ALBERT

Notes: Abstract The molecular weight distributions of chondroitin sulphate from normal articular cartilage of the lower femoral epiphysis from human autopsy cases of different ages and from osteoarthritic cartilage have been determined by gel chromatography on Sephadex G-200. The polysaccharide samples fell within the same molecular weight range but differed with respect to molecular weight distribution. The average molecular weight of chondroitin sulphate from normal articular cartilage was higher in newborn and young individuals than in adults or the aged. Chondroitin sulphate from osteoarthritic cartilage had a lower molecular weight than that from macroscopically normal cartilage. The observed variations in polysaccharide chain length are discussed.

Notes: Abstract The amounts of inorganic pyrophosphate (PPi), orthophosphate and calcium have been measured in resting, proliferating, hypertrophic and calcified cartilage from foetal calf epiphyses and also in cancellous, periosteal and compact bone. In the cartilage samples, the content of PPi increased progressively in the order named above, from values of 8.59 μg P/g dry weight in resting cartilage to 236 μg P/g dry weight in calcified cartilage. However, the ratio of PPi to orthophosphate followed the reverse relationship and was highest in the resting zone and fell dramatically as the tissue calcified. The possible role of PPi in inhibiting the precipitation of amorphous calcium phosphate (ACP) and in the slowing the transformation of ACP to hydroxyapatite (HA) in calcifying tissues is discussed in relation to other factors, such as collagen, magnesium, phospholipids and proteinpolysaccharides, which might also influence the processin vivo. At present, no single factor can be identified as a proven physiological regulator.

Notes: Abstract The acid glycosaminoglycans of resting, columnar, hypertrophic and calcified zones of calf epiphyseal growth plate and of nasal septum cartilage were extracted with a dissociative solvent, 3M GuCl, according to Sajdera and Hascall (1969), to separate the glycosaminoglycans into an extractable pool and an unextractable pool which remains bound within the tissue. Epiphyseal cartilage required longer extraction times than did nasal septum cartilage to extract comparable amounts of acid glycosaminoglycans suggesting a stronger binding of proteoglycans within the tissue. Towards the calcification front the glycosaminoglycans were extracted more easily while in calcified zone not more than 30% could be extracted. Data obtained by the CPC microfractionation procedure of Antonopouloset al. (1964) indicated similar distribution according to molecular weight and/or charge density for extractable and unextractable chondroitin sulphate in nasal septum cartilage and in resting and columnar zones of epiphyseal growth plate. Unextractable glycosaminoglycans in hypertrophic and calcified zones were of predominantly low molecular weight and/or charge density compared to the extractable pool. Hyaluronic acid was unextractable in nasal septum and in resting, columnar and hypertrophic zones with increasing concentrations towards the calcification front. In calcified zone a shift to mainly extractable hyaluronic acid occurred. The significance of these findings is discussed.

Notes: Abstract The effect of vitamin A on the synthesis of acid mucopolysaccharides has been studiedin vitro in cell cultures of chick embryo sternal chondrocytes. Vitamin A produces a concentration-dependent inhibition of acid mucopolysaccharide synthesis. the inhibitory effect of vitamin A on acid mucopolysaccharide synthesis is not the result of a general cytotoxicity, since, under the conditions studied here, while there is over a 50% inhibition of mucopolysaccharide synthesis, the incorporation of leucine into acid insoluble material or the synthesis of collagen is unaltered. Lysosomal activation does not appear to be involved in the inhibition of sulfate incorporation, since E-amino caproic acid (a protease inhibitor) and chloroquine (a lysosomal membrane stabilizer) do not reverse this inhibition. In addition, enhanced polysaccharide breakdown is too small to account for vitamin A-inhibited sulfate incorporation. The effect of vitamin A is not readily reversed and chondrocytes become fibroblastic after a 24 hour treatment with 3 μg/ml of vitamin A. Because the incorporation of glucose into polysaccharides is inhibited faster than glucosamine or sulfate, it is suggested that vitamin A acts by an unknown mechanism to inhibit a step early in the pathway for mucopolysaccharide biosynthesis.

Notes: Abstract Iliac crest cartilage biopsies from five children with spondyloepiphyseal dysplasia congenita contained increased amounts of chondroitin-6-sulfate and keratan sulfate. The increase was similar to that observed in the cartilage of patients with Morquio's disease. However, unlike patients with Morquio's disease, those with spondyloepiphyseal dysplasia congenita did not excrete an abnormal amount of keratan sulfate in urine. Spondyloepiphyseal dysplasia congenita can be considered a mucopolysaccharidosis without mucopolysacchariduria.

Notes: Abstract As rabbits increase in size from 165 grams to 2,205 grams body weight, cytochrome oxidase activity of epiphyseal cartilage homogenates does not change in manometric and spectrophotometric enzyme assays, whereas the oxidase activities of xiphisternal and ear cartilage homogenates decline. Light absorption spectra of cartilage tissues were made for the first time, using strips of rabbit ear and pieces of chick embryo cartilage. Such spectra reveal characteristic absorption maxima for the terminal respiratory complex of mitochondria, including b, c and oxidase components and flavoprotein.

Notes: Abstract Proteinpolysaccharide (PP-L) of “resting” and “ossifying” zones of calf scapular cartilage and of calf nasal septum cartilage was extracted with aqueous 0.15 M KCl and fractionated into a series of products: PP-L3, PP-L4, PP-L5, PP-L6. An additional proteinpolysaccharide fraction, PP-L2, was extracted from the cartilage residues with hydroxylamine. Differences in chemical composition among corresponding proteinpolysaccharides obtained from the three cartilage sources were, in general, small. The calcium binding capacity of the PP-L, PP-L2, and PP-L3 samples, as measured by equlibrium dialysis, appeared to reflect principally their uronic acid and exchangeable sulfate contents. Application of a cation exchange technique indicated that PP-L from “resting” and “ossifying” zones of scapular cartilage had similar affinities for tracer quantities of calcium. However, the affinity of PP-L from both zones of scapular cartilage for tracer quantities of calcium was greater than that of PP-L from nasal septum. The data obtained from this study do not indicate sufficient differences between the chemical composition and calcium affinity of proteinpolysaccharides from “resting” and “ossifying” scapular cartilage to account for the calcium uptake in the “ossifying” zone.

Notes: Abstract A study of the ultrastructure of the hypertrophic chondrocyte has been made possible by an improved method of fixation. In contrast to the general belief that the hypertrophic cells die, our results strongly suggest cell activity from the beginning of enlargement of the cell up to the stage where the lacuna breaks open. The sequence of alterations within the cell have been described, with the increase of endoplasmic reticulum contents, the appearance of many free ribosomes and the occurrence of electron-dense granules in the mitochondria as the most prominent characteristics. The possible functions of the cell have been discussed. Most interesting is the support for the hypothesis that hypertrophic chondrocytes may ultimately become bone cells.

Notes: Abstract A method of fixation has been developed for the cartilage cells of the epiphyseal plate. Improved preservation of the flattened chondrocytes reveals small vesicles that seem to originate in the rough endoplasmic reticulum and are found in large amounts in the Golgi area. Different types of vacuoles can be distinguished in the Golgi complex, e.g. “intermediate vacuoles” and “large vacuoles”, the latter usually containing inclusions. It seems likely that the vesicles and vacuoles each play a specific role in the transport and production of sulfated protein polysaccharides.

Notes: Abstract This paper reports studies of the epiphyseal cartilage in normal and vitamin A deficient chicks. The organic composition and the phosphatase activity in the resting cartilage, ossifying cartilage and new bone were measured. The ossifying cartilage and new bone had a higher content of inorganic material, phosphate and collagen than the resting cartilage. Vitamin A deficiency caused increase in the phospholipid content of all three tissues. The resting cartilage from vitamin A deficient tissue had, after homogenisation and centrifugation, a supernatant with an activity of alkaline phosphatase and glycerophosphatase higher than that in control samples. It is considered that effects of vitamin A deficiency on enzymes are related to defects of the lysosomal membrane with release of phosphatases, and that normal mineralisation also involves phosphatases activity.

Notes: Abstract Matrix vesicles are extracellular, membrane-bounded particles which are abundant in the matrix of calcifying cartilage and which are morphologically related to the deposition of mineral in this tissue. The matrix vesicles and cells of the epiphyses of the long bones of fetal calves were liberated by digestion with collagenase and separated by differential centrifugation. Vesicle preparations were found to be lipid-rich and to contain significantly more sphingomyelin and phosphatidyl serine than cellular fractions. The ratio, moles cholesterol to moles total phospholipid, was higher in vesicles than in cells, a finding which is consistent with vesicles having their origins in the plasma membranes of the chondrocytes.

Notes: Abstract The glycosaminoglycans in articular cartilage from the femoral condyles of 119 human autopsy cases have been isolated and subsequently characterized using cetylpyridinium chloride. Using the same column method in microscale the glycosaminoglycans were estimated quantitatively in normal articular cartilage from birth to 95 years of age and in osteoarthritic cartilage. In normal cartilage chondroitinsulphate occurred as chondroitin-4-sulphate and chondroitin-6-sulphate, decreasing in ratio with age. The chondroitinsulphate had a sulphate/hexuronic acid ratio below unity, below 20 years, above unity in the higher age groups. Two types of keratansulphate and hyaluronic acid were isolated. The glycosaminoglycans isolated from osteoarthritic cartilage showed no differences from normal cartilage as to the amount of their moities of chondroitin sulphate isomers. In normal cartilage there was a significant decrease of total hexosamines from birth to 11–20 years, due to a reduction of chondroitinsulphate. In adults, an increasing ratio of the keratansulphate plus glycoprotein fraction to chondroitinsulphate was observed. Osteoarthritic cartilage showed a significant reduction of all glycosaminoglycan fractions without change of distribution. Normal cartilage taken from osteoarthritic joints was unaltered. Solubility profiles indicated decreases in molecular weight of chondroitin sulphate in normal cartilage from birth to 21–30 years, and a trend towards lower molecular weight in osteoarthritic cartilage.

Notes: Abstract Monolayer cell cultures from the distal femoral epiphyses of embryo calves were studied following the first subculture, which was carried out after confluence in primary culture. Light microscopic examination revealed scattered deposits of metachromatic-staining material; on electron microscopy fine fibrils considered to be developmental collagen were seen. After several days in culture lacuna-like patterns of cells were seen. The nature of the cell secretions were studied by radioactive precursors, which were fractionated on Sephadex G200 and by ion exchange chromatography. Enzyme digestion with bacterial and testicular hyaluronidase and chondroitinase-AC and-ABC revealed that the cells synthesized 70% sulphated, and 30% non-sulphated glycosaminoglycans. Of the sulphated glycosaminoglycans 70% was chondroitin-4-sulphate, 20% chondroitin-6-sulphate, and the remainder probably keratansulphate. Studies were labelled amino acid precursors suggested that the cells synthesized a high-molecular weight protein containing hydroxyproline, as well as some non-collagenous protein, shown by tryptophan incorporation

Notes: Abstract Removal of the soluble proteoglycans from slices of bovine costal cartilage by extraction in 4 M guanidinium hydrochloride permitted the visualization of abundant amounts of dispersed and disaggregated collagen in the matrix. Proteoglycans which are resistant to extraction are seen as small granules which are concentrated in the perilacunar regions. Large proteoglycan granules appear to originate in the chondrocyte. As they come to occupy positions in the matrix distant from the chondrocyte, the granules become smaller. A non-granular, “amorphous” component masks the collagen fibers so that they cannot be readily seen in the intact cartilage.

Notes: Abstract A method for studying the growth of fetal rat long bones in a chemically defined medium in organ culture is described. Cartilage ends and bone shafts were analyzed separately for growth and mineralization by measuring the collagen, calcium, and phosphate content, dry weight, and incorporation of labeled proline into hydroxyproline. Growth and mineralization of the bone shaft were slower in a chemically defined medium thanin vivo. Growth could be enhanced by supplementation of the medium with non-essential amino acids, albumin or serum. Cartilage ends showed a greater increase in weight and collagen content than the shafts, and medium supplements had less effect on their growth. Bone shaft growth and mineralization were enhanced by increasing medium phosphate concentration over a range of 1.5 to 4.5 mM whether or not the medium was supplemented with serum or albumin. At a low medium calcium concentration (0.5 mM) bone shaft growth and mineralization were impaired. At a low magnesium concentration (0.5 mM) mineralization was enhanced, but growth was impaired.

Notes: Abstract Acid glycosaminoglycans were studied from isolated chondrocytes in suspension and the extracellular matrix obtained by sequential trypsin and collagenase treatment of 15- to 17-day-old embryonic chick bone. After papain digestion and removal of the nucleic acids by treatment with DNAse and RNAse, the glycosaminoglycans were precipitated by CPC and isolated as their sodium salt. Analyses of the CP-glycosaminoglycan solubility properties with the microfractionation procedure of Antonopouloset al. (1964) showed intracellular glycosaminoglycans of predominantly lower molecular weight and/or charge density than those of extracellular matrix. On micro-zone electrophoresis, only a minor part of the isolated intracellular glycosaminoglycans showed a mobility similar to that of the chondroitin sulphate standards while the major part moved only half the distance of hyaluronic acid and showed broad tailing, indicating a low negative charge. This was considered to be due to loss of a regulatory influence of the removed matrix components upon glycosaminoglycan synthesis and to release of originally synthesized glycosaminoglycans into the medium. For the first time, a small amount of intracellular hyaluronic acid was shown by typical electrophoretic mobility.

Notes: Abstract Short-term incubation of organ cultures of puppy scapulae in a medium enriched with lysozyme or protamine induces specific alterations in the histologic staining properties of the explant not induced by albumin or by ultraviolet-inactivated lysozyme. The induced changes are different for each two proteins. The lysozyme effect is anatomically distributed precisely while that of protamine is diffuse. If fluorescein conjugates of the proteins are used, identical histologic changes are induced in the cartilage and the distribution of fluorescence is precisely that of the altered tinctorial properties of the tissue. The observation, which shows that lysozyme, when added to cartilage, accumulates in specific anatomic sites suggests that it chemically interacts with specific components of cartilage matrix, presumably with negatively charged macromolecules.

Notes: Abstract Sections of the tibias from both intact and gonadectomized male and female rats were subjected to various histological and histochemical methods to determine the changes in the bone following gonadectomy. It was observed that both in castrated males and in ovariectomized females, the proximal epiphyseal cartilage became “sealed off” by bone after the fifth post-surgical month. In addition osteoporosis of the shaft of the bone was also observed. The epiphyseal cartilage of the gonadectomized rats exhibited intense PAS positive intercellular matrix in contrast to the alcianophilic ature of the matrix from control animals. The diaphyseal trabeculae from gonadectomized rats stained red with PAS and contained wide regions of calcified cartilage cores. In addition, the epiphyseal cartilage and bony trabeculae from these animals stained faintly in the ninhydrin-Schiff reaction. The staining reactions indicated that in gonadectomy there was an alteration in the state of aggregation of protein-mucopolysaccharide component of the organic matrix, a situation which would result in an interference in the mineralization of the bone.

Notes: Abstract The content and composition of lipids of chick epiphyseal tissue were investigated. The amounts of total lipids, triglycerides and phospholipids in bone were 7.50, 7.81 and 1.02 mg/g, respectively, and in cartilage, 5.20, 1.46 and 0.53 mg/g, respectively. The main fatty acids of both bone and cartilage were palmitic and oleic in phospholipids, and palmitic, oleic and linoleic in triglycerides. For the substrates studied, the order of incorporation into total lipids of both bone and cartilage was palmitate 〉 glucose 〉 acetate 〉 citrate. Acetate was the main precursor for iatty acid synthesis whereas only a minor portion of glucose was found in the fatty acids. Esterification appeared to be the predominant pathway of lipid synthesis in chick bone and cartilage.

Notes: Abstract Chondroitin-4-sulfate (C-4-S), chondroitin-6-sulfate (C-6-S) and keratan sulfate (KS) concentrations were measured in growth plate cartilage derived from rabbits of two different ages, and from three growth plate zones from rabbits of the same age. With increasing age, the concentration of C-4-S increased, C-6-S decreased and KS remained constant. There was no variation when three different growth plate zones were sampled. Radiosulfate uptake into chondroitan sulfate was also measured, and this showed an inverse relationship to concentration with increasing age.

Notes: Abstract Ten male weanling Holtzman rats, injected intraperitoneally with aqueous estradiol (Progynon, Schering), in daily doses of 1 μg. per g body weight, were sacrificed, simultaneously with controls receiving an equivalent amount of diluent, at intervals ranging from one hour to six days. Upper tibial epiphyseal cartilage plates (ECP), procesed for electron microscopy, revealed, as early as three hours after injection, appreciable enhancement of secretory activity, evidenced, in the zone of matrix secretion, by the abundance in Golgi cisternae of stippled material representing proteinpolysaccharide complexes. Disintegration of the lining membrane of individual Golgi vesicles was advanced after twenty-four hours; following three days of treatment, few vesicles remained intact, and pools of initially intravacuolar material were observable in the gound plasm. Long filaments, suggestive of primary or precursor collagen fibrils were apparent in this secretion. After six days, virtual lakes of this substance filled cells in the zone of prehypertophy, with consequent displacement of the rough endoplasmic reticulum against the cell periphery. Cytoplasmic vacuoles, containing mateerial similar to that found in the lacunar moat, and displaying finely beaded, radially arrayed filaments on the lining membrane were frequently encountered. Our observations suggest an initial acclleration of chondrocytic secretory activity, with subsequent retardation of transport. The resultant retention and intracellular polymerization of precollagenous products accelerates hypertrophy, thereby promoting early degeneration of chondrocytes. These ultrastructural alterations are apparently estrogen-specific.

Notes: Abstract The mechanism by which (HCO 3 − ) is elevated in extracellular cartilage fluids (Cfl) of rat tibial growth plates was investigated. Inin vitro studies, the pH- $$P_{CO_2 } $$ curves in a synthetic lymph were not detectably altered by proteinpolysaccharides or by a cationic protein. Also, (HCO 3 − ) in Cfl aspirated from isolated incubates of growth cartilage decreased rapidly as a function of time. Results of both of these experiments mitigated against a role for cartilage secretions as the cause of blood-Cfl (HCO 3 − ) gradientsin vivo. Acetazolamide administered to ratsin vivo reduced the blood-Cfl (HCO 3 − ) gradient to an undetectable level. This effect could not be attributed to systemic acidosis produced by acetazolamide since control rats with a similar degree of systemic acidosis resulting from NH4Cl treatment, maintained a substantial blood-Cfl (HCO 3 − ) gradient. The distribution of carbonic anhydrase activity in epiphyseal and metaphyseal tissues of similar rats was determined by microassay. Enzymatic activity was not detected in cartilage samples, but was found in significant amounts in adjacent structures. This carbonic anhydrase activity measured in adjacent structures was hypothesized to represent sites of HCO 3 − secretion. The possible involvement in HCO 3 − secretion of epiphyseal or metaphyseal capillaries and bone cells is discussed.

Notes: Abstract Electrolytes were analyzed in serum, and in mineralizing tissues at varying stages of calcification, in normal and vitamin D-deficient chickens and pigs. Moisture, ash, and organic matter were also measured. Results revealed that in addition to Ca, other serum electrolytes were altered in rickets. Mg and inorganic P were diversely affected by the vitamin deficiency. Larger changes in mineral level were seen in the tissues in the early stages of calcification than were seen at corresponding times in the serum, suggesting a direct effect of the vitamin deficiency on the calcifying tissue itself. Relative to the serum level, Ca was deposited in the tissues of rachitic animals to a greater extent than in normal animals. This was not true for Mg or inorganic P, indicating a preferential affinity for Ca in the rachitic tissue. Gravimetric analyses of organic and dry matter in epiphyseal zones revealed that the amounts of proliferating and hypertrophic cartilage were increased, and calcified cartilage decreased, in rickets, in accord with previous histological observations. Unexpectedly, the proportion of resting cartilage was also increased.

Notes: Abstract A limited, standardized osteochondral defect was created in adult rabbits and the regenerated tissue was examined histologically and autoradiographically after labellingin vitro with35S-sulphate, and microchemically for its content and composition of glycosaminoglycans. The principal findings were: 1. Between 1 week and 4 to 8 weeks, non-metachromatic connective tissue differentiated to metachromatically stained cartilage, and the proportion of the chondroitin sulphate increased significantly at the expense of the glycoproteins. 2. Up to 4 weeks, the major part of the defect area was filled with newly formed bone; after this time, the area lay above the level of the “tidemark”, towards the articular surface. 3. Hyaline cartilage with morphological, autoradiographic and chemical resemblance to the articular cartilage in terms of the distribution of glycosaminoglycans constituted the articular surface of the defect area in one-third of the cases at observation times after 8 weeks. Hyaline cartilage was observed mainly in areas where newly formed bone had closed the medullary cavity. 4. In two-thirds of the cases, after 8 weeks, parts of the articular surface of the defect consisted not only of cartilage but also of fibrous tissue, occurring mainly in the lateral parts of the defect and in areas where the medullary cavity facing the articular surface had not been sealed by bone tissue. The glycoprotein fraction increased relative to the chondroitin sulphate fraction. 5. In most cases after 20 weeks, newly-formed cartilage underwent degenerative changes, which were reflected in some reduction of the chondroitin sulphate.

Notes: Abstract Proteinpolysaccharides (PP) have been extracted by disruptive and dissociative methods from iliac crest cartilage of nine achondroplastic children, eight girls and one boy, from eight to thirteen years of age. Both high speed homogenization of the tissue in water or incubation with guanidinium chloride released from the cartilage of younger children normal amounts of PP of high molecular weight. Cartilage samples obtained from older children released instead little or no PP material by water extraction. The water-insoluble PP were found soluble in 4 M guanidinium chloride, unbuffered MgCl2 solution and 0.067 M phosphate buffer. While the guanidine extract contained, after dilution of guanidinium chloride to 0.4 M, a high molecular weight component, MgCl2 and phosphate buffer extracts contained only degraded PP material of low molecular weight. The insolubility in water of PP of cartilage of children over ten years of age is interpreted as a secondary manifestation related either to mucopolysaccharide changes occurring in the cartilage at that age, or to other unknown factors. The primary biochemical defect could be an abnormality of the PP macromolecule which, at a certain stage of maturation of the tissue, is reflected either in an altered state of aggregation or in an abnormal relationship with the collagen matrix.

Notes: Abstract Injection into chick embryos incubated for nine days, of β-aminopropionitrile (BAPN) at doses above 0.312 mg/embryo prevented the formation of the cartilage normally present on the membrane bones from the eleventh day of incubation onwards. The histogenesis of this inhibition is described. BAPN increased the proportion of soluble collagen without altering total collagen synthesis and decreased the synthesis of hexosamine-containing acid mucopolysaccharides within the membrane bones. The relationship between cross-linking of collagen, collagen solubility, synthesis of acid mucopolysaccharides and mechanical stimulation (the extrinsic factor initiating chondrogenesis in this system) in the formation of cartilage from bipotential germinal cells on the membrane bones is discussed.

Notes: Abstract Pieces of scapula were removed from young rats, decalcified, and treated with pronase, papain, hyaluronidase or trypsin. The epiphyseal cartilages of control scapulae took up the Alcian blue stain, but those treated with pronase, papain or hyaluronidase did not; trypsin did not abolish the staining reaction. When the bones were implanted subcutaneously into the animals from which they had been taken, the epiphyseal cartilages of those treated with the first three enzymes were rapidly invaded by capillaries and small round cells. The cartilages of the control, untreated bones and those from bones treated with trypsin were not invaded in this way. It is postulated that the removal of acid glycosaminoglycans from cartilage may be one of the factors that trigger the capillary invasion that occurs in endochondral calcification.

Notes: Abstract In adult rabbits, a transplant was made to an osteochondral defect on the femoral head measuring 3×4×3 mm of autologous costal cartilage which had first been implanted intramuscularly for 4–5 weeks and fresh autologous costal cartilage. After 6 different observation times between 2 and 52 weeks the animals were killed. The original material, the tissue in the transplant area on the articular surface and the articular cartilage above the “tidemark” were analysed in each individual animal, after microdissection of freeze-dried sections of these structures, by a quantitative microchemical technique for their content and composition of glycosaminoglycans and for their calcium content. The hexosamine content (% per organic dry weight) was generally higher in the transplants than in the articular cartilage at observation times of 2 and 6 weeks, after which time it was similar. The distribution of the different glycosaminoglycan fractions was different in the transplants compared with the articular cartilage. Thus the glycoprotein and/or keratan sulphate fraction was smaller in the transplant than in the articular cartilage, while the opposite held for the chondroitin sulphate. With increasing observation times, some resemblance became evident between the transplant and articular cartilage with respect to the relative size of these fractions. The solubility profiles of the chondroitin sulphate were similar. The calcium content was generally higher in the transplant than in the articular cartilage, after 12 weeks it showed a pronounced decrease in comparison with the original material. The chemical findings indicate that the cartilage in the transplant area did not undergo any marked degenerative changes.

Notes: Abstract The skeletal tissues of teleost (Scomber scombrus andGadus callaria) and elasmobranch (Raja batis andScyllium canicula) fish were examined for the presence of lipids. It was found that more phospholipid was removed from the bony tissues of the teleosts than was extractable from the cartilaginous tissues of the elasmobranchs. Apart from there being a greater proportion of lipid associated with teleost bone, differences were also noted in the lipid composition of the two tissues. Before demineralization, inGadus andScomber, the extracts were predominantly of neutral phospholipids. In contrast, the phosphatides of the cartilaginous tissues contained appreciable quantities of acidic phospholipids. After demineralization of piscine bone and cartilage, neutral lipid solvents eluted both acidic and neutral phosphatides from these tissues. When the demineralized lipid extracted tissues were treated with acidified solvents, lipid was again removed from the bone and cartilage. In this final extract, a wide variety of phospholipids was detected. At this stage, more phosphatide was extracted from the demineralized bone ofScomber, compared with the other piscine species. The principal phosphatide of this final extract was cardiolipin. More cardiolipin was present in the bone ofScomber than in any other hard tissue.

Notes: Abstract A solution of chromium sulphate was used as a combined fixative, stain and demineralizing agent for the ultrastructural study of mineralizing cartilage. This technique revealed the presence of an organic ‘crystal ghost’ associated with each crystallite. The effectiveness of chromium sulphate as a demineralizing agent is discussed.

Notes: Abstract Although experimental and clinical experience indicates that large doses of testosterone lead to premature cessation of growth, the exact mechanism and precise site of action of this hormone on the growth apparatus of long bones remain unknown. In this study, plateaued male rats were injected with supraphysiologic doses of testosterone to observe the submicroscopic effects on the various zones of the epiphyseal cartilage. In the zone of cell division there were increased numbers of dividing cells. The maturing cells accumulated larger amounts of secretory products at earlier stages of their life cycle, and appeared to undergo a more abrupt hypertrophy. In the zone of prehypertrophy, the interterritorial matrix contained foci of early and premature calcification and thicker and longer collagen fibers than at comparable levels in controls. It is concluded that in intact animals, even large doses of testosterone initially cause a stimulation of chondrocyte proliferation, prior to promoting maturation processes.

Notes: Abstract Articular cartilage from the lower femoral epiphysis of human autopsy cases was collected in ten age groups from birth to 95 years and in osteoarthrosis of two grades of severity. By chromatography on Ecteola cellulose columns, the different glycosaminoglycans were separated and keratan sulphate was fractionated. The results are consistent with earlier studies using CPC-cellulose column technique, i.e. a higher content of total hexosamine and chondroitin sulphate was found in early childhood. Furthermore, normal articular cartilage in adults showed a higher content and a higher degree of heterogeneity of keratan sulphate than in childhood. In osteoarthrosis, a decreased content of total hexosamine due to a decrease both of chondroitin sulphate and keratan sulphate was found.

Notes: Abstract 1. The quantitative distribution of alkaline phosphatase and glucose-6-phosphate dehydrogenase in proliferating and hypertrophic zones of rachitic rat cartilage during healing induced by vitamin D administration or starvation is presented. 2. Alkaline phosphatase activity in the hypertrophic zone is about three times greater than that of the proliferating zone of the normal weanling rat and approximately four times greater in the rachitic rats. 3. Following a single dose of vitamin D (8000 I. U.) there was a significant elevation in alkaline phosphatase activity of the proliferating zone. A less significant increase was observed in the proliferating zone of rats which had breen fasted 48 hours. 4. There was a significant decrease in the alkaline phosphatase activity of the central region of hypertrophic zone of cartilage after vitamin D treatment. No such decrease was observed in the fasted rats. 5. In the normal weanling rat glucose-6-phosphate dehydrogenase (G-6-PDH) activity was greater in the proliferating zone as compared to the hypertrophic zone. In the untreated rachitic rat the enzyme content was about the same in the two zones. 6. Following vitamin D treatment there was an appreciable decrease in the G-6-PDH activity. No such decrease was observed in the fasted rat.

Notes: Abstract The glycosaminoglycans of articular cartilage in 4 patients with CPPD crystal-deposition disease and 13 controls were fractionated on CPC-cellulose and ECTEOLA-cellulose columns. No difference in the total amount of hexosamines was found between the two groups. An increase in the relative concentration of keratan sulphate and a decrease of the ratio of 4 sulphated and 6-sulphated chondroitin sulphate were encountered in the patient group. Solubility profiles of chondroitin sulphate showed an increase in subfractions representing low molecular weight and/or sulphate content in CPPD crystal-deposition disease. On the other hand, the solubility profiles of keratan sulphate indicated high molecular weight and/or sulphate content in the diseased cartilage. These chemical changes might partly be a result of impaired metabolism of the glycosaminoglycans concomitant with, and possibly also preceding, crystal deposition. The changes are different from those found in osteoarthrosis and from normally mineralizing cartilage.

Notes: Abstract Comparison of mineralization in the hypertrophic zone of the tibial epiphyseal plate in immature rats was carried out after treatment with cortisone, propylthiouracil, or after fasting. Under normal conditions, in the extracellular matrix at the calcification front, calcium and phosphate increased, sulfated mucopolysaccharides decreased, and matrix vesicles, which serve as the locus for the formation of hydroxyapatite crystals, increased. In propylthiouracil-treated rats, hydroxyapatite crystals were prominent, related to an increase in calcium deposition, a decrease of mitochondrial granules (thought to contain calcium and phosphate), an increase in the number of matrix vesicles, and to a marked decrease in the amount of sulfated mucopolysaccharide. In cortisone-treated rats, hydroxyapatite crystals were present but they were not as numerous as in the propylthiouracil-treated rats. Correspondingly, calcium deposition was slightly reduced, mitochondrial granules were more numerous than in the previous groups of rats, matrix vesicles were less numerous, and sulfated mucopolysaccharide were more prominent than in the propylthiouracil-treated rats. In fasted rats, hydroxyapatite crystals were markedly reduced or absent, and related to a decrease in calcium deposition, an increase in the number of mitochondrial granules (suggesting a delay in transport to the extracellular matrix). Matrix vesicles were markedly reduced in number, and sulfated mucopolysaccharide much more prominent than in either the cortisone or the propylthiouracil-treated rats.

Notes: Abstract The incorporationin vitro of35S-sulphate into chondroitin sulphate fractions, quantitatively assessed and separated, was studied in different layers of bovine articular cartilage in relation to age. After incubationin vitro in Tyrode solution with35S-sulphate the specimens were washed for 12 hours in saturated sodium sulphate solution. No loss of chondroitin sulphate occurred in the young and adolescent articular cartilage after the incubation and washing procedures. Thus, the proteoglycans appear to be firmly bound within this cartilage In contrast, large losses of chondroitin sulphate were found from the epiphyseal cartilage. In the cow, no chondroitin sulphate was lost from the superficial layers but increasing losses of both chondroitinand keratansulphate occurred from the middle to the deep layers. In all ages, the articular surface layer 40 μ in thickness showed high “specific activities” in the different chondroitin sulphate fractions and incorporation of35S-sulphate into chondroitin sulphate of predominantly high molecular weight and/or charge density suggesting an increased turnover rate of these fractions. In the matrix of this layer, the chondroitin sulphate consisted of predominantly low molecular weight/low charged fractions. In the other layers, a pattern of35S incorporation was found largely reflecting the actual chondroitin sulphate distribution pattern. This suggests that sulphation is completeted intracellularly. Further, the data suggested similar turnover rates for the different chondroitin sulphates. Increased incorporation of35S-sulphate into chondroitin sulphate at higher ages indicated increased rate of synthesis, probably secondary to increased degradation.

Notes: Abstract The glycosaminoglycans in different layers of bovine articular cartilage from calves, heifers and cows were studied quantitatively and qualitatively using CPC microfractionation procedure and by subsequent microzone electrophoresis on different fractions; water, calcium and hydroxyproline were also estimated. Chondroitin-4-sulphate was the main glycosaminoglycan in all layers in calves and heifers while chondroitin-6-sulphate predominated in cows. Corresponding fractions from the microcolumns showed different electrophoretic mobility varying with the depth from the articular surface and with age. In general, increased concentration of glycosaminoglycans was found in all ages with increasing distance from the articular surface. In calves and heifers, chondroitin sulphate was responsible for this increase while in cows, keratan sulphate, and possibly glycoproteins, were the major components of the increased hexosamine concentrations found with increasing depth. In all ages chondroitin sulphate showed a relative increase of fractions with higher molecular weights/charge density with increasing depth. In the calves and heifers, the individual fractions showed electrophoretical homogeneity while in cows an additional chondroitin sulphate showing an extremely slow mobility was found. The chondroitin sulphate in the articular surface layer showed considerable heterogeneity in all ages. The ratio of the combined keratan sulphate and glycoprotein fractions to chondroitin sulphate increased significantly with increasing age.

Notes: Abstract Growth plates of rachitic, pair-fed control, normal and Vitamin D2- and phosphate-treated rachitic rats were studied with the electron microscope. Mitochondrial granule distribution was modified in the rachitic tissue; granules were noted only in a few cells adjacent to the zone of provisional calcification. Control rats demonstrated a gradient of granules throughout the growth plate. Supplementation of the rachitogenic diet with either phosphate, Vitamin D2, or both was able to re-establish the granule density and distribution found in control animals. It is suggested that one specific modification induced in mitochondria of chondrocytes by a low phosphate, Vitamin D2-deficient diet is the reduced ability to form mineral-containing mitochondrial granules.

Notes: Abstract This study provides quantitative microchemical data on the glycosaminoglycans of developing chick embryo cartilage. The major glycosaminoglycan was chondroitin sulfate, which appeared co-incidentally with cartilage in the limbs and rapidly increased as the amount of cartilage increased. Significant amounts of other glycosaminoglycans were not found in whole limbs, whole bone rudiments or microscopically-dissected cartilage samples. The characteristics of the chondroitin sulfate as revealed by cetylpyridinium chloride fractionation were the same for all of these samples, even cartilage samples differing markedly in morphology and known functional response. Significant age-related increases in tissue concentration of chondroitin sulfate were demonstrated in early whole bone rudiments, and in two of the three histologic zones of tibial cartilage. The higher concentrations observed in the hypertrophic zone at all stages of tibial development and the marked increase in the concentration in this region at a time of accelerated bone formation suggested that this increase is related to endochondral bone formation.

Notes: Abstract Sections of the tibias from intact and from aparathyroid animals were examined histologically and histochemically to determine the changes in bone following parathyroidectomy. The proximal epiphyseal cartilages of parathyroidectomized rats showed arelative increase of intercellular matrix, decrease in cellularity, and disorganization of the cellular arrangement. The epiphyseal cartilages of the aparathyroid rats exhibited an intense PAS-positive intercellular matrix in contrast to the alcianophilic nature of the matrix from control animals. The diaphyseal trabeculae from aparathyroid animals stained red with PAS and contained wide regions of calcified cartilaginous cores. In addition, the epiphyseal cartilages and bone trabeculae from the parathyroidectomized animals stained less intensely to the ninhydrin-Schiff reaction than did the bone from the intact animal. The response to the various histochemical reactions indicated that following parathyroidectomy there was a change in the collagen-protein mucopolysaccharide components. This condition would produce an alteration in the calcification of bone.

Notes: Abstract In 20 adult rabbits autologous costal cartilage was transplanted to an osteochondral defect on the femoral head. After 1, and 11/2 years the tissue of the transplant area and the articular cartilage from the operated and non-operated joints were studied. The histological and autoradiographic (35S-sulphate) study showed that the articular surface and the bulk of the transplant area consisted mainly of viable cartilaginous tissue showing signs of degeneration to varying degree. Also, fibrous tissue was found on the articular surface of the transplant area. The quantitative microchemical study of glycosaminoglycans, hydroxyproline and calcium was made on microdissected freeze-dried material. The total hexosamines and the keratan-sulphate and/or glycoprotein fraction were lower in the transplant than in the articular cartilage from the operated joints while the contrary was true for chondroitin sulphate. The articular cartilage from the operated joints showed a reduction of total hexosamines and of all glycosaminoglycan fractions compared to the non-operated joints. These changes were especially pronounced in samples showing signs of degeneration in Azur-Astained, freeze-dried sections. It is suggested that in early and slight superficial articular cartilage degeneration the glycoprotein and/or keratan-sulphate fraction is reduced while chondroitin-sulphate remains relatively unaltered.

Notes: Abstract The organic phase (or crystallite ghost) associated with each crystallite, together with the background material associated with each crystallite cluster, was demonstrated in calcified cartilage using basic chromium sulphate as a combined fixative, stain, and demineralizing agent. Subsequent treatment of the tissue with papain, or with hyaluronidase, suggests that the crystallite ghosts represented a protein-polysaccharide complex and that the background material was principally protein together with some acid polysaccharide. The relationship between inorganic and organic phases is discussed.

Notes: Abstract The effect of various degrees of simple, vitamin D-deficient rickets on the levels, composition and extractability of phospholipids from calcifying and soft tissues was studied in chickens and pigs. A variety of effects of rickets on the phospholipid composition of the calcifying tissues were detected; there was little change in the soft tissues. The quantitative effects on the lipid concentration of rachitic hard tissues were variable from zone to zone and appeared to reflect variations in other tissue constituents. However, the qualitative differences indicated a direct effect on specific phospholipids. Acidic phospholipids displayed the greatest percentage change, although numerous statistically-significant changes were also seen in the neutral phospholipids. Tissue zones displaying the greatest change in phospholipid composition in rickets were proliferating and hypertrophic cartilage, and cancellous bone, with calcified cartilage showing the least effect. Rickets also reduced the extractability of certain acidic and neutral phospholipids from zones of early mineralization. These findings are interpreted as indicating the presence of elevated levels of phospholipid complexed with calcium phosphate in rachitic tissue, presumably due to the blockage of normal mineralization.

Notes: Abstract Proteinpolysaccharides (PP) have been extracted from the iliac crest cartilage of skeletally normal children varying in age between a few days and sixteen years, with both disruptive and dissociative methods. Homogenization of the cartilage in water or stirring with guanidinium chloride yielded high molecular weight PP material of similar properties. The water extracts contained considerable amounts of collagen which was present only in traces in the guanidine extracts. Degraded products, probably due to the action of proteolytic enzymes, were obtained by extraction of the tissue with 0.067 M phosphate buffer pH 7.4 at 30°; 0.5 M or 3.0 M MgCl2 at 10°.

Notes: Summary Ultrastructural studies have been carried out on the innervation of the interstitial gland of the mouse ovary. In addition Falck's fluorescence method was applied. 1. Fluorescence microscopy: In the ovarian stroma green fluorescent nerve fibers are frequently to be found in the surroundings of large and small vessels. Seldom small fibers invade blocks of interstitial cells; however, their final ramification is not discernible. 2. Electron microscopy: Terminal fibers of the autonomic nervous system reach the cells of the interstitial gland from all sides. They may be independent from the course of the vessels. Many axons penetrate the basal membrane and come into close contact with interstitial cells, partly by forming large swellings (boutons), which may be deeply embedded into the cytoplasm of the innervated cell. The synaptic cleft is about 200 Å wide. Specialized pre- and postsynaptic membranes have not been found. The innervated cells show no peculiarities. The possible function of the synapses is discussed.

Notes: Summary The fine structure of the nerves of the ovarian stroma of the domestic fowl is described for the first time. In the fowl, the nerves are concentrated upon blood vessels, smoth muscles and mainly, the thecal gland with the steroid-producing cells. Myelinated as well as unmyelinated nerve fibers were observed. Numerous axon terminals representing adrenergic and also presumptive cholinergic nerve fibers are regularly seen in membranous contact with steroid-producing cells. In these axon terminals microvesicles are oriented towards the steroid-producing cells indicating a specialization of the surface from axon-to-cell contact. Evidence has been presented that there is a membranous neuro-humoral contact between the peripheral autonomie nervous system and the steroid-producing cells in the ovary. The present investigation has demonstrated that there is morphologic evidence for a nervous control of steroid-producing cells. The physiological importance of this neuro-humoral contact is discussed.

Notes: Summary Pituicytes of Rana pipiens could be classified into two types, pale and dense, according to their relative densities of cytoplasm and the populations of free ribosomes and cell organelles. An intermediate type of pituicyte was also recognized. Lipid droplet such as are typical in the cytoplasm of mammalian pituicytes, are not in the cytoplasm of either types of frog pituicyte. Both types have long cytoplasmic processes which run among the nerve fibers, and some of them end at the pericapillary space. Nerve endings making synapse-like contacts with the cell bodies or the processes of the pituicyte are frequent. According to the structures and sizes of granules and vesicles in the nerve endings, these endings are classified into one of three types: 1) A, which appears to be a peptidergic neuronal ending containing dense granules 1,200–2,000 Å in diameter and small clear vesicles 300–400 Å in diameter; 2) B, which appear to be monoaminergic endings containing cored vesicles 600–1,000 Å in diameter and small clear vesicles 300–500 Å in diameter; 3) C, which appear to be cholinergic endings containing only small clear vesicles. Type C endings are relatively rare. In the synaptic area the axonal membranes appose those of the pituicytes across a gap of about 200 Å and numerous “presynaptic” vesicles are clustered or accumulated near the presynaptic membranes.

Notes: Summary The pars tuberalis of the hypophysis of Rana temporaria presents the general structural and the cytological characteristics of an endocrine gland. It is composed of elongated cells with long, branching processes ending on the external basement membrane of the pericapillary space. The pars tuberalis cells produce secretory granules which are accumulated in the pericapillary endings of the processes. Corresponding to its separate localization, the pars tuberalis of Rana temporaria has a separate vascularization of which the efferent capillaries anastomose with the capillary plexus of the median eminence. The general direction of the blood flow of the pars tuberalis is towards the capillaries of the median eminence. Also, the secretory products of the pars tuberalis pass into the blood stream of the hypophysial portal system. Several characteristics of the pars tuberalis show that its function must be different from that of the pars distalis of the hypophysis. Moreover, in contrast with the pars distalis, the activity of the pars tuberalis is not regulated by neurohumoral factors. The results show that a role of the pars tuberalis in the regulation of the activity of the pars distalis of the hypophysis is not excluded.

Notes: Summary The innervation of the adrenal cortex of the rat and the pig is investigated with the electron microscope. Nerve fibers containing synaptic and two types of dense-cored vesicles come into contact with endocrine cells. There are no specialized pre- and postsynaptic membranes. The synaptic cleft is about 200 Å wide. Generally the basement membrane between nerve and cell is absent. These observations are discussed on the base of more recent experimental findings. Small fibers having an average diameter of about 0.2 to 0.5 μ and containing only tubules and filaments are considered to represent parts of an afferent nervous system.

Notes: Summary The innervation of the mesenteric microvasculature was studied in fetal and neonatal rabbits with the aid of methods demonstrating fluorescence of catecholamines and cholinesterase activity as well as a silver impregnation procedure. The results showed that: (1) adrenergic nerve fibers were present, coursing independently in the mesentery by day twenty-one of gestation, and were found routinely in the adventitia of arterioles and venules by day 25 of gestation; (2) cholinesterase positive cells and fibers of the myenteric plexus were present by day 18 of gestation but cholinergic fibers were not present in the mesentery until day 26; the latter not being associated with blood vessels; and (3) nerve fibers in the mesentery thought to be sensory stained positively with the Holmes silver method on day 18 of gestation.

Notes: Summary Quantitative electron microscopic studies have been carried out on the human thymus. According to the equation L v =(2n)/F (Hennig, 1963) we have calculated that there is less than 0.204 mm nerve per 1 mm3 thymus tissue inside the blood-thymus-barrier (level of significance of 0.95). This result is compared to the degree of innervation in brown adipose tissue, which contains more than 160 mm nerve per 1 mm3 tissue. The biological significance of the paucity of neuronal elements in the thymus is undetermined.

Notes: Summary 1. The Harderian gland in rabbits, representing the type of a tubulo-alveolar gland, is located on the medial and posterior aspect of the eyeball and consists of two different parts, a small white lobe and a larger red one. The secretory cells in the tubular endpieces of both lobes are lipids containing cells. The lipid droplets can be stained with Sudan IV and Sudan black B. The luminal surface of both cell types is characterized by an alcianophilia at pH 2,5. 2. The tubules of both lobes have a single layer of columnar epithelium. The lipid vacuoles in the cells of the red lobe are large, these of the white lobe small. The multilocular cytoplasm of all cells contains many free ribosomes and high amounts of mitochondria lying very closely together. All cells exhibit numerous and large Golgi-zones but only few ergastoplasm membranes. 3. The lateral surfaces of the secretory cells are connected by elaborate junctional complexes (Zonulae occludentes, zonulae adhaerentes, desmosomes). These lateral surfaces are increased by intercellular canaliculi. 4. Before being released into the glandular lumen, the limiting membranes of adjacent lipid droplets fuse, thus forming a large lipid vacuole. Extrusion generally is characterized by the coalescence of the limiting membrane with the plasmalemma, the formation of an opening at the cell surface and the discharge of the secretory lipid material. In the course of another mechanism of extrusion, the fat vacuoles are transported to the apical part of the cell where consequently the plasmamembrane bulges into the lumen. Eventually the fat vacuole is pinched off surrounded by a thin cytoplasmic envelope. 5. Terminal fibers of the autonomic nervous system penetrate the basal membrane and can be found closely attached to the secretory or myoepithelial cells, partly by forming large swellings, which may be deeply embedded into the cytoplasm of the innervated cell. These terminal parts of the axons contain groups of synaptic and dense-cored vesicles, mitochondria and neurotubuli. Specific pre- and postsynaptic membranes have not been observed. The possible function of the harderian gland is discussed.

Notes: Summary Electron microscopic studies have been carried out on the innervation of the mammalian anterior pituitary and parathyroids. The total area of grid squares (2.25·10−2mm2) examined was 2000 per gland and species. In the pituitary pars distalis and in the parenchyma of the parathyroid gland we did not observe a single axon profile. According to the equation $$L_V = \frac{{2n}}{F}$$ proposed by Hennig (1963) we have calculated that there might be—if any—0.133 mm of nerves per 1 mm3 tissue in those two endocrine glands (level of significance 0.95). Comparing these results to the degree of innervation in brown adipose tissue containing more than 160 mm nerve per 1 mm3 tissue we can not imagine that such a small degree of innervation is of any biological importance. In the pituitary pars tuberalis two types of axon terminals have been found both inside and outside the basement membrane surrounding the epithelial complexes. One type contains “synaptic” and two populations of smaller dense-cored vesicles, the other one contains a population of larger granules which have some properties of the classical elementary granules. Further investigations have to clarify the functional significance of those nerve endings.

Notes: Summary The penis retractor muscle of Helix pomatia is passed by thick nerve trunks, which probably are nerve nets. In the contracted muscle, they are folded meanderlike, in the extended they are pulled smooth. Four types of vesicles different in size and contents can be distinguished in the nerves. Varicose fibres accompany the muscle cells. Frequently the terminals are running in a groove of the muscle fibre. In certain axon types occur presynaptic accumulations of vesicles and thickenings of the terminal membrane. Terminal and muscle cell are separated by a cleft of 300 AE width. The muscle fibre membrane has no subsynaptic infoldings. Beside these axons thin, naked neurites are running through the connective tissue. They are characterized by a high content of neurotubuli. One part of the axons presumably possesses a monoaminergic transmitter. After the glutaraldehyde-dichromate-reaction they contain dense grana, whose diameters are mainly below 1000 AE. The nerve trunks fluoresce after exposure to paraformaldehyde vapour, excited with UV-light, green to green-yellow. The maximum of the excitation was determined at 413 nm and the maximum of emission at 496 respectively at 510 nm. It is concluded, that both, a catecholamine and 5-HT are responsible for the fluorescence. Extraction and paperchromatographic separation lead to the opinion, that the catecholamine is dopamine.

Notes: Summary Qualitative and quantitative studies were made to determine the amount of nerve fiber supplying corpora lutea (CL) of rats during the oestrous cycle and pregnancy and sow CL during days 4–6 after ovulation. Fluorescence microscopy of freeze-dried, paraformaldehyde treated (Falck-Hillarp method) rat ovaries reveals adrenergic nerve fibers which run along with vessels and form a network among interstitial gland cells. Nerve fibers do not enter the granulosa cell layer in follicles or CL. In the CL circumference both vascular and non-vascular nerves occur the latter being related to the fibromuscular layer and probably innervating smooth muscle cells. No striking differences exist between the innervation of the ovary in non-pregnant and pregnant rats. Bodian and methylene blue staining did not contribute to a more detailed knowledge of rat ovary nerve supply. Electron microscopic quantitative analysis of rat and pig CL (rat: day 18 of pregnancy; pig: day 4–6 after ovulation) revealed no axon profiles in 2.000 grid squares (one square measuring 2.25×10-2 mm2) of randomly taken CL sections. Thus it was possible to calculate an upper limit of 133 μm of nerve fibers per 1 mm3 CL tissue, in case there were any at all.

Notes: Summary The innervation of the pituitary gland of Carassius auratus was studied by light and electron microscopy under various physiological and experimental conditions to investigate whether or not neurosecretory fibres play a role in regulating pars distalis function. Two types of neurosecretory fibre (Type A and Type B) were distinguished. Prolactin, ACTH and TSH1 cells were innervated by Type B fibre terminals separated from the endocrine cells by a continuous basal lamina (“indirect contacts”). Gonadotropic, STH and TSH2 cells were innervated by Type A as well as Type B neurosecretory fibres, mostly without an intervening basal lamina (“direct contacts”). The assessment of the amount of neurosecretory granules and microvesicles in nerve terminals during the pre-spawning, spawning and postspawning seasons and following the administration of Oestradiol, Thyroxine, Thiourea and Metopiron respectively revealed convincing evidence for a participation in pars distalis control for Type A and Type B fibres innervating gonadotropic cells and STH cells and Type B fibres innervating TSH2 and ACTH cells. Immediately after spawning both nerve fibre types innervating gonadotropic cells and Type A fibres innervating STH cells showed a striking decrease in the amount of dense core vesicles. During the spawning season nerve fibres innervating somatotropic cells, TSH2 cells and ACTH cells also undergo changes suggesting that prior to spawning major changes in the endocrine system of the goldfish take place.—These results point to a dual control, by peptides and amines, of teleost pars distalis function.

Notes: Summary The olfactory mucosa of frog has been studied at an ultrastructural level to confirm previous light microscope observations in regard to the presence, in the sensory epithelium, of nerve fibres not belonging to the first cranial nerve proper. It has been observed that both myelinated and unmyelinated nerve fibres are present in the lamina propria and that eventually these fibres terminate inside the epithelium. Unmyelinated fibres usually contain dark core vesicles and similar content is seen in their intraepithelium terminals. Terminals containing only clear vesicles are also observed in the epithelium and they are believed to represent the terminals of the myelinated fibres. The significance of these ultrastructural findings is discussed in view of their functional meaning.

Notes: Summary The Octopus iris is composed of five different layers: A, the external epithelium; B, the chromatophore layer; C, the iridocyte layer; D, the layer of muscles and collagen strands; E, the pigment epithelium. The nerves innervating the sphincter and the chromatophore muscles are identified and their neuromuscular junction is described. The motor endings of chromatophore nerves have an additional ending in presynaptic position which probably functions as a modifier of neuromuscular transmission. The chromatophores are naked and exhibit a tubular channel system between plasmalemma and pigment container which looks similar to the T-system of muscle cells.

Notes: Summary In the tortoise Testudo graeca, the lizards Lacerta dugesi and Lacerta pityusensis, and the snake Natrix natrix, the innervation of the testicular interstitial tissue was studied by light and electron microscopy, the acetylcholinesterase (ache) technique, the Falck-Hillarp method for the detection of catecholamines, and the application of 6-hydroxydopamine. The intertubular spaces of the reptilian testes studied contain adrenergic nerve fibers the amount and distribution of which varies considerably both in various species and in various stages of the reproduction cycle. Nerve fibers do not enter the seminiferous epithelium. Fluorescence microscopy of the lizard testis reveals catecholaminergic varicosities which are mainly arranged around blood vessels, but do not show obvious connexions to Leydig cells. Ache-positive fibers are equally distributed in lizard testes surrounding each seminiferous tubule. In Natrix natrix ache-positive fibers are irregularly spread among groups of tubules, without showing a definite relation to Leydig cells either. By electron microscopy bundles of unmyelinated axons and axon terminals can be more easily detected in the testes of immature animals than in adult. Terminals of nerve fibers containing small (400–500 Å in diameter) and large (800–1400 Å) dense-cored vesicles and sometimes small clear vesicles establish contacts with Leydig cells. Three types of contact are described. 1. “Contacts” par distance at a distance of about 2000 Å and basal lamina interposed; 2. membranous contacts having a 200 Å gap only between axolemma and Leydig cell plasmalemma; 3. invaginations of terminals into Leydig cell perikarya. The latter may exhibit surface specialisations, which strongly resemble postsynaptic membrane thickenings. Experiments using 6-hydroxydopamine underline the adrenergic character of testicular nerve fibers, which can be regarded as another example of non-cholinergic, ache-positive neurons. In the testis of the immature tortoise profiles of axons occur which probably represent purinergic, ache-positive neurons.

Notes: Summary Terminal axons emerging from the inner plexiform layer of the cat retina reach the wall of the arteria centralis retinae, as revealed by electron microscopy. Numerous unusually large dense core vesicles (about 1000 Å in diameter), of different electron densities, occur in the varicosities of these axons. These observations may be compatible with the idea of an innervation of the central artery of the retina which is non-autonomic, possibly intrinsic in nature.

Notes: Summary Neural elements within the parenchyma of the sebaceous gland have not been reported previously. Nerve endings have been observed only in the connective tissue surrounding the gland or in close association with the undifferentiated basal cells. In this study, electron microscopy revealed the possible presence of nerve endings (or terminal portions of neural elements) in the suprabasal level of functional sebaceous glands of pinnae of white rats. Morphologically, there are two distinct types of nerve endings. Type 1 is bordered by a membrane of relatively irregular contour and contains a single mitochondrion, various-sized vesicles, numerous microtubules, fine neurofilament-like fibrils, and occasional ribosome-like granules. Type II is also bordered by a membrane, but its contour was relatively smooth and rounded. Moreover, Type II contains many mitochondria, varying in size, density, and the arrangement of cristae. While ribosome-like granules are scattered throughout the structure in relative abundance, there are scarcely any fine neurofilament-like fibrils or microtubules. Whether these two structures are sensory or autonomic fibers could not be determined by electron microscopic examination.

Notes: Summary The innervation of the brown, red and yellow chromatophore muscle cells in Loligo vulgaris shows quantitative and qualitative differences. The nerve bundles regularly contain axons with electronlucent vesicles of about 300 Å diameter, and occasionally axons with dense core vesicles of about 600 Å diameter. The myofibrils of the contractile cortex show a staggered arrangement and are wound in a spiral with respect to the axis of the muscle cell. In the axial sarcoplasm there is additionally a bundle of thin filaments of about 70 Å thickness. The bundle is orientated in parallel with the longitudinal axis of the muscle cell. Its function may be to maintain tonic contractions.

Notes: Summary The ultrastructure of the innervation of the anterior cerebral artery of the rat was studied in control animals and in animals after superior cervical ganglionectomy. Fluorescence histochemistry shows a periarterial network of intensely fluorescent fibers which are divided into two groups, adventitial and periadventitial. The fluorescence begins to decrease 26 hours after, and completely disappears about 32 hours after, ganglionectomy. Fine structural changes are first observed 18 hours after ganglionectomy, when the axoplasm of degenerating axons becomes electron dense. This density gradually increases up to about 32 hours. By 32 hours most axons with disintegrating axolemmas become inclusion bodies of the Schwann cells. At this stage, synaptic vesicles can still be distinguished as less dense areas, but the membrane structures of synaptic vesicles and mitochondria are difficult to recognize. The degenerating axons are gradually absorbed and by 38 hours dense, residual bodies are observed in the Schwann cells. Generally speaking, the degeneration occurs first in the adventitial fibers and then in the periadventitial fibers. The transient appearance of small, granular vesicles is noticed in axon terminals about 18 hours after denervation, although very few small, granular vesicles are seen in control tissue or at later stages of degeneration.

Notes: Summary Nerve fibers were demonstrated in the glycogen body of the avian lumbar spinal cord by using silver-impregnation and fluorescence microscopic (Falck-Hillarp) techniques. These nerve fibers originated from nuclear areas in lateral juxtaposition with the glycogen body. The fluorescence of the nuclear area was strongest near the polar regions of the glycogen body. It was suggested that the aminergic neurons of the avian lumbar spinal cord may influence the glycogen body. The avian glycogen body was compared with other storage sites of glycogen within the central nervous system.

Notes: Summary By means of electron microscopy “light” and “dark” pinealocytes can be distinguished in the guinea-pig pineal gland. Glial cells are rare. In the “light” pinealocyte. the most frequent cell type, some “vesicle-crowned rodlets” (VCR) show circumscribed thickenings. From these structures “vesicle-crowned balls” (VCB) have to be clearly distinguished. Furthermore “cylinders” occur, which, it is suggested, are precursors of VCB. “Dark” pinealocytes characterized by chromatin-rich nuclei and electron-dense cytoplasm are rare and contain fewer vesicles, VCR, VCB and “cylinders” than “light” pinealocytes. Numerous non-myelinated nerve fibres are situated within perivascular spaces, a few also in the parenchyma. Synapses between nerve fibres and pinealocytes were not observed. In the pineal gland of pregnant guinea-pigs the following changes can be observed in the second half of gestation. The “light” cells show many nuclear indentations and an increase of “active” zones, mitochondria, smooth ER, agranular vesicles, VCR, VCB, and “cylinders” respectively. The “dark” cells increase in number. After birth these changes reverse to normal within one week. Constant darkness leads to an activation of the “light” cells accompanied by an increase of the VCR and to an increase in number of the “dark” cells. Under constant illumination the “light” cells show a decrease of their organelles and a strong increase of the VCR. After 70 days the VCR also show a change in shape. Following reserpine treatment the VCR decrease in number and show signs of degeneration. It is discussed that the VCR function as pre- or postsynaptic structures and that they are involved either in transmitting impulses from nerve fibres to pinealocytes or from one pinealocyte to the other.

Notes: Summary This investigation is concerned with pineal organs of human embryos 60 to 150 days old. At every stage central nerve fibres enter the pineal organ by way of the habenular commissure, but are restricted to the pineal's proximal part. On about the 60th day of the development the sympathetic nervus conarii grows into the distal pole of the pineal organ from a dorso-caudal direction and plays the predominant part in the innervation of the pineal organ. After penetrating, it soon branches out and forms a network in the pineal tissue. Much later, not until the 5th embryonic month, sympathetic nerves appear accompanying the supplying vessels in the perivascular spaces. After a short time these nerves pierce the outer limiting basement membrane and penetrate the parenchyma. Towards the end of the 5th embryonic month the axons of the sympathetic nerves form varicosities containing clear and dense core vesicles. At this point large amounts of laminated granules appear primarily in cell processes, probably of pinealocytes. Isolated granules also occur in the varicosities of axons. The granules encountered here are most likely secretory granules.

Notes: Summary Nervous (sensory) pathways of the pineal complex were studied in Lampetra planeri. The following observations were made: (1) The pineal tract, containing more than 600 unmyelinated fibres, connects directly or indirectly to the posterior commissure. (2) Connections of the nervous region (NR) adjoining the parapinéal organ (“ganglion parapinéales” of Tretjakoff or “division of the left habenular ganglion” of Studnička) have been re-examined; our study shows that the rostral division of NR is connected to the parapineal organ by a short tract and to the left habenular ganglion (LHG) by a nervous layer (NL). The NR, NL, and LHG contain the same following elements: (1) A collection of typical neurones (which are rare in the NL) in an abundant meshwork of nerve fibres. (2) At least two main types of axons: (a) Type 1 axons, containing few granules (1,000–1,300 Å diameter). (b) Type 2 axons, containing numerous granules (900–2,500 Å diameter), and resembling neurosecretory axons. Type 2 axons are not observed in the pineal and parapinéal organs. (3) Axo-dendritic and axo-axonic(?) synapses. Our study concludes that NR and NL represent the rostral part of LHG; nevertheless, NR and NL may contain the parapinéal tract. The problem of possible incorporation of epithalamic elements (e.g., elements of the habenular ganglia) into the parapinéal and pineal organs in other animals has been considered phylogenetically.

Notes: Summary Umbilical vessels of guinea-pig fetuses were studied shortly before birth. In all umbilical cords investigated an innervation of the umbilical vessels is lacking. The intrafetal parts of the umbilical vessels on the other hand are richly innervated. A marked difference in the amount of nerve fibres and the pattern of innervation is found between artery and vein. The artery is supplied by a dense nerve plexus which spins around the media and which originates from nerve bundles within the outer adventitial layers. The comparatively scanty innervation of the vein exhibits a more coarsely meshed net pattern. The nerve bundles in the vein exhibit a close affinity to the vasa vasorum. Number and type of the close contacts between the muscle cells are different in the various sections of the umbilical vessels. Similar to the distribution of nerves they are almost absent in the vessels of the umbilical cord, numerously, however, in the intrafetal parts. Contrary to the innervation, the close contacts in the vein are developed more numerously and more broadly than in the corresponding artery.

Notes: Summary The development of adrenergic nerves to the anterior eye segment was studied in human and guinea-pig embryos. Adrenergic terminals had already appeared in the earliest human embryos available (4–6 cm). They first appeared mainly in nerve trunks in the primitive chorioid, especially in the region of the developing ciliary body. Adrenergic nerves then grow into different structures of the eye as these develop, but typical terminals in contact with effector cells appeared late during the development, about the 25–30 cm stage. No adrenergic nerves were observed in the chamber angle. Corneal adrenergic nerves (also intraepithelial terminals) appeared much more frequently in embryos than in adults. No adrenergic neurons were observed in the retina. In the guinea-pig, the first adrenergic fibres were observed at about gestation day 35. The general principle of the development was very similar to that of the humans. At gestation day 45 to 50, the supply of adrenergic fibres was essentially that of the adult animal, except that the corneal adrenergic fibres were increasing until just before birth and that the adrenergic terminals of the chamber angle appeared shortly before term.

Notes: Summary The intrinsic innervation of the anterior byssal retractor muscle in Mytilus edulis L. has been demonstrated using Maillet's osmium-zinc iodide technique (ZIO). Grouped and isolated osmiophilic cells, thought to be neurons, and nerve fibers forming neuromuscular contacts and nerve endings, have been observed. When the period of fixation is not overly prolonged, glio-interstitial cells may also be distinguished.

Notes: Summary The fine structure of the upper tibial epiphyseal plate of the normal guinea pig has been investigated. On morphological and functional grounds this cartilage can be divided into four zones. In all zones the chondrocytes are distributed in an electronlucent organic matrix composed of thin collagenous fibrils and small non-membrane-bounded granules representing proteoglycans. 1. The reserve cell zone consists of a primitive type of hyaline cartilage. The cells have a very high nucleo-cytoplasmic ratio. The endoplasmic reticulum is the most developed cellular organelle. 2. In the proliferative zone the flattened cells are arranged in distinct columns. The endoplasmic reticulum and the Golgi complex are prominent. Mitochondria and lysosomes are more numerous than in the reserve cells. Extracellularly, the collagen fibrils are brought together in longitudinal and transversal septa. 3. In the hypertrophying zone the cells are more rounded and the pericellular zones wider than in the preceding zones. The endoplasmic reticulum contains dilated cisternae. The Golgi complex includes many large vacuoles. Mitochondria and lysosomes are more abundant than previously. 4. There is a progressive mineralization of the longitudinal septa in the calcifying zone. Within the cells the amount of endoplasmic reticulum is decreased. Its cisternae mostly are short, rounded, and non-continuous. The Golgi apparatus is poorly developed, and the mitochondria few.

Notes: Summary The ultrastructure of the pars intermedia (PI) of the normal VII +/+ and hereditary nephrogenic diabetes inspidus DI Os/+ mice has been studied with particular reference to the morphology of the glandular cells and their innervation. Four types of cells were observed in both the genotypes of mice, 1) the light glandular cell, 2) the dark cell, 3) a type of cell similar to ependymal cells and 4) a small percentage of typical ACTH cells, observed mostly on the PI border of the cleft and rarely in the centre of PI. The predominant light glandular cells contain mainly two types of membrane bound granules: 1) electron dense core granules, which measure 1500–2500 Å and 2) electron lucent vesicles, which measure 3000–4000 Å in diameter. Granules of intermediate size with various density are also present in both types of mice. The electron dense core granules are predominant in DI Os/+ mice, whereas, electron lucent vesicles are predominant in the normal VII +/+ mice. Similar uniform size membrane bound electron dense granules have been observed in ACTH cells of PI and pars distalis. From earlier experimental evidences and the present observations, it is concluded that the dense core granules in PI may be synthesizing ACTH or ACTH-like substance. It is also discussed that these dense core granules may further mature and give rise to MSH in the form of electron lucent vesicles. If it is so, PI light glandular cells may have dual functions, of producing MSH and ACTH. One of the functions of ependymal-like cells, may be the transport of PI secretion. Three types of nerve endings are observed throughout the PI, making synaptic contact with the predominant cell type. The innervation is more in DI Os/+ mice than in normal mice. The classification of these nerves is according to Bargmann and co-workers 1) peptidergic neurosecretory fibers, contain mainly membrane bound dense core granules, measuring 1200 to 1800 Å, and are the classic neurosecretory granules; 2) adrenergic fibers, measuring 700–900 Å; 3) cholinergic fibers, measuring 300–400 Å. Adrenergic and cholinergic fibers are more towards the hypophysial cleft. The increased innervation, the synaptic contact, the extremely hypertrophied PI and the greater activity of its light glandular cells in the DI Os/+ mice show the PI is under the influence of the nervous system.

Notes: Summary The innervation of the salt gland of the goose, the duck and the swan was investigated by means of electron microscopy. Axonal swellings were observed in relationship to secretory cells as well as to central duct cells. The terminals contain synaptic and densecored vesicles. There are no specialized pre- and postsynaptic membranes.

Notes: Summary Morphological evidence is presented supporting the possibility of basal secretion into hypendymal capillaries of the adult rabbit subcommissural organ (SCO). The synthetic apparatus of the SCO cell is described as well as the heterogeneous granules and vesicles which are concentrated in the basal processes bordering a widened perivascular space. The origin of the electron dense granules, of which two fairly distinct subgroups are found, is discussed. A binding of secretory sacs to the lateral plasma membrane is seen. The possibility of a lateral secretion is supported by the presence of a system of extracellular channels between SCO cells which are filled with a flocculent material resembling that of the secretory sacs. Nerve perikarya which are separated from the SCO by only a few glial fibers are demonstrated. Synapses are described in nerve fascicles bordering on the hypendymal capillaries. The possibility of an innervation of the hypendymal region is discussed as well as possible nervous connections with the pineal gland.

Notes: Summary The salivary glands of the moth, Manduca sexta (Insecta: Sphingidae), are unlike most other salivary glands in that they are innervated from one source only. Vital staining of nerves with methylene-blue reveals numerous fine nerves extending to the glands from the oesophageal nerve, a part of the stomatogastric or visceral nervous system. Light and electron microscopy confirm that only the fluid-secreting cells, confined to a discrete region in these glands, are innervated. Axons with or without glial wrappings are found in intercellular spaces between fluid-secreting cells. Axons lacking a glial sheath contain, after glutaraldehyde-osmium tetroxide fixation, large granular and small agranular vesicles. In nerve endings in glands fixed with permanganate these smaller vesicles are granular, having the electron-dense cores characteristic of monoamine-containing neurons. These nerve endings with “synaptoid areas” are in close (“direct”) contact with the fluid-secreting cells.

Notes: Summary Electron microscope studies of axons, distributing singly or in small bundles in the human ventricular and atrial myocardium, indicate a few per-cent of the axon profiles to be significantly large in diameter (1.5–3.0 μ). They are characteristically packed with a profuse number of mitochondria along with large granular vesicles, glycogen rosettes, lysosomic bodies; and some of them terminate on a “specific terminal cell” (Knoche and Schmitt). These mitochondria-rich, large axons are assumed to be terminal portions of the cardiac afferents. About half of the axons encountered in the ventricle and 2/3 in the atrium are non-vesiculated, usually less than 0.5 μ. in diameter. The varicosities containing numerous vesicles are mostly 0.5–1.5 μ in diameter and are assumed to be terminal portions of the cardiac efferents. The ratio between the number of axon profiles containing small granular vesicles and that of axon profiles containing agranular vesicles without small granular vesicles is 2∶1 in the ventricular myocardium and 1∶1.7 in the atrial myocardium.

Notes: Summary The hilus cells in the ovary of the pig have been investigated with the electron microscope. These elements are identical with Leydig cells. The hilus cells contain an abundant agranular endoplasmic reticulum, which is either organized as a tubular network or as an onion like system of closely packed flattened cisternae sometimes filling up half of a cell and often being concentrated around lipid droplets and pigment granules. The mitochondria have cristae, tubuli and sacculi and a matrix of variable electron density. The golgi cisternae are poorly developed. The cells have centrioles, and there are hints of an amitotic cell division. The cytoplasm of the hilus cells contains lipid droplets, which have a diameter up to 5 μm. The pigment granules are extremely polymorphic, nearly always surrounded by a single membrane. They possibly may be derived from lipid droplets and mitochondria. Single axons of the hilus nerve occasionally penetrate the basement membrane and come into close contact with the hilus cells. Typical synapses were not observed. The results are discussed on the background of light microscopical, biochemical and pathological findings and compared with those obtained on Leydig cells of different species. Various cell images are interpreted to represent progressive stages of maturation.

Notes: Summary The rabbit SA-node was outlined electrophysiologically and its adrenergic and cholinergic innervation patterns were studied with the electron microscope. Differentiation between adrenergic and cholinergic terminals was achieved by fixation of the specimens in KMnO4 which produces dense-cored synaptic vesicles in adrenergic terminals, whereas synaptic vesicles in cholinergic terminals are empty. It was found that adrenergic and cholinergic nerve terminals often come in close apposition to each other, the distance between adjoining membranes being in the order of 250 Å. At times, faint membrane thickenings could be seen in these places. The available pharmacological, physiological and morphological evidence leaves little room for doubt that cholinergic terminal fibers can influence the adrenergic ones. From mainly morphological evidence it is also postulated that adrenergic terminals influence cholinergic terminals.

Notes: Summary The median eminence and the pars nervosa of Rana esculenta have a different structure. The median eminence has 2 different zones: the outer zone situated near the pars distalis and the inner zone under the ependyme. In the outer zone there are, according to the size and the shape of the granules, 5 types of nerve terminals. 1. Endings containing spherical fine dense granules of 800 to 1000 Å in diameter; 2. Endings with spherical granules from 1000 to 1200 Å in diameter; 3. Endings with granules of irregular shape which are bigger than the former (1200 to 1600 Å); 4. Endings with spherical dense granules of about 1200 to 1800 Å in diameter; 5. A few endings containing only clear vesicles. Type 3 and type 4 endings are probably neurosecretory. The inner zone contains numerous neurosecretory fibres. They are of two types: one with big granules (1600–2400 Å), the second with smaller granules (1300–2000 Å). Non-neurosecretory fibres have also been observed. The pars nervosa contains two principal types of neurosecretory fibres: one with dense granules of 1600 to 2400 Å in diameter, the other with lighter granules of about 1300 to 2000 Å. In the external zone lining the pars intermedia, aminergic fibres with fine granules have been observed.

Notes: Summary The innervation of the portal vein of the white rat was examined with light-, fluorescence-, and electronmicroscopic techniques. The results are as follows: 1. Paraldehyde treated vein preparations (Falck's fluorescence method) demonstrate a predominantly longitudinally orientated external nerve plexus, being situated on the outermost muscle cells. Near the liver the nerve net is characterized by broad meshes, near the intestinal tract by narrow ones. The circular subendothelial inner plexus originates in the outer plexus of the intestinal vascular bed. 2. Nerve bundles in the fibrous adventitia were demonstrated with Gomori's Acethylcholinesterase method. In other respects, the enzyme activity was only observed in the intercellular spaces of the muscle layer. The electronmicroscopic demonstration of Acetylcholinesterase (Karnovsky's method) further showed that the enzyme activity is restricted to the muscle cell membrane. 3. The electronmicroscopic examination verified the results obtained with fluorescence microscopic techniques. a) In the proximity of the liver, only isolated nerve bundles occur on the outer muscle layer. The individual nerves are entirely surrounded by Schwann cells. Only a few of the axons in the vicinity to the muscle cell have agranular vesicles. Evaginations of the vesicular axons occur infrequently. Their distance from the muscle cell amounts to 1000–2000 Å. b) In more than one thousand thin sections, no axons were found inside the thick muscular layer. c) Subendothelial axons (inner plexus) are either partially or totally evaginated from the Schwann cells. They are densely filled with empty vesicles (350–650 Å) and contain a few dense core vesicles of 800–1600 Å in diameter. Synaptic endings were not observed. d) A dense collection of vesicle-containing axons, that were partially in their entirety and partially only from the muscle cell proximal side evaginated from the Schwann cells, were observed in the single muscle layer at the junction of the superior mesenteric and the portal vein. From these bundles, smaller bundles and individual axons pass between the muscle cells and reach the endothelium. Typical synapses were not observed. No vesiclecontaining axon was nearer than 1000 Å to the muscle cell. 4. Those axons possessing vesicles and being evaginated are considered to be vegetative conducting pathways. The excitation of the effector structures by transmitter substances is discussed in connection with the post mortem autonomic vascular contractility.

Notes: Summary 1. There is a gradual proximo-distal increase in the thickness of the muscle coat of the human ductuli efferentes, duetus epididymidis and ductus deferens. Circularly arranged smooth muscle bundles predominate in the ductuli efferentes and ductus epididymidis of the caput section. Scanty strands of longitudinally and obliquely oriented smooth muscle bundles form an additional, incomplete outer muscle layer around the ductus epididymidis of the corpus. Small smooth muscle-like cells constitute the muscle elements of the upper sections of the excretory ducts (from the ductuli efferentes to the midcauda). At the transition of the corpus and cauda epididymidis ordinary large smooth muscle cells join the small contractile cells to form—in more distal sections of the cauda—a composed, thick subepithelial muscle coat. In most distal portions of the cauda, the two-layered muscle coat of the ductus epididymidis is transformed into a three-layered coat, a pattern of construction which is retained in the vas deferens. 2. Electron microscopically, three types of contractile cells are distinguished in the human ductuli efferentes and ductus epididymidis: a) contractile cells of medium transparency containing exclusively thin myofilaments (60 Å in diameter), b) dark contractile cells containing bundles of thin myofilaments (60 Å in diameter) and single coarse filaments (140 Å in diameter), c) light contractile cells with loosely dispersed, interweaving thin and thick myofilaments. Commutual diameter changes at regular intervals are seen in individual myofilaments, giving the impression of structural periodicity not unlike that of filaments of striated muscle. Ordinary smooth muscle cells of the cauda epididymidis and vas deferens are characterized by uniformly sized, closely packed but evenly distributed thin myofilaments with numerous dense patches. 3. Fluorescence microscopy performed on formaldehyde treated freeze dried tissues reveals that the contractile cells of the ductuli efferentes in man and monkey receive a low number of single adrenergic terminal fibres penetrating the depth of the muscle coat. The adrenergic innervation of the ductus epididymidis is restricted to small peritubular nerve fascicles running contiguous to the most superficially located bundles of smooth muscle-like cells. The adrenergic ground plexus is rather wide-meshed in the proximal cauda, becomes increasingly dense in more distal cauda sections and in initial, funicular portions of the vas deferens, and reaches maximum density in abdominal parts of the ductus. Perivascular and adventitial adrenergic plexuses are well developed at arteries of the caput and corpus epididymidis in man, monkey, rabbit, guinea-pig and rat. 4. Electron microscopically, noradrenergic nerves have been identified by the presence of small granular vesicles in preterminal varicose axon dilatations. Nerve fibre swellings filled with small empty spherical vesicles have been considered to belong to “cholinergic” neurons whereas occasional varicosities equipped with some large membrane bound granules and abundant mitochondria may represent local expansions of sensory axons. 5. Neuromuscular relationships in the upper sections of excretory ducts comprise adrenergic synapses by distance (more than 1000 Å), and a few intimate, ensheated close contacts, whereas the main type of contact of nerves to ordinary smooth muscle cells in the lower duct section is by means of close but not intimate approach (500–2000 Å). 6. Adrenergic synapses in the ductus epididymidis and ductus deferens of the monkey resemble—what concerns their morphology, relationship to effectors and distribution pattern—those of man. 7. In accordance with the total number of vascular and non-vascular adrenergic nerves, visualized by fluorescence microscopy, the amount of noradrenaline varied considerably in different sections of the human male internal genital organs: The lowest amounts were estimated in the testis (0.12±0.03 μg/g). Medium to high concentrations were detected in various sections of the caput and corpus epididymidis (ductuli efferentes 0.60±0.09 μg/g; ductuli efferentes and caput 0.72±0.13 μg/g; corpus epididymidis 1.04±0.25 μg/g; proximal cauda 0.95±0.17 μg/g; distal cauda 0.97±0.19 μg/g). The highest noradrenaline content was found in the human vas deferens (prox. vas deferens 1.11±0.21 μg/g; interm. vas deferens 1.20±0.42 μg/g; distal portion 1.43±0.39 μg/g). 8. For comparison, the noradrenaline content of the testis and epididymis of the rhesus monkey, the epididymis of the rabbit and the vas deferens of the rabbit, mouse, guinea-pig and rat has been determined. 9. Adrenaline of exogenous origin was detected in the vas deferens, cauda epididymidis and plexus pampiniformis of two cases who received this catecholamine as part of the local anaesthetic drug mixture. Due to methodological reasons, the presence of small amounts of adrenaline of endogenous source in adrenergic nerves of the human and monkey internal male genital organs cannot be excluded. 10. The differences in motility behaviour of the ductus epididymidis (spontaneous, rhythmic contractions) and ductus deferens (absence of any spontaneous movements under conditions at rest) in vivo and in vitro have been correlated with the occurrence of specialized contractile cells in the upper segment (ductuli efferentes, ductus epididymidis of the caput, corpus and initial cauda) and ordinary large smooth muscle cells in the lower segment (ductus epididymidis of the distal cauda and the vas deferens) and furthermore correlated with differences in the pattern of the adrenergic innervation; the concept is advanced that progressive cytological differentiation of smooth muscle cells and the development of a dense direct adrenergic innervation suppresses autocontractility and, that the reverse condition may favour spontaneous motility of smooth muscle elements.

Notes: Summary The glandula infraorbitalis buccalis in rabbits represents the type of a tubuloacinar mucous secreting gland. The secretory product of gland cells is electron lucent. Extrusion generally is characterized by the formation of an opening at the cell surface and the discharge of the secretory material. Intercellular canaliculi are absent. Mitochondria and arrays of granular endoplasmic reticulum are more numerous than in other mucous glands of rabbits. Intercalated ducts are short and connect the tubules with intralobular ducts. Striated ducts are absent in the infraorbital gland. Nerve terminals occur in some instances between gland cells and between glandular and myoepithelial cells.

Notes: Summary 1. Three species of the genusGnathonemus (Gn. petersii, moorii andstanleyanus) as all species of the family Mormyridae possess two pairs of electric organs situated symmetrically on each side of the vertebral column between the caudal, dorsal and anal fins. Each organ is composed of a series of 70–170 electroplaques. 2. The stalks of the electroplaques contain no nerves. Unbranched motor nerve fibres innervate the end sections of the stalks by synaptic knobs inserting into cavities of the electroplasma. 3. The electroplasma membrane surrounds the stalk (also in the region where the stalk penetrates through holes in the body of the electroplaque) and the electroplaque. The membrane is deeply indented on the cranial side as well as on the caudal side of the electroplaque. Through this the surface is greatly enlarged. In the inside of the electroplaque are striated muscles. 4. The motor and sensory nerve fibres are clearly different in diameter. 5. The number of the nerve cells in the spinal ganglions is, in the electric organ, about a multiple larger than in the segments of the body muscular system situated in front of the electric organ. 6. The reconstruction of the peripheral sensory nerves of one segment (in view of electron microscopical analyses), reveals that these, with the exception of two free nerve endings in the integument and the free nerve endings in the dorsal myoseptum, mainly innervate four large tendons on the side of the electric organs. 7. The innervation of the electric organs leads to the hypothesis that the tendons, in connection with the function of the electric organs, possess sensory functions.

Notes: Summary The fine structure of the pancreatic nerves of the domestic fowl has been studied. Naked axon beadings were found in membranous contact with endocrine as well as exocrine cells. From an anatomical point of view it seems reasonable to suggest that the endocrine glands might be subjected to some influence of the autonomic nervous system.

Notes: Summary Histological, histochemical, and ultrastructural investigations have been carried out on ageing costal and tracheal cartilage of rats. The following age groups of animals have been studied: 1, 7, 14, 20, 30, 45, 75 days, 6 months, and 2 years. Ageing induces cellular changes which are represented by a reduction in the number of chondrocytes, a progressive increase in glycogen deposition, and processes of degeneration, the most frequent of which is the accumulation of lipidic material within large cytoplasmic vacuoles. Changes in the intercellular matrix become evident after 20 days in costal cartilage and after 30 days in tracheal cartilage. Chondroitin sulphate decreases while keratan sulphate, whose presence is limited to the territorial matrix, increases. Glycoproteins increase slightly in young animals and then remain constant; they decrease in the subperichondrial areas in old animals. Ultrastructurally, the matrix of cartilage of young animals contains thin collagen fibrils, most of which have no periodic banding. Roundish electron dense granules are associated with these fibrils. Irregular filaments associated with small electron-dense circular bodies are present around chondrocytes as well as within cytoplasmic vacuoles. With increasing age, and coincident with the reduction of chondroitin sulphate, the thickness of collagen fibrils increases, their period becomes evident, and the associated matrix granules decrease in number and size. Areas containing these fibrils undergo calcification, which frequently starts within roundish bodies of cellular origin. Collagen fibrils with a period of 640 Å but a highly variable thickness are often present in cartilage of adult and old rats. These fibrils seem to be due to an abnormal synthetic activity of chondrocytes.

Notes: Summary The innervation of the Purkinje fibres in the atrium of the heart of the adult fowl was investigated by light microscopy, using the Champy-Maillet OsO4-ZnI2 technique and the cholinesterase reaction and by electron microscopy. After impregnation of the tissue with OsO4-ZnI2, the dark-stained nerve fibres were clearly visible on the unstained Purkinje fibres. In the upper part of the posterior wall of the right atrium, the diffuse portion of the conducting system is especially richly innervated by varicose and smooth nerve fibres. Some of these fibres are cholinesterase-positive. The terminal axons run in the space between the Purkinje fibres and the fibrocytic envelope. They are either naked or accompanied by Schwann cell processes. In addition to varicosities containing granular vesicles, there are varicosities containing agranular vesicles with oval profiles. In the “en passant” synapses, the width of the synaptic cleft between a varicosity and the Purkinje cell is about 600 Å. The innervation of the Purkinje fibres appears more like the innervation of smooth muscle than that of striated skeletal muscle. The possible role of Purkinje fibres as mechanical receptors is discussed.

Notes: Summary The pathways of axonal transport of secretions from neurosecretory cells (NSC) in the medial group (viz. A-, A1, B-, and C-type NSC) and the lateral group (L-type NSC) are described. Individual axons can be recognized in the electron microscope by the kind of neurosecretory particles they contain. In general, the secretions from the medial NSC are carried to the contralateral nervi corporis cardiaci (NCC), those from the lateral NSC to the ipsilateral NCC. Some axons from the A-type NSC, in addition, may run to ipsilateral NCC. All A-type axons have collaterals which run to the ipsilateral NCC. The medial and lateral bundles of “mixed” axons run through one paired NCC but remain separated spatially. Release of secretion from the C-type NSC can take place before the corpus cardiacum is reached. A- and A1-type NSC have additional collaterals that branch from the proximal part of the axons and penetrate deeply into the neuropile of the protocerebral lobes. Local swellings appear to be closely associated with fibers from non-neurosecretory neurons. The sites of contact are characterized by the accumulation of microvesicles (400 Å) near an electron-dense cleft of 150–200 Å width, and resemble regular chemical synapses. The microvesicles prove to be present within the neurosecretory fiber in most of the specimens studied, and within the non-neurosecretory fiber in only a few cases. It seems most likely that the collaterals in some phases convey afferent signals to the NSC which inhibit the release of an efferent neurochemical “messenger” of unknown nature into the neuropile.

Notes: Summary The cartilage matrix in which the early calcium salts are deposited has been studied in the tibial epiphyses and in the costo-chondral junctions of 30-day-old guinea pigs. The results may be summarized as follows: (1) Structures of globular shape (“globules”) are to be found throughout the entire epiphyseal plate. (2) They have a homogeneous matrix and are bounded by a membrane. (3) Early calcification occurs in globules. Calcification of collagen fibrils seems to occur later. (4) The earliest mineral deposited would seem to consist of tiny granules about 20 Å in diameter. Then apatite crystals are laid down, initially in small clusters and later filling the globules completely. (5) The globules are strongly osmiophilic. They seem to contain a fair amount of neutral polysaccharides, but no acid polysaccharides except a coating on their outer membrane. Hyaluronidase digestion does not affect globules. Papain digestion makes them more reactive to uranium and lead. (6) Globules are of cellular origin but they are almost certainly not pre-formed in the chondrocytes. Finally, the present paper advances the hypothesis that some globules derive from degenerating chondrocytes and others from the processes of normal chondrocytes.

Notes: Summary The nerve supply to the iridic melanophores of the rat was studied with the electron microscope. The adrenergic and cholinergic terminals were identified with the aid of 5-hydroxydopamine, which produces dense-cored 400–800 Å synaptic vesicles in adrenergic axon varicosities, whereas the synaptic vesicles of cholinergic axons remain empty. It was found that both adrenergic and cholinergic terminal axons come in close apposition (200–250 Å) with the melanophores. The appositions have the same appearance as synapses in peripheral tissues. It seems likely that the murine iridic melanophores have a double innervation, although its functional significance is obscure.