Rana Pipiens Tadpoles Research Articles

Binding of thyroxine with subcellular components of brain, liver and tail tissues of Rana pipiens tadpoles

Binding of radioiodinated thyroxine given in vivo was measured in subcellular fractions of liver, tail and brain tissues of premetamorphic tadpoles of Rana pipiens. Binding at 6 hr after hormone injection was measured in otherwise untreated tadpoles, or in animals given various additional quantities of unlabeled hormone up to 1000 times the dose of labeled hormone. In all tissues increasing doses of cold hormone depressed binding or accumulation of labeled hormone in cytosol or nuclear fractions, but some of the binding was not saturable. Thyroid hormone binding in brain cell nuclei was almost completely of the saturable type, and in this sense, it differed from liver and tail tissues.

Phagocytic capabilities of the pigment epithelium

The ability of the pigment epithelium (PE) to phagocytize polystyrene spheres, native Sarcina subflava and a mixture of these two particles was studied at short time intervals following injection into the subretinal space in Rana pipiens tadpoles. Polystyrene spheres were interiorized as quickly as 30 min following injection and by 3 hr polystyrene spheres were found throughout the cytoplasm of the PE cells. Sarcina subflava when presented alone were not phagocytized at any interval studied up to 24 hr post injection. Following presentation of the mixed suspension of bacteria and polystyrene spheres large numbers of bead profiles were present within the PE cells indicating that the PE is active phagocytically in the presence of the micrococcus. Occasionally mixed phagosomes containing polystyrene spheres and Sarcina subflava were observed, though the overwhelming majority of the experimental phagosomes contained only polystyrene spheres. These results indicate that the nature of the material presented to the PE can determine whether or not that material will be phagocytized.

Selective reinnervation in skin rotation grafts in Rana pipiens.

Skin rotation grafts were performed in Rana pipiens tadpoles producing misdirected reflexes in adult frogs. The pattern of innervation fields of dorsal and ventral rami of segmental nerves 3, 4, 5 and 6 were investigated by recording from the severed rami with suction electrodes. Innervation patterns of regenerated fields were influenced by graft borders and adjacent fields. Misdirected reflexes were found for ventral skin on the dorsal side only when innervated by ventral rami in a manner never seen in normal or sham operated animals. This suggested that selective reinnervation has occurred.

Pineal and serum melatonin at midday and midnight following pinealectomy or castration in male rats.

Melatonin has been extractkd by chloroform from rat serum and quantified by the Rana pipiens tadpole bioassay. Like pineal melatonin, serum melatonin was high at mid-dark and low at mid-light. This finding suggests a diurnal rhythm of serum melatonin in the rat. Serum samples obtained at mid-dark had melatonin levels ranging from 0.02 to 0.05 ng per ml of serum. When rats were pinealectomized, melatonin activity in the serum was abolished. Thus, rat serum melatonin may originate primarily from the pineal. There was no significant change in pineal melatonin content nor in serum melatonin concentrations 7 or 12 days following orchidectomy.

The Effects of Partitions on the Growth Rates of Crowded Rana pipiens Tadpoles

It was recognized at the outset that growth rates of tad- poles are retarded in proportion to the density of the population and that psychological stress is the major cause for retardation. This paper shows that the rate of growth is not dependent simply upon the volume of space, but also upon the shape of the space. The results support the contentions that space is psychological and that the stress of crowding is proportional to the frequency of physical encounters between tadpoles. Vision is not an important factor. Adolph's (1931) correlation of growth rates with the inverse of density can be improved by incorporating a term to represent the shape of the space or perhaps the length of the periphery.

MICROTUBULAR ORGANIZATION IN ELONGATING MYOGENIC CELLS

Microtubule organization has been studied in serially sectioned myogenic cells in the tail muscle regeneration blastema of Rana pipiens tadpoles. In mesenchymal cells and in some premyoblasts, microtubules radiate from centriolar satellites in a cell center, while in more mature myoblasts and myotubes the centrioles no longer appear to serve as organizing centers for microtubules. In all elongate, fusiform myogenic cells, the microtubules are predominately oriented in the longitudinal axis of the cell. Counts of microtubules in transverse sections spaced at regular intervals along the cells show that the absolute number of microtubules is greatest in the thickened midregions of the cells and decreases relatively smoothly toward the tapered ends of the cells. Close paraxial association of microtubules (within 40 nm surface-to-surface) occurs along the entire lengths of cells but appears with greatest frequency in their tapered ends. In two myoblasts, serial sections were used to trace all microtubules in 8-microm long segments of the cells located about midway between the nucleus and one end of the cell. Since tracings show that as many as 50% of the microtubules terminate within the 8-microm long segment, it seems unlikely that any microtubules extend the entire length of the cell. It is proposed that lateral interactions between paraxial microtubules stabilize the overall microtubular apparatus and contribute to maintenance of the bipolar form of the cells. A three-dimensional model of the complete microtubular array in one of the 8-microm long segments of a myoblast has been constructed. The model reveals that a few microtubules within the segment are bent into smooth curves and loops that could be generated by sliding interaction between paraxial microtubules.

Effects of methylmercury chloride on Rana pipiens tadpoles

Rana pipiens tadpoles were allowed to be raised in water containing various concentrations of methylmercuric chloride or were injected with 0.025 ml of 0.1% aqueous solutions of methylmercuric chloride (0.025 mg Hg), every other day for 10 days. All the tadpoles which were raised in water containing more than 0.05 ppm mercury died within 48 hours. The distention of the body cavities and eventual death of these tadpoles was believed to be the result of a disturbance of the osmotic regulatory system by the mercury. Total arrest of further development and differentiation was observed in tadpoles which were raised in water containing 0.001–0.01 ppm mercury. Extensive deposition of blood pigment (hemosiderosis) was observed in the livers of the mercury-injected tadpoles. Such hemosiderotic condition was thought to be predisposed by hemolysis of the red blood cells by mercury followed by severe peripheral edema and hemopoietic reactions in the kidneys of these tadpoles.

Phagocytic activity in the retinal pigment epithelium of the frog Rana pipiens: II. Exclusion of Sarcina subflava

In Rana pipiens embryos, tadpoles, and frogs, the ability of the pigment epithelium (PE) to phagocytize pasteurized bacteria ( Sarcina subflava ) was evaluated. The bacteria carcasses were injected into the space bounded by the retina and PE. Eyes were recovered at several intervals after injection and the location of the bacteria was determined. In each of the stages examined, phagocytic cells, probably leukocytes, invade the space between the retina and PE. Although most of the bacteria are engulfed by these invading phagocytic cells, numerous extracellular bacteria were observed in close association with the PE. Bacteria were never observed inside the PE cells, indicating that the PE failed to phagocytize this material.

Phagocytic activity in the retinal pigment epithelium of the frog Rana pipiens: I. Uptake of polystyrene spheres

In Rana pipiens embryos, tadpoles, and frogs, the ability of the retinal pigment epithelium (PE) to phagocytize polystyrene spheres was evaluated. Test spheres were injected into the space bounded by the retina and PE. Eyes were recovered at several intervals after injection, and the location of the beads was determined. The PE in each of the stages studied contained numerous bead profiles indicating the active uptake of the test material by this tissue. Bead profiles were not observed in the adjacent retina. In the embryo, the ingested polystyrene was toxic to the PE causing fragmentation of the endoplasmic reticulum and disruption of the mitochondria whereas in the PE of the tadpole and frog no toxicity to polystyrene was noted. Interaction between lysosomes and the polystyrene spheres was observed in each stage examined suggesting an attempt to degrade these ingested particles.

Temperature influence in developing tadpoles. III. Influence of temperature on some kinetic and thermodynamic parameters of partially purified glucose-6-phosphate dehydrogenase from spring and fall eggs and tadpoles of Rana pipiens.

1. 1. Some kinetic and thermodynamic parameters were studied in partially purified G6PDH preparations from spring and fall Rana pipiens eggs and tadpoles raised at 10 and 20 °C. 2. 2. Results suggest that the spring groups of G6PDH are metabolically more responsive because the energies of activation as determined from the initial velocity contants of G6PDH are lower than the corresponding fall groups of G6PDH. 3. 3. Thermodynamic considerations suggest that the fall tadpole enzyme preparations may be more efficient than the spring tadpole enzyme preparations because of an increased molecular flexibility. 4. 4. It appears that development during the fall season may be more stressful than development during the spring season. There would be need for both an increase in yield and thermodynamic efficiency in animals preparing to survive the stress of the cold winter environment.

Interaction of the sarcoplasmic reticulum with Z-lines during myogenesis in amphibian skeletal muscle.

AbstractElectron microscopic examination of developing myofibrils in regenerating skeletal muscle of Rana pipiens tadpole tails reveals that sarcoplasmic reticulum (SR) is in regular and close association with Z‐lines of young sarcomeres before transverse tubules have formed alongside the myofibrils. SR is also associated with M‐lines of young sarcomeres but on a less regular basis than with Z‐lines. Counts of the number of contact sites of SR with Z‐lines, M‐lines and combined A‐ and I‐bands of longitudinally sectioned sarcomeres of young myofibrils confirms the visual impression that the SR associates with a relatively much greater frequency with Z‐lines and M‐lines than with other regions of the sarcomere. This suggests the formation of a specific bond between SR and Z‐ and M‐lines.Serial transverse sections of developing myofibrils were examined in order to determine whether Z‐lines displayed SR association at all stages in their formation. Of fifty discrete Z‐lines that were examined, only four smaller Z‐lines appeared to lack SR association. An examination of a comparable number of more diffuse Z‐bodies representing an earlier stage of Z‐line formation suggests that no more than one‐half of these Z‐bodies were accompanied by SR. These observations support the conclusion that SR does not initiate formation of Z‐line precursor elements, but rather associates with Z‐lines as they condense from more diffuse Z‐bodies.

Temperature influences in developing tadpoles: I. Influence of temperature on the enzymatic activity of glucose-6-phosphate dehydrogenase

The ability of Rana pipiens embryos and tadpoles to acclimate to environmental temperature has been studied in animals obtained in the spring after the winter dormancy and in the fall before the winter dormancy period. Fall Rana pipiens embryos and tadpoles grow faster at 10 °C and 20 °C than do spring embryos and tadpoles. Enzymatic acclimation, as measured by changes in the specific activity of G6PDH, occurs in spring tadpoles raised at 10 °C and 20 °C, and not in fall tadpoles. It is postulated that development at 10 °C prevents the hybrid genome from being fully expressed. In the fall tadpoles, the maternal genetic influence has a more lasting effect.

Temperature influence in developing tadpoles: II. Influence of temperature on isozymes and energies of activation of partially purified glucose-6-phosphate dehydrogenase

Isozymic banding patterns and energies of activation were studied in partially puriled G6PDH preparations from spring and fall Rana pipiens eggs and tadpoles raised at 10 °C and 20 °C. G6PDH preparations from spring tadpoles raised at 20 °C demonstrated new isozyme patterns. However, in the spring 10 °C and fall 10 °C and 20 °C raised tadpoles G6PDH preparations conserved isozymes already present. The energies of activation of G6PDH determined from the maximum velocity constants in the spring groups are lower than in the corresponding fall groups.

Histochemical patterns in the tadpole tail during normal and thyroxine-induced metamorphosis: I. Alkaline phosphatase, acid phosphatase, esterase, and aminopeptidase

The distribution of alkaline phosphatase, acid phosphatase, esterase, and aminopeptidase was demonstrated by histochemical methods in sections of both growing and resorbing tails of Rana pipiens tadpoles. In general, main sites of localization of each of the enzymes were the epidermis and connective tissue, but not striated muscle. In addition, alkaline phosphatase, acid phosphatase, and esterase were present in the ependyma of the spinal cord and the endothelium of various blood vessels. The latter two enzymes were also found in spinal ganglia and, along with aminopeptidase, in the notochord. As the tail shortened during both normal and thyroxine-induced metamorphosis, activity of these enzymes increased; it became particularly prominent in the epidermis and in phagocytes in the connective tissue. These data on the differential distribution of four hydrolytic enzymes in the R. pipiens tail support histochemical and biochemical studies in other species.

Neurogenesis in frogs after early larval treatment with somatotropin or prolactin

Rana pipiens tadpoles were given six injections (over an 11-day period) of either prolactin, somatotropin, or saline, during early stages of larval development. Brain and body growth and aspects of brain nucleic acid metabolism were studied at frequent intervals through metamorphosis. The three groups showed no differences in morphology, rates of development, or general patterns of body growth—although the extent of body growth was increased in the experimental groups. In contrast, both hormones produced profound and very different effects on neurogenesis. Increases in brain weight and DNA and RNA content after prolactin treatment were modest during the early stages, but ultimately extensive; and this enhancement embraced all three major regions: forebrain, midbrain, and hindbrain. Labeling experiments with tritiated thymidine showed that prolactin caused both an increase in brain DNA synthesis (during the early and especially the mid-larval stages) and a decrease in neural cell death (during late stages). Somatotropin greatly enhanced brain weight and DNA content (in all three brain regions) during the period of its administration. Labeling experiments attributed this to a period of very rapid DNA synthesis. By the end of metamorphosis, however, the somatotropin-treated animals showed only slight advantages over the controls in brain weight and nucleic acid content—although the forebrain remained significantly enhanced. Possible mechanisms of hormone action and roles for these hormones in normal neurogenesis are discussed, as are some of the technical problems in experiments of this sort. Finally, comments are made on normal neurogenesis based on observations of the control population.

Diurnal Periodicity in the Effectiveness of Prolactin to Inhibit Metamorphosis in Rana pipiens Tadpoles

Diurnal Periodicity in the Effectiveness of Prolactin to Inhibit Metamorphosis in Rana pipiens Tadpoles

Some observations on the ultrastructure of the hepatocyte in the metamorphosing tadpole

The principal ultrastructural changes occurring during metamorphosis in the hepatocyte of Rana pipiens tadpoles are (1) the great degree of pleomorphism in the mitochondria, (2) the appearance of glycogen in some nuclei of late stages and (3) the disappearance of cytoplasmic glycogen, presumably due to its breakdown to glucose, in late stages. The latter may be related to the inhibition of tryptophan oxygenase activity during this developmental period. Differences between the hepatocyte of the tadpole and the adult include the above, plus the appearance of pinocytotic vesicles between adjacent cells during metamorphosis which are not seen in the adult.

Changes at the dermoepidermal junction in resorbing tadpole tail fin in Vitro

Tail fin pieces (“discs”) of Rana pipiens tadpoles were treated in vitro with thyroxine (T4, 50 μg/liter) and the morphological changes at the dermoepidermal junction were followed. After 5 days of T4 treatment the hemidesmosomal plaques were apparently dislodged from their normal site. The resulting “free” hemidesmosomes were vesicular, containing what looked like the components of the globular layer. The 2-cell-layered epithelium was transformed into a multilayered structure on the sixth day of T4 treatment. After this, cells containing free hemidesmosomes appeared in increasing numbers in the superficial layers of the epithelium. The free hemidesmosomes were often seen coalescing within each cell. The basal lamina (adepidermal membrane) lost its close association with the basal epithelial cells at several sites by day 6 of T4 treatment and was lost completely by day 8.

Effects of prolactin and growth hormone on growth and metamorphosis of tadpoles of the frog, Rana pipiens

Mammalian prolactin at doses of 1–50 μg/day promoted growth (increased wet weight, dry weight and body length) in Rana pipiens tadpoles. Mammalian growth hormone (GH) promoted growth only at the higher doses (50 μg/day and above) and was not so effective as prolactin. Prolactin also inhibited metamorphosis (at doses of 5 μg/day and above). Inhibition of metamorphosis did not appear to be due to reduction in tissue sensitivity to thyroxine since prolactin-treated tadpoles metamorphosed in response to exogenously administered thyroxine (4–100 μg/liter) to the same extent as controls. Prolactin promoted further growth in tadpoles in which the thyroid level had been reduced with thiouracil or by thyroidectomy. This indicates that growth promotion caused by prolactin is not just the result of thyroid inhibition, although it is possible that a portion of the growth stimulation is due to the antithyroid action of prolactin.

Light and electron microscopic study on thyroxine-induced in vitro resorption of the tadpole tail fin.

Tail fin pieces (“discs”) of Rana pipiens tadpoles were treated with thyroxine (T4) solution (50 μg/liter) and the induced changes were studied with the light and electron microscopes. Definitive effects first appeared after 4 days of treatment. Thereafter various resorptive processes proceeded at an accelerating pace. By the 9th or 10th day the discs were reduced to tiny spherules. The most conspicuous changes were those of the breakdown and disposal of the basement lamella. First there was a “loosening” of the collagen layers followed by massive infiltration by macrophages which engulfed the collagen fibrils. By the 8th day the phagocytosis of the basement lamella was completed for the most part and the macrophages were clumped into masses. After 6 days of T4 treatment the epidermis was transformed from a 2- or 3-cell layered epithelium to a multilayered one. This was due to the detachment of the basal epithelial cells from the basement lamella followed by their movement towards the surface. Later, the epidermal cells showed atrophy by extensive autolytic processes. Discs incubated in the control medium (devoid of T4) remained intact for the duration of the experiment. Their morphology was essentially the same as that described by others from studies in vivo.