Hypoxia affects sex differentiation and development,

leading to a male dominated population in fish

Rudolf Wu

Centre for Coastal Pollution & Conservation

City University of Hong Kong

Diaz & Rosenberg, 1995

What is “Hypoxia”?

• Shortage of oxygen

• < 2 ml /L

• <2.8 mg/L

Hypoxia is A Pressing Global Problem

<2.8 mgO2/L

Diaz & Rosenberg 1995Hypoxic area> 1 million Km2

150 “Dead Zones” all over the world

UNEP, 2004

High density(High salinity or low temperature)

ALGAL BLOOMALGAL BLOOM

Low density(Low salinity or high temperature)

Pycnocline

Dead algae shrink &decomposers use up oxygen

COD / BOD / SOD COD / BOD / SOD

Oxygen

Oxygen blocked

Mortality

Vessel waste discharge

Anthropogenic activitiesAnthropogenic activities

Organic matters & nutrients (EUTROPHICATION)Organic matters & nutrients (EUTROPHICATION)Sewage effluentRunoff

Organisms escape

Hypoxia: Some Trend

• Decrease in DO in many coastal areas in USA, China, Norway, UK, Sweden, Germany, Denmark, the Black Sea, Adriatic Seas in the last 30-80 years

Diaz & Rosenberg, 1995; Rabalais, 2001

Current situation

• Increases in nutrient concentration, algal blooms, large scale hypoxia, and change of species composition, mass mortality of fish and decline in fisheries production are found in large coastal areas all over the world

Diaz & Rosenberg, 1995; Gray & Wu, 2002

5.8 mg/L5.8 mg/L

0.8 mg/L0.8 mg/L

Experimental set up

• Eggs of zebrafish exposed to 0.8 and 5.8 mg O2/L for 3 months

• Studied growth, gonad wt. gametogenesis, malformation, sex hormones, sex ratio and various genes controlling synthesis of sex hormone and apoptosis over time

Oxygen controller

Endocrine disruption occurs as early as 48hpf

Testosterone in zebrafish embryos

**

**

0

5

10

15

20

25

30

35

40

48hpf 120hpf

Con

cent

rati

on (p

g/m

l)

Control

Hypoxia

Shang & Wu, 2004** p<0.01

Endocrine disruption occurs as early as 48hpf

Estradiol in zebrafish embryos

***

***

0

2

4

6

8

10

12

14

16

18

48hpf 120hpf

Con

cent

rati

on (p

g/m

l)

Control

Hypoxia

Shang & Wu, 2004*** p<0.005

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

Control Hypoxia

Apoptosis(24hpf) identified by AO staining

*

**

0

20

40

60

80

100

120

140

Head Tail

No.

ap

opto

tic

cells

Control 5.8mg/l

Hypoxia 0.8mg/l

Hypoxia alters normal pattern of apoptosis

♀ Female Zebrafish♀ Female Zebrafish

2 mm

1 cm

200 µm

2 mm

1 cm

200 µm

♂ Male Zebrafish♂ Male Zebrafish

Testis Ovary

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

Shang, Yu & Wu, 2006Shang, Yu & Wu, 2006

***

*** p < 0.001

Where the girl are?

Expression of selected genesunder hypoxia

Sex differentiation/

reversal is completed

Final maturation of the gonads

Gonads differentiate into

ovariesSpawning

Sex differentiation

begins

3-HSD (-)CYP11A (-)

CYP19A (ND)CYP19B (-)

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

Aromatase (-)

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 (+) Aromatase (-)

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

CYP19B (NC)T/E2 (+)

Aromatase (-)

(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

In vitro evidence

• In vitro studies using H295R cell line

Overall Conclusions

• Hypoxia affects very large areas and has caused major changes in aquatic ecosystems, as well as decline in fisheries production all over the world

• For the first time, we found that hypoxia is an endocrine disruptor, and impairs reproduction of fish

Overall Conclusions

• For the first time in science, we found that :

– Hypoxia alters various genes controlling synthesis of sex hormones

– Alternation of sex hormones affects sex differentiation and sex development, and favors a male-biased population in fish

• A higher male to female ratio will greatly reduce the chance of fish reproduction

Overall Conclusions

• Since the genes and hormones controlling sex differentiation and reproduction are highly conserved, the endocrine disrupting effects and biased sex ratio observed in fish may also occur in other vertebrates.

• These are likely to be the major factors leading to decline of natural populations in hypoxic waters

• The problem of hypoxia is likely to get worse in the coming years

Epilogue• Sex ratio in human depends on level of sex

hormones of father and mother during conception, and high hormone level during conception favors subsequent birth of sons (James, 2004)

• Patients suffering from sleeping apnea have lower sex drive and testosterone level (Saaresranta & Polo, 2003)

Acknowledgments

• This research was supported by the Area of Excellence Grant (AoE/P-04/04) from the University Grants Council, HK SAR government

Dr. Eva Shang