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EFECTOS DE LOS CONTAMINANTES METÁLICOS EN LA FISIOLOGÍA DE LA REPRODUCCIÓN EN PECES
Renata G. Moreira Whitton
Department of Physiology, Biosciences Institute, University of Sao Paulo, Brazil
renatagm@ib.usp.br
Effects of pollutants in the physiology of fish
POLLUTANTS Osmoregulation
Metabolism
Endocrine
Biochemical Cellular
Growth Behaviour
Reproduction Vitellogenesis & Steroidogenesis; Substrate deposition; egg development; Brain-Pituitary-Gonads axis alteration; sexual behaviour alteration; genetic damages.
Aluminum
The third element in abundance in the earth's crust;
Brazil is one of the largest producers;
Natural sources: rock erosion;
Anthropogenic sources: industry (metallurgy, textile, pulp and paper, pharmaceutical, manufacturing cosmetics) and water treatment to obtain potable water.
Alumínio bauxite feldspar aluminum
Manganese
• 11º element in abundance in the earth's crust;
• Present in the soil derived from oxides, carbonates, silicates and sulphates;
• The excess of manganese accumulated in the liver and central nervous system resulting from prolonged exposure by inhalation, causes symptoms such as "Parkinson“ disease (degenerative). For these and other adverse effects is that manganese is considered toxic.
Primary effects
• Gills
• Osmoregulation
• Ionoregulation
Secondary effects
• Metabolic
• Endocrine
• Reproduction
Manganese
Gonadal steroidogenesis
Fertility of males
Plasmatic concentration of FSH
and testosterone
Lipoperoxidation and metabolic
stress
Aluminum
Effects on HPG and HPI axis
Alters the concentrations of
sexual hormones (androgens,
estrogens and progestagens)
Acidic pH
Effects on plasma hormonal
concentration
Alters production on sexual
cells
Alterations caused by metals on hepatic vitellogenesis in teleosts
Al/Mn
VTG
PO4
E2
Mn+
RNAm
Mn+
Enz. quinases
lipídios
lipídios
lipídios
lipídios
X
X
X
Al/Mn
Percentages of non-compliant results with respect to the standards established for Class 2, in 2013 and in the period 2008-2012.
Main Basins in Brazil
Southeast region
São Paulo State Basins
1) Considering that aluminum affects fish physiology, can the brain-pituitary-gonads axis be impaired in the presence of this metal?
QUESTIONS RAISED:
3) In vivo and in vitro effects of aluminum are similar?
2) What are the effects of aluminum on the major energetic substrates of vitellogenic females?
4) Aluminum and manganese, isolated or combined, affect fish steroid levels during the breeding season?
Teleost fish species with different reproductive strategies
Astyanax altiparanae, “lambari” , group synchronous with high
plasticity in the reproductive tatics
Oreochromis niloticus, “tilapia”, asynchronous
Source: MPA
Tilapia and lambari production in Brazil
Production of 180.000.000 fingerlings: -Live bait -Research
(Sussel, 2016)
Experimental Groups:
1) Control (neutral; pH 7.0) (ctr – n) 2) Acidic pH (acidic; pH 5.5) 3) Aluminum (0.5 mg.L-1; pH 7.0) (Al-n) 4) Aluminum (0.5 mg.L-1; pH 5.5) (Al-ac)
1) Considering that aluminum affects fish physiology, can the brain-pituitary-gonads axis be impaired in the presence of this metal?
ACUTE EXPERIMENT – 96 hours
Brain
Neurotransmitters
neurohypophysis adenohypophysis
Gonads
Pituitary stalk
GnRH (+)
GnIH (-)
Dopamine (-)
FSH
Gonadal Steroids
LH
Fee
db
ack
(+
) o
r (-
)
(Medrado, 2014)
Brain-Pituitary-Gonads Axis controlling vertebrate reproduction
Comparing the amount of mRNA copy number FSH normalized by the number of mRNA copies EF1A in the experiment of acute exposure of Oreochromis niloticus to aluminum in neutral pH (7.0) and acid pH (5.5) and respective groups control. Different letters indicate significantly difference among treatments (P <0.05) (Narcizo, 2009).
a a
b
b
Estradiol plasma concentration of mature females of Oreochromis niloticus exposed for 96h to aluminum in water with neutral pH (Al-N) and acid pH (Al-Ac). Also fish were maintained in the absence of aluminum in water with neutral pH (CTR-N) and acid pH (CTR-Ac). Data are expressed as mean ± SEM. Different letters indicate significantly difference among treatments (P <0.05) (Correia et al. 2010).
Aluminum as endocrine disruptor
No effect
-B-P-G axis regulates E2 synthesis -females were already vitellogenic
Oreochromis niloticus
Plasma concentrations of 17α-hydroxyprogesterone of mature females of Oreochromis niloticus exposed for 96h to aluminum in water with neutral pH (Al-N) and acid pH (Al-Ac). Also fish were maintained in the absence of aluminum in water with neutral pH (CTR-N) and acid pH (CTR-Ac). Data are expressed as mean ± SEM. Different letters indicate significantly difference among treatments (P <0.05) (Correia et al. 2010).
Comparing the amount of mRNA copy number LH normalized by the number of mRNA copies EF1A in the experiment of acute exposure of Oreochromis niloticus to aluminum in neutral pH (7.0) and acid pH (5.5) and respective groups control. Different letters indicate significantly difference among treatments (P <0.05) (Narcizo, 2009).
a
b b
a
Aluminum as endocrine disruptor
Impairment in spawning!
Oreochromis niloticus
Lubzens et al., (2009)
LH
FSH
STEROIDOGENESIS IN FEMALE FISH
Maturation-inducing hormone (MIS)
- Energetic investment to accelerate spawn;
- Due to the mechanism of asynchronic oocyte development, O. niloticus has the physiological plasticity to accelerate the mobilization process of vitellogenesis, resulting in an increased lipid concentration in ovaries.
2) What are the effects of aluminum on the major energetic substrates of vitellogenic females?
Oreochromis niloticus
-Group synchronic species – Hepatic lipid also decreased but it was not mobilized to the ovaries; -Al increased liver lipoperoxidation in mature A. altiparanae
2) What are the effects of aluminum on the major energetic substrates of vitellogenic females?
No effects
Astyanax altiparanae
2) What are the effects of aluminum on the major energetic substrates of vitellogenic females?
Astyanax altiparanae
Incubation of pituitaries of O. niloticus in cell culture dish of 24 wells in 200ul of L15 medium in experimental conditions.
-Pituitaries:
Control: only put in L15
GnRH: L15 + GnRH (10um)
GnRH + Al: L15 + GnRH (10um) + AlCl3 (0.1 M)
Al: L15 + AlCl3 (0.1 M) (Hwang et al., 2000)
Analysis of gonadotropins gene expression by real time PCR after 24 hours
3) In vivo and in vitro effects of aluminum are similar?
-Ovaries: Fractions of 50mg of ovaries were incubated in Krebs-Ringer-HEPES-glucose buffer: Only Krebs: endogenous control Krebs + hCG (6ug/ml) Krebs + hCG (6ug/ml) + AlCl3 (0.1 M) Krebs + AlCl3 (0.1 M)
Oreochromis niloticus
Analysis of E2 and 17a-OHP after 4 hours
Comparing the amount of mRNA copy number of FSH in vitro experiments from mRNA extracted from pituitary glands of mature female O. niloticus after 24 hours exposure to GnRH 10mM, to GnRH 10mM + AlCl3 (0.1M) and only AlCl3 (0.1M) Data are presented as mean ± SEM. Different letters indicate significant differences between treatments (P <0.05).
ab
a
c bc
Nu
mb
er
of
cop
ies
of
mR
NA
of
FSH
Comparing the amount of mRNA copy number of LH in experiments in vitro from mRNA extracted from pituitary glands of mature female O. niloticus after 24 hours exposure to GnRH 10mM, to GnRH 10mM + AlCl3 (0.1M) and only AlCl3
(0.1M). Data are presented as mean ± SEM. Different letters indicate significant differences between treatments (P <0.05).
ab
a
c
bc
Nu
mb
er
of
cop
ies
of
mR
NA
of
LH
3) In vivo and in vitro effects of aluminum are similar?
-Al inhibited the cellular response of the pituitary to GnRH; - A similar effect comparing with the in vivo experiment
In vitro effects of Al in tilapia pituitary
a
Concentration of 17αOHP in Krebs-Ringer-HEPES-glucose buffer solution in vitro experiments using fragments of mature ovaries O. niloticus after 4 h of exposure to hCG 6μg/ml; 6μg/ml to hCG and 10 mM Al, and Al only to 10mM. Data are presented as mean ± SEM. Different letters indicate significant differences among treatments (P <0.05).
a a a
b
Concentration of E2 Krebs-Ringer-HEPES-glucose buffer solution in vitro experiments using fragments of mature ovaries O. niloticus after 4 h of exposure to hCG (6μg/ml); to hCG (6μg/ml) and Al (10 mM), and Al only to 10mM. Data are presented as mean ± SEM. Different letters indicate significant differences between treatments (P <0.05).
a a
b
a
3) In vivo and in vitro effects of aluminum are similar?
-Al inhibited the cellular response of the ovarian steroidogenic cells to gonadotropins; -A similar effect comparing with the in vivo experiment for 17α-OHP but different for E2 (no effect in vivo);
In vitro effects of Al in tilapia ovarian follicles
ab
a
b
0
500
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3000
CTRL - neutro CTRL - ácido Alumínio
pg/
mL
17 β ESTRADIOL
AGUDO
Neutral Acidic Aluminum
a
b
0,00
500,00
1000,00
1500,00
2000,00
2500,00
3000,00
CTRL - neutro CTRL - ácido Manganês
pg/
mL
17 β ESTRADIOL
AGUDO
Neutral Acidic Manganese
4) Aluminum and manganese, isolated or combined, affect fish steroid levels during the breeding season?
a
a
b
0,00
500,00
1000,00
1500,00
2000,00
2500,00
3000,00
CTRL - neutro CTRL - ácido Sinérgico
pg/
uL
17 β ESTRADIOL
AGUDO
Neutral Acidic Al + Mn Same effects of Al and Mn – isolated or combined
Astyanax altiparanae
a
b
0
5
10
15
20
25
CTRL - neutro CTRL - ácido Alumínio
pg/
mL
17 α OHP AGUDO
Neutral Acidic Aluminum
a a
a
b
0,00
5,00
10,00
15,00
20,00
25,00
CTRL - neutro CTRL - ácido Manganês
pg/
mL
17 α OHP AGUDO
Neutral Acidic Manganese
a
a
b
0,00
5,00
10,00
15,00
20,00
25,00
CTRL - neutro CTRL - ácido Sinérgico
pg/
mL
17 α OHP AGUDO
Neutral Acidic Al + Mn
Same effects of Al and Mn – isolated or combined
4) Aluminum and manganese, isolated or combined, affect fish steroid levels during the breeding season?
Astyanax altiparanae
a a
b
0,00
10,00
20,00
30,00
40,00
50,00
60,00
CTRL - neutro CTRL - ácido Manganês
ug/
dL
Cortisol AGUDO
Neutral Acidic Manganese
4) Aluminum and manganese, isolated or combined, affect fish steroid levels during the breeding season?
a a
b
0
10
20
30
40
50
60
CTRL - neutro CTRL - ácido Alumínio
ug/
dL
Cortisol AGUDO
Neutral Acidic Aluminum
0,00
10,00
20,00
30,00
40,00
50,00
60,00
CTRL - neutro CTRL - ácido Sinérgico
ug/
dL
Cortisol AGUDO
Neutral Acidic Al + Mn Acidic
When Al and Mn are combined, there is no effect
Astyanax altiparanae
4) Aluminum and manganese, isolated or combined, affect fish steroid levels during the breeding season?
Astyanax altiparanae
Acidic pH and Al increases androgens
Mn increases E2
Acidic pH increases cortisol
1) Aluminum can be considered an endocrine disruptor in pituitary – ovaries axis, both in
vivo and in vitro, decreasing GtH gene expression and sexual steroids in mature females;
CONCLUSIONS
5) In vitro, Al inhibited the cellular response of the pituitary to GnRH, and the ovarian
steroidogenic cells to gonadotropin, similar effects comparing with the in vivo experiment.
However, the inhibition for E2 synthesis was only demonstrated in vitro;
3) A. altiparanae males use reproductive tactics to trigger changes in testicular
steroidogenesis, by accelerating spermatogenesis and spermiogenesis. On the other hand,
A. altiparanae females prioritized the use of energetic resources during Al exposure
instead of prioritizing reproduction, thereby avoiding the ovulation of impaired eggs.
2) Estradiol levels decrease in group synchronous lambari but does not change in
asynchronous tilapia mature females;
4) The effect of Al and Mn, isolated or combined are not the same;
Thank you!!!!