ImmuneEndocrine control of Leydig cell function - PowerPoint PPT Presentation

Title: ImmuneEndocrine control of Leydig cell function


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Immune-Endocrine control of Leydig cell function

• Dale Buck Hales, PhD
• University of Illinois at Chicago
• Department of Physiology and Biophysics

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Cross section of rat testis Seminiferous tubules
and Interstitium where Leydig cells reside.
Kent Christensen, Univ. Michigan
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(No Transcript)
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Interstitium of rat testis showing endothelium,
Leydig cells (L), and macrophages (arrow). Note
close association of macrophages and Leydig
cells.
Scott Miller, Univ Utah
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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
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Macrophage-Leydig cell interactions
Cytokines, ROS
?
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LH
Extracellularlipoprotein
Cholesterolpool
acetate
ATP
cAMP
cholesterol
PKA
DYm
Pregnenolone
3bHSD
Progesterone
P450c17
Androstenedione
17bHSD
TESTOSTERONE
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IN 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 StAR
• Aanalyze DYm by fluorescent microscopy

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Effect of LPS on steroidogenic mRNA levels
P450scc
P450c17
3b-HSD
actin
- - - - -
LPS
2h 4h 6h 8h 24h
time
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LPS vs. serum testosterone 2-24 hours
control
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LPS
12
10
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Testosterone (ng/ml)
6

4
2
0
24 h
2 h
4 h
8 h
6 h
Time post LPS
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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

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StAR facilitates cholesterol transfer
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Domains and phosphorylation sites
of Steroidogenic Acute Regulatory protein (StAR)
signal peptides
critical region
cholesterol transfer
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Transfer across outer mitochondrial membrane and
cleavage of first peptide
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Transfer across inner membrane, formation of
contact sites for cholesterol transfer, and
cleavage of second peptide
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Mature 30 kDa protein associated with inner
mitochondrial membrane post cholesterol transfer
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N'-mutant protein associates only with outer
mitochondrial membrane and still facilitates
cholesterol transfer
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C'-mutant protein neither associates with outer
mitochondrial membrane nor facilitates
cholesterol transfer
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Current working model of StAR mechanism of action
via association of C with outer mitochondrial
membrane
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LPS vs. StAR protein expression 2 hr after
injection
37 kDa
30 kDa
con
LPS
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LPS vs. StAR mRNA expression
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LPS vs. StAR Synthesis 2 hr
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LPS vs. StAR synthesis 2 hr
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Effect of LPS on Steroidogenic Proteins
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What mediates the acute LPS inhibition?

• Tested numerous inflammatory mediators in Leydig
  cells in vitro-- none mimicked the LPS effect
• cytokines (TNFa, IL-1, IL-6, IFNg, TGFb)
• prostaglandins (PGF2a, PGE)
• catecholamines (norepi, isoproteranol)
• ceramide (C2, C8)
• nitric oxide donors (Sin-1, SNAP, SNP, Nor-3)
• Calcium inophore (A23187)

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Carbonyl cyanide m-chlorophenylhydrazone (cccp)

• Carbonyl cyanide m-chlorophenyl-hydrazone (cccp)
  potent uncoupler of oxidative phosphorylation
  protonophore, mitochondrial disrupter.
• Causes transient disruption of DYm

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Mitochondrial respiration, OX-PHOS and DYm
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CCCP vs. Progesterone in MA10s
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Effect of CCCP on StAR protein
37 kDa
30 kDa
Control cAMP cAMP cccp cccp
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Effect of CCCP on StAR synthesis
37kDa
30kDa
Control cAMP cccp cAMP cccp
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Effect of CCCP on protein synthesis
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Effect of CCCP on StAR synthesis
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Effect of CCCP on StAR mRNA
3.4 kB
2.9 kB
StAR
1.6 kB
cyclophilin
con
cA
cAcccp
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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.

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CCCP disrupts DYm in MA10s
control
CCCP-treated
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H2O2 vs. Progesterone in MA10s
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H202 vs. StAR and 3b-HSD protein in MA10s
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Effect of H2O2 on StAR mRNA
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TMRE staining of MA-10 cells exposed to H2O2
100mM H2O2
Control
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Summary StAR and 3b-HSD studies in MA10s

• CCCP transiently disrupts DYm
• CCCP inhibits StAR processing and progesterone
  production
• Disruption of DYm alone does not block 3b-HSD
  protein-- activity??
• H2O2 inhibits StAR and 3b-HSD protein and
  progesterone production
• H2O2 disrupts DYm

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Hypothesis-one

• LPS causes an abrupt inhibition of
  steroidogenesis by disrupting Leydig cell
  mitochondria
• LPS activates generation of reactive oxygen
  species (ROS) from testicular interstitial
  macrophages
• ROS from adjacent macrophages disrupts Leydig
  cell mitochondria

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Hypothesis-two

• LPS causes Leydig cell apoptosis
• Long term depolarization precedes initiation of
  apoptosis
• Apoptosis of Leydig cells results in long-term
  depression of serum testosterone
• Perturbation of 3b-HSD and StAR is an early
  indictor of apoptosis

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Summary 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 3b-HSD
  protein
• LPS causes disruption of Leydig cell DYm,
• Preliminary data supports the role of ROS in
  mediating LPS effects in vivo

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Hales Lab
collaborators
Thorsten Diemer Beth Nardulli Salil Ginde John
Allen John Choi
Bruce Bosmann Barbara Clark Jim Ferguson Lester
Lau Jean-Guy LeHoux Mark McLean Yossi Orly
Keith Parker Anita Payne Richard
Pestell Catherine Rivier Focko Rommerts Douglas
Stocco
Karen Held Hales
NIH HD25271 HD35544