Excretion and conservation of glycerol, and expression of aquaporins and glyceroporins, during cold acclimation in Cope's gray tree frog Hyla chrysoscelis

Cope's gray tree frog Hyla chrysoscelis accumulates glycerol during cold acclimation. We hypothesized that, during this process, gray tree frogs adjust renal filtration and/or reabsorption rates to retain accumulated glycerol. During cold acclimation, plasma concentrations of glycerol rose >200-fold, to 51 mmol/l. Although fractional water reabsorption decreased, glomerular filtration rate (GFR) and, consequently, urine flow were <5% of warm levels, and fractional glycerol reabsorption increased. In contrast, dehydrated frogs increased fractional water reabsorption, decreased GFR, and did not accumulate glycerol. We hypothesized that expression of proteins from the aquaporin (AQP)/glyceroporin (GLP) family was associated with changing patterns of water and glycerol movement. We cloned the cDNA for three such proteins, quantified mRNA expression in nine tissues using real-time quantitative PCR, and functionally characterized them using a Xenopus oocyte expression system. HC-1, an AQP1-like water channel conferring low glycerol permeability, is expressed ubiquitously in warm- and cold-acclimated tissues. HC-2, a water channel most similar to AQP2, is primarily expressed in organs of osmoregulation. HC-3, which is most similar to AQP3, is functionally characterized as a GLP, with low permeability to water but high permeability to glycerol. Aspects of expression levels and functional characteristics varied between cold and warm conditions for each of the three AQPs, suggesting a complex pattern of involvement in osmoregulation related to thermal acclimation.

Season and testosterone affect contractile properties of fast calling muscles in the gray tree frog Hyla chrysoscelis

In anurans, circulating levels of androgens influence certain secondary sexual characteristics that are expressed only during the breeding season. We studied the contractile properties of external oblique muscles (used to power sound production) in a species of North American gray tree frog, Hyla chrysoscelis, during the breeding season and also in testosterone-treated captive males and females after the breeding season. Compared with the muscles of breeding-season males, the trunk muscles of postbreeding-season males have 50% less mass, 60% longer twitches, and 40% slower shortening velocities. Testosterone levels similar to those found in breeding-season male hylid frogs restore the contractile speed and mass of male trunk muscles and also convert the small slow trunk muscles of females into larger fast-contracting muscles. We conclude that androgens likely play a key role in altering the contractile properties of these muscles in males during the annual cycle, allowing them to operate in the breeding season at the frequencies required to produce the characteristic rapidly pulsed calls of this species. Females as well as nonbreeding-season males do not produce advertising calls, and therefore the slower muscles found in these animals may allow more economic operation of these muscles. The effects of testosterone on female trunk muscles indicate the potential of this hormone in contributing to the sexual dimorphism in size and contractile properties of these muscles, but this dimorphism is likely due to the interaction of more than one hormone.

Contractile properties of muscles used in sound production and locomotion in two species of gray tree frog

The sound-producing muscles of frogs and toads are interesting because they have been selected to produce high-power outputs at high frequencies. The two North American species of gray tree frog, Hyla chrysoscelis and Hyla versicolor, are a diploid-tetraploid species pair. They are morphologically identical, but differ in the structure of their advertisement calls. H. chrysoscelis produces very loud pulsed calls by contracting its calling muscles at approximately 40 Hz at 20 degrees C, whereas, H. versicolor operates the homologous muscles at approximately 20 Hz at this temperature. This study examined the matching of the intrinsic contractile properties of the calling muscles to their frequency of use. I measured the isotonic and isometric contractile properties of two calling muscles, the laryngeal dilator, which presumably has a role in modulating call structure, and the external oblique, which is one of the muscles that provides the mechanical power for calling. I also examined the properties of the sartorius as a representative locomotor muscle. The calling muscles differ greatly in twitch kinetics between the two species. The calling muscles of H. chrysoscelis reach peak tension in a twitch after approximately 15 ms, compared with 25 ms for the same muscles in H. versicolor. The muscles also differ significantly in isotonic properties in the direction predicted from their calling frequencies. However, the maximum shortening velocities of the calling muscles of H. versicolor are only slightly lower than those of the comparable muscles of H. chrysoscelis. The calling muscles have similar maximum shortening velocities to the sartorius, but have much flatter force-velocity curves, which may be an adaptation to their role in cyclical power output. I conclude that twitch properties have been modified more by selection than have intrinsic shortening velocities. This difference corresponds to the differing roles of shortening velocity and twitch kinetics in determining power output at differing frequencies.