Differential survival of isolated portions of crayfish axons

Summary Electron microscopic studies show that transplanted segments of sensory axons of varying lengths degenerate within 7–14 days whereas transplanted segments of crustacean motor axons survive morphologically intact for 20–30 days. The middle portion of an isolated motor axon segment degenerates less rapidly than portions of the same axon located nearer the periphery or nearer the ventral nerve cord. One week after transplantation, glial cells appear to phagocytize sensory axons whereas glial cells around motor axons appear to hypertrophy and to have more rough endoplasmic reticulum. After three weeks, motor axons also appear to be phagocytized by glial cells.These data suggest that the glia surrounding isolated motor axons can change from a supportive to a destructive function, whereas glial cells surrounding severed sensory axons primarily have a destructive function. These and other data also indicate that crustacean motor axons receive significant trophic inputs from their own perikaryon, from post-synaptic contacts, and from adjacent glial cells. The possibility that adjacent healthy cells may supply metabolically deficient cells with needed substances could be a significant adaptive advantage for the evolution of multicellular organisms.Supported in part by an NIH grant (NS-1186101) to Dr. BittnerThe authors wish to thank Mr. Martis Ballinger and Mr. Robert Riess for their valuable assistance in all stages of this research     

Two types of neuromuscular junctions in the pharyngeal retractor muscle ofHelix lucorum

The pharyngeal retractor muscle of the snailHelix lucorum is innervated by a pair of nerves containing axons of two types, for which there are two corresponding types of myoneural junctions with the muscle cells. The junctions of type I correspond to the thick axons. The terminals of these axons, which contain numerous spherical transparent vesicles (41±5 nm) and fewer vesicles of the dense-core type (67±3 nm), make contact mainly with noncontracting sarcoplasmic projections of the muscle cells. Junctions of type II correspond to thin axons, containing many granules. The terminals of these axons make contact with contractile parts of the muscle cells and they contain a heterogeneous population of vesicles: small spherical clear vesicles (44±2 nm), granules with fine-grained contents (135±5 nm), and a few spherical dense-core vesicles. The distance between the muscle cells is usually great — over 50 nm, but in the region of the sarcoplasmic processes the surface membranes come together to form a gap which in some areas does not exceed 10 nm.N. K. Kol'tsov Institute of Developmental Biology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 5, pp. 539–542, September–October, 1977.     

Dorsal giant fibre septum of earthworm. Fine structural details and further evidence for gap junctions

The fine structure of the dorsal giant fibre septa of earthworm (Lumbricus terrestris and Eisenia foetida) was studied by thin sectioning. E-PTA, BIUL and ZIO impregnation were carried out to characterize the various membrane specializations found. A septum, which is formed by two axon membranes only ca. 7 nm apart, is a heterogeneous structure showing a number of different specializations (intermediate junctions, dense projection-like humps, septate regions, vesicles quite often associated with a widened intercellular gap and membrane thickening). Septal areas referred to as septal complexes are of particular interest. They comprise up to 20% of septal cross-sections and are characterized by membrane appositions (diameter ca. 25 nm) which bridge the septal gap, protrude 16-25 nm into the respective axoplasms, and form a more or less hexagonal array with a centre-to-centre spacing of ca. 33 nm (ca. 29 nm after E-EPTA treatment) in en face sections. We interpret the septal complexes as gap junctions, i.e. sites of electronic coupling, and emphasize their unusually large dimension. The vesicles found at the septum could not be stained by ZIO treatment.     

Regeneration of motor axons in crayfish limbs: Distal stump activation followed by synaptic reformation

Summary Servered distal stumps of limb motor axons in the crayfish Procambarus clarkii remain ultrastructurally intact for at least 2–3 ms after being severed from their cell body. Initial regeneration of a motor axon is associated with the appearance of up to 200 small profiles (satellite axons) having no glial sheath adjacent to the large surviving stump for about 1 cm distal to the lesion at 4–5 wks postoperatively. These satellite axons are seen 2–4 cm distally at the target muscles 3–4 ms postoperatively. By 14–15 ms postoperative, the motor sheaths from the lesion site to the target muscles contain small axonal processes having thick glial sheaths. Behavioral tests show that some axons that are reconnected to the CNS at 4–5 wks may not be connected at 14–15 ms, whereas other axons not connected by 3–4 ms may be connected at 14–15 ms when the original distal stumps have degenerated.We suggest that all these data can best be explained by the view that motor axons in crayfish limbs initially regenerate via activation of the surviving distal stump by satellite axons which grow out from proximal stump. In most cases, these satellite axons continue to activate the surviving distal stump as they slowly grow to the target muscle. Eventually the satellite axons reform synapses on the target muscle and the original distal stump degenerates.This work was supported by NSF grants BNS 77-27678 and 80-22248 and an NIH RCDA 00070 to GDB. The authors would like to thank Mr. Martis Ballinger, Mr. Robert Reiss, and Mrs. Mary Raymond for their excellent technical assistance. We would also like to thank Dr. Wesley Thompson and Mr. Douglas Baxter for helpful discussions.     

Restoration of gap junction-like structure after detergent solubilization of the proteins from liver gap junctions

Gap junctions isolated from rat liver were partially solubilized with a mixture of digitonin and octyl glucoside. After supplementation with lecithin and cholesterol, the octyl glucoside was removed from the soluble fraction by dialysis. The membranes of the reconstituted vesicles, observed in freeze-fracture, contained particles ranging from 7 to 12 nm diameter, more or less aggregated depending on the protein-to-lipid ratio. At every protein concentration, the arrangement of particles in contact areas between adjacent membranes closely resembles the organization of intact gap junctions. We conclude that the mixture of digitonin and octyl glucoside is able to solubilize the proteins of the liver gap junctions while preserving their property of restoring a gap junction-like structure.     

The distribution of orthogonal arrays and their relationship to intercellular junctions in neuroglia of the freeze-fractured hypothalamo-neurohypophysial system

Summary Using freeze-fracture techniques, we have investigated membrane specializations of the glia associated with the hypothalamo-neurohypophysial system of the rat. In the paraventricular (PVN) and supraoptic (SON) nuclei, astrocytes in areas of high neuronal density (i.e., magnocellular regions) display orthogonal arrays of 6–7 nm particles soley near gap junctions, while astrocytes in areas of lower neuronal density (i.e., parvocellular regions) contain additional arrays in membranes not displaying gap junctions. Arrays are especially numerous on astrocytic perivascular end-feet in both nuclei and in the laminations of the pial-glial limitans ventral to the SON. Ependymal cells near the PVN show arrays both on their lateral surfaces (displaying gap junctions) and on their apical surfaces (facing the CSF). Tight junctions are not noted on astrocytes or ependymal cells, but are noted on both the somas and myelin lamellae of oligodendroglia. Both of these latter membranes occasionally contain gap junctions as well; however, orthogonal arrays are never noted on oligodendroglia.The plasma membranes of pituicytes in the neurohypophysis display gap junctions, complex junctions, and tight junctions. Orthogonal arrays are noted near the first two of these, but not near the last. Arrays in the neural lobe appear most dense on membranes adjacent to subpial or perivascular spaces. Pituicyte membranes containing orthogonal arrays appear infrequently near the neural stalk, increasing towards the distal end of the neural lobe. The distribution of orthogonal arrays in this system, as well as in other systems in which they have been noted, suggests a polarization of membrane activity.     

LOW RESISTANCE JUNCTIONS IN CRAYFISH : I. Two Arrays of Globules in Junctional Membranes

The ultrastructure of low resistance junctions between segments of lateral giant fibers in crayfish is studied in sections from specimens fixed either by conventional methods or by glutaraldehyde-H₂O₂ or by glutaraldehyde-lanthanum. Cross sections through junctions fixed by conventional glutaraldehyde display the usual trilaminar profile of two parallel membranes separated by a narrow gap. Most of the junctional regions appear covered by 500–800 Å vesicles which lie on both sides of the junction in rows adjacent to the membranes. Gross sections through junctions fixed by glutaraldehyde-H₂O₂ display, in regions containing vesicles, membranes with a beaded profile. The beads correspond to globules ~125 Å in width and ~170 Å in height arranged in a hexagonal pattern with a unit cell of ~200 Å. The globules of one membrane match precisely with those of the adjacent membrane, and opposite globules seem to come in contact with each other at the center of the junction. The membrane of the vesicles also contains globules. Occasionally the globules of the vesicles seem to join with those of the junctional membranes, apparently forming intracellular junctions. Injunctions negatively stained by lanthanum the globules are seen organized into two arrangements. Areas containing globules in a hexagonal array with a unit cell of ~200 Å (swollen pattern) are seen adjacent to areas in which the globules are more closely and disorderly packed (close packing), the minimum center-to-center distance between adjacent globules being ~125 Å. At higher magnification each globule appears composed of six subunits arranged in a circle around a central region occupied by lanthanum (possibly a pit).     

Ultrastructural studies of severed medial giant and other CNS axons in crayfish

Summary The distal stumps of severed medial giant axons (MGAs) and of non-giant axons (NGAs) in the CNS of the crayfish Procambarus clarkii show long-term (5–9 months) survival associated with disorientation of mitochondria and thickening of the glial sheath. However, the morphological responses of the two axonal types differ in that neither the proximal nor the distal stump of severed MGAs ever fills with mitochondria as is observed in some severed NGAs. Furthermore, the adaxonal glial layer never completely encircles portions of MGA axoplasm as occurs in many severed NGAs; in fact, ultrastructural changes in the adaxonal layer around severed MGAs are often difficult to detect. No multiple axonal profiles are ever seen within the glial sheath of the proximal or distal stumps of severed MGAs whereas these structures are easily located within severed NGAs.This work was supported by NIH research grant #NS-14412 and an RCDA#00070 to GDB     

Fine structure of the ocellus of Sarsia tubulosa (Hydrozoa,Anthomedusae)

Summary The fine structure of the ocellus of Sarsia tubulosa is described. The ocellar cup is formed of pigment cells and receptor cells. The receptor cells outnumber the pigment cells in almost a 2:1 ratio. Lateral extensions of neighbouring pigment cells enclose a distal region of 2 to 10 receptor cells. The receptor cell body is 5–7 m in diameter with an apical extension (20–60 m long) that reaches the ocellar cavity. A cilium (9+2 microtubules) arises from the distal part of the receptor cell. The ciliary membrane forms lateral microvilli. The tips of a number of cilia are swollen into large vesicles forming a cornea. The central region of the ocellar cavity contains extracellular electron dense homogeneous material surrounded by swollen ciliary tips and small vesicles. The close apposition between the plasma membrane covering the distal part of adjacent receptor cells as well as the adjacent ciliary shafts suggests the presence of gap junctions. The basal part of each receptor cell forms an axon. The axons of receptor cells form 3 to 4 nerve bundles that join to form the optic nerve. Synapses occur between receptor cell bodies, between axons and receptor cell bodies and among axons.     

Long-term survival followed by degradation of neurofilament proteins in severed Mauthner axons of goldfish

The morphology and protein composition of intact and severed Mauthner axons (M-axons) from goldfish were examined on electron micrographs, sodium dodecyl sulfate gels, and immunoblots. Neurofilaments were the most common cytoskeletal element on electron micrographs, and neurofilament proteins (NFPs) were the most intensely silver-stained bands in M-axoplasm microdissected from control M-axons. NFPs at about 235, 145, 123, 105, 80, and 60 kD in M-axoplasm were identified with four monoclonal and three polyclonal antibodies. Similar immunoblots of samples of the M-axon myelin sheath (M-sheath) showed no reactivity to antibodies against NFPs. For up to 62 days following spinal cord severance in goldfish maintained at 15°C, the ultrastructure, protein banding pattern, and anti-NFP immunoreactivity of severed distal segments of M-axons did not change compared with control M-axons. At 62 to 81 days after severance, novel bands appeared in many silver-stained gels and anti-NFP immunoblots of distal M-axons. NFP bands completely disappeared from distal M-axon segments of some M-axons as early as 72 days after severance. However, NFP bands persisted in some distal segments for up to 81 days after severance. The degradation of NFPs occurred equally along the entire length of a distal M-axon segment, that is, there was no indication of a proximal-to-distal or distal-to-proximal sequence of NFP degradation in distal segments of severed M-axons. These biochemical data were consistent with morphological data that showed little change in the diameter or ultra-structure of severed M-axons held at 15°C for about 2 months followed by a rapid collapse of the entire distal segment at 72 to 85 days postseverance. 1994 John Wiley & Sons, Inc.     

Intercellular junctions during development and in tissue cultures ofDrosophila melanogaster: An electron-microscopic study

Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis.The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos.Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development.All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae.Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.     

Fine structure of the ependyma and intercellular junctions in the area postrema of the rat

Summary Ependymal cells and their junctional complexes in the area postrema of the rat were studied in detail by tracer experiments using horseradish peroxidase (HRP) and colloidal lanthanum and by freeze-etch techniques, in addition to routine electron microscopy. The ependyma of the area postrema is characterized as flattened cells possessing very few cilia, a moderate amount of microvilli, a well-developed Golgi apparatus and rough endoplasmic reticulum. Numerous vesicles or tubular formations with internal dense content were found to accumulate in the basal processes of ependymal cells; the basal process makes contact with the perivascular basal lamina. It is suggested that the dense material in the tubulovesicular formations is synthesized within the ependymal cell and discharged into the perivascular space. The apical junctions between adjacent ependymal cells display very close apposition, with a gap of 2–3 nm, but no fusion of adjacent plasma membranes; they thus represent a transitional form between the zonulae adhaerentes present in the ordinary mural ependyma and the zonulae occludentes in the choroidal epithelium. A direct intercommunication between the ventricular cerebrospinal fluid (CSF) and the blood vascular system indicates that a region exists lacking a blood-ventricular CSF barrier.     

A freeze-fracture study of exocytosis and reflexive gap junctions in human ovarian decidual cells

Fine-structural features of ovarian decidual cells and their mode of secretion were examined by means of freeze-fracture microscopy. Unique cortical peduncular processes contained secretory vesicles within the expanded peduncle tip, the membrane-leaflets of which exhibited a particle-poor E face adjacent to the vesicle lumen and a P face containing a greater particle number. Exocytosis from attached peduncles involved release of vesicular profiles 40-55 nm in diameter; small particles 8.5-11.5 nm in diameter were also observed at degranulation sites. In fractures revealing the E face of the plasmalemma, cytoplasmic portals at the bases of peduncular stalks were distinguishable from endocytic vesicles. The frequent occurrence of reflexive gap junctions associated with peduncles was shown by freeze-fracture. However, there appeared to be no consistent spatial relationship between gap junctions, secretory peduncles, or sites of exocytosis. Freeze-fracture analysis of the topography of reflexive gap junctional profiles revealed that such gap junctions share basic similarities with intercellular gap jum particle-free aisles. The finding in the present study of reflexive gap junctions occurring between peduncles and the cell soma, as well as between peduncles, suggests that the original definitiof the same cell should be broadened to include any gap junctional specialization formed between portions of the plasma membrane of one cell.     

Gap junctions are non-randomly distributed inDrosophila wing discs

Summary The distribution of gap junctions in mature larvalDrosophila melanogaster wing discs was analyzed by means of quantitative electron microscopy. Gap junctions are non-randomly distributed in the proximal-distal disc axis and in the apical-basal cell axis of the epithelium. In the epithelial cells, the surface density, number and length of gap junctions are greatest in the apical cell region and distal disc region. The average gap junction surface density is 0.0572 m^–1 and 2.77% of the lateral cell surface is composed of gap junctions. In the adepithelial cells, the gap junction surface density is 0.0005 m^–1 and 0.06% of the cell surface is composed of gap junctions. No gap junctions were observed between epithelial cells and adepithelial cells. The absolute area of gap junctions was estimated in a proximal-distal strip of cells in the disc and is considerably less in the folded regions of the epithelium compared to the flat notum and wing pouch regions. The results are discussed with respect to pattern formation and growth control in imaginal discs.     

Rhythmic contractions of the ampullar epidermis during metamorphosis of the ascidian Molgula occidentalis

Summary The ampullae of Molgula occidentalis are hollow, tubular extensions of the epidermis. They are ensheathed by a secreted tunic. When they grow out shortly after settlement, the ampullae spread the tunic over the substratum to form a firm attachment for the sessile juvenile. A simple squamous epithelium forms the thin ampullar walls. A glandular, simple columnar epithelium forms the distal tip of each ampulla. The glandular cells probably secrete the adhesive that attaches the tunic to the substratum.Repetitive, peristaltic contractions pass from the base to the distal end of each ampulla. Microsurgery, time-lapse cinemicrography and TEM have been used to analyze this phenomenon. The contractions are mediated by a layer of 4–8 nm microfilaments in the base of the ampullar epithelium.Each juvenile has 7–9 ampullae which contract at different frequencies. Isolated ampullae continue to contract normally for several days. Thus each ampulla has an intrinsic rhythm. Microsurgical experiments suggest that there is no specific region within an ampulla with unique pacemaker properties. It is proposed that communication via gap junctions allows the coordination of ampullar cells into a well organized peristaltic wave.     

Isolation and protein composition of gap junctions from rabbit hearts.

We have modified a method for isolating gap-junctional membrane from mouse hearts [Kensler & Goodenough (1980) J. Cell Biol. 86, 755-764] to isolate gap junctions of comparable purity from rabbit hearts more rapidly, with better yield, and without resort to non-ionic detergents. Purification was monitored by electron microscopy of thin-sectioned membrane pellets and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gap junctions were obtained as vesicles whose mean surface area approximated that of junctions in intact myocardial cells. About 10-20% of the vesicles were ferritin-impermeable. Approx. 125 micrograms of membrane protein was obtained per 8 g of rabbit heart. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of purified gap junctions showed five major protein bands of mol.wts. 46 000, 44 000, 33 000, 30 000 and 28 500 that co-purified with the junctions. This protein composition was nearly identical with that published for gap junctions of mouse hearts, and differed markedly from the protein composition of gap junctions from non-excitable cells (lens and liver). The constancy of junctional protein composition between hearts of two different species and its non-identity with that from liver and lens suggest that, although gap-junctional structure in mammalian tissues seems to be remarkably similar by electron-microscopic techniques, junctional-channel protein composition actually varies from tissue to tissue and may be adapted to the permeability requirements of the tissue.     

The ultrastructure of the innervation of the intestinal wall in the teleosts Myoxocephalus scorpius and Pleuronectes platessa

Summary The innervation of the intestinal wall in the teleosts Myoxocephalus and Pleuronectes was examined electron microscopically. Two classes of axons can be identified. The first, which is in the majority, contains numerous 50–150 nm granular vesicles as well as some 40–50 nm agranular vesicles while the second contains predominantly the 40–50 nm agranular vesicles. Chromate/dichromate staining methods suggest that the first type is aminergic. Both types lie in close association with the perikarya of intrinsic myenteric neurons but only axons containing predominantly agranular vesicles have synaptic membrane specialisations. No axon bundles pass into the longitudinal muscle layer in Myoxocephalus gut and though some do in Pleuronectes, they do not closely approach the smooth muscle cells. Axons containing large granular vesicles lie in intimate contact with the myocytes of the circular muscle layer. Both axon types pass through the submucosa to form a plexus underneath the mucosal epithelium. Varicosities containing agranular or granular vesicles are separated from the epithelial cells by a gap of about 200 nm in which lies a basal lamina.     

Quantitative gap junction alterations in mammalian heart cells quickly frozen or chemically fixed after electrical uncoupling

Summary The gap junction morphology was quantified in freeze-fracture replicas prepared from rat auricles that had been either quickly frozen at 6 K or chemically fixed by glutaraldehyde, in a state of normal cell-to-cell conduction or in a state of electrical uncoupling. The general appearance of the gap junctions was similar after both preparative procedures. A quantitative analysis of three gap junctional dimensions provided the following measurements in the quickly frozen conducting auricles (mean±sd): (a) P-face particles' diameter 8.27±0.74 nm (n =5709), (b) P-face particles' center-to-center distance 10.78±2.12 nm (n=4800), and (c) E-face pits' distance 9.99±2.19 nm (n=1600). Corresponding values obtained from chemically fixed tissues were decreased by about 3% for the particle's diameter and about 5% for the particles' and pits' distances. Electrical uncoupling by the action of either 1 mM 2–4-dinitrophenol (DNP), or 3.5 mMn-Heptan-1-ol (heptanol), induced a decrease of the particle's diameter, which amounted to –0.69±0.01 nm (mean ±se) in the quickly frozen preparations and –0.71±0.01 nm in the chemically fixed ones. The particles' distance was decreased by –0.96±0.04 nm in the quickly frozen samples and by –0.90 ±0.03 nm in the chemically fixed ones and the E-face pits' distance was similarly reduced. All differences were statistically significant (P<0.001 for all dimensions). Electrical recoupling after the heptanol effect promoted a return of these gap junctional dimensions towards normal values, which was about 50% complete within 20 min. It is concluded that very similar morphological alterations of the gap junctional structure are induced in the mammalian heart by different treatments promoting electrical uncoupling and that these conformational changes appear independently of the preparative procedure. The suggestion that the observed decrease of the particles' diameter is genuinely related to the closing mechanism of the unit cell-to-cell channel set in thei centers is thus confirmed.     

Gap junction formation and functional interaction between neonatal rat cardiocytes in culture: A correlative physiological and ultrastructural study

Summary The time course of gap junction formation and growth, following contraction synchronization of cardiac myocytes in culture, has been studied in a combined (electro)physiological and ultrastructural study. In cultures of collagenase-dissociated neonatal rat cardiocytes, pairs of spontaneously beating myocytes synchronized their contractions within one beat interval within 2–20 min after they apparently had grown into contact, 45 sec after the first synchronized beat an appreciable junctional region containing several small gap junctions was already present. In the following 30 min, neither the area of individual gap junctions nor their total area increased, 75 min after synchronization both the area of individual gap junctions and their total area had increased by a factor of 10–15 with respect to what was found in the first half hour. In the period between 75 and 300 min again no further increase in gap junctional area was found. In double voltage-clamp experiments, gap junctions between well-coupled cells behaved like ohmic conductors. In poorly coupled cells, in which the number of functional gap-junctional channels was greatly reduced, the remaining channels showed voltage-dependent gating. Their single-channel conductance was 40–50 pS. The electrophysiologically measured junctional conductance agreed well with the conductance calculated from the morphometrically determined gap-junctional area. It is concluded that a rapid initial gap junction formation occurs during the 2–20 min period prior to synchronization by assembly of functional channels from existing channel precursors already present in the cell membranes. It then takes at least another 30 min before the gap-junctional area increases possibly byde novo synthesis or by recruitment from intracellular stores or from nonjunctional membranes, a process completed in the next 45 min.     

Granulated folliculo-stellate cells and growth hormone cells immunostained with anti-S 100 protein serum in the pituitary glands of the goat

Summary Goat pituitary glands were immunohistochemically studied with antisera for bovine S-100 protein, rat LH, FSH, TSH, prolactin, ovine GH, and porcine ACTH^1–39 by use of the superimposition technique on adjacent sections. Folliculo-stellate (F-S) cells were divided into two categories on the basis of ultrastructural properties: One consisted of a mass of agranular cells in which the pseudolumina were equipped with microvilli and cilia. Elongate gap junctions were often observed among these cells. The other was a group of granulated cells with or without pseudolumina. In this group the gap junctions were shown to be disintegrated. The dense granules 150–250 nm in diameter began to accumulate in the cells. However, neither type of these F-S cells was immunostained for S-100 protein. On the other hand, numerous polygonal, elongate, irregular or stellate cells containing S-100 protein were distributed throughout the gland. Most of them were immunohistochemically identical with the GH cells laden with the secretory granules 250–450 nm in diameter, but some of them were identical to TSH and prolactin cells which immunostained faintly for S-100 protein. This appears to be the first demonstration of GH cells intensely immunostained for S-100 protein.