summary of my thesis research

1. Does an environmental toxicant (MEHP) target
signaling in spermatogonial stem cells?
Benjamin E Lucas
MS, PhD Candidate
Department of Comparative Biosciences
College of Veterinary Medicine
University of Illinois at Urbana-Champaign
Urbana, IL

2. Blood testis barrier
Myoid cell
Leydig cell
The Testis

3. Petersennd et al., 2000;
Barr et al., 2003; Duty et
al., 2004.

4. Myoid cell
Leydig cell
Steroid synthesis INSL3
FSH response
Junctional complexes
DEHP
MEHP
Richburg et al., 1996; Sharpe et al., 2003; Bhattacharya et al., 2008, Yao et al. 2007 and 2010.
MEHP targets
Cytoskeleton
Gene expression
ERK1/2
Germ cell
apoptosis
Blood testis barrier

5. Spermatogenesis / Stem cells
SSC markers
POU5F1
GFRA1
TEX14
KIT
Differentiation markers

6. Braydich-Stolle et al,. 2007; Simon et al., 2007; He et al,. 2008.
GFRA1
GFR
GDNF GDNF
ERK 1/2
• Proliferation in vitro
• Self-renewal in vivo
• Survival
P-RET P-RET
FOS
SRC
MYCN
GDNF signaling in SSCs
Early differentiation Maintenance
Sertoli cell
FGF

7. Hypothesis
MEHP
Reduced sperm production

8. Spermatogonial stem cell line C18-4
Hofmann et al, 2005

9. MEHP does not trigger apoptosis and triggers
necrosis only at higher concentrations
0 0.5 5 10 50 ST0.1
0%
20%
40%
60%
80%
100%
Apoptosis after overnight exposure to MEHP
MEHP concentration (µM)
%
of
total
population
*
0 0.5 5 10 50 ST0.1
0%
20%
40%
60%
80%
100%
Necrosis after overnight exposure to MEHP
MEHP concentration (µM)
%
of
total
population
**
**
0 0.5 5 10 50 ST0.1
MEHP concentration (μM)
MEHP does not trigger DNA laddering
Lucas et al., submitted 01/31/12

10. GFR-1
GFR-1
GDNF GDNF
ERK
• Proliferation in vitro
• Self-renewal in vivo
P-RET P-RET
FOS
SRC
MYCN
MEHP
Hypothesis

11. MEHP does not affect SRC/MYCN
signaling at the protein level
Lucas et al., submitted 01/31/12

12. MEHP does affect ERK/FOS
signaling at the protein level
Lucas et al., submitted 01/31/12

13. MEHP affects RNA expression of
Fos but not Mycn
Lucas et al., submitted 01/31/12

14. GFR-1
GFR-1
GDNF GDNF
ERK
• Proliferation in vitro
P-RET P-RET
FOS
SRC
MYCN
MEHP
Conclusion: MEHP affects ERK signaling in
the spermatogonial stem cell line C18-4
Lucas et al., submitted 01/31/12

15. Leydig cell
 GDNF is one of the most
important factors regulating
SSC in vivo
 We used 6- to 8-day-old
pups from the
DBAB6;CBA-Tg(Pou5f1-
EGFP)2MnnF1/J mouse
strain to isolate SSCs.
Myoid cell
Blood testis barier
Is the same pathway affected in fresh
SSCs ?

16. SSC isolation
Testes
Seminiferous tubules
Tunica and epididymis
Differential plating
GFP +
cells
SSC
GFP -
cells

17. Culture - Treatments
MEHP concentration
Seminal Plasma ≤ 1μM
Serum ≤ 1 μM
Urine ≤ 5 μM
Thereforewe'll work
with
0.5 μM MEHP
5.0 μM MEHP
 SSC's survival &
proliferation is greatly
enhanced by
 GDNF (20 ng/mL)
 GFRa1 (120 ng/mL)
 FGF (2 ng/mL)
 Gelatin coated dishes
GF

18. Does MEHP directly affect SSCs?
• Live GFP positive
counts
• TaliTM image
cytometer
• Direct observation
after 48 of culture
0
0.5
1
1.5
2
2.5
3
3.5
Single Double 4 cell Clump
Population
(X1
000cells
)
NOGF + 0 μM
GF + 0 μM
GF + 0.5 μM
GF + 5.0 μM
MEHP
b
a a
a
b
a aa
b
a aa
Lucas et al., in preparation
0
0.5
1
1.5
2
2.5
3
3.5
Fold
Change
GF - + + +
MEHP (μM) - - 0.5 5.0
b
a a a

19. MEHP affects Fos and other GDNF
-dependent genes expression
 PCR after 1 and 2
days of exposure to
MEHP
MEHP
0
1
10
100
0 1 2
Ccna2
fold
change
Time (days)
GF + 0 μM
GF + 0.5 μM
GF + 5.0 μM
**
*
MEHP
0
1
10
100
0 1 2
Cdk2
fold
change
Time (days)
GF + 0 μM
GF + 0.5 μM
GF + 5.0 μM
**
*
MEHP
0
1
10
100
0 1 2
Fos
fold
change
Time (days)
GF + 0 μM
GF + 0.5 μM
GF + 5.0 μM
**
*
**
**

20. MEHP affects proliferation but does not alter GDNF-dependent
change of differentiation related genes
0.1
1
10
100
0 1 2
Ccne1
fold
change
Time (days)
GF + 0 μM
GF + 0.5 μM
GF + 5.0 μM
MEHP
**
*
**
**
0
1
10
100
0 1 2
Pcna
fold
change
Time (days)
GF + 0 μM
GF + 0.5 μM
GF + 5.0 μM
MEHP
**
*

21. MEHP alters SSC markers
 48h exposure
 Staining for PLZF and
GFRA1
 Recording with TaliTM
cytometer
0
0.5
1
1.5
No GF GF GF + 0.5 GF + 5.0
Fold
change
PLZF+
in
GFP+
cells
GF - + + +
MEHP (μM) - - 0.5 5.0
b
a
c
a
0
0.5
1
1.5
2
2.5
3
No GF GF GF + 0.5 GF + 5.0
Fold
change
GFRA1+
in
GFP+
cells
GF - + + +
MEHP (μM) - - 0.5 5.0
b
a
c
a

22. MEHP also alters the expression of the
differentiation marker KIT
 48h exposure
 Staining for KIT
0
0.5
1
1.5
No GF GF GF + 0.5 GF + 5.0
Fold
change
KIT+
in
GFP+
cells
GF - + + +
MEHP (μM) - - 0.5 5.0
b
a
b
c

23. GDNF expression is affected in Sertoli cells
 Sertoli cells stay in the bottom of the dish that was used for
differential plating
 GDNF expression is stimulated in Sertoli cells by FGF2
(20ng/mL)
 MEHP decreases GDNF production even in presence of
FGFand MEHP
 Treatment :
48h
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 0.5 5
Gdnf
expression
MEHP concentration (µM)
b
a
b

24. Braydich-Stolle et al,. 2007; Simon et al., 2007; He et al,. 2008.
GFRA1
GFR
GDNF GDNF
ERK 1/2
• Proliferation in vitro
• Self-renewal in vivo
• Survival
P-RET P-RET
FOS
SRC
MYCN
GDNF signaling
Early differentiation Maintenance
SSC
Sertoli cell
FGF
?
?

25. SSC transplantation
6 to 8-day-old
GFP mice pups -
isolation
24h exposure to MEHP (0.5 and
5.0 µM)
Transplantation
45 days later:
• Colony count
• Histology
If stemness is
altered : lower or
no colonization

26. Acknowledgments
Committee members
Dr. Marie-Claude Hofmann
Dr. Jodi Flaws
Dr. Romana Nowak
Dr. Rex Hess
Dr. David Miller
Dr. Humphrey Yao (former member)
Lab members (current)
Dr. Thomas Garcia
Dr. Jaspreet Kaur
Daryl Melling
past
Phil Byun
Neeraj Joshi
George Li
Source of funds
Lilly Foundation
toxicology/pharmacology fellowship
NIH R01 HD044543
Other Lab
Dr. Sidonie Lavergne