Masataka Shiino

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

SummaryA clonal strain of anterior pituitary cells was derived from Rathkes 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.

Effects of Fetal Brain Extract on the Growth and Differentiation of Rat Pituitary Anlage Cells

Summary Using dissociated Rathkes pouch epithelial cells from 11- to 13-day-old fetal rats as starting material, we have successfully established 11 NRR (normal rat Rathke) cell lines. It was necessary to supplement the growth medium with a rat fetal brain extract in order to induce cell growth and differentiation. These NRR cell lines produced no detectable anterior pituitary hormones unless adult rat median eminence (MEE) extract was added to the culture medium. Following the addition of MEE to the culture medium the NRR cell lines continued to produce from one to six anterior pituitary hormones. The NRR cell lines all show a normal diploid karyotype and have exhibited good growth characteristics through 18 cell passages. From one of the NRR cell lines (NRR-5) 65 clones of normal diploid anterior pituitary cells have been established. Fifty-six of these clones have been examined for hormone production, and each clone examined has been shown to produce one, two, or three hormones.

Calcium-Dependent Changes in Electrical Properties of Prolactin-Secreting Anterior Pituitary (2B8) Clonal Cells

These studies utilized a prolactin-secreting clonal strain (2B8) of rat anterior pituitary cells. These cells secrete increasing amounts of prolactin in response to thyrotrophin releasing hormone (TRH) and do not produce any other pituitary hormone. In response to TRH and in the presence of calcium in the culture medium the cells demonstrated evoked responses without spike discharge following electrical stimulation. No spontaneous evoked response with spike discharge was observed in 2B8 cells. The evoked responses were not suppressed by tetrodotoxin (TTX) but were markedly suppressed by D-600, or lanthanum chloride. The results suggest that calcium ion flux across the cell membrane may be a necessary component of the intracellular events which lead to the release of prolactin.

Ultrastructural observations of growth hormone (STH) cells of anterior pituitary glands of partially hepatectomized rats

SummaryUltrastructural changes in pituitary growth-hormone cells were observed in partially hepatectomized rats. The hepatectomies were carried out during the afternoon after 3 p.m. The animals were sacrificed by decapitation at midnight at intervals of 32, 80, and 104 hours after the operation.The principal changes in the growth-hormone cells of anterior pituitary glands of partially hepatectomized rats were: (1) increased numbers of secretory granules in exocytosis, (2) increased numbers of microtubules, and (3) enlargement of endoplasmic reticulum and occurrence of dilated cisternae of the endoplasmic reticula. Many growth-hormone cells contained a reduced number of secretory granules. Exocytosis of growth-hormone granules was more frequently observed in animals sacrificed at 32 hours after the operation than in those killed at 80 or 104 hours after surgery.The above results in which appearance of numerous microtubules and active secretory granule extrusion in the growth-hormone cells were observed after hepatectomy indicate that ultrastructure of growth-hormone cells and growth hormone secretion were markedly stimulated by the operation.

Morphological changes of pituitary gonadotrophs and thyrotrophs following treatment with LH-RH or TRH in vitro.

SummaryTo investigate the relationship between LH-RH and TRH and the formation of castration cells and thyroidectomy cells, pituitary glands of 14-day old female rats were cultured with LH-RH or TRH for 10 days. Observed in these glands were external and internal zones, the former containing active, healthy appearing cells, and the latter consisting of many degenerative and necrotic cells. Gonadotrophs and thyrotrophs were readily demonstrated in the external zone of the organs by immunocytochemistry. Hypertrophie gonadotrophs (castration cells) and thyrotrophs (thyroidectomy cells) were observed only in the external zone by electron microscopy. Neither the typical signet-ring gonadotrophs nor intracisternal granules of thyroidectomy cells were seen in the present study. However, the hypertrophic gonadotrophs or thyrotrophs were similar to castration cells or thyroidectomy cells observed in the pituitary gland following castration or thyroidectomy. The results indicate that LH-RH and TRH directly stimulate the secretion of hormones and alter the morphological features of pituitary target cells.

Prolactin secretion from clonal pituitary cells following incubation with estradiol, progesterone, thyrotrophin releasing hormone and dopamine.

Summary Prolactin (PRL) release was assessed using clonal cells from the 2B8 strain grown in the presence of estradiol (sodium sulfate) (E2), progesterone (P), thyrotrophin-releasing hormone (TRH) and/or dopamine hydrochloride (DA). Using concentrations of E2 ranging from 10−11 M to 10−6 M, it was found that a significant amount of PRL was released with doses of 10−10 M to 10−8 M. No effect was observed with either higher or lower concentrations of the steroid. Approximately the same amount of PRL was released from pituitary cells incubated with 2 × 10−9 M TRH as was noted with 10−8 M E2. When these two secretagogues were combined, significantly more PRL was present in the medium than when either was employed alone. Prolactin release was significantly inhibited from cells cultured with 10−7 M-10−5 M DA; lower concentrations were either ineffective or were found to stimulate PRL secretion. In combination with 10−8 M E2 or 2 × 10−9 M TRH, 10−6 M DA significantly reduced the stimulatory effect of either of these two compounds. However, when E2 and TRH were incubtaed together in the presence of DA, less of an inhibition of PRL was noted than when DA was combined with either E2 or TRH. Inhibition of PRL release also occurred when the cells were incubated with 10−11 M-10−6 M P. In addition, the stimulatory effect of 10−8 M E2 was found to be suppressed by 10−6 M P. The results indicate that the 2B8 clonal strain is sensitive to agents known to effect PRL secretion in vivo.

Immunoreactive luteinizing hormone (ir-LH) cells in the lung and stomach of chick embryos

Abstract.Luteinizing hormone (LH) immunoreactivity was detected in the lung and stomach of chick embryos by the immunoperoxidase staining technique using specific antiserum to chicken LH. Immunoreactive LH (ir-LH) cells first appeared in the primordial cells of the epithelial layer of lung bud and foregut as well as of Rathke’s pouch in the 3-day-old embryo, Hamburger and Hamilton stage 21. Ir-LH cells increased in number with advancing age of embryos in the lung, stomach, and pituitary gland. In the lung of 7-day-old embryos, stage 31, the ir-LH cells were distributed in the epithelium of primary, secondary, and tertiary bronchi, and their shapes were pseudostratified columnar, simple columnar, and simple cuboidal, depending on their sites in the intrapulmonary airway. Ir-LH cells were more numerous in the median part than in the lateral part of the lung, and the population in the epithelial layer of entobronchi of the secondary bronchi was 4 times higher than that in ectobronchi and laterobronchi of the secondary bronchi and in the primary bronchi. The immunoreactive products were found, either in the entire cell or in the apical part, facing the lumina of bronchi. In the stomach, ir-LH cells were found in the epithelial layer of gastric glands. No ir-LH cells were observed in interstitial regions, which consisted of mesenchymal cells and blood vessels, in the lung and stomach tissues. With advancing age, ir-LH cells changed their shapes to flat or squamous, coincident with the formation of parabronchi. Other pituitary hormones were not observed immunohistochemically in either the lung or stomach before hatching. Preabsorption of the antiserum against avian LH with the purified chicken LH or the extract of pituitaries from 10-day-old embryos completely destroyed the immunoreactivity to the cells in the lung and the pituitary. A single band of the immunoreaction products, whose molecular weight was around 25 K daltons, was shown by the immunostaining of nitrocellulose membrane transblotted after sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified pituitary LH, extracts of pituitaries from 10-day-old embryos, and the extracts of lungs from 7-, 10-, and 14-day-old chick embryos. These results demonstrated that ir-LH cells are present in extrapituitary tissues, and may play an important role during the development of chick embryonic lung and stomach.

Effect of indoles, AVT, oxytocin, and AVP on prolactin secretion in rat pituitary clonal (2B8) cells.

Abstract The effects of several putative pineal hormones on prolactin (PRL) secretion from clonal pituitary cells were studied in vitro. The pituitary cells (2B8) were derived from Rathkes pouch epithelium of normal fetal rats, and they were shown earlier to produce only prolactin when grown in Hams F-10 medium. The substances tested were: 5-HTP (5-hydroxytryptophan), serotonin (5-HT; 5-hydroxytryptamine), NA-5-HT (N-acetyl serotonin), melatonin, and the nonapeptide AVT (arginine—vasotocin). For comparison with the effects of AVT, oxytocin, and AVP (arginine—vasopressin), the neurohypophyseal hormones which are analogs of AVT were also used. After 6 hr of incubation in the presence of these agents, the cells were separated and the media were collected. The media were later assayed for prolactin by radioimmunoassay. 5-HTP, 5-HT, and NA-5-HT (each 10-11 to 10-6 M) had no direct effect on PRL secretion from the 2B8 clonal cells. In contrast, melatonin clearly enhanced PRL secretion over the range of concentrations used. AVT also stimulated PRL secretion in low concentrations (10-11 and 10-10 M), although it inhibited PRL secretion in much higher concentrations (10-8 to 10-6 M). In contrast, oxytocin and AVP did not evoke any response in PRL secretion from these cells. We conclude that the pineal hormones melatonin and AVT, in physiological concentrations, may have direct stimulatory roles in PRL secretion.

Unique features of secretory granules observed in the pituitary growth hormone-secreting (GH) cells of the musk shrew (Suncus murinus L.)

SummaryUnique rod-shaped secretory granules were observed among oval or spherical secretory granules in GH cells of the anterior pituitary gland of musk shrew using the protein A-gold procedure combined with electron microscopy. The rod-shaped and spherical secretory granules were both immunoreactive by the immuno-gold method using antiserum to sheep GH. The rod-shaped secretory granules, which seem to be formed directly from the Golgi vesicles, extend from several hundred to several thousand nm in length. They often touch each other and fuse. The spherical secretory granules are also unique in that they may also fuse with loss of dense contents to leave “empty” circular membrane profiles in the cytoplasm. Both the rodshaped and spherical secretory granules are secreted from the cell by exocytosis.

The participation of corticosterone in luteinizing hormone releasing hormone (LHRH) action on luteinizing hormone (LH) release from anterior pituitary cells in vitro

Abstract Corticosterone alone was not able to stimulate release of luteinizing hormone (LH) from anterior pituitary cells in vitro , but corticosterone in combination with luteinizing hormone releasing hormone (LHRH) augmented the release of LH into the culture media. These results may indicate that corticosterone may have the capacity to activate membrane receptors for LHRH in the gonadotrophs.