Nongranulated cells in the mouse adenohypophysis

Summary Follicular cells in the mouse adenohypophysis were studied electron microscopically. These elements appear to be very similar to the marginal cells that delineate both sides of the hypophyseal cleft.The mouse differs from most other species in that the follicular cells in the pars distalis and the marginal cells look completely inactive in young, intact animals. This makes the mouse exceptionally favorable for correlating morphological changes in the cells of both types with changes in the physiological state of the animal. Different treatments applied in the present investigation all induced morphological reactions in the follicular and/or marginal cells; these reactions were generally similar. Thus, morphological changes in the follicular or marginal cells should be considered as general phenomena accompanying many changes in the physiological state of the animal, rather than as a specific result of the treatment applied.In three experiments, the follicular and marginal cells were involved in the digestion of waste material from other cells. It is suggested that the morphological changes in the other experiments should also be interpreted as signs of such an activity.In the pars tuberalis of the young, intact mouse the follicular cells may show characteristics that in the pars distalis are found only under experimental conditions. Therefore, the follicular cells in this part of the hypophysis are probably in an active state.     

Cell types in the fetal pars tuberalis of the human adenohypophysis at mid-gestation

Summary The pars tuberalis of the adenohypophysis was investigated in three human fetuses at mid-gestation by electron microscopy or immunohistochemistry. In addition to gonadotrophs and thyrotrophs, identified by immunohistochemistry and ultrastructural morphology, electron microscopy revealed the existence of an additional differentiated cell type closely resembling pars tuberalis-specific cells known from other species. The role of this cell type in the human endocrine regulation remains to be elucidated.     

The adenohypophysis of the flounder,Pleuronectes flesus,and the minnow,Phoxinus phoxinus

Summary The pituitary gland of the flounder, Pleuronectes flesus, showed several unusual cytological features. Between the RPD and the PPD was a zone of cells that stained purple with Alcian blue—PAS—orange G. Many of these cells were apparently degenerating. In the PPD the strands and coils of presumptive STH cells showed a tremendous variation in both size and staining properties. In the PI there were two cell types, the PAS-positive one bordering the neurohypophysis. Around the periphery of the PI was a zone of chromophobic cells, and throughout the PI were numerous intracellular and extracellular acidophil spheres.No well defined ACTH cells were found in the RPD of the minnow (Phoxinus phoxinus). The Alcian blue—PAS—orange G technique distinguishes between blue TSH cells and purple GTH cells in the RPD and PPD. GTH cells from animals collected in the winter were vacuolated. The PI contained two cell types whose staining reactions and ultrastructure were extremely variable. Intra- and extra-cellular acidophil spheres were present.I should like to thank Dr. T. Kerr of the Zoology Department, Leeds University, for his help and encouragement, and Mr. J. Dingley of the Department of Zoology, Aberystwyth, for collecting the flounders.     

Immuno-electron-microscopic localization of basic fibroblast growth factor in the dystrophic mdx mouse masseter muscle

Summary The localization of basic fibroblast growth factor (bFGF)-like immunoreactivity in the masseter muscle of dystrophic mdx mice on postnatal day 28 was investigated by immunoblot analysis and electron microscopy. Crude homogenate of the masseter muscle, when subjected to immunoblotting with a bFGF antiserum, exhibited a main band with the same molecular weight (18 kDa) as bovine bFGF. By electron microscopy, bFGF immunoreactivity was detected in small regenerating myocytes; the smaller cells were the premature myocytes, the most intense staining was the immunoreactivity within the cytoplasm. Putative precursors of the muscle cells with a few myofilaments, which were most intensely labeled with anti-bFGF, contacted each other and possibly developed into multinucleated myocytes through cell fusion. Mature myocytes with densely packed myofilaments and peripherally located nuclei did not exhibit bFGF immunoreactivity; they formed myoneural junctions with motor nerve endings immunoreactive for bFGF. Early differentiating myocytes with intense bFGF-like immunoreactivity did not make contact with immunoreactive nerve terminals. Degenerating large myocytes with a limited number of distorted and/or disrupted myofilaments exhibited electron-dense deposits in the cristae of mitochondria; these deposits were not abolished by immunoadsorption control experiments. Thus, the cell-size-dependent decrease in bFGF immunoreactivity in regenerating but not in degenerating myocytes provides a morphological basis for an autoregulatory role of bFGF in muscle regeneration. This study suggests that neuronal bFGF is not involved in initial muscle regeneration in the dystrophic mdx mouse.     

Ultrastructural observations on membranous structures in developing mouse oocytes

Summary Ultrastructural studies have revealed the presence of unusual membrane complexes within developing mouse oocytes. These structures, most obvious 18 days post fertilization, are found in the nucleus or cytoplasm of cells in meiotic prophase. The complexes, usually found in small groups, are characterized by a slightly bowed appearance, and a thin middle section that is vesiculated at each end. At high magnification the middle section exhibits a pentalaminar structure similar to tight junctional complexes, while the looped membranes of the vesiculated ends are trilaminar in appearance. In addition to being free in the nucleoplasm or cytoplasm, the complexes are also seen in continuity with the inner and outer leaflets of the nuclear envelope, and with typical membranes forming cytoplasmic tubular systems. The possible formation of these complexes from blebs or vesicles derived from the nuclear envelope is presented and the role that these structures may play in developing oocytes is discussed.Supported by Louisiana State University Medical School Institutional Grant.Dr. Skalko's current address is Birth Defects Institute, New York State Health Department, Albany, New York. The authors wish to thank Mr. Garbis Kerimian for his excellent photographic work, Mrs. Janell Buck, Mrs. Edna Burgess and Mrs. Eunice Schwartz for their excellent technical and secretarial assistance.     

Immunohistochemical localization of LH-producing cells in the adenohypophysis of the Japanese quail,Coturnix coturnix japonica

Summary The cells that produce luteinizing hormone (LH) in the adenohypophysis of the Japanese quail were identified immunohistochemically using anti-chicken LH serum and horseradish peroxidase-labeled goat anti-rabbit gamma globulin serum. The LH cells are localized in the caudal lobe of the pars distalis. They are elongate in shape and are polarized toward the sinusoids, especially in their active states. Alterations in size of LH cells are directly related to changes in circulating LH levels as induced by castration or photostimulation. The LH cells identified immunohistochemically were only stained by alcian blue with periodic acid-Schiff (PAS), alcian blue and orange G.PAS-positive gonadotropic cells in the cephalic lobe were stained immunohistochemically only slightly if at all using anti-chicken LH serum and consequently may be FSH producing cells. In the cephalic lobe another type of basophilic cell was stained with alcian blue. These cells were also stained immunohistochemically with anti-chicken LH serum. These cells may possibly be identified as TSH cells due to the characteristics of the antichicken LH serum used in this study which cross react with LH and TSH but only slightly with FSH, and also on the basis of previous light and electron microscopic studies.We express our gratitude to Professors Hideshi Kobayashi and Katsumi Wakabayashi for their valuable guidance during the experiment. We also express our cordial thanks to Professor B.K. Follett for the gift of anti-chicken LH serum and standard LH. This work was supported in part by Grants from Ministry of Education of Japan and from the Ford Foundation.     

Plasticity of interstitial cells of Cajal: a study of mouse colon

Ablation of the myenteric plexus in mouse colon with the detergent benzalkonium chloride (BAC) is followed by considerable recovery of the nerves, indicating that this plexus is capable of regeneration and has plasticity. Interstitial cells of Cajal (ICC) are closely associated with enteric nerves, and the acquisition and maintenance of their adult phenotype are nerve-dependent. Little is known about the regenerative processes of ICC or about the possible dependence of these processes on neurons. To address these questions, we ablated the myenteric plexus in the mouse colon with BAC and followed changes in the adjacent ICC (ICC-MP) from day 2 to day 70 after treatment, by using c-kit-immunohistochemistry and electron microscopy. In the untreated area, c-kit-positive cells and ICC-MP with normal ultrastructural features were always present. The region partially affected by BAC contained some c-kit-positive cells, and either normal or vacuolated ICC-MP were observed by electron microscopy. Moreover, at days 60–70, ICC-MP with particularly extended rough endoplasmic reticulum were present in this area. In the treated area, either denervated or reinnervated, c-kit-positive cells were always absent. By day 14 after BAC treatment, nerve fibers had started to grow back into the treated region and, in the reinnervated area, cells with fibroblast-like features appeared and were seen to contact both nerve endings and smooth muscle cells and to acquire some typical ICC features. Thus, ICC are vulnerable to external insult but appear to have some ability to regenerate.This work was supported by the US-Israel Binational Science Foundation (BSF, 98-00185; to M.H.) and University funds “quota di ateneo ex 60%” (M.-S. F.-P.).     

Collagen remodeling during decidualization in the mouse

Summary An ultrastructural study of the features and distribution of collagen fibrils was performed in the endometrium of virgin and pregnant (2nd to 11th day) mice. Collagen-containing structures were observed in the cytoplasm of fibroblasts on the 2nd day of pregnancy. Treatment of tissues with lanthanum nitrate established that these structures were intracytoplasmic. Their association with lysosome-like bodies suggested the occurrence of intracellular digestion of collagen, probably connected with remodeling of the endometrial stroma prior to decidualization. On the 4th day of pregnancy, very few collagen fibrils were present in the intercellular space. From the 6th day of pregnancy onwards, thick collagen fibrils were observed between decidual cells. The diameter of these fibrils measured up to 300 nm whereas the fibrils present in the endometrium of virgin mice measured 40–68 nm.     

Myelinated nerve cell perikaryon in mouse spinal cord

Summary In the thoracic cord (posterior horn region) of a wild mouse, we have observed a small nerve cell soma completely enveloped by a myelin sheath. The number of myelin lamellae varied between 7 and 12. In one place, the existence of an inner mesoperikaryon could also be shown. The significance of this fortuitous finding has not yet been explained.

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Zusammenfassung Im Thorakalmark (Hinterhornbereich) einer Wildmaus wurde ein kleines Nervenzellperikaryon beobachtet, das vollständig von einer Markscheide umhüllt war. Die Zahl der Markscheidenlamellen variierte zwischen 7 und 12. An einer Stelle konnte ein sogenanntes inneres Mesoperikaryon nachgewiesen werden. Die Bedeutung dieses zufällig erhobenen Befundes ist vorerst noch offen.

     

Effect of cyproterone acetate on cells of the pars distalis of the adenohypophysis in the Beagle Bitch

Summary The effects of oral administration of 100 mg per kg per day cyproterone acetate (CPA) for four weeks on cells of the pars distalis, as revealed by the immunoperoxidase technique and chemical staining, were studied in the ovariectomized beagle bitch. For immunochemical staining antisera to the following hormones were used: canine GH, canine PRL, porcine ACTH, bovine TSH, bovine LH and human FSH¹. The most striking effects of the treatment were an overall increase in the relative proportion of GH cells and a marked morphological indication of high secretory activity in these cells. In contrast, PRL cells were not affected significantly. In all ovariectomized control bitches a marked atrophy of the cells stained for FSH (FSH cells) and hypertrophy of the cells shown to contain LH(LH cells) were observed. FSH cells became enlarged, while LH cells appeared reduced in size by administration of CPA. In some treated bitches ACTH/MSH cells showed atrophy and regressive changes, whereas TSH cells seemed to become enlarged and were more densely arranged. These structural responses indicate that, in addition to its partial antigonadotropic properties, CPA as a synthetic progesterone derivative may stimulate GH secretion and possibly suppress CRH-ACTH activity in the ovariectomized beagle bitch.Abbreviations of Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin - CRH Corticotropin Releasing Hormone The authors are grateful to Dr. Christel Schoebel and Mrs. P. Kurth for carrying out the experimental work on animals, to Dr. H. Wiemann for the statistical analysis, to Mrs. B. Schilk, G. Soulioti and Miss U. Tüshaus for their excellent technical assistance, and to Dr. P. Guenzel for his advice and encouragementRecipient of a Research Scholarship from the Arabic Republic of Egypt     

Morphological evidence for a direct innervation of the mouse vomeronasal glands

Summary The morphological evidence for a direct autonomic innervation of the mouse vomeronasal glands is presented. Axonal varicosities containing a few densecore vesicles and numerous clear vesicles (36–60 nm in diameter) make synaptic contacts with the secretory cells at the base of the glandular acini. The axonal presynaptic membrane is associated with a distinct dense material and it is separated from the secretory cell by a synaptic cleft of about 12–14 nm. At the postsynaptical level, coated vesicles can be found. Additional postsynaptical specializations have not been observed.     

Effect of cholecystokinin-pancreozymin (CCK-PZ) on glycoprotein secretion from mouse gallbladder epithelium

Summary Structural changes in the gallbladder epithelial cells of the mouse were studied following in vivo and in vitro stimulation of the gallbladder with the gastrointestinal hormone cholecystokinin-pancreozymin (CCK-PZ). Signs of increased secretory activity were observed within the first 2–3 min after hormone administration. At the ultrastructural level, best visualized with the PA-CrA-silver technique, granule discharge was observed, as was an overall increase in size of the granules. After prolonged in vitro incubation or repeated in vivo stimulation, there was an almost total depletion of secretory granules. This phenomenon is accompanied by an enhanced uptake of extracellular thorium dioxide by endocytotic vesicles at the apical cell surface. An exocytosisendocytosis coupling mechanism may be important for membrane conservation in the gallbladder epithelial cells. The findings establish that the hormone CCK-PZ stimulates the secretion of glycoproteins from the mouse gallbladder epithelium.This investigation was supported by grants from the University of Umeå and from the Swedish Medical Research Council (grant No. B76-12X-04758-01). The authors wish to thank Miss K. Karlsson and Miss M. Borg for skilful technical assistance     

Ultrastructure of the adenohypophysis in the teleost Poecilia latipinna

Summary In the sailfin molly, Poecilia latipinna, seven morphological endocrine celltypes could be distinguished with the electron microscope. Each of these was identified with one of the seven cell-types distinguished with the light microscope, to most of which endocrine functions have previously been allocated. Corticotrophs and prolactin cells form the rostral pars distalis, and the proximal pars distalis consists of an outer layer of gonadotrophs and an inner zone containing growth hormone cells and thyrotrophs. The pars intermedia contains two cell-types, of uncertain function. Stellate cells (interstitial cells) occur throughout the adenohypophysis, but are most numerous and prominent in the rostral pars distalis. The inner proximal pars distalis contains a cell-type not previously distinguished in this species with the light microscope, the Z-cell, which could be aminergic.The ultrastructural features of each cell-type are described in detail, and discussed in comparisons with the homologous cells described in other teleosts. There is good agreement for different teleosts in the ultrastructural details of each cell type.We thank Mr. L. Ethridge, Mr. M. P. Hancock, Mr. D. Hollingworth and Mr. W. Thomson for technical assistance, and Mr. D. Taylor of the Nuffield Institute of the Zoological Society of London for permission to use the Nuffield Institute electron microscope. We are grateful to Dr. Harry Grier, who collected and embedded glands from P. latipinna in its natural fresh-water habitat in Florida, U.S.A. T. Batten is in receipt of an S.R.C. Research Studentship.     

Cell types in the adenohypophysis of the hagfish,Myxine glutinosa (Cyclostomata)

Summary The anterior pituitary of the Atlantic hagfish, Myxine glutinosa, was investigated by transmission electron microscopy. The cells of the adenohypophysis are arranged in follicles surrounded by connective tissue. Five cell types containing granules and one agranular cell type were identified. At the present state of the study the function of these cells remains open to discussion.This study was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (Grant no. 4070) and the Stiftungs- und Förderungsgesellschaft der Paris Lodron-Universität in Salzburg. The authors are grateful to the Director of the Biological Station in Drøbak (Norway), Amanuensis Finn Walvig, for procuring the material     

Intracellular oxygen measurements of mouse liver cells using quantitative fluorescence video microscopy

Our currently developed fluorescence video microscope can measure fluorescence intensities with an error of ±1.5% of full scale in 65 536 different positions of a microscope field. With a video frame freeze acquisition time of 33 ms, time-dependent changes of this order of time or slower can be followed. Using cells which have absorbed pyrene-1-butyrate to an intracellular concentration of 0.05 to 1 mM, the changes in fluorescence intensity with oxygen concentration are easily measured. The spatial resolution for data collection is 0.5 μm when a 54X objective is used. The individual Stern-Volmer quenching constants of each individual pixel were measured for agar slices and mouse liver cells treated with pyrenebutyric acid. The distribution of quenching constants for agar follows a normal curve about a mean value of 16 · 10^?4 torr^?1. The data for mouse liver cells gave a non-normal distribution of quenching constants with a mean value of 18 · 10^?4 torr^?1. The greater spread of the data from cells is interpreted as evidence for a real biological variation in the solubility coefficent of oxygen in different locations within the cell. In all the cells examined, this distribution has been observed to be non-random and appears to be associated with specific cell structures.     

Endothelial cell connecting filaments anchor endothelial cells to the subjacent elastic lamina in the developing aortic intima of the mouse

The ultrastructural association of endothelial cells with the subjacent elastic lamina was investigated in the developing mouse aorta by electron microscopy. In the 5-day postnatal aorta, extensive filament bundles extend along the subendothelial matrix connecting the endothelial cells to the underlying elastic lamina. The connecting filaments form lateral associations with the abluminal surface of the endothelial cells in regions of membrane occupied by membrane-associated dense plaques. On the intracellular face of each plaque, the termini of stress fibers penetrate and anchor to the cell membrane in alignment with the extracellular connecting filaments. Both the stress fibers and the connecting filaments are oriented parallel to the longitudinal axis of the vessel. High magnification electron micrographs of individual endothelial cell connecting filaments reveal features similar to those of elastin-associated microfibrils. Each connecting filament consists of a 9–10 nm linear core with an electron-lucent center and peripheral spike-like projections. From the filaments, small thread-like extensions span laterally, linking the filaments into a loose bundle and anchoring them to the endothelial cell membrane and the surface of the elastic lamina. The filaments also appear heavily coated with electron-dense material; often with some degree of periodicity along the filament length. During development, the number of endothelial cell connecting filaments decreases as the elastic lamina expands and the subendothelial matrix is reduced. In the aortic intima of mature mice, the elastic lamina is closely apposed to the abluminal surface of the endothelial cell and no connecting filaments are seen. These observations suggest that endothelial cell connecting filaments are developmental features of the aortic intima which, together with the intracellular stress fibers, aid to maintain the structural integrity of the endothelial cell layer during development by providing the cells with protection from intraluminal shear forces.     

Myelinated Herring bodies in the posterior part of the median eminence of the mouse

Summary The posterior part of the median eminence of the albino mouse (CF # 1-JCL) contains a cluster of myelinated axons beneath the tanycyte layer. Among them, small Herring bodies surrounded by myelin sheaths are revealed by electron microscopy. These structures contain electron-dense bodies, lamellar bodies, autophagic bodies, autophagic vacuoles, and neurofilaments. A few neurosecretory granules and mitochondria are also present. Some myelinated axons contain mostly accumulated neurosecretory granules.     

Abnormal recruitment of periendothelial cells to lymphatic capillaries in digestive organs of angiopoietin-2-deficient mice

The fine structure of lymphatic capillaries in the digestive organs of angiopoietin-2 (Ang2) knockout mice was studied by using both immunohistochemistry and electron microscopy. The genetic deletion of Ang2 yielded hypoplasia and disorganization of the lymphatic capillaries, with their shapes being irregular, and an aberrant recruitment of vascular periendothelial cells immunopositive for smooth muscle actin to the lymphatic capillaries. The abnormal lymphatic periendothelial cells were considered to be a type of pericyte for the lymphatic capillaries after the deletion of Ang2, because they were ultrastructurally characterized by abundant thin myofilaments in their cytoplasm and long cytoplasmic extensions similar to those shown by blood vascular pericytes. The genetic replacement of Ang2 with Ang1 rescued the defects, viz., the disorganization and disordered structure of the lymphatic capillaries. The present findings suggest that Ang2 serves the morphogenesis of lymphatic capillaries as an agonist for the receptor, Tie2, and that Ang1 can replace Ang2 in guiding lymphatic formation and development. H. Shimoda and M. Witte were supported by an Exchange Visitor Program Grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by Contract 6011 from the Arizona Disease Control Research Commission, respectively.     

Non-uniform distribution of mitochondria in pancreatic acinar cells

The distribution of mitochondria in pancreatic acinar cells was investigated using confocal fluorescence microscopy and transmission electron microscopy (EM). Acinar cells were studied either after enzymatic isolation or in small segments of undisassociated pancreatic tissue. Loading of isolated acinar cells with Mito Tracker Green or Red, a fluorescence mitochondrial probe, showed that mitochondria are predominantly situated in the perigranular, subplasmalemmal and perinuclear regions. Subsequent applications of EM fixatives induced a leak of the fluorescent indicator to the cytosol but did not change the distribution of mitochondria. EM was then performed on isolated acinar cells and on acinar cells of pancreatic tissue segments. The intracellular distribution of mitochondria was quantified by calculating the percentage of the cross-sectional area that was occupied by mitochondria. In isolated acinar cells the highest density of mitochondria was seen in the perigranular region, where mitochondria occupied 25.69±1.58% of the area, then the subplasmalemmal region with 12.61±0.77% and the perinuclear region with 9.07±0.97% (n=26). Similar results were obtained from acinar cells of pancreatic tissue segments: the perigranular 22.9±1.95%, subplasmalemmal 12.45±0.78% and perinuclear regions 9.07±0.97% (n=26). The outer mitochondrial membranes were frequently positioned close to membranes of the ER, which followed the outer contour of mitochondria. Mitochondria were never found in direct contact with the nuclear envelope: there were usually layers of ER between the mitochondrial and nuclear membranes. Subplasmalemmal mitochondria were found in a very close proximity to the plasma membrane with no ER layers between the mitochondrial and the corresponding plasma membranes. We conclude that in pancreatic acinar cells mitochondria are preferentially distributed to perigranular, subplasmalemmal and perinuclear regions and this distribution is not affected by isolation or fixation procedures.P.R. Johnson and N.J. Dolman contributed equally to the study. This work was supported by a Medical Research Council programme grant. P.R.J. is a Medical Research Council PhD student and N.J.D. is a Wellcome Trust PhD student.     

Basal cells in the mouse olfactory epithelium after axotomy: immunohistochemical and electron-microscopic studies

Summary The olfactory epithelium of mice after axotomy was investigated to clarify the stem cells of olfactory cells by double immunostaining using antikeratin (MA903) and anti-bromodeoxyuridine (BrdU) antibodies and by conventional electron microscopy. When a single dose of BrdU was given to mice 9 days after axotomy, immunostaining for BrdU was found in the globose basal cells which were negative for MA903, but not in the basal cells proper which were positive for MA903. The BrdU-immunoreactive cells increased 3-to 6-fold over the number of these cells in the controls, indicating active cell proliferation. At other postoperative days (4 and 14 days), fewer BrdU-immunoreactive cells were found. Furthermore, three pulses of BrdU resulted in numerous BrdU-immunolabelings in the globose basal cells and a few in the basal cells proper. There was no detectable difference in the number of labeled basal cells proper in operated and unoperated mice. In the electron micrographs 9 days after axotomy, the basal cells proper, flat-shaped in unoperated mice, appeared cylindrical or pyramidal in shape and the globose basal cells often lay between the basal cells proper. In unoperated controls, the globose basal cells were located above the flat-shaped basal cells proper. The results suggest that the stem cells of the olfactory cells are globose basal cells and not basal cells proper, and that the shape of basal cells proper changes in relation to the active proliferation of stem cells.