ACTH Cells Research Articles

Immunohistochemistry of the hypothalamic neuropeptides and anterior pituitary cells in the Japanese quail.

The pars distalis of the avian adenohypophysis consists of well-defined cephalic and caudal lobes which are distinct in their cellular constituents. Immunocytochemical investigations on the pituitary hormones of the pars distalis of the Japanese quail reveal five types of secretory cells, adenocorticotropin (ACTH) cells, prolactin (PRL) cells, thyroid-stimulating hormone (TSH) cells, growth hormone GH (STH) cells, and FSH/LH (gonadotropic) cells. The ACTH cells, TSH cells, and PRL cells are restricted to the cephalic lobe, and GH (STH) cells are confined to the caudal lobe, while FSH/LH cells are distributed throughout the cephalic and caudal lobes. The median eminence of birds has distinct anterior and posterior divisions, each with different neuronal components. The avian hypophysial portal vessels also consists of two groups, anterior and posterior. The peculiar arrangement and distribution of the avian hypophysial portal vessels are possibly related to the distribution of neuropeptides in the two divisions of the median eminence and to the cytological and functional differentiation of two lobes of the pars distalis. The localization of perikarya and fibers containing luteinizing hormone releasing hormone (LHRH), somatostatin, vasotocin, mesotocin, corticotropin-releasing factor (CRF), vasoactive intestinal polypeptide (VIP), glucagon, metenkephalin, and substance P in the hypothalamus and median eminence of the Japanese quail has been investigated by means of immunohistochemistry using antisera against the respective neuropeptides. LHRH-, somatostatin-, VIP-, met-enkephalin-, and substance P-immunoreactive fibers are localized in the external layer of the anterior and posterior divisions of the median eminence, while CRF- and vasotocin-reactive fibers are demonstrated only in the external layer of the anterior division of the median eminence. The metenkephalin fibers are thicker in the anterior median eminence but the substance P fibers are more abundant in the posterior division. Mesotocin fibers occur only in the internal layer of the median eminence and neural lobe.

The stimulation by ACTH of 17α-hydroxylation in cultures or rabbit adrenal cells

Adrenal cells from control rabbits (control-cells) and from rabbits that had been injected twice daily for 3 days prior to sacrifice with 25 IU ACTH (ACTH-cells) were cultured both in the absence and presence of 100 mIU ACTH. Culture durations varied from 24 to 120 h in 24 h increments. The culture medium was changed daily and fresh ACTH added to appropriate vessels. At the time of the final media change 0.1 μCi [4- 14C]pregnenolone was added. Twenty-four hours later the cultures were terminated and the products formed from the pregnenolone were isolated, quantified and identified by solvent extraction, chromatography and crystallization to constant specific activity. After 72 h ACTH-cells cultured in the presence of ACTH converted 18.5% of the pregnenolone substrate to 17-hydroxycorticosteroids (cortisol plus 11-deoxycortisol) while ACTH-cells cultured in the absence of ACTH converted a maximum of 1.6%. A similar but smaller difference, 10.9 vs 2.1%, was recorded with control cells cultured in the presence and absence of ACTH. Corticosterone production from [4- 14C]pregnenolone in the 72-h cultures was increased to a lesser degree by ACTH exposure. In ACTH-cells the conversion climbed from 5.9 to 10.5% and from 9.6 to 12.0% in control cells. Microscopic examination of parallel cultures showed no significant differences in cell density between cells cultured in the presence or absence of ACTH. 17α-Hydroxylase activity arising from the in vivo stimulation did not survive cell division in culture, but required the continual presence of ACTH. In conclusion, the data show that ACTH is capable of stimulating 17-hydroxycorticoid formation in rabbit adrenal cell cultures.

Ultrastructure of Crooke's cells with special reference to their secretory granules.

Twenty-seven postmortem pituitary glands were electron microscopically investigated. Eighteen of them revealed an accumulation of fine microfilaments in the cytoplasm ranging from 70 to 80 A in width, which is equivalent to a hyaline change in Crooke's cells seen with a light microscope. Since Crooke's changes were immunohistochemically evidenced to occur exclusively in ACTH cells, we examined their secretory granules quantitatively and concluded that the majority of them were ovoid, homogeneously electron dense and measured 200 to 450 nm in diameter averaging 332 nm. Occasionally, those irregularly shaped and larger than 600 nm were observed.

Immunocytochemical identification of CRF-like and SRIF-like peptides in the brain and the pituitary of cyprinid fish.

Single and double immunocytochemical techniques were applied to the brain and pituitary of carps and goldfish. With the use of antiserum raised against synthetic corticotropin-releasing factor (CRF), immunoreactive perikarya were observed in the nucleus praeopticus and the nucleus praeopticus periventricularis. CRF-like-immunoreactive hypothalamic nerve fibers reach the pituitary. In cyprinids, some fine fibers enter the rostral neurohypophysis bordered by prolactin- and ACTH cells. Other thicker fibers extend ventrocaudally into the neurointermediate lobe. This CRF-like system appears to differ from the SRIF-like system, which is restricted to the proximal pars distalis of the pituitary containing somatotrophs.

Synaptic regulation of hypothalamic neurons containing ACTH by substance P

Synaptic connections between substance P (SP)- and ACTH-containing neurons in the rat arcuate nucleus were studied by a technique of combined pre- and postembedding immunohistochemical staining. The results showed that immunoreactive (ir) SP fiber terminals form synapses with ir ACTH cell bodies. This strongly suggests that the activity of ACTH neurons is under neuronal regulation by SP neurons.

Immunolabeling of adenohypophysial cells with protein A-colloidal gold--antibody complex for electron microscopy: use of the freeze-substitution technique in tissue preparation.

The value of the freeze-substitution (FS) method for preparing tissues for electron microscopic immunohistochemistry was studied by comparing anterior pituitary cells prepared by this method and by a conventional method. Ultrathin sections of tissues embedded in Epon were subjected to immunostaining. The antigens adrenocorticotropin (ACTH) and prolactin (PRL) in a single ultrathin section were demonstrated by a simple double-labeling technique using a protein A-colloidal gold-antibody (pAG-Ab) complex. The preservation of cellular ultrastructure was superior in preparations obtained by FS. Gold-labeling was seen over secretory granules, and in ACTH cells also over the cytoplasmic matrix. The labeling was more intense in preparations obtained by FS, judging from the numbers of gold particles. In the double-labeling procedure, in which the pA-small colloidal gold-anti-PRL complex and pA-large colloidal gold-anti-ACTH complex were applied sequentially to sections, no cross-labeling with small and large gold particles was observed. It is concluded that if the antisera are sufficiently specific, the use of FS and the pAG-Ab complex is very effective in peptide immunohistochemistry. However, in the double-labeling procedure it is essential that the Fc-binding sites of pAG are saturated by the use of excess amounts of antibodies.

The prolonged stimulatory effect of ACTH on 11 beta-hydroxylation, and its contribution to the steroidogenic potency of adrenocortical cells.

The mechanism of the prolonged stimulatory influence of corticotropin (ACTH) on the capacity of adrenocortical cells to produce cortisol in response to ACTH and more specifically the role of 11 beta-hydroxylation, was studied on guinea-pig adrenocortical cells dispersed from control and ACTH-treated animals. As a result of the previous in vivo exposure to ACTH, the net maximal production of glucocorticoids in response to ACTH (by 10(5) cells and 2 h incubation) increased from 660 +/- 33.9 ng (control group) to 1105 +/- 117.9 ng for cells from ACTH-treated animals (P less than 0.001), whereas the apparent affinity of the steroidogenic response remained unchanged. In addition there occurred an increased conversion of exogenous pregnenolone into cortisol by cells from ACTH-treated animals, indicating a prolonged stimulatory influence of ACTH on the post-pregnenolone pathway of cortisol biosynthesis. The activity of 11 beta-hydroxylation step was therefore examined by incubating the adrenocortical cells from control and ACTH-treated animals in the presence of increasing amounts of 11-deoxycortisol. The maximal capacity of 11-deoxycortisol conversion into cortisol was increased as a result of the in vivo exposure to ACTH, averaging 3423 +/- 211 ng cortisol formed from 5 micrograms 11-deoxycortisol by 10(5) cells from ACTH-treated animals vs 2074 +/- 185 ng for cells from control guinea-pigs (P less than 0.001). However, the conversion of lower amounts of 11-deoxycortisol into cortisol, reproducing quantitatively the maximal effect of ACTH on cortisol biosynthesis, was only barely increased in cells from ACTH-treated animals (P greater than 0.05). Therefore it was concluded that ACTH increases in a lasting way not only the overall steroidogenic capacity of adrenocortical cells but also the maximal efficiency of 11 beta-hydroxylation. Since the latter effect cannot account quantitatively for the magnitude of the lasting effect of ACTH on the maximal capacity of adrenocortical cells to produce cortisol in response to ACTH, it appears that the prolonged influence of ACTH on cortisol biosynthesis should also involve a stimulatory influence of the peptide on earlier step(s) of steroidogenesis.

A histometrical study of immunohistochemically identified mitotic adenohypophysial cells in immature and mature castrated rats.

Two weeks after operation the effects of castration on all types of mitotic adenohypophysial cells in 30-day-old (immature) and 75-day-old (mature) male rats were studied immunohistochemically by light microscopy. The total number of mitotic cells in gland specimens increased 1.4 times in immature rats, but remained unchanged in mature animals; mitotic activity (mitotic cell number/mm2) was not affected in either age group. The population of mitotic gonadotrophs increased five to six times in comparison with intact controls. In addition, the percentage of mitotic gonadotrophs and their mitotic activity were raised in both age groups. Castration affected the mitosis of prolactin cells. The percentage of mitotic prolactin cells was reduced by one-third in castrated immature rats, but the total number and percentage of mitotic prolactin cells, as well as their mitotic activity, was reduced by about two-thirds in mature castrated rats. No significant changes in mitotic ACTH, TSH, GH and immunonegative cells were found after castration in either group of animals. Mitotic cells were more numerous in the anterior than in the posterior region of the gland in normal (uncastrated) immature rats, but were uniformly scattered in both regions in normal mature rats. On the other hand, castration induced a high population of mitotic cells in the anterior region regardless of age. This high population was the result of the enhanced mitosis of gonadotrophs. It is concluded that 2 weeks after castration the increment of mitotic divisions of all types of pituitary cells is age-dependent, and that mitosis takes place frequently in gonadotrophs and less frequently in prolactin cells.

The effects of tunicamycin on anterior pituitary hormone producing cells in the rat.

An electron microscopic study was performed to clarify the effects of tunicamycin, a glycosylation inhibitor, on rat anterior pituitary cells. Tunicamycin (10, 50, and 100 micrograms/250 g B.W.) was intraperitoneally injected into rats, which were sacrificed 24 hrs later. Protein hormone producing GH and prolactin cells, and ACTH cells which are known to have a glycosylated precursor, showed no recognizable ultrastructural changes. TSH cells and gonadotrophs, both of which secrete glycoprotein hormones consisting of alpha and beta subunits, showed remarkable dilatation of the rough endoplasmic reticulum, and decreased numbers of secretory granules. These results suggest that the role of glycosylation in TSH cells and gonadotrophs may have a different biological significance from that in ACTH cells.

Study of growth hormone in spontaneous dwarf rat

In 1977, in the Research Laboratories, Morishita Pharmaceutical Co. Ltd., we were fortunate to discover that small mutant rats named Spontaneous Dwarf Rat (gene symbol dr), inherited an autosomal recessive. In previous papers, we reported that these rats might be characterized by a defect of growth hormone (GH). In this paper, we carried out the studies of the secretory cells and GH deficiency in the pituitary glands of +/dr and dr/dr rats using immunohistochemistry, electron microscopy and acrylamide gel electrophoresis. ACTH, PRL and GH secretory cells in the pituitary gland respectively identified with antiserum to porcine ACTH, rat PRL and ovine GH by indirect method of immunohistochemistry. In the pituitary of dr/dr rats, ACTH and PRL secretory cells were confirmed, but none of the cells were stained with ovine GH antiserum. Using electron microscopy, GH secretory cells could not be identified precisely although the other cells were identified in the pituitary gland of dr/dr rats. With the analysis of pituitary homogenate by acrylamide gel electrophoresis, PRL band appeared in dr/dr rats but GH did not. These results obviously indicated that the inhibition of body weight in dr/dr rats was caused by the absence of GH in the anterior pituitary. Therefore, the Spontaneous Dwarf Rat may be a useful animal model for human dwarfism caused by isolated GH deficiency.

Hypothalamic ACTH-related peptides containing cells vs pituitary ACTH cells.

Hypothalamic ACTH-related peptides containing cells vs pituitary ACTH cells.

Substance P-like immunoreactive neurons in the tuberoinfundibular area of rat hypothalamus. Light and electron microscopy

Immunoreactive substance P (ir SP) neurons were examined with special reference to their links with other neurons and blood vessels in the tuberoinfundibular hypothalamic area of normal and colchicine-treated male rats. On intraventricular administration of colchicine, ir SP neuronal cell bodies appeared in both the arcuate nucleus and the ventral portion of the ventromedial nucleus. These ir fibers were seen in the nuclei of untreated animals closely surrounding ir methionine-enkephalin and ACTH cell bodies, suggesting the presence of synaptic junctions. Electron microscopic examination of the arcuate nucleus stained with anti-SP serum showed synapses of ir SP fiber terminals on unlabeled neuronal cell bodies and fibers, some of which might be methionine-enkephalin and ACTH neurons. The ir fiber terminals contained numerous small clear and/or cored vesicles and a few large cored vesicles. Fiber terminals with the same ultrastructural characteristics were seen protruding into the pericapillary space in the external and subependymal layers of the median eminence. The possible role of the SP neurons in the tuberoinfundibular area is discussed.

Co-occurrence of immunoreactive corticotrop in-like and α-melanotropin-like material in pituitary cells: Differences between young and adult rats

We examined to which extent adrenocorticotropin (ACTH)-like substances are cleavable into α-melanotropin (MSH)-like material by application of exogenous enzymes on pituitary sections prior to immunocytochemical stainings. In pro-opiomelanocortin (POMC) cells of the anterior lobe from developing rats (1 to 21 postnatal days) at least one of two cells contained α-MSH-like immunoreactivity besides ACTH-like immunostaining. Pretreatment of these tissue sections with tryptic enzymes enhanced remarkably α-MSH-like immunostaining. In contrast, α-MSH-lik immunoreactivity was almost not detectable in anterior lobes from adult rats, also after treatment with enzymes. After adrenalectomy (ax) POMC cells in anterior lobes behaved similarly: a few days after the operation almost all ACTH cells contained also α-MSH immunostaining, whereas after more than two weeks α-MSH immunostaining was nearly absent, also after application of enzymes. In contrast to rats, in young and adult guinea pig pituitaries all ACTH-like immunoreactivity was prevented by treatment with tryptic enzymes. These observations are consistent with the assumption that occurrence of ACTH- and α-MSH-like substances in rat pituitaries is regulated by a protection mechanism against proteolytic cleavage in the ACTH sequence which appears with delay in differentiating and maturing cells.

Ontogenesis of hypothalamic immunoreactive ACTH cells in vivo and in vitro: Role of Rathke's pouch

The ontogenesis of immunoreactive (ir) ACTH cells and ir α-MSH cells in rat hypothalamus was studied in vivo and in vitro. Ir ACTH cells first appeared in the neuroepithelial cell layer lining the floor of the third ventricle on Day 13.5 of gestation, whereas ir α-MSH first appeared in the cytoplasm of several ir ACTH cells in the basal part of the arcuate nucleus of the hypothalamus on Day 19.5. When the medial-basal hypothalamus of 12.5-day embryos was cultured alone, a few ir ACTH cells were found after culture for 10 days, but not 3 days, and no ir α-MSH cells were observed in the cultures. When the hypothalamus was cultured with Rathke's pouch (intact or without the intermediate lobe anlage), ir ACTH cells appeared within 3 days. In these cultures on Days 6 and 10, long beaded fibers were seen projecting from cells in the neuronal tissue, and some cells showed immunolabeling for α-MSH. When the hypothalamus was cocultured with oral epithelium instead of Rathke's pouch, the appearance of neuronal ir ACTH cells was like that in cultures of hypothalamus alone. These in vitro findings suggest that stimulus from the anterior lobe anlage of the pituitary is necessary for normal development of ir ACTH α - MSH cells in the hypothalamus.

ACTH-immunoreactive buotons form synaptic contacts in the hypothalamic arcuate nucleus of rat: evidence for local opiocortin connections

ACTH-like immunoreactive structures were localized in rat hypothalamic arcuate nucleus using the unlabeled antibody, peroxidase-antiperoxidase method. At the ultrastructural level, immunoreactive presynaptic nerve terminals were observed forming symmetrical synaptic contacts with unlabeled dendrites and with ACTH-like immunoreactive perikarya. The results obtained are consistent with the hypothesis that ACTH in the brain acts as a synaptic regulator or transmitter, contributing not only to long projection pathways but also to a local circuit in the arcuate nucleus where ACTH cell bodies are localized.

Ultrastructural aspects of the effect of calcium ionophore A23187 on incubated anterior pituitary cells of rats.

In order to study the fine structural effect of calcium influx on secretory activity of rat anterior pituitary cells, small pieces of anterior pituitary were incubated in Krebs' medium containing the calcium ionophore A23187 (0.15 mM) and were examined electron microscopically. Marked changes were present in all types of secretory cells incubated for 3, 12 and 20 min in the medium containing calcium and A23187. Secretory granules tended to accumulate in the peripheral cytoplasm of the secretory cells, and more numerous images of granule release by exocytosis were observed in somatotroph (STH cell), luteotroph (LTH cell), thyrotroph (TSH cell), corticotroph (ACTH cell), type 1 gonadotroph (Type 1 GTH cell), and type 2 gonadotroph (Type 2 GTH cell). In addition to the increase in the number of exocytosis of single granules, the simultaneous extrusion of multiple granules, "multigranular exocytosis", was often observed in all kinds of secretory cells, especially the ACTH-cells. Large numbers of granule cores were often located in large vacuole-like or channel-like structures, irregular in shape and size, which were open to the intercellular or pericapillary space. Some parts of the membrane of the vacuole-like or channel-like structures were coated. These observations are interpreted to suggest that the calcium influx stimulates the extrusion of the secretory granules by single or multigranular exocytosis.

Pituitary hyperplasia. Definition, light and electron microscopical structures and significance in surgical specimens.

Surgical specimens of 15 normal and 106 para-adenomous anterior pituitaries were studied immunocytochemically and in part electron microscopically for the presence of hyperplasia. GH cell hyperplasia was found in 13% of all normal pituitaries, in 6% of the cases with Prolactin secreting adenomas and in 9% of the cases with ACTH secreting adenomas. Prolactin cell hyperplasia occurred in nearly equal percentages (17-23%) in normal pituitaries and in areas adjacent to GH-, Prolactin- or ACTH-secreting adenomas or adjacent to inactive adenomas. Previous findings of relatively more frequent Prolactin cell hyperplasia occurring together with Prolactin producing adenomas have to be revised. Prolactin cell hyperplasia as a primary source of hyperprolactinemia is very rare and almost always occurs in conjunction with oncocytic adenomas. ACTH cell hyperplasia was found in 13% of the normal pituitaries, in 14% of the cases with Prolactin secreting adenomas, in 58% of the cases with ACTH producing adenomas and in 40% of the pituitaries with GH secreting adenomas. We have no explanation for the latter result. ACTH cell hyperplasia may be the primary cause of Cushing's disease (18% of all Cushing cases). Hyperplasia of TSH cells in normal pituitaries was rare (7%) and with the exception of Prolactin producing adenomas (22%) was not found near adenomas. Clinical-pathological correlations are discussed.

Ultrastructural characterization of human corticotrophs (ACTH cells).

Ultrastructural characterization of human corticotrophs (ACTH cells) was performed by 'superimposition technique', which enabled detailed ultrastructural observation of immunoreactive ACTH cells in adjacent semi-thin light microscopic immunoperoxidase and routine electron microscopic sections. The human corticotrophs were large and round or polygonal and were not stellate. They had scanty rough endoplasmic membranes and were packed with numerous large secretory granules measuring from 250 to 500 nm in diameter. The sizes of secretory granules in 6 human pituitaries were 448 +/- 128, 344 +/- 86, 448 +/- 117, 244 +/- 65, 316 +/- 76, and 340 +/- 93 nm, respectively. The granules were not seen in a single row along the plasma membrane as is the case in the rat. They possessed somewhat irregular outlines with a rarely discernible halo. Different densities of granule matrices were occasionally found. The cells often contained a few large heterogeneous vacuoles. From these findings, the human ACTH cells were recognized to be remarkably different in cell shape and size, properties of secretory granules and cytoplamic inclusions from those of the rat pituitary gland. In respect to secretory granule properties, the human ACTH cells are similar to those of some other mammals (fox, young pig, and lerot). More data is required to elucidate the relationship between human ACTH cell morphology and functional state.

An organ culture study on the site of determination of ACTH and LH cells in the rat adenohypophysis.

In fetal rats, immunoreactive ACTH and LH cells are known to appear first near the ventral periphery of the pars distalis. The objective of this study was to test whether the developmental fate of these types of cells is actually determined in the ventral region of the developing adenohypophysis. Before the appearance of any immunoreactive cells (days 12.5–13.5), the adenohypophysial primordia of fetal rats were cut at different levels into dorsal and ventral parts and were then cultivated with surrounding tissues. When separated on day 12.5, ACTH and LH cells were immunohistochemically demonstrated almost exclusively in expiants of the dorsal half. Furthermore, a considerable number of ACTH and LH cells were found even in cultures of the most dorsal small region, i.e., the primordial roof accompanied by the brain. Cultivation of “roofless” primordia on the other hand, resulted in a marked decrease in the number of both types of cells. When adenohypophysial primordia were isolated and cut on day 13.5, a number of ACTH cells were observed alike in expiants of both the dorsal and ventral halves. On the other hand, LH cells were found almost exclusively in those expiants derived from the ventral half. The fact that the majority of future LH cells were included within the ventral bulgings called the lateral lobes was also shown either by their cultivation or removal. These results suggest that the developmental fate of ACTH and LH cells is determined in the dorsal region. A possible change in the localization of such determined cells from the dorsal to ventral portion of the pars distalis is discussed in relation to adenohypophysial growth during development.

Effects of brain and mesenchyme upon the cytogenesis of rat adenohypophysis in vitro. I. Differentiation of adrenocorticotropes.

This study investigates the role of the developing diencephalic floor or mesenchymal tissue in the differentiation of ACTH-producing cells. The adenohypophysial primordia of fetal rats on days 12.5 and 13.5 of gestation were treated with collagenase; some primordia were allowed to retain an association with the brain and mesenchyme, but in others the brain and/or mesenchyme were removed. These different combinations of tissues were cultured and examined by immunohistochemical techniques using antisera against pACTH and synthetic alpha-MSH. Removal of mesenchyme alone had little effect on the development of ACTH cells as compared to primordia maintained with brain and mesenchyme. In contrast, removal of the brain with or without mesenchyme on day 12.5 resulted in a marked decrease of ACTH cells accompanied by a mal-growth of adenohypophysial tissue. Such changes were slight when the brain was separated from day 13.5 primordia. Immunoreactive alpha-MSH cells were sparse or absent in all cases. These results suggest that in fetal rats the developing diencephalic floor is essential for differentiation of ACTH cells before day 13.5 of gestation whereas mesenchyme has no apparent effect.