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Mitochondrial Control of Leydig Cell Steroidogenesis. Dale Buchanan Hales, PhD University of Illinois at Chicago Department of Physiology and Biophysics. Cross section of rat testis Showing Seminiferous Tubules and Interstitium where Leydig cells reside. Kent Christensen, Univ. Michigan.

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Mitochondrial Control of Leydig Cell Steroidogenesis

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Mitochondrial Control of Leydig Cell Steroidogenesis

Dale Buchanan Hales, PhD

University of Illinois at Chicago

Department of Physiology and Biophysics


Cross section of rat testisShowing Seminiferous Tubules and Interstitium where Leydig cells reside

Kent Christensen, Univ. Michigan


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

?


transcription

DYm

ATP

LH

Extracellularlipoprotein

Cholesterolpool

acetate

ATP

cAMP

cholesterol

PKA+

pbr

Pregnenolone

3bHSD

Progesterone

P450c17

Androstenedione

17bHSD

TESTOSTERONE


CytokinesPKC agonists

ROS/mitochondrial disruptors

-

-

+

+

PKA

Acute regulation at the level of substrate availability

Chronic regulation at the level of gene transcription

+

testosterone

mitochondrial

nuclear

Mitochondrial vs. Nuclear control of steroidogenesis

cAMP


P450scc

P450c17

3b-HSD

actin

- + - + - + - + - +

LPS

2h 4h 6h 8h 24h

time

Effect of LPS on steroidogenic mRNA levels


Effect of LPS on P450c17 protein levels 2 and 24 h post injection

control

LPS

control

LPS

2 hours

24 hours


LPS vs. serum testosterone: 2-24 hours

control

14

LPS

12

10

8

Testosterone (ng/ml)

6

4

2

0

2 h

4 h

6 h

8 h

24 h

Time post LPS


LPS vs. StAR protein expression: 2 hr after injection

37 kDa

30 kDa

con

LPS


LPS vs. StAR mRNA expression


Steroidogenic Acute Regulatory 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 DYm


StAR facilitates cholesterol transfer


N'

32 kDa

Inner- mitochondrial membrane

N'

30 kDa

37

StAR Processing

32

30

Inner-mitochondrial forms

Cytosol

37 kDa

N'

cholesterol transfer

critical region

signal peptides

Outer mitochondrial membrane

matrix


Time course of StAR decay


Time course of StAR decay


StAR

?


StAR N-terminal localization expression clones

MTS

1-37

ITS

38-47

pCMV-StAR

TAA

StAR-stop

MTS

1-37

StAR D-ITS

StAR D-N47

Tom20

OMTS

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)


LPS vs. StAR protein expression: 2 hr after injection

37 kDa

30 kDa

con

LPS


Carbonyl cyanide m-chlorophenylhydrazone (cccp)

  • Carbonyl cyanide m-chlorophenyl-hydrazone (cccp): potent uncoupler of oxidative phosphorylation; protonophore, mitochondrial disrupter.

  • Causes transient disruption of DYm


H+

DYm

e-

Mitochondrial respiration, OX-PHOS and DYm


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


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 disruptsDYmin MA10s

control

CCCP-treated


Effect of mitochondrial agents on progesterone production


Effect of mitochondrial agents on StAR protein expression

37 kDa

30 kDa

cAMP

Control

+ CCCP

+ arsenate

+ oligomycin


Effect of mitochondrial agents on StAR mRNA expression

3.2 kB

StAR

1.6 kB

cyclophilin

Con

cAMP

+ CCCP

+ oligm.

+ aresn.


Effect of H2O2 on StAR protein


Northern Blot

StARmRNA

Contr.

cAMP.

100

200

250

500

Cyclophilin mRNA

Effect of H2O2 on StAR mRNA


Effect of H2O2 on P450scc protein


Effect of xanthine/xanthine oxidase on StAR protein


cAMP + Xanthine Ox. (mU)

a

a

IOD StAR

a

IOD Ratio

37/30+30 kDa StAR

b

a

a

b

a

b

b

b

a

a

b

a

b

b

con. cAMP +10 +50 +100

con. cAMP +10 +50 +100

cAMP + Xanthine Ox. (mU)

cAMP + Xanthine Ox. (mU)

Effect of xanthine/xanthine oxidase on StAR forms


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


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


LPS inhibits Leydig cells in vivo via ROS

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


What is the Dym-sensitive component of steroidogenesis?

  • Protein import into matrix is Dym-dependent– but likely not responsible for inhibition of StAR

  • PBR?

  • Perturbation of intra-mitochondrial Ca2+ and/or ATP levels?


Ca2+ transport systems in mitochondria

Ruthinium Red

e-

H+

Ca2+ uniporter (U) facilitates the transport of Ca2+ inward down the electrochemical gradient.

Ca2+ activated permeability transition pore (PTP) also is shown


Potential role for mitochondrial Ca2+

Ru360 is a cell permeable derivative

of Ruthinium Red-- a specific

Mitochondrial Ca2+ uptake blocker

Con cAMP +H202 +5 +10

uM Ru360

Con cAMP +H202 +5 +10

uM Ru360


CCCP disruptsDYmin MA10s

control

CCCP-treated


Excitation/Emission Spectra: Control vs. CCCP

Fluorescence intensity

nm


Excitation/Emission Difference Spectra


Time-based dual emission spectra

Fluorescence intensity

seconds


Ratiometric Fluorometry: Estimation of DYm

Ratio 575/549

seconds


Sites in the electron transport chain that inhibitors act


Determination of NADH/NAD+ ratio


Effect of cAMP and Antimycin A on DYm


Effect of cAMP and Antimycin A on NADH/NAD+


Effect of mito compounds on StAR


Steroidogenic machinery


Sites of immune inhibition

ROS


Hales Lab

Collaborators

Fred Lepore

Neil Iyengar

Tristan Shankara

Marika Wrzosek

John Allen

Thorsten Diemer

Paul JanusSteinunn Thorardottir

Judy Bolton—UIC

Colin Jefcoate—UW Madison

Jean-Guy Lehoux—Sherbrooke

Yossi Orly—Hebrew Univ

Anita Payne—Stanford

Mariann Piano—UIC

Catherine Rivier—Salk Inst

Douglas Stocco—Texas Tech

Gregory Thatcher—UIC

Karen Held Hales

StARoxidative stressalcoholsteroidogenesis

NIH: HD25271 HD35544


“It takes balls to work on Leydig cells”

Anita Payne circa 1984


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