Hypoxia: Hypoxia: Problems and Scientific ChallengesProblems and Scientific Challenges
Prof. Rudolf WuSchool of Biological SciencesThe University of Hong Kong
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