Sex and seasonal differences in mRNA expression of estrogen receptor α (ESR1) in red-sided garter snakes (Thamnophis sirtalis parietalis).

Estrogens are important regulators of reproductive physiology including sexual signal expression and vitellogenesis. For the regulation to occur, the hormone must bind and activate receptors in target tissues, and expression of the receptors can vary by sex and/or season. By simultaneously comparing circulating hormone levels with receptor expression, a more complete understanding of hormone action can be gained. Our study species, the red-sided garter snake (Thamnophis sirtalis parietalis), provides an excellent opportunity to study the interaction between sex steroid hormones and receptor expression in addition to sexual dimorphism and seasonality. During the spring mating season, male garter snakes rely exclusively on the female's skin-based, estrogen-dependent sex pheromone to direct courtship. Males can be stimulated to produce this sexual attractiveness pheromone by treatment with estradiol (E2), which also induces male vitellogenesis. Estrogen receptors (ESRs) are required to transduce the effects of estrogens, thus we used quantitative RT-PCR to analyze expression of ESR alpha (ERα; gene ESR1) mRNA in the skin and liver of wild caught male and female garter snakes across simulated spring and fall conditions in the laboratory. While ESR1 was present in the skin of both sexes, there were no sex or seasonal differences in expression levels. Liver expression of ESR1, however, was sexually dimorphic, with females showing greatest expression in fall when circulating E2 concentrations were lowest. There were no statistically significant correlations between E2 and ESR1 expression. Our data suggest that the skin of both sexes is sensitive to estrogen signaling and thus the production of sex pheromone is dependent on bioavailable levels of E2. Female expression of ESR1 in the liver may increase in the fall to prime energy storage mechanisms required for vitellogenesis the following year.

Testosterone Mediates Seasonal Growth of the Song Control Nuclei in a Tropical Bird.

In mid- to high-latitude songbirds, seasonal reproduction is stimulated by increasing day length accompanied by elevated plasma sex steroid levels, increased singing, and growth of the song control nuclei (SCN). Plasticity of the SCN and song behavior are primarily mediated by testosterone (T) and its metabolites in most species studied thus far. However, the majority of bird species are tropical and have less pronounced seasonal reproductive cycles. We have previously documented that equatorial rufous-collared sparrows (Zonotrichia capensis) exhibit seasonal neuroplasticity in the SCN. Manipulating T in these birds, however, did not alter singing behavior. In the current study, we investigated whether T mediates plasticity of the SCN in a similar manner to temperate songbirds. In the first experiment, we treated captive male birds with T or blank implants during the nonbreeding season. In a second experiment, we treated captive male birds with either blank implants, T-filled implants, T with flutamide (FLU; an androgen receptor antagonist) or T with FLU and 1,4,6-androstatriene-3,17-dione (ATD; an estrogen synthesis inhibitor) during the breeding season. In both experiments, the volumes of the brain areas high vocal center (HVC), Area X, and robust nucleus of the arcopallium (RA) were measured along with singing behavior. In summary, T stimulated growth of HVC and RA, and the combined effect of FLU and ATD reversed this effect in HVC. Area X was not affected by T treatment in either experiment. Neither T-treated birds nor controls sang in captivity during either experiment. Together, these data indicate that T mediates seasonal changes in the HVC and RA of both tropical and higher- latitude bird species even if the environmental signals differ. However, unlike most higher-latitude songbirds, we found no evidence that motivation to sing or growth of Area X are stimulated by T under captive conditions.

Low cost audiovisual playback and recording triggered by radio frequency identification using Raspberry Pi.

Playbacks of visual or audio stimuli to wild animals is a widely used experimental tool in behavioral ecology. In many cases, however, playback experiments are constrained by observer limitations such as the time observers can be present, or the accuracy of observation. These problems are particularly apparent when playbacks are triggered by specific events, such as performing a specific behavior, or are targeted to specific individuals. We developed a low-cost automated playback/recording system, using two field-deployable devices: radio-frequency identification (RFID) readers and Raspberry Pi micro-computers. This system detects a specific passive integrated transponder (PIT) tag attached to an individual, and subsequently plays back the stimuli, or records audio or visual information. To demonstrate the utility of this system and to test one of its possible applications, we tagged female and male tree swallows (Tachycineta bicolor) from two box-nesting populations with PIT tags and carried out playbacks of nestling begging calls every time focal females entered the nestbox over a six-hour period. We show that the RFID-Raspberry Pi system presents a versatile, low-cost, field-deployable system that can be adapted for many audio and visual playback purposes. In addition, the set-up does not require programming knowledge, and it easily customized to many other applications, depending on the research questions. Here, we discuss the possible applications and limitations of the system. The low cost and the small learning curve of the RFID-Raspberry Pi system provides a powerful new tool to field biologists.

Variation in the gonadotrophin-releasing hormone-1 and the song control system in the tropical breeding rufous-collared sparrow (Zonotrichia capensis) is dependent on sex and reproductive state.

Seasonal breeding in temperate zone vertebrates is characterised by pronounced variation in both central and peripheral reproductive physiology as well as behaviour. In contrast, many tropical species have a comparatively longer and less of a seasonal pattern of breeding than their temperate zone counterparts. These extended, more "flexible" reproductive periods may be associate with a lesser degree of annual variation in reproductive physiology. Here we investigated variation in the neuroendocrine control of reproduction in relation to the changes in the neural song control system in a tropical breeding songbird the rufous-collared sparrows (Zonotrichia capensis). Using in situ hybridization, we show that the optical density of GnRH1 mRNA expression is relatively constant across pre-breeding and breeding states. However, males were found to have significantly greater expression compared to females regardless of breeding state. Both males and females showed marked variation in measures of peripheral reproductive physiology with greater gonadal volumes and concentrations of sex steroids in the blood (i.e. testosterone in males; estrogen in females) during the breeding season as compared to the pre-breeding season. These findings suggest that the environmental cues regulating breeding in a tropical breeding bird ultimately exert their effects on physiology at the level of the median eminence and regulate the release of GnRH1. In addition, histological analysis of the song control system HVC, RA and Area X revealed that breeding males had significantly larger volumes of these brain nuclei as compared to non-breeding males, breeding females, and non-breeding females. Females did not exhibit a significant difference in the size of song control regions across breeding states. Together, these data show a marked sex difference in the extent to which there is breeding-associated variation in reproductive physiology and brain plasticity that is dependent on the reproductive state in a tropical breeding songbird.

The relationship between plasma steroid hormone concentrations and the reproductive cycle in the Northern Pacific rattlesnake, Crotalus oreganus.

We describe the reproductive cycle of Northern Pacific rattlesnakes (Crotalus oreganus) by quantifying steroid hormone concentrations and observing reproductive behaviors in free-ranging individuals. Additionally, we examined reproductive tissues from museum specimens. Plasma steroid hormone concentrations were quantified for both male and female snakes throughout the active season (March-October). We measured testosterone (T), 5alpha-dihydrotestosterone (DHT), and corticosterone (B) concentrations in both sexes and 17beta-estradiol (E2) and progesterone (P) in females only. We observed reproductive behaviors (e.g., consortship, courtship, and copulation) in the field and measured testis and follicle size in male and female snakes from museum collections to relate steroid hormone concentrations to the timing of reproductive events. Our study revealed that C. oreganus in central California exhibits a bimodal pattern of breeding, with most mating behavior occurring in the spring and some incidences of mating behavior observed in late summer/fall. Each breeding period corresponded with elevated androgen (T or DHT) levels in males. Testes were regressed in the spring when the majority of reproductive behavior was observed in this population, and they reached peak volume in August and September during spermatogenesis. Although we did not detect seasonal variation in female hormone concentrations, some females had high E2 in the spring and fall, coincident with mating and with increased follicle size (indicating vitellogenesis) in museum specimens. Females with high E2 concentrations also had high T and DHT concentrations. Corticosterone concentrations in males and females were not related either to time of year or to concentrations of any other hormones quantified. Progesterone concentrations in females also did not vary seasonally, but this likely reflected sampling bias as females tended to be underground, and thus unobtainable, in summer months when P would be expected to be elevated during gestation. In females, P was positively correlated with T and DHT, and E2 was positively correlated with T.

Effects of testosterone on morphology, performance and muscle mass in a lizard.

Because sexual selection pressures are high in sexually dimorphic organisms, morphological, physiological and performance traits are often studied in a sexual selection context. The proximate mechanisms underlying evolutionary change in these traits, however, remain largely unstudied. Here, we examined the role of steroids in shaping morphology and physiological performance in males of a sexually dimorphic lizard (Gallotia galloti). We compared morphology and physiological performance of males with experimentally elevated testosterone levels to sham-operated males. Before surgery, inter-individual variation in plasma testosterone levels correlated positively with bite force capacity. Administration of exogenous testosterone resulted in an increase of the mass of both jaw closing and locomotory muscles compared with sham-operated individuals, but the responsiveness varied considerably among muscle groups. In contrast to our expectations, the dramatic testosterone-induced changes in muscle masses did not result in concordant changes in bite force performance or sprint speed.

Hormonal response of male green anole lizards (Anolis carolinensis) to GnRH challenge.

Circulating plasma levels of testosterone often differ among social classes of sexually mature males within a population, but the general physiological mechanisms underlying such differences remain unclear. Within sexually mature male green anole lizards (Anolis carolinensis), smaller "lightweight" males have on average relatively smaller heads, lower bite-forces, and lower testosterone levels compared with larger "heavyweight" males. We conducted gonadotropin-releasing hormone (GnRH) challenges on lightweight and heavyweight males to determine if lightweight males were capable of producing comparable levels of circulating testosterone to heavyweight males but are socially or physiologically suppressed from doing so. We challenged lightweight and heavyweight males with chicken I and II GnRH and measured their resulting levels of testosterone and corticosterone. Neither lightweights nor heavyweights increased circulating testosterone levels after GnRH challenge, suggesting they are already at maximal production levels, consistent with the Challenge Hypothesis. Instead, testosterone levels tended to decrease and corticosterone levels increased, most likely owing to the stress response associated with handling. Our results are dramatically different from GnRH challenges conducted in bird species, suggesting that more field studies are needed in reptilian systems.

Steroid hormones act transsynaptically within the forebrain to regulate neuronal phenotype and song stereotypy.

Steroid sex hormones induce dramatic seasonal changes in reproductive related behaviors and their underlying neural substrates in seasonally breeding vertebrates. For example, in adult white-crowned sparrows, increased Spring photoperiod raises circulating testosterone, causing morphological and electrophysiological changes in song-control nuclei, which modify song behavior for the breeding season. We investigated how photoperiod and steroid hormones induce these changes in morphology, electrophysiology, and behavior. Neurons in a song premotor nucleus, the robust nucleus of the arcopallium (RA), show increased intrinsic spontaneous firing rate and soma size when birds are in breeding condition. Using combinations of systemic and unilateral local intracerebral hormonal manipulations, we show that long-day photoperiod accelerates the effects of systemic testosterone on RA neurons via the estradiol-synthesizing enzyme aromatase (CYP19A1); these changes require inputs from the afferent song control nucleus HVC (used as a proper name) and steroid receptor activation within HVC; local coactivation of androgen and estrogen receptors (ARs and ERs, respectively) within HVC, but not RA, is sufficient to cause neuronal changes in RA; activation of ARs in RA is also permissive. Using bilateral local intracerebral hormone-receptor blockade, we found that ARs and ERs in the song-control nucleus HVC mediate systemic testosterone-induced changes in song stereotypy but not rate. This novel transsynaptic effect of gonadal steroids on activity and morphology of RA neurons is part of a concerted change in key premotor nuclei, enabling stereotyped song.

Interactions of gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH) in birds and mammals.

Gonadotropin-releasing hormone (GnRH) regulates secretion of both of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone. Thus, it is a key hormone for vertebrate reproduction. GnRH was considered to be unusual among hypothalamic neuropeptides in that it appeared to have no direct antagonist, although some neurochemicals and peripheral hormones (opiates, GABA, gonadal steroids, inhibin) can modulate gonadotropin release to a degree. Five years ago, a vertebrate hypothalamic neuropeptide that inhibited pituitary gonadotropin release in a dose-dependent manner was discovered in quail by Tsutsui et al. (2000. Biochem Biophys Res Commun 275:661-667). We now know that this inhibitory peptide, named gonadotropin-inhibitory hormone, or GnIH, is a regulator of gonadotropin release in vitro and in vivo. Its discovery has opened the door to an entirely new line of research within the realm of reproductive biology. In our collaborative studies, we have begun to elucidate the manner in which GnIH interacts with GnRH to time release of gonadotropins and thus time reproductive activity in birds and mammals. This paper reviews the distribution of GnIH in songbirds relative to GnRHs, and our findings on its modes of action in vitro and in vivo, based on laboratory and field studies. These data are simultaneously compared with our findings in mammals, highlighting how the use of different model species within different vertebrate classes can be a useful approach to identify the conserved actions of this novel neuropeptide, along with its potential importance to vertebrate reproduction.

Photoperiod-independent changes in immunoreactive brain gonadotropin-releasing hormone (GnRH) in a free-living, tropical bird.

Timing of seasonal reproduction in high latitude vertebrates is generally regulated by photoperiodic cues. Increasing day length in the spring is associated with changes in the brain that are responsible for mediating reproductive activities. A primary example of this is the increased content of gonadotropin-releasing hormone (GnRH) in the preoptic area of the hypothalamus in birds as they enter the spring breeding season. Increased GnRH activity stimulates the release of luteinizing hormone and follicle-stimulating hormone from the anterior pituitary. These gonadotropins induce growth of the gonads and release of sex steroids which act on the brain to mediate reproductive behaviors. By contrast, seasonal breeding in the tropics can occur in the absence of significant changes in photoperiod. To our knowledge, no studies have investigated whether seasonal breeding in free-living tropical vertebrates is associated with seasonal changes in the GnRH system. We studied two populations of rufous-collared sparrows (Zonotrichia capensis) at the equator, separated by only 25 km, but with asynchronous reproductive phenologies associated with local climate and independent of photoperiodic cues. We collected brains and measured GnRH immunoreactivity (GnRH-ir) during each population's breeding and non-breeding periods. Breeding males had larger, but not more, GnRH-ir cells than non-breeding birds. The plasticity of the GnRH system was associated with local climate, such that the two populations exhibited asynchronous changes in GnRH-ir despite experiencing identical photoperiod conditions. Our results demonstrate that tropical birds can exhibit neural changes similar to those exhibited in higher latitude birds. However, these tropical populations appear to be using supplementary cues (e.g., rainfall, temperature, food availability) in a similar way to higher latitude species using an initial predictive cue (photoperiod). These results raise questions about the evolution of reliance upon photoperiodism and the strength of reproductive responses to other environmental cues in congeners from higher latitudes. The ability to respond to a multitude of environmental cues might be part of the ancestral condition, and the subsequent obligate photoperiodism in high-latitude congeners could reflect a loss of flexibility in response to environmental signals.

Seasonal-like plasticity of spontaneous firing rate in a songbird pre-motor nucleus.

Many animals exhibit seasonal changes in behavior and its underlying neural substrates. In seasonally breeding songbirds, the brain nuclei that control song learning and production undergo substantial structural changes at the onset of each breeding season, in association with changes in song behavior. These changes are largely mediated by photoperiod-dependent changes in circulating concentrations of gonadal steroid hormones. Little is known, however, about whether changes in the electrophysiological activity of neurons accompany the dramatic morphological changes in the song nuclei. Here we induced seasonal-like changes in the song systems of adult white-crowned sparrows and used extracellular recording in acute brain slices from those individuals to study physiological properties of neurons in the robust nucleus of the arcopallium (RA), a pre-motor nucleus necessary for song production. We report that: RA neurons from birds in breeding condition show a more than twofold increase in spontaneous firing rate compared to those from nonbreeding condition; this change appears to require both androgenic and estrogenic actions; and this change is intrinsic to the RA neurons. Thus, neurons in the song circuit exhibit both morphological and physiological adult seasonal plasticity.

Evidence for a novel gonadotropin-releasing hormone in hypothalamic and forebrain areas in songbirds.

One or more of three distinct forms of gonadotropin-releasing hormones (GnRHs), has been found in all vertebrates studied. Birds are known to contain two types of GnRH, chicken GnRH-I and -II (cGnRH-I and -II), but to date nobody has investigated the possibility of the presence of lamprey GnRH-III in birds. Thus, our objective was to investigate the presence and distribution of immunoreactive lamprey (ir-lamprey) GnRH-III in hypothalamic and forebrain areas in house sparrows (Passer domesticus) and Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii). In addition to its distribution in the CNS, we investigated whether or not exogenous lamprey GnRH-III peptide could elicit luteinizing hormone (LH) release in vivo. This study presents two novel findings: 1) The widespread presence in the central nervous system of an immunoreactive third GnRH in birds that is clearly hypophysiotropic and has gonadotropin-releasing capabilities. Intravenous injection of 100 ng lamprey-GnRH-III peptide elicited a rapid rise in circulating luteinizing hormone as compared to controls. This third GnRH, ir-lamprey GnRH-III, likely has multiple functions, as suggested by its widespread distribution. 2) Ir-lamprey GnRH-III is present in abundance in telencephalic areas, including the hippocampal formation and the song control system. In no vertebrate has a GnRH been localized in these 'higher' control regions before. The fact that ir-lamprey-GnRH-III is present in auditory processing areas (higher vocal center, or HVc, area X, caudomedial neostriatum) and song-producing areas (HVc, robust nucleus of the neostriatum, or RA) suggests a direct way in which auditory processing and song production can affect circulating gonadotropins.

Latitudinal variation in plasma testosterone levels in birds of the genus Zonotrichia.

Birds breeding in northern latitudes generally have elevated plasma testosterone levels throughout the breeding season with a peak at the onset of the breeding season. In contrast, tropical birds tend to have extremely low plasma testosterone levels year round with only a slight increase during breeding. While these patterns have been consistent in the species investigated, closely related species have not been investigated across a range of latitudes. Birds of the genus Zonotrichia present an ideal opportunity to investigate latitudinal variation in plasma testosterone levels as breeding populations occur from northern Alaska to southern Argentina. We studied three taxa of Zonotrichia: (1) Gambel's white-crowned sparrows, Zonotrichia leucophrys gambelii, breeding at high latitude in northern Alaska, (2) Puget Sound white-crowned sparrows, Z. l. pugetensis, breeding at mid-latitude in Washington state, and (3) an equatorial population of the rufous-collared sparrow, Z. capensis, in Ecuador. To compare both baseline breeding and maximal testosterone levels, males from the three taxa were either bled immediately upon capture during the breeding season or first challenged with gonadotropin-releasing hormone (GnRH) and then bled. As a control, another group of birds experienced a saline challenge. Two-way ANCOVA, with treatment and taxa as factors and testis volume as a covariate, showed a significant effect of treatment on plasma testosterone levels. There was also a significant interaction between taxa and treatment. Contrary to expectations, the equatorial species exhibited greater plasma testosterone levels in response to the GnRH challenge than the high latitude species. There were no differences between the mid- and high-latitude species. The equatorial species had the smallest average testis size and within each taxa there were no relationships between plasma testosterone and testis size. These data suggest that latitudinal clines in plasma testosterone levels in Zonotrichia do not follow previously described patterns and that tropical birds can have levels of testosterone similar to northern latitude species.

Steroid hormone interrelationships with territorial aggression in an Arctic-breeding songbird, Gambel's white-crowned sparrow, Zonotrichia leucophrys gambelii.

The breeding season is very brief for arctic-breeding passerines, and any interruptions of parental care by aggressive interactions over territory may reduce reproductive success. We tested both the "testosterone insensitivity" and "corticosterone insensitivity" hypotheses in the arctic-breeding Gambel's white-crowned sparrow, Zonotrichia leucophrys gambelii. Additionally, we tested whether simulated territorial intrusions (STIs), known to stimulate increases in luteinizing hormone (LH) and testosterone (T) in mid-latitude breeding Z. l. pugetensis, would also be effective in either the early or late phases of the brief breeding season of Z. l. gambelii. Plasma levels of T and LH were high early in the breeding season and declined as egg laying began. Exposure of free-living males to 10 min of STI significantly increased LH but not T secretion. Nonetheless, the pituitary-gonadal axis is sensitive as jugular injection of gonadotrophin-releasing hormone increased plasma T at 10 min relative to saline-challenged controls. T implants failed to increase territorial aggression following STI during incubation. These data are consistent with the T insensitivity hypothesis and contrast sharply with the response of the southerly breeding subspecies, Z. l. pugetensis, in which the territorial response to T administration is retained throughout its relatively long breeding season. However, corticosterone implants during the incubation period decreased territorial aggression during STI. This responsiveness to corticosterone is not consistent with the corticosterone insensitivity hypothesis of stress modulation. Z. l. gambelii retain sensitivity to corticosterone levels that may occur naturally in response to environmental perturbations resulting in suppression of territorial behavior.