Mitochondrial Control of Leydig Cell Steroidogenesis

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

2. Cross section of rat testisShowing Seminiferous Tubules and Interstitium where Leydig cells reside Kent Christensen, Univ. Michigan

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

4. 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

5. Macrophage-Leydig cell interactions Cytokines, ROS ?

6. transcription DYm ATP LH Extracellularlipoprotein Cholesterolpool acetate ATP cAMP cholesterol PKA+ pbr Pregnenolone 3bHSD Progesterone P450c17 Androstenedione 17bHSD TESTOSTERONE

7. 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

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

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

10. 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

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

12. LPS vs. StAR mRNA expression

13. 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

14. StAR facilitates cholesterol transfer

15. 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

16. Time course of StAR decay

17. Time course of StAR decay

18. StAR ?

20. 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

21. 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)

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

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

24. H+ DYm e- Mitochondrial respiration, OX-PHOS and DYm

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

26. Effect of CCCP on StAR mRNA 3.4 kB 2.9 kB StAR 1.6 kB cyclophilin con cA cA+cccp

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

28. 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.

29. CCCP disruptsDYmin MA10s control CCCP-treated

30. Effect of mitochondrial agents on progesterone production

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

32. Effect of mitochondrial agents on StAR mRNA expression 3.2 kB StAR 1.6 kB cyclophilin Con cAMP + CCCP + oligm. + aresn.

33. Effect of H2O2 on StAR protein

34. Northern Blot StARmRNA Contr. cAMP. 100 200 250 500 Cyclophilin mRNA Effect of H2O2 on StAR mRNA

35. Effect of H2O2 on P450scc protein

36. Effect of xanthine/xanthine oxidase on StAR protein

37. 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

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

39. 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

40. 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

41. 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?

42. 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

43. 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

44. CCCP disruptsDYmin MA10s control CCCP-treated

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

46. Excitation/Emission Difference Spectra

47. Time-based dual emission spectra Fluorescence intensity seconds

48. Ratiometric Fluorometry: Estimation of DYm Ratio 575/549 seconds

49. Sites in the electron transport chain that inhibitors act

50. Determination of NADH/NAD+ ratio