Inflammation and Male Inflammation and Male Reproductive Function Reproductive Function Dale Buchanan Hales, PhD Dale Buchanan Hales, PhD University of Illinois at University of Illinois at Chicago Chicago Department of Physiology and Department of Physiology and Biophysics Biophysics
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Inflammation and Male Reproductive Function Dale Buchanan Hales, PhD University of Illinois at Chicago Department of Physiology and Biophysics Dale Buchanan.
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Inflammation and Male Inflammation and Male Reproductive FunctionReproductive Function
Inflammation and Male Inflammation and Male Reproductive FunctionReproductive Function
Dale Buchanan Hales, PhDDale Buchanan Hales, PhDUniversity of Illinois at ChicagoUniversity of Illinois at Chicago
Department of Physiology and BiophysicsDepartment of Physiology and Biophysics
Dale Buchanan Hales, PhDDale Buchanan Hales, PhDUniversity of Illinois at ChicagoUniversity of Illinois at Chicago
Department of Physiology and BiophysicsDepartment of Physiology and Biophysics
Kent Christensen, Univ. Michigan
Cross section of rat testisCross section of rat testisShowing Seminiferous Showing Seminiferous
Tubules and Interstitium Tubules and Interstitium
Cross section of rat testisCross section of rat testisShowing Seminiferous Showing Seminiferous
Tubules and Interstitium Tubules and Interstitium
Functional and Anatomical Functional and Anatomical Compartments of the TestisCompartments of the TestisFunctional and Anatomical Functional and Anatomical
Compartments of the TestisCompartments of the Testis
Testicular interstitium and Testicular interstitium and seminiferous epitheliumseminiferous epithelium
Testicular interstitium and Testicular interstitium and seminiferous epitheliumseminiferous epithelium
Schematic Schematic viewview of the testis of the testisSchematic Schematic viewview of the testis of the testis
• Macrophage elaborated cytokines are potent inhibitors of Leydig cell steroidogenesis
• Macrophage and endothelium-derived reactive oxygen species (ROS) are deleterious in interstitium and seminiferous tubule– Singlet oxygen and peroxynitrile radicals
• Intracellularly -derived ROS during Ca2+ overload and/or oxidative stress
cholesterol
Extracellularlipoprotein
Cholesterolpool
LH
ATP
cAMPPKA+
Pregnenolone
Progesterone
Androstenedione
TESTOSTERONE
m
3HSD
P450c17
17HSD
acetate
IL-1, TNF and PMA vs. IL-1, TNF and PMA vs. Testosterone productionTestosterone productionIL-1, TNF and PMA vs. IL-1, TNF and PMA vs. Testosterone productionTestosterone production
0
500
1000
1500
20002500
3000
3500
4000
4500
con
cAM
P
cAM
P+IL1
cAM
P+TNF
cAM
P+PMA
ng
/10
6 LC
/24
h
IL-1, TNFIL-1, TNF and PMA vs. and PMA vs. steroidogenic mRNA expressionsteroidogenic mRNA expression
IL-1, TNFIL-1, TNF and PMA vs. and PMA vs. steroidogenic mRNA expressionsteroidogenic mRNA expression
cAMP+IL-1
+TNF+PMA
P450c17
P450scc
• Of all the steroidogenic enzymes, P450c17 is the most sensitive to repression
• Environmental disruptors such as dioxin, pthalates, PAHs, etc. are inhibitory
• Androgen-mediated feedback repression
P450c17 is sensitive to P450c17 is sensitive to transcriptional repression transcriptional repression
P450c17 is sensitive to P450c17 is sensitive to transcriptional repression transcriptional repression
IN VIVO METHODSIN VIVO METHODSIN VIVO METHODSIN VIVO METHODS
• Inject mice ip with LPS• Sacrifice mice at various times• Collect blood for serum hormone analyses by RIA• Collect testes, adrenals, and other organs• Isolate Leydig cells and testicular macrophages
– RNA and Protein analyses
• Metabolically label Leydig cells ex vivo with 35S-methionine and immunoprecipitate
• Aanalyze m by fluorescent microscopy
Effect of LPS on steroidogenic mRNA levelsEffect of LPS on steroidogenic mRNA levels Effect of LPS on steroidogenic mRNA levelsEffect of LPS on steroidogenic mRNA levels
P450scc
P450c17
3-HSD
actin
LPS - + - + - + - + - +
time 2h 4h 6h 8h 24h
0
2
4
6
8
10
12
14
LPS vs. serum testosterone: 2-24 hoursLPS vs. serum testosterone: 2-24 hoursLPS vs. serum testosterone: 2-24 hoursLPS vs. serum testosterone: 2-24 hours
Tes
tost
ero
ne
(ng
/ml)
control
LPS
Time post LPS
24 h2 h 4 h 8 h6 h
Steroidogenic Acute Steroidogenic Acute Regulatory Protein: StARRegulatory Protein: StAR
Steroidogenic Acute Steroidogenic Acute Regulatory Protein: StARRegulatory Protein: StAR
• Essential for steroid hormone biosynthesis• Cyclic-AMP dependent expression• Facilitates cholesterol transfer across inner-
mitochondrial (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 m
StAR facilitates cholesterol transferStAR facilitates cholesterol transfer StAR facilitates cholesterol transferStAR facilitates cholesterol transfer
Pulsatile nature of cholesterol Pulsatile nature of cholesterol flux into the mitochondriaflux into the mitochondria
Pulsatile nature of cholesterol Pulsatile nature of cholesterol flux into the mitochondriaflux into the mitochondria
StAR ProcessingStAR ProcessingStAR ProcessingStAR Processing
signal peptides
37 kDa
Outer mitochondrial membrane
Inner- mitochondrial membrane
critical regioncholesterol transfer
matrix
Cytosol
37
3230
Inner-mitochondrial forms
N'
32 kDaN'
30 kDaN'
Transfer across outer mitochondrialTransfer across outer mitochondrialmembrane and cleavage of first peptidemembrane and cleavage of first peptide
Transfer across outer mitochondrialTransfer across outer mitochondrialmembrane and cleavage of first peptidemembrane and cleavage of first peptide
Transfer across inner membrane, Transfer across inner membrane, formation of contact sites for cholesterolformation of contact sites for cholesteroltransfer, and cleavage of second peptidetransfer, and cleavage of second peptide
Transfer across inner membrane, Transfer across inner membrane, formation of contact sites for cholesterolformation of contact sites for cholesteroltransfer, and cleavage of second peptidetransfer, and cleavage of second peptide
Mature 30 kDa protein associated withMature 30 kDa protein associated withinner mitochondrial membrane postinner mitochondrial membrane post
cholesterol transfercholesterol transfer
Mature 30 kDa protein associated withMature 30 kDa protein associated withinner mitochondrial membrane postinner mitochondrial membrane post
cholesterol transfercholesterol transfer
N'-mutant protein associates only withN'-mutant protein associates only withouter mitochondrial membrane and outer mitochondrial membrane and still facilitates cholesterol transferstill facilitates cholesterol transfer
N'-mutant protein associates only withN'-mutant protein associates only withouter mitochondrial membrane and outer mitochondrial membrane and still facilitates cholesterol transferstill facilitates cholesterol transfer
C'-mutant protein neither associates with C'-mutant protein neither associates with outer mitochondrial membrane nor outer mitochondrial membrane nor
facilitates cholesterol transferfacilitates cholesterol transfer
C'-mutant protein neither associates with C'-mutant protein neither associates with outer mitochondrial membrane nor outer mitochondrial membrane nor
facilitates cholesterol transferfacilitates cholesterol transfer
sc cAd x-re d
a d x
3 H SD
M ito c ho nd ria lm a trix
C yto so l
chol
cholchol
StARN ’ C ’
PBR
M ito c ho nd ria lm a trix
C yto so l
TO MTIM
PBRVDAC
ANT
HKC KC p hD
Effect of LPS on Steroidogenic ProteinsEffect of LPS on Steroidogenic ProteinsEffect of LPS on Steroidogenic ProteinsEffect of LPS on Steroidogenic Proteins
LPS vs. StAR mRNA expressionLPS vs. StAR mRNA expressionLPS vs. StAR mRNA expressionLPS vs. StAR mRNA expression
What mediates the acute LPS What mediates the acute LPS inhibition?inhibition?
What mediates the acute LPS What mediates the acute LPS inhibition?inhibition?
Ethyl Ester (TMRE): Uptake is dependent on m. Rapidly and reversibly taken up by allowing dynamic measurement of membrane potential by fluorescent microscopy and flow cytometry.
• 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
Do reactive oxygen species Do reactive oxygen species (ROS) mediated the acute (ROS) mediated the acute inhbitory effects of LPS?inhbitory effects of LPS?
Do reactive oxygen species Do reactive oxygen species (ROS) mediated the acute (ROS) mediated the acute inhbitory effects of LPS?inhbitory effects of LPS?
*
*
*
cAMP + H2O2 (in цM) cAMP + H2O2 (in M)
H2O2 vs. Testosterone
production in Leydig cells*
* H2O2 vs. Progesterone
production in MA-10 cells
44%
47%
Pro
ges
tero
ne
ng
/ 106 c
ells
/ hr.
0
10
20
30
40
50
60
Pro
ges
tero
ne
ng
/ 106 c
ells
/ hr.
Con cAMP +100 + 250 + 500
cAMP + H2O2 (M)
47%
*
**
0
100
200
300
400
500
600
700
800
Tes
tost
ero
ne
ng
/ 106 c
ells
/ hr.
Con cAMP +100 + 250
cAMP + H2O2 (M)
** 44%
Effects of ROS on Steroidogenesis Effects of ROS on Steroidogenesis
in MA-10 cells and Leydig cellsin MA-10 cells and Leydig cells
Effects of ROS on Steroidogenesis Effects of ROS on Steroidogenesis
in MA-10 cells and Leydig cellsin MA-10 cells and Leydig cells
0
1000
2000
3000
4000
0
200
400
600
800
1000
0
1000
2000
3000
4000
5000
Con cAMP +100 +250 +500
cAMPH2O2 (M)
IOD
StA
RIO
D 3
-H
SD
IOD
P4
50
sc
c
Con. cAMP +100 + 250 +500
Con cAMP +100 + 250 + 500
Con cAMP +100 + 250
**
n.s
cAMP+H2O2 (M)
cAMP+H2O2 (M)
cAMP+H2O2 (M)
n.s n.s
*
StAR protein
3-HSD protein
P450scc protein
90%
35%
Con cAMP +100 +250 +500
cAMPH2O2 (M)
Con cAMP +100 +250 +500
cAMPH2O2 (M)
HH22OO2 2 effects on steroidogenic proteins in MA-10 cellseffects on steroidogenic proteins in MA-10 cells
Effect of HEffect of H22OO22 on StAR mRNA on StAR mRNAEffect of HEffect of H22OO22 on StAR mRNA on StAR mRNA
Northern Blot
StAR mRNA
Contr. cAMP. 100 200 250 500
Cyclophilin mRNA
TMRE staining of MA-10 cells TMRE staining of MA-10 cells exposed to H2O2exposed to H2O2
TMRE staining of MA-10 cells TMRE staining of MA-10 cells exposed to H2O2exposed to H2O2
John Allen John Allen Ed Lewin Ed Lewin Beth NardulliBeth NardulliAlice KimAlice KimMarika WrzosekMarika WrzosekSalil GindeSalil GindeJohn Choi John Choi Thorsten DiemerThorsten Diemer
John Allen John Allen Ed Lewin Ed Lewin Beth NardulliBeth NardulliAlice KimAlice KimMarika WrzosekMarika WrzosekSalil GindeSalil GindeJohn Choi John Choi Thorsten DiemerThorsten Diemer
Hales LabHales LabHales LabHales Lab
Bruce Bosmann Bruce Bosmann Barbara ClarkBarbara ClarkJim FergusonJim FergusonLarry Jamison Larry Jamison Jean-Guy LeHoux Jean-Guy LeHoux Artur MayerhofferArtur MayerhofferMark McLean Mark McLean Yossi Orly Yossi Orly Keith Parker Keith Parker Anita Payne Anita Payne Richard PestellRichard PestellCatherine Rivier Catherine Rivier Focko RommertsFocko RommertsDouglas StoccoDouglas Stocco
Bruce Bosmann Bruce Bosmann Barbara ClarkBarbara ClarkJim FergusonJim FergusonLarry Jamison Larry Jamison Jean-Guy LeHoux Jean-Guy LeHoux Artur MayerhofferArtur MayerhofferMark McLean Mark McLean Yossi Orly Yossi Orly Keith Parker Keith Parker Anita Payne Anita Payne Richard PestellRichard PestellCatherine Rivier Catherine Rivier Focko RommertsFocko RommertsDouglas StoccoDouglas Stocco