Kosuke Kawamura

Induction from posterior hypothalamus is essential for the development of the pituitary proopiomelacortin (POMC) cells of the toad (Bufo japonicus)

The role of the posterior hypothalamus in the development of the epithelial hypophysis was studied in Bufo embryos. In animals from which the central part of the neural plate (NP) had been surgically removed at the open neurula stage, the infundibulum did not develop, and the epithelial hypophysis was formed away from the normal site without morphological connection with the brain. Immunoreactive MSH cells and ACTH cells, i.e, the pituitary POMC cells, were not detected in any of the surgically treated animals while other types of secretory cells (PRL, GH, TSH and GTH cells) were invariably present. In view of the fact that POMC cells originate in the anterior neural ridge, and not in the neural plate, the embryonic brain seems to exert an inductive influence upon the primordial pituitary POMC cells. Since these cells differentiate in a tail graft, isolated from the brain at a later stage (tail-bud stage), the inductive stimuli must be conveyed from/via the posterior hypothalamus to the pituitary anlage between the open neurula and the tail-bud stages.

Isolation and sequence of a novel amphibian pancreatic chitinase

An approximately 60-kDa protein with chitinase activity was purified from the pancreas of the toad Bufo japonicus. Its specific activity was 4.5 times higher than that of a commercial bacterial chitinase in fragmenting crab shell chitin, and its optimal pH was approximately 6.0. A cDNA clone encoding a protein consisting of 488 amino acid residues, including part of the peptide sequence determined from the isolated protein, was obtained from a toad pancreas cDNA library. The deduced amino acid sequence indicated that the protein contained regions with high homology to those present in chitinases from different species, with the amino acid residues for the chitinase activity and the chitin-binding ability being completely conserved. We designate the protein as toad pancreatic chitinase (tPCase). Northern blot analysis revealed the mRNA of this enzyme to be expressed exclusively in the pancreas. Toad PCase is the first amphibian chitinase to be identified as well as the first pancreatic chitinase identified in a vertebrate.

Rapid and selective removal of larval erythrocytes from systemic circulation during metamorphosis of the bullfrog, Rana catesbeiana

Mechanisms of hemoglobin transition during bullfrog metamorphosis were investigated by labeling red blood cells from larvae (L‐RBC) and from froglets (A‐RBC) with a fluorescent dye, PKH26. The life span of the labeled L‐RBC in systemic circulation was significantly shorter when they were injected into the animals at the metamorphic climax, compared to injection into pre‐ or postmetamorphic animals. The A‐RBC had a long life span regardless of the metamorphic stage of the recipient animal. Therefore, L‐RBC were selectively removed from the systemic circulation at the time of metamorphic climax. During climax, the labeled L‐RBC were ingested by hepatic and splenic macrophages, indicating that macrophages are involved in the specific elimination of L‐RBC.

Molecular Cloning of a Putative Gastric Chitinase in the Toad Bufo japonicus

Abstract On the basis of our preliminary observation that a crude extract of the stomach of the toad Bufo japonicus exhibited a chitinase activity with its optimum pH around 3.0, we undertook molecular cloning of a cDNA encoding this putative gastric chitinase. By use of 2 degenerate oligonucleotide primers derived from the 2 conserved regions of the vertebrate chitinases, a reverse transcription-PCR product was obtained. This product was used as a probe to screen a cDNA library constructed from the toad stomach. The longest positive clone was revealed to contain an open reading frame for a putative chitinase protein of 484 amino acids, which protein exhibited sequence similarity to the known vertebrate chitinases. Our data also revealed this putative gastric chitinase to be distinct from the chitinase that we had previously isolated from the pancreas of the same species. In this putative gastric chitinase, both the N-terminal catalytic domain and the C-terminal chitin-binding domain were perfectly conserved, suggesting this protein to function as chitinase in the toad stomach.

Genomic DNA fragmentation in red blood cells of the bullfrog during metamorphosis

Red blood cells (RBC) of the bullfrog (Rana catesbeiana) contain larval‐type hemoglobin (Hb) during the larval period. At the beginning of metamorphosis, RBC containing adult‐type Hb appear and two types of RBC coexist in the systemic circulation. During the metamorphic climax, RBC with larval‐type Hb disappear from the circulation and, simultaneously, RBC containing adult‐type Hb begin to circulate. These two types of RBC were separated by Percoll density gradient centrifugation to examine the molecular size of the genomic DNA of each population. DNA fragmentation was detected only in new RBC with adult‐type Hb that appeared in the systemic circulation and remained throughout post‐metamorphic life. Semiquantification of DNA on agarose gel showed that the degree of DNA fragmentation was highest at the metamorphic climax. As the existence of DNA fragments suggested endonucleolytic cleavage, nuclease activity was examined by an activity gel system and in vitro circular plasmid DNA digestion assays. The latter revealed that both types of RBC possess endonucleolytic activity throughout the pre‐ and post‐metamorphic periods. Assays of endogenous endonucleolytic activities under different divalent ionic conditions suggested that mobilization of intracellular Ca2+‐Mg2+ induces genomic DNA fragmentation in adult‐type RBC.

Localization and characterization of prolactin-like immunoreactivity in the pituitary of the frog Rana ridibunda

Distribution and quantification of PRL in the pituitary gland of the frog Rana ridibunda were investigated using a high-affinity antiserum raised against bullfrog prolactin (PRL). The immunoreactive PRL-producing cells were distributed throughout the pars distalis, the highest density of cells being observed in the rostral region of the adenohypophysis facing the neurointermediate lobe. The dorsal region of the pars distalis contained only a few scattered PRL-immunoreactive cells. At the electron microscopic level, PRL-containing cells were visualized using the immunogold procedure. PRL-immunoreactive material was exclusively stored in secretory granules (size ranging from 200 to 700 nm in diameter). Neither the rough endoplasmic reticulum nor the Golgi apparatus were immunolabeled. Using a radioimmunoassay method we have compared the displacement curves obtained with bullfrog PRL and acetic extracts from Rana ridibunda pituitary. The two binding curves were not completely parallel, suggesting the existence of slight variations of the amino acid sequences of PRL in the two species. The concentration of PRL in the green frog adenohypophysis appeared somewhat higher (35.3 +/- 8.8 micrograms/mg protein) than that in the bullfrog pituitary. These results validate the use of an antiserum to bullfrog PRL to investigate the regulation of PRL secretion in Rana ridibunda.

Lack of Inhibitory Control of Melanophore-Stimulating Hormone Secretion in the Larval Toad, Bufo japonicus

Abstract In general, both larval and adult amphibians are known to change their skin color depending on the background color. However, in the Japanese toad Bufo japonicus, the larvae were found to be devoid of the ability of responding to the background color. Melanin granules in the dermal melanophores were in a state of constant dispersion independently of the color of the background to which they were subjected. Pharmacological and histochemical studies revealed that in the toad tadpoles, melanophore-stimulating hormone is being released without inhibition, presumably because innervation of catecholaminergic fibers in the intermediate lobe is incomplete until they finish metamorphosis. Biological significance of the dark color of Bufo tadpoles is discussed in conjunction with another characteristic that they have a tendency to aggregate to form a dense black mass.

Morphogenesis of the Hypothalamus and Hypophysis

Publisher Summary This chapter describes the morphogenesis of the hypothalamus and hypophysis. The pituitary gland is a secretory organ endowed with a crucial role in the regulation of endocrine systems. This central role is enabled by circuits that receive signals from the brain and blood and secrete several tropic hormones that stimulate the activity of other endocrine organs such as the thyroid gland, the adrenal cortex, and the gonads. In every vertebrate species the gland is situated under the diencephalic floor with a structural connection with the brain through the ventral protrusion of the diencephalic floor, the infundibulum. The pituitary gland is histologically subdivided into a non-neural epithelial part and a neural part. The former part, the adenohypophysis, consists of the anterior lobe (pars distalis) and the intermediate lobe (pars intermedia), both of which are compactly folded epithelial sheets containing a mixed population of secretory cells. The latter part, the neurohypophysis (pars nervosa), is an aggregate of nerve endings with intervening supporting cells (pituicytes).