Hypoxia caused by human activities in regard to organic and nutrient enrichment is becoming one of the major threats to aquatic ecosystems worldwide, and this problem is expected to worsen in the coming years. Previous studies have shown that hypoxia can affect reproductive activities in animals via disruption of steroid hormone production and regulation. However, the molecular mechanisms underlying hypoxia-induced impairment of reproductive functions have not been systematically elucidated. The Hypoxia Inducible Factors (HIFs) are principal transcription regulators of various hypoxia-responsive genes which assist animals to adapt and survive under hypoxia. Although over 100 different genes are now known to be controlled by HIF-1 in vertebrates, whether genes encoding steroidogenic enzymes (that control sex hormone production) are regulated by HIF-1 remains little known.
In this dissertation, the hypothesis that HIFs are involved in regulating genes that control the steroidogenesis pathway in vertebrates was tested using zebrafish (Danio rerio) as a model. Experiments were performed to determine the effects of hypoxia, overexpression and knockdown of zHIF-1α, zHIF-2α, zHIF-3α and 3β-HSD on steroidogenesis at various embryonic stages in zebrafish. Nine steroidogenic enzyme genes (StAR, CYP11a, CYP11b2, 3β-HSD, HMGCR, CYP17a1, 17β-HSD2, CYP19a and CYP19b) and four regulatory genes (IGFBP-1, CITED-2, ERα and zlep-a) were investigated by quantitative real-time PCR (qRT-PCR). Hypoxia was found to differentially regulate the expression of all steroidogenic enzyme genes and the four regulatory genes in zebrafish embryos. It is speculated that reduced expression of StAR, HMGCR, 17β-HSD2 and CYP19b under hypoxia might cause impairments in steroid hormone biosynthesis and in turn specific reproductive functions in fish. The downregulation of ERα indicates that hypoxia might influence reproductive processes via regulating steroid hormone receptors in zebrafish.
Following zHIF-1α over-expression, with the exception of StAR (which is reduced), all of the steroidogenic genes examined were up-regulated. Interestingly, the reduced expression of StAR in hypoxic embryos strongly suggests that the expression of StAR may be regulated by the HIF-1 signaling system. Since StAR catalyzes the transfer of cholesterol from the outer to the inner mitochondrial membrane, and is a rate-limiting step in the steroid biosynthesis pathway, downregulation of StAR might result in a reduction of steroid hormones, which has implications in the regulation of reproductive processes in fish. Moreover, upregulated expression of StAR, CYP11a and 3β-HSD in zHIF-1α knockdown zebrafish embryos as compared to the hypoxic embryos suggest that zHIF-1α be might be implicated in the regulation of these genes.
Following zHIF-2α over-expression, StAR, CYP11b2, HMGCR, CYP17a1 and CYP19b were down-regulated in zebrafish embryos at various developmental stages. The similar expression pattern of steroidogenic enzyme genes in zebrafish embryos under hypoxia and following zHIF-2α over-expression indicate that zHIF-2α might have a role in the regulation of steroidogenesis under hypoxia. Downregulation of ERα in zebrafish embryos at 24 hpf and 48 hpf following zHIF-2α over-expression suggests that zHIF-2α might modulate reproduction via steroid hormone receptors. All of the investigated genes were down-regulated in zebrafish embryos at almost all developmental stages examined following zHIF-2α knockdown treatment. The predominant downregulation of steroidogenic enzyme genes in zHIF-2α knockdown zebrafish embryos suggests that the constitutive expression of zHIF-2α under normoxia is essential for the steroidogenesis processes.
In zHIF-3α over-expression treatment, the expression patterns of steroidogenic genes are variable at different developmental stages. CYP11a, CYP11b2, 3β-HSD and HMGCR showed similar regulated expression pattern at 48 hpf under hypoxia and in zHIF-3α over-expression treatment. The observation implies that zHIF-3α might be involved in the regulation of steroidogenic enzyme gene expression under hypoxia.
The expression of 3β-HSD was up-regulated in zebrafish embryos under hypoxia, and embryos subjected to zHIF-1α, zHIF-2α and zHIF-3α over-expression treatment. Down regulation of StAR, CYP17a1 and 17β-HSD2 in 3β-HSD over-expression treatment implies that hypoxia might modulate steroidogenesis via the regulation of 3β-HSD expression. The stimulated expression of StAR, CYP11b2, HMGCR, CYP17a1, 17b-HSD2 and CYP19b in 3β-HSD knockdown treatment strongly indicate that 3β-HSD might act as a negative regulator in steroidogenesis regulation.
Among the four regulatory factor genes examined, IGFBP-1 and CITED-2 were upregulated in hypoxic embryos, and in embryos microinjected with zHIF-1α and -2α mRNAs, but were downregulated in zebrafish embryos microinjected with zHIF-3α mRNAs. ERα was downregulated in hypoxic fish embryos, and embryos microinjected with zHIF-1α or -2α mRNAs, but upregulated in embryos microinjected with zHIF-3α mRNAs. A reverse expression pattern was observed for IGFBP-1, CITED-2 and ERα in fish embryos microinjected with zHIF-3α mRNAs as compared to hypoxic embryos. This may be due to the negative regulatory activity of HIF-3α on HIF-1α. The zlep-a gene was upregulated under hypoxia, and in embryos microinjected with zHIF-1α and -3α mRNAs. The expression pattern of these four regulatory genes indicates there is crosstalk between HIF and CITED-2, leptin and estrogen receptors in the regulation of steroidogenesis.
Overall, this is the first comprehensive and systematic study demonstrating that HIFs can differentially affect steroidogenesis. The results of this study provide some important insights into the molecular mechanisms underpinning the disruption of steroid hormones and reproductive impairment in fish.
| Date of Award | 14 Feb 2014 |
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| Original language | English |
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| Awarding Institution | - City University of Hong Kong
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| Supervisor | Yuen Chong Richard KONG (Supervisor) |
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