Cross section of rat testis Showing Seminiferous Tubules and Interstitium

transcript

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

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

cholesterol

Extracellularlipoprotein

Cholesterolpool

cAMPPKA+

Pregnenolone

Progesterone

Androstenedione

TESTOSTERONE

P450c17

acetate

• 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

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

20002500

IL-1, TNFIL-1, TNF and PMA vs. and PMA vs. steroidogenic mRNA expressionsteroidogenic mRNA expression

cAMP+IL-1

+TNF+PMA

P450c17

P450scc

cAMP responsive regions of the cAMP responsive regions of the CypCyp1717 promoterpromoter

-2500 -1021 -346 -245

Control

TNF inhibits Cyp17 promoter activityTNF inhibits Cyp17 promoter activityTNF inhibits Cyp17 promoter activityTNF inhibits Cyp17 promoter activity

Calphostin reverses TNF inhibition of Calphostin reverses TNF inhibition of Cyp17 promoterCyp17 promoter

TNF and PMA stimulate translocation of PKC from cytoplasm to membrane

control

PMA TNF

No antibody

Putative transfactor binding sitesPutative transfactor binding sitesPutative transfactor binding sitesPutative transfactor binding sites

-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

Comparison of cAMP-responsive sequences in mouse and rat

Binding sites (ATF2/cjun-like, Steroidogenic factor 1, StF-IT-2, and StF-IT-1/COUP-TF1) 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.

Characterization of the Cyp17 Promoter Characterization of the Cyp17 Promoter Revealed a Region Between -245 and -346 Revealed a Region Between -245 and -346

Responsible for the Minimal cAMP Responsible for the Minimal cAMP Responsiveness of the GeneResponsiveness of the Gene

Characterization of the Cyp17 Promoter Characterization of the Cyp17 Promoter Revealed a Region Between -245 and -346 Revealed a Region Between -245 and -346

Responsible for the Minimal cAMP Responsible for the Minimal cAMP Responsiveness of the GeneResponsiveness of the Gene

-2500 -1021 -346 -245

Control

Site-directed mutagenesis of Site-directed mutagenesis of Cyp17Cyp17 CRR CRR

(-346 to –245)(-346 to –245)

Site-directed mutagenesis of Site-directed mutagenesis of Cyp17Cyp17 CRR CRR

(-346 to –245)(-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 mutantsCyclic-AMP induction of CRR mutantsCyclic-AMP induction of CRR mutantsCyclic-AMP induction of CRR mutants

cAMP Induction of CRR mutantscAMP Induction of CRR mutantscAMP Induction of CRR mutantscAMP Induction of CRR mutants

f Wild

Putative sites revealed by Putative sites revealed by mutantsmutants

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 mut 9 mut 10

AhR/Arnt (core sequence)

-440 -250

ATF2 mutants 2,5,9

C/EBP– upstream site

AhR/ARNT mutant 6

SF-1 mutant 7

Putative regulatory motifs revealed by mutagenesis

The Minimal cAMP Responsive Region of The Minimal cAMP Responsive Region of the the Cyp17Cyp17 Promoter (CRR): Promoter (CRR):

-346 -245

-346 TGATGACAT TAATTATTAA CTGTGCAGCA

CTTTTGACAT TACAGCACGC ACTCTGAAAC

CTTGATCTTA ATCTAGCATT TGCCTCTGGG

AGGATCCATA GCG -245

Putative ATF-2 binding site

Binding of Nuclear Proteins to the CRR Probe is Binding of Nuclear Proteins to the CRR Probe is Augmented by Treatment of MA-10 Cells with cAMPAugmented by Treatment of MA-10 Cells with cAMP

Nuclear Proteins from Primary Leydig Cells Nuclear Proteins from Primary Leydig Cells Form Two Complexes with the CRR ProbeForm Two Complexes with the CRR Probe

The Upstream ATF-2 C/EBPThe Upstream ATF-2 C/EBP Binding Site Binding SiteThe Upstream ATF-2 C/EBPThe Upstream ATF-2 C/EBP Binding Site Binding Site

-450 TTGTGTGACC TTATGCAAAC TAACCCA -423

-450 -245

Nuclear Proteins from Control and cAMP-Nuclear Proteins from Control and cAMP-Treated MA-10 cells Bind to the Upstream Treated MA-10 cells Bind to the Upstream

ATF-2 C/EBPATF-2 C/EBP Probe Probe

Nuclear Proteins from Control and cAMP-Nuclear Proteins from Control and cAMP-Treated MA-10 cells Bind to the Upstream Treated MA-10 cells Bind to the Upstream

ATF-2 C/EBPATF-2 C/EBP Probe Probe

Incubation of the Upstream ATF-2 C/EBPIncubation of the Upstream ATF-2 C/EBP probe probe with Nuclear Proteins Isolated from Primary with Nuclear Proteins Isolated from Primary Leydig Cells Results in Complex FormationLeydig Cells Results in Complex Formation

Formation of the Higher Order Complex Formed Formation of the Higher Order Complex Formed by the CRR Complex is Decreased by Addition of by the CRR Complex is Decreased by Addition of Unlabeled ATF-2 or C/EBPUnlabeled ATF-2 or C/EBP Competitor Oligos Competitor Oligos

Binding of Nuclear Proteins to the Upstream Binding of Nuclear Proteins to the Upstream ATF-2 C/EBPATF-2 C/EBP Probe can be Inhibited by Probe can be Inhibited by

Addition of ATF-2 or C/EBPAddition of ATF-2 or C/EBP Competitor Oligos Competitor Oligos

Binding of Nuclear Proteins to the Upstream Binding of Nuclear Proteins to the Upstream ATF-2 C/EBPATF-2 C/EBP Probe can be Inhibited by Probe can be Inhibited by

Addition of ATF-2 or C/EBPAddition of ATF-2 or C/EBP Competitor Oligos Competitor Oligos

Overexpression of C/EBPOverexpression of C/EBP Induces Induces Transcription of the -491/-255 Transcription of the -491/-255 CypCyp 17 reporter 17 reporter

Cyp 17 Cyp 17 + ATF-2 Cyp 17 +C/EBPb

Cyp 17 + ATF-2+ C/EBPb

Control

+ cAMP

Western Analysis of ATF-2 Expression

0.000.200.400.600.801.001.201.40

12 hSFM

1 hcAMP

2 hcAMP

4 hcAMP

8 hcAMP

12 hcAMP

ATF-2 Expression in MA-10 Cells is Not ATF-2 Expression in MA-10 Cells is Not Affected by cAMP TreatmentAffected by cAMP Treatment

Western Analysis of C/EBP Expression

12 hSFM

1 hcAMP

2 hcAMP

4 hcAMP

8 hcAMP

12 hcAMP

C/EBPC/EBP Expression is Significantly Expression is Significantly Increased in MA-10 Cells with cAMPIncreased in MA-10 Cells with cAMPC/EBPC/EBP Expression is Significantly Expression is Significantly

Increased in MA-10 Cells with cAMPIncreased in MA-10 Cells with cAMP

Summary of Cyp17 studySummary of Cyp17 studySummary of Cyp17 studySummary of Cyp17 study

• TNF-mediated inhibition of transcription involves activation of PKC

• ATF2 and C/EBP participate cooperatively in cAMP-induction of transcription

• ATF2 is constitutively expressed

• C/EBP expression is induced by cAMP

HypothesesHypothesesHypothesesHypotheses

• ATF2 and C/EBP interact as heterodimers binding to the “ATF2” sites in the promoter

• The stoichiometry of C/EBP and ATF2 interaction is critical to driving transcription

• Repressors may act by inhibiting C/EBP expression or through post-translational modifications that inhibit its activity

• C/EBP phosphorylation by PKC may block it from interacting with ATF2

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

LPS - + - + - + - + - +

time 2h 4h 6h 8h 24h

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

control

Time post LPS

24 h2 h 4 h 8 h6 h

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

StAR ProcessingStAR ProcessingStAR ProcessingStAR Processing

signal peptides

37 kDa

Outer mitochondrial membrane

Inner- mitochondrial membrane

critical regioncholesterol transfer

matrix

Cytosol

Inner-mitochondrial forms

32 kDaN'

30 kDaN'

sccAdx-red

3 H SD

M itochondria lm atrix

C ytosolchol

cholchol

S tARN ’ C ’

sccAdx-red

3 H SD

M itochondria lm atrix

C ytosol

cholchol

S tARN ’ C ’

M ito c ho nd ria lm a trix

C yto so l

TO MTIM

HKC KC p hD

MTS1-37

ITS38-47

pCMV-StAR

StAR-stop

Tom20OMTS

StAR/CCHL

CCHLIMSS

StAR/Tom20

StAR -N47

StAR N-terminal localization expression clones

StAR -ITS

MTS1-37

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

30 kDa

37 kDa

conLPS

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

Mitochondrial respiration, OX-PHOS and Mitochondrial respiration, OX-PHOS and mm

CCCP Vs. Progesterone in MA10s

0100200300400500

con cAMP cAMP +cccp

R22 R22 +cccp

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

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

Effect of CCCP on StAR synthesisEffect of CCCP on StAR synthesisEffect of CCCP on StAR synthesisEffect of CCCP on StAR synthesis

Control cAMP cccp cAMP + cccp

Effect of CCCP on StAR Effect of CCCP on StAR synthesissynthesis

37 kDa30 kDa

Effect of cccp on StAR decayEffect of cccp on StAR decayEffect of cccp on StAR decayEffect of cccp on StAR decay

500000

1000000

1500000

2000000

2500000

3000000

3500000

0 60 120 180 240 300 360 420 480

minutes

control

Time course of StAR decayTime course of StAR decayTime course of StAR decayTime course of StAR decay

100000

0 15 30 45 60 75 90 105 120

minutes

30 kDa

32 kDa

37 kDa

con cAMP +Oligom +arsen +CCCP

Effect of mitochondrial agents Effect of mitochondrial agents on progesterone productionon progesterone production

Effect of mitochondrial agents Effect of mitochondrial agents on StAR protein expressionon StAR protein expression

37 kDa

Control

cAMP+ oligomycin

+ arsenate

+ CCCP

30 kDa

3.2 kB

1.6 kB

cyclophilin

Effect of mitochondrial agents Effect of mitochondrial agents on StAR mRNA expressionon StAR mRNA expression

ConcAM

P+ oligm.

+ aresn.

+ CCCP

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

• 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?

ROS causes increase in 37 kDa StAR in ROS causes increase in 37 kDa StAR in Leydig cells in vitroLeydig cells in vitro

Con cAMP +10 +25 +100 +250

cAMP + H2O2 (M)

30/32 kDa

37 kDa

Con cAMP +10 +25 +100 +250

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

ControlControlControlControl 100100M HM H22OO22100100M HM H22OO22

TMRE staining of MA-10 cells TMRE staining of MA-10 cells exposed to H2O2—time lapseexposed to H2O2—time lapse

PFC Increases DHR-123 FluorescencePFC Increases DHR-123 FluorescencePFC Increases DHR-123 FluorescencePFC Increases DHR-123 Fluorescence

+PFC Brightfield +PFC Merge+PFC Fluorescence

Control Brightfield Control Fluorescence Control Merge

PFC: 4-phenyl-3-furoxancarbonitrile

Lipid peroxides

control LPS

LPS inhibits Leydig cells in vivo via ROSLPS inhibits Leydig cells in vivo via ROSLPS inhibits Leydig cells in vivo via ROSLPS inhibits Leydig cells in vivo via ROS

Increased lipid Increased lipid peroxidation and peroxidation and depolarization of Leydig depolarization of Leydig cell mitochondria support cell mitochondria support involvement of ROS in involvement of ROS in LPS action in vivoLPS action in vivo

cholc ho le ste ro lp o o l

c ha p e ro nin

m ito c ho nd ria

c yto so l

ROSROSROSROS

InflammationInflammationLPS, sepsisLPS, sepsis

Ischemia/Ischemia/reperfusionreperfusionIschemia/Ischemia/

reperfusionreperfusion

AgingAgingAgingAging

AlcoholAlcoholAlcoholAlcohol

XenobioticsXenobioticsPAHs, PPsPAHs, PPs

AdenosineAdenosineAdenosineAdenosine

MitochondriaMitochondria

NucleusNucleus

ArsenateArsenateArsenateArsenate

CytokinesCytokinesCytokinesCytokines

NONO°°NONO°°

UVaUVaUVaUVa

DiabetesDiabetesDiabetesDiabetes

Steroidogenic machinerySteroidogenic machinerySteroidogenic machinerySteroidogenic machinery

Sites of immune inhibitionSites of immune inhibitionSites of immune inhibitionSites of immune inhibition

But what does it all But what does it all mean, anyway?mean, anyway?

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

Importance of Immune-Importance of Immune-endocrine interactionsendocrine interactions

• Females are more susceptible to autoimmune diseases than males

• Estradiol and prolactin are both immuno-stimulatory

• 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 StuartiiAntechinus Stuartiivictim of his own testosteronevictim of his own testosterone

NIH: HD25271 HD35544NIH: HD25271 HD35544NIH: HD25271 HD35544NIH: HD25271 HD35544

John Allen John Allen Paul JanusPaul JanusFred LeporeFred Lepore

Beth NardulliBeth NardulliSalil GindeSalil GindeJohn Choi John Choi Thorsten DiemerThorsten Diemer

John Allen John Allen Paul JanusPaul JanusFred LeporeFred Lepore

Beth NardulliBeth NardulliSalil 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 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

Cross section of rat testis Showing Seminiferous Tubules and Interstitium

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