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Inflammation and Male Reproductive Function

Inflammation and Male Reproductive Function. Dale Buchanan Hales, PhD University of Illinois at Chicago Department of Physiology and Biophysics. Cross section of rat testis Showing Seminiferous Tubules and Interstitium. Kent Christensen, Univ. Michigan.

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Inflammation and Male Reproductive Function

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  1. Inflammation and Male Reproductive Function Dale Buchanan Hales, PhD University of Illinois at Chicago Department of Physiology and Biophysics

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

  3. Functional and Anatomical Compartments of the Testis

  4. Testicular interstitium and seminiferous epithelium

  5. Schematic view of the testis

  6. Spermatogenesis

  7. The wave of the seminiferous epithelium

  8. Stages of spermatogenesis {Y. Claremont, 1952}

  9. Stage-Specific gene expression FSH-R/cAMP Testosterone/ABP {Adapted from M. Parvinen}

  10. Intra-tubular regulatory factorsproduced by Sertoli and germ cells • Cytokines (IL-1, IL-6, TGFb, IFNs, TNFa) • Growth factors (IGF-1, TGFa, bFGF, NGF, Steel factor, PDGF) • Inhibin, activin, MIS • Nitric Oxide, endothelin, VGEF {Adapted from B. Jégou}

  11. Spermiation

  12. Phagocytosis of residual body by Sertoli cell

  13. Physiological Immune-endocrine interactions • Intra-tubular production of cytokines, growth factors and other inflammatory mediators orchestrate autonomous control of germ cell development • Role of macrophages in Leydig cell development • Required for development and regeneration • May produce positive regulatory factors

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

  15. 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

  16. Macrophage-Leydig cell interactions Cytokines, ROS ?

  17. Pathophysiological immune-endocrine interactions • Macrophage elaborated cytokines are potent inhibitors of Leydig cell steroidogenesis • Macrophage and endothelium-derived reactive oxygen species (ROS) are deleterious in interstitium and seminiferous tubule • Singlet oxygen and peroxynitrile radicals • Intracellularly -derived ROS during Ca2+ overload and/or oxidative stress

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

  19. IL-1, TNF and PMA vs. Testosterone production

  20. IL-1, TNFa and PMA vs. steroidogenic mRNA expression P450scc P450c17 +IL-1 cAMP +PMA +TNFa

  21. P450c17 is sensitive to transcriptional repression • Of all the steroidogenic enzymes, P450c17 is the most sensitive to repression • Most cytokines tested inhibit c17 transcription: • IL-1a/b, IL-2, IL-6, TNFa, TGFb, INFa/b, INFg • Inflammatory mediators: PGF2a, ceramide, vasopressin, PKC agonists • Environmental disruptors such as dioxin, pthalates, PAHs, etc. are inhibitory • Androgen-mediated feedback repression

  22. 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 • Aanalyze DYm by fluorescent microscopy

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

  24. 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

  25. 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

  26. StAR facilitates cholesterol transfer

  27. Pulsatile nature of cholesterol flux into the mitochondria

  28. N' 32 kDa N' 30 kDa 37 StAR Processing 32 30 Inner-mitochondrial forms Cytosol 37 kDa N' cholesterol transfer critical region signal peptides Outer mitochondrial membrane Inner- mitochondrial membrane matrix

  29. Transfer across outer mitochondrial membrane and cleavage of first peptide

  30. Transfer across inner membrane, formation of contact sites for cholesterol transfer, and cleavage of second peptide

  31. Mature 30 kDa protein associated with inner mitochondrial membrane post cholesterol transfer

  32. N'-mutant protein associates only with outer mitochondrial membrane and still facilitates cholesterol transfer

  33. C'-mutant protein neither associates with outer mitochondrial membrane nor facilitates cholesterol transfer

  34. Effect of LPS on Steroidogenic Proteins

  35. LPS vs. StAR mRNA expression

  36. 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) • ceramide (C2, C8) • Most nitric oxide donors (Sin-1, SNAP, SNP, Nor-3) • Calcium inophore (A23187)

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

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

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

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

  41. 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.

  42. CCCP disruptsDYmin MA10s control CCCP-treated

  43. Analysis of DYm in Leydig cells:Post-staining protocol • Inject mice with LPS and purify Leydig cells as usual • Incubate purified cells with TMRE • Examine by fluorescent microscopy • Indicates if cells suffered long-term depolarization

  44. LPS disrupts Leydig Cell DYmlong-term effect

  45. 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

  46. Effects of ROS on Steroidogenesis in MA-10 cells and Leydig cells H2O2 vs. Progesterone production in MA-10 cells H2O2 vs. Testosterone production in Leydig cells 47% * 47% * 44% * * * 44% * Testosterone ng/ 106cells/ hr. Progesterone ng/ 106cells/ hr. Progesterone ng/ 106cells/ hr. * * * * Con cAMP +100 + 250 + 500 cAMP + H2O2 (in цM) Con cAMP +100 + 250 cAMP + H2O2 (in M) cAMP + H2O2 (M) cAMP + H2O2 (M)

  47. H2O2 effects on steroidogenic proteins in MA-10 cells n.s IOD StAR 90% StARprotein * * Con cAMP +100 +250 +500 cAMP + H2O2 (mM) Con. cAMP +100 + 250 +500 cAMP+H2O2 (M) 35% IOD 3-HSD n.s n.s 3-HSD protein Con cAMP +100 +250 +500 cAMP + H2O2 (mM) * Con cAMP +100 + 250 + 500 cAMP+H2O2 (M) IOD P450scc P450scc protein Con cAMP +100 +250 +500 cAMP + H2O2 (mM) Con cAMP +100 + 250 cAMP+H2O2 (M)

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

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