Microspectrofluorometric study of monoamines in the auricle of the heart of Protopterus aethiopicus

Summary The auricle of the heart of Protopterus aethiopicus contains large numbers of chromaffin cells, often lying immediately adjacent to the endothelium and displaying a bright blue-white fluorescence characteristic for catecholamines after formaldehyde treatment (Falck and Owman 1965). These results combined with X-ray microanalysis after initial fixation with glutaraldehyde and subsequent treatment with dichromate established that these chromaffin cells are the storage site of primary catecholamines (Scheuermann 1978, 1979, 1980; Scheuermann et al. 1980). The aim of the present pilot study was to demonstrate in these cells noradrenaline (NA) or dopamine (DA), or a mixture of both. The evaluation of the excitation spectra of the catecholamine fluorophore transformed by treatment with HCl vapour (excitation maxima at 320 and 370 nm) and the excitation-peak ratio analysis (peak ratio 370/320 nm =1.05–1.5; and 320/280 nm >1.5) identify DA as the primary catecholamine stored in these chromaffin cells. The low fading rate of the monoamine fluorescence after acidification confirms the presence of DA. These microspectrofluorometric findings demonstrate that chromaffin cells in the auricle of the Protopterus heart, which are a part of the medullary homologue of the adrenal gland of higher vertebrates, contain a primary catecholamine, namely DA.     

Increased urea synthesis and/or suppressed ammonia production in the African lungfish, Protopterus annectens, during aestivation in air or mud

The objective of this study was to elucidate how the African lungfish, Protopterus annectens, ameliorated ammonia toxicity during 12 or 46 days of aestivation in air or in mud. Twelve days of aestivation in air led to significant increases in contents of urea, but not ammonia, in tissues of P. annectens. The estimated rate of urea synthesis increased 2.7-fold despite the lack of changes in the activities of hepatic ornithine–urea cycle enzymes, but there was only a minor change in the estimated rate of ammonia production. After 46 days of aestivation in air, the ammonia content in the liver decreased significantly and contents of urea in all tissues studied increased significantly, indicating that the fish shifted to a combination of increased urea synthesis (1.4-fold of the day 0 value) and decreased ammonia production (56% of the day 0 value) to defend against ammonia toxicity. By contrast, 12 days of aestivation in mud produced only minor increases in tissue urea contents, with ammonia contents remained unchanged. This was apparently achieved through decreases in urea synthesis and ammonia production (40 and 15%, respectively, of the corresponding day 0 value). Surprisingly, 46 days of aestivation in mud resulted in no changes in tissue urea contents, indicating that profound suppressions of urea synthesis and ammonia production (2.6 and 1.2%, respectively, of the corresponding day 0 value) had occurred. This is the first report on such a phenomenon, and the reduction in ammonia production was so profound that it could be the greatest reduction known among animals. Since fish aestivated in mud had relatively low blood pO₂ and muscle ATP content, they could have been exposed to hypoxia, which induced reductions in metabolic rate and ammonia production. Consequently, fish aestivating in mud had a lower dependency on increased urea synthesis to detoxify ammonia, which is energy intensive, than fish aestivating in air.     

The ammonotelic African lungfish,<Emphasis Type="Italic"> Protopterus dolloi</Emphasis>, increases the rate of urea synthesis and becomes ureotelic after feeding

This study aimed to elucidate the role of urea synthesis in the slender African lungfish Protopterus dolloi in detoxifying ammonia after feeding. There were significant increases in the rate of ammonia excretion in P. dolloi between hours 6 and 15 after feeding. Simultaneously, there were significant increases in urea excretion rates between hours 3 and 18. Consequently, the percentage of total nitrogen (N) excreted as urea N increased to ~60% between hours 12 and 21 post-feeding. Hence, after feeding, the normally ammonotelic P. dolloi became ureotelic. Approximately 41% of the N intake from food was excreted within 24 h by P. dolloi, 55% of which was in the form of urea N. At hour 12 post-feeding, the accumulation of urea N was greater than the accumulation of ammonia N in various tissues, which indicates that feeding led to an increase in the rate of urea synthesis. This is contrary to results reported previously on the infusion of ammonia into the peritoneal cavity of the marine dogfish shark, in which a significant portion of the exogenous ammonia was excreted as ammonia. In contrast, feeding is more likely to induce urea synthesis, which is energy intensive, because feeding provides an ample supply of energy resources and leads to the production of ammonia intracellularly in the liver. The capacity of P. dolloi to synthesize urea effectively prevented a postprandial surge in the plasma ammonia level as reported elsewhere for other non-ureogenic teleosts. However, there was a significant increase in the glutamine content in the brain at hour 24, indicating that the brain had to defend against ammonia toxicity after feeding.Communicated by I.D. Hume     

Immunocytochemical demonstration of mammalian lutropin-like material in the pituitary of the lungfish,Lepidosiren paradoxa

Summary The presence of lutropin (LH)-like material in the pituitary gland of the South-American lungfish, Lepidosiren paradoxa, has been demonstrated by means of the unlabeled antibody-enzyme method, by use of rabbit antiovine LH as first antibody. The LH-like substance was revealed in a single PAS-positive cell type primarily located in the anterior part of the distal lobe. Dot blot tests as well as conventional liquid-phase absorption experiments indicate that the anti-ovine LH antibodies possess specificity against the oLH/gb subunit. These observations indicate that dipnoans (Lepidosiren) share a number of antigenic determinants with those of mammalian LH/gb and support the concept that mammalian LH/gb, or part of it, was established early in evolution. The exact nature and physiological function of the substance detected remains to be defined.     

Ultrastructural study on nuclear-cytoplasmic relationships in oocytes of the African lungfish,Protopterus aethiopicus

Summary Growing oocytes of Protopterus, like those of some amphibians and teleosts, show an impressive development of the nucleolar apparatus. Numerous nucleolus-like bodies establish close spatial relationships with the nuclear envelope by extending pedicels and streams of finely dispersed material towards the inner membrane.At such contact points, gaps in the perinuclear cistern are more frequent than elsewhere along the nuclear boundary. Expansion of the outer nuclear membrane gives rise to blebs, with or without visible content, and these become pinched off to form small vesicles in the perinuclear cytoplasm.Small, electron dense aggregates, indistinguishable from nucleolar material occur on both sides of the nuclear envelope opposite to each other, some being connected by a slender portion of the same material within a nuclear pore. Such accumulations are interpreted as detached parts of nucleolar bodies in transit to cytoplasmic sites where they presumably participate in the biogenesis of ribosomes. At the height of nucleolar emission, nucleoplasm and perinuclear cytoplasm are so rich in small electron dense particles that they are almost indistinguishable from each other.At this stage of massive transport, the route provided by the nuclear pores seems to be insufficient and another, more spacious, gateway may be in operation. The latter involves direct passage of material across the nuclear membranes preferentially where these form blebs.This view is supported not only by the overt spatial relationships between nucleolar pedicels and blebs, but by the occurrence within perinuclear lacunae and blebs of particles that seem to be derived from nucleolar bodies. Furthermore, frequent interruptions in the nuclear membranes preferentially located where they expand into outpocketings suggest that at these sites temporary gateways may exist in the living cell that permit easy access of intranuclear components to the cytoplasm.Supported by grants AM-3984, NB-00840, and NB-05219 from the U.S.P.H.S.     

Immunocytochemical localization and characterization of mammalian prolactin- and somatotropin-like material in the pituitary of the Australian lungfish,Neoceratodus forsteri

The aim of the present study was to define at the light-microscopic level expression of prolactin and somatotropin material in the pituitary gland of the Australian lungfish, Neoceratodus forsteri, by use of polyclonal antibodies against ovine prolactin (oPRL) and bovine somatotropin (bSTH). Substances immunologically related to mammalian oPRL as well as bSTH were detected in two morphologically different cell types in the distal lobe, corresponding to the acidophilic cells. The specificity of the antibodies was initially confirmed in a porcine tissue control system. First, our absorption studies confirm that in Neoceratodus the anti-oPRL identifies part of an oPRL-like molecule different from bSTH. Secondly, the anti-bSTH identifies both part of a bSTH-like molecule proper to bovine and Neoceratodus STH, and part of a bSTH-like molecule having antigenic determinants in common with both bSTH and oPRL. This part of the oPRL is, however, not shared with the Neoceratodus PRL as revealed by the anti-oPRL. Altogether these observations support the concepts: (1) that mammalian PRL and STH, or part of those, were established early in evolution, and (2) that dipnoans as living sarcopterygians have an ancestor in common with the early amphibians. The exact nature and physiological functions of the substances detected remain to be defined.     

Reproductive biology of the East African lungfish (Protopterus aethiopicus) in Mwanza Gulf, Lake Victoria

The population of lungfish (Protopterus aethiopicus (Heckel)) was sampled by bottom trawling for 12 months from June 1984 to May 1985. A total of 576 lungfish were investigated. The sex ratio in the population was 1 male : 1 -98 females. Average size at maturity was found to be 960cm total length (TL) but the smallest matured male measured 72-9 cm and the female 840 cm TL. It was not possible to establish spawning times and reproductive activity trends either from the variation in relative condition factor ‘kn’ or from gonadosomatic index (GSI). Whereas male lungfish seem to show no further increase in GSI after gonadal maturity stage III, females exhibit GSI values that vary directly with oocyte maturation and ovarian weights. Fat deposited along the gonads was found to be a better basis for assessing maturation and spawning in lungfish. The potential spawning period was found to be from September to May. Absence of lungfish with spent gonads over the 12-month period and the presence of different egg sizes in ovaries, suggest that the fish is a multiple spawner. Fecundity was found to range from 705 eggs (in 84-cm TL fish) to 14,922 eggs (in 130-cm TL Fish).     

Insulin-, glucagonand somatostatin-like immunoreactivity in the endocrine pancreas of the lungfish,Neoceratodus forsteri

Summary The endocrine pancreas of the Australian lungfish,Neoceratodus forsteri, was investigated immunocytochemically for the presence of polypeptide hormone-producing cells. Three cell types were identified, namely insulin-, glucagon-, and somatostatin-immunoreactive elements. The insulin cells are confined solely to the center of the islets. Glucagon and somatostatin cells are distributed peripherally around the central mass of the insulin cells. Isolated cells or clusters of glucagon and somatostatin cells are also dispersed within the exocrine parenchyma. The immunoreactive cell types are compared with those staining with standard histological procedures. The spatial relationships of the different cell populations are examined.     

Taurine,amino acid transmitters,and related molecules in the retina of the Australian lungfish Neoceratodus forsteri: a light-microscopic immunocytochemical and electron-microscopic study

The morphology of the retina of the Australian lungfish Neoceratodus forsteri was investigated by means of light- and electron microscopy, whilst immunocytochemical studies were performed to determine the cellular distributions of the major amino acid neurotransmitters and other amino acids. The distributions of glycine and GABA were similar to those previously described for teleost, amphibian and mammalian retinae. Labelling was abundant in amacrine cells, whilst GABA was also present in one layer of horizontal cells and some bipolar cells. Taurine was present in both rods and cones, but, unlike the mammalian or avian retina, was absent from other cellular structures, including glial elements. Unexpectedly, the photoreceptor terminals lacked an apparent content of the excitatory amino acid transmitter glutamate. The glutamate that was present in the rods and cones occupied a crescentic arc corresponding to the location of glycogen-rich paraboloids. Asparagine was also present in rods, albeit in the modified mitochondria that formed the elipsoids of the rod inner segments. Arginine, the precursor for formation of nitric oxide, was present in glial cells, and in the paraboloids of both rods and cones.     

Effects of hypoxia on the energy status and nitrogen metabolism of African lungfish during aestivation in a mucus cocoon

We examined the energy status, nitrogen metabolism and hepatic glutamate dehydrogenase activity in the African lungfish Protopterus annectens during aestivation in normoxia (air) or hypoxia (2% O(2) in N(2)), with tissues sampled on day 3 (aerial exposure with preparation for aestivation), day 6 (entering into aestivation) or day 12 (undergoing aestivation). There was no accumulation of ammonia in tissues of fish exposed to normoxia or hypoxia throughout the 12-day period. Ammonia toxicity was avoided by increased urea synthesis and/or decreased endogenous N production (as ammonia), but the dependency on these two mechanisms differed between the normoxic and the hypoxic fish. The rate of urea synthesis increased 2.4-fold, with only a 12% decrease in the rate of N production in the normoxic fish. By contrast, the rate of N production in the hypoxic fish decreased by 58%, with no increase in the rate of urea synthesis. Using in vivo (31)P NMR spectroscopy, it was demonstrated that hypoxia led to significantly lower ATP concentration on day 12 and significantly lower creatine phosphate concentration on days 1, 6, 9 and 12 in the anterior region of the fish as compared with normoxia. Additionally, the hypoxic fish had lower creatine phosphate concentration in the middle region than the normoxic fish on day 9. Hence, lowering the dependency on increased urea synthesis to detoxify ammonia, which is energy intensive by reducing N production, would conserve cellular energy during aestivation in hypoxia. Indeed, there were significant increases in glutamate concentrations in tissues of fish aestivating in hypoxia, which indicates decreases in its degradation and/or transamination. Furthermore, there were significant increases in the hepatic glutamate dehydrogenase (GDH) amination activity, the amination/deamination ratio and the dependency of the amination activity on ADP activation in fish on days 6 and 12 in hypoxia, but similar changes occurred only in the normoxic fish on day 12. Therefore, our results indicate for the first time that P. annectens exhibited different adaptive responses during aestivation in normoxia and in hypoxia. They also indicate that reduction in nitrogen metabolism, and probably metabolic rate, did not occur simply in association with aestivation (in normoxia) but responded more effectively to a combined effect of aestivation and hypoxia.     

Electron microscopic studies of the lung of the frog

Summary Scanning and transmission electron microscopy were used to study the inner architecture of the frog lung. In some specimens the alveolar surface mucus layer was removed to permit the examination of underlying features. The inner surface of the frog's lung is covered by a layer of microvilli belonging to only one type of epithelial cells. The boundaries of these epithelial cells are demarcated by small ridges. Different degrees of lung expansion cause variations of the surface topography. The morphology of certain surface features is examined in detail. Several methods of drying the specimens are compared.The author wishes to thank Dr. I. E. Richter, Institut für allgemeine und experimentelle Pathologie der Bundeswehr, Mainz, for the opportunity to do these investigations and for helpful discussions.     

Translocation of marbled African lungfish,Protopterus aethiopicus (Telostei: Protopteridae), and its fishery in Lake Baringo,Kenya

The translocation of the marbled African lungfish Protopterus aethiopicus into Lake Baringo created a new fishery for the local community, who capture them primarily in a bottom-set long line fishery. Its introduction, development and current fishery status in the lake are documented. Annual catch data were obtained from the District Fisheries Office, while catch and effort data of the long line fishery were recorded at one active fish-landing site between February and October 2001. Lungfish comprise a significant component of commercial landings, sometimes exceeding catches of the Baringo tilapia Oreochromis niloticus baringoensis as the most landed species by weight. Daily catch rates and effort varied considerably, ranging from 5.51–15.18kg day^?1 and from 50–590 baited hooks day^?1, respectively. Daily catch per fisher ranged from 0–35.72kg, while the overall mean catch per unit effort (CPUE) was only 0.003kg per hook-hour. This study provides the only baseline CPUE data for a lungfish long line fishery that the authors are aware of, for comparison with future studies in this or other lakes where lungfish are similarly exploited. Recommendations for improved management and the maintenance of a viable lungfish long line fishery in Lake Baringo are presented.     

The morphology of cultured melanophores from tadpoles of Xenopus laevis: Scanning electron microscopical observations

Summary Tail-fin melanophores of tadpoles of Xenopus laevis (Daudin) in primary culture were examined scanning electron microscopically in the aggregated and in the dispersed state. After isolation, the melanophores are spherical, but within 24 h they develop thin filopodia for attachment to the substratum. Subsequently, cylinder-like as well as flat sheet-like processes are formed, which adhere to the substratum with terminal pseudopodia and filopodia. The processes of adjacent melanophores contact each other, thus forming an interconnecting network between the melanophores.In the aggregated state the central part of the melanophore is spherical and voluminous. Both the central part and the processes bear microvilli. In melanophores with dispersed melanosomes the central part is much flatter; the distal parts have a thickness that equals a monolayer of melanosomes. The surface of the cell bears only scarce microvilli.These features indicate that melanophores do not have a fixed shape and that pigment migration is accompanied by reciprocal volume transformation between the cell body and its processes.     

The outer segments of photoreceptive pinealocytes in the pineal organ of the funa,Carassius gibelio langsdorfi

Summary The form and size of the outer segments of photoreceptive pinealocytes in the pineal organ of the funa, Carassius gibelio langsdorfi, were observed with the scanning electron microscope. The height of the outer segments measures between 1 and 3 m and the diameter varies widely from 1.5 to 8 m. Various forms of outer segments, i.e. a slender type, a dome-like type, a cap-like type and a helical type, were demonstrated. The parallel-oriented filamentous processes of the inner segments have the same length as the outer segments and a diameter of approximately 100 nm; they are projections from the apical border of the inner segment and surround the cone-like outer segments. The processes make a right angle with the lamellar disks. The distance between two processes averages 100 nm. The lamellar disks of the outer segments are oriented at right angles to the modified cilium in the basal part, but the angle often changes in the peripheral part, where the lamellar disks are raised and become parallel to the cilium.Supported by a fellowship from the Japan Society for the Promotion of Science and a grant from the Deutsche Forschungsgemeinschaft to M. UeckSupported by a grant from the Ministry of Education of Japan to K.Wake     

Gonadotropin-releasing hormone-immunoreactive neuronal structures in the brain and pituitary of the African catfish,Clarias gariepinus (Burchell)

Summary The distribution of gonadotropin-releasing hormone (GnRH) immunoreactivity was studied in the African catfish, Clarias gariepinus, by means of immunofluorescence and immunoperoxidase techniques. Immunoreactive neurons were found throughout the preoptic nucleus (NPO). However, only a portion of the secretory perikarya in the NPO showed a positive reaction by use of an anti-LHRH serum. Numerous immunoreactive fibres were found to enter the pituitary and to terminate in its proximal pars distalis, the site of concentration of the gonadotropic cells. Since GnRH is present in the brain and pituitary of the African catfish, the lack of spontaneous ovulation in captivity is apparently due to an insufficient release of GnRH.     

The structure of the lung of the newborn marsupial bandicoot,Isoodon macrounus

Summary In bandicoots, implantation takes place 9.5 days after mating and birth occurs 3 days later. It is therefore of interest to examine the lungs of newborn marsupials to ascertain the stage of lung development with respect to other organs and other mammals in view of the short period of time allowed for maturation of lung tissue. The newborn bandicoot lung is composed of a primitive system of branching airways which terminate in large blind sacs; alveolar ducts or alveoli are not present. The terminating sacs, which are delineated by thick connective-tissue septae, have a highly vascularised internal lining. Thin squamous cells and numerous type-II surfactant-secreting cells constitute the air interface of the sac. Corticosteroids influence the development of the eutherian fetal lung by accelerating pulmonary maturation and increasing the synthesis of surfactant. Since recent evidence indicates that the adrenal of the bandicoot is active at, or before birth, it is possible that this organ also plays an important role in preparing the lungs of the marsupial for extra-uterine life in the pouch.     

Evidence for a frontal-organ homologue in the pineal complex of the salamander,Hynobius dunni

The pineal complex of larval and adult salamanders, Hynobius dunni, was examined by light and scanning electron microscopy. This pineal complex displays an anterior and a posterior portion, both of which possess a lumen. The anterior lumen is small and closed, whereas the posterior lumen is in open communication with the third ventricle. Cell processes of the photoreceptor cells and microvilli of the supportive cells are visible in both lumina. The anterior part of the complex is formed by an independent, second evagination from the common pineal anlage; this process takes place immediately after hatching. The anterior body of the pineal complex of H. dunni appears to be homologous to the frontal organ of anurans.     

Isolation of gonadotrops from the pituitary of the African catfish,Clarias lazera

Summary Dispersed pituitary cells from male African catfish, Clarias lazera, were fractionated in a density gradient of Percoll. Five fractions were isolated, consisting of about 6, 19, 39, 95 and 83% gonadotrops, respectively. The gonadotrops were identified by their ultrastructural characteristics, by immunocytochemistry, and by measuring their hormone content. After one day in culture, in each fraction the secretion of gonadotropin could be stimulated by a luteinizing hormone-releasing hormone analogue, indicating that the cells had retained their functional integrity. Since the regulatory mechanisms of different cell types from the pituitary have some similarity, purification of the gonadotrops provides a model to study the regulation of gonadotropin secretion.A portion of the results was presented as a poster at the XII^th Conference of the European Society of Comparative Endocrinology, Sheffield, July 31–August 5, 1983     

Thymic vascular system of the European common frog,Rana temporaria: a scanning electron-microscopic study of vascular casts

Vascular corrosion casts of the thymus of adult individuals of the European common frog, Rana temporaria, were analysed by scanning electron microscopy. The main arterial vessel, which is derived either from the temporal artery or from the auricular ramus, approaches the central territory of the gland and branches into twigs that, on penetrating the parenchyma, give rise to capillaries. Most of these capillaries run vertically towards the surface of the gland; they either join the superficial capillary plexus or follow this plexus for a variable distance and then run back towards the medulla, forming capillary loops. The former capillaries link with the extensive venous plexus composed of irregular meshes, whereas the latter capillaries join the venules at the cortico-medullary boundary and finally escape into collecting veins on the gland surface. The venous twigs, which join together near the gland, form the main thymic vein, which empties into the external jugular vein. The details of the thymic vasculature of the anuran amphibian, R. temporaria, are compared with those described in mammalian species, viz. the mouse, rat and guinea pig.