Tomoaki Nakada

Amphibian pheromones and endocrine control of their secretion

Abstract: Amphibian sex pheromones of 3 urodele (Cynops pyrrhogaster, C. ensicauda, and Plethodon jordani) and 1 anuran (Litoria splendida) species have been isolated and characterized and found to be either small peptides or larger proteins. Each pheromone secreted by the male acts on conspecific females. Endocrine control of pheromone secretion has been best studied in Cynops. The C. pyrrhogaster pheromone, sodefrin, and the C. ensicauda pheromone, silefrin, are generated from their precursor proteins. The sodefrin and silefrin precursor mRNA levels in the abdominal gland of the cloaca are elevated by prolactin and androgen. An increase in the level of both immunoassayable pheromones caused by treatment with these hormones has also been demonstrated. Receptors for both of these hormones have been localized in the abdominal gland. The discharge of sodefrin into the water is elicited by arginine vasotocin. The responsiveness of the female vomeronasal epithelial cells to sodefrin, as estimated by electro‐olfactography, is enhanced markedly by a combination of prolactin and estrogen. Sodefrin elevates intracellular calcium levels in vomeronasal epithelial cells. The population of the sodefrin‐responsive cells increases during the breeding period.

Processing of multiple forms of preprosodefrin in the abdominal gland of the red-bellied newt Cynops pyrrhogaster: regional and individual differences in preprosodefrin gene expression.

Peptides derived from the post-translational processing of preprosodefrin were isolated from an extract of the abdominal glands of male red-bellied newts Cynops pyrrhogaster obtained 5 months prior to the onset of the breeding season. Structural characterization of the peptides showed that the pheromone sodefrin (SIPSKDALLK) is stored in a biologically inactive COOH-terminally extended form (SIPSKDALLKISA). It follows, therefore, that the activation of a protease that cleaves at a Lys-Ile bond to generate the active pheromone must occur by the time of onset of reproductive behavior. Additional peptides (representing preprosodefrin-(146-175)-peptide and preprosodefrin-(159-173)-peptide), that are derived from the precursor by cleavage at monobasic and dibasic processing sites, were also purified from the extract. The isolation of paralogs of these peptides, including an inactive COOH-terminally extended form of [Asn10]sodefrin, provides evidence for the expression of multiple genes encoding preprosodefrin. PCR products derived from total RNAs from the abdominal gland of individual newts collected from three different regions of Japan were analyzed. The data confirm the existence of multiple genes encoding sodefrin and its variants whose expression varied according to the individuals and the regions. However, genes encoding sodefrin were found to be expressed in all the specimens sampled.

Responsiveness of vomeronasal cells to a newt peptide pheromone, sodefrin as monitored by changes of intracellular calcium concentrations.

A peptide pheromone of the red-bellied male newt, sodefrin was tested for its ability to increase intracellular concentrations of Ca(2+) ([Ca(2+)]i) in the dissociated vomeronasal (VN) cells of females by means of calcium imaging system. The pheromone elicited a marked elevation of [Ca(2+)]i in a small population of VN cells from sexually developed females. The population of cells exhibiting sodefrin-induced elevation of [Ca(2+)]i increased concentration-dependently. A pheromone of a different species was ineffective in this respect. The VN cells from non-reproductive females or from reproductive males scarcely responded to sodefrin in terms of elevating [Ca(2+)]i. In the cells from hypophysectomized and ovariectomized females, the sodefrin-inducible increase of [Ca(2+)]i never occurred. The cells from the operated newts supplemented with prolactin and estradiol exhibited [Ca(2+)]i responses to sodefrin with a high incidence. Thus, sex- and hormone-dependency as well as species-specificity of the responsiveness of the VN cells to sodefrin was evidenced at the cellular level. Subsequently, possibility of involvement of phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3) and/or PLC-diacylglycerol (DAG)-protein kinase C (PKC) pathways in increasing [Ca(2+)]i in VN cells in response to sodefrin was explored using pharmacological approaches. The results indicated that PLC is involved in generating the Ca(2+) signal in all sodefrin-responsive VN cells, whereas IP3 in approximately 50% of the cells and DAG-PKC in the remaining cells. In the latter case, the increase of [Ca(2+)]i was postulated to be induced by the influx of Ca(2+) through the L-type channel. The significance of the finding is discussed.

Evidence for processing enzymes in the abdominal gland of the newt, Cynops pyrrhogaster, that generate sodefrin from its biosynthetic precursor.

Abstract Sodefrin (Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys) is a female-attracting peptide pheromone secreted by the abdominal gland of the male red-bellied newt, Cynops pyrrhogaster. Sequence analysis of a cDNA encoding sodefrin revealed that the peptide is located in the C-terminal region of its precursor protein (residues 177–186 of preprosodefrin) and extended from its C-terminus by the tripeptide sequence Ile187-Ser188-Ala189 and flanked at its N-terminus by Leu174-Gly175-Arg176. This suggests that sodefrin is generated by enzymatic cleavage at monobasic (Lys and Arg) sites within the precursor molecule. To demonstrate the presence in the abdominal gland of proteolytic enzymes capable of generating sodefrin, an enzymatic assay was developed using t-butoxycarbo-nyl (Boc)-Leu-Gly-Arg–4methylcoumaryl-7-amide (MCA) and Boc-Leu-Leu-Lys-MCA as synthetic substrates. A crude extract of the abdominal gland hydrolyzed both substrates to liberate 7-amino-4- methylcoumarin, suggesting that enzymes that generate sodefrin from its precursor molecule are present in the gland. The activity in the extract for cleaving Boc-Leu-Gly-Arg-MCA was optimal at pH 9.0 and 45°C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40°C. The effects of a range of specific inhibitors on activities in the extract suggest an involvement of enzymes belonging to the serine protease family. It was also demonstrated that enzymatic activity in an extract of the abdominal glands of sexually developed males was significantly (three- to six-fold; p<0.01) higher than that of sexually undeveloped males.

Imorin: a sexual attractiveness pheromone in female red-bellied newts ( Cynops pyrrhogaster )

The male red-bellied newt (Cynops pyrrhogaster) approaches the female’s cloaca prior to performing any courtship behaviour, as if he is using some released substance to gauge whether she is sexually receptive. Therefore, we investigated whether such a female sexual attractiveness pheromone exists. We found that a tripeptide with amino acid sequence Ala-Glu-Phe is secreted by the ciliary cells in the epithelium of the proximal portion of the oviduct of sexually developed newts and confirmed that this is the major active substance in water in which sexually developed female newts have been kept. This substance only attracted sexually developed male newts and acted by stimulating the vomeronasal epithelial cells. This is the first female sexual attractiveness peptide pheromone to be identified in a vertebrate.

Regionally specific occurrence of an active sodefrin variant in the red-bellied newt.

Abstract: Sodefrin (SIPSKDALLK) is a female‐attracting pheromone that is secreted by the abdominal gland of the male red‐bellied newt. We found that mRNA encoding a sodefrin variant, [Val8] sodefrin, is expressed exclusively in specimens captured in the Nara area of Japan. The synthetic peptide was tested for its activity. It attracted females from Nara, but not those from other regions, suggesting that there is a geographic variation in the pheromone molecule and in the responsiveness to the pheromone. Employing an abdominal gland extract and synthetic substrates, the possibility of generation of the putative pheromone, [Val8] sodefrin, from the precursor molecule was demonstrated.

Sodefrin and Related Pheromones

ABSTRACT This article describes the current state of understanding of sodefrin, a peptide pheromone from the newt Cynops pyrrhogaster, and [Leu3, Gln8] sodefrin (silefrin) from the congeneric species, C. ensicauda. Both pheromones are composed of 10-amino-acid residues and were isolated from the abdominal glands of the male newts. They exhibit a potent female-attracting activity only to the conspecific females, suggesting a contribution of the pheromones to maintaining the reproductive isolation of the species. Both pheromones are generated from 20 kDa precursor molecules. These pheromones are emitted through the cloaca of the male and are directed toward the female partners snout during courtship to act primarily on the lateral nasal sinus cells. Hormone dependency of secretion of and response to the pheromones has been demonstrated.