Endocrine regulation of reproductive behavior in the newt Cynops pyrrhogaster

Hormonal control of the expression of courtship behavior and of secretion of the female-attracting pheromone sodefrin by the male red-bellied newt, Cynops pyrrhogaster, together with the hormonal influence on the responsiveness to the pheromone in the female, is reviewed.Expression of the initial stage of the courtship behavior, i.e., tail vibration by the male in front of the female, is dependent on prolactin (PRL) and androgen. During the courtship, sodefrin seems to be released from the cloaca through the ducts of the abdominal gland. Both content of immunoreactive sodefrin and preprosodefrin mRNA levels in the abdominal gland are elevated by a combination of PRL and androgen, indicating that the pheromone synthesis is stimulated by these two hormones. On the other hand, the discharge of sodefrin is accelerated by AVT, its action being mediated by V1 receptor. In female newts, responsiveness of the vomeronasal epithelium to the pheromone is elevated by a combination of PRL and estrogen. Thus, it can be concluded that PRL, AVT, and sex steroids are key hormones for the reproductive performance in the red-bellied newt. In this article, the significance of the structure of the pheromone molecule as a peptide is also discussed in terms of its species-specificity and its effectiveness in an aquatic environment.     

Hormonal control of urodele reproductive behavior

Hormonal control of expression of courtship behavior and of development of structures related to the reproductive behavior in two species of Japanese newts, Cynops pyrrhogaster and Cynops ensicauda, was described. Prolactin (PRL) and androgen were essential factors for eliciting courtship behavior. In addition, arginine vasotocin markedly enhanced the expression of courtship behavior. PRL induced migration to water, in which courtship and oviposition take place, and converted the integument from the terrestrial type to the aquatic one. PRL also stimulated the growth of the tail fin, which was blocked by estrogen. Cellular and nuclear size and number of synapses on the somata of Mauthner cells, which are involved in tail movement, were also increased by PRL and androgen. Synthesis of sodefrin, a female-attracting pheromone, in the abdominal gland as well as that of mucopolysaccharides constituting the sac of sperm in the lateral gland was enhanced by PRL and androgen. Structural development of oviducts was elicited by estrogen or PRL to a certain extent, and full oviducal development by the combination of these two hormones, PRL being indispensable for the oviducal jelly secretion.     

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.     

Hormonal influence on the olfactory response to a female-attracting pheromone, sodefrin, in the newt, Cynops pyrrhogaster

Sodefrin is a female-attracting pheromone isolated from the abdominal glands of male newts, Cynops pyrrhogaster. Previously, the preference of conspecific female newts for sodefrin was shown to be completely abolished by plugging the bilateral nostrils, indicating that it acts on the olfactory organ. To determine the sensitivity of the olfactory receptor cells to sodefrin, electro-olfactograms (EOGs) in response to sodefrin solution were recorded from the ventral nasal epithelium of sexually developed female newts. Sodefrin elicited marked EOG responses in a dose-dependent manner on the epithelium of the lateral nasal sinus (LNS) region, a putative vomeronasal organ. In ovariectomized females, treatment with prolactin (PRL) and estrogen markedly enhanced the EOG response to sodefrin. The EOG response to the pheromone was also enhanced considerably by treatment with either PRL or estrogen alone. A slight but significant elevation was observed in castrated males receiving PRL plus estrogen or estrogen alone. It was concluded that the main site of action of sodefrin resides in the lateral sinus region and that sensitivity to sodefrin is under the control of PRL and estrogen. The presence of a sex difference in olfactory sensitivity to the hormones and/or pheromone was also suggested.     

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.     

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.     

Silefrin, a sodefrin-like pheromone in the abdominal gland of the sword-tailed newt, Cynops ensicauda

Sodefrin-like female-attracting pheromone was purified from the abdominal glands of male sword-tailed newts, Cynops ensicauda, by gel-filtration chromatography and reversed-phase high-performance liquid chromatography. The final product comprises 10 amino acid residues with the sequence SILSKDAQLK which coincided with the sequence deduced from its precursor cDNA. This peptide was designated silefrin. The sequence of silefrin was different from that of sodefrin by two amino acid residues, with substitutions Leu for Pro and Gln for Leu at positions 3 and 8, respectively. Both native and synthetic silefrin exerted an equipotent activity in attracting conspecific females.     

Multiple mechanisms of phenotype development in the bluehead wrasse

Despite having detailed information on mechanisms mediating sex-typical behavior in many species, we have little understanding of whether the same mechanisms regulate these behaviors when they are performed in the same species under different social contexts. In the five field experiments of this study of bluehead wrasses (Thalassoma bifasciatum), a sex-changing fish, we examined the roles of arginine vasotocin (AVT) and the potent teleost androgen 11-ketotestosterone (11KT) in mediating sexual and aggressive behaviors typical of dominant males. We demonstrated that AVT appears necessary for the assumption of dominant territorial status in males and females, but is sufficient only in the socially dominant terminal phase (TP) male phenotype. Specifically, an AVP V(1) receptor antagonist prevented both TP males and females from gaining dominance over recently vacated territories. However, unlike TP males in a previous study, neither females nor initial phase males responded to AVT treatment with increases in display of TP male typical behaviors when under social conditions that inhibit sex change. Treating females with 11KT did not alter responsiveness to AVT, but did induce male coloration and courtship behavior that was not observed in oil-treated females. Combined with the results of a previous study, these results indicate that the ability of AVT to induce male-typical behavior differs among sexual phenotypes and that this differential responsiveness appears to be dependent on social context and not directly on exposure to 11KT. Furthermore, since 11KT can induce courtship behavior in females that is not affected by AVT, there may be different hormonal mechanisms mediating courtship behavior under different social contexts.     

Manipulations of the AVT system shift social status and related courtship and aggressive behavior in the bluehead wrasse

Arginine vasotocin (AVT) and its mammalian homologoue arginine vasopressin (AVP) influence male sexual and aggressive behaviors in many species. We tested the effects of AVT and an AVP-V(1a) receptor antagonist on the display of alternative male tactics in a tropical coral reef fish, the bluehead wrasse Thalassoma bifasciatum. We gave AVT injections to territorial and nonterritorial males of the large and colorful phenotype (terminal phase) and an AVP-V(1a) receptor antagonist, Manning compound, to territorial males in the field. AVT increased courtship independent of status, while its effects on territoriality and aggression were dependent upon male status. In territorial males, AVT increased courtship and tended to decrease the number of chases toward initial phase individuals. In nonterritorial males, AVT increased courtship, chases toward initial phase individuals, and territorial behavior while decreasing feeding. These are all behaviors rarely seen in nonterritorial males, so AVT made these males act like territorial TP males. The AVP-V(1a) receptor antagonist had opposite effects. It decreased courtship and territorial defense, making these males act more like nonterritorial males. Manipulations of the AVT system shifted males within a single phenotype from the nonterritorial social status to the territorial social status and vice versa. Since the entire suite of behaviors related to territoriality was affected by AVT system manipulations, our results suggest that the AVT system may play a key role in motivation of behaviors related to mating.     

Male Courtship Pheromones Induce Cloacal Gaping in Female Newts (Salamandridae)

Pheromones are an important component of sexual communication in courting salamanders, but the number of species in which their use has been demonstrated with behavioral evidence remains limited. Here we developed a behavioral assay for demonstrating courtship pheromone use in the aquatically courting Iberian ribbed newt Pleurodeles waltl. By performing an in-depth study of the courtship behavior, we show that females invariably open their cloaca (cloacal gaping) before engaging in pinwheel behavior, the circling movement that is the prelude to spermatophore uptake. In contrast, cloacal gaping was not observed in failed courtships, where females escaped or displayed thanatosis. Since gaping mainly occurred during male amplexus and cloacal imposition, which is the obvious period of pheromone transfer, we next investigated whether male courtship water (i.e., water holding courtship pheromones) alone was able to induce this reaction in females. These tests showed that courtship water induced cloacal gaping significantly more than water, even in the absence of a male. Cloacal gaping thus provides a simple and robust test for demonstrating courtship pheromone use in the Iberian ribbed newt. Since opening the cloaca is an essential prerequisite for spermatophore pick-up in all internally fertilizing salamanders, we hypothesize that variations on this assay will also be useful in several other species.     

Vasotocin treatment inhibits courtship in male zebra finches; concomitant androgen treatment inhibits this effect

Zebra finches evolved in arid areas of Australia. Their reproduction is stimulated by water availability, which is unpredictable. Cheng (Poult. Sci. Rev. 5 (1993) 37) hypothesized that the primary mechanism controlling reproduction in species relying on unpredictable cues should be inhibitory. The onset of stimulatory environmental conditions terminates the inhibition, allowing rapid initiation of reproduction. As the primary hormone regulating water balance in birds, arginine vasotocin (AVT) appears a likely candidate to modulate reproduction in finches. Drought conditions cause sustained AVT release, which in other species inhibits androgen production. To determine whether increased AVT inhibits reproductive behavior, intact males were tested with females and divided into three groups matched for courtship behavior. Osmotic minipumps containing (a) saline, (b) 264 ng AVT, or (c) 1320 ng AVT in saline were implanted subcutaneously and males tested 48 h later. AVT-treated males socialized with females, but the high dose significantly reduced singing and courtship displays. To determine whether AVT acted by depressing androgen secretion, additional males were given subcutaneous androgen implants and divided into two groups matched for courtship behavior. Males were then implanted with minipumps containing (a) saline or (b) the high AVT dose. Males treated with AVT plus androgen showed no deficits in courtship behavior. These data suggest that AVT secretion during periods of drought may inhibit reproduction by inhibiting androgen production. Inhibition of reproductive behavior by AVT may be a more general phenomenon. Large quantities of AVT or, in mammals the closely-related peptide vasopressin (VP), are released when animals are stressed, and high levels of AVT/VP may inhibit reproductive behavior. The extremely short half-life of these peptides means that once proximal factors become more favorable, the gonads should rapidly be released from the peptides' inhibitory actions.     

Courtship Behavior of the Cumberland Plateau Woodland Salamander,Plethodon kentucki (Amphibia: Plethodontidae), with a Review of Courtship in the Genus Plethodon

Complete catalogs of courtship behavior are available for only seven of the 42 currently recognized species of Plethodon. Additional detailed studies of courtship behavior in Plethodon species are needed to analyze the evolution of courtship behavior in this genus. We investigated the courtship behavior of the Cumberland Plateau woodland salamander (Plethodon kentucki) and compared it to previous accounts of courtship in other Plethodon species. In the laboratory, we videotaped the complete courtship of 30 different P. kentucki pairs, which included 46 tail-straddling walks that resulted in spermatophore deposition. From a transition matrix of observed motor patterns, we constructed a flow diagram of significant motor-pattern transitions during courtship. In general, the courtship behavior of this species is very similar to that of other large eastern Plethodon; however, there are some notable differences. Prior to the tail-straddling walk, the male transfers courtship pheromone from his mental gland to the female's nasolabial grooves (via mental-gland tapping and ‘chin-to-chin’ behavior patterns) more frequently than in other Plethodon. In most courtships, the female initiates contact leading to the tail-straddling walk. Males exhibit a greater propensity to deposit multiple spermatophores per courtship (two deposited in 33% of courtships, three deposited in 10% of courtships, X = 1.53 spermatophores per courtship) than males of other plethodontids. Mean size of the spermatophore is smaller than in other large eastern Plethodon. Overall, the courtship behavior of this species is most similar to that of P. jordani.     

Duration of androgen treatment modifies behavioral response to arginine vasotocin in Taricha granulosa

Five and eighteen days after surgery, injection of arginine 8-vasotocin (AVT) stimulated clasping behavior in castrated controls (unimplanted or cholesterol-implanted) but not in androgen-implanted, castrated newts (Taricha granulosa). Conversely, AVT injected 33 days after castration increased the incidence of courtship behavior of androgen-implanted males, but not of unimplanted castrates. Plasma androgen concentration of androgen-implanted castrates (as determined by radioimmunoassay) was maintained at a level typical of intact males at the peak of the breeding season. Because the AVT-androgen interaction could occur at the level of the pituitary gland, castrated, androgen-implanted newts were hypophysectomized and injected with AVT. Hypophysectomy did not abolish the behavioral response to AVT.     

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.     

Male indifference to female traits in an arctiid moth (Utetheisa ornatrix)

Abstract.  1. Female Utetheisa ornatrix (Lepidoptera: Arctiidae) mate selectively with large males able to transmit sizeable quantities of nutrient and defensive pyrrolizidine alkaloid with the spermatophore. The female gauges male size indirectly by assessment of the male's courtship pheromone.
2. Male Utetheisa invest upward of 10% of body mass in spermatophore production, and could therefore have been expected to be choosy; however, when offered females differing in alkaloid content, body mass, or mating status, males showed disregard of these parameters, both in their choice of partner and in their allocation of resources to the spermatophore.
3. It is concluded that Utetheisa males do not have the option to select females by comparison shopping . Females broadcast their attractant pheromone for less than an hour per day. Given this time constraint and the potentially high cost of mate localisation, males may have no choice but to mate on an opportunistic basis.     

Exogenous vasotocin alters aggression during agonistic exchanges in male Amargosa River pupfish (Cyprinodon nevadensis amargosae)

Pupfishes in the Death Valley region have rapidly differentiated in social behaviors since their isolation in a series of desert streams, springs, and marshes less than 20,000 years ago. These habitats can show dramatic fluctuations in ecological conditions, and pupfish must cope with the changes by plastic physiological and behavioral responses. Recently, we showed differences among some Death Valley populations in brain expression of arginine vasotocin (AVT). As AVT regulates both hydromineral balance and social behaviors in other taxa, these population differences may indicate adaptive changes in osmoregulatory and/or behavioral processes. To test whether AVT is relevant for behavioral shifts in these fish, here we examined how manipulations to the AVT system affect agonistic and reproductive behaviors in Amargosa River pupfish (Cyprinodon nevadensis amargosae). We administered exogenous AVT (0.1, 1, and 10 microg/g body weight) and an AVP V1 receptor antagonist (Manning compound, 2.5 microg/g body weight) intraperitoneally to males in mixed-sex groups in the laboratory. We found that AVT reduced the initiation of aggressive social interactions with other pupfish but had no effect on courtship. The effects of AVT were confirmed in males in the wild where AVT (1 microg/g body weight) reduced the aggressive initiation of social interactions and decreased aggressive responses to the behavior of other males. Combined, these results show that AVT can modulate agonistic behaviors in male pupfish and support the idea that variation in AVT activity may underlie differences in aggression among Death Valley populations.