Lipocortin-1 immunoreactivity in the human pituitary gland

We evaluated the distribution of lipocortin-1 immunoreactivity in 118 immature or mature human hypophyses using the peroxidase-antiperoxidase (PAP) technique with a polyclonal rabbit antiserum against lipocortin-1. Serial sections were evaluated for five pituitary hormones and S-100 protein immunoreactivity to compare their distributions with that of lipocortin-1. Scattered or moderate numbers of cells exhibited lipocortin-1 immunoreactivity in the pars distalis of 89 subjects ranging in age from 27 weeks' gestation to 83 years. Seven immature and seven aged specimens exhibited no immunostaining, while 15 specimens from older individuals exhibited only rare immunostaining. Immunostaining did not appear to co-localize selectively with any specific pituitary hormone, although the distribution of immunoreactivity did overlap that of some corticotrophs and was seen in elongated processes of S-100-containing folliculostellate cells. Lipocortin-1 was also found in epithelial cells lining colloid cysts of the residual pars intermedia in 115 of 118 pituitaries ranging in age from 23 weeks' gestation to 83 years. In many intermediate lobe cysts, lipocortin-1 exhibited a pattern of immunoreactivity that partially overlapped the distribution of S-100 protein immunostaining, although the pattern was not identical. Pre-absorption of anti-lipocortin-1 antiserum with lipocortin-1-coupled Sepharose-4B immunoreactivity resulted in loss of immunoreactivity in both lobes. No lipocortin-1 immunoreactivity was seen in the neurohypophysis.     

Localization of interleukin-18 and its receptor in somatotrophs of the bovine anterior pituitary gland

A pro-inflammatory cytokine, interleukin 18 (IL-18), induces intracellular expression of IL-1 and the release of IL-6. IL-1 and IL-6 has been detected in anterior pituitary cells, suggesting that IL-18 is produced in anterior pituitary cells and may serve to aid immuno-endocrine regulation. In the present study, we addressed this hypothesis by investigating the intracellular localization of IL-18 and its receptor in bovine anterior pituitary gland. IL-18 mRNA and its protein were detected in the anterior pituitary gland by RT-PCR and Western blotting. In situ hybridization showed that IL-18 mRNA was expressed in the anterior pituitary cells. Immunohistochemistry of IL-18 and specific hormones revealed the presence of IL-18 in somatotrophs. Furthermore, the expression of GH mRNA in IL-18 immunoreactive cells was confirmed by immuno-laser microdissection. These results first demonstrated that somatotrophs produced IL-18. Subsequently, the distribution of the IL-18 receptor alpha (IL-18Rα) was investigated in order to understand IL-18 signaling among the anterior pituitary cells. Bovine IL-18Rα cDNA was partially sequenced and detected in the anterior pituitary gland by RT-PCR. Immunohistochemistry of IL-18Rα, IL-18 and GH showed that IL-18Rα was co-localized in IL-18 immunoreactive cells or somatotrophs. These data suggest that IL-18 acts on somatotrophs as an immuno-endocrine mediator through the autocrine pathway. This work was supported by a Grant-in-Aid for Scientific Research (B) (No.13460122) from the Ministry of Education, Science and Culture of Japan     

Presence of dynorphin-like immunoreactivity in rat pituitary gland and hypothalamus

Using a highly specific and sensitive radioimmunoassay for dynorphin(1-13), dynorphin-like immunoreactivity (dynorphin-LI) was detected in rat pituitary and hypothalamus. Gel chromatographic studies on Sephadex G-50 revealed three components of dynorphin-LI with molecular weights of approximately 7500-9500 (big dynorphin), 3500-5500 (intermediate dynorphin) and 1600-1900 (small dynorphin), the latter of which eluted at the same position as authentic dynorphin contamination in porcine ACTH extracts (Sigma). Dynorphin-LI in rat anterior pituitary existed mainly as big dynorphin, whereas dynorphin-LI in rat intermediate-posterior pituitary and hypothalamus eluted mainly at the position of authentic small dynorphin.     

Localization of gonadotrophic hormones in the dog pituitary gland

Summary Using the immunoperoxidase technique and antisera to the specific beta () subunits of FSH and LH¹, selective immunochemical staining was localized mostly in the same cell type in the pars distalis and pars tuberalis of the dog pituitary gland. However, some cells were consistently shown to react solely with antisera to either LH or FSH. The cells stained for FSH were at least 1.5 times less numerous than those shown to contain LH. In the pars distalis of adult male dogs the immunoreactive gonadotrophs varied greatly in their relative proportion and were mostly shown to be much less numerous than in bitches in the anestrus phase of the sexual cycle. These cells were found to be positive to aldehyde fuchsin, alcian blue, periodic acid-Schiff (PAS) and aniline blue. The performic acid-alcian blue (pH 0.2)-PAS-orange G procedure stained the FSH/LH cells blue or turquoise, demonstrating TSH cells (blue-purple), ACTH/MSH cells (red-purple) and PRL cells (orange-red). The FSH/LH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not apply to gonadotrophic hormones, although some cells seem to be the source of either FSH or LH.Abbreviations for Pituitary Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin The authors are grateful to Dr. H. Wiemann for the statistical evaluation and to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistanceRecipient of a Research Scholarship from the Arabic Republic of Egypt     

Localization of CRF-like immunoreactivity in the brain and pituitary of teleost fish

Immunocytochemical techniques were applied to brain and pituitary sections of eleven teleost species. A corticotropin-releasing factor (CRF)-antiserum allowed the identification of a CRF-like system in these species. Perikarya were labeled in the preoptic nucleus. Labeled fibers were traced laterally, then ventrally close to the optic chiasma, forming two symmetrical tracts running through the basal hypothalamus. These ended in the rostral neurohypophysis (NH) close to ACTH cells as shown by double immunostaining. Other fibers, often more variquous, ended in the caudal NH close to melanocorticotropic cells. In Salmo fario, small perikarya also stained in the nucleus lateralis tuberis. The CRF-like system appears distinct from that of somatostatin. In Anguilla, adjacent sections stained with CRF- and vasotocin (AVT)-antisera respectively showed that these two peptides coexist in some perikarya. As few fibers containing only AVT end in the rostral NH, they probably do not control ACTH cells directly. AVT fibers terminate mostly in the caudal NH close to melanocorticotropic cells. Some extra-hypothalamic fibers suggest that CRF may also act as a neurotransmitter. The plurality of hormones showing a CRF-like activity in teleosts is considered.     

Distribution and characterization of synenkephalin immunoreactivity in the bovine brain and pituitary

The distribution of synenkephalin, the N-terminal fragment of proenkephalin, was studied in various parts of the bovine brain (globus pallidus, caudate nucleus, hypothalamus) and in the posterior pituitary by the use of a radioimmunoassay. The distribution of synenkephalin-immunoreactivity (IR) was compared to the distribution of Met-enkephalin-IR. Gel exclusion chromatography was used to examine the molecular forms of the immunoreactivities present in the tissues. The distribution of synenkephalin-IR was similar to the distribution of Met-enkephalin-IR, with a molar ratio of Met-enkephalin/synenkephalin ranging between 2.7 and 5.9. In all regions tested except the hypothalamus the synenkephalin-IR was present as a single species. However, in the hypothalamus a small amount of IR material (3% of the total synenkephalin-IR) was detected in fractions where larger Met-enkephalin-containing peptides eluted. Based on the concordance between the molar ratio of Met-enkephalin to synenkephalin found in the tissues and the molar ratio present in the sequence of adrenal proenkephalin, it is concluded that the brain and adrenal glands utilize a similar precursor for enkephalin biosynthesis.     

Localization of thyrotropin (TSH) in the dog pituitary gland

Summary Using the immunoperoxidase technique and antisera to the specific beta () subunits of bovine and rat TSH¹, selective immunocytochemical staining was localized in a specific cell population in the pars distalis of the dog pituitary gland. These TSH cells were found to be positive to aldehyde fuchsin, alcian blue, periodic acid-Schiff (PAS) and aniline blue. With the performic acidalcian blue (pH 0.2) -PAS-orange G procedure these cells stained blue-purple, demonstrating FSH/LH cells (blue or turquoise), ACTH/MSH cells (redpurple) and PRL cells (orange-red). The TSH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunocytochemical double staining. In the pars distalis of adult male dogs the TSH cells were mostly shown to be smaller in size and less numerous than in bitches in the anestrous phase of the sexual cycle. Moreover, cytological alterations in the immunoreactive thyrotrophs in the pituitary of male and female dogs generally paralleled the spontaneous changes in thyroid function associated with thyroid atrophy and/or pituitary insufficiency, and thyroid hyperplasia or goiter. In conclusion, because of their specificity and high potency, the antisera to the -subunits of bovine and rat TSH represent an effective tool for the selective immunocytochemical localization of TSH in the dog pituitary. This allows the study of the morphology and function of TSH cells under different physiological, pathological and experimental conditions.Abbreviations for Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin - TRH TSH Releasing Hormone - CG Chorionic Gonadotropin The authors are grateful to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistance     

Ornithine decarboxylase immunoreactivity in the pituitary gland. A comparative lightmicroscopical study

In the present study efforts are made to localize ornithine decarboxylase enzyme protein--the key enzyme of polyamine biosynthesis--in the adenohypophysis of different vertebrates by means of immunocytochemistry. The antigenic expression of ornithine decarboxylase was revealed in the pituitary of the clawed frog (Xenopus laevis D.), but not in rat and human adenohypophysis. The immunocytochemical results are compared with the staining pattern of the periodic acid-Schiff-reaction. No correlation between these results and the immunocytochemically obtained data has been found. Conclusions are drawn from the location of the enzyme and possible phylogenetic and humoral regulation mechanism.     

Localization of acetylcholinesterase and choline acetyltransferase in the rat pituitary gland

Summary Experiments were conducted to determine the presence of two cholinergic biomarkers, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the rat pituitary. A histochemical procedure for AChE was used to provide visualization of structures containing this enzyme. Radiochemical methods provided a sensitive assay for measuring ChAT activity. Nerve fibres staining for AChE activity were observed in the neurointermediate lobe, with the greatest concentrations appearing at the junction region with the pituitary stalk. Cells staining for AChE were found in the pars distalis and pars intermedia. ChAT activity correlated well with AChE distribution in pars nervosa and pars intermedia but not in pars distalis. The greatest levels of ChAT activity were in pars intermedia and the region where the stalk joins the pituitary. Significant values were also found for the pars nervosa. The presence of AChE and ChAT in pars intermedia and pars nervosa is evidence for a cholinergic innervation to these regions. In pars distalis, where other investigators have found muscarinic receptors, intense staining for AChE and absence of ChAT activity may indicate non-innervated, acetylcholine-sensitive sites.     

Ultrastructural evidence for endogenous testosterone immunoreactivity in the pituitary gland of the rat

Summary Several attempts have been made to localize steroids by means of immunocytological techniques. However, these methods were found inadequate for detecting steroids bound to their receptors. To localize endogenous testosterone (T) in its target cells at the ultrastructural level, an immunocytological technique was performed on ultrathin sections obtained by cryo-ultramicrotomy. T was detected in the pituitary glands obtained from intact male or female rats and castrated rats, but not in castrated + adrenalectomized rats. Animals were also injected either with testosterone, with other steroids (estradiol, progesterone, corticosterone) or with an androgen antagonist (cyproterone acetate). In addition, some ultrathin sections were preincubated either with phosphate buffers of various pH, corticosterone, cyproterone acetate solution, or with T solution. The content of T in the pituitary before and after fixation was measured by radioimmunoassay; it decreased after fixation. T immunoreactivity was localized in the gonadotropic cells only, both in the male and female rats. At the subcellular level, the immunoreactivity was detected in the cytoplasmic matrix and in the nucleus. Immunoreactive T disappeared 1) in rats after castration+adrenalectomy; by means of radioimmunoassay no T was measured in these pituitary glands; 2) in rats injected with 25 (g/rat of cyproterone acetate; 3) after preincubation of pituitary sections on a drop of cyproterone acetate (1 × 10^-6 M). The immunocytological reaction was not modified when the rats were injected with estradiol, progesterone or corticosterone (1 mg/rat), or after preincubation of the sections with corticosterone (1 × 10^-3 M), or a buffer solution at pH 7.6. Lower or higher pH values led to a strong decrease in the immunoreactivity. After injection of T (15 g/rat) the immunocytological reaction was more abundant in the nucleus and less in the cytoplasm. The immunoreactivity was again observed when the sections were preincubated with cyproterone acetate solution and then with T solution. These data suggest that T can be detected by means of immunocytochemistry. It is probably bound to a specific binding site.This work was presented in part at the VI^th International Congress on Hormonal Steroids, Jerusalem, 1982     

Identification and immunohistochemical localization of various bovine pancreatic trypsin inhibitor-isoforms in bovine pituitary gland

Summary Three isoinhibitors of bovine pancreatic trypsin inhibitor (BPTI) have been identified and isolated from bovine pituitary gland. The results of the purification process by affinity chromatography on immobilized trypsin, the electrophoretic mobility in non-denaturing conditions, the antiproteolytic activity and the immunochemical reactions indicate that these inhibitors correspond to those previously isolated from bovine spleen and lung. In addition, immunohistochemical experiments show that the isoinhibitors and BPTI are exclusively localized in the mast cells, and not in the endocrine cells, of the pars intermedia and posterior lobe (neurohypophysis) of the pituitary gland. The physiological implications of these findings are discussed.     

Localization of microfilaments in prolactin cells of the rat anterior pituitary gland

Summary Prolactin cells from anterior pituitary glands of normal non-lactating female rats, and lactating animals, some of which were separated from their pups for 48 hours, were examined ultrastructurally for the presence of microfilaments. Microfilaments were found in specific intracellular locations in all cells examined. They were in association with the nuclear envelope, the Golgi complex, the endoplasmic reticulum, small vesicles of the endoplasmic reticulum, and secretory granules. The possible role of microfilaments in the movement of intracellular organelles is considered.This investigation was supported by the National Institutes of Health grants AM 12583 and TW 02023.The authors wish to express their gratitude to Mr. M. G. Williams and Miss Pauline Cisneros for their excellent technical assistance.     

Localization of 3H-dihydrotestosterone in the pituitary gland of the rhesus monkey

Summary The uptake and retention of radiolabelled dihydrotestosterone by the pituitary gland was examined in the rhesus monkey. Two animals were given an intravenous injection of 1.0g/kg ³H-dihydrotestosterone (DHT) alone while one monkey received both the labelled androgen and 100g/kg of unlabelled steroid. One and a half hours later, they were sacrificed. The pituitary glands were removed and processed for autoradiography and immunocytochemistry. Autoradiographic localization of DHT was discernible in the partes nervosa, intermedia and distalis, albeit the highest concentration of radiolabelled cells was noted in the pars distalis. Immunocytochemical staining with antibodies to rat PRL, human TSH and ovine LH revealed a population of steroid-concentrating cells that contained TSH and a second group that contained LH. None of the cells that reacted with the anti-PRL serum were radiolabelled.     

Characterization of a thermostable LH-immunoreactive material from bovine pituitary gland

A thermostable peptide, immunologically similar to the beta-subunit of ovine lutropin, was isolated from a heated extract of bovine pituitary glands. The relative molecular mass of the peptide as well as its amino acid composition differed significantly from the lutropin subunits. Antibodies were raised to a preparation of this peptide.     

Dissociation between ACTH and beta endorphin immunoreactivity in cells of the rat pituitary gland

We have studied by immunoperoxidase histology the pituitary gland of the rat with rabbit antisera to unconjugated (human) beta endorphin and to ACTH 1–24. All of the intermediate lobe cells were positive with both antisera. ACTH- and beta endorphin-positive cells were present in the anterior lobe but there was considerable dissociation between them and the latter were more numerous. These results suggest that there can be differential processing of the 31K precursor by pituitary cells.     

3':5'-cyclic-nucleotide phosphodiesterase in the bovine pituitary gland

Cyclic-AMP phosphodiesterase activity in the homogenate of the anterior pituitary gland was 2-fold higher than that in the homogenate of the posterior pituitary, whereas cyclic-GMP phosphodiesterase activity was dominant in the posterior homogenate. There were two peaks of cyclic-AMP phosphodiesterase activity with different isoelectric points of 4.3 and 5.2. Fraction I had a molecular weight of 240 000 and a sedimentation coefficient of 6.2 S; fraction II had a molecular weight of 180 000 and a sedimentation coefficient of 3.1 S. Cyclic AMP hydrolytic activity in the supernatant of the posterior lobe corresponded to fraction I in the anterior lobe. Cyclic GMP hydrolytic activity in both the anterior and posterior lobes (activated by Ca2+/calmodulin) had an isoelectric point of 5.2, a molecular weight of 240 000 and a sedimentation coefficient of 6.2 S. Cyclic AMP and GMP hydrolytic activities in both the anterior and posterior lobes appeared in fraction I and did not separate when the preparations were mixed before electric focusing or sucrose density gradient procedures. Cyclic AMP hydrolytic activity in fraction II could be separated from cyclic GMP hydrolytic activity.     

Localization of melanin-concentrating hormone-like immunoreactivity in the brain and pituitary of the frog Rana ridibunda

The distribution of melanin-concentrating hormone (MCH) in the central nervous system of the frog Rana ridibunda was determined by the indirect immunofluorescence technique using antibodies against synthetic salmon MCH, generated in rabbits. The most prominent group of MCH-like containing perikarya was detected in the preoptic nucleus. Comparatively, a moderate number of cell bodies was observed in the dorsal infundibular nucleus and in the ventral thalamic area. Brightly immunofluorescent nerve bundles were found in the preoptic nucleus and in the ventral infundibular nucleus, coursing towards the internal zone of the median eminence and the pituitary stalk. An intense network of immunofluorescent fibers was localized in the neural lobe of the pituitary. The subcellular localization of MCH-like material was studied in the neurohypophysis using the immunogold technique. It was demonstrated that MCH-like material was contained in dense core vesicles (80–90 mm in diameter) within specific nerve terminals. The present findings indicate that, in amphibians, MCH-like peptide is located in specific hypothalamic neurons. Our data suggest that MCH may be released by neurohypophyseal nerve endings as a typical neurohormone.     

Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg²⁺-dependent, (b) Ca²⁺-dependent and (c) Mg²⁺+Na⁺+K⁺-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg²⁺+Na⁺+K⁺-dependent enzyme, whereas the Mg²⁺- and Ca²⁺-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg²⁺- and Ca²⁺-ATPases; however, the activity of the Mg²⁺+Na⁺+K⁺-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg²⁺- and Ca²⁺-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg²⁺ or Ca²⁺ and the other activated only by Ca²⁺. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.