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Mitochondrial Control of Leydig Cell Steroidogenesis Dale Buchanan Hales, PhD University of Illinois at Chicago Department of Physiology and Biophysics.

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Presentation on theme: "Mitochondrial Control of Leydig Cell Steroidogenesis Dale Buchanan Hales, PhD University of Illinois at Chicago Department of Physiology and Biophysics."— Presentation transcript:

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2 Mitochondrial Control of Leydig Cell Steroidogenesis Dale Buchanan Hales, PhD University of Illinois at Chicago Department of Physiology and Biophysics Dale Buchanan Hales, PhD University of Illinois at Chicago Department of Physiology and Biophysics

3 Kent Christensen, Univ. Michigan Cross section of rat testis Showing Seminiferous Tubules and Interstitium where Leydig cells reside

4 Scott Miller, Univ Utah Interstitium of rat testis showing endothelium, Leydig cells (L), and macrophages (arrow). Note close association of macrophages and Leydig cells.

5 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.

6 Cytokines, ROS ? Macrophage-Leydig cell interactions

7 cholesterol Extracellular lipoprotein Cholesterol pool LH ATP cAMP PKA+ Pregnenolone Progesterone Androstenedione TESTOSTERONE 3  HSD P450c17 17  HSD acetate transcription  m ATP pbr

8 cAMP PKA + + testosterone + Chronic regulation at the level of gene transcription nuclear ROS/mitochondrial disruptors - Cytokines PKC agonists - mitochondrial Acute regulation at the level of substrate availability Mitochondrial vs. Nuclear control of steroidogenesis

9 Effect of LPS on steroidogenic mRNA levels P450scc P450c17 3  - HSD actin LPS time 2h 4h 6h 8h 24h

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

11 LPS vs. serum testosterone: 2-24 hours Testosterone (ng/ml) control LPS Time post LPS 24 h2 h4 h8 h6 h

12 LPS vs. StAR protein expression: 2 hr after injection 30 kDa 37 kDa con LPS

13 LPS vs. StAR mRNA expression

14 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  m

15 StAR facilitates cholesterol transfer

16 StAR Processing signal peptides 37 kDa Outer mitochondrial membrane Inner- mitochondrial membrane critical regioncholesterol transfer matrix Cytosol Inner-mitochondrial forms N'N' 32 kDa N'N' 30 kDa N'N'

17 Time course of StAR decay

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19 StAR ?

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21 MTS 1-37 ITS pCMV-StAR StAR-stop Tom20 OMTS StAR/CCHL CCHL IMSS StAR/Tom20 StAR  -N47 StAR N-terminal localization expression clones StAR  -ITS MTS 1-37 TAA

22 What mediates the acute LPS 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)

23 LPS vs. StAR protein expression: 2 hr after injection 30 kDa 37 kDa con LPS

24 Carbonyl cyanide m- chlorophenylhydrazone (cccp) Carbonyl cyanide m-chlorophenyl- hydrazone (cccp): potent uncoupler of oxidative phosphorylation; protonophore, mitochondrial disrupter. Causes transient disruption of  m

25 Mitochondrial respiration, OX-PHOS and  m H+H+ e-e-  m

26 Effect of CCCP on StAR protein Control cAMP cAMP + cccp cccp 37 kDa 30 kDa

27 concAcA+cccp StAR cyclophilin 3.4 kB 1.6 kB 2.9 kB Effect of CCCP on StAR mRNA

28 Effect of CCCP on StAR synthesis Control cAMP cccp cAMP + cccp 37kDa 30kDa

29 Tetramethylrhodamine 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.

30 controlcontrol CCCP-treatedCCCP-treated CCCP disrupts  m in MA10s

31 Effect of mitochondrial agents on progesterone production

32 Effect of mitochondrial agents on StAR protein expression 37 kDa Control cAMP + oligomycin + arsenate + CCCP 30 kDa

33 3.2 kB 1.6 kB StAR cyclophilin Effect of mitochondrial agents on StAR mRNA expression ConcAMP + oligm.+ aresn. + CCCP

34 Effect of H 2 O 2 on StAR protein

35 Effect of H 2 O 2 on StAR mRNA Northern Blot StAR mRNA Contr.cAMP Cyclophilin mRNA

36 Effect of H 2 O 2 on P450scc protein

37 Effect of xanthine/xanthine oxidase on StAR protein

38 IOD StAR cAMP + Xanthine Ox. (mU) a b b a bb a a a con. cAMP cAMP + Xanthine Ox. (mU) a b b bb a a a con. cAMP cAMP + Xanthine Ox. (mU) IOD Ratio 37/30+30 kDa StAR Effect of xanthine/xanthine oxidase on StAR forms

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

40 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 (ROS) mediated the acute inhbitory effects of LPS?

41 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

42 What is the  m-sensitive component of steroidogenesis? Protein import into matrix is  m- dependent– but likely not responsible for inhibition of StAR PBR? Perturbation of intra-mitochondrial Ca 2+ and/or ATP levels?

43 Ca2+ transport systems in mitochondria Ca 2+ uniporter (U) facilitates the transport of Ca 2+ inward down the electrochemical gradient. Ca 2+ activated permeability transition pore (PTP) also is shown e-e- H+H+ Ruthinium Red

44 Potential role for mitochondrial Ca2+ Con cAMP +H uM Ru360 Con cAMP +H uM Ru360 Ru360 is a cell permeable derivative of Ruthinium Red-- a specific Mitochondrial Ca 2+ uptake blocker

45 controlcontrol CCCP-treatedCCCP-treated CCCP disrupts  m in MA10s

46 Excitation/Emission Spectra: Control vs. CCCP nm Fluorescence intensity

47 Excitation/Emission Difference Spectra

48 Time-based dual emission spectra seconds Fluorescence intensity

49 seconds Ratiometric Fluorometry: Estimation of  m Ratio 575/549

50 Sites in the electron transport chain that inhibitors act

51 Determination of NADH/NAD+ ratio

52 Effect of cAMP and Antimycin A on  m

53 Effect of cAMP and Antimycin A on NADH/NAD+

54 Effect of mito compounds on StAR

55 Steroidogenic machinery

56 Sites of immune inhibition ROS

57 Fred Lepore Neil Iyengar Tristan Shankara Marika Wrzosek John Allen Thorsten Diemer Paul Janus Steinunn Thorardottir Fred Lepore Neil Iyengar Tristan Shankara Marika Wrzosek John Allen Thorsten Diemer Paul Janus Steinunn Thorardottir Hales Lab 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 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 CollaboratorsCollaborators Karen Held Hales StAR oxidative stress alcohol steroidogenesis NIH: HD25271 HD35544

58 “It takes balls to work on Leydig cells” Anita Payne circa 1984


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