Involvement of Activin and Follistatin in the Pathogenesis of Chronic Testicular Inflammation in Mice Inaugural Dissertation submitted to the Faculty of Medicine in partial fulfilment of the requirements for the Ph.D. Degree of the Faculty of Medicine of the Justus Liebig University Giessen as part of the joint award Ph.D. program with Monash University by Nour NICOLAS from Sofia, Bulgaria Giessen, 2016 From the Institute of Anatomy and Cell Biology Director/Chairman: Prof. Dr. Eveline Baumgard-Vogt Biology of Reproduction Group Head: Prof. Dr. Andreas Meinhardt Faculty of Medicine Justus-Liebig-University of Giessen, Germany First supervisor and committee member: Prof. Dr. Andreas Meinhardt/Dr. Monika Fijak Second supervisor and committee member: Prof. Dr. Martin Bergmann Committee Members: Date of Doctoral defense: 20/03/2017 Contents CONTENTS ABBREVIATIONS .......................................................................................................... vii LIST OF FIGURES AND TABLES ..................................................................................xi 1. INTRODUCTION .................................................................................................. - 1 - 1.1. The male reproductive tract............................................................................ - 1 - 1.2. The testis: structure and function ................................................................... - 1 - 1.2.1. Spermato- and spermiogenesis ............................................................... - 2 - 1.2.2. Steroidogenesis and the hypothalamic-pituitary-gonadal axis ................. - 4 - 1.3. The immune system ....................................................................................... - 7 - 1.4. The immune privilege of the testis ................................................................ - 11 - 1.4.1. The blood-testis-barrier (BTB) ............................................................... - 12 - 1.4.2. Immunoregulatory factors ...................................................................... - 12 - 1.4.3. Testicular leukocytes ............................................................................. - 13 - 1.5. Activin .......................................................................................................... - 15 - 1.5.1. Structure ................................................................................................ - 15 - 1.5.2. Sites of production ................................................................................. - 16 - 1.5.3. Activin signaling ..................................................................................... - 16 - 1.5.4. Function ................................................................................................. - 17 - 1.6. Follistatin ...................................................................................................... - 19 - 1.6.1. Forms of follistatin ................................................................................. - 19 - 1.6.2. Sites of production ................................................................................. - 19 - 1.6.3. Function and therapeutic applications ................................................... - 20 - 1.7. Male infertility ............................................................................................... - 20 - 1.7.1. Experimental autoimmune epididymo-orchitis (EAEO) .......................... - 21 - i Contents 1.8. Hypothesis and aim of the study .................................................................. - 23 - 2. MATERIALS AND METHODS ............................................................................ - 24 - 2.1. Animals ........................................................................................................ - 24 - 2.2. Human testicular biopsies ............................................................................ - 25 - 2.3. Induction of EAEO ........................................................................................ - 25 - 2.4. Therapeutical elevation of circulating follistatin levels in mice ...................... - 28 - 2.5. Biotin tracer study for analysis of the integrity of the blood-testis-barrier ..... - 29 - 2.6. Paraffin-embedding ...................................................................................... - 30 - 2.7. Histochemistry .............................................................................................. - 30 - 2.7.1. Periodic acid Schiff (PAS) staining ........................................................ - 30 - 2.7.2. Azo-carmine and aniline blue (azan) staining ........................................ - 31 - 2.7.3. Masson’s trichrome staining .................................................................. - 31 - 2.7.4. Toluidine blue staining of mast cells ...................................................... - 32 - 2.7.5. Proliferating cell nuclear antigen (PCNA) staining ................................. - 32 - 2.7.6. TUNEL staining of apoptotic cells .......................................................... - 33 - 2.8. Immunofluorescence microscopy ................................................................. - 34 - 2.8.1. Immunofluorescence staining of testicular macrophages ...................... - 34 - 2.8.2. Activin A immunofluorescence staining ................................................. - 35 - 2.8.3. Alpha smooth muscle actin (α-SMA) immunofluorescence staining ...... - 35 - 2.8.4. Immunofluorescence staining for detection of the integrity of the BTB .. - 36 - 2.8.5. Evaluation of Sertoli and peritubular cells purity by α-SMA and Sox9 double staining .............................................................................................................. - 36 - 2.9. Flow cytometric analysis of T cells ............................................................... - 37 - 2.10. Analysis of gene expression by quantitative RT-PCR ............................... - 39 - 2.10.1. RNA isolation ......................................................................................... - 39 - ii Contents 2.10.2. Test for presence of genomic DNA contamination ................................ - 39 - 2.10.3. Reverse transcription (RT) by PCR ....................................................... - 40 - 2.10.4. Quantitative RT-PCR ............................................................................. - 41 - 2.11. ELISA (enzyme-linked immunosorbent assay) and RIA (radioimmunoassay) .. .................................................................................................................. - 44 - 2.11.1. Preparation of protein lysates ................................................................ - 44 - 2.11.2. Measurement of protein concentration .................................................. - 45 - 2.11.3. Activin A and B ELISA ........................................................................... - 45 - 2.11.4. Measurement of follistatin, inhibin and hormones (FSH, LH and testosterone) by RIA ........................................................................................... - 46 - 2.12. Isolation of mouse testicular cells ............................................................. - 47 - 2.12.1. Isolation of peritubular cells ................................................................... - 47 - 2.12.2. Isolation of Sertoli cells .......................................................................... - 49 - 2.13. Effect of TNF and activin A on Sertoli and peritubular cells in vitro ........... - 50 - 2.14. Statistical analysis ..................................................................................... - 51 - 3. RESULTS ........................................................................................................... - 52 - Study I .................................................................................................................... - 52 - 3.1. Induction rate of EAEO in C57BL/6N mice ................................................... - 52 - 3.2. Morphological changes in EAEO testes ....................................................... - 56 - 3.2.1. Altered histology in EAEO testes ........................................................... - 56 - 3.2.2. Strong fibrotic responses in EAEO testes .............................................. - 59 - 3.3. Inflammatory responses in EAEO testes ...................................................... - 62 - 3.3.1. Expression of inflammatory mediators was increased in EAEO testes .. - 62 - 3.3.2. Immune cell populations were increased in EAEO testes ..................... - 63 - 3.4. Peritubular cells did not proliferate in EAEO testes ...................................... - 67 - iii Contents 3.5. Expression of activin, inhibin and follistatin was altered in EAEO testes ...... - 69 - 3.5.1. Localization of activin A in normal and inflamed mouse testes ............ - 69 - 3.5.2. Activin A and B but not inhibin expression was changed in EAEO testes ..... .............................................................................................................. - 71 - 3.5.3. Activin A receptor expression was decreased in EAEO testes .............. - 73 - 3.5.4. Follistatin expression was upregulated in EAEO mouse testes ............. - 73 - Study II ................................................................................................................... - 75 - 3.6. Serum levels of follistatin were elevated after FST315 vector injection ........ -