Immunocytochemical localization of neuropeptide Y (NPY) in the human hypothalamus

Summary In order to study the distribution of neuropeptide Y-like immunoreactivity in the human hypothalamus, an immunocytochemical localization of this peptide was performed. Using antibodies developed against synthetic porcine neuropeptide Y (NPY), we have been able to localize immunoreactivity in neuronal cell bodies located exclusively in the infundibular nucleus. Immunostained fibers were found in several regions in the hypothalamus with a high concentration in the periventricular areas. Fibers were also found in the neurovascular zone of the median eminence, the pituitary stalk and the posterior pituitary. These results suggest that immunoreactive material related to porcine NPY is present in the human hypothalamus, with a distribution similar to that observed in the rat.     

Immunoreactive neuronal pathways of growth hormone-releasing hormone (GRH) in the brain and pituitary of the teleost Gadus morhua

Summary Using an antiserum directed against the C-terminus of hGRH(1–44)NH₂ and another recognizing the mid portion to C-terminal of hGRH(1–40)OH, we identify two immunocytochemically distinct GRH-immunoreactive systems in the brain of the codfish, Gadus morhua. The antiserum directed against GRF(1–44)NH₂ stains cell bodies exclusively in the rostral pars distalis. The other antiserum immunoreactive with GRF(1–40)OH reacts with a population of parvocellular and magnocellular neuronal cell bodies in the hypothalamus and with two major axonal pathways which project toward the median eminence and terminate primarily in the pars nervosa. These results indicate the presence of at least two forms of hGRH-like peptides in the teleost which may have different roles in the regulation of pituitary function.     

Ontogenetic appearance of immunoreactive GRF-containing neurons in the rat hypothalamus

Summary Ontogenetic development of GRF-containing neurons in the rat hypothalamus was studied employing antisera which were generated against hpGRF (1–44)NH₂ and rhGRF(1–43)OH: anti-hpGRF-C and -rhGRF sera recognize the species-specific C-terminal portions of the peptides, and anti-hpGRF-MC and -N sera recognize hpGRF(27–44)NH₂ and the N-terminal portion of hpGRF(1–44)NH₂, respectively. The anti-hpGRF-C and-rhGRF sera stained different neuronal cell bodies, which were localized in distinct hypothalamic areas. The former serum did not stain the axonal terminals in the median eminence, but the latter stained them strongly. The antihpGRF-MC and -N sera stained neuronal cell bodies, some of which corresponded to those immunolabelled with antihpGRF-C or -rhGRF serum. The anti-rhGRF serum first demonstrated immunoreactive perikarya in the ventral-lateral border of the arcuate nucleus of 19.5-day-old fetuses that had received an intraventricular colchicine administration 24 h previously. The immunoreactive fibers were recognized first in the external layer of the median eminence of untreated fetuses on day 19.5 of gestation, and then they increased in amount with development. No immunore-active fibers, however, were found in the median eminence of colchicine-treated animals during the fetal period. It is concluded that in rats GRF may be synthesized in the perikarya on day 18.5 of gestation and conveyed to the median eminence without delay via axonal flow.     

Ultrastructural localization of prolactin,growth hormone and luteinizing hormone by immunocytochemical techniques in the bovine pituitary

Summary The technique of ultrastructural immunocytochemistry involving the unlabeled antibody and the soluble peroxidase-antiperoxidase complex was used to identify and describe the prolactin (P) cells, somatotropic (STH) cells and luteinizing hormone (LH) cells in the bovine anterior pituitary gland. This method was used to localize the three hormones at the electron microscopic level. Staining of varying intensity was found on the secretory granules and on the small granules and vesicles within the Golgi complex. No stain was found in nuclei, on mitochondria or in the endoplasmic reticulum.     

Topographical and ontogenetic study of the neurons producing growth hormone-releasing factor in human hypothalamus

Neurons producing growth hormone-releasing factor have been characterized and analyzed by immunohistochemistry in the hypothalami of human fetuses, neonates, infants and adults, using two antibodies against human pancreatic GRF (hpGRF). One of the antibodies recognized both the hpGRF(1-40)OH and hpGRF(1-44)NH2 in the mid portion (between the 28th and 39th amino acid), the other one specifically recognized the C-terminal end of hpGRF(1-44)NH2. These two antibodies stain a single neuronal system with cell bodies mainly located in the infundibular (arcuate) nucleus, and in the ventromedial and lateralis tuber nuclei. These neurons project to the median eminence where they give numerous endings in contact with portal vessels. These neurons are distinct from those containing LH-RH, somatostatin, CRF or pro-opiocortin. In fetuses, neurons immunoreactive with hpGRF antibodies are first detected at the 29th week. They display a neuroblastic aspect which persists after birth. Immunoreactive fibers are detectable in the median eminence after the 31st week. These results demonstrate that the infundibular nucleus plays a major role in control of GH secretion in man and that secretion of GRF appears late during fetal life; this suggests that the first stages of differentiation and development of GH producing cells in the human fetus do not depend on hypothalamic GRF secretion.     

Ontogeny of the response to growth hormone-releasing factor

Primary cell cultures were prepared from fetal, neonatal and adult rat pituitaries and evaluated for their ability to secrete growth hormone (GH) in response to growth hormone-releasing factor (GRF). Pituitary cells prepared from fetuses at days 19 and 21 of gestation, neonatal animals at the day of birth (day 0) or the following day (day 1) and peripubertal male rats showed full dose response curves to GRF with maximal GH release when stimulated with 1 X 10(-10) M rat GRF. At this concentration of GRF, the amount of GH released was not different from that elicited by activation of adenylate cyclase with 1 X 10(-5) M forskolin. In contradistinction, a preparation of cells from fetuses at day 18 of gestation did not show the same release of GH when challenged with 1 X 10(-10) M GRF and forskolin (0.057 +/- 0.001, compared to 0.076 +/- 0.003 micrograms/10(5) cells per 4.5 h), although the cells clearly responded to both secretagogues (basal levels of GH, 0.029 +/- 0.002 micrograms/10(5) cells per 4.5 h). While cells prepared from fetuses at day 21 of gestation or from animals after birth released 5-10% of their total cellular GH content, those prepared from 18- and 19-day fetuses released as much as 40% of their total GH suggesting there is a maturation of intracellular GH processing that occurs late in gestation. The results show that, in late pregnancy, the rat fetal pituitary is highly responsive to growth hormone-releasing factor and suggest that this peptide participates in regulating GH levels during the perinatal period.     

Ultrastructural immunocytochemical localization of prolactin and growth hormone in the porcine pituitary

Summary The immunocytochemical peroxidase-antiperoxidase technique was used to identify prolactin- and growth hormone-producing cells in the porcine pituitary at the ultrastructural level. The growth hormone-producing cells contain round secretory granules (300 nm to 500 nm in diameter). The prolactin-producing cells can be identified by their distinct round and ovoid secretory granules which vary in size. Most of these cells contain large granules (450 nm to 750 nm in diameter), but some prolactin-producing cells display smaller secretory granules (250 nm to 500 nm). The two hormones were localized exclusively in the secretory granules. Staining for prolactin was observed in round and ovoid granules, as well as in small and polymorphic granules within the Golgi complex. This study confirmed (i) that the two hormones are located in different cells, and (ii) that under normal physiological conditions no one cell can synthesize and store both hormones simultaneously.     

Immunohistochemical localization of corticotropin-releasing factor (CRF)-containing neurons in the hypothalamus of the Japanese quail,Coturnix coturnix

Immunohistochemical localization of corticotropin-releasing factor (CRF)-like immunoreactivity in the brain of the Japanese quail was studied by means of the peroxidase anti-peroxidase (PAP) method. CRF-immunopositive perikarya of parvocellular neurons were observed mainly in the nucleus praeopticus medialis and nucleus paraventricularis. Additional perikarya were also detected in the nucleus hypothalamicus posterior medialis in the hypothalamus and in the non-hypothalamic nucleus accumbens, nucleus septalis lateralis and nucleus dorsomedialis and dorsolateralis thalami. No CRF immunoreaction was found to coexist with the vasotocin (Vt)-containing system in comparative examination of consecutive sections treated with anti-vasopressin (Vp) serum. The CRF-immunoreactive fibers were detected mainly in the external layer of the anterior median eminence but not in its posterior division. Unilateral adrenalectomy induced the marked reduction in number of the CRF immunopositive fibers in the anterior median eminence.     

In vitro pituitary hormone releasing activity of 40 residue human pancreatic tumor growth hormone releasing factor

The hypophysiotropic activities of a synthetic human pancreatic growth hormone releasing factor (hpGRF) with 40 residues was examined in vitro using rat pituitary halves. At concentrations from 10(-10) M to 10(-7) M the peptide stimulated GH release in a dose-dependent manner with the ED50 being 1.2 x 10(-9) M. The concentration of 10(-10) M hpGRF is comparable to the basal hypophyseal portal blood levels of other known hypothalamic hypophysiotropic hormones. However, GH release was enhanced three-fold by concentration as low as 10(-12) M, though no dose-response relationship was observed up to 10(-10) M. Thus, this peptide not only stimulates the release of GH in a dose-dependent manner, but at lower concentrations also maintains elevated GH levels. The release of ACTH, beta-endorphin, LH, and FSH was not affected by hpGRF at any of the concentrations tested. At hpGRF concentrations less than 10(-7) M, the release of TSH and PRL were unaffected. However, at 10(-6) M, TSH release was enhanced about 2.5 fold and prolactin release was elevated slightly.     

Immunocytochemical localization of growth hormone releasing factor (GHRF)-containing structures in the rat brain using anti-rat GHRF serum

The distribution of growth hormone releasing factor (GHRF) immunoreactive structures in the rat hypothalmus was studied after colchicine treatment with PAP immunocytochemistry in vibratome sections using an antiserum directed to rat hypothalamic GHRF. The majority of the GHRF-immunoreactive cell bodies were found in the arcuate nucleus, the medial perifornical region, and the ventral premammillary nuclei of the hypothalamus. Scattered cells were seen in the lateral basal hypothalamus, the medial and lateral portions of the ventromedial nucleus, and the dorsomedial and paraventricular nuclei. Immunoreactive fibers were observed in all the regions mentioned above. GHRF terminals were located in the central region of the median eminence. In addition, GHRF-immunoreactive neuronal processes were seen in the ventral region of the dorsomedial nucleus, the medial preoptic and suprachiasmatic regions, dorsal portion of the suprachiasmatic nucleus, bed nucleus of the stria terminals and the hypothalamic portion of the stria terminals. The localization of GHRF-immunoreactive terminals in the median eminence reinforces the view that GHRF plays a physiological role in the regulation of pituitary function. In addition, the localization of GHRF-immunoreactive structures in areas not usually considered to project to the median eminence suggest that GHRF may act as a neuromodulator or neurotransmitter.     

Light-microscopic immunocytochemical localization of fibronectin in the developing rat lung

Summary The development of the rat lung is a process of continuing morphological change. Indications from work in other mammalian systems suggest that fibronectin may be important in the control of this process. The present study has examined embryonic, neonatal, and adult lung tissue of the rat by means of the peroxidase-antiperoxidase (PAP) technique to demonstrate fibronectin at the light-microscopic level. Positive reaction was observed with anti-fibronectin serum in all stages examined. Control sections treated with pre-immune serum or no primary serum gave negative results in each case. Fibronectin in adult tissue was localized to the alveolar surface and alveolar basal lamina. Neonatal tissue showed fibronectin on pulmonary tubule walls and in basal lamina while embryonic tissue revealed localization of the protein in the basal lamina and in association with small groups of cells at the base of septal buds. These findings suggest a role for fibronectin in the control of rat lung development. The results are discussed in terms of the known functions of fibronectin as a preliminary matrix for the subsequent deposition of collagenous connective tissue, as a cellular adhesion protein, and as surface-bound material for cellular migration.     

Tilapia adropin: the localization and regulation of growth hormone gene expression in pituitary cells

The peptide hormone adropin, encoded by the energy homeostasis-associated (Enho) gene, plays a role in energy homeostasis and the control of vascular function. The aim of this study was to examine the role of adropin in growth hormone (GH) gene expression at the pituitary level in tilapia. As a first step, the antiserum for the tilapia adropin was produced, and its specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the tilapia pituitary. Adropin could be detected immunocytochemically in the proximal pars distalis (PPD) of the tilapia pituitary. In primary cultures of tilapia pituitary cells, tilapia adropin was effective in increasing GH mRNA levels. However, removal of endogenous adropin by immunoneutralization using adropin antiserum inhibited GH gene expression. In parallel experiments, pituitary cells co-treated with ovine pituitary adenylate cyclase activating polypeptide 38 (oPACAP38) and adropin showed a similar increase level compared to those treated with oPACAP38 alone, whereas insulin-like growth factor 1 (IGF1) not only had an inhibitory effect on basal GH mRNA levels, but also could abolish adropin stimulation of GH gene expression. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was enhanced by adropin. Taken together, these findings suggest that adropin may serve as a novel local stimulator for GH gene expression in tilapia pituitary.     

Effects of growth hormone-releasing factor, somatostatin and dopamine on growth hormone and prolactin secretion from cultured ovine pituitary cells

A synthetic form of human pancreatic growth hormone releasing factor (GRF-44-NH2) was shown to be a potent stimulator of growth hormone (GH) secretion and cellular cyclic AMP levels in cultured sheep pituitary cells. A small dose-dependent stimulation of prolactin secretion was also observed. Somatostatin (0.5 microM) completely blocked the maximal GRF (1 nM)-stimulated secretion without a significant effect on cyclic AMP levels. Dopamine (0.1 microM) inhibited the GRF-elevated GH secretion by 50% and lowered cyclic AMP levels by 30%. Dopamine (0.1 microM) inhibition of basal prolactin secretion was not affected by GRF (1 nM). The data support the hypothesis that cyclic AMP is involved in the action of GRF but suggest that somatostatin can inhibit GRF-induced secretion of GH independently of cyclic AMP.     

Dynamics of secretory granules in somatotrophs of rats after stimulation with growth hormone-releasing factor: a stereological analysis

The anterior pituitary tissue of male rats injected with growth hormone-releasing factor (GRF) was either processed for stereology at the light-and electron-microscopic levels, or homogenized for growth hormone (GH) assay 2–60 min after GRF injection. Secretory granules of somatotrophs became smaller but increased in numerical density 2 min after GRF injection. Their volume density began to increase at 5 min. The frequency of exocytosis of the granules was most prominent as early as 2 min after GRF injection and reduced thereafter. GH levels in the tissue were lowest at 2–5 min, and returned to the control value by 60 min. Serum GH levels were highest at 15 min; even at 60 min, this value was higher than in the controls. These findings suggest that secretory granules in somatotrophs are stimulated to divide by GRF, resulting in a decrease in size and an increase in number. The discrepancy between the earlier formation of new secretory granules and the later restoration of intracellular GH levels implies that GRF first stimulates the synthesis of constituents of granules other than GH, and only later the synthesis of GH, and that newly formed small secretory granules contain less GH. From the clearance rate of serum GH and the frequency of granule exocytosis, it can be estimated that about a half million granules are released to maintain 1 ng/ml of serum GH in rats.     

Culture of human pituitary prolactin and growth hormone cells

Summary Fragments of pituitary tissue obtained from a total of 37 patients with either breast cancer, diabetic retinopathy, galactorrhea, or acromegaly were dissociated into single cell suspensions prior to cell culture. Release of human growth hormone (hGH) and human prolactin (hPRL) into the culture medium was measured by radioimmunoassay. During a 3-week culture period, prolactin cells released 9–13 times the intracellular levels of hPRL at the time of seeding, whereas hGH release from growth hormone cells was only 1–2 times that of their initial intracellular level during this same time. Both growth hormone and prolactin cells retained distinctive ultrastructural features during culture. The prolactin cells responded to TRH stimulation by elevated release of PRL into the medium. No evidence for mitotic division of prolactin cells in vitro was found.This work was supported by NCI Contract NO 1-CB-23863     

Immunocytochemical localization of CRF in the ovine hypothalamus

A population of neuronal cell bodies and their fiber pathways have been elucidated within the ovine hypothalamus. The immunoreactive neurons were located in the anterior and dorsal hypothalamus interspersed throughout the paraventricular nucleus. These perikarya were only observed when an antiserum that was generated against the C-terminal of CRF was employed. A dense fiber projection traversed the medial-basal hypothalamus and ended within the palisade-contact zone of the median eminence and neural stem. Fibers were revealed by antisera generated against both the N-terminal and the C-terminal of CRF. Antisera pre-absorbed with synthetic CRF failed to yield immunoreactivity.     

Immunohistochemical detection of insulin-like growth factor-I, transforming growth factor-beta2, basic fibroblast growth factor and epidermal growth factor-receptor expression in developing rat ovary

The aim of this study was to determine the immunohistochemical expression and localization of insulin-like growth factor-I (IGF-I), transforming growth factor-β2 (TGF-β2), basic fibroblast growth factor (bFGF) and epidermal growth factor-receptor (EGF-R) in developing rat ovaries.Eighteen female Wistar rats were enrolled in this study; newborn (n = 6), one-month-old (n = 6) and adult (n = 6) rats. Formalin-fixed and parafin-embedded ovarian tissues were stained with antibodies against IGF-I, TGF-β2, bFGF and EGF-R, immunohistochemically. The ovarian cells were evaluated by semi-quantitative scoring system under light microscope.The staining of IGF-I, TGF-β2, bFGF and EGF-R were most intense in the oocytes and were heavily at one-month-old rats. A moderate immunostaining in theca cells and corpus luteii reacted with IGF-I in adult rats. Furthermore the staining intensity for IGF-I was moderate in granulosa cells of newborn rat ovaries. We detected also a moderate staining for TGF-β2 in corpus luteii of adult rats. In addition, we found a bFGF immunostaining mainly in oocytes of follicles of young and adult rats. Immunostaining for EGF-R was moderate in granulosa cells of one-month-old rats.In conclusion, this study suggests that growth factors play a pivotal role in ovarian function, especially in follicular development. The role of growth factor in controlling degeneration or growth (or both) of ovary follicles remain as explained.     

人生长激素研究进展

综述了人生长激素的结构,及最近几年国内外对人生长激素在分子结构、基因改造、制备、提纯及检测等方面的最新进展和研究趋势,对各种可能影响生长激素分泌的因素进行了分析、讨论。     

Ultrastructural immunocytochemical localization of lysozyme in human monocytes and macrophages

Summary In order to investigate the mechanism of synthesis and secretion of lysozyme (LZ) by human mononuclear phagocytes, the ultrastructural localization of LZ was studied by a pre-embedding direct immunoperoxidase method. Blood monocytes showed a reaction product for LZ in cytoplasmic granules, whereas cultured monocytes showed the reaction product in phagosomes as well as granules at 5 h of culture and in numerous large granules at 3 days of culture. In Kupffer cells, LZ was present in cytoplasmic granules, vacuoles and phagosomes. Some Kupffer cells showed a positive reaction for LZ in the rough endoplasmic reticulum, perinuclear cisterna and Golgi apparatus. Macrophages in the lymph nodes contained LZ in cytoplasmic granules. Bone marrow macrophages contained numerous phagosomes with electron-dense degradation products of erythrocytes, but the reaction product for LZ could not be clearly identified. The present study demonstrated that LZ is present in the granules of human mononuclear phagocytes and released into phagosomes. An in-vitro culture study, furthermore, demonstrated that macrophages produce LZ-containing large granules distinct from those of monocytes. However, findings that indicate the synthesis and secretion of LZ by cultured monocytes, as suggested previously by other investigators, were not observed in this study.