Multiple molecular mechanisms of hypoxia-induced reproductive endocrine disruption in zebrafish (Danio rerio)

Abstract

Hypoxia/anoxia caused by eutrophication and organic pollution is now one of the most pressing problems in aquatic ecosystems worldwide, and this problem is likely to be exacerbated in the coming years. Laboratory and field studies have shown that hypoxia can disrupt sex hormones, leading to reproductive impairment in fish. However, the specific molecular mechanisms underpinning these effects have not been systematically elucidated. In this dissertation, the zebrafish (Danio rerio) was employed as a model species to test the hypothesis that hypoxia can act on multiple targets of the brain-pituiary-gonad (BPG) axis and liver, by a variety of molecular mechanisms to disrupt reproductive endocrine function in fish. The expression profiles of key genes associated with reproductive endocrine pathways in brain, pituitary, gonad and liver, as well as circulating sex hormones were studied in adult fish exposed to normoxia (6.4 mg O2l-1) and hypoxia (0.6 mg O2l-1) for 21 days. For the first time, we show that hypoxia can simultaneously disrupt expression of key regulatory genes at different levels along the BPG axis (at all three levels in females and in pituitary and gonad in males), and decrease circulating sex hormones (estradiol in females and testosterone in males) in fish. In female zebrafish exposed to hypoxia, downregulation of sGnRH in the brain, FSH􀈕 and LH􀈕 in the pituitary, together with a decrease in CYP19A and an increase in HMG-CoA reductase (HMGR) and FSH receptor (FSH-R) transcripts in the ovary, were observed. In males exposed to hypoxia, downregulation of LH􀈕 in the pituitary, as well as LH-receptor (LH-R) and genes controlling steroidogenesis including HMGR, StAR, CYP11A, CYP11􀈕 and 20􀈕-HSD were clearly evident in the testis. Given that the regulation of reproductive hormones and the BPG axis are highly conserved in fish and other vertebrates, our observations provide important insight into mechanisms and pathways through which hypoxia might affect neuroendocrine functions, steroidogenesis, sex hormone production and subsequently reproduction in vertebrates. Hypoxia induced sex and tissue-specific changes in the expression of estrogen (ERs: esr1, esr2a and esr2b), androgen (ARs) and membrane progestin (mPRs) receptors along the BPG axis. esr1, AR and mPR􀄮 were upregulated, while esr2b was downregulated in brains of hypoxic males, but no noticeable changes in expression of these genes and receptors were observable in brains of hypoxic females. In the pituitary, all three subtypes of ERs (esr1, esr2a, esr2b) and mPRß were downregulated by hypoxia in both sexes. Sex hormone receptors in the brain-pituitary complex are involved in the feedback regulation of sex hormones, which is important to achieve the synchronization between the different elements of the BPG axis. Thus, the alterations in the mRNA levels of sex hormone receptors in the brain and pituitary of hypoxic fish observed in this study suggests that hypoxia can alter sex hormone signals at the brain and pituitary levels by disrupting the transcription of these sex hormone receptors. This may disrupt the synchronization along the BPG axis, leading to reproductive disorders. The significant, positive correlations found between pituitary receptors (esr1, esr2a, esr2b and mPRß) and pituitary GtHß (FSHβ and LHβ) in both sexes offer further evidence to this postulation. In gonads, differential sex response of receptor genes to hypoxia was observed, except mPRß in both sexes. In ovaries, hypoxia downregulated esr1, but upregulated AR. However, in testes, hypoxia downregulated esr2a, esr2b and mPR􀄮. Gonadal estrogens and androgens are instrumental in regulating ovarian and testicular functions. Thus, the changes of ERs and AR mRNA levels in the gonad of hypoxic fish indicate that hypoxia disrupts the responsiveness of gonads to sex hormone signals, which may impair oogenesis and spermatogenesis. Testicular mPR􀄮, an intermediary in progestin stimulation of sperm hypermotility in fish, was downregulated by hypoxia, which provides a plausible mechanism underlying hypoxia-reduced sperm motility in fish. In female liver, upregulation of esr1 and esr2a and downregulation of esr2b were observed, and this was associated with a marked downregulation in two forms of vitellogenins (Vtg: Vtg1 and Vtg2). These findings indicate hypoxia may disrupt vitellogenesis via interfering with hepatic ERs and Vtgs. In liver, hypoxia treatment was found to produce remarkable inductions of sex hormone-binding globulin (SHBG), CYP3A65 and CYP1A mRNAs in females. In males exposed to hypoxia, hepatic SHBG mRNA was moderately increased, but CYP3A65 and CYP1A mRNAs were not significantly changed. SHBG is a plasma glycoprotein able to bind and transport sex steroids in the blood, and CYP3A65 and CYP1A encode enzymes for metabolizing sex hormones. Our data thus indicate the increased sex hormone transport and clearance could be alternative mechanisms for reduction of sex hormone levels caused by hypoxia. Hypoxia suppressed the expression of genes relating to neurotransmitters, neuropeptides and neurosteroidogenesis in fish brain. Neuropeptide Y (NPY), two forms of tryptophan hydroxylase (TPH1 and TPH2, rate-limiting enzymes of serotonin), tyrosine hydroxylase (TH1, rate-limiting enzyme of dopamine) and HMGR were downregulated in the brain of both sexes by hypoxia. Moreover, hypoxia downregulated serotonin transporter (serta) and CYP19B, but upregulated CYP11􀈕 in female brain. Given the important roles of these neuroendocrine factors in regulating GnRH, GtHs and feedback of sex hormones in the brain-pituitary complex, hypoxia may impair fish reproduction through disrupting multiple neuroendocrine signaling in the fish brain. This is the first comprehensive and systematic study demonstrating that hypoxia can act on multiple, specific target sites, including brain, pituitary, gonad, liver and feedback systems of sex hormones along the entire reproductive axis, leading to disruption of reproductive endocrine function in fish. The results of this study provide new insight into the molecular mechanisms underpinning the disruption of sex hormones and reproductive impairment in fish and possibly other higher vertebrates.