Hypoxia: Hypoxia: Problems and Scientific ChallengesProblems and Scientific Challenges

Prof. Rudolf WuSchool of Biological SciencesThe University of Hong Kong

(rudolfwu@hku.hk)

Hypoxia: An Old Problem with New Insights

Hypoxia: TrendHypoxia: 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: TrendHypoxia: 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 km2

Number of Dead Zones doubled Number of Dead Zones doubled

every 10 years since the 1960severy 10 years since the 1960s

Diaz & Rosenberg, 2008

Hypoxia Has Caused Major Changes in Hypoxia Has Caused Major Changes in Structure and Functions of EcosystemsStructure 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., 2002Wu, 2002; Gray et al., 2002

Normoxia Hypoxia

Diversity, Species richnessDemersal fishLarger body sizePredator

Suspended feeders

Deposit feedersMeiofauna, NanoplanktonPelagic fishShort life cycle

Generalized Changes in Structure Generalized Changes in Structure and Functions of Ecosystemsand Functions of Ecosystems

Wu, 2002

New Scientific EvidenceNew Scientific Evidence

further show that…..further show that…..

Hypoxia is Hypoxia is an endocrine disruptoran endocrine disruptor

Common carp (Cyprinus carpio)

MaleMale

0

0.1

0.2

0.3

7.0 mg/L

1.0 mg/L

***

(ng/ml)

** p<0.01; *** p<0.001

Wu et al, 2003

0

1

2

3

4

5

6

7

8

9

10

7.0 mg/L

1.0 mg/L

EstradiolEstradiolTestosteroneTestosterone

**

Female Female

0

2

4

6

8

10

Testosterone Estradiol

7.0 mg/L

1.0 mg/L

*** *

(ng/ml)

* p<0.05; *** p<0.001

Wu et al, 2003

• Gonadal

development

• Gametogenesis

• Spawning

• Fecundity

Hypoxia impairs fish reproductionHypoxia impairs fish reproduction

• Fertility

• Gamete quality

• Offspring survival

• Reproductive behavior

Common carp

(Wu et al. 2003)

Zebrafish

(Shang et al. 2006)

Atlantic croaker

(Thomas et al. 2006,2007)

Gulf killifish

(Landry et al. 2007)

Sperm MotilitySperm Motility

0

10

20

30

40

50

60

70

80

7 mg/L

1 mg/L

Curvilinearvelocity

StraightLine velocity

ActualPath velocity

*

*

*

* p < 0.05

µmS-1

Wu et al, 2003

Reproductive ImpairmentReproductive Impairment

0

10

20

30

40

50

60

70

80

90

100

7 mg/L

1 mg/L

Fertilization Hatching LarvalSurvival

Egg to Larvae

***

***

***

***

*** p < 0.001

%

Wu et al, 2003

Follow-up questions: What caused the Follow-up questions: What caused the observed endocrine disruption?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 vitroIn vitro evidence evidence

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

Cholesterol

CYP11A

Pregnenolone

3β-HSD

Progesterone

CYP21

11-Deoxy-corticosterone

CYP11B1

Corticosterone

CYP11B2

Aldosterone

CYP17

17α-OH-

Pregnenolone

3β-HSDCYP17

17α-OH-

Progesterone

CYP21

11-Deoxycortisol

CYP11B1

Cortisol

CYP17

DHEA

3β-HSDCYP17

Androstene-dione

17β-HSD

Testosterone

CYP19

17β-Estradiol

Zona glomerulosaZona fasciculata

Zona reticularis

StAR

Hypoxia is a teratogenHypoxia is a teratogen

Zebrafish (Danio rerio)

Hypoxia delays developmentHypoxia delays development24hpf

Control

Hypoxia (0.5mg/l)

48hpf

Shang & Wu, 2004

Hypoxia caused spine and cardiac malformationsHypoxia caused spine and cardiac malformations

Shang & Wu, 2004

Mal

form

atio

n (

%)

Time (hour)

* p < 0.05; ** p < 0.01

5.8 mg/L0.8 mg/L* **

**

Under hypoxia, apoptosis concentrates in the head Under hypoxia, apoptosis concentrates in the head but not the tailbut not the tail

Control Hypoxia

Shang & Wu, 2004

Sex differentiation/

reversal completed

Final maturation

of the gonads

Gonads differentiate into ovaries

SpawningSex

differentiation begins

3-HSD (-)CYP11A (-)

CYP19A (ND)CYP19B (-)

3-HSD (-)CYP11A (-) CYP19A (-)CYP19B (-)

Male3-HSD (-)CYP11A (-)CYP19A (+)CYP19B (+)

T/E2 (NC)

Male3-HSD (-)CYP11A (-)CYP19A (-)

CYP19B (NC)T/E2 (NC)

Female 3-HSD (+)CYP11A (-)CYP19A (+)CYP19B (+)

T/E2 (+)

Female 3-HSD (-)CYP11A (-)CYP19A (+)

CYP19B (NC)T/E2 (+)

(C) 60 dpf (D) 120 dpf

(B) 40 dpf (A) 10 dpf

10-12 dpf 23-25 dpf 42 dpf 60 dpf 120 dpf

Juvenile AdultLarval 3 dpf 90 dpf 30 dpf

Shang et al, 2006

Hypoxia tips sex balance & Hypoxia tips sex balance & favors a male biased populationfavors a male biased population

***

*** p < 0.001

Shang, Yu & Wu, 2006

Where the girls are? Where the girls are?

Will this affect reproductive success of Will this affect reproductive success of the natural population?the natural population?

Is the observed male biased Is the observed male biased phenotypic or geneotypic?phenotypic or geneotypic?

Will the same happens to Will the same happens to species with XY species with XY chromosomes?chromosomes?

Phenotypic & Phenotypic & gonadal Sex in genotypic onadal Sex in genotypic females (females (O. latipes)O. latipes) are altered by are altered by hypoxiahypoxia

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

Wu & Orr, 2005

7.50 4.50 3.500

20

40

60

80

100

A

% c

op

ula

tion

***

7.50 4.50 3.500

2

4

6

8

10

B

No

.of o

ffsp

rin

g

***

7.50 4.50 3.500.0

0.5

1.0

C

Dissolved oxygen (mg O2 L-1)

No

. of b

roo

ds

***7.50 4.50 3.50

0

10

20

30

40

50

***

D

Dissolved oxygen (mg O2 L-1)

Fec

un

dity

*** p<0.005

Methyl farnesoate?

Ecdysteroid?

Will the same occur in invertebrates?

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

Increase TemperatureIncrease freshwater input

Increase nutrient flux Increase stratification

Nutrient enrichmentEnhanced productivity

Global Warming

HypoxiaSediment

Increase metabolic rate

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)

Brain

Pituitary

Ovary

Blood

sGnRH

FSH

LH

(+)

(+) FSH-R

HMGR

(-)

(-)Progestin ?

(+)?

Summary of effects of hypoxia on The HPG axis in female zebrafish

ER

GnRH FSH CYP19A E2

CYP19A

E2(+)(+)

(+)

Proposed WorkCollaborate 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 WorkCollaborate 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 spermatogenesisHypoxia affects spermatogenesis

Normoxia Hypoxia

** p < 0.01; *** p < 0.001

0

1

2

3

4

5

6

7

8

9

SPG SPC SPD

***

*****

common carpcommon carp

No

. of

cyst

s/lo

bu

le

0

10

20

30

40

50

60

70

SPG SPC SPD

******

***

zebrafishzebrafish

% o

f ea

ch s

tag

e in

tes

tis

Wu et al., 2003; Shang et al., 2006

0

50

100

150

200

250

300

Dia

met

er (m

)/L

ob

ule

***

common carpcommon carp

HypoxiaNormoxia

0

10

20

30

40

50

60

70

Oo PreV Vit PreO

Ovarian cell stage

% o

f ea

ch s

tag

e in

oo

cyte

***

***

*** ***

zebrafishzebrafish

Shang et al., 2006; Thomas et al., 2007 Landry et al., 2007

% o

f o

oc

yte

s

Atlantic croakerAtlantic croaker

Normoxia Hypoxia

*** p < 0.001

Hypoxia affects OogenesisHypoxia affects Oogenesis