neogenesis, and are localized in the same secretory granules. No topographic segregation of their respective immunoreactive moieties are seen within the secretory granule.