Darcy B. Kelley

Historical perspective: Hormonal regulation of behaviors in amphibians

This review focuses on research into the hormonal control of behaviors in amphibians that was conducted prior to the 21st century. Most advances in this field come from studies of a limited number of species and investigations into the hormonal mechanisms that regulate reproductive behaviors in male frogs and salamanders. From this earlier research, we highlight five main generalizations or conclusions. (1) Based on studies of vocalization behaviors in anurans, testicular androgens induce developmental changes in cartilage and muscles fibers in the larynx and thereby masculinize peripheral structures that influence the properties of advertisement calls by males. (2) Gonadal steroid hormones act to enhance reproductive behaviors in adult amphibians, but causal relationships are not as well established in amphibians as in birds and mammals. Research into the relationships between testicular androgens and male behaviors, mainly using castration/steroid treatment studies, generally supports the conclusion that androgens are necessary but not sufficient to enhance male behaviors. (3) Prolactin acts synergistically with androgens and induces reproductive development, sexual behaviors, and pheromone production. This interaction between prolactin and gonadal steroids helps to explain why androgens alone sometimes fail to stimulate amphibian behaviors. (4) Vasotocin also plays an important role and enhances specific types of behaviors in amphibians (frog calling, receptivity in female frogs, amplectic clasping in newts, and non-clasping courtship behaviors). Gonadal steroids typically act to maintain behavioral responses to vasotocin. Vasotocin modulates behavioral responses, at least in part, by acting within the brain on sensory pathways that detect sexual stimuli and on motor pathways that control behavioral responses. (5) Corticosterone acts as a potent and rapid suppressor of reproductive behaviors during periods of acute stress. These rapid stress-induced changes in behaviors use non-genomic mechanisms and membrane-associated corticosterone receptors.

Molecular cloning of androgen receptors from divergent species with a polymerase chain reaction technique: Complete cDNA sequence of the mouse androgen receptor and isolation of androgen recepter cDNA probes from dog, guinea pig and clawed frog

We have cloned and sequenced 2.8 kilobases of cDNA encoding the mouse androgen receptor by RNA amplification with transcript sequencing. Sequence analysis predicts that this cDNA contains an open reading frame of 2697 nucleotides encoding a polypeptide of 899 amino acids. Androgen receptor cDNA probes of dog, guinea pig, and frog were also isolated and sequenced using consensus primers derived from human and rat androgen receptor cDNAs. Northern blot analysis with the species-specific probes revealed similarities in size between amphibian and mammalian mRNAs. These results demonstrate the utility of this technique in obtaining nucleic acid probes and sequence information of steroid receptors from different species. The sequence data and the Northern blot analysis of the receptors in different species demonstrate that the androgen receptor has been well-conserved during evolution.

Vocal communication in frogs

The robust nature of vocal communication in frogs has long attracted the attention of natural philosophers and their biologically inclined successors. Each frog species produces distinctive calls that facilitate pre-mating reproductive isolation and thus speciation. In many terrestrial species, a chorus of simultaneously calling males attracts females to breeding sites; reproductive females then choose and locate one male, using distinctive acoustic cues. Males compete with each other vocally and sometimes physically as well. Anuran acoustic signaling systems are thus subject to the strong pressures of sexual selection. We are beginning to understand the ways in which vocal signals are produced and decoded by the nervous system and the roles of neurally active hormones in both processes.

Androgen-induced myogenesis and chondrogenesis in the larynx of Xenopus laevis☆

We investigated a possible role for testosterone-induced cell proliferation in the development of sexual dimorphism in the larynx of South African clawed frogs, Xenopus laevis. Androgen-induced cell proliferation was studied using [3H]thymidine autoradiography. Nuclei of cartilage, perichondrium, and muscle were labeled in the larynx of sexually immature frogs of both sexes but not in adults. Cell proliferation did not occur with estradiol treatment nor was it seen in nonlaryngeal muscle or cartilage. Electron microscopic/autoradiographic studies of laryngeal muscle indicate that testosterone stimulates satellite cell division which later results in formation of myonuclei. We conclude that testosterone induces both chondrogenesis and myogenesis in juvenile larynx and that this process may contribute to the pronounced sexual dimorphism of the adult vocal organ.

Development and hormone regulation of androgen receptor levels in the sexually dimorphic larynx of Xenopus laevis

Development of the sexually dimorphic larynx in African clawed frogs is controlled by secretion of androgenic steroids (D. Sassoon and D. Kelley, 1986, Amer. J. Anat. 177, 457-472). Adult laryngeal muscle shows high levels of androgen binding relative to other skeletal muscles and binding activity in males is three times that in females (N. Segil, L. Silverman, and D. Kelley, 1987, Gen. Comp. Endocrinol. 66, 95-101). To determine when androgen sensitivity and sex differences arise, we assayed [3H]dihydrotestosterone (DHT) binding activity in larynges from metamorphic and postmetamorphic male and female frogs. Scatchard analyses indicate that DHT binds to a saturable component with high affinity. At metamorphosis, male and female juveniles have average binding levels of 262 and 269 fmoles/mg protein, respectively, approximately 7 to 20 times their adult values. At 3 months postmetamorphosis (PM), sexually dimorphic binding levels are observed. Binding activity declines gradually in females from metamorphosis to 9 months PM. In males, levels of binding activity remain high throughout the first 6 months PM and then decrease to near adult levels by 9 months PM. Administration of exogenous DHT to 3 months PM juveniles decreases average binding activity from 180 (male) or 74 fmoles/mg (female) to 33.5 fmoles/mg in both sexes. Testosterone has a less pronounced effect on binding activity in males than DHT and is ineffective in females. We conclude that sexually dimorphic adult levels of androgen binding in larynx arise by differential decrease from initially high, sexually monomorphic levels and that high titers of circulating androgens normally present by 6 months PM in males are responsible for the marked decrease in binding activity observed during laryngeal development.

Androgen receptor mRNA expression in Xenopus laevis CNS: Sexual dimorphism and regulation in laryngeal motor nucleus

Using Northern analysis, in situ hybridization, and nuclease protection assays, the expression and regulation of androgen receptor messenger RNA (AR mRNA) was examined in the CNS of juvenile Xenopus laevis. Only one of the AR mRNA isoforms expressed in X. laevis is transcribed in the CNS as shown by Northern blot analysis. Nuclease protection assays demonstrate that the expression of AR mRNA is higher in the brain stem than in the telencephalon and diencephalon. Although expression of AR mRNA is widespread throughout the CNS, cells of cranial nerve nucleus IX-X (N.IX-X) and spinal cord display the highest in situ hybridization signals in their cytoplasm. Double labeling using horseradish peroxidase and digoxigenin labeled AR probes reveals that laryngeal and anterior spinal cord motor neurons express AR mRNA. More cells express AR mRNA in N.IX-X of males than of females. The number of AR expressing cells in N. IX-X decreases following gonadectomy in both sexes, and dihydrotestosterone (DHT) treatment for 1 month reverses this effect. Increased expression of AR mRNA in the brain of DHT treated animals is also apparent in nuclease protection assays. Sex differences in number of AR expressing cells and hormone regulation of AR mRNA expression in motor nuclei may influence neuromuscular systems devoted to sexually differentiated behaviors.

Direct action of gonadotropin in brain integrates behavioral and reproductive functions

Essential roles for gonadotropins in gonadal development and reproduction are well established. Over the past decade, however, the expression of luteinizing hormone receptor (LHR) has also been reported in the brain of various mammals and birds. Although suggestive, it has not yet been determined whether this expression pattern supports a novel function for gonadotropins. Here, we demonstrate a CNS-mediated role of gonadotropins in a reproductive behavior: the courtship songs of the South African clawed frog, Xenopus laevis. Male advertisement calling in this species depends on a nongonadal action of gonadotropin. To determine whether this effect is due to action on the CNS, we administered gonadotropin intracerebroventricularly (ICV) or systemically to intact or castrated males with or without concomitant androgen replacement. In intact and androgen-replaced gonadectomized males, gonadotropin significantly increased calling within 1 h after ICV injection. The effective dosage via ICV injections was less than one hundredth of the effective systemic dose. In situ hybridization with a cloned fragment of Xenopus LHR revealed strong expression in ventral forebrain areas important for vocal control. Further, gonadotropin treatment of brain in vitro up-regulates immunoreactivity for the LHR downstream target, egr-1, specifically in these vocal forebrain areas. Up-regulation occurs even when synaptic transmission is suppressed by incubation in Ca2+ free/high magnesium saline. These results demonstrate a neural role for gonadotropin in the control of calling behavior, potentially mediated via LHRs in forebrain vocal nuclei. Gonadotropin may play a novel integrative role in modulating both reproductive physiology and behavior.

Auditory and Lateral Line Inputs to the Midbrain of an Aquatic Anuran; Neuroanatomic Studies in Xenopus laevis

Computation of rate in auditory signals is essential to call recognition in anurans. This task is ascribed to a group of central nervous sytem nuclei in the dorsal midbrain or torus semicircularis, homologous to the inferior colliculus of mammals. We have mapped the connections of the subnuclei of the torus semicircularis in Xenopus laevis to determine which receive auditory and which receive lateral line information. Relative to terrestrial anurans, the torus of X. laevis is hypertrophied and occupies the entire caudal, dorsal midbrain. Auditory input to the torus, that arising directly from the dorsal medullary nucleus, is present only in the laminar nucleus. The principal and magnocellular nuclei receive their input from the lateral line nucleus of the medulla. All three nuclei of the torus also have reciprocal connections with the superior olive and the nucleus of the lateral lemniscus. Ascending efferents from all three nuclei of the torus innervate central and lateral thalamic nuclei, and all have a weak reciprocal connection with the posterior thalamus. The laminar and magnocellular nuclei have reciprocal connections with the ventral thalamus, and all three nuclei of the torus receive descending input from the anterior entopeduncular nucleus. The laminar and magnocellular nuclei also receive descending input from the preoptic area. Based on our identification of toral nuclei and these results we assign a major function for the detection of water‐borne sounds to the laminar nucleus and a major function for the detection of near field disturbances in water pressure to the principal and magnocellular nuclei. J. Comp. Neurol. 438:148–162, 2001.

androgen-binding levels in a sexually dimorphic muscle of Xenopus laevis

The larynx of adult South African clawed frogs, Xenopus laevis, is larger in males than in females and hypertrophies in adult females and juveniles in response to androgen. Sexual dimorphism and androgen sensitivity suggest that the larynx is a testosterone target tissue. Saturation analysis of androgen (R1881) binding in laryngeal cytosol revealed an approximately threefold quantitative difference between male and female androgen-binding levels (36.4 vs 11.5 fm/mg protein). By contrast, as measured by one-point assay, androgen-binding levels in thigh muscle of either males or females were between 0 and 4 fm/mg protein with no apparent sex difference. Competition studies indicated that dihydrotestosterone was the most effective competitor for R1881 binding activity in the larynx. Saturation analysis showed the binding activity to be saturable and of high affinity (apparent Kd 0.46 nM in the male and 0.38 nM in the female). After 1 month of testosterone treatment, female binding levels averaged 16.6 fm/mg protein with a Kd of 0.49 nM, within the range for normal females. In males castrated for 4 months, binding levels were 52 fm/mg protein. After 1 year of castration, binding levels were 25 fm/mg protein. We conclude that laryngeal muscle is an androgen target tissue with sexually dimorphic levels of binding in adults.

Sexually dimorphic expression of a laryngeal-specific, androgen-regulated myosin heavy chain gene during Xenopus laevis development

Masculinization of the larynx in Xenopus laevis frogs is essential for the performance of male courtship song. During postmetamorphic (PM) development, the initially female-like phenotype of laryngeal muscle (slow and fast twitch fibers) is converted to the masculine form (entirely fast twitch) under the influence of androgenic steroids. To explore the molecular basis of androgen-directed masculinization, we have isolated cDNA clones encoding portions of a new Xenopus myosin heavy chain (MHC) gene. We have detected expression of this gene only in laryngeal muscle and specifically in males. All adult male laryngeal muscle fibers express the laryngeal myosin (LM). Adult female laryngeal muscle expresses LM only in some fibers. Expression of LM during PM development was examined using Northern blots and in situ hybridization. Males express higher levels of LM than females throughout PM development and attain adult levels by PM3. In females, LM expression peaks transiently at PM2. Treatment of juvenile female frogs with the androgen dihydrotestosterone masculinizes LM expression. Thus, LM appears to be a male-specific, testosterone-regulated MHC isoform in Xenopus laevis. The LM gene will permit analysis of androgen-directed sexual differentiation in this highly sexually dimorphic tissue.