Kinji Inoue

Immunocytochemical Delineation of Thyrotrophic Area: Origin of Thyrotropin-Releasing Hormone in the Median Eminence

To identify the specific thyrotropin-releasing hormone (TRH)-containing neurons projecting to the median eminence (ME), a retrograde tracing method with horseradish peroxidase (HRP) was combined with immunocytochemical staining for TRH. Three days after HRP injection restricted to the ME, several TRH-positive neuronal perikarya were found to contain HRP. Such double-stained cells were exclusively distributed in the anterior parts of the periventricular nucleus and the most medial parts of the paraventricular nucleus. Few double-stained cells were observed in other parts of the brain examined. The present observations appear to demonstrate that the specific TRH neurons projecting to the ME are located along the border of the third ventricle, anterior to the ME.

Ultrastructural immunocytochemical localization of LH and FSH in the pituitary of the untreated male rat

SummaryRapid freeze-substitution fixation was employed in immunocytochemical studies on the localization of LH and FSH in the typical gonadotrophs of the anterior pituitary in the untreated male rat; a modification of a recently described ferritin antibody method (Inoue et al. 1982) was used in these studies. It was shown that rapid freeze-substitution fixation provides good preservation not only of the ultrastructure but also of the antigenicity. Both LH and FSH were clearly demonstrated in the same gonadotrophic cells, but the subcellular localization of these gonadotrophins differed: (i) LH was mainly located in small secretory granules, 250–300 nm in diameter; (ii) FSH was mainly present in large secretory granules, up to 500 nm in diameter. In the pituitary gland of the adult male rat, all gonadotrophs that react to antibodies against gonadotrophins are characterized by small and large secretory granules. Other types of cells of the anterior pituitary containing either small secretory granules or resembling corticotrophs with secretory granules assembled at cell periphery did not react to either anti-LH beta or anti-FSH beta serum.For light microscopy, the peroxidase antibody method was used. All of the gonadotrophin-positive cells contain both LH and FSH. None of the pituitary cells reacted to antibody against only one gonadotrophin. However, some cells are “LH-rich” while other cells are “FSH-rich”.

Immunohistochemical characterization of thyrotropin-releasing hormone-containing neurons in rat septum.

A sensitive and specific immunohistochemical technique has been used to reveal the distribution of thyrotropin-releasing hormone (TRH) in extrahypothalamic regions. Numerous cell bodies of the lateral septum were surrounded by profiles of dense, spotted TRH-containing axon terminals, suggesting the presence of synaptic contacts. The immunoreactive TRH was found to decrease markedly after electrolytic lesion of the medial preoptic area. The same lesions also lowered the septal content of TRH to 65%, as estimated by radioimmunoassay.

Immunohistochemical localization of thyrotropin-releasing hormone in the brain of carp, Cyprinus carpio

The localization of immunoreactive thyrotropin-releasing hormone (IR-TRH) in the forebrain and pituitary of carp was studied immunohistochemically using the peroxidase-antiperoxidase technique. In the hypothalamus. IR-TRH was present in the neuronal processes extending from the preoptic nucleus (NPO) to the nucleus recessus lateralis (NRL). Cell bodies appeared to be present in the inside of the medial NRL. Most of these neurons were fusiform and bipolar. Immunoreactive-beaded fibers streamed from the anterior part of the NRL toward the nucleus posterioris periventriculas and nucleus lateral tuberis pars posterioris. Vertical strands of the beaded fibers ran in the nucleus lateral tuberis pars anterioris. In the pituitary, the reaction product was found in the neural lobe, where intense immunoreactivity was evident along neural fibers entering the intermediate lobe. Staining could be detected only rarely in the anterior lobe. IR-TRH-beaded fibers were present in the olfactory stalk as well as in the caudal and inner parts of the olfactory bulb. In contrast to the high concentration of IR-TRH in the olfactory bulb, immunohistochemical data from this work indicated weak immunoreactivity in this region.

Conversion of growth hormone-secreting cells into prolactin-secreting cells and its promotion by insulin and insulin-like growth factor-1 in vitro

The newly established rat pituitary cell line, MtT/S, has pituitary somatotroph (growth hormone-producing cell)-like characteristics, i.e., the cells produce growth hormone (GH), possess GH-immunopositive secretory granules, and respond to GH-releasing hormone. When MtT/S cells were cultured in regular medium no prolactin (PRL) cells were observed and PRL was not detected, by radioimmunoassay or Western blot analysis, in the medium or the cells. However, GH production and the GH cell population decreased markedly when the cells were incubated with insulin or insulin-like growth factor-1 (IGF-1). After stimulation with insulin or IGF-1 there was a 2-day lag period, then some PRL was detected in the medium; after 5 days a number of PRL cells appeared. Double immunocytochemistry indicated clearly that no cell contained both PRL and GH. These results show that insulin and IGF-1 stimulate conversion of MtT/S cell line GH cells to PRL cells. This suggests that the MtT/S cell line is an excellent model system which shows the GH-cell/PRL-cell lineage.

Insulin-like growth factor-I stimulates endothelin-3 secretion from rat anterior pituitary cells in primary culture

Since we found relatively high concentrations of immunoreactive (ir-) ET-3 in the rat pituitary gland (190 pg/g tissue), we have investigated the possible ET-3 secretion from the primary culture of anterior pituitary cells and the effects of various growth factors on the ET-3 secretion. The ir-ET-3 was detected in the incubation medium within 2 h, and 24 h of culture attained the concentrations of 1.15 +/- 0.26 pg/well/6 x 10(5) cells. The ir-ET-3 secretion was stimulated by insulin, insulin like growth factor-II (IGF-II), and most effectively by insulin like growth factor-I (IGF-I) in a dose- and time-dependent manner, whereas the production of ir-ET-1 and ir-big ET-1 was slightly inhibited by IGF-I and IGF-II. In reverse-phase HPLC, the ir-ET-3 released into the culture media showed identical retention time with authentic ET-3. Although ir-ET-1 and ir-big ET-1 secretion was stimulated by transforming growth factor-beta (TGF-beta), ir-ET-3 secretion was inhibited. These results indicate that the anterior pituitary cells secrete ET-3 and the secretion is stimulated by IGF-I.

Role of brain lysosomes in the development of manganese toxicity in mice

To study the mechanism of development of manganese toxicity, the manganese content in blood, brain, liver, and subcellular fractions of brain was measured at several intervals following ip injection of a single dose of manganese (245 mg Mn(CH3COO)2 X 4H2O/kg body wt) in mice. The ultrastructural alterations in neurons were correlated with the data for manganese content. Peak concentrations of manganese in blood and liver occurred and disappeared in less than 24 hr. In brain, however, manganese concentration was maintained for 4 days and decreased very little during the 10-day postdose interval studied. Most of the absorbed manganese in brain was recovered in mitochondria and lysosome-rich fractions separated by density gradient centrifugation. Lysosomes took up manganese to a greater extent than mitochondria when compared to controls. Electron microscopy revealed that by 24-hr postdose the number of lysosomes in neurons increased in corpus striatum and midbrain in mice given manganese by ip injection. These results suggested that brain lysosomes play an important role in the cellular metabolism of manganese and in the development of manganese toxicity.

Co-localization of LHβ and FSHβ mRNAs in the porcine anterior pituitary by insitu hybridization with biotinylated probes

Abstract The localizations of mRNAs encoding LHβ and FSHβ in porcine pituitary were investigated by in situ hybridization technique. Biotinylated porcine LHβ and FSHβ cDNA probes were used on frozen sections of paraformaldehyde-fixed pituitary specimens. Hybridizations to both mRNAs were observed specifically in cytoplasm with unstained nuclei. Furthermore, cells hybridized for LHβ mRNA were demonstrated to be identical to those for FSHβ mRNA. This study provided the first morphological evidence that both gonadotropin β genes are expressed in the same cell.

Mitotic activity of gonadotropes in the anterior pituitary of the castrated male rat

SummaryThe proliferation of gonadotropes in the anterior pituitary of the castrated male rat was examined immunohistochemically after colchicine treatment. The results show a more than 10-fold increase in mitotic frequency in gonadotropes 1 or 2 weeks after castration, as compared with controls. This result explains the increase in the population of immunoreactive LH cells in castrated male rats. The gonadotropes decreased significantly 1 month after castration. The mitotic activity of gonadotropes was almost completely suppressed in castrates implanted with a silastic tube filled with testosterone.

Immunocytochemical demonstration of skeletal muscle type peptidylarginine deiminase in various rat tissues.

We have performed an immunocytochemical study of peptidylarginine deiminase (EC 3.5.3.15) in various rat tissues using an antiserum to the enzyme purified from rat skeletal muscle. Staining was observed in skeletal muscle fibers, glia cells of the central nervous system, serous cells of submandibular gland, demilunar cells (serous cells) of sublingual gland, uterine endometrium and myometrium, and certain cells in the lamina propria of intestinal villi. Possible involvement of the enzyme in multiple cellular processes were discussed.