Ultrastructure of the Non-granulated Hypophysial Cells in the Teleost Pseudocrenilabrus philander (Hemihaplochromis philander), with Particular Reference to Cytological Changes in Culture

Abstract In the intact gland, non-granulated cells are most numerous in the rostral pars distalis; they are characterized by a lack of typical secretory granules, a ribosome-rich cytoplasm and long cytoplasmic processes. The latter extend between the granulated cells and often form a layer beneath the capsule and at the zone of contact between adenohypophysis and neurohypophysis. Study of cultured hypophysial tissue provides unequivocal evidence for the phagocytosis of cytoplasm of granulated cells by non-granulated cells. In short-term cultures (1.5 h), non-granulated cells envelop small portions of granulated cell cytoplasm and their processes extend into intercellular spaces and contact tissue fragments located there. In longer term cultures (6—120 h), large areas of cytoplasm belonging to presumptive prolactin and somatotropic cells are engulfed by non-granulated cells; these phagosomes undergo progressive breakdown to form residual bodies. It is suggested that an accumulation of cellular debris and/or stale secretory material may be important factors promoting phagocytic activity in cultured non-granulated cells.     

The relationship between the folliculo-stellate network and the thyrotropic cells of the avian adenohypophysis

Summary The folliculo-stellate network of the avian adenohypophysis consists of stellate cells surrounding colloid-containing follicular cavities into which cilia and microvilli project. Other identifying criteria are agranularity, junctional complexes at the apical pole, presence of cytoplasmic processes ramifying between adjacent secretory cells, and close appositions of plasma membranes linking folliculo-stellate cells and presumptive thyrotropic cells.Transmission electron microscopy reveals that TRH and L-DOPA induce simultaneous ultrastructural changes in the folliculo-stellate network and in the thyrotropic cells. TRH transforms at cell of the cephalic lobe into a highly hypertrophic cell in which enlargement of cisterns of rough endoplasmic reticulum containing secretory granules, development of a large Golgi complex, presence of newly synthesized secretory granules, and granulation of the cytoplasm are the main features. In the meantime, the follicular cavities become dilated by large amounts of homogeneous colloid. The administration of L-DOPA also leads to the development of dilated cisterns in presumptive thyrotropic cells of the cephalic lobe. Intracisternal granules, immature secretory granules, and large Golgi complexes, however, are not observed. Degranulation of the cytoplasm is obvious. The follicular cavities of both cephalic and caudal lobes are enlarged and filled with colloid in which granular elements are noted.The ultrastructural changes observed in thyrotropic cells and in the folliculo-stellate network reflect functional changes induced by the experimental manipulation. These changes may be related, directly or indirectly, or completely independent.     

An electron microscopic study of stellate cells and cavities in the pars distalis of frog pituitary

Stellate cells in the pars distalis of adult Rana ridibunda were observed electron microscopically under normal and experimental conditions (TRH injection). The stellate cells have lengthy processes extending into the intercellular spaces between the secretory cells and scanty cytoplasm surrounding the nucleus. Occasional desmosomes link stellate cells to adjacent secretory cells. In the pars distalis of animals injected with thyrotropic-releasing hormone (TRH), the stellate cells form large cavities (2-6 mum) filled with heterogeneous material. Their cytoplasm contains well-developed Golgi complexes and some lysosomes; these are the principal morphological alterations as compared to those observed in control animals. It is suggested that stellate cells could play an active role in addition to providing a structural framework for the pars distalis.     

Ultrastructure of the adenohypophysis in the larval anadromous sea lamprey,Petromyzon marinus L.

The ultrastructure of the adenohypophysis (AH) in the larval anadromous sea lamprey, Petromyzon marinus L., was examined. The AH is subdivided into three regions, the pro-, meso-, and meta-AH. Cells of the nasopharyngeal stalk extend directly beneath the pro- and meso-AH to form the ventral surface of the gland. Some cells in the pro- and meso-AH are arranged into small follicles. Each region of the AH is characterized by a single granulated (secretory) cell type. Granulated cells constitute 80–90% of the pro-AH and contain secretory granules that range from 800 to 2400 Å in diameter. Only 10–20% of the cells in the meso-AH are granulated and they contain much smaller secretory granules (400 to 1250 Å diameter) than those in the pro-AH. Granulated cells constitute 80–90% of the meta-AH and contain only a few secretory granules, ranging from 1000 to 2500 Å in diameter, and many vesicles containing either a loose flocculent or dense granular material. Nongranulated (stellate) cells are found in all regions. They are characterized by their long cell processes, abundant cytoplasmic filaments, and variable electron density. The appearance of organelles in these cells suggests they are nonsecretory. They may play a role in maintaining the structural integrity of the gland and the regulation of granule release in the pro-AH. Two types of nongranulated cells make up 80–90% of the meso-AH. Type I are stellate cells, type II may be undifferentiated cells. The functional significance of the secretory cells in the larval AH is discussed.     

Stellate cells as phagocytes of the anuran pars distalis

When the stellate cells (nongranulated cells) from dissociated-cell preparations of the anuran pars distalis were examined, they were seen to contain debris within phagocytic vacuoles (phagosomes). These phagosomes were variable; some contained granules from secretory cells while others were similar to lipid-like bodies and myelin figures. In situ partes distales from frogs were examined at the breeding season. The tissues were divided into lobules that were bounded by processes of stellate cells located between the secretory cells. Processes of stellate cells in the interior of a lobule interdigitate with processes extending inward from the stellate cells forming the border of the lobule. When these processes come together, a small cavity is formed. In many of the intact frogs the spaces between the stellate and secretory cells were greatly enlarged. At this particular time the processes of the stellate cells were attenuated and enclosed secretory granules that were also present as debris in these dilated, intercellular spaces. Within the cytoplasm of these stellate cells were not only phagosomes containing secretory granules but also organelles that appeared to be lipid bodies and lysosomes. Thus, the stellate cells of the pars distalis function in vivo, as well as in vitro, as phagocytes. In addition, macrophage-like cells moving from the blood may form another component of this system of phagocytes.     

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.     

Distribution of microtubules and microfilaments in exocrine (ventral prostatic epithelial cells and pancreatic exocrine cells) and endocrine cells (cells of the adenohypophysis and islets of Langerhans). The relationship between cytoskeletons and epithelial-cell polarity

We investigated the distribution of microtubules and microfilaments in some exocrine and endocrine cells in rats. Microtubules were stained by applying an immunofluorescent technique using antibodies against beta-tubulin, while microfilaments were stained with rhodamine-phalloidin, which binds selectively to polymerized actin filaments. In the cytoplasm of some exocrine cells (pancreatic acinar cells and ventral prostatic epithelial cells), the microtubules were distributed longitudinally from the apical region to the basal region, but no microtubules were found in the nuclear region. In exocrine cells, most of the microfilaments were localized beneath the apical plasma membrane. In some endocrine cells (those of the adenohypophysis and the islets of Langerhans), the microtubules exhibited a radial or reticular distribution in the cytoplasm, and intense fluorescence was observed in the perinuclear region. The immunofluorescence produced by the antibodies against beta-tubulin was more intense in endocrine cells than in exocrine cells. The microfilaments observed in the endocrine cells studied were homogenously distributed beneath the plasma membrane. Dot-like rhodamine-phalloidin staining was often observed in the cytoplasm of both the exocrine and endocrine cells. The present study clearly demonstrated marked differences in the distribution of cytoskeletal elements in exocrine and endocrine cells, and these may reflect differences in the secretory direction of such cells as well as in epithelial-cell polarity.     

An immunocytochemical study of effects of light deprivation on prolactin cells in the adenohypophysis of the golden hamster.

Population ratio and morphology of prolactin cells were studied by employing immunohistochemical methods in the adenohypophysis of normal and experimental golden hamsters of both sexes at 16 weeks of age. Prolactin cells occupied 29% of the total adenohypophyseal cells in the intact males exposed to 14/10 h light/dark schedule. After stimulation of the pineal activity by binding or exposure of males to continuous darkness for eight weeks, prolactin cells became atrophic and were reduced in population to 17% and 13%, respectively. Pinealectomy prevented to some extent the effects of the above treatments; thus, prolactin cells constituted 27% in the pinealectomized and blinded hamsters, and 19% in the pinealectomized and darkness-treated group; and their morphology was comparable with that of the intact controls. Prolactin cells in the normal females were apparently larger in size and more numerous as compared with those of the normal males, comprising 47% of cell population in the anterior pituitary. In response to light deprivation, prolactin cells were atrophic with a diminished cytoplasm and decreased in cell number as reflected in the population ratio of 27% in the blinded and 21% in the darkness-treated groups. In pinealectomized females combined with binding or darkness-treatment, prolactin cells contained an abundance of secretory granules in the cytoplasm and maintained the population ratio comparable to that in the intact females. The present study revealed that light deprivation exerts an inhibitory effect on the secretory activity of prolactin cells and also causes hypoplasia of prolactin cells in the hamster adenohypophysis, the effects being mediated by the pineal gland.     

Hepatic stellate cell (vitamin A-storing cell) and its relative--past, present and future

HSCs (hepatic stellate cells) (also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells or Ito cells) exist in the space between parenchymal cells and liver sinusoidal endothelial cells of the hepatic lobule and store 50-80% of vitamin A in the whole body as retinyl palmitate in lipid droplets in the cytoplasm. In physiological conditions, these cells play pivotal roles in the regulation of vitamin A homoeostasis. In pathological conditions, such as hepatic fibrosis or liver cirrhosis, HSCs lose vitamin A and synthesize a large amount of extracellular matrix components including collagen, proteoglycan, glycosaminoglycan and adhesive glycoproteins. Morphology of these cells also changes from the star-shaped SCs (stellate cells) to that of fibroblasts or myofibroblasts. The hepatic SCs are now considered to be targets of therapy of hepatic fibrosis or liver cirrhosis. HSCs are activated by adhering to the parenchymal cells and lose stored vitamin A during hepatic regeneration. Vitamin A-storing cells exist in extrahepatic organs such as the pancreas, lungs, kidneys and intestines. Vitamin A-storing cells in the liver and extrahepatic organs form a cellular system. The research of the vitamin A-storing cells has developed and expanded vigorously. The past, present and future of the research of the vitamin A-storing cells (SCs) will be summarized and discussed in this review.     

The structure and differentiation of granulated metrial gland cells of the pregnant mouse uterus

Summary A study was made with the light and electron microscopes of the granulated metrial gland cells of the decidua basalis of the pregnant mouse uterus, up to day 11 of pregnancy. The granulated metrial gland cells are large, up to 50 in diameter, mono- or binucleate and the glycogen rich cytoplasm typically contains many large glycoprotein granules which may be up to 5 in diameter. Morphological evidence is described in support of a lymphocyte-like cell being the precursor to the granulated metrial gland cell. This differentiation sequence is similar to that already proposed in the rat but differences between the ultrastructure of the mature metrial gland cells of rats and mice were noted.     

Extravascular circulation in the pituitary of Mugil cephalus (Teleostei)

Summary Extravascular circulation in the pituitary of Mugil cephalus was investigated by injecting live fish with horseradish peroxidase and studying the distribution of the enzyme in the gland. The principal components of the extravascular circulatory system are the pericapillary spaces, and, arising from them, the interlobular and circumhypophyseal spaces. Extensions of these spaces penetrate the glandular parenchyma of the pars distalis, where they merge with pericellular spaces. In the neurohypophysis, pericapillary spaces are connected to the periaxonal spaces.Capillaries penetrating from the proximal neurohypophysis into the pars distalis are accompanied by neurosecretory axons. These axons form a mass of tissue which is limited near the capillaries by the pericapillary spaces and near the adenohypophysis by the interlobular spaces. Toward the interior of the adenohypophysis the amount of nervous tissue accompanying the capillaries progressively diminishes, thus reducing the distance between pericapillary and interlobular spaces. Within the pars distalis, the neurosecretory axons accompanying the capillaries are sparse, and the secretory and stellate cells are mostly located directly adjacent to the pericapillary spaces. In the neuro-intermediate lobe, interlobular spaces outline the neuro-adenohypophyseal boundary.The relationship between extravascular spaces and hormone-secreting cells varies in the different regions of the adenohypophysis depending upon the type of neurosecretory innervation in the respective region. In the regions of prolactin and gonadotropin cells, where neurosecretory axons are in direct contact with the secretory cells, the hormone-secreting and stellate cells are adjacent to the pericapillary spaces. In the regions of ACTH and STH cells, secretory and stellate cells are found adjacent to the interlobular spaces, which are interposed between the cells and the neurosecretory axons.Abbreviations AH adenohypophysis - CH circumhypophyseal - DNH distal neurohypophysis - HRP horseradish peroxidase - NH neurohypophysis - NS neurosecretory - PD pars distalis - PI pars intermedia - PPD proximal pars distalis - RNH rostral neurohypophysis - RPD rostral pars distalis This research was supported by a grant from the National Council for Research and Development, Israel, and the GKSS Geesthacht-Tesperhude, Federal Republic of Germany     

Tooth formation and the 28,000-dalton vitamin D-dependent calcium-binding protein: an immunocytochemical study

Antiserum to the 28-kilodalton vitamin D-dependent calcium-binding protein (CaBP) was used to localize CaBP in histologic sections of the continuously erupting incisor in mandibles obtained from normal rats. With the peroxidase--anti-peroxidase technique, no CaBP was detected in undifferentiated ameloblasts or in those which had become columnar and were facing pulp. Calcium-binding protein was first noted in the cytoplasm of random ameloblasts facing dentin in the presecretion zone. As the ameloblasts became more mature in the zone of enamel secretion, CaBP was uniformly present in their cytoplasm. Ameloblasts with Tome's processes clearly contained CaBP in these processes as well as in the cell-body cytoplasm. Near the later developmental stages of the zone of enamel secretion, some of the adjacent underlying cells of the stratum intermedium also contained CaBP in their cytoplasm. In some stratum intermedium cells and papillary cells, CaBP extended into the zone of enamel maturation, but not to the end of that zone. Cytoplasmic CaBP continued to be present in ameloblasts as they progressed through the zone of enamel maturation to the final, shortened cells at the gingival margin of the erupting incisor. No CaBP was detected in odontoblasts, pulpal cells, the stellate reticulum, or the outer dental epithelium.     

Differentiation of liver epithelial (stem-like) cells into hepatocytes induced by coculture with hepatic stellate cells

The liver is believed to contain stem cells that can differentiate into either hepatocytes or biliary epithelial cells. In the present study, we established a nonhepatocytic epithelial cell line from the normal livers of adult rats. The established cells, designated HSL cells, were immunoreactive against alpha-fetoprotein, but neither albumin nor cytokeratin 19. To demonstrate the differentiation potential of HSL cells in vitro, the cells were cocultured with hepatic stellate cells as a mixture or separately using insert wells. Consequently, although coculture with hepatic stellate cells rendered HSL cells able to produce albumin, the mixed coculture system mimicking the hepatic environment elicited this phenomenon more effectively than the separated coculture system. In conclusion, HSL cells have immature properties and the potential to differentiate into mature cells. Not only the extracellular matrices but also soluble factors, which are produced by hepatic stellate cells, induce this maturation, demonstrating the importance of the hepatic environment for hepatocyte differentiation.     

Immunohistochemical characterization of pituitary stellate cells in rats

Pituitary stellate cells from the normal adult male rats were immunohistochemically investigated at the light microscopical level by the use of rat TSH-beta, porcine ACTH1-39, porcine ACTH17-39, rat FSH and ovine FSH antisera. They were characterized by the stellate shape and a mimic engulfment of acidophils. In the present study, they were identified to be the ACTH cells but some were TSH cells. Although most of the corticotrophs showed a peripheral fringe immunostained with the porcine ACTH17-39 antiserum, some others were stained diffusively throughout the cytoplasm. The latter cells coincided, in shape and in homogenous stainability of the cytoplasm, with the stellate TSH cells. Both cells did not correspond but were independent in distribution at the same site of the gland on the adjacent two sections. The stellate type of FSH cells could react with the ovine FSH antiserum, but not with the rat FSH antiserum. Absorption tests of the ovine FSH, procine ACTH1-39 and procine ACTH17-39 antisera were carried out by an application of procine ACTH. In consequence, the porcine ACTH)-39 and porcine ACTH17-39 antisera were absorbed efficaciously by the ACTH antigen at the dose of 10 micrograms/ml, but the ovine FSH antiserum was not enough absorbed by ACTH in the doses of less than 1 mg/ml. It was not finally concluded whether or not the single stellate cells produced ACTH and FSH.     

Access and distribution of exogenous substances in the intercellular clefts of the rat adenohypophysis

In model experiments with the use of horseradish peroxidase (HRP), two pathways of transport of substances to the adenohypophysis were studied, as well as the distribution of the tracer in the latter organ. The first pathway allows the tracer to penetrate from the intercellular milieu of the median eminence below the meningeal sheath covering the adenohypophysis to the surface of the pituitary gland. The second pathway transports the tracer via the capillaries of the hypophysial portal circulation to the interior of the glandular parenchyma. These results show (i) that the meningeal sheath establishes a barrier between the hemal milieu of the pituitary and the hemal milieu of the general circulation, and (ii) that the tracer reaching the adenohypophysis via both routes is found in the intercellular clefts of the glandular parenchyma only to a limited extent. By means of conventional electron microscopy, intercellular contacts between hormone-producing adenohypophysial cells are observed resembling focal tight junctions. Between the membranes of entwined processes of stellate cells, only small maculae adhaerentes are found. Freeze-etch studies on unfixed adenohypophyses reveal zonulae occludentes between the durafacing layers of the meningeal sheath and focal maculae occludentes between parenchymal cells. Additional tissue-culture experiments with adenohypophysial cells directly exposed to HRP reveal a gradual cessation of the labeling process in the intercellular clefts in accord with the observations from the in-vivo experiments, as well as intercellular focal tight junctions between individual hormone-producing cells.     

Histochemical and cytochemical demonstration of Ca++-ATPase activity in the stellate cells of the adenohypophysis of the guinea pig

The histo- and cytochemical localization of Ca++-ATPase activity in the adenohypophysis of the guinea pig was studied utilizing a newly developed method (Ando et al. 1981). An intense reaction was observed in the wall of the blood vessels and between non-secretory cells (stellate cells) and endocrine cells of the pars distalis. Under the electron microscope the Ca++-ATPase reaction product was located extracellularly in relation to the plasmalemma of the stellate cells. This reaction was dependent on Ca++ and the substrate, ATP, and reduced by the addition of 0,1 mM quercetin to the standard incubation medium. Preheating of the sections before incubation completely inhibited the enzyme activity. When Mg++ in different concentrations were substituted for Ca++ in the incubation medium the reaction was always reduced. Both Ca++ and Mg++ in the incubation medium also reduced the reaction. The plasmalemma of the endocrine cells contains no demonstrable amount of Ca++-ATPase activity. The function of the Ca++-ATPase activity is discussed in relation to the regulation of the extracellular Ca++ concentration which seems to be important with respect not only to the secretory process of the endocrine cells but also to the metabolism of the adenohypophysis.     

Ultrastructure of the nerve cells and fibres in the urinary bladder wall of the cat.

The nerve elements in the urinary bladder of the cat were studied by electron microscopy. According to their ultrastructure, nerve cell somata can be classified into three types: the large cells with a cytoplasm rich in organelles, several processes and numerous synaptic contacts on their surface; the cytoplasm contained 80- 120-nm granulated vesicles. The second type is poor in cytoplasmic organelles and has very few processes and virtually no synaptic contacts on the soma. The third type contains numerous large 160- to 220-nm 'neurosecretory' vesicles in the cytoplasm. According to the morphology of the vesicle population, four types of nerve processes could be distinguished: Type a, with a dominant population of small (40-60 nm) agranular vesicles. These are thought to be sacral parasympathetic fibres. Type b, with small (40-60 nm) granular vesicles, which may be the noradrenergic sympathetic fibres. Type c, with 80- to 120-nm granulated vesicles, probably of local origin. Typed d, with large 160- to 220-nm 'neurosecretory' vesicles also of local origin. Different types of nerve fibres are converging on the local nerve cells. This suggests that the local circuits can play an important role in coordinating the function of the bladder. Therefore, ganglia may be considered as an elementary functional unit.     

Classifications of somatotropes, lactotropes and corticotropes in the mouse adenohypophysis with immunohistochemistry

Somatotropes, lactotropes and corticotropes of adult male mice were identified with immunohistochemistry in the adenohypophysis fixed by OsO4 alone. Somatotropes were classified into type I somatotropes that contain large (350 nm in diameter) round secretory granules and type II somatotropes that contain small (100-200 nm in diameter) round secretory granules. Most somatotropes were type I somatotropes. Lactotropes were also classified into type I lactotropes that contain irregularly shaped secretory granules and type II lactotropes containing small (100-200 nm in diameter) round secretory granules. Corticotropes are irregular stellate or slender cells with little cytoplasm. They contain round solid secretory granules in various densities along the cell periphery. Most of these are low-density granules (200-300 nm in diameter) and a few are high-density granules (200-250 nm in diameter). These data were compared with the classical data of mouse adenohypophysial cells that were fixed in OsO4 alone and identified only by conventional electron microscopy.     

Distribution of microtubules and microfilaments in exocrine (Ventral prostatic epithelial cells and pancreatic exocrine cells) and endocrine cells (Cells of the adenohypophysis and islets of Langerhans)

Summary We investigated the distribution of microtubules and microfilaments in some exocrine and endocrine cells in rats. Microtubules were stained by applying an immunofluorescent technique using antibodies against -tubulin, while microfilaments were stained with rhodamine-phalloidin, which binds selectively to polymerized actin filaments. In the cytoplasm of some exocrine cells (pancreatic acinar cells and ventral prostatic epithelial cells), the microtubules were distributed longitudinally from the apical region to the basal region, but no microtubules were found in the nuclear region. In exocrine cells, most of the microfilaments were localized beneath the apical plasma membrane. In some endocrine cells (those of the adenohypophysis and the islets of Langerhans), the microtubules exhibited a radial or reticular distribution in the cytoplasm, and intense fluorescence was observed in the perinuclear region. The immunofluorescence produced by the antibodies against -tubulin was more intense in endocrine cells than in exocrine cells. The microfilaments observed in the endocrine cells studied were homogeneously distributed beneath the plasma membrane. Dot-like rhodamine-phalloidin staining was often observed in the cytoplasm of both the exocrine and endocrine cells. The present study clearly demonstrated marked differences in the distribution of cytoskeletal elements in exocrine and endocrine cells, and these may reflect differences in the secretory direction of such cells as well as in epithelial-cell polarity.     

A new approach to three-dimensional reconstructed imaging of hormone-secreting cells and their microvessel environments in rat pituitary glands by confocal laser scanning microscopy.

There has been considerable interest in the relationship between hormone- secreting endocrine cells and their microvessels in human pituitary gland. However, microcirculatory networks have rarely been studied in three dimensions (3D). This study was designed to visualize and to reveal the relationship between hormone-secreting endocrine cells and their microvessel environment in 3D, using rat pituitary glands under various (hyper/hypo) experimental conditions by confocal laser scanning microscopy (CLSM). Female adult Wistar rats were used after bilateral adrenalectomy or ACTH administration for 2 weeks. Clear 3D reconstructed images of ACTH cells, the microvessel network and counterstained nuclei were obtained at a maximal focus depth of 1 mm by CLSM without any background noise. In the hyperfunctional state, slender cytoplasmic processes of hypertrophic stellate ACTH cells frequently extended to the microvessels. In the hypofunctional state, ACTH cells appeared atrophic and round with scanty cytoplasm, and cytoplasmic adhesions to microvessel network patterns were inconspicuous. Therefore, 3D reconstructed imaging by CLSM is a useful technique with which to investigate the microvessel environment of hormone-secreting cells and has the potential to reveal dynamic hormone-secreting pathways.