M.J. DARIAS*, D. MAZURAIS, G. KOUMOUNDOUROS, E. GISBERT C CAHU J L ZAMBONINO-INFANTEGISBERT, C. CAHU, J.L. ZAMBONINO-INFANTE
IFREMER Centre de Brest, France
University of Patras, Greece
* Present address
larvi 2009
MALFORMATIONSMALFORMATIONS
NUTRITION
VITAMINS A An overdose produceMixLarvae need more VM VITAMINS A
D C
An overdose produce growth delay and induce
cephalic malformations and one vertebra less
(Gisbert et al 2005;
MixLarvae need more VM than juvenilesA small amount of VM induce malformations(Mazurais et al., 2008) (Gisbert et al., 2005;
Villeneuve et al., 2005, 2006; Mazurais et al., 2009)
(Mazurais et al., 2008)? ?
larvi 2009
Calcium and phosphate Co-substrate for hydroxylase dhomeostasis
Protection of skeletal integrity
and oxygenase enzymes involved in the biosynthesis
of pro-collagen
Antioxidant
Pro-oxidant
Hilton & Ferguson, 1982Graff et al., 2002Haga et al., 2004
Halver, 1957,1989Andrews & Murai, 1975
Lim & Lovell, 1978Chávez de Martínez et al., 1990
Soliman et al., 1986
Studies at larval stageNo studies at larval stageDabrowski, 1990
Gapasin et al., 1998
larvi 2009
Cartilage damagePugheadness
Cartilaginous vertebraeHaemal arch not formed
Deformities of the caudal finPugheadnessPoor mineralization Poor mineralization
One vertebrae lost
Mineralization delay Poor mineralization
Vertebral deformities (Kyphosis, scoliosis)Branchiostegal rays deformities Epurals, uroneural, specialized neural arch
Epurals, uroneural, specialized neural archDeformities of dentaryDeformities of the dorsal and anal fin
Vertebral deformities (kyphosis, scoliosis)Mineralization delay Poor mineralization
Branchiostegal rays deformities
80evel
/ lar
vae)
Supernumerary vertebrae
120140 24 vertebrae
15000 15000
(%)
8 times the requirements of juvenil fish (NRC, 1993)
40
60
80
sific
atio
nle
pixe
lnum
ber/
406080
100120
cuen
cy (%
) 25 vertebrae
40
60
80 CONTROLCONTROL
5000
10000
5000
10000
orm
atio
ns(
0
20
VD-0 VD-19.2 VD-38.4 VD-140
Oss
(Tot
al p
02040
VC-15 VC-30 VC-50 VC-400
Frec
0
20
VC-15 VC-30 VC-50 VC-4000
VD-0 VD-19.2 VD-38.4 VD-1400
VC-15 VC-30 VC-50 VC-400
Mal
fo
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60
80
60
80
ons
(%)
0
20
40
60
0
20
40
60
Mal
form
atio
0VD-0 VD-19.2 VD-38.4 VD-140
0VC-15 VC-30 VC-50 VC-400
Mev
el/ l
arva
e) 15000 15000
sific
atio
nle
pixe
lnum
ber/
5000
10000
5000
10000
Oss
(Tot
al p
0VD-0 VD-19.2 VD-38.4 VD-140
0VC-15 VC-30 VC-50 VC-400
MOLECULAR PATHWAYS INVOLVED IN MINERALIZATION
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Vitamin D Receptor(VDR)VD3 VC
?
MOLECULAR PATHWAYS INVOLVED IN MINERALIZATION
Regulation of osteoblasts
differentiationINTESTIN BONE
VDR
Calcium absorption differentiation, proliferation and
mineralization
BONE
TRPV-6SVCT-1
indirectly directly
BMP-4/PPAR γ IGF-1,RARγ
Regulation of bone mineralization Malformations ?
Osteocalcin
CONTROL OF INTESTINAL ABSORPTION
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TRPV-6
Ca2+ absorption
1,6 VC 0
1,2
1,6
RPV
-6 VD-0VD-19.2VD 38 4
1,2
,
f TR
PV-6
VC-0VC-5VC-15VC-30VC-50VC 400
a a a
b
0 4
0,8
,
essi
onof
TR VD-38.4
VD-140 a
c
b bc
0,4
0,8
xpre
ssio
n of VC-400
0,0
0,4
Day 11 Day 22 Day 45
Expr
e
0,0Day 11 Day 25 Day 45
Ex
EFFECT ON GROWTHAND DEVELOPMENT
INDIRECT EFFECT ON BONE MINERALIZATION
CONTROL OF INTESTINAL ABSORPTION
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SVCT-1
VC absorption
50
60Strong blueLigth Blue
/ lar
vae Day 45
1,0
1,2
1,4
SVC
T-1
VC-0VC-5VC-15VC-30
a
ab
b ab
20
30
40
xeln
umbe
r/
0,4
0,6
0,8
ress
ion
of S VC-50
VC-400
aaba
0
10
VC-15 VC-30 VC-50 VC-400
Tota
l pix
0,0
0,2
0,4
Day 11 Day 15 Day 25 Day 45
Exp bbb
Day 11 Day 15 Day 25 Day 45
CONTROL OF BONY TISSUE DEVELOPMENT
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Osteoblasts proliferation and
IGF-1
proliferation and stimulation of its function
1 5
2,5
3,0
IGF-
1 VD-0VD-19.2VD-38 4
c1,0
1,5
of IG
F-1
VC-0 VC-5VC-15 VC-30VC-50 VC-400
a
bc
ab
1,0
1,5
2,0
pres
sion
of I VD-38.4
VD-140
ab bb
ab
b 0,5xp
ress
ion
oab
aabb b b
c
bc
0,0
0,5
Day 11 Day 22 Day 45
Exp
aab
0,0Day 11 Day 25 Day 45
Ex b
CONTROL OF BONY TISSUE DEVELOPMENT
larvi 2009
Osteoblasts mineralization
Osteocalcin
mineralization
1 00 a
1,00
eoca
lcin VD-0
VD-19.2VD-38 4
a a a
b0,10
1,00
steo
clac
in
VC-0 VC-5VC-15 VC-30VC-50 VC-400
bb b
0,01
0,10
ssio
n of
ost
e VD 38.4VD-140
a bcbc 0,01
ress
ion
of o
s
a aabbbb
0,00Day 11 Day 22 Day 45
Expr
es
0,00Day 11 Day 25 Day 45
Expr
CONTROL OF BONY TISSUE DEVELOPMENT
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Stimulation f TRPV 6 d
VDR
1,8VC-0 VC-5VC 15 VC 30
of TRPV-6 and osteocalcin expression
1,21,41,61,8
of V
DRβ
VC-15 VC-30VC-50 VC-400
b
aa
abab ab
a
a
* * * * * *
** *
*
1,0
1,2
1,4
VDRβ
VD-0VD-19.2
VD-38.4abab b
a* * * *
0,40,60,81,0
Expr
essi
on o b
b b
bc
c c
bb
b
****
**
****
**
0,4
0,6
0,8
ress
ion
of V VD-140ab b
aa a
** ** ** **
** *********
0,00,20,4
Day 11 Day 25 Day 45
E
0,0
0,2
Day 11 Day 22 Day 45
Expr a a
b bb ***
CONTROL OF BONY TISSUE DEVELOPMENT
larvi 2009
Osteoblasts diff ti ti
BMP-4
differentiation
1,5 VC-0 VC-5
1,5
MP-
4 VD-0VD-19.2VD 38 4
abab
a1,0
,
BM
P-4
VC-0 VC-5VC-15 VC-30VC-50 VC-400
ab
bbb
bab
b
0,5
1,0
ress
ion
of B VD-38.4
VD-140b
ba ab a
0,5pr
essi
on o
f B a b
0,0Day 11 Day 22 Day 45
Expr
b
0,0Day 11 Day 15 Day 25 Day 45
Exp
CONTROL OF CARTILAGE TISSUE DEVELOPMENTCONTROL OF ADIPOCITIC TISSUE DEVELOPMENT
larvi 2009
Cartilage formation
RARγ
formationPPARγAdipocyte
differentiation
2,0 VC 0 VC 52,0
1 2
1,6
2,0
of R
ARγ
VC-0 VC-5VC-15 VC-30VC-50 VC-400 a
a
b
c
1,2
RA
Rγ
VD-0
VD-19.2
VD-38.4
a
1 2
1,6
2,0
PPA
Rγ
VC-0VC-5VC-15VC-30VC-50
a
aa
0 4
0,8
1,2
xpre
ssio
n o c
0,4
0,8
pres
sion
of VD-140 b
bb
b b b
a
0 4
0,8
1,2
ress
ion
of P VC-400
aa a
a
b
a
baab
a ababb
0,0
0,4
Day 11 Day 25 Day 45
E
0,0Day 11 Day 22 Day 45
Exp
0,0
0,4
Day 11 Day 15 Day 25 Day 45
Expr b
Before day 22 …Before day 22 …Skeletal elements that developed during this period were
sensitive to low VD3 levels And also those that developed later on were
sensitive to low VD3 levelsSkeletal elements that developed during this period were less sensitive to high VD3 levelsSkeletal elements that developed later on were
more sensitive to high VD3 levels
larvi 2009
OSSIFICATIONDELAY
After day 22
59%
pugheadness & caudal fin deformities (5 mm TL, 11 dph)branchiostegal rays (8 mmTL, 25 dph)After day 22 Vertebral column & branchiostegal rays deformities
BMP-4 OSTEOBLASTS
MALFORMATIONS
DELAY
ACCELERATION
Multipotentcells
RARγ+IGF-1
+BMP-4
VDR OST
ACCELERATIONOSSIFICATION
VITAMIN D OPTIMAL
49%
20%
+ +VDR OSTVITAMIN D
OSSIFICATION
11.2 IU g-1ENTEROCYTES
Ca+2TRPV-642 120 IU g-1
11.2 IU g
27.6 IU g-1
~16 IU g-1
~14 IU g-1
42-120 IU g 1
At day 22 …Before day 45 …Skeletal elements that developed early were
sensitive to low VC levelsAnd also those that developed later on
Epurals & specialized neural archSkeletal elements that developed early were less
sensitive to high VC levelsSkeletal elements that developed later on were more sensitive to
high VC levels
larvi 2009
At day 22 …
ADIPOCYTESOSSIFICATION
DELAYPPARγ
At day 45…
50%
Before day 45 …At day 45… Pugheadness & haemal arches
Epurals & specialized neural archgEpurals, specialized neural arch, dentary, dorsal & anal fins,
supernumerary vertebrae
OSTEOBLASTS MALFORMATIONS
γMultipotent
cells IGF-1
BMP 4
+
RARγ
++
VDR OST
MALFORMATIONS
OPTIMAL 30%VITAMIN C
BMP-4 RARγ
VDROSSIFICATION
> 30 mg Kg-1
VITAMIN C
ENTEROCYTES
Ca+2TRPV-6400 mg kg-1
30 mg Kg
50 mg kg-1
SVCT 1 VC 400 mg kg 1SVCT-1 VC
CONCLUSIONSlarvi 2009
No evident effect on larval developmentMaturation of the intestinal functions delayed => effect on larval development
Disruption of the expression of genes Disruption of the expression of genes
Disruption of intestinal Ca2+ absorption (TRPV-6)
Disruption of intestinal Ca2+ & VC absorption (TRPV-6, SVCT-1)
p p ginvolved in skeletogenesis (BMP-4, IGF-1,
RARγ) and bone mineralization (VDR, osteocalcin)
p p ginvolved in skeletogenesis (IGF-1, RARγ) and
bone mineralization (VDR, osteocalcin) in favor of adipocytic tissue formation (PPARγ)
Sk l t l l t th t d l d i l Sk l t l l t th t d l d i l
Skeletal elements that developed in early and later stages were equally sensitive to low VD3
levels
Skeletal elements that developed in early and later stages were equally sensitive to low VC
levels
Optimal VD is very restricted
Skeletal elements that developed in early stages were more resistant to high VD3 than
those that developed later on
Skeletal elements that developed in early stages were more resistant to high VC than
those that developed later on
Optimal VC is restricted
27.6 IU VD3/g diet(11.5 x dose of juveniles; NRC,1993)
MALFORMARTIONS ~30%
Optimal VD3 is very restricted
50 mg VC/kg diet(0.5 x dose of juveniles; NRC, 1993)
Optimal VC is restricted
PERSPECTIVESlarvi 2009
Cartilage damagePugheadness
Cartilaginous vertebraeHaemal arch not formedDeformities of the caudal fin
Pugheadness ↓ osteocalcin↓ RARg Pugheadness
One vertebrae lostDeformities of the caudal finVertebral deformitiesBranchiostegal rays deformities Epurals, specialized neural arch
↓ osteocalcin↓ osteocalcin
↓ RARg↓ osteocalcin
↑ PPARg
Epurals, specialized neural archDeformities of dentaryDeformities of the dorsal and anal fin
Vertebral deformitiesBranchiostegal rays deformities
↑↑ osteocalcin↑↑ osteocalcin
↓ osteocalcin↑ PPARg
≠ DISRUPTIONSDIFFERENT FUNCTION
DIFFERENT MODE OF ACTION
INTRAMEMBRANOUSCHONDRAL
DIFFERENT ACTION ON THE RATE OF BONE MINERALIZATION
DIFFERENT TYPE OF BONE MINERALIZATION
STUDY OF THE SPECIFIC MOLECULAR MARKERS OF EACH TYPE OF OSSIFICATION
ADAPTATION OF THE AMOUNT OF VITAMINS TO THE DEVELOPMENTAL STAGE
ACKNOWLEDGEMENTSL b f Ad t ti R d ti & N t iti f i fi h
larvi 2009
Centre de BrestF
Lab of Adaptation Reproduction & Nutrition of marine fish
Unit of Fish Biology & Quality in Aquaculture
France
Biology DepartmentBiology DepartmentUniversity of PatrasGreece
Thank You for Your Attention !
Before day 22 …Before day 22 …Skeletal elements that developed during this period
were very sensitive to low VD3 levels And also those that developed later on were
sensitive to low VD3 levelsSkeletal elements that developed during this period were less sensitive to high VD3 levelsSkeletal elements that developed later on were
more sensitive to high VD3 levels
larvi 2009
OSSIFICATIONDELAY
After day 22
59%
pugheadness & caudal fin deformities (5 mm TL, 11 dph)branchiostegal rays (8 mmTL, 25 dph)After day 22 Vertebral column & branchiostegal rays deformities
BMP-4 OSTEOBLASTS
MALFORMATIONS
DELAY
ACCELERATION
Multipotentcells
RARγ+IGF-1
+BMP-4
VDR OST
ACCELERATIONOSSIFICATION
VITAMIN D OPTIMAL
49%
20%SCL
+ +VDR OSTVITAMIN D
OSSIFICATION
11.2 IU g-1
OSTEOCYTES
SCL
?
ENTEROCYTES
Ca+2TRPV-642 120 IU g-1
11.2 IU g
27.6 IU g-1
~16 IU g-1
~14 IU g-1
42-120 IU g 1
