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 km2
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
Suspended feeders
Diversity, Species richness
Demersal fish
Larger body size
Predator
Hypoxia
Deposit feeders
Meiofauna,
Nanoplankton
Pelagic fish
Short life cycle
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
(ng/ml)
0.2
6
5
4
3
**
7.0 mg/L
7.0 mg/L
1.0 mg/L
1.0 mg/L
0.1
***
2
1
0
0
** p<0.01; *** p<0.001
Wu et al, 2003
Female
10
8
(ng/ml)
6
7.0 mg/L
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
1 mg/L
40
30
*
20
*
10
0
Curvilinear
velocity
Straight
Line velocity
Actual
Path velocity
* p < 0.05
Wu et al, 2003
Reproductive Impairment
100
90
80
%
70
60
50
40
***
***
30
20
10
0
7 mg/L
1 mg/L
***
***
Fertilization
Hatching
Larval
Survival
Egg to
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
17α-OH-
CYP11A
Pregnenolone
CYP17
DHEA
Pregnenolone
3β-HSD
3β-HSD
3β-HSD
Progesterone
CYP17
CYP17
Androstene-dione
17α-OHProgesterone
17β-HSD
CYP21
CYP21
11-Deoxy-corticosterone
Testosterone
11-Deoxycortisol
CYP11B1
CYP19
CYP11B1
Corticosterone
17β-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
**
Malformation (%)
20
*
**
15
5.8 mg/L
0.8 mg/L
* p < 0.05;
** p < 0.01
10
5
0
48h
72h
96h
Time (hour)
120h
168h
Shang & Wu, 2004
Under hypoxia, apoptosis concentrates in the head
but not the tail
Control
Hypoxia
Shang & Wu, 2004
Gonads
differentiate
into ovaries
Sex
differentiation
begins
3-HSD (-)
CYP11A (-)
CYP19A (ND)
CYP19B (-)
Sex
differentiation/
reversal
completed
Female
3-HSD (-)
CYP11A (-)
CYP19A (+)
CYP19B (NC)
T/E2 (+)
Female
3-HSD (+)
CYP11A (-)
CYP19A (+)
CYP19B (+)
T/E2 (+)
(B) 40 dpf
(C) 60 dpf
23-25 dpf
10-12 dpf
Larval
3 dpf
42 dpf
Spawning
Male
3-HSD (-)
CYP11A (-)
CYP19A (+)
CYP19B (+)
T/E2 (NC)
Male
3-HSD (-)
CYP11A (-)
CYP19A (-)
CYP19B (NC)
T/E2 (NC)
3-HSD (-)
CYP11A (-)
CYP19A (-)
CYP19B (-)
(A) 10 dpf
Final
maturation
of the
gonads
(D) 120 dpf
60 dpf
Juvenile
30 dpf
120 dpf
Adult
90 dpf
Shang et al, 2006
Hypoxia tips sex balance &
favors a male biased population
male
female
***
100%
90%
80%
70%
% in F1
60%
61.9%
74.4%
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 & gonadal 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?
10
A
80
60
40
20
0
No.of offspring
% copulation
100
***
7.50
4.50
1.0
4
2
***
7.50
4.50
3.50
50
D
C
Fecundity
No. of broods
6
0
3.50
B
8
0.5
Methyl farnesoate?
40
30
20
10
0.0
***
7.50
4.50
0
3.50
-1
Dissolved oxygen (mg O2 L )
***
7.50
4.50
3.50
Dissolved oxygen (mg O2 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 nonpoint source are significant
• Trans-boundary issues are difficult to resolve
Global
Warming
Increase Temperature
Increase freshwater input
Increase nutrient flux
Increase stratification
Nutrient enrichment
Enhanced productivity
Sediment
Hypoxia
Increase
metabolic rate
Risk Assessment
•
•
•
•
•
•
Ecological consequence
Area affected
Socioeconomic loss
No. people affected
Probability of occurrence
Trend
Very serious
Very large
Very big
Very large
Very high
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)
Summary of effects of hypoxia on
The HPG axis in female zebrafish
ER
GnRH
FSH
Pituitary
FSH
CYP19A
(+)
E2
Blood
E2
Ovary
CYP19A
Brain
sGnRH
FSH-R
HMGR
LH
(+)?
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
70
9
***
50
***
40
30
***
20
10
No. of cysts/lobule
zebrafish
60
8
common carp
***
7
6
5
4
3
2
***
1
0
SPG
SPC
SPD
0
SPG
SPC
**
SPD
common carp
300
Diameter (m)/Lobule
% of each stage in testis
Hypoxia affects spermatogenesis
250
200
***
150
Normoxia
Hypoxia
** p < 0.01; *** p < 0.001
100
50
0
Normoxia
Hypoxia
Wu et al., 2003; Shang et al., 2006
70
zebrafish
Atlantic croaker
PNS
***
60
50
CA
PYS
SYS
TYS
100%
40
80%
***
30
20
***
10
0
Oo
PreV
Vit
***
*** p < 0.001
60%
40%
PreO
Ovarian cell stage
Normoxia
% of oocytes
% of each stage in oocyte
Hypoxia affects Oogenesis
20%
Hypoxia
0%
Control
2.7 mg/L
1.7 mg/L
Shang et al., 2006; Thomas et al., 2007 Landry et al., 2007