Changes in the fine structure of the human testicular interstitial cells after treatment with human gonadotrophins

Summary Study of the fine structure of the human interstitial cells after prolonged stimulation with human gonadotrophin reveals a striking increase in the quantity of the agranular endoplasmic reticulum. This is accompanied by an increase in the number of mitochondria which exhibit more extensive cristae, collections of intramitochondrial lipid and aggregations of electron-dense granular deposits. A rise is also evident in the number of lipofuscin pigment deposits and granular membrane-bounded bodies, both of which exhibit acid phosphatase activity. These changes after gonadotrophic stimulation are discussed in relation to steroid biosynthesis.In the pretreatment biopsies of these patients aged between 25–35 years, some interstitial cells contain intranuclear crystals which exhibit a hexagonal structure. The relationship of these intranuclear crystals to the cytoplasmic crystals of Reinke is discussed.The author is indebted to Dr. J. W. Johnstone and Dr. A. Long for the human material used in this study. Thanks are also due to Dr. H. P. Taft for helpful suggestions in the management of these patients, to Professor B. Hudson for the estimations of plasma testosterone and to Dr. J. B. Brown for the supply of human pituitary gonadotrophin and the estimations of urinary oestrogens. The technical help of Mr. T. Mezciems and the photographic assistance of Mr. J. S. Simmons F. R. P. S. and Miss S. Flett is gratefully acknowledged.     

On lipid droplets in renal interstitial cells

Summary By examining Epon sections of the renal papilla, it is demonstrated that a brief period of salt repletion in salt-depleted rats induces a considerable increase in the number of lipid droplets in the renal interstitial cells. It is supposed that this difference indicates that the lipid droplets contain substances which are physiologically important for the kidneys. Possibly, these substances are prostaglandins, which have been demonstrated previously by other authors in extracts from the renal medulla.This work was supported by a grant from the Danish State Research Foundation.The author wishes to acknowledge the support and inspiration given by E. Bojesen, M.D., and P. Leyssac, M.D., the Institute of Experimental Medicine.     

Hormonal control of leydig cell differentiation

Summary The fine structure of the testicular interstitial cells of the 9-day-old mouse submitted to stimulation with human chorionic gonadotropin (HCG) is reported. As was previously described (Baillie 1964) the interstitial tissue of the prepubertal mouse testis is characterized by the presence of well differentiated epithelioid cells at a quiescent stage. They are characterized by large cytoplasmic depots of lipid droplets and glycogen particles in contrast to poorly developed membranous organelles. These cells are highly sensitive to the action of gonadotropins. Five daily injections of HCG cause their differentiation into cells with active secretory characteristics. The gonadotropin induces a marked depletion of the lipid droplets and glycogen content of the cytoplasm, concurrent with an unusual development of the membranes of the agranular endoplasmic reticulum. Golgi complexes and rough reticulum are prominent. Several changes also appear in the nucleus, especially in the nucleolus.The correlation of the present electron microscopic study of the interstitial cells under HCG stimulation with previous biochemical and physiological findings tentatively suggests that the immature Leydig cells exhibit the basic organization necessary for biosynthesis of steroid hormones.     

On lipid droplets in renal interstitial cells

Summary By ultracentrifugation of homogenates of rat renal papillae, a low density layer composed of small primary fluorescing lipid droplets was isolated. Probably the isolated lipid material originates mainly from the primary fluorescing lipid droplets of the renal interstitial cells. The isolated lipid droplets were mainly triglycerides, cholesterol esters and free longchain fatty acids. The long-chain fatty acid composition of the main components differed markedly from that of the analogous components of plasma and depot fat. The findings suggest that the complex lipids of the isolated lipid droplets are synthetized by the renal papilla and probably by the renal interstitial cells. The prostaglandin precursor arachidonic acid constitutes a significant fraction of the triglyceride long-chain fatty acids. Usually also small quantities of prostaglandins were present. Altogether the results suggest that the lipid droplets of the renal interstitial cells may function as a storage site for prostaglandin precursor.This work was supported by a grant from the Danish State Research Foundation. — The authors wish to acknowledge the support and inspiration given by E. Bojesen, M.D., Ph.D., the Institute of Experimental Medicine, Division of Endocrinology. — The authors wish to thank Dr. John E. Pike of the Upjohn Company, Kalamazoo, Michigan, for supplying the Prostaglandins used in this study.     

On the localization of a prostaglandin-dehydrogenase activity in the kidney

Summary Using the kidneys from white rats a method for the localization of a prostaglandin-dehydrogenase activity is presented.The activity demonstrated showed NAD dependence and displayed a high substrate specificity.The most pronounced activity was observed in the thick ascending limb of the loop of Henle and in the distale tubule. Lesser activity was found in the collecting tubules of the inner medulla, in the interstitial cells of the medulla, in the epithelial cells of the pelvis, in the tunica media of the cortical arteries and arterioles and in the visceral epithelium of the renal corpuscles.The pitfalls of the cytochemistry of the co-enzyme linked dehydrogenases are discussed and correlated to the present study. Similarly the observations noticed is discussed in relation to the metabolism and biological activity of the prostaglandins.This work was supported by a grant from the Danish State Research Foundation.The authors thank Dr. John E. Pike of the Upjohn Company, Kalamazoo, Michigan, for the gift of prostaglandins used in these studies.     

An Outwardly Rectifying and Deactivating Chloride Channel Expressed by Interstitial Cells of Cajal from the Murine Small Intestine

Recent studies have suggested a role for a chloride current in the modulation of pacemaker potentials generated by interstitial cells of Cajal. Patch-clamp recordings were made from inside–out patches of cultured interstitial cells of Cajal from the murine small intestine. The majority of patches were quiescent immediately after excision, but in some patches currents activated spontaneously after a period of 10 min to 1 h. Currents could also be activated by strongly polarizing the patch. It was found that the currents activated in both cases included a chloride channel. This channel could also be activated by ATP and the catalytic subunit of protein kinase A. The channel had conductance states (±SD) of 53 ± 25.35, 126 ± 21.44, 180 ± 12.57 and 211 ± 8.86 pS. It was outwardly rectifying (as a function of open probability) and deactivated (i.e., gave a tail current) but showed no inactivation. The permeability sequence of the channel was I⁻>>Br⁻≥Cl⁻>Asp⁻. It was unaffected in magnitude or rectification by changing the free Ca²⁺ concentration of the bath between <10 nm, 100 nm (control) and 2 mm.     

Macrophages containing langerhans cell granules in normal lymph nodes of the rabbit

Summary Light and electron microscopic studies on macrophages of normal rabbit lymph nodes showed two types, one with little phagocytic activity and many features similar to those of epidermal Langerhans cells. Among these are characteristic Langerhans cell granules. From these findings it is concluded that the Langerhans cells may be derived from lymph node macrophages.The helpful advice and criticism of Dr. Toshio Nagano, Department of Anatomy, are gratefully acknowledged. The discussion with Dr. Mitsumasa Itoh, Department of Dermatology, was also helpful.     

Fluorescence studies on neural structures and endocrine cells in the pancreas of the cat

Summary With the use of the Falck-Hillarp histochemical technique for the detection of monoamines, nerve fibre fluorescence is observed throughout the tail of the pancreas of the cat and the arrangement and distribution of the nerve fibres can be studied in both the exocrine and endocrine tissue. In the exocrine pancreas, adrenergic nerve fibres innervate arterioles, larger veins and major pancreatic ducts. Adrenergic nerve fibres also appear to terminate on the non-adrenergic nerve cell bodies of the intrapancreatic ganglia. In the islets of Langerhans, adrenergic nerve fibres innervate both the endocrine cells and blood vessels. Some of the islet cells exhibit fluorescence with the Falck-Hillarp technique and these cells have been identified as alpha cells. In animals treated with reserpine, the fluorescence in nerve fibres and in alpha cells is absent.The author wishes to thank ProfessorG. C. Schofield and Dr.G. C. Smith for their encouragement and valuable criticism during the course of this study. The assistance of MissJ. Bennett and MissW. Kemp and the photographic help of Mr.J. S. Simmons, F.R.P.S., are gratefully acknowledged. The diagram was drawn by MissS. Flett.     

Ultrastructure of the human submaxillary gland

Summary Myoepithelial cells in the human submaxillary gland are stellate in form, with long, tapering processes. They are interposed between the base of the secretory cells and the basement membrane, and are bound to the secretory cells by desmosomes. Their cytoplasm contains numerous myofilaments measuring approximately 40 Å in diameter, which frequently aggregate to form structures similar to the dark bodies seen in smooth muscle cells. The myofilaments are anchored to the plasma membrane by attachment devices. The myoepithelial cells are often accompanied by cells which have a similar shape, but possess an extremely electron-lucent cytoplasm that contains almost no organelles or inclusions. Electron microscopical observations indicate that these cells, termed clear cells, are transformed directly into myoepithelium, since all morphological intergrades between the two cell types have been recognized.This work was supported in part by a grant from the Henry Spenadel Trust. The technical assistance of Mr. Roy R. Keppie and Mrs. Mona Brandreth is gratefully acknowledged.     

Submicroscopic structure of the membranous labyrinth

Summary Investigations were performed by light and electron microscope on the submicroscopic structure of the epithelium of Corti's organ in the white rat.Morphological and structural differences between the inner hair cells and the outer hair cells are revealed.The inner hair cells are closely inter-related with the inner supporting cells and have a polyhedral shape, whereas the outer hair cells look like cylinders and are surrounded by an intraepithelial fluid.The structural peculiarities consist of differences in the dimensions of the hairs, in the arrangement of cytoplasmic organoids and in the aspect of the receptoneural junction. In both sensory hair cells 4 zones of different structure can be distinguished from the surface inwards: apical zone, intermediate zone, perinuclear zone and receptoneuronal junction. The functional value of these different zones is discussed and compared with what has been demonstrated in other receptors.The pillar cells and the Deiters' cells are supporting cells which have a filamentous skeleton, composed of submicroscopic individual filaments. These filaments have a diameter of about 215 Å and present some analogies with the tonofilaments of the stratified squamous epithelium. The filaments are arranged differently in the pillar cells and in the Deiters' cells. Possible functional differences between these patterns are discussed.The reticular membrane is not an extracellular cuticle. It consists of intracellular cementitious material (like the terminal bars of the epithelial cells).The Hensen's and Claudius cells, the Böttcher's cells, the inner supporting cells, the inner and outer spiral sulcus cells are regular prismatic cells with few endoplasmic organoids and without filaments.This work is dedicated to the memory of the late Prof. L. Pietrantoni.     

The ultrastructure of the test of the tadpole larva ofCiona intestinalis

Summary The test of the tadpole larva ofCiona intestinalis consists of an amorphous background substance in which are long 4–7 nm fibrils. There is a narrow outer dense region of the test where both the background material and the test fibrils are more concentrated and orientated. Fibrils of similar dimensions are found in the epithelial cells below the test, and also in cells associated with the outside surface of the test. No such fibrils are found in the cells located within the test substance. There are some regions where the external cell membrane of the epithelial cells is indistinct and in these regions the fibrils of the test are continuous with the fibrils within the epithelial cells. No large or small vesicles opening from the epithelial cells into the test have been seen.This evidence has been interpreted as support for the tunicization theory of test formation. It is suggested that the cells within the test are the source of the polyphenols that are necessary for quinone tanning that gives rigidity to the test.The fibril-containing cells outside the test probably add fibres to the cuticular region of the test.I wish to thank ProfessorJ. Z. Young, F.R.S. for much advice and encouragment, also Dr.R. Bellairs for the use of electron microscope facilities and Mr.R. Moss and Mrs.J. Hamilton for skillful technical assistance.     

Cytotoxicity of glucose analogues in V79 multicell spheroids

Summary 2-Deoxy-d-glucose (2DG) and 5-thio-d-glucose (5TG) are glucose antimetabolites that are known to be selectively toxic to hypoxic cells grown as single cells or as monolayer cultures. These analogues were toxic to Chinese hamster V79 cells grown as multicell spheroids even under aerobic conditions. When spheroids, 500- to 600-μm diameter, were exposed to 7.5mm of these chemicals for 3 days, the number of clonogenic cells per spheroid dropped to 50% for 5-thio-d-glucose and 20% for 2-deoxy-d-glucose, relative to control values. Survivals were reduced to less than 1% when the experiment was repeated in glucose-free medium. Scanning electron photomicrographs of spheroids treated with 7.5mm of either analogue showed extensive damage to the outer cells. The cell killing observed was much more than could be predicted on the basis of the hypoxic fraction known to be present in these spheroids. The crowded tumor-like environment may make the cells vulnerable to the cytotoxic action of glucose analogues and other glycolytic inhibitors. Supported by the Ontario Cancer Treatment and Research Foundation, London Clinic.     

Regulation of angiogenesis in vitro by collagen metabolism

Summary The role of collagen in microvascular growth was investigated using the aortic ring model of angiogenesis. Collagen production by vasoformative outgrowths in plasma clot culture of rat aorta was either stimulated with ascorbic acid or inhibited with the proline analogue cis-hydroxyproline. Microvessels proliferating in the absence of ascorbic acid supplements became ectatic and developed large lumina. In contrast, newly formed microvessels in the presence of ascorbic acid remained small and maintained thin lumina throughout the angiogenic process. Biochemical studies demonstrated enhanced collagen production and deposition in cultures treated with ascorbic acid. Ultrastructural studies of these cultures showed a marked increase in newly formed interstitial collagen in the perivascular matrix and in regions of the plasma clot containing nonendothelial mesenchymal cells. Small microvessels with thin lumina similar to the ones observed in ascorbic acid-treated plasma clot cultures were obtained by growing aortic explants in gels of interstitial collagen in the absence of ascorbic acid. Inhibition of collagen production with the proline analogue cis-hydroxyproline had a marked anti-angiogenic effect in both plasma clot and collagen gel cultures. The anti-angiogenic effect of cis-hydroxyproline was abolished by addingl-proline to the culture medium, thereby restoring normal metabolism. These results support the hypothesis that angiogenesis is regulated by collagen production and suggest that the size of newly formed microvessels is influenced by the degree of collagenization of the extracellular matrix.     

In-vitro cleavage of a fusion protein bound to cellulose using the soluble yscFs (Kex2) variant

Summary Intra- and extracellular metabolite concentrations were determined for hybridoma cells grown in tissue culture flasks in batch culture. Significant differences were found for intracellular lactate and amino acid concentrations depending on the culture medium. AB2-143.2 cells cultivated in Dulbecco's modified Eagle's (DME) medium supplemented with 50 mm lactate exhibited intracellular lactate and glutamine levels of 40 mm and 4 mm, respectively, whereas cells grown in standard DME medium had low intracellular glutamine and 20 mm lactate concentrations. For cells cultivated in medium supplemented with 6 mm pyruvate, intracellular lactate levels were estimated at approx. 40 mm, but these cells also showed an increased intracellular alanine concentration of 22 mm. The higher alanine pools probably result from increased transamination of pyruvate under these conditions.     

Histochemistry of some acid hydrolases in striated muscles of the rat

Summary The distribution of acid phosphatase, -n-acetylglucosaminidase, -glucuronidase, and acid -galactosidase was studied in mm. extensor digitorum longus, soleus, and diaphragm of rats. Using the technic of semipermeable membranes activities of these enzymes were demonstrated beside cells of the interstitial tissue in muscle fibers themselves as well. Acid phosphatase displayed the highest activity which appeared in many small dots dispersed in the fiber. The activity of acid phosphatase was about 1.2 x higher in the m. soleus than in the m. extensor digitorum longus. In the latter muscle a somewhat higher activity was often found in muscle fibers displaying a higher staining for NADH tetrazolium reductase. The activity of -n-acetylglucosaminidase was slightly lower, that of -glucuronidase very weak but still discernible. The activity of acid -galactosidase was not ascertained in the majority of fibers. The ratio of activities measured in an area of the same size in cells of the interstitial tissue and in muscle fibers amounted in average to 2.6: 1 in the case of acid phosphatase, 2.5:1 in the case of -n-acetylglucosaminidase, 5.7: 1 in the case of -glucuronidase, and 44.3:1 in the case of acid -galactosidase. The importance of the histochemical technic in studies concerned with acid hydrolases in striated muscle fibers in normal and pathological conditions is pointed out.     

Fluorescence microscopy of the 5-HTP turnover in the exocrine pancreas of mice and rats

Summary The turnover ofl-5-HTP,d-5-HTP and 5-HT in the exocrine pancreas have been studied by means of the fluorescence method ofFalck andHillarp. l- andd-5-HTP are easily taken up by the acinar cells, whereas 5-HT seems to pass into the cells only to a minor extent. After the administration ofl-5-HTP (and in some cases after 5-HT administration), specific fluorescence is seen in the form of apically located granules (probably identical with the zymogen granules) for a short period, which is prolonged, if the animals are pretreated with a MAO inhibitor. Decarboxylase inhibition prevents the appearance of these fluorescent granules. Administration ofd-5-HTP does not give rise to this granular fluorescence but to a diffuse fluorescence throughout the cells. Thus, there are reasons to assume that the granular fluorescence derives from 5-HT. The results obtained in this work correspond well with those from a similar study withl-DOPA and some of its analogues.abbreviations DOPA 3,4-dihydroxyphenylalanine - DA dopamine - NA noradrenaline - A adrenaline - 5-HTP 5-hydroxytryptophan - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase This work was supported by grants from the Swedish Medical Research Council (B68-12X-712-03B and B68-14X-56-04B), the United States Public Health Service (06701-02) and the Faculty of Medicine, University of Lund, Lund, Sweden.     

A study of the effect of radium upon the eggs of Ascaris megalocephala univalens

Conclusions My observations support the conclusions ofPerthes andHertwig that the achromatic figure appears perfectly normal; that the first segmentation divisions are regular; and that irregularities enter sooner or later which cause the death of the embryo. They further support the conclusions ofHertwig that the effect of the radium manifests itself in the first segmentation division and that the chromosomes are broken up into irregular masses and granules. In addition to these, I find that in the first segmentation division, the enlarged masses of chromatin which are similar to the cast off ends in the somatic cells of normal segmentation, may or may not take part in nuclear reconstruction and the succeeding division; that the chromatin of the first segmentation division is more greatly disturbed than in the divisions which follow. More important, however, than these is the fact that the chromatin of the sex and somatic cells in the second and third segmentation divisions behaves differently. That is, while the chromosomes of both cells break up, the fragments in the sex cells are generally larger.     

A study of the dependence of the human erythrocyte glucose transport system on membrane sulfhydryl groups

Summary A brief review of the data relating the glucose transport system and other membrane functions of red cells to surface sulfhydryl groups is presented. The effect of a variety of sulfhydryl reagents on glucose efflux rates from loaded red cells was studied. Neither iodoacetate nor iodoacetamide at 5mm inhibited efflux. Several maleimide derivatives and disulfides inhibited efflux in 0.7 to 2.0mm concentrations. Organomercury compounds, on the other hand, were active in the 0.07 to 0.1mm range. These data suggest that, if sulfhydryl groups are important in the glucose efflux process, they are not equally accessible to the above reagents; and that the primary effect of these reagents may be on structural elements near membrane sulfhydryl groups.     

Stimulation of olfactory receptors alters regulation of [Cai] in olfactory neurons of the catfish (Ictalurus punctatus)

Summary Intracellular calcium was measured in single olfactory neurons from the channel catfish (Icatalurus punctatus) using the fluorescent Ca²⁺ indicator fura 2. In 5% of the cells, olfactory stimuli (amino acids) elicited an influx of calcium through the plasma membrane which led to a rapid transient increase in intracellular calcium concentration. Amino acids did not induce release of calcium from internal stores in these cells. Some cells responded specifically to one stimulus (l-alanine,l-arginine,l-norleucine andl-glutamate) while one cell responded to all stimuli. An increase in intracellular calcium could also be elicited in 50% of the cells by direct G-protein stimulation using aluminum fluoride. Because the fraction of cells which respond to direct G-protein stimulation is substantially larger than the fraction of cells responding to amino acids, we tested for possible damage of receptor proteins due to exposure of the olfactory neurons to papain during cell isolation. We find that pretreatment with papain does not alter specific binding ofl-alanine andl-arginine to olfactory receptor sites in isolated olfactory cilia. The results are discussed in terms of their relevance to olfactory transduction.     

Electron microscope observations of some peripheral synapses in the sensory pathway of the sucker of Octopus vulgaris

Summary A synaptic axo-dendritic linkage is described between primary receptors lying in the epithelia of the sucker of Octopus and encapsulated nerve cells found near the rim of the sucker in the subepithelial connective tissue. These synapses are postulated to perform a drastic reduction of inputs between the primary receptors of the order of more than ten thousand and the subjacent encapsulated nerve cells of the order of some hundreds. The morphology of these cells as well as that of the synaptic structures are described from electron microscope studies. Aknowledgement. This work was done at University College London, while I was in receipt of a Medical Research Council grant.I am deeply indebted to Prof. J. Z. Young F. R. S. for support and criticism, and to Dr. E. G. Gray for advice and discussion. My thanks are due to Mr. A. Aldrich for the photographs.