Development of functional sex differences in the larynx of Xenopus laevis

Three laryngeal properties associated with the production of masculine song--laryngeal muscle tension, fiber twitch type, and fiber recruitment--are markedly sexually dimorphic in adult Xenopus laevis frogs. To elucidate the pattern of sexual differentiation, tension and fiber recruitment in male and female larynges and fiber twitch type in male larynges were examined throughout postmetamorphic development. Masculinization of male laryngeal properties begins early in postmetamorphic development and continues until adulthood. In contrast, tension and fiber recruitment in females do not change after the end of metamorphosis. Laryngeal muscle tension and fiber type are gradually and progressively masculinized; the temporal pattern of masculinization is very similar for these properties. Fiber recruitment, on the other hand, appears to masculinize in a stepwise manner. Masculinization of all three properties is highly correlated with larynx weight in males. We have used this relation to divide postmetamorphic development into seven stages associated with key events in sexual differentiation. This staging scheme provides an important experimental tool for studying the hormonal regulation of sexual differentiation, the subject of the accompanying paper.     

Hormone-sensitive stages in the sexual differentiation of laryngeal muscle fiber number in Xenopus laevis

The number of muscle fibers in the vocal organ of the adult male African clawed frog, Xenopus laevis, exceeds that of adult females. This sex difference is the result of rapid fiber addition in males between the end of metamorphosis, post-metamorphic stage 0 (PM0) and PM2. At PM0, male and female frogs have similar numbers of laryngeal muscle fibers. Males then add more muscle fibers than females and achieve an adult value that is 1.7 times the female number. Males castrated at PM0 have the same fiber number as females. Ovariectomy at PM0 does not alter muscle fiber addition in females. Gonadectomy at PM2 has no effect on fiber addition in either sex. Females attain masculine muscle fiber number if their ovaries are replaced with a testis at metamorphosis. Exogenous testosterone treatment at PM0 significantly increases fiber number in females but not in males. Exogenous testosterone given at PM2 has no effect on fiber number in females but decreases fiber number in males. We conclude that the testes are necessary for the marked addition of laryngeal muscle fibers seen in male X. laevis between PM0 and PM2. The masculine pattern of muscle fiber addition can be induced in females provided with a testis. Androgen secretion from the testes most probably accounts for masculinization of laryngeal muscle fiber number. After PM2, androgens are no longer necessary for muscle fiber addition and cannot increase fiber number in females.     

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.     

Androgen directs sexual differentiation of laryngeal innervation in developing Xenopus laevis

In adult Xenopus laevis, innervation of the vocal organ is more robust in males than in females. This sex difference originates during tadpole development; at stage 56, when the gonads first differentiate, the number of axons entering the larynx is the same in the sexes, but by stage 62, innervation is greater in males. To determine if androgen secretion establishes sex differences in axon number, we treated tadpoles with antiandrogen or androgen beginning at stage 48 or 54 and counted laryngeal nerve axons at stage 62 using electron microscopy. When male tadpoles were treated with the antiandrogen hydroxyflutamide, axon numbers were reduced to female-typical values; axon numbers in females were unaffected by antiandrogen treatment. When female tadpoles were treated with the androgen DHT (dihydrotestosterone), axon numbers were increased to male-like values. These findings suggest that endogenous androgen secretion during late tadpole stages in males is required for the sexual differentiation of laryngeal innervation observed from stage 62 on. Because androgen treatment and laryngeal innervation affect myogenesis in postmetamorphic frogs, numbers of laryngeal dilator muscle fibers were determined for hormonally manipulated tadpoles. At stage 62, vehicle-treated males had more laryngeal axons than females; laryngeal muscle fiber numbers did not, however, differ in the sexes. Both male and female tadpoles, treated from stage 54 with DHT, had more muscle fibers at stage 62 than vehicle-treated controls. Thus, while endogenous androgen secretion during late tadpole stages is subthreshold for the establishment of masculinized muscle fiber numbers, laryngeal myogenesis is androgen sensitive at this time and can be increased by suprathreshold provision of exogenous DHT. A subgroup of tadpoles, DHT treated from stage 54 to 62, was allowed to survive, untreated, until postmetamorphic stage 2 (PM2: 5 months after metamorphosis is complete). Androgen treatment between tadpole stages 54 and 62 does not prevent the ontogenetic decrease in axon numbers characteristic of laryngeal development. In addition, the elevation in stage 62 axon numbers produced by DHT-treatment at late tadpole stages was not associated with elevated numbers of laryngeal muscle fibers at PM2. Juvenile males normally maintain elevated axon numbers (relative to final adult values) through PM2 and the presence of these additional axons may result from-rather than contribute directly to—laryngeal muscle fiber addition. 1994 John Wiley & Sons, Inc.     

Transient masculinization in the fossa, Cryptoprocta ferox (Carnivora, Viverridae)

In at least 9 mammalian species, females are masculinized throughout life, but the benefits of this remain unclear despite decades of thorough study, in particular of the spotted hyaena (Crocuta crocuta) in which the phenomenon has been associated with a high fitness cost. Through examination of wild and captive fossas (Cryptoprocta ferox, Viverridae), androgen assays, and DNA typing for confirmation of gender, we made the first discovery of transient masculinization of a female mammal. Juvenile female fossas exhibited an enlarged, spinescent clitoris supported by an os clitoridis and a pigmented secretion on the underpart fur that in adults was confined to males. These features appeared to diminish with age. The majority of adult females lacked them, and os clitoridis length was inversely related to head-body length. No evidence was found to link this masculinization to elevated female androgen levels. Circulating concentrations of testosterone and androstenedione, but not dihydrotestosterone, were significantly lower in females than in males. No significant differences in testosterone, androstenedione, or dihydrotestosterone levels were found between juvenile (masculinized) and adult (nonmasculinized) females. There are several possible physiological mechanisms for this masculinization. None of the hypotheses so far proposed to explain the evolutionary basis of female masculinization in mammals are applicable to our findings. We present 2 new hypotheses for testing and development.     

Differential effects of prenatal testosterone timing and duration on phenotypic and behavioral masculinization and defeminization of female sheep

The process of sexual differentiation leaves genetically female individuals at risk of being masculinized by exogenous androgens. Previous research with sheep indicates that exposure to excess testosterone from Gestational Day (GD) 30 to GD 90 of the 147-day gestation masculinizes and defeminizes behavior as well as genitalia. Lower doses and shorter durations produce animals with varying degrees of genital virilization and alterations of the hypothalamic-pituitary-gonadal axis, but to our knowledge, the effects on complex behavior and its prediction by the amount of external virilization have not been explored. Previous research in rodents has suggested that sexual differentiation of the central nervous system and the external genitalia can be dissociated. Therefore, we hypothesized that the extent of virilization of external genitalia would not be predictive of the lack of female-typical, or the presence of male-typical, mating behavior. To test this hypothesis, we compared control females, females exposed to exogenous testosterone from GD 30 to GD 90 (T60 females) that have virilized genitalia, and females exposed to testosterone from GD 60 to GD 90 (T30 females) that have female-typical genitalia. Both natural behavioral estrus in the flock and hormonally controlled behavioral tests were used to explore reproductive behavior. The T60 and T30 females exhibited more masculinized reproductive behavior than the controls; however, the T30 females also exhibited feminine behavior. Neither testosterone-treated group was receptive or was mounted at rates comparable to those of controls. These data illustrate that variation in the timing or duration of exposure to prenatal testosterone during a critical period for masculinization can have variable effects on defeminization and that the effects of testosterone on genitalia are not entirely predictive of behavior.     

The effects of peripubertal testosterone on ethylmorphine demethylase activity in adult rats testectomized neonatally

The physiologic activity of ethylmorphine demethylase (EMDM) is 2.5 times greater in adult male rats than in females. Defeminization, effected neonatally by testicular androgens, causes 65% of the increase while masculinization causes the rest. Castration of the intact adult does not alter the defeminized activity of the enzyme but abolishes its masculinized activity. Testosterone restores the latter. Adults that are not defeminized, because of neonatal castration, have enzymes unresponsive to testosterone. Thus, defeminization seems necessary for the expression of masculine activity. This was studied by castrating males at birth (day 1) and implanting capsules of testosterone propionate on day 35. On day 71, the activity had increased 4.4-fold to physiologic levels, compared with rats receiving no steroid (1.9 nmol.min-1.mg-1). Removal of the implant on day 71 decreased the activity on day 84 to that of the no-steroid rats. Thus, defeminization is neither caused by androgen in peripubertal rats nor needed to effect full physiologic activity of the enzyme. The activation of EMDM by peripubertal testosterone may reflect its regulation of growth hormone; first, testosterone (days 35-71) failed to increase enzyme activity in males hypophysectomized on day 35; second, growth hormone infused (5 micrograms.h-1) over days 71-76 blocked the activation, actually decreasing activity 3.7-fold; third, the activity induced by testosterone was further increased (26%) by growth hormone release-inhibiting hormone (5 micrograms.h-1, days 71-76).     

The sexually dimorphic larynx of Xenopus laevis: development and androgen regulation

The aims of this study were to characterize sexual dimorphism in the larynx of adult Xenopus laevis and to determine how sex differences arise during postmetamorphic development. The larger male larynx is a result of greater cell numbers in both cartilage and muscle. The dilator laryngis muscle of the male larynx has 6-7 times more muscle fibers than that of the female. At metamorphosis, the larynx is sexually monomorphic and feminine in phenotype. The DNA content of the male larynx doubles during the first 6 months following metamorphosis; there is no net DNA increase in the female larynx during this time. Both sexes experience a marked increase in laryngeal DNA content and mass between 6 months and adulthood. The number of muscle fibers in the male larynx increases at an average rate of 150 fibers a day during the first 10 months of postmetamorphic development. There is no net change in fiber numbers in the female larynx from metamorphosis to adulthood. Administration of the antiandrogen Flutamide to metamorphic frogs prevents the net addition of laryngeal muscle fibers in males. Thus, we propose that addition of postmetamorphic laryngeal muscle fibers in males is dependent upon the presence of circulating androgens. Exogenous testosterone administration results in an increase in laryngeal mass, DNA content, and cellular proliferation in juvenile frogs. Using [3H]thymidine injections to probe ongoing, as well as testosterone-induced, cell proliferation, we conclude that cellular proliferation is regulated differently in males and females during development. Thus androgen-induced proliferation is one cellular mechanism responsible for the sexual dimorphism observed in adults.     

The role of androgen receptors in the masculinization of brain and behavior: what we've learned from the testicular feminization mutation

Many studies demonstrate that exposure to testicular steroids such as testosterone early in life masculinizes the developing brain, leading to permanent changes in behavior. Traditionally, masculinization of the rodent brain is believed to depend on estrogen receptors (ERs) and not androgen receptors (ARs). According to the aromatization hypothesis, circulating testosterone from the testes is converted locally in the brain by aromatase to estrogens, which then activate ERs to masculinize the brain. However, an emerging body of evidence indicates that the aromatization hypothesis cannot fully account for sex differences in brain morphology and behavior, and that androgens acting on ARs also play a role. The testicular feminization mutation (Tfm) in rodents, which produces a nonfunctional AR protein, provides an excellent model to probe the role of ARs in the development of brain and behavior. Tfm rodent models indicate that ARs are normally involved in the masculinization of many sexually dimorphic brain regions and a variety of behaviors, including sexual behaviors, stress response and cognitive processing. We review the role of ARs in the development of the brain and behavior, with an emphasis on what has been learned from Tfm rodents as well as from related mutations in humans causing complete androgen insensitivity.     

Testicular masculinization of vocal behavior in juvenile female Xenopus laevis reveals sensitive periods for song duration,rate, and frequency spectra

Summary In Xenopus laevis, adult males but not females produce courtship songs comprised of rapid trills. Two experiments were conducted to determine whether male-typical singing could be induced in females. At 6 different juvenile stages, male and female frogs were gonadectomized and implanted with testes, grown to sexual maturity, and tested for vocal behavior. All frogs with functional testicular implants sang; females sang as much as males. The frequency spectra of the clicks within trills were fully masculinized in females implanted at PM0, PM1, and PM2. There were deficiencies in song quality in females implanted late in juvenile life. Females receiving testis implants at PM3, PM4, and PM5 did not produce clicks with masculine spectral qualities. In a concurrent experiment, adult males and females were gonadectomized and implanted with testes or silicone tubes containing testosterone proprionate. When tested for vocal behavior 10 to 15 months after implantation, 8/10 androgen-treated males, 3/12 androgen-treated females, 5/5 testes-implanted males, and 2/4 testes-implanted females sang. The females that did sing spent much less time singing than males. The click rates of females were uniformly slower than males and no female produced clicks with a masculine frequency spectrum. Thus, testicular secretions can induce male-typical singing in females until late in juvenile development. However, females exhibit a progressive decline in vocal potential with increasing age, culminating in an almost complete loss of singing ability by adulthood.Abbreviations FFT fast Fourier transform - ICI inter-click interval - PM post-metamorphic - TP testosterone proprionate     

Effects of prenatal treatment with antiandrogens on luteinizing hormone secretion and sex steroid concentrations in adult spotted hyenas,Crocuta crocuta

Prenatal androgen treatment can alter LH secretion in female offspring, often with adverse effects on ovulatory function. However, female spotted hyenas (Crocuta crocuta), renowned for their highly masculinized genitalia, are naturally exposed to high androgen levels in utero. To determine whether LH secretion in spotted hyenas is affected by prenatal androgens, we treated pregnant hyenas with antiandrogens (flutamide and finasteride). Later, adult offspring of the antiandrogen-treated (AA) mothers underwent a GnRH challenge to identify sex differences in the LH response and to assess the effects of prenatal antiandrogen treatment. We further considered the effects of blocking prenatal androgens on plasma sex steroid concentrations. To account for potential differences in the reproductive state of females, we suppressed endogenous hormone levels with a long-acting GnRH agonist (GnRHa) and then measured plasma androgens after an hCG challenge. Plasma concentrations of LH were sexually dimorphic in spotted hyenas, with females displaying higher levels than males. Prenatal antiandrogen treatment also significantly altered the LH response to GnRH. Plasma estradiol concentration was higher in AA-females, whereas testosterone and androstenedione levels tended to be lower. This trend toward lower androgen levels disappeared after GnRHa suppression and hCG challenge. In males, prenatal antiandrogen treatment had long-lasting effects on circulating androgens: AA-males had lower T levels than control males. The sex differences and effects of prenatal antiandrogens on LH secretion suggest that the anterior pituitary gland of the female spotted hyena is partially masculinized by the high androgen levels that normally occur during development, without adverse effects on ovulatory function.     

Digit Ratio (2D∶4D) Differences between 20 Strains of Inbred Mice

The second to fourth digit ratio (2D∶4D) is sexually differentiated in a variety of species, including humans, rats, birds, and lizards. In humans, this ratio tends to be lower in males than in females. Lower digit ratios are believed to indicate increased prenatal testosterone exposure, and are associated with more masculinized behavior across a range of traits. The story seems more complicated in laboratory mice. We have previously shown that there is no sex difference in the digit ratios of inbred mice, but found behavioral evidence to suggest that higher 2D∶4D is associated with more masculinized behaviors. Work examining intrauterine position effects show that neighbouring males raise pup digit ratio, suggesting again that higher digit ratios are associated with increased developmental androgens. Other work has suggested that masculinization is associated with lower digit ratios in lab mice. Here, we examine the fore- and hindlimb digit ratios of 20 inbred mouse strains. We find large inter-strain differences, but no sexual dimorphism. Digit ratios also did not correlate with mice behavioral traits. This result calls into question the use of this trait as a broadly applicable indicator for prenatal androgen exposure. We suggest that the inbred mice model presents an opportunity for researchers to investigate the genetic, and gene-environmental influence on the development of digit ratios.     

Antiandrogen blocks estrogen-induced masculinization of the song system in female zebra finches

Song behavior and the neural song system that serves it are sexually dimorphic in zebra finches. In this species, males sing and females normally do not. The sex differences in the song system include sex differences in the proportion of neurons that express androgen receptors, which is higher in specific brain regions of males. Estradiol (E2) administered in early development profoundly masculinizes the song system of females, including the proportion of neurons expressing androgen receptors. We examined whether or not the expression of these androgen receptors was causally related to the E2-induced masculinization of this system by co-administering Flutamide, which blocks androgen action at the receptor, along with E2 at hatching. E2 alone had its usual masculinizing effect on the female song system, measured in adulthood: increasing the size of song nuclei, the size of neurons in HVC, RA, and 1MAN, and the number of neurons in HVC. E2's masculinizing action, however, was significantly diminished on all measures by co-administering Flutamide. Indeed, females receiving both E2 and Flutamide were never significantly more masculine than controls on any measure. Flutamide alone had no effect. Our results strongly suggest that the activation of androgen receptors is necessary for the E2-induced masculinization of the song system in females.     

Masculinized female yellow-bellied marmots initiate more social interactions

The presence of male siblings in utero influences female morphology and life-history traits because testosterone transferred among foetuses may masculinize females. Similarly, litter sex composition might alter the display of sexually dimorphic behaviour, such as play and allogrooming, since they are modulated by androgens. We explored whether masculinization alters the frequency of play and sociopositive behaviour in female yellow-bellied marmots (Marmota flaviventris). We found that masculinized juvenile females were more likely to initiate play and allogrooming, but yearling females exhibited higher levels of oestrogen-modulated sociopositive behaviours. Additionally, the more they interacted, the greater number of different partners they interacted with. Our results suggest that masculinization increases the rate of age-dependent social behaviour. This probably works by increasing exploration that predisposes individuals to higher encounter rates. Further support comes from previous findings showing that masculinized females were more likely to disperse. Our study stresses the importance of considering litter sex composition as a fitness modulator.     

Neonatal neural organizing effects of exogenous corticosteroids on sexual differentiation of the brain in the female rat

Testosterone, deoxycorticosterone, or vehicle was administered neonatally to female Long-Evans rats. Parameters expressing the reproductive physiology and behavior of the adult animals were studied. It was found that neonatal administration of testosterone produced the expected "defeminization" and "masculinization" of the brain, affecting both the reproductive behavior and cyclicity of these females. In contrast, neonatal administration of the adrenal steroid did not affect cyclicity although it "defeminized" and "masculinized" sexual behavior, albeit to a lesser degree than testosterone. The results suggest a dichotomy in the neuroregulation of reproductive physiology and sexual behavior.     

Sexual dimorphism and androgen regulation of satellite cell population in differentiating rat levator ani muscle

The bulbocavernosus (BC) and levator ani (LA) muscles of rats show remarkable androgen-dependent sexual dimorphism. These muscles are additionally of interest because they are thought to indirectly mediate sexual differentiation of innervating spinal motoneurons. This sexual differentiation of the BC/LA is thought to be due to an increase in muscle units in the male rat during the first week after birth. We examined the cellular basis of this differentiation by studying satellite cells in the LA of postnatal day 2.5 rats, when sexual dimorphism is already prominent. Two experiments were performed in which LA satellite cells were measured: (1) wild-type (WT) males were compared with females and to Tfm androgen receptor mutant males, which are androgen insensitive despite producing masculine amounts of testosterone, and (2) females treated prenatally and/or postnatally with testosterone proprionate were compared with females receiving vehicle injections. Our results indicate that WT males have a larger LA and a greater number of satellite cells in the LA muscle than females or Tfm males. However, satellite cell density was similar for all three groups. Prenatal testosterone treatment masculinized LA size and resulted in a corresponding increase in satellite cell populations, while postnatal TP treatment resulted in a tendency for increased satellite cell density without a significant increase in LA size. Taken together, these studies indicate that satellite cells in the neonatal LA muscle are sexually dimorphic, and that this dimorphism likely results from perinatal actions of androgens on androgen receptors.     

Laryngeal muscle and motor neuron plasticity in Xenopus laevis: Testicular masculinization of a developing neuromuscular system

In Xenopus laevis, the sexual differentiation of the neuromuscular system responsible for courtship song is controlled by testicular androgen secretion. To explore the sensitivity of this system to androgenic stimulation, male and female frogs were gonadectectomized and given testis transplants at seven different developmental stages between the end of metamorphosis and adulthood, grown to sexual maturity, and the laryngeal muscle fibers and motor axons were counted. Muscle fiber and axon numbers in males were not affected by the testicular transplant at any stage. In females, testicular transplants at all developmental stages increased muscle fiber numbers in adulthood. Values attained were, however, significantly less than those of adult intact or testis-transplanted males. Testis transplantation increased laryngeal axon numbers in females to levels equivalent to those of intact males; this effect was obtained at every stage of postmetamorphic development including adulthood. To further explore androgen regulation in adults, males and females were gonadectomized and implanted with silicone tubes containing testosterone propionate for 1.5–3 years and laryngeal muscle fibers and axon numbers compared to those of gonadectomized or sham-operated adult controls. Neither treatment with exogenous androgen nor gonadectomy had any effect on laryngeal muscle fiber or axon number in either males or females; values did not differ from those of sham-operated controls. We conclude that testicular secretions can induce laryngeal muscle fiber and axon addition in females throughout postmetamorphic life. This degree of plasticity, exhibited after the period when adult values are normally attained, stands in contrast to the effects of administration of synthetic androgen and suggests that the degree of plasticity in adult females may be underestimated if exogenous hormones rather than testicular transplants are provided. © 1993 John Wiley & Sons, Inc.     

Transient genital abnormalities in striped hyenas (Hyaena hyaena)

The highly masculinized genitalia of female spotted hyenas Crocuta crocuta is unique among mammals: Crocuta have no external vagina so urination, penile intromission and parturition take place through the clitoris, which mimics a fully erectile male penis. Among hyenids, virilization of external female genitalia has previously been observed only in Crocuta, so functional explanations of masculinization have focused on aspects of social ecology unique to the species. Here we first show that the striped hyena Hyaena hyaena exhibits both unusual similarity in male and female androgen concentrations and transient genital anomalies characterized by a convergence in genital appearance among young males and females. We then evaluate hypotheses regarding the evolution of genital masculinization in the Hyaenidae and other taxa. Hyaena are behaviorally solitary, so discovery of unusual genital development patterns in this species does not support any current evolutionary models for masculinization in Crocuta, which all rely on the trait originating within a highly social species. Some hypotheses can be modified so that masculinization in Crocuta represents an extreme elaboration of a preexisting trait, shared as a homology with Hyaena.     

Sex difference in whole-body androgen content in rats on fetal days 18 and 19 without evidence that androgen passes from males to females

The whole-body content of androgen (testosterone + 5 alpha-dihydrotestosterone) was significantly higher on Fetal Days 18 and 19 in male than in female rats; androgen content was equivalent in the two sexes at other fetal ages, including Days 16, 17, 20, and 21, and prior to parturition on Fetal Day 22. These results partially corroborate previous data of Weisz and Ward (Endocrinology 1980; 106:306-316), who measured testosterone in pooled plasma from rat fetuses and suggest that androgens contribute to masculine brain sexual differentiation only briefly during fetal life. No significant differences in whole-body androgen content were observed among groups of females situated in utero between 0, 1, or 2 males on each side (contiguous male model) or among groups of females with 0, 1, or 2 or more males located caudally (on the cervical side) in the same uterine horn, regardless of whether combined data from Fetal Days 17-22 or only Fetal Days 18 and 19 were considered. These results provide no evidence that androgens from males reach female fetuses in the same uterine horn.     

Neuroeffectors for vocalization in Xenopus laevis: hormonal regulation of sexual dimorphism

South African clawed frogs use sex-specific vocalizations during courtship. In the male, vocalizations are under the control of gonadal androgen. Though females have moderate levels of circulating androgen, they do not give male-typical mate calls. Both muscles of the vocal organ and neurons of the central nervous system (CNS) vocal pathway are sexually dimorphic and androgen-sensitive. Recent studies suggest that the failure of androgen to masculinize adult females results from a male-specific, androgen-regulated developmental program. At metamorphosis the larynx is sexually monomorphic and feminine in morphology, muscle fiber number and androgen receptor content. During the next six months, under the influence of increasing androgen titers and high receptor levels, myoblasts proliferate in the male and muscle fibers increase at an average rate of 100/day. Females have much lower hormone levels, receptor values decline and they display no net addition of fibers. At metamorphosis, both males and females have approximately 4000 muscle fibers. By adulthood, males have eight times the female fiber number. In the CNS, adult laryngeal motor neurons are more numerous with larger somata and dendritic trees in males than in females. Certain connections of neurons in the vocal pathway are also less robust in females. Unlike the periphery, motor neuron number does not appear to be established by androgen-induced proliferation. Our current hypothesis is that androgen acts at the level of laryngeal muscle to produce more muscle fibers and thus provide more target for motor neurons in the male. This process could regulate cell number by ontogenetic cell death. In the CNS, androgen-target neurons become capable of accumulating hormone shortly before metamorphosis. Androgen receptor in laryngeal motor neurons may permit the dendritic growth characteristic of males by increasing sensitivity to afferent stimuli. Such a process could account for the observed differences in CNS vocal "circuitry" in X. laevis and thus behavioral differences between the sexes.