Transcript Cross section of rat testis Showing Seminiferous Tubules and Interstitium Kent Christensen, Univ.

Cross section of rat testis
Showing Seminiferous
Tubules and Interstitium
Kent Christensen, Univ. Michigan
Functional and Anatomical
Compartments of the Testis
Interstitium of rat testis
showing endothelium,
Leydig cells (L), and
macrophages (arrow).
Note close association of
macrophages and
Leydig cells.
Scott Miller, Univ Utah
Close association of
Leydig cell and
macrophage, lower panel
shows close up of
“digitation” of Leydig cell
process extending onto
macrophage surface.
Scott Miller, Univ. Utah
Macrophage-Leydig cell interactions
Cytokines, ROS
?
Extracellular
lipoprotein
acetate
DYm
LH
Cholesterol
pool
ATP
cAMP
cholesterol
PKA+
Pregnenolone
3bHSD
Progesterone
P450c17
Androstenedione
17bHSD
TESTOSTERONE
P450c17 is sensitive to
transcriptional repression
• Of all the steroidogenic enzymes, P450c17 is the
most sensitive to repression
• Most cytokines tested inhibit c17 transcription:
– IL-1a/b, IL-2, IL-6, TNFa, TGFb, INFa/b, INFg
• Inflammatory mediators: PGF2a, ceramide,
vasopressin, PKC agonists
• Environmental disruptors such as dioxin,
pthalates, PAHs, etc. are inhibitory
• Androgen-mediated feedback repression
P+
P
M
A
F
cA
M
P+
TN
cA
M
L1
P+
I
cA
M
cA
M
P
4500
4000
3500
3000
2500
2000
1500
1000
500
0
co
n
ng/106 LC/24 h
IL-1, TNF and PMA vs.
Testosterone production
IL-1, TNFa and PMA vs.
steroidogenic mRNA expression
P450scc
P450c17
Relative CAT Activity
cAMP responsive regions of the Cyp17
promoter
100
80
Control
cAMP
60
40
20
0
-2500
-1021
-346
-245
TNF inhibits Cyp17 promoter activity
Calphostin reverses TNF inhibition of
Cyp17 promoter
TNFa and PMA stimulate translocation of
PKCa from cytoplasm to membrane
control
No antibody
PMA
TNFa
Putative transfactor binding sites
Comparison of cAMP-responsive
sequences in mouse and rat
-436 mouse
GTGACCTTAT GCAAACTAAC CCTAAAAGAC CTCTCTCTCC TCAACTATCA GATAATAAGA
GTGACCTTAT GCCGACTAAC CTTTGAAGAT CTCTTTCTCC TCAACTGTCA GATAGTAAGA
-447 rat
-376 mouse
CTGAAGTCTC TTTGACAGCT TTGGCTAGCT GCAACCTGAT GACATTAATT ATTAACTGTG
CTGCAGTCTC T--------- ---------- GAAACCCGAT GGCAGTAATT ATTAACCGTA
-387 rat
-316 mouse
CAGCACTTTT GACATTACAG CACGCACTCT GAAACCTTGA TCTTAATCTG ATAGCATTTG
TAGCACTT T GACATTACA CACAGACTCT AAAACCTTGA TCTCACTCTG ATAGCATTTG
-346 rat
-59 mouse
CACGTCTTCAAGGTGA
CTCGACGTCAAGGTGA
-73 rat
Binding sites (ATF2/cjun-like, Steroidogenic factor 1, StF-IT-2, and StF-IT-1/COUPTF1) are shown in bold color in the sequence for the species in which it was identified.
Those that are conserved between species are underlined. Sequence differences are shown
in blue for mouse and pink for rat.
Relative CAT Activity
Characterization of the Cyp17 Promoter
Revealed a Region Between -245 and -346
Responsible for the Minimal cAMP
Responsiveness of the Gene
100
80
Control
cAMP
60
40
20
0
-2500
-1021
-346
-245
Site-directed mutagenesis of Cyp17 CRR
(-346 to –245)
•Oligos were designed to place an XhoI once every
ten base pairs within the 100 base pair CRR.
•This resulted in changing as few as three (mutant
6) to as many as six (mutant 1 and 7) of every ten
nucleotides.
•Mutagenesis was performed with Altered Sites
(Promega) and all mutants were verified by
sequencing.
Cyclic-AMP induction of CRR mutants
W
ild
Ty
M pe
ut
an
M t1
ut
an
M t2
ut
an
M t3
ut
an
M t4
ut
an
M t5
ut
an
M t6
ut
an
M t7
ut
an
M t8
ut
an
t9
% of Wild Type Induction
by cAMP
cAMP Induction of CRR mutants
250
200
150
100
50
0
Putative sites revealed by
mutants
gcaacctgat gacattaatt attaactgtg cagcactttt gacattacag
CTCGAGtgat CTcGAGaatt CtCGaGtgtg cTCGaGtttt CTcGAGacag
mut 1
mut 2
mut 3
mut 4
mut 5
cacgcactct gaaaccttga tcttaatctg atagcatttg cctctgggag
cTcgAGctct CTCGAGttga CTCGaGtctg CtCgAGtttg cACGAgggag
mut 6
mut 7
mut 8
ATF2
AhR/Arnt (core sequence)
SF-1
mut 9
mut 10
Putative regulatory motifs revealed by mutagenesis
?
-250
-440
ATF2 mutants 2,5,9
C/EBPb– upstream site
AhR/ARNT mutant 6
SF-1 mutant 7
ARE
The Minimal cAMP Responsive Region of
the Cyp17 Promoter (CRR):
-346 TGATGACAT
CTTTTGACAT
CTTGATCTTA
AGGATCCATA
TAATTATTAA CTGTGCAGCA
TACAGCACGC ACTCTGAAAC
ATCTAGCATT TGCCTCTGGG
GCG -245
-346
-245
Putative ATF-2 binding site
Binding of Nuclear Proteins to the CRR Probe is
Augmented by Treatment of MA-10 Cells with cAMP
Nuclear Proteins from Primary Leydig Cells
Form Two Complexes with the CRR Probe
The Upstream ATF-2 C/EBPb Binding Site
-450 TTGTGTGACC TTATGCAAAC TAACCCA -423
-245
-450
ATF-2 Binding Sites
C/EBPß Binding Site
Nuclear Proteins from Control and cAMPTreated MA-10 cells Bind to the Upstream
ATF-2 C/EBPb Probe
Incubation of the Upstream ATF-2 C/EBPb probe
with Nuclear Proteins Isolated from Primary
Leydig Cells Results in Complex Formation
Formation of the Higher Order Complex Formed
by the CRR Complex is Decreased by Addition of
Unlabeled ATF-2 or C/EBPb Competitor Oligos
Binding of Nuclear Proteins to the Upstream
ATF-2 C/EBPb Probe can be Inhibited by
Addition of ATF-2 or C/EBPb Competitor Oligos
Overexpression of C/EBPb Induces
Transcription of the -491/-255 Cyp 17 reporter
7
Fold Induction
6
**
5
*
4
Control
3
+ cAM P
2
1
0
Cyp 17
Cyp 17 + ATF-2
Cyp 17 +
C/EBPb
Cyp 17 + ATF-2
+ C/EBPb
ATF-2 Expression in MA-10 Cells is Not
Affected by cAMP Treatment
Fold Induction
Western Analysis of ATF-2 Expression
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
12 h
SFM
1h
2h
4h
8h
12 h
cAMP cAMP cAMP cAMP cAMP
C/EBPb Expression is Significantly
Increased in MA-10 Cells with cAMP
p38
p34
Western Analysis of C/EBPb Expression
Fold Induction
10
8
**
6
4
2
0
12 h
SFM
1h
cAMP
2h
cAMP
4h
cAMP
8h
cAMP
12 h
cAMP
Summary of Cyp17 study
• TNFa-mediated inhibition of transcription
involves activation of PKC
• ATF2 and C/EBPb participate cooperatively
in cAMP-induction of transcription
• ATF2 is constitutively expressed
• C/EBPb expression is induced by cAMP
Hypotheses
• ATF2 and C/EBPb interact as heterodimers
binding to the “ATF2” sites in the promoter
• The stoichiometry of C/EBPb and ATF2
interaction is critical to driving transcription
• Repressors may act by inhibiting C/EBPb
expression or through post-translational
modifications that inhibit its activity
• C/EBPb phosphorylation by PKC may block it
from interacting with ATF2
Effect of LPS on steroidogenic mRNA levels
P450scc
P450c17
3b-HSD
actin
LPS
- + - + - + - +
time
2h
4h 6h
- +
8h 24h
LPS vs. serum testosterone: 2-24 hours
control
LPS
14
Testosterone (ng/ml)
12
10
8
6
4
2
0
2h
4h 6h
8 h 24 h
Time post LPS
Steroidogenic Acute
Regulatory Protein: StAR
• Essential for steroid hormone biosynthesis
• Cyclic-AMP dependent expression
• Facilitates cholesterol transfer across innermitochondrial (aqueous) space
• Translated as a 37 kDa precursor protein that
is processed to the 30 kDa mature form as it
translocates into the mitochondria
• Cholesterol transport activity depends on
intact DYm
StAR facilitates cholesterol transfer
Pulsatile nature of cholesterol
flux into the mitochondria
37
StAR Processing
32
30
Inner-mitochondrial
forms
Cytosol
37 kDa
N'
signal peptides
cholesterol transfer
critical region
Outer mitochondrial membrane
Inner- mitochondrial membrane
N'
32 kDa
matrix
N'
30 kDa
chol
chol
adx
Mitochondrial
matrix
Adx-red
scc
3bHSD
N’
StAR
C’
chol
Cytosol
chol
adx
Mitochondrial
matrix
Adx-red
scc
chol
Cytosol
PBR
chol
3bHSD
N’
StAR
C’
TOM
TIM
PBR
Cytosol
VDAC
ANT
Mitochondrial
matrix
CphD
CK
HK
StAR N-terminal localization expression clones
MTS
1-37
ITS
38-47
pCMV-StAR
TAA
StAR-stop
MTS
1-37
Tom20
OMTS
StAR D-ITS
StAR D-N47
StAR/Tom20
CCHL
IMSS
StAR/CCHL
What mediates the acute LPS
inhibition?
• Tested numerous inflammatory mediators in
Leydig cells in vitro-- none mimicked the acute
LPS “effect”
–
–
–
–
–
–
cytokines (TNFa, IL-1, IL-6, IFNg, TGFb)
prostaglandins (PGF2a, PGE)
catecholamines (norepi, isoproteranol)
ceramide (C2, C8)
Most nitric oxide donors (Sin-1, SNAP, SNP, Nor-3)
Calcium inophore (A23187)
LPS vs. StAR protein
expression: 2 hr after injection
37 kDa
30 kDa
Carbonyl cyanide mchlorophenylhydrazone (cccp)
• Carbonyl cyanide m-chlorophenylhydrazone (cccp): potent uncoupler
of oxidative phosphorylation;
protonophore, mitochondrial
disrupter.
• Causes transient disruption of DYm
Mitochondrial respiration, OX-PHOS and DYm
H+
DYm
e-
ng/ml
CCCP Vs. Progesterone in MA10s
500
400
300
200
100
0
con
cAMP
cAMP +
cccp
R22
R22 +
cccp
Effect of CCCP on StAR protein
37
kDa
30
kDa
Control
cAMP
cAMP + cccp cccp
Effect of CCCP on StAR mRNA
3.4 kB
2.9 kB
StAR
1.6 kB
cyclophilin
con cA cA+cccp
Effect of CCCP on StAR synthesis
37kDa
30kDa
Control
cAMP
cccp
cAMP + cccp
Effect of CCCP on StAR
synthesis
80
60
50
37 kDa
30 kDa
40
30
20
10
C
P
cA
+C
C
P
C
C
C
cA
M
P
nt
ro
l
0
co
Corrected density
70
Effect of cccp on StAR decay
3500000
3000000
2500000
cccp
contro
2000000
1500000
1000000
500000
0
0
60 120 180 240 300 360 420 480
minutes
Time course of StAR decay
Time course of StAR decay
density
100000
80000
30 kDa
32 kDa
37 kDa
60000
40000
20000
0
0
15
30
45
60
minutes
75
90 105 120
Effect of mitochondrial agents
on progesterone production
900
800
700
ng/ml
600
500
400
300
200
100
0
con
cAMP
+Oligom
+arsen
+CCCP
Effect of mitochondrial agents
on StAR protein expression
37 kDa
30 kDa
Effect of mitochondrial agents
on StAR mRNA expression
3.2 kB
StAR
1.6 kB
cyclophilin
Tetramethylrhodamine
Ethyl Ester (TMRE)
• Tetramethylrhodamine
Ethyl Ester (TMRE):
Uptake is dependent on DYm.
Rapidly and reversibly taken
up by allowing dynamic
measurement of membrane
potential by fluorescent
microscopy and flow
cytometry.
CCCP disrupts DYm in MA10s
control
CCCP-treated
Do reactive oxygen species
(ROS) mediated the acute
inhbitory effects of LPS?
• Testicular Macrophages are known to
produce ROS when activated
• ROS are produced rapidly after exposure to
LPS
• Many potential sources of ROS in testicular
interstitium
ratio {37/30 x 1000}
ROS causes increase in 37 kDa StAR in
Leydig cells in vitro
120
100
80
60
40
20
0
Con
cAMP
+10
+25
+100
+250
44%
37 kDa
30/32 kDa
Con
cAMP
+10
+25
+100
+250
cAMP + H2O2 (mM)
Effect of H2O2 on StAR mRNA
Northern Blot
StAR mRNA
Contr.
cAMP.
Cyclophilin mRNA
100
200
250
500
TMRE staining of MA-10 cells
exposed to H2O2
Control
100mM H2O2
TMRE staining of MA-10 cells
exposed to H2O2—time lapse
PFC Increases DHR-123 Fluorescence
B
Control Brightfield
Control Fluorescence
Control Merge
C
+PFC Brightfield
+PFC Fluorescence
+PFC Merge
PFC: 4-phenyl-3-furoxancarbonitrile
LPS inhibits Leydig cells in vivo via ROS
MDA + HNE (uM/10e6 LC)
Increased lipid
peroxidation and
depolarization of Leydig
cell mitochondria support
involvement of ROS in
LPS action in vivo
Lipid peroxides
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
control
LPS
PKA
mitochondria
ROS
H+
Dm
?
cytosol
chol
chaperonin
chol
chol
cholesterol
pool
Inflammation
LPS, sepsis
Aging
NO°
Nucleus
Ischemia/
reperfusion
ROS
Diabetes
Cytokines
Alcohol
UVa
Mitochondria
Xenobiotics
PAHs, PPs
Adenosine
Arsenate
Steroidogenic machinery
Sites of immune inhibition
ROS
But what does it all
mean, anyway?
Conclusions
• Inflammation and infection may contribute to, or
cause decreased male reproductive function
• There is a push-pull system between the immune
and endocrine systems
– During times of sickness the immune system
suppresses the reproductive system
(testosterone behavior vs. sickness behavior)
– During times of normal health testosterone
suppresses the immune response
Importance of Immuneendocrine interactions
• Females are more susceptible to autoimmune
diseases than males
• Estradiol and prolactin are both immunostimulatory
• Testosterone is immuno-inhibitory
• Castration results in marked increase in thymic
cell proliferation
• Higher concentration of androgen receptors in
thymus than all other tissues except prostate
Antechinus Stuartii
victim of his own testosterone
Hales Lab
John Allen
Paul Janus
Fred Lepore
Beth Nardulli
Salil Ginde
John Choi
Thorsten Diemer
Karen Held Hales
NIH: HD25271 HD35544
collaborators
Bruce Bosmann
Barbara Clark
Jim Ferguson
Larry Jamison
Jean-Guy LeHoux
Artur Mayerhoffer
Mark McLean
Yossi Orly
Anita Payne
Richard Pestell
Catherine Rivier
Focko Rommerts
Douglas Stocco