Download the cell context influences rainbow trout gonadotropin receptors

THE CELL CONTEXT INFLUENCES RAINBOW TROUT GONADOTROPIN
RECEPTORS SELECTIVITY
Saha A.*, Sambroni E.1, Bogerd J.2, Schulz R.W.2, Le Gac F.1, Lareyre J.J.1
*Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, 751002, India
Fax:+91-674-2465407 email:[email protected]
1 INRA SCRIBE, UPR 1037, 35042 Campus de Beaulieu, Rennes, France
2 Utrecht University, Utrecht, The Netherlands
INTRODUCTION
BACKGROUND: The presence of two distinct gonadotropin receptors (GtHRs) in a single fish species was confirmed by the molecular cloning of two different cDNAs in
several fish species including trout. In mammals, GtHRs show little cross-activation (0.01–0.1%). In contrast, the bioactivity of fish gonadotropins seems to be less well
separated as a result of promiscuous hormone–receptor interactions.
OBJECTIVE: The present study aimed at investigating whether the cellular context modulate the ligand selectivity of trout gonadotropin receptors.
METHOD: trout GtHRs expression vectors were transfected in two different mammalian cell lines (COS-7 and HEK 293 cells). The activation of trout GtHRs was induced in
presence of salmonid or non salmonid gonadotropins for six hours and monitored using a cAMP responsive luciferase reporter gene.
RESULTS
FSHR COS
FSHR HEK
125
cFSH
cLH
100
% maximum induction
% maximum induction
125
75
50
25
0
1
10
100
1000
cFSH
cLH
100
75
50
FSH receptor is activated by both cFSH and
cLH in the HEK293 cell line but only by cFSH
in COS-7 cell line
25
0
10000
1
10
100
GtH ng/ml
1000
10000
GtH ng/ml
Figure 1: Dose response curve of rainbow trout FSHR in the two different cell lines
LHR COS
LHR HEK
125
cFSH
cLH
100
% maximum induction
75
50
25
cFSH
cLH
100
LH receptor is mainly stimulated by cLH in
both HEK293 and COS-7 cell lines.
75
50
25
0
0
1
10
100
1000
10000
1
10
100
GtH ng/ml
1000
10000
GtH ng/ml
Figure 2: Dose response curve of rainbow trout LHR in the two different cell lines
The cell context modulates ligand-receptor selectivity to
salmonid and non salmonid gonadotropins
HEK cells
COS cells
FSHR
FSHR
*
80000
70000
*
60000
50000
300
9000
*
8000
7000
*
40000
6000
5000
4000
*
30000
20000
*
10000
3000
2000
1000
0
0
0
cFSH
Ex vivo, trout GtHR show much higher
selectivity for their cognate salmonid
gonadotropins
cLH
zfFSH
zfLH
hFSH
hLH
hCG
0
cFSH
cLH
ZfFSH
ZfLH
hFSH
hLH
hCG
800 ng/ml
800 ng/ml
11 KT production (ng/ml)
% maximum induction
125
13.7
250
200
150
5.9
100
LHR
900000
LHR
*
800000
50
40000
700000
*
35000
600000
30000
500000
*
400000
300000
//
//
*
40000
30000
*
//
25000
Control
*
15000
*
20000
*
*
10000
*
20000
//
10000
0
0
cFSH
cLH
zfFSH
zfLH
800 ng/ml
hFSH
hLH
hCG
1
1
0
cFSH
cLH
ZfFSH
ZfLH
hFSH
hLH
cFSH
cLH
hFSH
hLH
hCG
800 ng/ml
5000
0
0.8
0
hCG
800 ng/ml
Figure 4: Trout testicular explants collected from pre-spermiating animals were cultured
in vitro and stimulated for 18 hours in presence of salmonid or human gonadotropins. 11ketotestosterone (11-KT) production was determined by radioimmunoassay. Fold
changes are indicated on the top of the bars.
Figure 3: The responsiveness of rainbow trout GtHRs to non salmonid gonadotropins
depends on the cell line used. Note that FSHR is responsived to LH and hCG in HEK293
cells but not in COS cells
CONCLUSION
We demonstrate that cross-selectivity of the trout GtHRs’ responsiveness is modulated by the cellular context. Trout GtHRs showed higher ligand selectivity when expressed in
COS-7 cells, but not when expressed in HEK cells. However, trout receptors selectivity was more prononced in the homologous cellular context. Altogether, we propose that
trout GtHRs are highly selective in vivo and that the mammalian cell lines used reflect only partially this high selectivity.