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Biomedical subjects

D B Kelley

Publications and source records attributed to D B Kelley.

At least 19 recordsLinked to original sources

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

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 propionate. 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.

Acoustic Stimulation

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.

Amino Acid Sequence

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.

Animals

Temporal constraints on androgen directed laryngeal masculinization in Xenopus laevis.

Temporal constraints on androgen regulated masculinization of three sexually dimorphic laryngeal properties--tension, fiber type, and fiber recruitment--were examined in Xenopus laevis frogs. Endocrine state was manipulated at PM0 when the larynx is similar in males and females, at PM2 when the larynx begins sexual differentiation, and at PM6 when sexual differentiation is complete. Removing the testes in developing males (PM0 or PM2) completely arrests laryngeal masculinization. Masculinization resumes when testosterone is replaced later in development (PM2 or PM6, respectively). Thus, testicular secretions, in particular androgens, are required for laryngeal masculinization. The ability of androgens to masculinize tension, fiber type, and fiber recruitment in developing and adult larynges was also determined. Five weeks of testosterone treatment in PM0 or PM2 males and females completely masculinizes laryngeal tension and fiber type, but only partially masculinizes fiber recruitment. However, fiber recruitment can be fully masculinized in PM6 males castrated at PM2. We conclude that androgen induced masculinization of tension and fiber type are not temporally constrained but that androgen induced masculinization of fiber recruitment is. Prolonged androgen treatment can override the temporal constraints on masculinization of the larynx. Testosterone treatment for more than 6 months fully masculinizes fiber recruitment in developing (PM0 or PM2) females. In addition, prolonged treatment (greater than 9 months) completely masculinizes tension, fiber type, and fiber recruitment in adult females; these properties were not fully masculinized by shorter (1-3 months) treatments in adult females. Testosterone induced masculinization in females is maintained for up to 8 months following testosterone removal; thus androgen effects are long lasting and possibly permanent.

Aging

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 receptor 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.

Amino Acid Sequence

The vocal motor neurons of Xenopus laevis: development of sex differences in axon number.

Sex differences in the number of muscle fibers in the larynx of clawed frogs (Xenopus laevis) develop after metamorphosis. In order to examine possible contributions of neural innervation to this process, we prepared sections of the laryngeal nerve from tadpole stage 56, when the sexes can first be distinguished, through adulthood, and counted axons on electron micrographs. The adult number of axons is achieved by a sexually differentiated pattern of axonal addition and loss. Axon numbers are high at tadpole stage 56 and equal for males and females; thereafter, males have more axons. Sex differences are most pronounced at tadpole stage 62 because between stages 59 and 62 the number of laryngeal axons in males increases by an average of 119 per nerve. Ultrastructural evidence is congruent with the hypothesis that new axons are added to the laryngeal nerve between tadpole stages 56 and 62. The loss of axons from the laryngeal nerve is greater for females than for males. Between tadpole stages 56 and adulthood, overall axon number decreases by 47% in males and by 64% in females. Signs of axonal degeneration are present in both sexes before metamorphosis but are rare at juvenile or adult stages. The numbers of axons in juvenile frogs do not differ from those in adults and continue to be greater in males than in females. In contrast to the amount of axon addition and loss, the timing of axon loss and the percentage of myelinated axons is the same for males and females throughout development. Thus sex differences in the innervation of laryngeal muscle originate before metamorphosis and could contribute to the marked sex differences in muscle fiber addition that occur thereafter.

Animals

An improved method for mounting frozen-section specimens.

We describe an efficient method for embedding frozen-section specimens. Transparent tape is used to produce a well on the surface of the tissue holder ("chuck"). This well contains a quantity of nonmotile mounting media in which a tissue specimen can be easily oriented. After freezing, the tape is removed, leaving a relatively smooth surface.

Specimen Handling

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.

Androgens

Connective tissue disease in southeastern Georgia: a case-control study of etiologic factors.

The authors conducted a population-based case-control study of connective tissue disease in a four-county area in southeastern Georgia. Interviews with 44 cases and 88 controls were conducted in September and October of 1986. The association with a family history of these disorders was reconfirmed, and a positive association with the use of hair dyes was found. A negative association was found with a family history of diabetes mellitus.

Amines

Sex differences in the motor nucleus of cranial nerve IX-X in Xenopus laevis: a quantitative Golgi study.

In the clawed frog (Xenopus laevis), motor neurons in cranial nerve nucleus IX-X control contraction of laryngeal muscles responsible for sexually dimorphic vocal behaviors. We examined sex differences in dendritic arbors of n.IX-X cells using the Golgi-Cox method. Three morphological classes of somal types (ovoid, triangular, and elongate) are present in similar frequencies in n.IX-X of both males and females. The male n.IX-X neuron is a more complex and hypertrophied version of the female n.IX-X cell. The number of primary dendrites is the same for both sexes, but males have more total dendritic segments. The overall dendritic length of male n.IX-X neurons is two to three times that of the female. Males have longer dendritic segments between all branch points. Male and female frogs differ in levels of circulating androgens; neurons of n.IX-X are targets for androgenic steroids. To determine if androgen can affect dendritic morphology in adult females, we examined Golgi-impregnated cells in n.IX-X from ovariectomized females treated with testosterone for 1 month. The total number of dendritic segments was reduced by androgen treatment due to reduction in the number of higher order dendritic segments; the number of primary dendritic segments was unchanged. Androgen treatment may induce resorption of higher order dendritic branches. The overall dendritic length of androgen-treated female n.IX-X neurons was unchanged, and dendritic segments were longer. Thus, although androgen can alter dendrites of n.IX-X cells in adult females, this short-term treatment does not produce a masculine dendritic architecture.

Animals

Sexually dimorphic behaviors.

Sex differences in behavior are the result of natural and sexual selection. The dimorphic classes of behavior described here, courtship, copulatory, and parental behaviors, reflect both kinds of evolutionary selective pressures. We can further distinguish two kinds of mechanisms that produce differences in male and female behaviors. In one, both sexes can perform a behavior but one does not because of sex differences in the external stimuli or the endocrine milieu. Maternal behavior in rodents falls into this category, as do certain other reproductive behaviors. In the other, the sensory, CNS, or motor components that produce behaviors are different in males and females. Many courtship and copulatory behaviors are in this category. I have considered some cellular mechanisms that generate sex differences in behavioral effector neurons, including sensitivity to hormones, cell number, and synaptic connectivity. A common feature of many such systems is a degree of developmental arrest: sexually dimorphic, hormone-sensitive neurons or muscles are immature at stages when other cells have completed differentiation. The cellular and molecular processes whereby hormones harness the developmental programs of behavioral effector cells remain largely unknown and are the focus of active investigation.

Animals

Electrophysiology and dye-coupling are sexually dimorphic characteristics of individual laryngeal muscle fibers in Xenopus laevis.

Sex differences at the laryngeal neuromuscular junction of Xenopus laevis were examined by recording intracellularly from muscle fibers in response to nerve stimulation. Male laryngeal muscle contains 2 physiologically distinct fiber types. Type I fibers generate postsynaptic potentials in response to low-magnitude stimulus pulses and action potentials in response to higher-magnitude stimulus pulses. Type II muscle fibers require repetitive stimulation for action potential production, probably because of facilitation. Subthreshold events in type I and II fibers suggest that these neuromuscular synapses have low safety factor junctions. Female laryngeal muscle contains one fiber type (III), which is physiologically distinct from those found in the male. Type III fibers produce an action potential in response to a single-stimulus pulse of suprathreshold voltage delivered to the laryngeal nerve; subthreshold events were not observed. Iontophoretic injection of Lucifer yellow into a single female muscle fiber resulted in as many as 43 labeled fibers. In males, only one fiber was labeled. Dye-coupling was not observed in adult females treated with the androgenic steroid hormone, testosterone. We have previously reported that laryngeal muscle fibers are recruited throughout a stimulus train presented to the laryngeal nerve in males, but are not recruited in females (Tobias and Kelly, 1987). Sex differences in the frequency of electrophysiological fiber types described here may account for sex differences in fiber recruitment. Synchronous activity of dye-coupled fibers may increase the effectiveness of muscle contraction in females.

Animals

Neurogenesis in the vocalization pathway of Xenopus laevis.

We examined possible contributions of neurogenesis to sex differences in the vocalization pathway of the South African clawed frog, Xenopus laevis. Birthdates of neurons were obtained from autoradiograms of animals receiving tritiated thymidine from gastrulation through 1 month after metamorphosis. Thymidine availability studies showed that 80% of the [3H]-thymidine injected into embryos and tadpoles was incorporated into the DNA of dividing cells within 3 hours. We observed 3 patterns of neurogenesis: late-short, a short burst of proliferation occurred late in development in the anterior preoptic area, the ventromedial nucleus of the thalamus, and the pretrigeminal nucleus of the dorsal tegmental area of the medulla; protracted-bimodal, a prolonged period of proliferation with an early and a late peak in the number of labeled cells occurred in the ventral striatum and in the ventrolateral and posterior nuclei of the thalamus; protracted-unimodal, a prolonged period of proliferation with a single early peak occurred in the inferior reticular formation and in the medial and lateral nucleus IX-X (containing laryngeal motor neurons). There were no differences between sexes in the number of tritiated thymidine labeled cells in any nucleus. The difference in nucleus IX-X neuron number in adults does not appear to result from sex differences in the proliferation of these cells during development. Since neurons in the vocalization pathway do not exhibit androgen receptors until after neurogenesis is complete, we also conclude that androgen probably does not regulate the genesis of these cells.

Animals

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.

Androgens

Vocalizations by a sexually dimorphic isolated larynx: peripheral constraints on behavioral expression.

The clawed frog Xenopus laevis uses sexually dimorphic vocalizations, mate calling and ticking, to advertise reproductive state. The basic unit of vocalization is a brief click, produced by the movement of cartilagenous disks located within the larynx. The rate of click production in the male-specific mate call (71 Hz) is an order of magnitude faster than the rate of click production in female typical ticking (6 Hz). To determine if vocalization rate is constrained by the periphery, male and female larynges were isolated and response of the muscles to nerve stimulation was studied. Laryngeal muscle response is markedly dimorphic in the 2 sexes, both in the amplitude potentiation of electromyograms and in the rate at which discrete tension transients can be produced. At 6 Hz (ticking), both sexes generate discrete tension transients in response to each stimulus pulse. In response to nerve stimulation at 71 Hz (mate calling), male laryngeal muscle generates discrete tension transients while female laryngeal muscle does not. Since expression of sex-specific vocalizations is regulated by androgenic hormones, responses of laryngeal muscle to nerve stimulation in androgen-treated adult females and castrated adult males were also examined. The responses of laryngeal muscle from castrated and intact males are similar. Androgen-treated female larynx is partially masculinized but does not produce tension transients at the mate call rate. These physiological results are in close agreement with behavioral observations. Sounds produced by the isolated larynx were nearly identical in spectral properties to those produced by an intact male. We determined that the production of a discrete tension transient is prerequisite to click production. Thus, one reason females do not mate call, even when treated with androgens, is that female laryngeal muscle cannot produce discrete tension transients at a rapid rate.

Androgens

Androgen regulation of muscle fiber type in the sexually dimorphic larynx of Xenopus laevis.

We used histochemical techniques [assays for adenine triphosphatase (ATPase) and succinate dehydrogenase (SDHase) activity] to identify muscle fiber types in the larynx of Xenopus laevis. Male muscle is made up of one fiber type, medium-sized fibers (approximately 9 microns2) that stain lightly for acid-stable ATPase and intensely for SDHase activity. In contrast, the female has 3 fiber types: small fibers (approximately 6 microns2) that stain intensely for ATPase and SDHase, medium-sized fibers (approximately 13 microns2) with moderate staining for ATPase and dark staining for SDHase, and large fibers (approximately 15 microns2) with little SDHase or ATPase activity. Long-term castration (6 months) has no effect on histochemical staining of adult male fibers. Long-term testosterone treatment (5 months) increases the proportion of medium-sized, moderately staining fibers in adult females, and reduces the proportion of both the small, darkly staining fibers and the large, lightly staining fibers. At metamorphosis, both males and females have 3 fiber types whose ATPase activity is similar to that of the adult female. However, no SDHase activity is observed. Treatment of juveniles for 3 weeks with testosterone results in nearly complete masculinization of muscle fibers, as judged by increased cross-sectional area, homogeneous ATPase staining, and a marked increase in SDHase activity. Thus, juvenile muscle is considerably more responsive to testosterone than is adult female muscle. We propose that the uniform metabolic properties of male laryngeal muscle contribute to the production of the rapid (66 Hz) mate call vocalizations characteristic of this species. Further, our results suggest that androgens direct the masculinization of laryngeal muscle fibers during postmetamorphic development.

Androgens