Inflammation and Male Reproductive Function Dale Buchanan Hales, PhD University of Illinois at...
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Transcript of Inflammation and Male Reproductive Function Dale Buchanan Hales, PhD University of Illinois at...
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
SpermatogenesisSpermatogenesisSpermatogenesisSpermatogenesis
The wave of the seminiferous The wave of the seminiferous epitheliumepithelium
The wave of the seminiferous The wave of the seminiferous epitheliumepithelium
Stages of spermatogenesisStages of spermatogenesisStages of spermatogenesisStages of spermatogenesis
{Y. Claremont, 1952}
Stage-Specific gene expressionStage-Specific gene expressionStage-Specific gene expressionStage-Specific gene expression
FSH-R/cAMP Testosterone/ABP
{Adapted from M. Parvinen}
Intra-tubular regulatory Intra-tubular regulatory factorsfactors
produced by Sertoli and germ cellsproduced by Sertoli and germ cells
Intra-tubular regulatory Intra-tubular regulatory factorsfactors
produced by Sertoli and germ cellsproduced by Sertoli and germ cells
• Cytokines (IL-1, IL-6, TGF, IFNs, TNF)
• Growth factors (IGF-1, TGF, bFGF, NGF, Steel factor, PDGF)
• Inhibin, activin, MIS
• Nitric Oxide, endothelin, VGEF
{Adapted from B. Jégou}
SpermiationSpermiationSpermiationSpermiation
Phagocytosis of residual body by Phagocytosis of residual body by Sertoli cellSertoli cell
Phagocytosis of residual body by Phagocytosis of residual body by Sertoli cellSertoli cell
Physiological Immune-Physiological Immune-endocrine interactions endocrine interactions Physiological Immune-Physiological Immune-endocrine interactions endocrine interactions
• Intra-tubular production of cytokines, growth factors and other inflammatory mediators orchestrate autonomous control of germ cell development
• Role of macrophages in Leydig cell development– Required for development and regeneration– May produce positive regulatory factors
Scott Miller, Univ Utah
Interstitium of rat testis Interstitium of rat testis showing endothelium, showing endothelium, Leydig cells (L), and Leydig cells (L), and
macrophages (arrow). macrophages (arrow). Note close association of Note close association of
macrophages and macrophages and Leydig cells. Leydig cells.
Interstitium of rat testis Interstitium of rat testis showing endothelium, showing endothelium, Leydig cells (L), and Leydig cells (L), and
macrophages (arrow). macrophages (arrow). Note close association of Note close association of
macrophages and macrophages and Leydig cells. Leydig cells.
Scott Miller, Univ. Utah
Close association of Close association of Leydig cell and Leydig cell and
macrophage, lower panel macrophage, lower panel shows close up of shows close up of
“digitation” of Leydig cell “digitation” of Leydig cell process extending onto process extending onto macrophage surface.macrophage surface.
Close association of Close association of Leydig cell and Leydig cell and
macrophage, lower panel macrophage, lower panel shows close up of shows close up of
“digitation” of Leydig cell “digitation” of Leydig cell process extending onto process extending onto macrophage surface.macrophage surface.
Cytokines, ROS
?
Macrophage-Leydig cell interactionsMacrophage-Leydig cell interactionsMacrophage-Leydig cell interactionsMacrophage-Leydig cell interactions
Pathophysiological immune-Pathophysiological immune-endocrine interactionsendocrine interactions
Pathophysiological immune-Pathophysiological immune-endocrine interactionsendocrine interactions
• 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
• Most cytokines tested inhibit c17 transcription: – IL-1, IL-2, IL-6, TNF, TGF, INF, INF
• Inflammatory mediators: PGF2, ceramide, vasopressin, PKC agonists
• 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?
• Tested numerous inflammatory mediators in Leydig cells in vitro-- none mimicked the acute LPS “effect”– cytokines (TNF, IL-1, IL-6, IFN, TGF)– prostaglandins (PGF2, PGE) – catecholamines (norepi, isoproteranol) – ceramide (C2, C8)– Most nitric oxide donors (Sin-1, SNAP, SNP, Nor-3)– Calcium inophore (A23187)
LPS vs. StAR protein LPS vs. StAR protein expression: 2 hr after injectionexpression: 2 hr after injection
LPS vs. StAR protein LPS vs. StAR protein expression: 2 hr after injectionexpression: 2 hr after injection
30 kDa
37 kDa
conLPS
Carbonyl cyanide Carbonyl cyanide mm--chlorophenylhydrazone (cccp)chlorophenylhydrazone (cccp)
Carbonyl cyanide Carbonyl cyanide mm--chlorophenylhydrazone (cccp)chlorophenylhydrazone (cccp)
• Carbonyl cyanide m-chlorophenyl-hydrazone (cccp): potent uncoupler of oxidative phosphorylation; protonophore, mitochondrial disrupter.
• Causes transient disruption of m
Effect of CCCP on StAR proteinEffect of CCCP on StAR proteinEffect of CCCP on StAR proteinEffect of CCCP on StAR protein
Control cAMP cAMP + cccp cccp
37 kDa
30 kDa
con cA cA+cccp
StAR
cyclophilin
3.4 kB
1.6 kB
2.9 kB
Effect of CCCP on StAR mRNAEffect of CCCP on StAR mRNAEffect of CCCP on StAR mRNAEffect of CCCP on StAR mRNA
Tetramethylrhodamine Tetramethylrhodamine Ethyl Ester (TMRE)Ethyl Ester (TMRE)
Tetramethylrhodamine Tetramethylrhodamine Ethyl Ester (TMRE)Ethyl Ester (TMRE)
• Tetramethylrhodamine
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.
controlcontrolcontrolcontrol CCCP-treatedCCCP-treated CCCP-treatedCCCP-treated
CCCP disruptsCCCP disrupts mm in MA10sin MA10sCCCP disruptsCCCP disrupts mm in MA10sin MA10s
Analysis of Analysis of m in Leydig cells:m in Leydig cells:Post-staining protocolPost-staining protocol
Analysis of Analysis of m in Leydig cells:m in Leydig cells:Post-staining protocolPost-staining protocol
• Inject mice with LPS and purify Leydig cells as usual
• Incubate purified cells with TMRE
• Examine by fluorescent microscopy
• Indicates if cells suffered long-term depolarization
LPS disrupts Leydig Cell LPS disrupts Leydig Cell mmlong-term effectlong-term effect
LPS disrupts Leydig Cell LPS disrupts Leydig Cell mmlong-term effectlong-term effect
• 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
ControlControlControlControl 100100M HM H22OO22100100M HM H22OO22
H2O2 disrupts m
0
500
1000
1500
2000
2500
3000
30/32 kDa
0
1000
2000
3000
4000
5000
Con cAMP +10 +25 +100 +250
cAMPH2O2 (M)
StAR protein
Con cAMP +10 + 25 +100 +250 cAMP+H2O2 (M)
IOD
StA
R
Con cAMP +10 +25 +100 +250 cAMP+H2O2 (M)
IOD
3-
HS
D Con cAMP +10 +25 +100 +250
cAMPH2O2 (M)
3-HSD protein
*
n.s. n.s.
H2O2 effects on steroidogenic proteins in Leydig cellsH2O2 effects on steroidogenic proteins in Leydig cells
44%
HH22OO2 2 disrupts disrupts mm in Leydig cells in Leydig cellsHH22OO2 2 disrupts disrupts mm in Leydig cells in Leydig cells
ControlControlControlControl 250uM H2O2 250uM H2O2 250uM H2O2 250uM H2O2
Summary: StAR and 3Summary: StAR and 3-HSD -HSD studies in MA10sstudies in MA10s
Summary: StAR and 3Summary: StAR and 3-HSD -HSD studies in MA10sstudies in MA10s
• CCCP transiently disrupts m
• CCCP inhibits StAR processing and progesterone production
• Disruption of m alone does not block 3-HSD protein-- activity??
• H2O2 inhibits StAR and 3-HSD protein and progesterone production
• H2O2 disrupts m
Analysis of lipid peroxidationAnalysis of lipid peroxidationAnalysis of lipid peroxidationAnalysis of lipid peroxidation
• Lipid peroxidation is a mechanism of cellular injury that leads to production of lipid peroxides and their by-products.
• Malonaledyde (MDA) and 4-hydroxy-2-nonenal (HNE) are end products derived from peroxidation of polyunsaturated fatty acids and related esters.
• Measurement of these aldehydes is an index of lipid peroxidation.
Lipid peroxidation in Leydig Lipid peroxidation in Leydig cellscells
Lipid peroxidation in Leydig Lipid peroxidation in Leydig cellscells
Lipid peroxides in Leydig cells
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
control LPS
MD
A +
HN
E (
uM
/10
e6
LC
)
Summary: in vivo studiesSummary: in vivo studiesSummary: in vivo studiesSummary: in vivo studies
• LPS causes an abrupt and prolonged decrease in serum testosterone levels
• Abrupt decreases in testosterone are correlated to inhibition of Leydig cell StAR and 3-HSD protein
• LPS causes disruption of Leydig cell m,
• Preliminary data supports the role of ROS in mediating acute effects of LPS in vivo
chol
chol
cholc ho le ste ro lp o o l
PKA
ROS
?
c ha p e ro nin
m ito c ho nd ria
c yto so l
H+
m
ROSROSROSROS
InflammationInflammationLPS, sepsisLPS, sepsis
Ischemia/Ischemia/reperfusionreperfusionIschemia/Ischemia/
reperfusionreperfusion
AgingAgingAgingAging
AlcoholAlcoholAlcoholAlcohol
XenobioticsXenobioticsPAHs, PPsPAHs, PPs
XenobioticsXenobioticsPAHs, PPsPAHs, PPs
AdenosineAdenosineAdenosineAdenosine
MitochondriaMitochondria
NucleusNucleus
ArsenateArsenateArsenateArsenate
CytokinesCytokinesCytokinesCytokines
NONO°°NONO°°
UVaUVaUVaUVa
Steroidogenic machinerySteroidogenic machinerySteroidogenic machinerySteroidogenic machinery
Sites of immune inhibitionSites of immune inhibitionSites of immune inhibitionSites of immune inhibition
ROS
ConclusionsConclusionsConclusionsConclusions
• 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
NIH: HD25271 HD35544NIH: HD25271 HD35544NIH: HD25271 HD35544NIH: HD25271 HD35544
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
collaboratorscollaboratorscollaboratorscollaborators
Karen Held HalesKaren Held HalesKaren Held HalesKaren Held Hales
Ryder Buchanan Hales