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.     

DNA SYNTHESIS AND CELL TURNOVER IN RANA PIPIENS TADPOLE LIVER DURING SPONTANEOUS METAMORPHOSIS1

The relationship of DNA synthesis and cellular turnover to biochemical differentiation during metamorphosis of R. pipiens liver was investigated. Average DNA/cell was constant at 11.6 pg/ nucleus through stage XXV; but increased during juvenile growth; during metamorphosis stages, changes in total DNA content must correspond to changes in cell number. Rates of DNA synthesis were estimated by rates of ³H-thymidine incorporated into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. DNA content increased steadily from premetamorphosis until late prometamorphosis; at preclimax stages XVIII and XX there were two successive decreases in DNA content of approximately 30%. Fluctuations in synthesis rates preceded corresponding fluctuations in content; DNA synthesis was maximal at stages XVI and XVIII, decreased nearly ten-fold at metamorphic climax, and then gradually rose again during late climax stages. The size of the endogenous thymine pool increased transitorily during spontaneous metamorphosis corresponding to a stage of maximal DNA synthesis. These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthesis rates and content during metamorphosis. Metamorphosis of the tadpole liver appears to be associated with both proliferation and cellular death, perhaps a replacement of “larval” by “adult” cells. Metamorphosis of the liver cannot be occuring in a “fixed population of cells” as is commonly assumed. An interpretation of the population dynamics of the metamorphic liver is presented.     

DNA SYNTHESIS IN RANA PIPIENS TADPOLE LIVER DURING TRIIODOTHYRONINE-INDUCED METAMORPHOSIS1

The relationship of DNA synthesis and cellular turnover to biochemical differentiation during Ts-induced metamorphosis of R. pipiens liver was investigated. Rates of DNA synthesis were estimated by rates of 3 H-thymidine incorporation into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. During T3 -induced metamorphosis, periods of DNA synthesis and fluctuations in DNA content preceded expression of biochemical differentiation as measured by the enzyme arginase, and fluctuations in synthesis rates preceded corresponding fluctuations in content. The earliest response to T3- , was a 50% decrease in liver DNA, followed by increases in thymidine incorporation at 16 hr, 2 days, and 5-8 days. The size of the endogenous thymine pool was not significantly altered by T3 These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthetic rates and content during metamorphosis. Expression of thyroxin-induced development of the tadpole liver appears to be associated with both proliferation and cellular death, and metamorphosis of the liver cannot be occurring in a “fixed population of cells.”     

粤北山区饰纹姬蛙胚后发育的初步观察

初步观察记录了饰纹姬蛙Microhyla ornata胚后发育过程。饰纹姬蛙的卵采于广东韶关小坑国家森林公园,置于人工实验缸中自然孵化,在23.0～28.7℃水温条件下观察饰纹姬蛙蝌蚪胚后发育的过程。该蛙的胚后发育可以分为19期,历时31.1d,对其后肢芽、趾、前肢的发育,以及肛管及尾的变化等方面做了系统的描述,饰纹姬蛙蝌蚪的头长、体长、尾长和后肢长度随发育时间的增加而显著增长。     

The formation, distribution and function of ribosomes and microsomal membranes during induced amphibian metamorphosis

1. A lag period of about 4 days preceded the onset of metamorphosis precociously induced by tri-iodothyronine in tadpoles of the giant American bullfrog (Rana catesbeiana). It was established by the accelerated synthesis or induction of carbamoyl phosphate synthetase and cytochrome oxidase in the liver, serum albumin and adult haemoglobin in the blood, acid phosphatase in the tail, and the increase in the hindleg/tail length ratio. 2. A 4- to 6-fold stimulation, 2 days after the induction of metamorphosis, of the rate of synthesis of rapidly labelled nuclear RNA in liver cells was followed by an increasing amount of RNA appearing in the cytoplasm. Most of the newly formed RNA on induction of metamorphosis was of the ribosomal type. An accelerated turnover at early stages of development preceded a net accumulation of RNA in the cytoplasm, with no change in the amount of DNA per liver. 3. Most hepatic ribosomes of the pre-metamorphic tadpoles were present as 78s monomers and 100s dimers; metamorphosis caused a shift towards larger polysomal aggregates with newly formed ribosomes that were relatively more tightly bound to membranes of the endoplasmic reticulum. 4. The appearance of new polyribosomes in the cytoplasm on induction of metamorphosis was co-ordinated in time with a stimulation of synthesis of phospholipids of the smooth and rough endoplasmic reticulum, followed by a gradual shift in preponderance from the smooth to the rough type of microsomal membranes. 5. Electron- and optical-microscopic examination of intact hepatocytes revealed a striking change in the distribution and nature of ribosomes and microsomal membranes during metamorphosis. 6. Ribosomes prepared from non-metamorphosing and metamorphosing animals were identical in their sedimentation coefficients and in the structural ribosomal proteins. The base composition and sedimentation coefficients of ribosomal RNA were also identical. Induction of metamorphosis also did not alter the incorporation of (32)P into the different phospholipid constituents of microsomal membranes. 7. Nascent (14)C-labelled protein with the highest specific activity was recovered in the ;heavy' rough membrane fraction of microsomes, whereas little (14)C was associated with ;free' polysomes. Protein synthesis in vivo was most markedly stimulated during metamorphosis in the tightly membrane-bound ribosomal fraction after the appearance of new ribosomes. 8. The rate of synthesis of macromolecules in vivo could not be followed beyond 7-8 days after induction because of variable shifts in precursor pools due to regression of larval tissues. 9. The stimulation of RNA and ribosome formation was specifically associated with the process of metamorphosis since no similar response to thyroid hormones occurred in those species (Axolotl and Necturus) in which the hormones failed to induce metamorphosis.     

Changes in amino acid uptake and protein synthesis of bullfrog tadpole liver and tail tissues during triiodothyronine-induced metamorphosis

The effect of triiodothyronine (T₃′) on the uptake of several amino acids into the amino acid pools and into proteins of Rana catesbeiana tadpole liver and tail muscle and tail fin has been studied. Labeling of the alanine and glycine pool was stimulated in the liver more than the leucine pool. After exposure to T₃ for 3 days, uptake of α-aminoisobutyric acid (a transport model substrate) into liver was stimulated about 55%. In tail tissues uptake of leucine was stimulated but uptake of alanine was depressed by T₃. Incorporation of leucine and alanine into tissue protein was stimulated in the liver but inhibited in tail tissues after T₃ injection.Changes in other macromolecules and ATP and ADP levels in liver and tail muscle were also investigated during induced metamorphosis. In the liver, the total DNA content did not change, but the RNA and protein content per liver increased significantly. The increase in RNA/DNA and protein/DNA ratios, suggested that liver cells underwent hypertrophy during induced metamorphosis. The ATP level showed a transient decrease after 3 days of T₃ treatment. In tail muscle, protein and RNA content decreased as the muscle regressed, but the DNA content and ATP level remained unchanged throughout the experimental period.     

Changes in the pattern of RNA synthesis in different tissues of Xenopus larvae during induced metamoprhosis

The pattern of newly synthesized RNA in liver, tail muscle, brain and muscle of hindlimbs during induced metamorphosis was analysed by electrophoretic separation of RNA extracts on exponential polyacrylamide gels. During the first 24 h of thyroxine treatment the distribution of labelled RNA was identical in treated animals and controls. Four days after induction of metamorphosis relatively more radioactivity was observed in stable RNA, especially rRNA, whereas unstable RNA, mainly HnRNA, was relatively less labelled. Apart from these general effects, in tail muscle thyroxine treatment caused the disappearance of a definite high molecular weight RNA. Moreover maturation of 28 S rRNA was accelerated and degradation of newly synthesized rRNA was no more detectable.     

The dynamics of proliferation and differentiation of osteogenic cells under supportive unloading

With the use of radioactive marker of DNA synthesis--3H-thymidine we have studied the dynamics, peculiarities of proliferation and differentiation of osteogenic cells under hind limb unloading of white rats ("tail suspension" method at an angle 35 degrees) during 28 days. The 3H-thymidine was administered at a single dose at the end of the experiment, the biosamples were taken from femoral bones in 1, 48, 96 hr. Light and electron-microscopic radioautography with 3H-thymidine (in 1 hour) have shown, that basic fraction of DNA synthesizing cells in the zones of adaptive remodelling of bone tissue is represented by little-differentiated perivascular cells (that include osteogenic cell precursors). A tendency for a decrease of a labelling index in the 3H-thymidine osteogenic cells on metaphyseal bone trabeculae under hind limb unloading has been established. The dynamics of labelled cells during various time intervals after 3H-thymidine injection testifies to a delay in the differentiation precursors in osteoblasts and their transformation to osteocytes in experiment animals. The obtained data have shown that a long-term supportive unloading leads to lowering the intensity of osteogenetic processes in long bones and reducing bone mass.     

Simultaneous induction of ectopic pelvic zone and duplication of regenerated limbs in tadpoles of Polypedates maculatus by vitamin A

With a view to determine ectopic limb developing capacity along with normal hind limb regeneration in response to vitamin A palmitate in well-differentiated hind limb stage tadpoles of P. maculatus, higher doses of vitamin A (30 IU/ml and 20 IU/ml) were administered for a longer period (120 hr) to the tadpoles following tail amputation through middle and hind limb amputation through middle of thigh. Simultaneous development of ectopic pelvic zone was observed along with hind limbs from the cut end of tail and duplication of regenerated hind limbs in the same tadpole for the first time. Besides, development of double ectopic pelvic girdle was also reported in one case. Results also indicate that induction of pelvic zone and duplication of regenerated limbs are concentration dependent.     

Molecular cloning of hepatic mRNAs in Rana catesbeiana responsive to thyroid hormone during induced and spontaneous metamorphosis

Amphibian metamorphosis affords a useful experimental system in which to study thyroid hormone regulation of gene expression during postembryonic vertebrate development. In order to isolate gene-specific cDNA probes which correspond to thyroid hormone-responsive mRNAs, we employed differential colony hybridization of a cDNA library constructed from poly(A)+ RNA of thyroxine-treated premetamorphic tadpole liver. From an initial screening of about 6000 transformants, 32 "potentially positive" colonies were obtained. The recombinant cDNA-plasmids from 13 of these colonies plus two "potentially negative" colonies were purified for further study. Southern blot analysis of the plasmid DNA was employed to determine whether different cDNAs encoded for the same mRNA. The effect of thyroid hormone on the relative levels of specific mRNA species was examined by Northern analysis of liver RNA from premetamorphic tadpoles, thyroxine-treated tadpoles, and adult bullfrogs. Three independent cDNA clones were obtained which encoded thyroid hormone-enhanced mRNAs. We also obtained two independent cDNA clones encoding thyroid hormone-inhibited mRNAs and three independent clones encoding thyroid hormone-unresponsive mRNAs. The levels of two thyroid hormone-enhanced mRNAs and one thyroid hormone-inhibited mRNA were essentially the same in the thyroid hormone-treated tadpole liver and adult liver, suggesting that thyroid hormone induces stable changes in liver gene expression during spontaneous metamorphosis. Using selected cDNAs, RNA dot blot analysis of liver mRNA from tadpoles at different stages of metamorphosis showed that the level of one thyroid hormone-enhanced mRNA increased during late prometamorphosis and metamorphic climax. Similarly, a mRNA which was strongly inhibited by thyroid hormone treatment was observed to decline during prometamorphosis and reach undetectable levels during metamorphic climax. One mRNA was detected which was reproducibly inhibited by thyroid hormone treatment but which remained essentially unchanged during spontaneous metamorphosis. These results provide the first direct evidence for the coordinate and selective pretranslational regulation by thyroid hormone of several liver genes during the developmental process of metamorphosis.     

Regulation of mitochondrial protein turnover by thyroid hormone(s)

1. The effect of thyroidectomy on turnover rates of liver, kidney and brain mitochondrial proteins was examined. 2. In the euthyroid state, liver and kidney mitochondria show a synchronous turnover with all protein components showing more or less identical half-lives compared with the whole mitochondria. The brain mitochondrial proteins show asynchronous turnover, the soluble proteins having shorter half-lives. 3. Mitochondrial DNA (m-DNA) of liver and kidney has half-lives comparable with that of whole mitochondria from these tissues. 4. Thyroidectomy results in increased half-lives of liver and kidney mitochondria, with no apparent change in the half-life of brain mitochondria. 5. A detailed investigation of the turnover rates of several protein components revealed a significant decrease in the turnover rates of mitochondrial insoluble proteins from the three tissues under study. 6. The turnover rates of m-DNA of liver and kidney show a parallel decrease. 7. Thus it is apparent that thyroid hormone(s) may have a regulatory role in maintaining the synchrony of turnover of liver and kidney mitochondria in the euthyroid state. Turnover of brain mitochondria may perhaps be regulated by some other factor(s) in addition to thyroid hormone(s). 8. It seems likely that during mitochondrial turnover m-DNA and insoluble proteins may constitute a major unit. 9. The mitochondrial protein contents of the three tissues are not affected by thyroidectomy. 10. No correlation was seen between the turnover rate of mitochondria and cathepsin activity in any of the tissues under study in normal or thyroidectomized animals. 11. On the other hand, mitochondrial proteinase activity shows good correlation with the turnover rates of mitochondria in normal animals, and a parallel decrease in activity comparable with the decreased rates of turnover is observed after thyroidectomy. 12. It is concluded that mitochondrial proteinase activity may play a significant role in their protein turnover.     

Increased SCE levels in Mediterranean Italian buffaloes affected by limb malformation (transversal hemimelia)

In recent years some buffalo farms in Campania have reported the birth of calves with limb malformation, especially with transversal hemimelia. We investigated 20 Mediterranean Italian buffaloes (8 males and 12 females) from one day to six months of age, of which 10 were affected by transversal hemimelia (group 1) and 10 were healthy controls (group 2). The following clinical and radiological patterns were observed in the malformed animals: hind limbs amputated, the right amputated off the second tarsus bones and the left amputated off the proximal epiphysis metatarsus, and the right thoracic limb hypoplasic (1 female); left hind limb amputated off the proximal epiphysis metatarsus (2 females and 1 male); left hind limb amputated off the third tarsus bones (1 female); left hind limb amputated off the tibia (1 female and 1 male); left hind limb amputated off the distal epiphysis metatarsus (1 female); left hind limb amputated off the first phalanx (1 male); right hind limb amputated off the proximal epiphysis metatarsus (1 male). In their malformed limbs all the animals presented more or less developed outlines of claws. The mean rate of SCE/cell in animals with transversal hemimelia was 8.80 +/- 3.19, that of the controls 6.61 +/- 2.73. The difference was statistically significant (P < 0.001).     

Thyroxine-binding proteins in liver and tail of Rana catebeiana undergoing metamorphosis.

Presence of a thyroxine-binding protein was demonstrated in vivo in cell sap of tail and liver of metamorphosing Rana catesbeiana tadpoles. Thyroxine-binding protein was not present in tail of prematamorphic tadpoles while it appeared during progressing metamorphosis roughly coinciding with the beginning of tail resorption. Susceptibility to pronase indicates that this thyroxine-binding macromolecule is protein in nature. Thyroxine-binding in liver was already present during premetamorphic stages and increased further during metamorphosis. A further difference between tail and liver thyroxine-binding protein was evidenced by molecular sieve chromatography on Sephadex G-200 indicating a molecular weight of thyroxine-binding protein in the tail of 60 000 as opposed to 42 000 for liver. Scatchard analysis of tail cell sap of tadpoles in metamorphic climax revealed a high affinity thyroxing binding site (Kd of 2 - 10(-10) M) of low capacity (1.7 pmol per mg protein) while tadpoles in premetamorphic stage had a thyroxine-binding site of lower affinity (9 - 10(-10) M) and higher capacity (4.8 pmol per mg protein). Thus affinity of thyroxine binding is 4-fold in metamorphic climax and appears to reflect the appearance of thyroxine binding observed in vivo.     

Role of type III iodothyronine 5-deiodinase gene expression in temporal regulation of Xenopus metamorphosis

To elucidate the role of type III iodothyronine 5-deiodinase (5-D) in the temporal regulation of amphibian metamorphosis, the regulation of gene expression of 5-D and thyroid hormone receptor beta (TRbeta) in organs of Xenopus laevis was investigated. High levels of TRbeta mRNA in the respective organs were observed at the times of their major morphological changes. Expression of the 5-D gene was highly regulated among the organs during metamorphosis, including up-regulation in the tail and down-regulation in the liver. The tail and liver expressed 5-D gene before their metamorphic changes. These precocious expressions correlated with the lower responsiveness to exogenously added triiodo-L-thyronine (T3) for inducing a high level of TRbeta mRNA expression. However, the same organs responded to lower doses of T3 to regulate 5-D gene expression as seen in spontaneous metamorphosis. The induction of 5-D gene expression was considerably delayed in the intestine, even at an excess dose of T3. Thus, the two genes in a given organ appeared to respond to T3 either with different dose dependencies or with different timetables. The results obtained are also discussed in respect to recent findings in Rana catesbeiana.     

Immunocytochemistry of luteinizing hormone-releasing hormone during spontaneous and thyroxine-induced metamorphosis of bullfrogs

Double-bridge peroxidase-antiperoxidase immunocytochemistry was used to compare the developmental appearance of immunoreactive LH-RH (ir-LH-RH) in brains of bullfrog (Rana catesbeiana) tadpoles during either spontaneous or thyroxine-induced metamorphosis. During spontaneous metamorphosis, ir-LH-RH was localized in fibers of the external layer of the median eminence (ME) of stage XIII-XXV animals, while immunoreactive perikarya and other immunostained brain structures were absent. The extent and intensity of ME immunostaining increased concomitantly with measured ME morphological development. Tadpoles induced with thyroxine to metamorphic stages XIX-XXI exhibited ME structural development and neurohypophysial neurosecretory staining similar to spontaneously metamorphosed individuals of equal stages. However, comparable ME ir-LH-RH immunostaining and gonadal size were both less developed in thyroxine-treated animals, although increased relative to non-metamorphic vehicle-injected controls. These results indicate that the hypothalamic LH-RH system changes concurrently with ME structural development during spontaneous metamorphosis. Reduced ME ir-LH-RH staining and gonadal size in thyroxine-treated animals suggest that during prometamorphosis, factors other than thyroxine alone may coordinate the normal maturation of the hypothalamo-pituitary-gonadal axis of the bullfrog.     

Sexual size dimorphism in Darevskia raddei (Sauria: Lacertidae) from northwestern Iran

We examined sexual size dimorphism of the rock-dwelling lizard Darevskia raddei (Boettger, 1892) with the help of 30 specimens that were provided from various sources. Eleven metric and seven meristic features were examined. Seven characters (gulars, length of basal tail, femoral pores, length of head, width of head, length of fore limb and length of hind limb) were identified as dimorphic between the two sexes. Some of these characters have important roles in copulation for males, especially the hind limb and the tail base. The number of femoral pores is important in the release of signal components because females release these components to attract males during the mating season. The length of the hind limb as locomotor performance plays an important role during mating, so that the male can grasp the female and adopt the correct position during copulation.     

Corticotropin-releasing factor accelerates metamorphosis in Bufo arenarum: effect on pituitary ACTH and TSH cells

The actions of several neuropeptides as hypothalamic mediators in the regulation of Bufo arenarum metamorphosis were investigated. Prometamorphic larvae were injected with 1.5 microg thyrotropin-releasing hormone (TRH), 2 microg ovine corticotropin-releasing factor (oCRF), 2 microg mammalian gonadotropin-releasing hormone (mGnRH), 2 microg human growth hormone-releasing hormone (hGHRH), or Holtfreter solution (control group). Larvae received two injections with the same dose: one at the beginning of the experiment and the other 7 days later. Several morphologic parameters (total length, tail length, wet weight, hind limb length, and metamorphic stages) were measured as indicators of growth and metamorphic development. These measurements were taken in 20 larvae per treatment or control group at the beginning of the experiment, at day 7 and at day 14 when the experiment ended. We observed that only the administration of exogenous CRF stimulated resorption of the tail and accelerated the rate of metamorphosis. In the pituitary of CRF-treated larvae we observed that thyrotropin (TSH) and adrenocorticotropic hormone (ACTH) producing cells showed a weaker immunoreactivity, a decrease in cell number and a reduction of volume density when compared with normal larvae. In conclusion, the results obtained indicate a possible role for CRF in Bufo arenarum metamorphosis. CRF may regulate interrenal and thyroid activity by acting directly upon TSH and ACTH cells. On the other hand, TRH, GnRH and GHRH were inactive in stimulating growth or metamorphosis of Bufo arenarum. J. Exp. Zool. 286:473-480, 2000.     

Effect of electromagnetic radiofrequency radiation on the rats' brain, liver and kidney cells measured by comet assay

The goal of study was to evaluate DNA damage in rat's renal, liver and brain cells after in vivo exposure to radiofrequency/microwave (Rf/Mw) radiation of cellular phone frequencies range. To determine DNA damage, a single cell gel electrophoresis/comet assay was used. Wistar rats (male, 12 week old, approximate body weight 350 g) (N = 9) were exposed to the carrier frequency of 915 MHz with Global System Mobile signal modulation (GSM), power density of 2.4 W/m2, whole body average specific absorption rate SAR of 0.6 W/kg. The animals were irradiated for one hour/day, seven days/week during two weeks period. The exposure set-up was Gigahertz Transversal Electromagnetic Mode Cell (GTEM--cell). Sham irradiated controls (N = 9) were apart of the study. The body temperature was measured before and after exposure. There were no differences in temperature in between control and treated animals. Comet assay parameters such as the tail length and tail intensity were evaluated. In comparison with tail length in controls (13.5 +/- 0.7 microm), the tail was slightly elongated in brain cells of irradiated animals (14.0 +/- 0.3 microm). The tail length obtained for liver (14.5 +/- 0.3 microm) and kidney (13.9 +/- 0.5 microm) homogenates notably differs in comparison with matched sham controls (13.6 +/- 0.3 microm) and (12.9 +/- 0.9 microm). Differences in tail intensity between control and exposed animals were not significant. The results of this study suggest that, under the experimental conditions applied, repeated 915 MHz irradiation could be a cause of DNA breaks in renal and liver cells, but not affect the cell genome at the higher extent compared to the basal damage.