Kouhei Matsuda

Sodefrin: a female-attracting peptide pheromone in newt cloacal glands

A decapeptide called sodefrin was isolated from the abdominal gland of the cloaca of the male red-bellied newt, Cynops pyrrhogaster. The native peptide, as well as the synthetic one, had a female-attracting activity. Sodefrin was found in the apical portion of the epithelial cells of the abdominal gland. Sodefrin is apparently species specific because it did not attract females of Cynops ensicauda. This is the first amphibian pheromone to be identified and the first peptide pheromone identified in a vertebrate.

Hormonal control of response to and secretion of sex attractants in Japanese newts.

Hormonal control of sexual chemoattraction in the newt, Cynops pyrrhogaster, was investigated by a water preference test. Sexually inert newts injected with prolactin (PRL) and human chorionic gonadotropin (HCG) in combination preferred the water in which newts of the opposite sex had been kept, whereas saline-injected specimens did not. This indicates that PRL and HCG enhance the response of the newts to attractant(s) secreted by the opposite sex. The water in which PRL plus HCG-treated newts had been kept attracted the opposite sex more intensely than the water in which PRL-, HCG-, or saline-injected newts had been kept. In castrated males, PRL plus testosterone propionate (TP), and in ovariectomized females, PRL plus estradiol induced the secretion of substance(s) that attracted newts of the opposite sex. Removal of the abdominal gland of the cloaca in the male or the oviduct in the female attenuated the attracting activity of the water in which they were kept. Involvement of PRL and gonadal steroids in the secretion of sex attractants by the male abdominal gland and female oviduct was suggested.

Purification and properties of newt prolactin

A highly purified prolactin (PRL) was obtained from pituitary glands of newts, Cynops pyrrhogaster, by extraction of acetone-dried powder with acid acetone and high-performance liquid chromatography (HPLC) on Mono-Q (anion exchange), Superose-12 (gel filtration), and TSK-gel ODS-120T (reverse-phase) columns with a yield of 4.5 mg per 325 mg of protein starting material. Purification was monitored by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting analysis employing antiserum against bullfrog PRL. Newt PRL thus obtained has a molecular weight of 23,000 as determined by SDS-PAGE. The isoelectric point is 4.7 as determined by isoelectric focusing. The amino acid composition closely resembles that of anuran PRLs. This hormone was as potent as bovine PRL in stimulating collagen synthesis in the bullfrog tadpole tail fin. Antiserum against newt PRL was produced by immunizing a rabbit. Histological studies on newt adenohypophyses revealed that the cells that immunologically reacted with the antiserum against newt PRL correspond to the ones positively stained with the antiserum against bullfrog PRL.

Development and application of homologous radioimmunoassay for newt prolactin.

A specific and sensitive homologous radioimmunoassay (RIA) for newt (Cynops pyrrhogaster) prolactin (PRL) was developed. PRL isolated from newt pituitary glands was used for generating antiserum in a rabbit, for radioiodination, and for the standard. Several dilutions of plasma and pituitary homogenate of newts yielded dose-response curves which were parallel to the standard curve. Plasma from hypophysectomized newts showed the least amount of cross-reaction. Pituitary homogenates of other species of urodeles such as Ambystoma mexicanum and Onychodactylus japonicus gave inhibition curves which were parallel to the standard curve. Purified PRLs of anurans such as Rana catesbeiana and Bufo japonicus gave inhibition curves which did not parallel the standard. Bovine PRL and ovine PRL showed no inhibition of binding even at relatively high doses in this RIA. The RIA was applied to the determination of plasma and pituitary PRL levels in the adult newts treated with dopamine agonist (bromocriptine) and/or antagonist (pimozide). Pimozide enhanced PRL levels and bromocriptine antagonized it, while pituitary PRL levels were not appreciably changed.

Amphibian prolactins: Activity in the eft skin transepithelial potential bioassay

The effects of purified prolactins isolated from frogs (fPRL; Rana catesbeiana) and newts (nPRL; Cynops pyrrhogaster) were compared with those of ovine prolactin (oPRL) and thyrotropin releasing hormone (TRH) in the eft (Notophthalmus viridescens) skin transepithelial potential (TEP) bioassay. At total doses as low as 0.4 micrograms/animal, both fPRL and nPRL were as effective as oPRL in reducing eft skin TEP. By contrast, TRH at total doses as high as 400 micrograms/animal was ineffective in altering eft skin TEP. These data provide the first direct evidence that amphibian PRLs can exert significant physiological control over salamandrid integumental sodium transport.

Involvement of prolactin in the regulation of plasma calcium levels in the newt, Cynops pyrrhogaster

In the newt, Cynops pyrrhogaster, parathyroidectomy (PTX) brought about a marked decrease in the concentration of plasma calcium. The animals recovered from the hypocalcemia by 15 days after the operation if the pituitary gland was left intact. After PTX, no significant changes in the plasma sodium concentration were observed. Experiments were then conducted to obtain direct evidence that endogenous prolactin (PRL) is involved in this recovery process. Recovery of the calcium level after PTX was blocked by administration of an antiserum raised against newt PRL. In newts deprived of both the pituitary and parathyroid glands, no recovery from hypocalcemia was observed. Administration of newt or ovine PRL to parathyroidectomized-hypophysectomized newts significantly elevated the blood calcium level. After PTX, the concentration of immunoassayable PRL in the blood rose to 10 times the value in sham-operated animals. These results indicate the involvement of PRL in calcium homeostasis in newts with a shortage of parathormone.