Gene expression and activity of urea cycle enzymes of rat hepatocytes cold stored up to 120h in University of Wisconsin solution

Urea cycle (UC) is the main pathway of ammonium removal. A deficiency in any of the five classical enzymes of the pathway causes a urea cycle disorder. Hepatocellular transplantation is one of the techniques applicable to treat this disorder. In the present work, we investigated the activities and the relative expression levels of two of the UC enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), in isolated hepatocytes preserved up to 120 h in University of Wisconsin (UW) solution at 0 degrees C, and during the rewarming of these suspensions. During preservation, CPSI showed differences in mRNA levels respect to time 0, while ornithine transcarbamylase remained unchanged. At the end of the rewarming, CPSI showed values of enzymatic activity and relative mRNA level comparable with the control, meanwhile, there was an increment in OTC activity. In line with these results, we found that hepatocytes cold preserved up to 120h in UW solution maintained their ability to remove an ammonium load comparable to freshly isolated hepatocytes. These data indicated that our preservation conditions up to 120h in UW solution followed by rewarming, preserves UC enzymes at levels similar to freshly isolated hepatocytes, allowing the use of these cells in bioartificial liver devices or hepatocellular transplantation.     

Rhesus glycoprotein and urea transporter genes are expressed in early stages of development of rainbow trout (Oncorhynchus mykiss)

The objective of this study was to determine if the genes for the putative ammonia transporters, Rhesus glycoproteins (Rh) and the facilitated urea transporter (UT) were expressed during early development of rainbow trout, Oncorhynchus mykiss Walbaum. We predicted that the Rh isoforms Rhbg, Rhcg1 and Rhcg2 would be expressed shortly after fertilization but UT expression would be delayed based on the ontogenic pattern of nitrogen excretion. Embryos were collected 3, 14 and 21 days postfertilization (dpf), whereas yolk sac larvae were sampled at 31 dpf and juveniles at 60 dpf (complete yolk absorption). mRNA levels were quantified using quantitative polymerase chain reaction and expressed relative to the control gene, elongation factor 1alpha. All four genes (Rhbg, Rhcg1, Rhcg2, UT) were detected before hatching (25-30 dpf). As predicted, the mRNA levels of the Rh genes, especially Rhcg2, were relatively high early in embryonic development (14 and 21 dpf), but UT mRNA levels remained low until after hatching (31 and 60 dpf). These findings are consistent with the pattern of nitrogen excretion in early stages of trout development. We propose that early expression of Rh genes is critical for the elimination of potentially toxic ammonia from the encapsulated embryo, whereas retention of the comparatively benign urea molecule until after hatch is less problematic for developing tissues and organ systems.     

Mechanisms and Consequences of Developmental Acceleration in Tadpoles Responding to Pond Drying

Many amphibian species exploit temporary or even ephemeral aquatic habitats for reproduction by maximising larval growth under benign conditions but accelerating development to rapidly undergo metamorphosis when at risk of desiccation from pond drying. Here we determine mechanisms enabling developmental acceleration in response to decreased water levels in western spadefoot toad tadpoles (Pelobates cultripes), a species with long larval periods and large size at metamorphosis but with a high degree of developmental plasticity. We found that P. cultripes tadpoles can shorten their larval period by an average of 30% in response to reduced water levels. We show that such developmental acceleration was achieved via increased endogenous levels of corticosterone and thyroid hormone, which act synergistically to achieve metamorphosis, and also by increased expression of the thyroid hormone receptor TRΒ, which increases tissue sensitivity and responsivity to thyroid hormone. However, developmental acceleration had morphological and physiological consequences. In addition to resulting in smaller juveniles with proportionately shorter limbs, tadpoles exposed to decreased water levels incurred oxidative stress, indicated by increased activity of the antioxidant enzymes catalase, superoxide dismutase, and gluthatione peroxidase. Such increases were apparently sufficient to neutralise the oxidative damage caused by presumed increased metabolic activity. Thus, developmental acceleration allows spadefoot toad tadpoles to evade drying ponds, but it comes at the expense of reduced size at metamorphosis and increased oxidative stress.     

Xenopus laevis serum albumin: sequence of the complementary deoxyribonucleic acids encoding the 68- and 74-kilodalton peptides and the regulation of albumin gene expression by thyroid hormone during development

In adult Xenopus serum, albumin gene expression is regulated by estrogen through the selective destabilization of its mRNA during the vitellogenic response. The present study reports the cDNA sequence of both the 68K and 74K Xenopus albumin mRNAs, their derived amino acid sequence, and the regulation of albumin gene expression during embryogenesis. Albumin mRNA has a 39 nucleotide 5' untranslated region terminating in a consensus translation initiation site. The derived amino acid sequence yields a 24-amino acid hydrophobic leader sequence (terminating in Lys-Arg) that shares significant homology with the leader peptide of rat albumin. Overall there is 37% sequence identity between rat and frog albumin, with exact conservation of all but one Cys residue and the Pro residues responsible for the three domain structure of the mature protein. The 74K albumin (unlike the 68K albumin) is glycosylated; a point mutation converting Lys256 to Asn introduces an N-linked glycosylation site that is similar to one found in the sequence of mammalian alpha-fetoproteins. A larval albumin-like protein was not detectable by silver staining in serum of tadpoles before the beginning of metamorphosis at stage 48. Albumin mRNA is absent from early tadpoles (stages 22-47); however, it is rapidly induced at stage 48 as one of the earliest manifestations of metamorphosis. Exposure of embryos to 10(-8) M T3, which regulates amphibian metamorphosis, resulted in the premature induction of albumin mRNA, such that it is evident by stage 43.     

Sequential up-regulation of thyroid hormone beta receptor, ornithine transcarbamylase, and carbamyl phosphate synthetase mRNAs in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis.

During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TR beta-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TR beta gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TR beta, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TR beta and OTC mRNAs.     

Cloning and thyroid hormone regulation of albumin mRNA in Rana catesbeiana tadpole liver.

Thyroid hormones are responsible for the specific biochemical and structural changes that occur during amphibian metamorphosis. In this study we screened a series of cDNAs from a library constructed from T4-treated premetamorphic tadpole liver poly(A)+ RNA in order to identify a clone that could be used to study the influence of T3 on liver-specific gene expression during Rana catesbeiana metamorphosis. The cDNA from one clone exhibited a greater degree of hybridization to liver RNA from thyroid hormone-treated tadpoles than untreated tadpoles and no hybridization to RNA from tail fins of tadpoles of either group. On Northern blots, the mRNA to which the cDNA hybridized was 2.3 kilobases in size. The pattern of hybridization to genomic DNA digested by various restriction enzymes was consistent with the presence of a single gene. Using slot blot analysis we found that the mRNA levels first rose above basal levels only after 5 days of immersion of tadpoles in 12.5 micrograms/liter T3. The mRNA levels increased approximately 10-fold after 7 and 9 days of treatment. Frog livers had mRNA levels that were intermediate between those in untreated tadpoles and tadpoles immersed in T3 for 7 days. Sequence analysis revealed a significant degree of homology to serum albumin and alpha-fetoprotein. While it is known that serum albumin levels rise dramatically during metamorphosis in Rana species, presumably playing a critical role in maintaining water and electrolyte balance during the animals' terrestrial phase, the molecular basis of the induction has not been fully explained.     

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.     

High temperatures influence sexual development differentially in male and female tadpoles of the Indian skipper frog, <Emphasis Type="Italic">Euphlyctis cyanophlyctis</Emphasis>

Although sex determination in amphibians is believed to be a genetic process, environmental factors such as temperature are known to influence the sex differentiation and development. Extremely low and high temperatures influence gonadal development and sex ratio in amphibians but the mechanism of action is not known. In the present study, effect of different temperatures on gonadal development, sex ratio and metamorphosis was studied in the Indian skipper frog, Euphlyctis cyanophlyctis. The embryos of Gosner stage 7 were exposed to 20, 22, 24, 26, 28, 30 and 32°C up to tadpole stage 42. The embryos (stage 7) were also exposed to 20 and 32°C up to tadpole stage 25 (non-feeding stages). Tadpoles of stage 25 were reared at 20 and 32°C up to stage 42 (feeding stages). The results show that exposure to higher temperatures (28, 30 and 32°C) during stages 7–42 produced male-biased sex ratio. Rearing of tadpoles at 32°C during stages 25–42 produced male-biased sex ratio, while exposure during stages 7–25 did not affect sex ratio. Embryos and tadpoles exposed to lower temperatures (20 and 22°C) died during the early stages. High temperatures stimulated testis development, and disturbed ovary development. Exposure to high temperatures resulted in the early metamorphosis of tadpoles with reduced body size. These results demonstrated that high temperatures influence gonadal development differently in male and female tadpoles, leading to male-biased sex ratio. These results suggest that high temperature probably acts through stress hormones and favours the small-sized sex.     

Sequential up-regulation of thyroid hormone β receptor,ornithine transcarbamylase,and carbamyl phosphate synthetase mRNAs in the liver of Rana catesbeiana tadpoles during spontaneous and thyroid hormone-induced metamorphosis

During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TRβ-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TRβ gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TRβ, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TRβ and OTC mRNAs. © 1992 Wiley-Liss, Inc.     

Searching for the physiological mechanism of density dependence: does corticosterone regulate tadpole responses to density?

Density-dependent population regulation is important in many natural populations, and in those populations, high population density is a likely stressor. In amphibians, previous laboratory studies with tadpoles suggest that corticosterone, the main glucocorticoid stress hormone, is one of the key regulators of density-dependent growth. To test this relationship in natural settings, we manipulated wood frog (Rana sylvatica) tadpole density at three levels in outdoor mesocosms and used a capture stress protocol to examine the hormonal stress response. In addition, we used the same capture protocol in six natural ponds (three high density and three low density). In the mesocosms, there was an increase in corticosterone levels in tadpoles following 1 h of confinement at weeks 1, 2, and 5. However, while tadpoles maintained at higher densities were smaller after metamorphosis, density did not alter mean levels of corticosterone obtained during confinement, and baseline levels of corticosterone did not differ between the densities. Similarly, in natural ponds, density did not correlate with baseline corticosterone or mean corticosterone levels obtained during confinement. We suggest that the physiological response to density may vary across the range of natural densities and that the role of corticosterone may be limited to periods of extreme high density, such as during pond-drying events.     

Effects of hydroperiod duration on developmental plasticity in tiger frog(Hoplobatrachus chinensis) tadpoles

While developmental plasticity can facilitate evolutionary diversification of organisms,the effects of water levels as an environmental pressure on tiger frogs remains unclear.This study clarifies the relationship by studying the responses of tiger frog(Hoplobatrachus chinensis)tadpoles to simulated hydroperiods(i.e.,constant low water levels,constant high water levels,increasing water levels,decreasing water levels,rapid changes in water levels and gradual fluctuations in water levels)in a laboratory setting.ANOVA analysis showed that none of the water level treatments had any significant effect on the total length,body mass,or developmental stages of H.chinensis tadpoles half way through development(11 days old).Tadpoles raised in rapidly fluctuating water levels had protracted metamorphosis,whereas tadpoles raised under low and gradually fluctuating water levels had shortened metamorphosis.None of the water level treatments had a significant effect on the snout-vent length(SVL)or body mass of H.chinensis tadpoles at Gosner stage 42,or on the body mass of tadpoles at Gosner stage 45.However,the tadpoles raised in high levels and rapidly fluctuating water levels,significantly larger SVL at Gosner stage 45,while ones under gradually fluctuating water levels had smaller SVL than the other groups.Time to metamorphosis was positively correlated with body size(SVL)at metamorphosis in H.chinensis tadpoles.H.chinensis tadpoles under constant low water level had the highest mortality rate among all the treatments(G-test).Moreover,ANOVA and ACNOVA(with body length as the covariate)indicated that water levels had no significant effect on either the morphology(i.e.head length,head width,forelimb length,hindlimb length and body width)or the jumping ability of juvenile H.chinensis.These results suggest that the observed accelerated metamorphosis and high mortality of H.chinensis tadpoles under decreasing water level treatment was driven by density-induced physical interactions among increasing conspecifics.     

Retardation of thyroxine-induced metamorphosis by Amphenone B in toad tadpoles

The effect of Amphenone B, an inhibitor of corticoid synthesis, on thyroxine (T4)-induced metamorphosis was studied in toad tadpoles kept in thiourea. Amphenone injections retarded T4-induced tail resorption markedly. The effect of Amphenone was nullified by aldosterone and corticosterone added to the water in which tadpoles were kept. Steroidogenic cells of adrenals in Amphenone-injected animals were enlarged markedly as compared with those in the saline-injected tadpoles or the Amphenone-injected tadpoles which were supplemented with corticoids. The results strongly suggest that endogenous corticoids act together with thyroid hormone to accelerate metamorphosis.     

Developmental expression of the creatine kinase isozyme system of Xenopus: maternally derived CK-IV isoform persists far beyond the degradation of its maternal mRNA and into the zygotic expression period

The differential expression of the multilocus CK isozyme system throughout development of the two Xenopus species X. laevis and X. borealis was investigated. A cDNA containing the nearly complete coding sequence of the CK-IV subunit of X. laevis was isolated and sequenced. Early development of X. laevis proceeds with a stock of maternally derived CK-IV/IV isozyme. While the mRNA declines rapidly after fertilization and disappears before neurulation, maternal CK-IV/IV isozyme is active far beyond the onset of zygotic expression and is still detectable when tadpoles start feeding. Zygotic expression of CK-IV begins after neurulation, at stage 22/24, and seems to start simultaneously with that of another gene, CK-III. Modulation in the expression of these two genes and the appearance of two other isoforms, the CK-I and CK-II/III isozymes, take place during development in a tissue-specific manner. During metamorphosis, the CK phenotypes of eyes and skeletal musculature undergo additional changes. The final adult pattern only appears several weeks after metamorphosis. The presumed orthologous CK isozymes of X. borealis show a developmental profile similar to that of X. laevis, except that CK-II/II is equally present in oocytes and during early development, in addition to CK-IV/IV isozyme. These results show that the expression of each of the four CK genes of Xenopus is under differential developmental control.     

Exposure to coal combustion residues during metamorphosis elevates corticosterone content and adversely affects oral morphology, growth, and development in Rana sphenocephala

Coal combustion residues (CCRs) are documented to negatively impact oral morphology, growth, and development in larval amphibians. It is currently unclear what physiological mechanisms may mediate these effects. Corticosterone, a glucocorticoid hormone, is a likely mediator because when administered exogenously it, like CCRs, also negatively influences oral morphology, growth, and development in larval amphibians. In an attempt to identify if corticosterone mediates these effects, we raised larval Southern Leopard Frogs, Rana sphenocephala, on either sand or CCR substrate and documented effects of sediment type on whole body corticosterone, oral morphology, and time to and mass at key metamorphic stages. Coal combustion residue treated tadpoles contained significantly more corticosterone than controls throughout metamorphosis. However, significantly more oral abnormalities occurred early in metamorphosis when differences in corticosterone levels between treatments were minimal. Overall, CCR-treated tadpoles took significantly more time to transition between key stages and gained less mass between stages than controls, but these differences between treatments decreased during later stages when corticosterone differences between treatments were greatest. Our results suggest endogenous increase in corticosterone content and its influence on oral morphology, growth and development is more complex than previously thought.     

Changes in the rate of production of superoxide anion in tails of Rana catesbeiana tadpoles during spontaneous and triiodothyronine-induced metamorphosis

1. The time course of changes in the rate of production of superoxide anion (O2) was compared with that of beta-glucuronidase in tails of Rana catesbeiana tadpoles during spontaneous and triiodothyronine (T3)-induced metamorphosis. 2. Superoxide production increases in tadpole tail tissue undergoing regression in vivo during spontaneous metamorphosis and in response to T3 treatment. 3. The specific activity of beta-glucuronidase rises three-fold relative to control levels in tadpoles treated with T3 in vivo. 4. The time of onset of the rise in beta-glucuronidase activity precedes the onset of tissue regression and the onset of the increase in superoxide production precedes the rise in beta-glucuronidase activity.     

Effects of prenatal dexamethasone on spatial learning and response to stress is influenced by maternal factors

The present study investigated the effect of prenatal dexamethasone (Dex) exposure on early perinatal events, hippocampal function, and response to stress. Pregnant rats received Dex in the evening water (2.5 microg/ml) or tap water (Veh) from gestational day 15 until delivery. On the day of parturition, pups were randomized, cross-fostered, and reduced to eight or nine per dam. Four groups resulted: Veh-Veh (offspring exposed to Veh in utero, rearing mother treated with Veh during gestation), Veh-Dex, Dex-Veh, and Dex-Dex. Spatial visual memory was evaluated with the Morris water maze. The corticosterone response to restraint stress was examined, and the expression of hippocampal glucocorticoid and mineralocorticoid receptors mRNA was determined by in situ hybridization. Exposure to Dex caused restlessness in mothers, low birth weights, and poor weight gain in the offspring. The Dex-Dex males had impaired spatial learning, inability to rapidly terminate the adrenocortical response to stress, and decreased hippocampal glucocorticoid receptor (GR) mRNA expression. In contrast, Dex-exposed animals reared by Veh-treated mothers had adequate spatial learning, enhanced glucocorticoid feedback, and increased hippocampal GR mRNA. We conclude that the environment provided by a healthy mother during the postnatal period can prevent the detrimental effects of prenatal Dex administration on cognition, GR mRNA expression of the hippocampus, and the quality of the stress response.     

Changes in the expression of soluble and integral-membrane trehalases in the midgut during metamorphosis in Bombyx mori

To elucidate the relationship between soluble trehalase (Treh1) and integral-membrane trehalase (Treh2) in the Bombyx mori midgut, expression profiles for both proteins and mRNAs were examined during metamorphosis by using Western-blotting and quantitative real-time PCR analyses. Two bands of Treh2 (about 74 kDa) were detected in the midgut of 0-day-old 5th (last) instar larvae. Levels of Treh2 decreased as the developing larvae approached spinning (8 days old). In contrast, towards the onset of the spinning stage, Treh1 (68 kDa) was clearly observed, and levels increased until the middle of the pupal stage. Treh2 mRNA expression relative to Bmrp49 mRNA expression was almost constant, although fluctuations were detected. Treh1 mRNA expression relative to Bmrp49 mRNA increased sharply just after spinning. To further examine the expression mechanism of the Treh1 gene in midgut, actively feeding larvae (4 days old) were starved or ligated between the 4th and 5th segments. Injection of a molting hormone into the larval-isolated abdomen led to activation of Treh1, demonstrating that molting hormone acts on the midgut and activates this gene.