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Potential Roles of Urea and Phosphodiesters in Eggs of the Antarctic Naked
Dragon Fish (Gymnodraco acuticeps)
J.M. Carlson1, Marina Marjanovic1, Barbara A. Lawrence2
1 Department of Biological Sciences, Eastern Illinois
University
2 Chemistry Department, Eastern Illinois University
Introduction
Near the Ross Ice Shelf at McMurdo Sound, Antarctica, eggs of the
Antarctic naked dragonfish (Gymnodraco acuticeps) develop in the vicinity
of anchor ice where the water temperature is stable at -1.9oC. As
expected, freshly laid eggs are isoosmotic to adult blood, but strongly
hypoosmotic to seawater and therefore have the potential to freeze. Previous
studies have investigated the physiology of freeze avoidance in the adult fish
of this species, but a full characterization of the eggs was never completed.
In this study, colorimetric assay and Nuclear Magnetic Resonance (NMR)
spectroscopy were used to characterize and quantify urea and phosphodiesters (PDE)
in dragonfish eggs. The potential role of PDEs as metabolic intermediates or
osmoprotectants in the eggs of freeze avoidant fishes has never been
investigated. Since the function of PDEs has remained elusive in the study of
most other vertebrates, further investigation of these compounds is required to
determine their significance in eggs of the Antarctic dragonfish.
Materials and Methods
Fresh samples of eggs from the dragonfish were harvested (October
- December 2003) at the McMurdo Sound Station in Antarctica (Figure 2).
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Figure 2. Location of McMurdo
Sound, Antarctica. |
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Nuclear Magnetic Resonance (NMR) Spectroscopy.
1H and 31P-NMR spectra were acquired using a 300 MHz G.E.
QE300 FT-NMR to characterize and quantify different osmolytes in whole-egg
extracts from G. acuticeps eggs. 1H-NMR spectra were acquired
continuously over 64 scans with 16,384 data points using a recycle delay of 1.0
seconds and pulse width of 3.10 µseconds with an average probe temperature of
22.0oC. Standard 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS,
200.2mM) was used as an external chemical shift and concentration reference for
trimethylamine oxide (TMAO) and urea.
31P-NMR spectra were acquired continuously over 1,000 scans with
16,384 data points using a recycle delay of 0.5 seconds and pulse width of 10
µseconds with an average probe temperature of 22.8oC. Methylene
diphosphonic acid (MDPA, 103.2mM) was used as an external reference. All spectra
were acquired using 5mm NMR tubes. Chemical shifts were used to identify
different phosphate metabolites (Figure 3) and peak areas relative to the
reference were used to determine their concentration (Figure 4).
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Figure 3. Molecular structures
of six noncyclic phosphodiesters: 1.) glycerophosphorylcholine
(GPC); 2.) glycerophosphorylethanolamine (GPE); 3.)
glycerophosphorylglycerol (GPG); 4.) threonine ethanolamine
phosphodiester (TEP); 5.) serine ethanolamine phosphodiester (SEP);
6.) lombricine. Redrawn from Van den Thillart and Van Waarde 1996;
the serine groups on SEP and lombricine are in L- and
D-conformations, respectively. |
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Figure 4. 31P-NMR
spectrum of Gymnodraco acuticeps egg extracts.
methylene-diphosphonic acid (MDPA) was used as an external
reference. Peaks corresponding to at least three phosphodiesters (PDEs)
were identified by “spiking” with known compounds. |
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Quantitative Colorimetric Urea Assay. The concentration of urea in pooled ooplasm extracts was measured by
quantitative colorimetric assay using the QuantiChromTM Urea Assay
Kit (DIUR-01K, BioAssay Systems). Optical density was measured at a wavelength
of 520nm using a spectrophotometer.
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Table 1. Osmolality, ion
concentrations and phase change temperatures in whole-egg extracts
from G. acuticeps.
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Table 2. Concentrations of
antifreeze glycoproteins I-VIII in whole egg extracts from G.
acuticeps (Sidell, 2000). |
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Table 3. Osmolality of pooled,
whole egg extracts from G. acuticeps accounted for by primary
osmolytes. |
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Results
31P-NMR spectra show the presence of
phosphodiesters previously observed in freeze tolerant frogs and turtles. The
eggs of G. acuticeps contained 137 ± 26 mM of different PDEs.
Spiking the ooplasm with known PDEs showed that serine ethanolamine
phosphodiester (SEP), Glycerol phosphorylcholine (GPC) and threonine
ethanolamine phosphodiester (TEP) are among the
cytosoluble PDEs found in eggs.
Following aqueous and organic extractions, 31P-NMR showed that in
addition to cytosoluble PDEs, the ooplasm may contain up to three phospholipid
compounds that have never been described in
G. acuticeps eggs.
Biochemical assay revealed a significant concentration of urea (20mM ± 2) in the
ooplasm.
Discussion
The precise mechanism that prevents phosphodiesters from being
degraded by phosphodiesterase is poorly understood and previous investigators
have suspected urea as an inhibitor of this enzyme (Burt and Ribolow 1994). Urea
may also function as a minor osmolyte in the ooplasm of G. acuticeps
eggs.
The combination of ions (Na+/Cl-/K+), TMAO, PDEs and urea in the ooplasm
accounts for 96.9 ± 3.0% of the total osmolality.
Studies by Van den Thillart and colleagues (1996) suggest that SEP plays a
significant role in neural function. SEP and other PDEs may also function in
phosphorus mobilization during embryonic growth and development to increase
soluble phosphates and contribute to neurological development.
Further study of the ooplasm composition is necessary to determine if these PDEs
are products of phospholipid catabolism or if they play a role as precursor
metabolites in phospholipid assembly.
Despite being strongly hypoosmotic, the presence of antifreeze glycoproteins, PDEs, ions and
organic osmolytes depresses the freezing point of eggs to a temperature well
below the freezing point of the aquatic environment.
References
Burt, C.T., and H. Ribolow. Glycerol phosphorylcholine (GPC) and
serine ethanolamine phosphodiester (SEP): evolutionary mirrored metabolites and
their potential metabolic roles. Comparative Biochemistry and Physiology. B,
Biochemistry and Molecular Biology 108(1):11-20, 1994.
Sidell, B. Life at Body Temperatures below 0°C: The physiology and biochemistry
of Antarctic fishes. Gravitational and Space Biology Bulletin
13(2):25-34, 2000.
Van den Thillart, G. and A. Van Waarde. Nuclear magnetic resonance spectroscopy
of living systems: Applications in comparative physiology. Physiological
Reviews 76(3):799-837, 1996.
Acknowledgements
Dr. Arthur L. DeVries (Dept. of Animal Biology; University of
Illinois, Urbana) for donating specimens of G. acuticeps from Antarctica
2004 Scholars in Undergraduate Research at Eastern Award (SURE)
2004 Undergraduate Research Award
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