Female-attracting pheromone in newt cloacal glands

It has been postulated that male newts emit pheromones that attract females of the same species. Female newts of the species Cynops pyrrhogaster were attracted to water in which sexually active conspecific males had been kept, but not to water in which abdominal gland-ablated males had been kept, indicating that the attracting pheromone was secreted by or through the abdominal gland of the cloaca. An attempt has been made to isolate and characterize the female-attracting pheromone in the abdominal glands of male newts. Female-attracting activity was monitored using a preference test. The active substance was isolated by two steps of purification using reverse-phase high-performance liquid chromatography. Direct sequencing of the final product revealed that it is a decapeptide with the amino acid sequence Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys. Its minimum effective concentration was estimated to be between 0.1 pM and 1.0 pM. The synthetic peptide showed a female-attracting activity similar to that of the native peptide. It seems to act through the olfactory organ of female newts, because the effect of the peptide was blocked by bilateral nostril plugging with cotton balls soaked in melted vaseline. An antiserum against sodefrin was generated in a rabbit. An immunoelectron microscopic study using this antiserum revealed that sodefrin exists predominantly within secretory granules in the epithelial cells of the abdominal glands. A radioimmunoassay for sodefrin was developed in which the antiserum was used along with sodefrin which was N-terminally extended with a tyrosine residue as a radioligand. The immunoassayable sodefrin content in C. pyrrhogaster males was diminished by castration and hypophysectomy. The sodefrin content was increased markedly in the castrated and hypophysectomized newts after treatment with both testosterone and prolactin. Testosterone but not prolactin increased the sodefrin content to a lesser extent. Aqueous extract of the abdominal glands of C. ensicauda showed no inhibition of binding in this assay. Moreover, C. ensicauda females were insensitive to sodefrin, although they were attracted to a water extract of abdominal glands from males of their own species. On the other hand, C. pyrrhogaster females responding to sodefrin were not attracted to the water extract of the abdominal glands from C. ensicauda males. Sequence analyses of sodefrin cDNA clones obtained from a C. ensicauda abdominal gland cDNA library revealed that the cDNA encoded a variant type of sodefrin peptide with substitutions of Leu3 and Gln8. The synthetic [Leu3, Gln8]-sodefrin attracted C. ensicauda females but not C. pyrrhogaster females. These results indicate that the female-attracting pheromone differs between these two species of genus Cynops.