缺氧對斑馬魚發育的致畸效應及內分泌干擾效應 | Que yang dui ban ma yu fa yu de zhi ji xiao ying ji nei fen mi gan rao xiao ying | Teratogenic and endocrine-disrupting effects of hypoxia on development of zebrafish (Danio rerio)

Hypoxia/anoxia caused by eutrophication and organic pollution occurs over thousands of km2 and has caused mass mortality, population decline and major changes in community structure and function in many aquatic ecosystems worldwide. Hypoxia is now being considered as one of the major threats to aquatic ecosystems, and the problem is expected to worsen in the coming years. In this thesis, the zebrafish (Danio rerio) was employed as a model species to test the hypotheses that hypoxia is teratogenic to fish embryos, and may affect sex determination via endocrine disruption during fish development. Malformation, growth, apoptosis pattern, gonad development, hormones and sex ratio were measured, and temporal and spatial responses of hypoxia-inducible genes, genes controlling the synthesis of sex hormones, as well as genes controlling apoptosis in fish exposed to normoxia (5.8 mg O2 l-1) and hypoxia (0.8 mg O2 l-1) were compared over time to elucidate molecular mechanisms underpinning the effects observed. Heart rate was initially increased when embryos were exposed to hypoxia, but was followed by a rapid decrease after 96 hours post fertilization (hpf) (t-test, p<0.001). It appears that enhancing oxygen uptake and maintaining oxygen delivery were employed only as short-term strategies to deal with hypoxia, while reducing energy expenditure was used as a long term strategy to better survive under hypoxic conditions. Embryos exposed to hypoxia showed a delay in their development, and body length of fish in the hypoxic treatment was 12.3% shorter than their counterparts’ in the normoxic control after 168 h of development (t-test, p<0.001). Upon exposure to hypoxia, embryos lost synchronization in their development, with their tails developing much faster than their heads. Skeletal deformities, predominantly manifested as altered axial spinal curvature, were clearly evident in the hypoxic fish. After 168 h, percentage of malformation in the hypoxic treatment was significantly higher (+77.4%) than that of the normoxic control (t-test, p<0.01). Concomitantly, a significant reduction in percentage of apoptotic cells in the tails (-63.7%), and an increase in percentage of apoptotic cells in the brain (+116%), were found in hypoxic embryos, indicating that malformation may be mediated through the alteration of the normal apoptotic pattern. In addition, a higher percentage (+121.1%) of embryos in the hypoxic group failed to develop their vascular systems when compared with the normoxic control, and died after 3 to 5 days. Hypoxia retards fish growth and gonad development. After two months, body length, body weight, gonad weight and gonadosomatic index (GSI) in female were significantly reduced by hypoxia (t-test, p<0.01). After 4 months, body length, body weight, gonad weight and GSI were all significantly reduced in both hypoxic females and males. Histological examinations further confirmed that gonadal development in both males and females was retarded after exposure to hypoxia for 4 months. Hypoxia disrupts the balance of sex hormones at very early stages of fish development. At 48 hpf, levels of testosterone (T) significantly increased while estradiol (E2) concentrations significantly decreased in hypoxic fish, resulting in a marked increase (+357%) of the testosterone/estradiol (T/E2) ratio in sexually undifferentiated fish (t-test, p<0.05), but this pattern was reversed at 120 hpf. After 2 months of development, T and E2 were significantly reduced in hypoxic males (t-test, p<0.01, p<0.001, respectively), but not in hypoxic females. After 4 months, an increase in T was clearly observed in hypoxic females (t-test, p<0.05). The increased T/E2 ratio observed in hypoxic females but not hypoxic males indicated that disruption of sex hormones was more severe in hypoxic females than that in hypoxic males, although sex differentiation and sexual development in both sexes were similarly affected by hypoxia. The sex ratio of zebrafish was altered upon chronic exposure to hypoxia, resulting in a male-biased population in the F1 generation (74.1±1.7% males in the hypoxic group vs 61.9±1.6% males in the normoxic groups; Chi-square test, p<0.05). Vitellogenin (VTG) was significantly reduced by hypoxia in both females (t-test, p<0.01 for 2 months and p<0.001 for 4 months) and males (t-test, p<0.001 for 2 months and p<0.01 for 4 months) at 2 and 4 months, indicating that oocyte development and egg production were also inhibited by hypoxia. Expression of various sex hormone control genes (viz. VTG, 3β-HSD, CYP11A, CYP19A and CYP19B) were determined at 10dpf, 40dpf, 2 months and 4 months. At the onset of gonad differentiation at 10 dpf, all selected genes were significantly down-regulated by hypoxia (t-test, p<0.05 for VTG and p<0.001 for other genes). At 40 dpf, when sex differentiation and sex reversal occurred, VTG was up-regulated (t-test, p<0.001) while all other genes were down-regulated (t-test, p<0.05). Expression of CYP11A, CYP19A, CYP19B, VTG, and 3β-HSD were altered at different developmental stages and in both sexes, showing that hypoxia can disrupt key steps in sex hormone synthesis in both developing and adult fish. Our results therefore indicate that synthesis of sex hormone and CYP aromatase in zebrafish were disrupted by hypoxia during sexual differentiation and development, which may account for the male-biased ratio observed in the hypoxic treatment. HIF-1a was significantly up-regulated by hypoxia at 2 hpf (t-test, p<0.001), but down-regulated thereafter. This cascaded into subsequent up-regulations of EPO, VEGF (fold-changes ranging from 1.4±0.3 to 5.0±0.2 and 1.2±0.1 to 5.7±0.2, respectively) and p53 at later stages of development. In particular, the ratio of Bax/Bcl-2 in hypoxic fish was twice as high as that in normoxic fish at all time points except 24 hpf, indicating that apoptosis was promoted in hypoxic fish. The overall results suggested that 48hpf was the most sensitive window, at which time all of the genes studied responded to hypoxia. At 36 hpf, up-regulation of Bax coupled with down-regulation of Bcl-2 was found in the head region; while the reversed pattern was observed in the tail, suggesting a higher apoptotic potential in the head region as compared to the tail region. This offered further evidence to support the differential apoptotic patterns observed earlier in hypoxic embryos. At two months, the ratio of Bax/Bcl-2 in hypoxic males was 198.2% of that measured in hypoxic females, implicating that apoptosis in males might potentially be more susceptible to hypoxic effects than in females. For the first time, this study provided experimental evidence to show that hypoxia is a teratogen, which may induce premature death, growth retardation, malformation and functional defects in zebrafish. We also report, for the first time, that hypoxia can change the activity of P450 aromatase and the expression of various genes controlling the synthesis of sex hormones, which in turn disrupt the balance of testosterone and estradiol during fish development and sex differentiation, resulting in a male-biased F1 generation. The increase in males and reduction of females in fish populations caused by hypoxia may subsequently reduce reproductive success in natural populations. Taken together with an increase in incidences of malformation and pre-mature death, we conclude that hypoxia poses a significant threat to the sustainability of natural fish populations over large areas worldwide. Because of the similarity in genome and organ development between zebrafish and other mammals including human and also because of the large scale and high frequency of occurrence of hypoxia in the natural environment, it is possible that hypoxia may also cause similar effects to other species.

Includes bibliographical references (leaves 190-246)

Thesis (Ph.D.)--City University of Hong Kong, 2005