Hypoxia: Problems and Scientific Challenges Prof. Rudolf Wu School - PowerPoint PPT Presentation

Hypoxia: Problems and Scientific Challenges Prof. Rudolf Wu School of Biological Sciences The University of Hong Kong (rudolfwu@hku.hk)

Hypoxia: An Old Problem with New Insights

Hypoxia: Trend During the last few decades, anthropogenic input of nutrients into our coastal environment has increased ca. three folds, and is expected to double or triple if no action is taken

Hypoxia: Trend Decrease in dissolved oxygen recorded over large coastal areas worldwide (including USA, China, Norway, UK, Sweden, Germany, Denmark, the Black Sea, Adriatic Seas) in the last 30-80 years Diaz & Rosenberg, 1995; Rabalais, 2001

Total area>245,000 km 2

Number of Dead Zones doubled every 10 years since the 1960s Diaz & Rosenberg, 2008

Hypoxia Has Caused Major Changes in Structure and Functions of Ecosystems – Mass mortality of fish and benthos – Changes in species composition – Changes in trophic relationships – Decrease in biodiversity and species richness – Decrease in fisheries production Phil, 1994; Wu, 2002; Gray et al., 2002 Wu, 2002; Gray et al., 2002

Generalized Changes in Structure and Functions of Ecosystems Normoxia Hypoxia Deposit feeders Meiofauna, Nanoplankton Pelagic fish Suspended feeders Short life cycle Diversity, Species richness Demersal fish Larger body size Predator Wu, 2002

New Scientific Evidence further show that…..

Hypoxia is an endocrine disruptor Common carp ( Cyprinus carpio )

Male Estradiol Testosterone 10 0.3 9 8 7 0.2 ( ng/ml) 6 7.0 mg/L 7.0 mg/L 1.0 mg/L 5 1.0 mg/L 4 ** *** 0.1 3 2 1 0 0 ** p <0.01; *** p <0.001 Wu et al, 2003

Female 10 8 6 7.0 mg/L ( ng/ml) 1.0 mg/L 4 *** 2 * 0 Testosterone Estradiol * p <0.05; *** p <0.001 Wu et al, 2003

Hypoxia impairs fish reproduction • Gonadal • Fertility development • Gamete quality • Gametogenesis • Offspring survival • Spawning • Reproductive behavior • Fecundity Common carp Zebrafish Atlantic croaker Gulf killifish (Wu et al. 2003) (Shang et al. 2006) (Thomas et al. 2006,2007) (Landry et al. 2007 )

Sperm Motility 80 70 60 * 50 µmS -1 7 mg/L 40 1 mg/L 30 * 20 * 10 0 Curvilinear Straight Actual velocity Line velocity Path velocity * p < 0.05 Wu et al, 2003

Reproductive Impairment 100 90 80 70 *** % 60 *** 7 mg/L 50 1 mg/L 40 30 *** 20 *** 10 0 Fertilization Hatching Larval Egg to Survival Larvae *** p < 0.001 Wu et al, 2003

Follow-up questions: What caused the observed endocrine disruption? • A smaller gonad and reduced hormone production due to reduced energy intake and reduced growth? • Hypoxia affects synthesis and metabolism of sex hormones? • Hypoxia affects GnRH and gonadotropins?

In vitro evidence • In vitro studies using H295R human adrenocortical carcinoma cell line and primary cell culture of medaka gonads

StAR Cholesterol CYP17 CYP17 17 α -OH- DHEA CYP11A Pregnenolone Pregnenolone 3 β -HSD 3 β -HSD CYP17 CYP17 3 β -HSD Androstene-dione 17 α -OH- Progesterone Progesterone 17 β -HSD CYP21 CYP21 Testosterone 11-Deoxy-corticosterone 11-Deoxycortisol CYP11B1 CYP19 CYP11B1 Corticosterone 17 β -Estradiol Cortisol CYP11B2 Zona reticularis Aldosterone Zona fasciculata Zona glomerulosa

Hypoxia is a teratogen Zebrafish ( Danio rerio )

Hypoxia delays development 24hpf 48hpf Control Hypoxia (0.5mg/l) Shang & Wu, 2004

Hypoxia caused spine and cardiac malformations ** 20 5.8 mg/L ** * 0.8 mg/L Malformation (%) 15 * p < 0.05; ** p < 0.01 10 5 0 48h 72h 96h 120h 168h Shang & Wu, 2004 Time (hour)

Under hypoxia, apoptosis concentrates in the head but not the tail Control Hypoxia Shang & Wu, 2004

Sex Final Gonads Sex differentiation/ maturation differentiate differentiation Spawning reversal of the into ovaries begins completed gonads Male Male 3 β -HSD (-) 3 β -HSD (-) CYP11A (-) CYP11A (-) CYP19A (-) CYP19A (+) CYP19B (NC) CYP19B (+) T/E 2 (NC) T/E 2 (NC) 3 β -HSD (-) 3 β -HSD (-) CYP11A (-) CYP11A (-) Female Female CYP19A (ND) CYP19A (-) 3 β -HSD (-) 3 β -HSD (+) CYP19B (-) CYP19B (-) CYP11A (-) CYP11A (-) CYP19A (+) CYP19A (+) CYP19B (NC) CYP19B (+) T/E 2 (+) (A) 10 dpf (B) 40 dpf T/E 2 (+) (C) 60 dpf (D) 120 dpf 10-12 dpf 23-25 dpf 42 dpf 60 dpf 120 dpf Larval Juvenile Adult 3 dpf 30 dpf 90 dpf Shang et al, 2006

Hypoxia tips sex balance & favors a male biased population *** male 100% female 90% 80% 70% 74.4% 61.9% 60% % in F1 50% 40% 30% 20% 10% 0% Normoxia Hypoxia *** p < 0.001 Shang, Yu & Wu, 2006

Where the girls are? Will this affect reproductive success of the natural population?

Is the observed male biased phenotypic or geneotypic? Will the same happens to species with XY chromosomes?

Phenotypic & g onadal Sex in genotypic females ( O. latipes) are altered by hypoxia Cheung, 2006; Wu, 2009

Will the same occur in higher vertebrates? • Salamanders ( Ambystoma sp .): delayed development and hatching, less developed and deformed embryos • Australian frog ( Crinia georgiana ): delayed embryonic development, increased malformation • Male albino rats: reduced numbers of sertoli cells and Leydig cells in testis • Male Wistar rats: lower levels of LH and testosterone Seymonr et al., 2000; Shevantaeva & Kosyuga, 2006; Farias et al., 2007

Will the same occur in invertebrates? 100 10 A No.of offspring B % copulation 80 8 60 6 40 4 20 2 *** *** 0 0 7.50 4.50 3.50 7.50 4.50 3.50 1.0 50 Methyl farnesoate? D C No. of broods 40 Fecundity 30 0.5 20 10 *** *** 0.0 0 7.50 4.50 3.50 7.50 4.50 3.50 Dissolved oxygen (mg O 2 L -1 ) Dissolved oxygen (mg O 2 L -1 ) Ecdysteroid? *** p <0.005 Wu & Orr, 2005

Is the situation getting better or getting worse?

The situation is likely to get worse because….. • Growth of treatment facilities is unlikely to catch up with growth of population and industry, especially in developing counties • Contributions from atmospheric fallout and non- point source are significant • Trans-boundary issues are difficult to resolve

Global Warming Increase Temperature Increase freshwater input Increase Increase nutrient flux Increase stratification metabolic rate Nutrient enrichment Enhanced productivity Hypoxia Sediment

Risk Assessment • Ecological consequence Very serious • Area affected Very large • Socioeconomic loss Very big • No. people affected Very large • Probability of occurrence Very high • Trend Getting worse The new scientific evidence presented here calls for an urgent re-assessment of this old problem

Will the same happen in humans? • Patients suffering from sleeping apnea have lower sex drive and testosterone level (Saaresranta & Polo, 2003) • Sex ratio in human depends on level of sex hormones of father and mother during conception, and high testosterone level during conception favors subsequent birth of sons (James, 2004)

Sum m ary of effects of hypoxia on The HPG axis in fem ale zebrafish ER GnRH FSH CYP19A E2 Pituitary Blood (+) FSH β E2 Ovary Brain CYP1 9 A sGnRH FSH-R HMGR LH β Progestin ? (+)?

Proposed Work Collaborate with fisheries authorities and undertake a scientific global review, focusing on : – Trend analysis (spatial and level in the last 50 years) – Changes in structure and trophodynamics of marine communities (plankton, benthos, fish) – Identification of sensitive groups (bioindicators)

Proposed Work Collaborate with fisheries authorities and undertake a scientific global review, focusing on : – Reproductive status and reproductive impairment of fish in hypoxic areas vs normoxic areas – Endocrine disruption, malformation, sex ratio – Deciphering effects of hypoxia from those caused by other anthropogenic activities (chemicals) prevailing simultaneously in the marine environment?? – Identifying information gaps and further studies

Hypoxia affects spermatogenesis common carp % of each stage in testis 70 9 zebrafish *** 8 No. of cysts/lobule 60 *** 7 50 6 *** 40 5 4 30 3 *** 20 2 *** 10 1 ** 0 0 SPG SPC SPD SPG SPC SPD common carp 300 Diameter ( µ m)/Lobule 250 Normoxia Hypoxia 200 *** 150 ** p < 0.01; *** p < 0.001 100 50 0 Normoxia Hypoxia Wu et al., 2003; Shang et al., 2006

Hypoxia affects Oogenesis 70 zebrafish Atlantic croaker % of each stage in oocyte *** PNS CA PYS SYS TYS 60 50 100% 40 80% *** 30 % of oocytes 20 60% *** 10 *** 40% 0 Oo PreV Vit PreO Ovarian cell stage 20% Normoxia Hypoxia 0% Control 2.7 mg/L 1.7 mg/L *** p < 0.001 Shang et al., 2006; Thomas et al., 2007 Landry et al., 2007