Ontogenesis of cells producing polypeptide hormones (ACTH,MSH, LPH,GH, Prolactin) in the fetal hypophysis of the rat: Influence of the hypothalamus

Summary The ontogenesis of cells containing polypeptide hormones (ACTH, MSH, LPH, GH and Prolactin) was investigated in the fetal rat hypophysis by immunohistochemistry using the peroxidase-antiperoxidase complex.Corticotrophs, melanotrophs and lipotropic cells were revealed earlier in the pars distalis than in the pars intermedia. In the pars distalis, cells producing LPH were found in the morning of day 15 of gestation using anti-- or anti--LPH sera, and in the afternoon using anti-- or -endorphin sera. Cells containing -MSH were observed from the afternoon of day 15. The cells stainable with the anti--MSH, anti--(17-39)ACTH and anti--(l-24)ACTH sera appeared on day 16. In the pars intermedia, the cells producing -MSH, MSH, - and -endorphin, and -LPH were observed in the morning of day 17, while cells containing ACTH were only revealed in the afternoon of the same day of gestation. Based on the treatment of serial paraffin sections with various antisera, it was clearly shown that MSH, ACTH, and LPH occur in the same cells located in the pars distalis as in the pars intermedia.The development of the corticotrophs, melanotrophs and lipotropic cells does not require the presence of the fetal hypothalamus or other central nervous structures. The pituitary glands of 21 day-old fetuses encephalectomized on day 16 showed as many reactive cells as those of the littermate controls.The somatotrophs were first revealed in the pars distalis in the afternoon of day 19. The cells producing prolactin were not observed before day 21 of gestation. On some cases GH and prolactin were found together in one cell. The cytodifferentiation of GH and prolactin cells is apparently not under hypothalamic control.     

Human foetal anterior pituitary immunofluorescent evidence for corticotropin and melanotropin activities

Summary Histological sections of seven to twenty-four-week-old human foetal anterior pituitaries were studied. The exploration was done by immunofluorescent methods with rabbit anti--(17–39) ACTH, anti--(1–24) corticotropin, anti--MSH, anti--MSH, anti--LPH, anti--(1–58) LPH, anti--(1–46) LPH serums and fluorescent sheep anti-rabbit . globulines. Corticotropin activity was observed to occur early on each side of Rathke's pouch in the youngest foetus, but a specific fluorescence occured only when using the antiserum to -(17–39) ACTH. At later stages, only corticotropin and -melanotropin activities were revealed in the human foetal anterior pituitary. These activities were located in the same cells. There was no fluorescence with the other sera. The specificity of each serum was determined by studying each serum against homologous and heterologous antigens. Cytological studies showed that these fluorescent cells are chromophobes. A possible participation by the human foetal anterior pituitary in the development and function of foetal adrenal zone is discussed.We would like to thank Professors M. Dumont and Garmier whose kind cooperation allowed us to carry out the rapid removals of hypophyses necessary for these experiments. We also thank Dr. E. Rabe for the translation.     

Concanavalin a binding and cytodifferentiation in pancreatic acinar carcinoma of rat

The binding of concanavalin A to the plasmalemma of acinar carcinoma cells was characterized by electron microscopy utilizing horseradish peroxidase. Heavy labeling due to specific concanavalin A binding was detected on the plasmalemma of undifferentiated carcinoma cells lacking zymogen maturation, neoplastic cells of intermediate differentiation with only occasional zymogen granules, and highly differentiated acinar carcinoma cells containing numerous cytoplasmic zymogen granules. The plasmalemma of acinar carcinoma cells was also compared to the normal pancreatic acinar cell plasmalemma by measurement of specific ¹²⁵I-labeled concanavalin A binding. Although only about one-third of pancreatic acinar carcinoma cells demonstrate mature zymogen differentiation, the acinar carcinoma had a full complement of normal plasmalemma receptors for ¹²⁵I-labeled concanavalin A. It is concluded that, unlike normal pancreas, the presence of concanavalin A receptors on the plasmalemma of acinar carcinoma cells is not a specific membrane marker for differentiated cells containing zymogen granules.     

Embryology and cytodifferentiation of the pituitary gland in the lizard Anolis carolinensis

Anolis embryos have limb buds at the time eggs are laid and require about 39 days to complete development at 28°C. Rathke's pouch is present at five days, and the subdivisions of the adenohypophysis are differentiated by ten days after oviposition. The cells of the rostral half of the pars distalis (PD) are derived from the anterior face of Rathke's pouch; cells of the caudal half from the posterior face. Lateral lobe cells differentiate on the lateral margins of the developing caudal PD, and knob-like outgrowths of this tissue attach to the walls of the diencephalon to form the pars tuberalis (PT). Subsequently, the cells of the PT lose their connection with the PD and become a pair of flattened oblong plaques. They reach maximal size in midincubation, and are gradually invaded by nervous elements and incorporated into the walls of the hypothalamus. Electron micrographs demonstrate that the embryonic PT is secretory. Ultrastructurally the pars intermedia (PI) and PD are composed of parenchymous secretory cells in a framework of stellate cells. Stellate cells surround the lumen of Rathke's pouch and are connected laterally by complex junctions that exclude the secretory cells from the luminal surface. They extend in sheet-like processes among the secretory cells to the outer margin of the gland where they form a partial sheath within the basal lamina around the secretory tissue. As development proceeds, the lumen becomes subdivided and the resulting reduced lumina are recognizable as the forerunners of the follicles of the adult adenohypophysis. The cells of the PI are differentiated into secretory or stellate cells halfway through incubation. At this time only half of the cells of the PD can be so classified. Four of the five granulated cell types described in the adult are recognizable by mid-incubation; the fifth cell type (prolactin cell) becomes distinguishable within ten days thereafter, and at hatching appears to be actively synthesizing secretory products.     

An alternative pathway for clearance of dead lactotropes from rat pituitary gland

In the pituitary glands of lactating rats there is a striking proliferation of lactotropes during pregnancy and lactation. After the cessation of lactation, the population of lactotropes experiences an extensive programmed cell death of redundant cells until it reaches prepregnancy levels. The regressive changes occurring in lactotropes are autolytic in nature and usually these cells are engulfed by stellate cells. Other involuting lactotropes are mobilized toward the blood capillaries and some go into the capillary lumen for final disposal, presumably in the mononuclear phagocyte system. In this study we report a sequence of events occurring in regressing lactotropes, investigated by a systematic electron microscope analysis of the rat pituitary gland at different periods after interruption of lactation. Direct disposal of degenerating lactotropes and their cell remnants appears to be an alternative pathway for the clearance of the regressing cells that occur extensively among surplus lactotropes following ablactation.     

Growth hormone-releasing peptide-2 stimulates secretion and synthesis of adrenocorticotropic hormone in mouse pituitary

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides which induce strong GH release in both animals and humans. Among them, GHRP-2 is known to stimulate GH release by acting at both hypothalamic and pituitary sites, but also induces adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRP-2 may stimulate ACTH release directly via GHRP receptor type 1a in ACTH-producing tumors. GHRP-2 increases ACTH secretion in rat in vivo, but not ACTH release from rat primary pituitary cells. In the present study, in order to elucidate the mechanism underlying ACTH secretion by GHRPs, mouse pituitary cells were stimulated by GHRP-2. GHRP receptor mRNA was expressed in the mouse pituitary, and GHRP-2 directly stimulated secretion and synthesis of ACTH in the mouse anterior pituitary cells. GHRP-2 increased intracellular cyclic AMP production. H89, a potent protein kinase A (PKA) inhibitor, and bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, inhibited the GHRP-2-induced ACTH release, and that H89, but not bisindolylmaleimide I, inhibited the GHRP-2-induced proopiomelanocortin mRNA levels. Together, the GHRP-2-induced ACTH release was regulated via both PKA and PKC pathways in the mouse pituitary cells, while ACTH was synthesized by GHRP-2 only via the PKA pathway.     

Evidence that lipolytic peptide B occurs in the ACTH/MSH-cells of the pituitary and in the brain

Summary Several lipid-mobilizing peptides occur in the pituitary, among them -lipotropin and lipolytic peptide A and peptide B. The latter two peptides are distinct from -lipotropin and appear to be chemically related to the neurophysins. Immunohistochemistry has now revealed that the lipolytic peptide B of the pituitary is localized in the ACTH- and MSH-cells. In addition, immunoreactive peptide B was found in axons of the posterior lobe of the pituitary. Immunoreactive peptide B was found also in nerve fibers and nerve cell bodies in the hypothalamus, particularly in the hypothalamo-hypophyseal tract and in the magnocellular neuronal system. Immunoreactive nerve fibers were numerous also in the periventricular nucleus of the thalamus. The antiserum against peptide B cross-reacts with neurophysin I, and hence, it cannot be excluded that at least part of the immunostaining in the brain reflects the presence of the latter component. However, the regional distribution of immunoreactive peptide B and neurophysin was not identical. Therefore, it is possible that authentic peptide B occurs not only in the pituitary but also in the brain.     

The rostral pars distalis of the pituitary gland of the freshwater and marine alewife (Alosa pseudoharengus)

Summary In the alewife the orohypophyseal duct, a remnant of Rathke's pouch, persists in adults as a tube passing from the rostral pars distalis to the pharyngeal region. Its lumen is not open to the buccal cavity. The prolactin cells are situated around the bifurcations of this duct in the rostral pars distalis. Contents from prolactin cells, such as granules, nuclei, mitochondria and Golgi structures were found in these bifurcations. These contents were indistinguishable from those of intact prolactin cells. Evidence of actual release into the duct was often noted. At the presumptive point of release, the cells lining the lumen separate and the contents, probably of an entire prolactin cell, are extruded. The cilia usually found at the point of extrusion arise from prolactin cells. The prolactin cells of freshwater fish were more heavily granulated than those from a marine environment. Prolactin cells of fish entering freshwater streams were not heavily granulated but showed evidence of increased activity. Granule size was not affected by salinity. The ACTH cells are arranged in bands along branches of the neurohypophysis in the rostral pars distalis. No differences in ACTH cells from fish of different salinities were noted.We would like to thank Mr. D. D. Zumwalt of the John G. Shedd Aquarium in Chicago, Dr. E. D. Warner, Mr. R. L. Flayter, Dr. J. G. Stanley of the University of Wisconsin, Milwaukee, Mr. L. Wells of the U. S. Fish and Wildlife Service, Great Lakes Fisheries Laboratory, Ann Arbor, and Mr. L. N. Flagg of the Department of Sea and Shore Fisheries, Augusta, Maine, for their assistance in obtaining the fish used in this study. Dr. T. N. Tahmisian and Mr. G. T. Chubb of Argonne National Laboratories, and Dr. L. M. Srivastava and Dr. V. Bourne of Simon Fraser University, Canada, kindly made electron microscope facilities available. Finally, we wish to thank Mr. W. Goossens and Mr. D. J. DeJong for valuable assistance. This project was supported by a grant from the National Marine Fisheries Service.     

In vitro and in vivo studies on cytodifferentiation of pituitary clonal cells derived from the epithelium of Rathke's pouch

Summary A clonal strain of anterior pituitary cells was derived from Rathke's pouch of the rat. These cells were shown to secrete ACTH, growth hormone and prolactin but no glycoprotein hormones, when grown in vitro. Cells from the 2A8 clone were implanted for one month under kidney capsules or into hypothalami of hypophysectomized female rats. Under the kidney capsule, prominent prolactin cells and poorly developed cells of other types were differentiated as seen in usual pituitary grafts. In hypophysiotrophic areas of the hypothalamus, the grafts were cytodifferentiated into various types of anterior pituitary cells with rich vascularization. These cells had the ultrastructural features indicative of hormone secretion. Increases in body and ovarian weights reflected the secretion of somatotrophic and gonadotrophic hormones. The results obtained indicate that implants of 2A8 clonal cells may differentiate into all types of anterior pituitary cells under the influence of hypothalamic hormones or perhaps some unknown factors present in the general systemic circulation of the rat.Supported by USPHS Grant AM 12583The authors wish to thank Mrs. Martha Castilleja and Mrs. Pauline Polette for their skillful technical assistance     

Comparative Effects of Ethanol and Malnutrition on the Development of Catecholamine Neurons: A Long-Lasting Effect in the Hypothalamus

Abstract: Exposure to ethanol during only the last week of gestation in the rat resulted in significantly decreased weight and levels of norepinephrine in the hypothalamus of the offspring after 26 weeks on standard diet compared with appropriate controls.     

Developmental Expression of Go in Neuronal Cultures from Rat Mesencephalon and Hypothalamus

The developmental expression of the alpha-subunit of Go was examined in neuronal cultures derived from rat mesencephalon (MES) and hypothalamus (HYP). These cultures were essentially free of contaminating glia and were maintained as a stable population for periods up to 3 weeks. Immunoblotting utilizing specific antisera against Go indicated that in neurons from both brain regions, membrane concentrations of Go increased dramatically during the first 2 weeks in vitro. Thereafter, increases in the amount of Go per neuron kept pace with increasing process (axons and dendrites) formation. Multiple forms of immunoreactive Go were detected in MES and HYP neurons, and the proportions of these forms changed between 4 and 14 days in culture. Finally, increasing neuron density significantly increased membrane levels of Go in MES but not HYP cultures.     

Ultrastructure of the pituitary gland (pars distalis) in sockeye salmon (Oncorhynchus nerka) during gonad maturation

Summary The fine structure of the various hormone-producing cell types (with the exclusion of the prolactin cells) in the pituitary gland (pars distalis) of migratory sockeye salmon is described. All fish were in an advanced stage of sexual maturation. In the proximal pars distalis five cell types were distinguished: growth hormone cells, ACTH cells, gonadotrops, vesicular cells, and chromophobe cells. Gonadotrops were also found throughout the rostral pars distalis. A conspicuous feature of the gonadotrops was the presence of two kinds of secretory inclusions: small electron-dense granules (200–375 m) and large, relatively electron-translucent globules (400–2 000 m). The large vesicular cells, so called because of their conspicuous vesicular endoplasmic reticulum, were numerous and often appeared to contain some small granules. It is argued that they may represent a second type of gonadotropic cell, which, in earlier stages of gonad development, contains many granules but becomes largely degranulated near the time of reproduction when the other gonadotrops (globular gonadotrops) abound. The chromophobes, which were smaller and far less abundant than the vesicular cells, also appeared to contain small granules (120–280 m). They are probably thyrotrops.The assistance of Mr. S. Killick, of the International Pacific Salmon Fisheries Commission, who helped in the collection of salmon, is gratefully acknowledged.     

Immunocytochemical demonstration of the hypothalamo-hypophysial vasotocinergic system of Lampetra fluviatilis

Summary With the use of the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the light microscopic level, it was shown that the preoptico-hypophysial neurosecretory system of the adult migrating Lampetra fluviatilis is a vasotocinergic system. It does not synthesize vasopressin. The results are entirely consistent with earlier chromatographic and pharmacological indications that it produces little or no oxytocin-like peptide hormone. In the adenohypophysis, immunoreactive neurohypophysial peptidergic fibres are absent.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Involvement of the hypothalamus in opiate-stimulated prolactin secretion

Administration of opiate agonists to rats is known to elevate plasma prolactin, an effect which is antagonised by the opiate antagonist naloxone. However, this appears not to be a result of a direct action at the pituitary gland. We report here that opiate agonists stimulate prolactin secretion from isolated adenohypophysial cells when they are coincubated with hypothalamic fragments. Both morphine and Met-enkephalin stimulated prolactin secretion by 1.84 fold and 1.50 fold respectively, and this was antagonised by naloxone. These findings support the hypothesis that one site of action of opioid compounds on pituitary hormone secretion is at the level of hypothalamus.     

Isolation and characterization of neurosecretory granules of the rat posterior pituitary gland

Summary Neurosecretory granules (NSG) of rat posterior pituitary glands were prepared by differential centrifugation techniques mainly according to the procedure as described by Barer, Heller and Lederis (1963). As revealed by electron microscopy, the recovery of neurophysin and the contents of enzymes, purified NSG were obtained in a pellet at 30 000 g/60 min (0.44 M sucrose). Eighteen h after injection of (³⁵S) cysteine into the supraoptic nucleus 60% of the recovered radioactivity in the neural lobe was found in the NSG, whereas 20% was found in the final supernatant (100 000 g/120 min). Sixteen days after injection the NSG and the final supernatant fraction contained fairly equal amount of (³⁵S) cysteine (approximately 40%). It is suggested that after a period of intragranular maturation neurophysin is extruded into an extragranular pool of neurosecretory material.With the use of conventional polyacrylamide-gel electrophoresis it was shown that the predominating proportion of radioactivity in the NSG after a hypothalamic injection of (³⁵S) cysteine was located within the neurophysin fraction A and in fraction B. Fraction B is suggested to be partly bound to the NSG membranes. When the NSG soluble and NSG insoluble proteins, obtained after lysis of NSG, were separated on polyacrylamide gels in the presence of sodium dodecylsulphate, the highly radioactive soluble protein was shown to consist of two components with average molecular weights of 12 300 and 14 600. Most of the proteins in the lysate were found in the NSG membranes, though less radioactive. A component with a mol.wt. of 37 000 was enriched in the membrane fraction. At longer times after isotope injection the high mol.wt. proteins, particularly those of the NSG membranes, contained increased amounts of radioactivity.Abbreviations NSG neurosecretory granule - NSM neurosecretory material - SON supraoptic nucleus The present investigation was supported by grants from Svenska Livförsäkringsbolags nämnd för medicinsk forskning from Svenska Sällskapet för Medicinsk forskning, from the Medical Faculty, University of Göteborg, and from the Swedish Medical Research Council (B 72-12X-2543-04A).We are indebted to Mrs. Marie-Louise Eskilsson, Mrs. Wally Holmberg and Mrs. Ulla Svedin for technical assistance, and to Miss Gull Grönstedt for careful secreterial work.     

Immunohistochemical study on the development of CRF-containing neurons in the hypothalamus of the rat

Summary Appearance of immunoreactive corticotropin-releasing factor (CRF)-containing neurons was studied in developing hypothalamus of the rat by use of antisera against rat- and ovine CRF. These neurons were first recognized in the lateral and paraventricular nuclei on days 15.5 and 16.5 of gestation, respectively, when antiserum against rat CRF was employed. Antiserum against ovine CRF revealed the cells two days later exclusively in the latter nucleus. In both nuclei, the neurons increased in number with development. The neurons in the paraventricular nucleus appeared to project their immunoreactive processes to the median eminence via the periventricular and lateral pathways. In the median eminence, the immunoreaction with antiserum to rat CRF was first recognized in its anterior portion in the form of dots on day 16.5 of gestation but as beaded fibers in the external layer on day 17.5; these structures increased in amount with development in rostro-caudal direction. Although antiserum to ovine CRF was less potent in immunostainability than antiserum to rat CRF, it also revealed the beaded fibers in the median eminence on day 17.5 of gestation. Since evidence is available that the paraventricular nucleus is involved in corticotropin release, it is concluded that, in rats, the hypothalamic regulatory mechanism controlling the release of corticotropin initially appears on days 16.5–17.5 of gestation.     

Are learning and attention related to the sequence of amino acids in ACTH/MSH peptides?

Learning and attention were examined in rats after injections of one of several molecules related to adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH). The initial phase of the learning process was linearly related to the length of the peptide with the smallest fragment (MSH/ACTH 4–10) improving learning the most and the largest molecule (ACTH 1–24) exerting no effect. Later stages of the learning problem, which were sensitive to the attentional state of the organism, resulted in U-shaped relations with the length of the same peptides. Enhancement of attention was significant only for the MSH compounds. These data indicate that some behaviors may be influenced as a function of the redundant information contained in the molecule while other behaviors may be discretely related to the specific conformation of the molecule.     

Transfer of carbonic anhydrase-positive particles from the follicle-associated epithelium to lymphocytes of Peyer's patches in foetal sheep and lambs

Summary Carbonic anhydrase cytochemistry of the ileal Peyer's patch in foetal and neonatal lambs has indicated secretion from the follicle-associated epithelium to the follicles. Reaction for carbonic anhydrase in the follicle-associated epithelium was found in the luminal plasma membrane, in cytoplasmic vesicles, and in vacuoles containing 50-nm membrane-bounded particles that seemed to be shed to the intercellular space. The lateral plasma membrane was negative for carbonic anhydrase, indicating that formation of carbonic anhydrase-positive particles was restricted to vacuoles. Administration of ferritin to ileal loops of sheep foetuses showed ferritin localized in vesicles and vacuoles of the follicle-associated epithelium followed by exocytosis, together with carbonic anhydrase-positive particles, into the indentations of the lateral cell border. The carbonic anhydrase-positive particles seemed to be transported to the centres of lymphoid follicles where many were attached to the plasma membrane of lymphocytes. Carbonic anhydrase-positive particles were also seen in vesicles and sometimes free in the cytoplasm of the lymphocytes or attached to their nuclear envelope. Light microscopically, carbonic anhydrase reactivity of the follicle-associated epithelium was associated with the early formation of the ileal Peyer's patch at about 100 days gestation. At this time the follicle-associated epithelium showed a strong luminal but at most a week lateral staining. With further foetal development there was a progressive increase in the amount of carbonic anhydrase-positive reaction product in extracellular particles, both along the lateral cell borders of the follicle-associated epithelium and among the lymphocytes of the follicle centres.     

Effect of the acute heat stress on the rat pituitary gland. Morphological and stereological study

1.

Morphological and stereological changes were examined in the pituitary gland of the rats exposed to heat stress (38 °C) for 60 min.