Martha L. Tobias

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.

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.

Evolution of advertisement calls in African clawed frogs.

For most frogs, advertisement calls are essential for reproductive success, conveying information on species identity, male quality, sexual state and location. While the evolutionary divergence of call characters has been examined in a number of species, the relative impacts of genetic drift or natural and sexual selection remain unclear. Insights into the evolutionary trajectory of vocal signals can be gained by examining how advertisement calls vary in a phylogenetic context. Evolution by genetic drift would be supported if more closely related species express more similar songs. Conversely, a poor correlation between evolutionary history and song expression would suggest evolution shaped by natural or sexual selection. Here, we measure seven song characters in 20 described and two undescribed species of African clawed frogs (genera Xenopus and Silurana) and four populations of X. laevis. We identify three call types - click, burst and trill - that can be distinguished by click number, call rate and intensity modulation. A fourth type is biphasic, consisting of two of the above. Call types vary in complexity from the simplest, a click, to the most complex, a biphasic call. Maximum parsimony analysis of variation in call type suggests that the ancestral type was of intermediate complexity. Each call type evolved independently more than once and call type is typically not shared by closely related species. These results indicate that call type is homoplasious and has low phylogenetic signal. We conclude that the evolution of call type is not due to genetic drift, but is under selective pressure.

Estrogen receptors in Xenopus: duplicate genes, splice variants, and tissue-specific expression.

The estrogenic steroid hormones, acting primarily through the nuclear estrogen receptors ERalpha and ERbeta, regulate sexual differentiation in a wide variety of vertebrates. In the frog Xenopus laevis, estrogen regulates the strength of vocal neuromuscular synapses and contributes to the physiological basis of sexually differentiated songs. To understand the mechanisms by which estrogen produces these effects, we have characterized the ERs of X. laevis and their expression in laryngeal muscle and other tissues. We found a remarkable molecular diversity in the estrogen receptor population within individuals. First, we have identified two distinct ERalpha genes, xlERalpha1 and xlERalpha2, which represent, to our knowledge, the first discovery of retained duplicates of the ERalpha gene in any species. These two genes are highly conserved at the amino acid level but have distinct nucleotide sequences; moreover, ERalpha2 has no N-terminal domain. Cloning of ERalpha and ERbeta in the related species Xenopus tropicalis and phylogenetic analysis indicate that the two xlERalpha loci were generated by a duplication specific to the X. laevis lineage-most likely the genome duplication that led to a doubling of the X. laevis chromosome number about 30 million years ago. The primary ER expressed in X. laevis laryngeal muscle is the novel gene xlERalpha2; ERalpha1 is primarily expressed in liver, forebrain, and oviduct. Alternatively spliced transcripts of both xlERalpha1 and xlERalpha2 are also expressed in a tissue-specific manner. We propose that complementary spatial expression of these two genes and their alternatively spliced transcripts contributes to their conservation over such a long period of time, consistent with the subfunctionalization model for evolution after gene duplication.

Estrogen and Laryngeal Synaptic Strength in Xenopus laevis: Opposite Effects of Acute and Chronic Exposure

Synaptic transmission at the vocal synapse, the laryngeal neuromuscular junction, of Xenopus laevis has been shown to be regulated by long-term changes in circulating estrogen. In females, high levels of circulating estrogen also accompany gonadotropin-induced ovulation and oviposition and the switch from sexually unreceptive to receptive states, including changes in vocal behaviors (ticking to rapping). Here we examine the effects of gonadotropin injection on laryngeal synaptic strength and call type. Gonadotropin acutely reduced quantal content values of laryngeal synapses in intact, adult females; the lowest values were attained by 12 h post-injection. Estrogen and progesterone levels increased following human chorionic gonadotropin (hCG) injection; the time course was similar to, but negatively correlated with, changes in synaptic strength. In ovariectomized frogs, exogenous estrogen, but not progesterone or hCG, mimicked the acute effects of hCG in weakening laryngeal synapses of intact frogs. hCG injection suppressed ticking and sometimes induced rapping. Females could tick with either strong or weakened laryngeal synapses while rapping was only produced during the weakening action of hCG. The normally strong synapses of females may enable vocal production even when laryngeal synapses are weakened by hormones that induce ovulation. In contrast to the acute effect of estrogen on weakening laryngeal synapses, juveniles required more than 2 weeks of estrogen treatment to strengthen laryngeal synapses while at least 4 weeks postovariectomy were required to weaken synapses in adult females. We conclude that acute (hours) increases in circulating levels of estrogen weaken synapses while chronic (weeks) increases strengthen laryngeal synapses.

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.

Genetics, Morphology, Advertisement Calls, and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog ( Xenopus , Pipidae) from West and Central Africa

African clawed frogs, genus Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon) to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana) and three species groups within the subgenus Xenopus (amieti, laevis, and muelleri species groups).

Attaining and maintaining strong vocal synapses in female Xenopus laevis

Synaptic efficacy at the laryngeal neuromuscular synapse differs markedly in adult male and female Xenopus laevis. Here, we examined the relation between circulating estrogen and synapse strength in developing and adult female frogs. Circulating estrogen levels in males and females during juvenile and adult stages were measured using radioimmunoassays. Synaptic strength was determined by quantal analysis in isolated female larynges. In males, estrogen levels are low (<40 pg/mL) throughout development. In females, estrogen levels are similar to those in males until 9 months after metamorphosis is complete and then increase throughout development. Female laryngeal synapses have low quantal contents until 24 months; quantal content increases significantly between 24 and 26 months, and high quantal contents are maintained thereafter. Measures of reproductive maturation, ovary, and oviduct weights, are strongly and positively correlated with estrogen level in 16- to 26-month females, while oocyte maturation is age dependent. Estrogen level and quantal content are not well correlated in these females. Ovariectomy at 24 months prevents the expected increase in quantal content and ovariectomy at 28 months results in a decrease in quantal content. Thus, the sex difference in efficacy of the laryngeal synapse develops under the influence of the ovary and requires the ovary for maintenance of strong synapses in adulthood. While the influence of the ovary is most likely due to estrogen secretion, the pattern of estrogen secretion required for maturation of the synapse in females is not known.

Estrogen Receptor Expression in Laryngeal Muscle in Relation to Estrogen-Dependent Increases in Synapse Strength

In Xenopus laevis, the laryngeal neuromuscular synapse is the final effector for sexually differentiated song production. Females have stronger laryngeal synapses than males, and synapse strength is estrogen dependent. Estrogen-induced increases in synaptic strength require at least 3 weeks of exposure, suggesting that the hormone acts via a classical genomic mechanism involving the estrogen receptor (ER). The locus of the sex difference in synapse strength, determined using quantal analysis, is presynaptic, leading to the prediction that estrogen acts directly on vocal motor neurons. However, laryngeal motor neurons do not accumulate estrogen. Estrogen might instead affect motor neuron transmitter release via a retrograde signal from its target muscle. To test this hypothesis, we determined whether laryngeal muscle expresses ER. With RT-PCR using primers that recognize highly conserved domains of the ERα, mRNA products of the predicted size were amplified from laryngeal muscle as well as from other classical target tissues (forebrain and oviduct). Northern blots using a portion of the PCR product as primer revealed the same-sized band in oviduct and laryngeal muscle. Immunocytochemistry and Western blots confirmed the presence of ER protein in laryngeal muscle fibers and revealed several proteins in laryngeal muscle, brain and liver; among these was an approximately 66-kD protein – presumed to be full-length ER – that was the only one found in oviduct. Estrogen treatment of juveniles resulted in an upregulation of the 66-kD ER protein concomitant with an increase in quantal content. Taken together, these experiments strongly suggest that the ER is expressed by laryngeal muscle; this receptor could mediate estrogen-dependent changes in synaptic strength via retrograde signaling.

Evolution of male and female release calls in African clawed frogs

In anurans, male clasps can elicit release calls from either sex. Male release calls have been observed in many anuran genera and this vocal response is thus highly conserved. Female release calls, however, are not as prevalent, suggesting that evolutionary trajectories for anuran release calls differ by sex. We analyzed male and female release calls in all available species of Xenopus, a fully aquatic African genus. Phylogenetic relationships in this genus include three species groups, two of which are clades and one of which is characterized by a reticulated phylogeny due in part to hybridizations between species with different ploidy levels (Evans et al., 2004; Evans, 2008). In all species, males produce release calls when clasped by another male. Females in the reticulated group do not produce release calls, but females in the rest of the genus do. Release calls consist of click trains of variable durations and inter-call intervals. In both sexes, inter-click interval divides the genus into groups with different click rates and these groups are phylogenetically related. In general, inter-click interval is shorter in male than in female release calls. Across species and sexes, release calls are characterized by a single, low (∼1000 Hz) dominant frequency. In X. laevis Congo and X. borealis, clasp duration is longer for male–female than for male–male pairs and clasp duration is correlated with the number, but not the duration, of release calls in male–male pairs. We discuss evolutionary scenarios for release call traits as well as the sex difference in occurrence.