Hormonal and pheromonal studies on amphibians with special reference to metamorphosis and reproductive behavior.

In this article, we review studies which have been conducted to investigate the hormonal influence on metamorphosis in bullfrog (Rana catesbeiana) and Japanese toad (Bufo japonicus) larvae, in addition to studies conducted on the hormonal and pheromonal control of reproductive behavior in red-bellied newts (Cynops pyrrhogaster). Metamorphosis was studied with an emphasis on the roles of prolactin (PRL) and thyrotropin (TSH). The release of PRL was shown to be regulated by thyrotropin-releasing hormone (TRH) and that of TSH was evidenced to be regulated by corticotropin-releasing factor. The significance of the fact that the neuropeptide that controls the secretion of TSH is different from those encountered in mammals is discussed in consideration of the observation that the release of TRH, which stimulates the release of PRL, is enhanced when the animals are subjected to a cold temperature. Findings that were made by using melanin-rich cells of Bufo embryos and larvae, such as the determination of the origin of the adenohypophyseal primordium, identification of the pancreatic chitinase, and involvement of the rostral preoptic recess organ as the hypothalamic inhibitory center of α-melanocyte-stimulating hormone (α-MSH) secretion, are mentioned in this article. In addition, the involvement of hormones in eliciting courtship behavior in male red-bellied newts and the discovery of the peptide sex pheromones and hormonal control of their secretion are also discussed in the present article.

Cell proliferation and neurogenesis in the adult telencephalon of the newt Cynops pyrrhogaster.

Urodele amphibians have the ability to regenerate several organs, including the brain. For this reason, the research on neurogenesis in these species after ablation of some parts of the brain has markedly progressed. However, detailed information on the characteristics and fate of proliferated cells as well as the function of newly generated neurons under normal conditions is still limited. In this study, we focused on investigating the proliferative and neurogenic zones as well as the fate of proliferated cells in the adult brain of the Japanese red-bellied newt to clarify the significance of neurogenesis in adulthood. We found that the proximal region of the lateral ventricles in the telencephalon and the preoptic area in the diencephalon were the main sites for continuous cell proliferation in the adult brain. Furthermore, we characterized proliferative cells and analyzed neurogenesis through a combination of 5-ethynyl-2'-deoxyuridine (EdU) labeling and immunohistochemistry using antibodies against the stem cell marker Sox2 and neuronal marker NeuN. Twenty-four hours after EdU injection, most of the EdU-positive cells were Sox2-immunopositive, whereas, EdU-positive signals and NeuN-immunoreactivities were not colocalized. Two months after EdU injection, the colocalization ratio of EdU-positive signals with Sox2-immunoreactivities decreased to approximately 10%, whereas the ratio of colocalization of EdU-positive signals with NeuN-immunoreactivities increased to approximately 60%. Furthermore, a portion of the EdU-incorporated cells developed into γ-aminobutyric acid-producing cells, which are assumed to function as interneurons. On the basis of these results, the significance of newly generated neurons was discussed with special reference to their reproductive behavior.

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.

Possible hormonal interaction for eliciting courtship behavior in the male newt, Cynops pyrrhogaster.

Reproductive behavior in amphibians, as in other vertebrate animals, is under the control of multiple hormonal substances. Prolactin (PRL), arginine vasotocin (AVT), androgen, and 7α-hydroxypregnenolone (7α-OH PREG), four such substances with hormonal activity, are known to be involved in the expression of the tail vibration behavior which is the initial step of courtship performed by the male newt, Cynops pyrrhogaster. As current information on the interaction(s) between these hormones in terms of eliciting tail vibration behavior is limited, we have investigated whether the decline of expression of tail vibration behavior due to suppression of the activity of any one of these hormones can be restored by supplying any one of the other three hormones exogenously. Expression of the behavior was determined in terms of incidence (% of test animals exhibiting the behavior) and frequency (number of times that the behavior was repeated during the test period). Neither PRL nor androgen restored the decline in the incidence and frequency of the tail vibration behavior caused by the suppression of the activity of any one of other three hormones. AVT completely restored both the anti-PRL antibody-induced and flutamide (an androgen receptor antagonist)-induced, but not ketoconazole (an inhibitor of the steroidogenic CYP enzymes)-induced decline in the incidence and frequency of the tail vibration behavior. The neurosteroid, 7α-OH PREG, failed to restore flutamide-induced decline in the incidence and frequency of the behavior. However, it was able to restore both anti-PRL antibody-induced and AVT receptor antagonist-induced decline in the incidence, but not in the frequency of the behavior. In another experiment designed to see the activity of hormones enhancing the frequency of the tail vibration behavior, AVT was revealed to be more potent than 7α-OH PREG. The role of each hormonal substance in determining the expression of the tail vibration behavior was discussed based on the results.

Expression of G proteins in the olfactory receptor neurons of the newt Cynops pyrrhogaster: their unique projection into the olfactory bulbs.

We analyzed the expression of G protein α subunits and the axonal projection into the brain in the olfactory system of the semiaquatic newt Cynops pyrrhogaster by immunostaining with antibodies against Gαolf and Gαo , by in situ hybridization using probes for Gαolf , Gαo , and Gαi2 , and by neuronal tracing with DiI and DiA. The main olfactory epithelium (OE) consists of two parts, the ventral OE and dorsal OE. In the ventral OE, the Gαolf - and Gαo -expressing neurons are located in the apical and basal zone of the OE, respectively. This zonal expression was similar to that of the OE in the middle cavity of the fully aquatic toad Xenopus laevis. However, the Gαolf - and Gαo -expressing neurons in the newt ventral OE project their axons toward the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively, whereas in Xenopus, the axons of both neurons project solely toward the MOB. In the dorsal OE of the newt, as in the principal cavity of Xenopus, the majority of the neurons express Gαolf and extend their axons into the MOB. In the vomeronasal organ (VNO), the neurons mostly express Gαo . These neurons and quite a few Gαolf -expressing neurons project their axons toward the AOB. This feature is similar to that in the terrestrial toad Bufo japonicus and is different from that in Xenopus, in which VNO neurons express solely Gαo , although their axons invariably project toward the AOB. We discuss the findings in the light of diversification and evolution of the vertebrate olfactory system.

Roles of arginine vasotocin receptors in the brain and pituitary of submammalian vertebrates.

This chapter reviews the functions of arginine vasotocin (AVT) and its receptors in the central nervous system (CNS) of primarily submammalian vertebrates. The V1a-type receptor, which is widely distributed in the CNS of birds, amphibians, and fish, is one of the most important receptors involved in the expression of social and reproductive behaviors. In mammals, the V1b receptor of arginine vasopressin, an AVT ortholog, is assumed to be involved in aggression, social memory, and stress responses. The distribution of the V1b-type receptor in the brain of submammalian vertebrates has only been reported in an amphibian species, and its putative functions are discussed in this review. The functions of V2-type receptor in the CNS are still unclear. Recent phylogenetical and pharmacological analyses have revealed that the avian VT1 receptor can be categorized as a V2b-type receptor. The distribution of this newly categorized VT1 receptor in the brain of avian species should contribute to our knowledge of the possible roles of the V2b-type receptor in the CNS of other nonmammalian vertebrates. The functions of AVT in the amphibian and avian pituitaries are also discussed, focusing on the V1b- and V1a-type receptors.

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.

Involvement of the neurosteroid 7α-hydroxypregnenolone in the courtship behavior of the male newt Cynops pyrrhogaster.

Reproductive behavior in amphibians, as in other vertebrate animals, is controlled by multiple hormones. A neurosteroid, 7α-hydroxypregnenolone, has recently been found to enhance locomotor activity in the male newt, Cynops pyrrhogaster. Here, we show that this neurosteroid is also involved in enhancing the expression of courtship behavior. Intracerebroventricular (ICV) injection of 7α-hydroxypregnenolone enhanced courtship behavior dose-dependently in the sexually undeveloped males that had been pretreated with prolactin and gonadotropin, which is known to bring the males to a sexually developed state. But, unlike the case in the locomotion activity, 7α-hydroxypregnenolone did not elicit the behavior in males receiving no prior injections of these hormones. ICV administration of ketoconazole, an inhibitor of the steroidogenic enzyme cytochrome P450s, suppressed the spontaneously occurring courtship behavior in sexually active males. Supplementation with 7α-hydroxypregnenolone reversed the effect of ketoconazole in these animals. It was also demonstrated that the effect of the neurosteroid on the courtship behavior was blocked by a dopamine D2-like, but not by a D1-like, receptor antagonist. These results indicate that endogenous 7α-hydroxypregnenolone enhances the expression of the male courtship behavior through a dopaminergic system mediated by a D2-like receptor in the brain.

Localization of three types of arginine vasotocin receptors in the brain and pituitary of the newt Cynops pyrrhogaster.

The distribution of three types of arginine vasotocin (AVT) receptors in the brain and pituitary of the newt Cynops pyrrhogaster, namely, the V1a-, V2-, and V3/V1b-type receptors, was studied by means of in situ hybridization and immunohistochemistry. mRNA signals and immunoreactive cells for the V1a-type receptor were observed in the telencephalon (mitral layer of the olfactory bulb, dorsal and medial pallium, lateral and medial amygdala, bed nucleus of the decussation of the fasciculus telencephali, bed nucleus of the stria terminalis), diencephalon (anterior preoptic area, magnocellular preoptic nucleus, suprachiasmatic nucleus, ventral thalamus, dorsal and ventral hypothalamic nucleus), mesencephalon (tegmentum, interpeduncular nucleus), and medulla oblongata (median reticular formation, nucleus motorius tegmenti). Cells expressing the V2-type receptor were found in the telencephalon (medial pallium, lateral and medial amygdala, bed nucleus of the decussation of the fasciculus telencephali), and mesencephalon (tegmentum trigemini and facialis). In the paraphysis (possibly the main site of cerebrospinal fluid production), only V2-type receptor mRNA signal and immunoreactivity were detected. V3/V1b-type receptor mRNA was expressed in the diencephalon (dorsal hypothalamic nucleus, nucleus tuberculi posterioris), mesencephalon (tegmentum, interpeduncular nucleus), and medulla oblongata (raphe nucleus), whereas V3/V1b-type-receptor-like immunoreactivity was scarcely detectable in the entire brain. The V3/V1b-type receptor was predominantly expressed in the anterior pituitary. V3/V1b-type receptor and proopiomelanocortin mRNAs were co-localized in the distal lobe of the pituitary. This is the first report of the distribution of three types of AVT receptor in the brain and pituitary of non-mammalian vertebrates.

Hormone-mediated reproductive behavior in the red-bellied newt.

Hormonal involvement in the performance of reproductive behavior of the red-bellied newt Cynops pyrrhogaster is described. The sexually developed male newt is likely to recognize the sexually responsive female newt by a yet unidentified substance released from the oviduct, secretion of this substance being stimulated by prolactin (PRL) and estrogen. At the initial stage of courtship behavior, the male newt vibrates his tail vigorously in front of the female partner. This action is elicited by PRL and androgen and is enhanced by another hormonal factor, arginine vasotocin (AVT). Both PRL and AVT were shown to act centrally to elicit this behavior. A recently discovered neurosteroid, 7alpha-hydroxypregnenolone, was also revealed to be an important factor for eliciting tail vibrating behavior. During courtship, the male newt emits a decapeptide pheromone that attracts the female partner. The synthesis of this attractant by the abdominal gland is promoted by PRL and androgen and its release from the cloaca is elicited by AVT. The responsiveness to the pheromone of the vomeronasal epithelial cells of the female newt is enhanced by PRL and estrogen. Toward the final stage of courtship, the male newts deposits spermatophores, which are picked up through the cloacal orifice of the female newt. AVT induces the discharge of spermatophores from the cloaca. Thus, PRL, AVT, androgen, estrogen, and the neurosteroid, 7alpha-hydroxypregnenolone, are considered to be important factors for the performance of reproductive behavior in the red-bellied newt.

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

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 Ile(187)-Ser(188)-Ala(189) and flanked at its N-terminus by Leu(174)-Gly(175)-Arg(176). 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 degrees C and for Boc-Leu-Leu-Lys-MCA at pH 9.0 and 40 degrees 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.

Molecular cloning of three types of arginine vasotocin receptor in the newt, Cynops pyrrhogaster.

Three types of cDNA encoding the arginine vasotocin (AVT) receptors from the newt, Cynops pyrrhogaster were cloned and the gene expression of each receptor analyzed in the organs and tissues of the newt. The deduced amino acid sequence of one type of AVT receptor, consisting of 418 amino acid residues, showed a high degree of sequence identity with the mammalian arginine vasopressin (AVP) V1a receptors (61-68%). The second type of cDNA, encoding an amino acid sequence consisting of 367 amino acid residues, exhibited a relatively high sequence identity with mammalian AVP V2 receptors (50-51%). The third cDNA, encoding a sequence of 415 amino acid residues, possessed high sequence identity with mammalian AVP V3/V1b receptors (59-63%). Phylogenetic analysis revealed that the first, second and third types of receptor were close to mammalian AVP V1a, V2 and V3/V1b receptors, respectively, and RT-PCR using gene specific primers for each type of receptor indicated that the first and second types of receptor mRNA were expressed in various organs and tissues, including the circulatory, osmoregulatory, and reproductive organs of both male and female newts. In contrast, mRNA expression of the third cDNA was mainly detected in the brain and pituitary, and its expression pattern was distinctly different from that of the other two. We suggest that the first, second and third types of newt AVT receptor obtained in the present study are counterparts of mammalian AVP V1a, V2 and V3/V1b receptors, respectively.

Isolation, characterization and bioactivity of a region-specific pheromone, [Val8]sodefrin from the newt Cynops pyrrhogaster.

Previous analysis of PCR products derived from total RNA from the abdominal gland of the male newt, Cynops pyrrhogaster, inhabiting the Nara area of Japan led to the identification of a gene encoding [Val(8)]sodefrin, as well as the female-attracting peptide pheromone, sodefrin. In this study, purification of this sodefrin variant from the abdominal glands of male newts from the Nara area was accomplished using gel-filtration chromatography and reversed-phase HPLC. Amino acid sequence analysis and mass spectrometry confirmed that the final product was [Val(8)]sodefrin. A full-length cDNA encoding the biosynthetic precursor of [Val(8)]sodefrin was cloned and characterized. The deduced amino acid sequence of prepro[Val(8)]sodefrin showed 86.2% identity with that of the sodefrin precursor. The [Val(8)]sodefrin variant potently attracted females from the Nara area, but the variant was much less or not effective in attracting females captured in the Niigata and Chiba areas. The term aonirin ("aoni" from "aoni-yoshi", the conventional epithet of Nara) is proposed to designate this region-specific pheromone. It is speculated that the coevolution of a novel pheromone and its complementary receptor in the Nara newts may lead to reproductive isolation and eventual differentiation into a separate species.

Amphibian pheromones and endocrine control of their secretion.

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.

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

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, [Val(8)] 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, [Val(8)] sodefrin, from the precursor molecule was demonstrated.

Localization of prolactin receptor in the newt brain.

In the male newt Cynops pyrrhogaster, prolactin (PRL) acts directly on the central nervous system and induces courtship behavior. As a step to elucidate the localization of neurons on which PRL acts, we developed a polyclonal antibody against an oligopeptide having a sequence completely identical with a part of the sequence of PRL receptors (PRLRs) of two species of newts, C. pyrrhogaster and C. ensicauda, and performed an immunohistochemical study with this antibody. PRLR-immunoreactive cells were observed in the medial amygdala, anterior preoptic area, magnocellular preoptic nucleus, suprachiasmatic nucleus, nucleus of the periventricular organ, ventral hypothalamic nucleus, and choroid plexus. We also performed in situ hybridization with a (35)S-labeled newt PRLR antisense RNA probe and detected signals in the preoptic area and choroid plexus. Colocalization of both PRLR-like immunoreactivity and arginine vasotocin-like or mesotocin-like immunoreactivity was demonstrated in the magnocellular preoptic nucleus. This is the first report of PRLR localization in the amphibian brain.

Prolactin acts centrally to enhance newt courtship behavior.

The effects of intracerebroventricular (ICV) and intraperitoneal (IP) injections of ovine prolactin (PRL), antiserum against newt PRL, and antibody against the newt PRL receptor on the expression of courtship behavior of male newts, Cynops pyrrhogaster, were studied to see whether PRL acts centrally or peripherally to induce the behavior. Injections of PRL by either route into gonadotropin-primed males enhanced the expression of the behavior dose-dependently. The minimum effective amount of ovine PRL administered intracerebroventricularly was 0.1 microg, whereas it was 100 microg when injected intraperitoneally. ICV injection of antiserum against newt PRL blocked the spontaneously occurring male courtship behavior when the anti-newt PRL serum was given either intracerebroventricularly or intraperitoneally. The minimum effective dose of the antiserum administered intracerebroventricularly was 0.05 microl, whereas it was 20 microl when injected intraperitoneally. Neither ICV nor IP injection of preimmune serum affected the expression of the behavior. Furthermore, ICV, but not IP, administration of 0.3 microg of anti-newt PRL receptor antibody, purified from antiserum against newt PRL receptor by use of an antigen-conjugated affinity column, blocked the spontaneously occurring courtship behavior in sexually developed males. Neither ICV nor IP injection of the same amount of normal rabbit IgG affected the expression of the behavior. The results strongly suggest that endogenous PRL enhances the behavior by acting centrally through the PRL receptors localized in the brain area.

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.

Peptide pheromones in newts.

This article reviews the current state of understanding of reproductive pheromones in amphibians, focusing mainly on the purification and characterization of peptide pheromones in newts of the genus Cynops, molecular cloning of cDNAs encoding the pheromone molecules, and hormonal control of secretion of these pheromones. Pheromones that attract sexually developed female Cynops pyrrhogaster and C. ensicauda newts were isolated from the male abdominal glands. The C. pyrrhogaster and C. ensicauda pheromones are peptides, designated sodefrin and silefrin, with the amino acid sequences SIPSKDALLK and SILSKDAQLK, respectively. Each pheromone attracts only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed the presence of precursor proteins that are considered to generate these pheromone peptides. Pheromone precursor mRNA levels and radioimmunoassayable pheromone concentrations in the abdominal glands were elevated by prolactin and androgen. Sexual dimorphism and hormone dependency of the responsiveness of vomeronasal epithelium to sodefrin were noted. Significance of pheromones in the form of peptide for those performing reproductive behavior in an aquatic environment was also discussed.

Peptide and protein pheromones in amphibians.

Purification, characterization and biological activity of urodele and anuran sex-pheromones were reviewed. Female-attracting pheromones obtained from the abdominal gland of Cynops pyrrhogaster and C. ensicauda males are peptides consisting of 10 amino acid residues being designated sodefrin and silefrin, respectively. Each pheromone attracted only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed that both are generated from precursor proteins. Synthesis of these pheromones is regulated by prolactin (PRL) and androgen. Responsiveness of the female vomeronasal epithelium to sodefrin is enhanced by PRL and estrogen. The submandibular gland of the male terrestrial salamander, Plethodon jardani secretes a 22-kD proteinaceous pheromone that enhances female receptivity. It was revealed that every salamander synthesizes multiple isoforms of this pheromone, Plethodontid receptivity factor. The magnificent tree frog, Litoria splendida breed in an aquatic environment. The skin glands of the male secrete a female-attracting peptide pheromone, splendipherin, comprising 25 amino acid residues. The significance of the structure of the amphibian sex-pheromone as peptide and protein is discussed in terms of their species specificity.