Immunocytochemical localization of Vitamin A in the retina and pineal organ of the frog,Rana esculenta

Summary Vitamin A immunoreactive sites were studied in the retina and pincal organ of the frog,Rana esculenta, by the peroxidase antiperoxidase, avidin-biotinperoxidase and immunogold methods. Indark-adapted material, strong immunoreaction was found in the outer and inner segments of the photoreceptor cells of both retina and pineal organ, as well as in the pigment epithelium, retinal Müller cells and pineal ependymal cells. Inlight-adapted retina, cones and green (blue-sensitive) rods were immunopositive.At the electron microscopic level, immunogold particles were found on the membranes of the photoreceptor outer segments as well as on the membranes of the endoplasmic reticulum and mitochondria. Individual retinal photoreceptor cells exhibited strong immunoreaction in the distal portion of the inner segment, the ciliary connecting piece and the electron-dense material covering the outer segment. In the pigment epithelium, the immunolabeling varied in intensity in the basal and apical cytoplasm and phagocytosed outer segments.The immunocytochemical results indicate that retinoids (retinal, retinol and possibly retinoic acid) are present not only in the photoreceptor cells of the retina but also in those of the pineal organ. The light-dependent differences in the immunoreactivity of vitamin A underlines its essential role in the visual cycle of the photopigments. Our results suggest that the pineal ependyma plays a role comparable to that of the Müller cells and pigment epithelium of the retina with regard to the transport and storage of vitamin A. The presence of a retinoid in nuclei, mitochondria and cytoplasmic membranes suggests an additional role of vitamin A in other metabolic processes.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the Hungarian OTKA grant Nr. 1619 to B.V., and a grant from the Pardee Foundation to G.H.W.     

Cytological and histochemical investigations on the pineal organ of the adult frog (Rana esculenta)

Cell and Tissue Research -     

Photoreceptor layer composition in the retina of the frog (Rana esculenta)

Retinas of Rana esculenta frogs were studied by light and electron microscopy in order to establish the photoreceptor layer composition. We found 56% red rods, 9% green rods, 19% single cones, and 16% double cones. This work provides the morphological basis for electrophysiological investigations concerning the mass receptor potential of isolated Rana esculenta retinas.     

Immunohistochemical investigation of the nitrergic system in the taste organ of the frog,Rana esculenta

We have studied by immunocytochemistry, the taste discs of the frog, Rana esculenta, with the aim of providing morphological and neurochemical data on the nitrergic system and of assessing the eventual presence of intrinsic neurons associated with the gustatory organs. In taste discs, antibodies against neuronal nitric oxide synthase (nNOS) revealed a positive immunoreaction in the taste receptor cell bodies and processes. The basal cells were also stained. All the fungiform papillae contained intragemmal nerve fibers showing nNOS immunoreactivity; these fiber were mainly located in the basal plexus. Immunoreactive nerve fibers were also visible at the periphery of the papilla-contacting ciliate cells, which form a ring around the taste disc. In conclusion, the findings obtained in this study suggest that the occurrence of nNOS-immunoreactivity in basal cells, taste cells and nerves might reflect a role for nitric oxide in taste mechanisms of Amphibia. The results may also sustain the physiological implication of NO as a molecule involved in the local target function of maintaining the taste bud mucosal integrity and in regulating the blood flow to the epithelium.     

Ovarian activity and reproduction in the frog, Rana esculenta

Rana esculenta specimens were collected, during the last 13 years, in well-defined areas around Naples. The annual ovarian cycle shows distinct phases of recrudescence (starting September; vitellogenesis), breeding (late March-early July; egg deposition and active oogenesis) and quiescence (July-August; no follicular growth). Previtellogenic follicles are recruited for vitellogenesis in early September and in between two successive ovulatory waves. Breeding congregations are generally formed after a heavy rain fall and eggs are laid in standing waters, temporary or permanent. A maximum of three clutches of eggs is produced during the breeding season, at roughly monthly intervals. All mature females reproduce to some extent. Ovarian weight and clutch size are positively correlated to body weight. Depending upon the body size, the potential clutch size ranges from 1000 to 3500 eggs during the first wave of ovulation and it is notably smaller in the successive wave(s) of ovulation. Egg masses and tadpoles are left unprotected and mortality is high. The life cycle from the fertilized egg to completion of metamorphosis is 2 months and oogenesis in the ovary starts in the larva before the onset of metamorphic climax. Young females hatching from the first clutch of eggs may reach sexual maturity and breed in May the following year; those hatching from the last clutch require nearly 20 months to reach sexual maturity. The importance of some endocrine and exocrine factors for the regulation of ovarian activity and reproduction is discussed.     

A comparative study of melatonin production in the retina,pineal gland and Harderian gland of Bufo viridis and Rana esculenta

1. The circadian patterns of melatonin and of its synthesizing enzyme N-acetyltransferase (NAT) were investigated in the serum, retina, pineal gland and Harderian gland (HG) of two amphibian species, Bufo viridis and Rana esculenta.2. Serum melatonin levels showed no diurnal fluctuations in Bufo viridis, whereas, in Rana esculenta, they exhibited a circadian rhythm, with the highest values occurring during the night. Retina melatonin exhibited characteristic circadian patterns in both species, with the highest values occurring during the day, in Bufo, and the highest concentrations occurring at night in Rana.3. In the retina, NAT activity peaked at night in both amphibians, but in Bufo the levels were up to 30 times higher than in Rana. In the HG and in the pineal gland, NAT activity showed different patterns in the two species with no diurnal variations in Bufo, and characteristic circadian rhythms in Rana.4. In the HG and pineal gland of both species, melatonin was only occasionally detectable over the 24-hr period.5. This is the first report exploring melatonin production in Bufo viridis and Rana esculenta. In our experimental conditions, marked differences emerged between the two species.     

The annual cycle of activity of the subfornical organ in the green frog Rana esculenta

Abstract

The nuclear volume in cells of the subfornical organ of Rana esculenta shows a cyclic annual size change. It is pronounced both in the so‐called Gomori‐positive cells and the ependymal covering of the organ. There appears a maximum in April in the epen‐dyma and in May in the Gomori‐positive cells. The amount of neurosecretory material present in the subependymal layer also shows a maximum in April.     

Intermitochondrial junctions in the heart of the frog,Rana esculenta

Summary In muscle fibers of the frog heart, junctions between outer membranes of adjacent mitochondrial profiles are occasionally found. In thin sections of embedded tissue and of mitochondrial pellets, the intermitochondrial junctional space is 5.4±0.15 nm; the external leaflets of the membranes are joined by periodic structures separated from each other by 16.3±0.29 nm. There are 65.3±2 periodic structures per m of membrane measured on a section perpendicular to the junction. After cryofracture, the outer membrane is cleaved into two parts. Closely packed, parallel rows of large particles and furrows are found either on the P-, or on the E-faces. The rows of particles are 11±0.3 nm thick and are separated from each other by 16.5±0.46 nm, their density being 65±2.28 per m of the membrane. In junctional areas, rows of particles on one membrane correspond with the furrows on the other membrane. Intermitochondrial junctions appear to be real structures and not artifacts due to preparation procedures. The conditions of their occurrence are discussed.     

Detection and localization of gonadotrophin-releasing hormone (GnRH)-like material in the frog,Rana esculenta,ovary

GnRH-like material has been identified using HPLC followed by RIA in the ovary of Rana esculenta. During the reproductive cycle three immunoreactive GnRH peaks were eluted. One of them coeluted with s-GnRH, the other two forms between GnRH and cII-GnRH. During the recovery phase s-GnRH immunoreactivity disappears. By immunocytochemistry, cII-GnRH immunostaining was localized to granulosa cells while s-GnRH was present in the perinuclear zone of the oocytes.     

Intratesticular control of spermatogenesis in the frog, Rana esculenta.

Adult intact and hypophysectomized (PDX) frogs, Rana esculenta, were treated with a gonadotropin releasing hormone agonist (GnRHA, HOE 766) and/or cyproterone acetate (CPA), the antiandrogen, in order to investigate the regulation of primary spermatogonial (I SPG) multiplication in vertebrates. Treatment with GnRHA (injections containing 900 ng administered for 12 days on alternate days) caused a significant increase of the mitotic index (MI) of I SPG in PDX animals and a further MI increase of SPG was observed when 0.66 mg CPA was given concomitantly with GnRHA. The treatment with 0.66 mg CPA in combination with GnRHA also increased secondary spermatocyte (II SPC) appearance. Moreover, number of nests containing spermatids (SPT) decreased as CPA, in combination with GnRHA, was administered in increasing doses (0.33 and 0.66 mg/injection). Intact animals treated with CPA (0.66 mg/injection) showed a time-dependent I SPG multiplication increase which reached highest values after 28 days. Secondary SPC also proliferated until day 28; meanwhile the number of nests containing SPT decreased. Neither testosterone nor R5020 (a progestin which is not converted to androgens) modified the basal and GnRHA-induced spermatogonial proliferation. These results confirm that in the frog, Rana esculenta, spermatid formation is impaired by CPA treatment and that I SPG multiplication is enhanced by a direct effect of GnRHA; moreover, we suggest that the absence of spermatids constitutes a signal promoting spermatogonial proliferation.     

Ultracytochemical localization of Ca++-ATPase activity in the paraphyseal epithelial cells of the frog,Rana esculenta

Summary Ca⁺⁺-ATPase activity was studied ultracytochemically (cf. Ando et al. 1981) in the paraphysis cerebri of the frog. An intense reaction was demonstrated on the plasmalemma of the microvilli at the apical pole of paraphyseal cells; in contrast, the basolateral plasmalemma showed only a slight staining. In addition, mitochondria, gap junctions, cilia, and cytoplasmic elements (e.g., microfilaments) displayed Ca⁺⁺-ATPase activity. Variation of the Ca⁺⁺-concentration in the incubation medium from 0.1 mM to 100 mM altered the Ca⁺⁺-ATPase activity of the cell organelles. The substitution of Ca-by Mg-ions resulted in a conspicuous decrease in the enzyme activity, especially on the apical plasmalemma. Ca⁺⁺-ATPase activity is claimed to be involved in a number of extra-and intracellular functions. In comparison to the epithelium of the adjacent choroid plexus the paraphyseal epithelial cell is thought to be a principal Ca-ion regulator of the cerebrospinal fluid in frogs.Fellow of the Alexander von Humboldt Foundation     

Immunohistochemical localization of chromogranin A and B in the endocrine cells of the alimentary tract of the green frog,Rana esculenta

Novel monoclonal antibodies to human chromogranin A (CgA) and chromogranin B (CgB) were used to investigate the presence of immunoreactive (-IR) elements in the alimentary tract of the green frog Rana esculenta. Numerous CgA-IR and a few CgB-IR endocrine cells were found within the gut mucosa, from the oesophagus to the cloaca, with some local differences in density. Co-localization studies demonstrated that they were costored in almost all the serotonin-IR, the amylin-IR or islet amyloid polypeptide-IR cells and in the peptide tyrosine tyrosine-IR cells located proximal to the pylorus, but not in those located in more caudal tracts. No other co-localization was demonstrated; substances investigated included somatostatin, substance P, gastrin/cholecystokin, glucagon, glycentin, bombesin, secretin and neurotensin. CgA-IR and CgB-IR cells nearly always displayed argyrophilia with the Grimelius silver method     

Haematological studies on the prewintering and wintering frog, Rana esculenta

1. A study of the haematology of the frog Rana esculenta including erythrocyte count (RBC), haemoglobin content (Hb), haematocrit (HCT), mean cell volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and erythrocyte size as a function of prewinter and winter was made. 2. The RBC count and Hb were significantly higher in contrast to MCV and MCH values during prewinter in both sexes. 3. The surface area to volume ratio was higher in prewinter whereas the length to width ratio (eccentricity) of the cytosome and nucleus was significantly higher during winter in both sexes. 4. Sexual differences in the erythrocyte count, Hb content and the surface area to volume ratio were also observed. 5. The physiological significance of these observations are reported for Rana esculenta.     

Immunocytochemical observations on pineal organ and retina of the Antarctic teleosts Pagothenia borchgrevinki and Trematomus bernacchii

In spite of the unique conditions they have to operate under, the pineal organs of Antarctic fishes have not previously been examined. We determined immunohistochemically that in the end-vesicles and the pineal stalks of Pagothenia borchgrevinki (a species found directly beneath the sea-ice) as well as Trematomus bernacchii (a species preferring somewhat deeper water than the former) at least two populations of physiologically-different cells occurred that displayed reactions indicative of typical vertebrate photoreceptors. Comparisons with immunocytochemically treated retinal sections from the eyes of the same two species showed that anti-opsin reactivity, characteristic of rods, was particularly strong in the lumina of the pineal stalks of both species. Anti-visinin reactions stained cones in the retinal sections of both fishes and occurred throughout the pineal organs, but in particular in the end vesicles of the pineals of both species. The difference in preferred habitat depth between the two species appears to have had very little influence on both retinal and pineal immunocytochemistry. It is concluded that the pineal organs of both species, at least during the austral summer, exhibit signs of being directly photo-sensitive.