Thyroxine-induced Metamorphosis Research Articles

Histone metabolism during amphibian metamorphosis: Isolation, characterization, and biosynthesis

The amino acid incorporation rates of several classes of liver protein from Rana catesbeiana tadpoles were examined at different stages of spontaneous and thyroxine-induced metamorphosis, particular attention being given to histones. Incorporation data were corrected for the specific radioactivity of the free amino acid pools in tadpole liver. Little change was observed in the overall incorporation rates for the crude mitochondrial and total liver proteins during thyroxine treatment or at selected stages of spontaneous metamorphosis, except that the incorporation rates for these proteins were approximately twofold greater for the newly metamorphosed froglet than for the other stages. However, an increase in the ratio of the specific radioactivities of the total and crude mitochondrial liver protein within each set of animals was observed during late stages of spontaneous metamorphosis, as well as during the second through sixth days of thyroxine treatment. The amino acid incorporation rates of the histones for the late metamorphic and froglet stages of spontaneous metamorphosis were three- to fourfold higher than those of premetamorphic animals, but no significant changes were observed during thyroxine treatment. Thyroxine treatment also produced no detectable changes in the relative amounts or incorporation rates of the histone fractions or subfractions. Apparently the developmental changes induced by thyroxine do not involve a reorganization of the histone complement of chromatin at this level of analysis. Furthermore, since histone and DNA syntheses are tightly coupled, our results show that the extensive metabolic changes induced in tadpole liver by thyroxine occur in the absence of significant levels of cell division.

Alterations in enzyme and cytochrome profiles of Rana catesbeiana liver organelles during thyroxine-induced metamorphosis. Changes in membrane-localized phosphohydrolases, oxidoreductases, and cytochrome levels in response to in vivo thyroxine administration.

A primary objective of the present study has been to determine the changes which occur in Rana catesbeiana liver organelle membranes during thyroxine-induced metamorphosis. To this end, enzyme and cytochrome profiles were determined for mitochondria, microsomes, and nuclear membrane fractions isolated from livers of R. catesbeiana tadpoles which had been fasted for 6 days at 15 +/- 0.5 degrees and then immersed in thyroxine, 2.6 X 10(-8) M, for periods of up to 12 days at 23.5 +/- 0.4 degrees. The ratio of total succinate-cytochrome c reductase activity in the initial homogenate fraction to the total activity of this mitochondrial "marker" enzyme recovered in the final mitochondrial fraction remained constant, approximately 0.5, throughout the course of thyroxine treatment; however, after a 3- to 4-day latency the mitochondrial protein mass recovered per unit mass of initial homogenate protein was found to increase significantly (approximately 2-fold by Day 10 of thyroxine treatment). A similar increase was also observed in the yield of microsomal, but not nuclear membrane, protein mass as a function of thyroxine treatment. Prolonged thyroxine treatment (12 days) resulted in approximately 50% decreases in tadpole liver homogenate and microsomal NADH-cytochrome c reductase specific activities; in contrast, mitochondrial and nuclear membrane NADH-cytochrome c reductase specific activities were not altered under the same conditions. In addition, homogenate and microsomal NADPH-cytochrome c reductase specific activities were found to have increased significantly after 12 days of thyroxine treatment; however, the specific activity of NADPH-cytochrome c reductase in the mitochondrial fraction was unchanged. It was also observed that thyroxine treatment resulted in increases in homogenate and microsomal glucose-6-phosphatase specific activities, whereas the mitochondrial as well as nuclear membrane glucose-6-phosphatase specific activities remained unchanged. Furthermore, in contrast to homogenate and mitochondrial monoamine oxidase specific activities, which decreased 30 and 40%, respectively, as a consequence of thyroxine treatment (12 days), the succinate-cytochrome c reductase and oligomycin-sensitive Mg2+ ATPase specific activities determined for these fractions increased significantly. In all instances, changes as a result of thyroxine treatment in membrane-localized homogenate or organelle enzyme specific activities were apparent only after a 3- to 4-day initial latent period. The in vitro effects of thyroxine (10(-10) - 10(-5) M) on the membrane-localized enzyme activities examined in this study were either negligible or, as in the case of mitochondrial succinate-cytochrome c reductase and microsomal NADH-cytochrome c reductase, opposite to the changes observed in response to in vivo thyroxine treatment, with the exception of microsomal NADPH-cytochrome c reductase activity which was enhanced approximately 2-fold by 10(-5) M thyroxine...

Incorporation of [5- 3H]uridine into ribonucleotide pools and RNA during thyroxine-induced metamorphosis of Xenopus laevis tadpoles

Incorporation of [ 3H|uridine into the ribonucleoside triphosphates UTP and CTP, total RNA, and nuclear and cytoplasmic RNA was followed in Xcnopus laevis tadpole liver during thyroxine (T 4)-induced metamorphosis. Pool sizes of UTP and CTP were found to remain unchanged, although turnover of the ribonucleoside triphosphates was found to be greatly stimulated after 4 days of hormone treatment. The time course of labeling of the 40-S pre-rRNA was very similar to that of UTP in both thyreostatic and T4-treatcd tadpoles, thus reflecting a direct relationship between turnover of the immediate precursors and labeling of RNA. Although a faster depletion of labeled UTP and prc-rRNA (precursor ribosomal RNA) was noted in T 4-treated tadpoles, labeled cytoplasmic rRNA continued to accumulate almost linearly for 24 h. In thyreostatic larvae no further increase in labeled cytoplasmic rRNA occurred beyond 4 h of labeling. From these results we conclude that both enhanced transcription and more effective utilization of pre-rRNA arc responsible for the net accumulation of rRNA observed on the 4th day of T4-induced metamorphosis.

Acceleration of thyroxine-induced metamorphosis by prolactin antiserum

The role of extrathyroidal hormones in amphibian growth and metamorphosis is as yet unclear. The involvement of prolactin in these processes has been suggested. In the present study a possible competition between thyroxine and prolactin in metamorphosis was analyzed by administration to tadpoles of a highly purified rabbit prolactin antiserum. The antiserum exhibited no cross-reactivity with ovine or bovine tissue, mammalian plasma proteins, or other mammalian pituitary hormones, as determined by the Ouchterlony double-diffusion precipitin test. Hind limb growth, tail resorption, and transformation of body form were the criteria employed in the analysis of induced metamorphosis. Control tadpoles treated with normal rabbit serum (NRS) or with prolactin antiserum (PR A S ) remained unchanged throughout the experiments. T 4 injected into tadpoles 1 day after the prolactin antiserum accelerated metamorphosis as compared to T 4 alone. We conclude that there is normally present in tadpoles a prolactin-like hormone which exerts an inhibitory effect on the peripheral action of T 4.

Studies on prolactin inhibition of thyroxine-induced metamorphosis in Rana catesbeiana tadpoles

Simultaneously administered prolactin partially prevents the induction by thyroxine of β-glucuronidase in tadpole tail, but does not affect the increases in hepatic ornithine transcarbamylase and tyrosine aminotransferase activities resulting from exposure to thyroxine. Prolactin also decreases the concentration of water-soluble protein of the tail below the levels found in tadpoles in thyroxine or control groups. The decrease in water-soluble protein appears to be mainly due to an increased water content of the tails of tadpoles treated with prolactin.

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.

Alterations in physical parameters and proteins of lens from Rana catesbeiana during development

Lens proteins and lens gross morphology were examined during tadpole and adult development of the bullfrog, Rana catesbeiana. Significant increases in the lens physical parameters of diameter, wet weight, dry weight (94–97% protein), and percent water were observed to accompany both natural and thyroxine-induced metamorphosis. These increases in lens parameters were not accompanied by growth of tadpoles during metamorphic change. Lens proteins were isolated from whole lenses and also from specified layers within whole lenses by a new fractionation method. Electrophoretic examination of whole lenses showed that the lens proteins did not change rapidly, one for another, prior to or during metamorphosis. However, changes became apparent during post metamorphic development. These changes included an increase in the relative concentration and mobility of alpha crystallin, a decrease in the relative concentration of gamma crystallin and an increase in the relative concentration of beta crystallin. Examination of specified layers within tadpole and frog lenses demonstrated that changes in the patterns of lens protein synthesis and modification may occur during development. Rapid and reproducible methods for quantitating changes in lens gross morphology and lens proteins, and for fractionating both tadpole and frog lenses into a number of definable layers were devised in the course of this study.

Unchanged number of rRNA genes in liver and tail muscle of Xenopus larvae during thyroxine induced metamorphosis

Using hybridization experiments the number of rRNA genes was determined in liver and tail muscle of Xenopus larvae during thyroxine-induced metamorphosis. In both tissues the amount of rDNA remained unchanged and was identical to that of adult erythrocyte DNA. Hence, an increase in the number of rDNA templates cannot account for the enhanced synthesis of rRNA during hormone-induced metamorphosis.

Anuran pancreas development during thyroxine-induced metamorphosis of Rana catesbeiana: RNA metabolism during the regressive phase of pancreas development

During thyroxine-induced metamorphosis of Rana catesbeiana tadpoles, the pancrease undergoes extensive regression, due to cell death, before regenerating and differentiating in the adult organ. During pancreatic regression, there is a biphasic stimulation of apparent RNA synthesis. The initial peak of apparent RNA synthesis is due to an increase in the specific activity of the RNA precursor pool without a change in the rate of RNA synthesis. The second phase of stimulation of RNA synthesis is due to an increase in both the specific activity of the precursor pool and the rate of RNA synthesis. A disproportionate increase in the amount of 4 S RNA occurs just prior to the period of maximum pancreatic regression.

A concerted response of the enzymes of urea biosynthesis during thyroxine-induced metamorphosis of Rana catesbeiana.

Abstract During natural metamorphosis, concomitant, linear increases were found in tadpole liver for the activities of the five enzymes in the ornithine-urea cycle: carbamyl phosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, and arginase. Upon immersion in 2.6 x 10-8 m l-thyroxine for two weeks, the biochemical changes which occur during natural metamorphosis were simulated with an elevation of the same enzymes by factors of 8.0, 3.3, 2.4, 2.3, and 3.0, respectively. The lag period prior to the initial rise of each enzyme activity was about 4 days. On the basis of the rate of change of the leg to tail ratio, the thyroxine-exposed tadpoles had an earlier, steeper rise in the levels of each of the five enzymes than that observed during natural metamorphosis. Argininosuccinate synthetase had the lowest specific activity of the ornithine-urea cycle enzymes, even though the assay was improved by the inclusion of urease in the incubation mixture, yielding higher activities than earlier reported. Plotting one enzyme against each of the other ornithine-urea cycle enzymes gave straight lines with high correlation coefficients and similar slopes for both natural and thyroxine-induced metamorphosis. These results demonstrate the simultaneous, concerted response of both mitochondrial and extramitochondrial enzymes in the ornithine-urea cycle enzymes during thyroxine-induced metamorphosis.

Temperature-dependent intracellular distribution of thyroxine in amphibian liver.

Rana catesbeiana tadpoles were injected with [(14)C]thyroxine, and the subcellular distribution of the labeled hormone was determined. At 25 degrees the amount of isotope found in the liver was maximal after 1-2 hr and then rapidly decreased to a relatively constant value. A large percentage of the hormone was found associated with the purified nuclei isolated 24 hr after injection of [(14)C]thyroxine. Injection of [(14)C]thyroxine into tadpoles maintained at 5 degrees resulted in a much slower but constant accumulation of isotope in the liver, with virtually no movement of thyroxine into the cell nucleus. Thyroxine was bound very tightly to the chromatin fraction of the nucleus, but extraction and chromatography revealed no chemical modification of the thyroxine itself. These results suggest the presence of two temperature-dependent processes: one concerned with the transport of thyroxine into the liver cell and a second concerned with the transport of the intracellular thyroxine into the cell nucleus. It is proposed that the latter process is involved in the low-temperature inhibition of thyroxine-induced metamorphosis.

A Concerted Response of the Enzymes of Urea Biosynthesis during Thyroxine-induced Metamorphosis of Rana catesbeiana

A Concerted Response of the Enzymes of Urea Biosynthesis during Thyroxine-induced Metamorphosis of Rana catesbeiana

Alteration of Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase Activities in Amphibian Liver Nuclei during Thyroxine-induced Metamorphosis

Abstract DNA-dependent RNA polymerase activity was determined in isolated tadpole liver nuclei. RNA polymerase I and II activities could be differentiated in situ by the use of α-amanitin. The nuclear RNA polymerases were shown to respond to Mn2+ and Mg2+ in a manner similar to that reported for the same enzymes from other eukaryotes and revealed a biphasic activity curve in the presence of increased ammonium sulfate concentration. It was shown that the two peaks of activity were the result of different optimal activities in the presence of ammonium sulfate of RNA polymerase I (50 mm) and of RNA polymerase II (300 mm). The nearest neighbor and base composition analyses revealed a marked change in the ratio of AMP + UMP: GMP + CMP when the isolated nuclei were assayed in the presence of α-amanitin. While the rate of RNA synthesis with isolated nuclei was nonlinear, this appeared not to be the result of RNA polymerase inactivation or of degradation of the product by ribonuclease. Thyroxine administration, either by injection or immersion of tadpoles, caused marked stimulation of both RNA polymerases I and II. The increases in activities of both enzymes were not detectable until at least 4 days after tadpole immersion in 2.0 x 10-8m thyroxine. An increase in mitochondrial carbamyl phosphate synthetase paralleled that of the RNA polymerases. There was no significant time lag between the increase in RNA polymerase I, RNA polymerase II, and carbamyl phosphate synthetase activities. The increase in RNA polymerase activities was also observed during natural metamorphosis.

Change in Na, K-ATPase activity of Rana catesbeiana tadpole epidermal tissue during thyroxine-induced metamorphosis.

The effect of accelerating metamorphosis with two different doses of L-thyroxine on the epidermal ATPase activity of Rana catesbeiana tadpoles was examined. Na, KATPase activity progressively increased in epidermis of tadpoles treated with 10ng/ml of thyroxine and at the end of the experimental period the value was around 5 times the control group. In the group of tadpoles treated with 100ng/ml of thyroxine, activity of this enzyme changed more rapidly reaching values 7 times the control on the 10th day of treatment. The effect of thyroxine treatment on epidermal content of Mg-ATPase in tadpoles was considerably less than the changes noted above for Na, K-ATPase; levels after 10 days of treatment were only twice control values. In contrast to its effect in tadpoles, thyroxine treatment effected neither ATPase activity nor oxygen consumption of epidermal tissue from adult Rana pipiens frogs. From these observations it might be concluded that Na, K-ATPase activity of frog skin is specifically dependent on thyroid hormones for its initial activation (during metamorphosis) but not its subsequent control.

ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS

Rabbit antibodies were prepared against the major hemoglobin components of the larval and adult stages of R. catesbeiana. The properties of the antisera were studied by double immunodiffusion, precipitation, and complement fixation. The antisera to tadpole and frog hemoglobins did not cross-react with either hemoglobin or apohemoglobin. The anti-serum against frog hemoglobin was used for the detection of frog hemoglobin in tadpoles undergoing either natural or thyroxine-induced metamorphosis. It was shown that frog hemoglobin is detectable first in the liver, indicating that the liver is the site of erythrocyte maturation during metamorphosis.

Autoradiographic studies on metabolism of DNA during thyroxine-induced tadpole metamorphosis

The amount of DNA and RNA, and the rate of orotic 14C-6-acid incorporation into these nucleic acids in tadpole liver do not change during thyroxine-induced metamorphosis. However, autoradiographic studies on liver with 3H-thymidine revealed that thyroxine treatment increased the number of liver cells in DNA synthesis. On the other hand, 3H-thymidine incorporation into nuclei of tail cells was not affected. Evidence is also presented to indicate that tadpole liver contains cytoplasmic DNA which incorporated the 3H-thymidine twice as fast as nuclear DNA.

Changes in the fine structure of tadpole ( Rana catesbeiana) liver during thyroxine-induced metamorphosis

Summary Alterations in the fine structure of tadpole ( Rana catesbeiana ) liver cells have been studied during the period of extensive biochemical modification caused by l -thyroxine. The nuclei, euchromatic in control cells, become heterochromatic. The mitochondria increase significantly in size with slight alterations in the cristae. The rough endoplasmic reticulum proliferates, and the staining density of the cisternal lumens increases. The Golgi complex and its cisternal contents increase in staining density, and the number of Golgi regions increases. These changes provide cytological criteria for the differentiation of liver cells during thyroxine-induced metamorphosis.

Effect of prolactin on thyroxine-induced metamorphosis.

Prolactin inhibits thyroxine-induced tail regression, a metamorphic event which has been unequivocally shown to be caused directly by thyroxine. On the other hand, thyroxineinduced pancreatic regression, and acid phosphatase increase and biochemical differentiation in the liver are not inhibited or counteracted by prolactin treatment. Therefore, prolactin does not antagonize the entire process of metamorphosis. These studies also indicate that some metamorphic events are not directly induced by thyroxine (Endocrinology85: 1213, 1969)

The Effect of Thyroxine on Ribonucleic Acid Synthesis by Premetamorphic Tadpole Liver Cell Suspensions

Abstract Suspensions of tadpole liver cells have been developed as a model to further understanding of the biochemical differentiation of this tissue during thyroxine-induced metamorphosis. The cell population is homogeneous and stable; no cell division or DNA synthesis occurs in these isolated cells. RNA synthesis in cell suspensions is stimulated by thyroxine and triiodothyronine. The optimum concentration of both thyroxine and triiodothyronine for stimulation of RNA synthesis is about 10-7 m. Rapidly labeled RNA isolated from thyroxine-treated and nontreated cell suspensions sediments in the region between 4 and 10 S. The base composition of RNA synthesized by liver cells treated in vitro with thyroxine differs significantly from that of untreated liver cells and from bulk tadpole liver RNA. The ratio of cytosine plus guanine to total bases of RNA from thyroxine-treated cells is similar to that of DNA from this species. Neither thyroxine nor triiodothyronine had any effect on the incorporation of amino acids into protein. Frog serum stimulates RNA and protein synthesis. Rana catesbeiana serum causes the aggregation of isolated liver cells.

The effect of thyroid function on hemoglobin differentiation in the chick

ABSTRACT The switching off of the synthesis of embryonic hemoglobin (EHb) and the turning on of adult hemoglobin (AHb) synthesis has served as one of several major examples of biochemical differentiation. However, the control mechanisms underlying this transformation remained elusive, except in the case of Rana catesbeiana (Moss &Ingram, 1965, 1968a, b) where the classical thyroxine-induced metamorphosis effected a precocious shift from EHb to AHb. In the chick embryo such a dramatic metamorphosis does not occur, but a change from EHb to AHb can be detected electrophoretically on the sixth day of incubation (Hashimoto &Wilt, 1966; Manwell, Baker &Betz, 1966).