Dogmas and controversies in the handling of nitrogenous wastes: osmoregulation during early embryonic development in the marine little skate Raja erinacea; response to changes in external salinity.
15 May 2004-The Journal of Experimental Biology (The Company of Biologists Ltd)-Vol. 207, Iss: 12, pp 2021-2031
TL;DR: The data indicate that little skate embryos synthesize and retain urea, as well as a suite of other osmolytes, in order to regulate osmotic balance with the external environment.
Abstract: SUMMARY Marine elasmobranchs retain relatively high levels of urea to
counterbalance the osmotic strength of seawater. Oviparous species, such as
the little skate Raja erinacea , release encapsulated embryos that
hatch after about 9 months on the seafloor. To study the ureosmotic capability
of skate embryos, we measured a variety of possible osmolytes and
ornithine–urea cycle (OUC) enzyme activities in little skate embryos,
and determined their physiological response to dilute seawater (75% SW)
exposure relative to controls (100% SW). The urea:trimethylamine oxide (TMAO)
+ other osmolytes ratio was 2.3–2.7:1. At the earliest stage of
development investigated (4 months), there were significant levels of the key
OUC enzyme, carbamoyl phosphate synthetase III, as well as ornithine
transcarbamoylase, arginase and glutamine synthetase, providing evidence for a
functional OUC. Embryos (4 and 8 months) survived and recovered from exposure
to 5 days of 75% SW. There was a significant increase in the rate of urea
excretion (five- to tenfold), no change in OUC enzyme activities, and
significant decreases in the tissue content of urea, TMAO and other osmolytes
in embryos exposed to 75% SW compared to 100% SW. Taken together, the data
indicate that little skate embryos synthesize and retain urea, as well as a
suite of other osmolytes, in order to regulate osmotic balance with the
external environment. Interestingly, these ureosmotic mechanisms are in place
as early as 4 months, around the time at which the egg capsule opens and the
embryo is in more direct contact with the external environment.
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TL;DR: The presence of ornithine carbamoyl transferase (OCT) in all developmental phases of pacu and in the adult liver and kidney suggests that this enzyme is performing different metabolic pathways.
Abstract: The present work reports the activities of urea cycle enzymes during the ontogenic development of the teleost pacu (Piaractus mesopotamicus). Urea cycle enzymes from the kidney and liver of adult fish were compared with those from the fish's embryonic phases. Samples were evaluated over all phases of embryonic development, the larval period and alevin. Ammonia and urea concentrations were determined during embryogenesis and in the plasma of adult fish. Except for carbamoyl phosphate synthetase-III (CPS-III), all enzymes of the urea cycle were expressed in the larvae and alevins as well as in the liver and kidney of adult fish. In spite of the low level of activity of the ornithine urea cycle (OUC) enzymes compared to those in mammals, and the low levels of tissue urea concentration compared to ammonia, the ureogenesis was evaluated in pacu. Ammonia seems to be the main nitrogenous waste during embryonic development. In this phase glutamine synthetase (GS) may play a role in ammonia detoxification, and the OUC enzymes can be individually involved in functions other than urea production. The presence of ornithine carbamoyl transferase (OCT) in all developmental phases of pacu and in the adult liver and kidney suggests that this enzyme is performing different metabolic pathways. OCT in the kidney, wherein the activity is less than in the liver, should work in the biosynthesis of polyamines and control the arginine plasma concentration given that renal arginase and argininosuccinate synthetase-argininosuccinate lyase are more active than from the liver. We suppose that OCT during the embryogenesis is a control step regulating the cellular concentration of ornithine for polyamines synthesis.
7 citations
Cites background from "Dogmas and controversies in the han..."
...…are the marine elasmobranches and the Crossopterygii, the coelacanth Latimeria, which produce urea as an excreted nitrogen waste rather than as an osmolite to maintain the osmoregulation either when adults (Mommsen and Walsh 1991) or during the early embryonic development (Steele et al. 2004)....
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...The most representative among those species are the marine elasmobranches and the Crossopterygii, the coelacanth Latimeria, which produce urea as an excreted nitrogen waste rather than as an osmolite to maintain the osmoregulation either when adults (Mommsen and Walsh 1991) or during the early embryonic development (Steele et al. 2004)....
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TL;DR: Evidence is provided that the YSM contributes to embryonic urea homeostasis until the liver and other extrahepatic organs become fully functional, and that urea-producing tissue shifts from the Y SM to the embryonic liver in the late developmental period of oviparous marine cartilaginous fishes.
Abstract: Urea is an essential osmolyte for marine cartilaginous fishes Adult elasmobranchs and holocephalans are known to actively produce urea in the liver, muscle and other extrahepatic organs; however, osmoregulatory mechanisms in the developing cartilaginous fish embryo with an undeveloped urea-producing organ are poorly understood We recently described the contribution of extraembryonic yolk sac membranes (YSM) to embryonic urea synthesis during the early developmental period of the oviparous holocephalan elephant fish (Callorhinchus milii) In the present study, to test whether urea production in the YSM is a general phenomenon among oviparous Chondrichthyes, we investigated gene expression and activities of ornithine urea cycle (OUC) enzymes together with urea concentrations in embryos of the elasmobranch cloudy catshark (Scyliorhinus torazame) The intracapsular fluid, in which the catshark embryo develops, had a similar osmolality to seawater, and embryos maintained a high concentration of urea at levels similar to that of adult plasma throughout development Relative mRNA expressions and activities of catshark OUC enzymes were significantly higher in YSM than in embryos until stage 32 Concomitant with the development of the embryonic liver, the expression levels and activities of OUC enzymes were markedly increased in the embryo from stage 33, while those of the YSM decreased from stage 32 The present study provides further evidence that the YSM contributes to embryonic urea homeostasis until the liver and other extrahepatic organs become fully functional, and that urea-producing tissue shifts from the YSM to the embryonic liver in the late developmental period of oviparous marine cartilaginous fishes
7 citations
TL;DR: In this article, the authors used the round stingray (Urobatis halleri) as a model elasmobranch species and captured adult males and pregnant females (matrotrophic histotrophy) and their embryos from two populations differing in their environmental exposure to organic contaminants.
Abstract: Organic contaminants are known to affect a suite of physiological processes across vertebrate clades. However, despite their ancient lineage and important roles in maintaining healthy ecosystems, elasmobranchs (sharks, skates, and rays) are understudied with regard to sublethal effects of contaminant exposure on metabolic processes. Perturbations resulting from contaminant exposure can divert energy away from maintaining physiological homeostasis, particularly during energetically challenging life stages, such as pregnancy and embryonic development. Using the round stingray (Urobatis halleri) as a model elasmobranch species, we captured adult males and pregnant females (matrotrophic histotrophy) and their embryos from two populations differing in their environmental exposure to organic contaminants (primarily polychlorinated biphenyls (PCBs)). Pregnant females from the PCB-exposed population experienced significant decreases from early- to late-pregnancy in tissue mass and quality not seen in reference females. PCB-exposed pregnant females also failed to maintain plasma urea concentrations as pregnancy progressed, which was accompanied by a loss in muscle protein content. Despite the energetic demands of late-term pregnancy, females had significantly greater liver lipid content than reproductively inactive adult males. PCB-exposed adult males also had high metabolic capacity (i.e., enzyme activity) for most substrate groupings of all sex-site groups, suggesting that males may be even more negatively impacted by contaminant exposure than pregnant females. Evidence that in utero exposure to PCBs via maternal offloading impairs embryo outcomes is accumulating. Embryos from the PCB-contaminated site had lower tissue quality measures and indications that sex-based differences were manifesting in utero as males had higher metabolic capacities than females. This study indicates that accumulated PCB contaminants are not physiologically inert in the stingray.
5 citations
TL;DR: These findings are contrary to previous starvation studies demonstrating constant levels of TMAO for up to 56days in elasmobranchs and suggest that all species rely to a degree on absorption of T MAO from the diet or that alternate synthetic or regulatory pathways play a larger role than previously thought.
Abstract: Trimethylamine oxide (TMAO) is an organic osmolyte and universal protein stabilizer. Its role as a cytoprotectant is particularly important in ureosmotic elasmobranchs that accumulate high levels of urea, a macromolecular perturbant. Feeding is a key component in the turnover and maintenance of these nitrogenous compounds. However, previous studies examining TMAO regulation have been largely completed using starved individuals, when nitrogen balance is altered. Here, under fed conditions, we test the importance of dietary TMAO on long-term maintenance in three elasmobranch species with differing endogenous synthetic capacities. Smoothhounds (Mustelus canis), spiny dogfish (Squalus acanthias), and little skates (Leucoraja erinacea) exhibited species- and tissue-specific differences in their ability to conserve TMAO when fed a low TMAO diet for 56 days. Smoothhounds, a species with the capacity for endogenous production, exhibited a decrease in muscle TMAO. Spiny dogfish and little skates, species with no reported ability for synthesis, exhibited decreases in plasma and liver TMAO, respectively. Our findings are contrary to previous starvation studies demonstrating constant levels of TMAO for up to 56 days in elasmobranchs. Further, the previously reported synthetic capacity of these species did not correlate with their ability to conserve TMAO and cannot be used to predict a species reliance on dietary contributions for prolonged maintenance. It is possible that all species rely to a degree on absorption of TMAO from the diet or that alternate synthetic or regulatory pathways play a larger role than previously thought.
4 citations
01 Jan 2016
TL;DR: TMAO’s role in combatting the environmental stress associated with increasing hydrostatic pressure was shown to increase with increasing depth of occurrence across all species of Hawaiian mid-water fishes studied, and environmental factors impart a larger influence on TMAO retention than evolutionary history.
Abstract: Trimethylamine oxide (TMAO) was first described in marine organisms as an osmolyte, involved in the balance of water and solutes. After its discovery, it was found to be part of a subset of osmotic constituents termed counteracting solutes. These solutes exhibit stabilizing properties and can preserve protein functionality against biological and environmental perturbations. TMAO acts as a universal stabilizer, protecting macromolecular structure and function in response to numerous stressors, including urea destabilization, hydrostatic pressure, temperature and salinity. The studies presented in this dissertation address the regulatory and environmental factors affecting TMAO accumulation. Both exogenous and endogenous sources are involved in the maintenance of TMAO. Exogenous TMAO accumulates through absorption from the diet while endogenous TMAO is synthesized from dietary or cellular precursors with the flavincontaining monooxygenase trimethylamine oxidase (TMAoxi). Species without a physiologically relevant synthetic capacity are hypothesized to rely entirely on dietary contributions for accumulation. Chapter 1 examines the necessity of an exogenous TMAO source on long-term maintenance in elasmobranch species with and without the ability for endogenous synthesis. These data show that presence or absence of TMAoxi cannot be used as a proxy to determine the importance of dietary TMAO on prolonged conservation. It seems that all species, regardless of synthesizing potential, rely to an extent on contributions from the diet. Chapter 2 further examines the regulatory factors affecting TMAO. This study provides evidence for endogenous production via an understudied synthetic pathway whereby TMAO is accumulated as a byproduct during lipid storage. The existence of this pathway is supported by a correlation between TMAO content and total lipid in a variety of Hawaiian mid-water fishes. The regulatory role of evolutionary relatedness on accumulation potential is also addressed in this chapter. Phylogenetic independent contrasts (PIC) showed no relationship between phylogeny and TMAO content across 27 species spanning nine orders. This suggests that environmental factors impart a larger influence on TMAO retention than evolutionary history. Chapter 2 goes on to examine TMAO’s role in combatting the environmental stress associated with increasing hydrostatic pressure. TMAO was shown to increase with increasing depth of occurrence across all species of Hawaiian mid-water fishes studied. These data support previous reports of TMAO accumulation as an environmental adaptation to combat the destabilizing effects of elevated hydrostatic pressure. Chapter 3 explores TMAO’s ability to counteract environmental fluctuations in temperature. Previous in vitro studies showed intracellular transport and accumulation of TMAO with increasing temperature in elasmobranch red blood cells. Further, this was shown to suppress the traditional heat shock response of heat shock protein 70 (HSP70) upregulation. However, we saw no increase in plasma or tissue TMAO in response to elevated temperature for two shark species in vivo. Either mechanisms established in vitro are not applicable at the organismal level or additional regulatory factors are limiting TMAO accumulation. Lastly, a brief study examining regulation of TMAO through ontogeny in an elasmobranch species, Squalus acanthias, is presented in the Appendix. Pups of this species exhibit low levels of urea and TMAO, their two primary osmolytes. However, total osmotic pressure is maintained at adult levels. Therefore, a shift in the osmotic milieu occurs sometime between birth and adulthood. These findings are in contrast to those reported for the little skate, Leucoraja erinacea, which expresses adult levels of these osmotic constituents early in development. These data point to divergence in the early osmoregulatory strategies of differing elasmobranch groups. In the enclosed chapters, key objectives regarding the regulatory and environmental factors influencing TMAO are addressed. Specifically, this research examines how contributing sources, evolutionary restrictions and environmental stress affect TMAO accumulation. These studies elucidate TMAO’s multifaceted role in marine organisms and provide insight into the factors regulating its adaptive potential.
2 citations
Cites background from "Dogmas and controversies in the han..."
...TMAO accumulation patterns are different through spiny dogfish and little skate ontogeny, perhaps supporting disparities in the way this molecule is utilized between the two species (Steele et al., 2004; Bockus and Seibel, submitted)....
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References
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TL;DR: The sensitivity of the colorimetric reaction and stability of the colour are enhanced over existing related procedures and the serum blank diminished, enabling urea to be precisely measured in micro amounts (1--5 microliters) of serum.
443 citations
"Dogmas and controversies in the han..." refers methods in this paper
...Water sample analysis Water samples were analyzed for urea content using a colorimetric assay described by Rahmatullah and Boyde (1980)....
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...Urea was measured using the method described by Rahmatullah and Boyde (1980)....
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TL;DR: Phylogenetic position, geographical distribution, benthic vs. pelagic habitat, adult size, egg-embryo size, feeding ecology, and embryonic osmoregulation are factors in the retention of oviparity or the evolution of viviparity.
Abstract: Patterns of chondrichthyan reproduction and development are diverse. Species either are reproductively active throughout the year, or have a poorly defined annual cycle with one or two peaks of activity, or have a well defined annual or biennial cycle. Based on embryological origin and adult morphology, their reproductive system is more similar to tetrapods than to teleosts. Primordial germ cells are of endodermal origin. The Wolffian ducts in males and Mullerian ducts in females become the functional urogenital ducts. Differentiation is under hormonal control. Unusual features of the reproductive system include an epigonal organ in males and females. It contains lymphoid and hemopoietic tissue. Leydig's gland, a modified region of the kidney, produces seminal fluid. In some species, sperm passing through the vas deferens, is enclosed in spermatophores. Rotating about their long axis, helical spermatozoa can move forward or reverse direction. Spermatogenesis often occurs in bicellular units, spermatocysts. These consist of a spermatogonium enclosed in a Sertoh cell. Fertilization is internal. Claspers, modified portions of the pelvic fins act as intromittent organs. In many viviparous sharks and rays, the female reproductive system is asymmetrical. Eggs of some sharks are the largest known cells. Yolk platelets contain lipovitellin. Oocytes have lampbrush chromosomes. Eggs released from the ovary into the body cavity are transported by ciliary action to the ostium of the oviduct. There they are fertilized. Physiological polyspermy is normal. The shell gland, a specialized region of the anterior oviduct, functions both in long term sperm storage and in egg case production. Egg cases of sharks and skates consist of unique collagenous protein with a 400 A period, organized as a cholesteric liquid crystal. Chimaeroid egg cases contain 550 A pseudotubules in orthogonal lattices. In small sharks, males copulate by coiling around the female. A parallel position is assumed by large sharks. Skates and rays copulate with ventral surfaces apposed or by a dorsal approach. Biting is a pre-copulatory release mechanism. Parental care, except for selective oviposition, is lacking. Heavily yolked eggs undergo meroblastic, discoidal cleavage. Development is lengthy, shortest (2–4 months) in rays, longer in skates (3–8 months) and longest (9–22 months) in sharks and chimaeras. Most sharks and all rays are viviparous. Chimaeras, skates, and some sharks are oviparous. Viviparity either involves a yolk sac placenta or is aplacental. If aplacental, the embryo derives nutrients either from yolk reserves, or by intra-uterine embryonic cannibalism, or from placental analogues which secrete “uterine milk.” Phylogenetic position, geographical distribution, benthic vs. pelagic habitat, adult size, egg-embryo size, feeding ecology, and embryonic osmoregulation are factors in the retention of oviparity or the evolution of viviparity.
436 citations
"Dogmas and controversies in the han..." refers background in this paper
...Embryos of oviparous species, however, are exposed directly to seawater well before hatching (Wourms, 1977) and may be able to independently ureosmoregulate by altering the rate of urea excretion and/or the rate of urea synthesis....
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TL;DR: Experiments indicate that evolutionary changes in intracellular solute compositions as well as in protein amino acid sequences can have important roles in intrACEllular protein function.
Abstract: Intracellular fluids of marine elasmobranchs (sharks, skates and rays), holocephalans and the coelacanth contain urea at concentrations averaging 0.4m, high enough to significantly affect the structural and functional properties of many proteins. Also present in the cells of these fishes are a family of methylamine compounds, largely trimethylamine N-oxide with some betaine and sarcosine, and certain free amino acids, mainly β-alanine and taurine, whose total concentration is approx. 0.2m. These methylamine compounds and amino acids have been found to be effective stabilizers of protein structure, and, at a 1:2 molar concentration ratio of these compounds to urea, perturbations of protein structure by urea are largely or fully offset. These counteracting effects of solutes on proteins are seen for: (1) thermal stability of protein secondary and tertiary structure (bovine ribonuclease); (2) the rate and extent of enzyme renaturation after acid denaturation (rabbit and shark lactate dehydrogenases); and (3) the reactivity of thiol groups of an enzyme (bovine glutamate dehydrogenase). Attaining osmotic equilibrium with seawater by these fishes has thus involved the selective accumulation of certain nitrogenous metabolites that individually have significant effects on protein structure, but that have virtually no net effects on proteins when these solutes are present at elasmobranch physiological concentrations. These experiments indicate that evolutionary changes in intracellular solute compositions as well as in protein amino acid sequences can have important roles in intracellular protein function.
382 citations
"Dogmas and controversies in the han..." refers background in this paper
...As well as having protein stabilizing properties (Yancey and Somero, 1979, 1980), these compounds are arguably some of the most important free amino acids involved in cell volume regulation in fish (King and Goldstein, 1983)....
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TL;DR: In this article, the colorimetric indophenol blue method was used for ammonia determination in natural waters by Solorzano's procedure, with phenol-alcohol, nitro-prusside, alkaline citrate and hypochlorite as reagents, was shown to be appropriate for routine work.
356 citations