Fish growth is one of the important indicators for individual fitness as well
as population health. Telomerase is a protein complex that is closely linked to growth
promotion and cell proliferation both in vitro and in vivo in mammalian tumor cell
systems. High level and constitutive telomerase activities have also been previously
reported, not only in tissues with robust cell division (e.g. gonads) but also in muscle
tissue of fish, which is in contradiction to the situation in humans. Different from
mammals, the growth of many fish species is indeterminate under a continuous food
supply and with no space limits. Skeletal muscle of fish constitutes as much as 80%
of body weight. The biological significance for constitutive expression of telomerase
activity (TA) in skeletal muscle of fish remains virtually unknown. The present study
was set to investigate the usefulness and limitations of muscle TA as an indicator for
predicting organismal growth of fish.
First, a cost-effective and high throughput real-time quantitative telomeric
repeat amplification protocol (RTQ-TRAP) assay that was specifically optimized for
quantification of telomerase activity in fish tissues. Telomerase activity was detected
ubiquitously in all fish species studied, which are of different sizes (marine medaka
Oryzias melastigma ≈ zebrafish Danio rerio << common carp Cyprinus carpio,
grouper Epinephelus coioides, mangrove red snapper Lutjanus argentimaculatus),
ages (fry, juvenile, adult) and sexual maturation stages.
In the second part of the study, change of muscle TA, body weight and
length were examined in fish subjected to different environmental stressors (namely
chronic starvation, dietary benzo-a-pyrene, B[a]P exposure and chronic hypoxia).
Our findings show that in vivo TA in fish muscle is responsive to environmental
stresses, but the response is stress-dependent. Muscle TA was significantly reduced in the fasted mangrove red snapper as well as B[a]P fed orange-spotted grouper,
before any change in body weight or body length was observed. The growth rate of
B[a]P fed fish was significantly slower as compared to that of the control fish, and
TA suppression was sustained in muscle until the end of 4-week exposure. We also
observed a rapid induction of muscle TA in the 8-week starved orange-spotted
grouper shortly after three days re-feeding. However, muscle TA in both
orange-spotted grouper and marine medaka exposed to hypoxia did not differ
significantly from the normoxic fish, suggesting muscle TA is insensitive to hypoxia
stress. Otherwise, in vivo response of fish muscle TA is generally sensitive and
persistent to environmental stresses. Reduction of muscle TA is likely indicative of a
fish being subjected to stresses, while TA induction is indicative of fish under stress
relief.
Using the marine fish Oryzias melastigma as a model, TERT gene of
Oryzias melastigma (omTERT) gene and protein expressions and the enzyme activity
profile in the muscle during fish growth and development were studied, which were
further linked to muscle cell proliferation and apoptosis in vivo. Medaka growth was
also measured using other biochemical markers of fish growth, i.e. muscle
RNA:DNA ratio and protein:DNA ratio. The change of telomerase profile along
medaka growth was studied in two separate phases. Before the medaka reached
sexual maturity (i.e. 8 weeks post-hatching), whole-body TA rose along with
increasing body weight, but the omTERT mRNA expression showed an opposite
pattern. Beyond 8 weeks of growth, a sexually dimorphic muscle TA pattern was
demonstrated, with males showing a decreasing trend but females showing no
change. A more in-depth investigation of omTERT gene and protein expression was
conducted in the adult male medaka. Telomerase activity was dissociated from omTERT gene expression but was in good correlation with omTERT protein level.
Therefore, muscle TA is most likely controlled by post-transcriptional mechanisms.
In agreement with the decreasing telomerase protein expression and activity after
sexual maturation in male medaka, diminishing cell proliferation, as measured by
immuno-reactivity of the proliferation marker PCNA, was also observed in medaka
muscle. Apoptosis, on the other hand, showed no significant relationship with muscle
TERT expression and TA.
In maturing marine medaka, muscle protein:DNA ratio, which is a marker
for muscle hypertrophy, is a better indicator of growth than muscle RNA:DNA ratio
(representing hyperplasia). The contribution of hypertrophic muscule growth is more
significant than hyperplastic muscule growth in mature fish. Due to technical
difficulty in isolating muscle from fry and juvenile of small size fish like medaka, the
relationship between muscle telomerase activity and fish growth at early
developmental stages requires further study using fish of larger final size. Fish
species that are well recognized as indeterminate growers, e.g. sturgeon and rockfish,
are good models for study in this aspect.
Overall, this study provided evidence for the ubiquitous occurrence of TA
in fish muscles. The results also clearly demonstrated the different sensitivity of
muscle telomerase activity, as a predictive growth indicator in fish, under three types
of frequent environmental stresses. The usefulness of telomerase in the study of fish
growth was also revealed, and was found to depend on (i) the type of stressor under
investigation; (ii) the sex; and (iii) the growth stage of the fish species under study.
Apart from these practical considerations, the results reported here also advanced our
understanding and knowledge of the behavior of telomerase in fish, which is
important for further exploration of telomerase function in non-mammals.
| Date of Award | 2 Oct 2008 |
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| Original language | English |
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| Awarding Institution | - City University of Hong Kong
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| Supervisor | Wai Ting Doris AU (Supervisor) |
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