FEMALE-ATTRACTING PHEROMONE IN NEWT CLOACAL GLANDS

It has been postulated that male newts emit pheromones that attract fe males of the same species, Female newts of the species Cynops pyrrhoga ster were attracted to water in which sexually active conspecific male s had been kept, but not to water in which abdominal gland-ablated mal es had been kept, indicating that the attracting pheromone was secrete d by or through the abdominal gland of the cloaca, An attempt has been made to isolate and characterize the female-attracting pheromone in t he abdominal glands of male newts, Female-attracting activity was moni tored using a preference test. The active substance was isolated by tw o steps of purification using reverse-phase high-performance liquid ch romatography, Direct sequencing of the final product revealed that it is a decapeptide with the amino acid sequence Ser-lle-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-at tracting activity similar to that of the native peptide. It seems to a ct through the olfactory organ of female newts, because the effect of the peptide was blocked by bilateral nostril plugging with cotton ball s soaked in melted vaseline, An antiserum against sodefrin was generat ed in a rabbit, An immunoelectron microscopic study using this antiser um revealed that sodefrin exists predominantly within secretory granul es 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 co ntent was increased markedly in the castrated and hypophysectomized ne wts 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 inhi bition of binding in this assay, Moreover, C. ensicauda females were i nsensitive to sodefrin, although they were attracted to a water extrac t of abdominal glands from males of their own species, On the other ha nd, 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 Leu(3) and Gln(8). The synthetic [Leu(3), Gln(8)]-sodefrin attracted C. ensicauda females bu t not C. pyrrhogaster females, These results indicate that the female- attracting pheromone differs between these two species of genus Cynops . (C) 1997 Elsevier Science Inc.