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