Role of peroxisomes in granulosa cells, follicular development and steroidogenesis in the mouse ovary Inaugural Dissertation submitted to the Faculty of Medicine in partial fulfillment of the requirements for the PhD-Degree of the Faculties of Veterinary Medicine and Medicine of the Justus Liebig University Giessen by Wang, Shan of ChengDu, China Gießen (2017) From the Institute for Anatomy and Cell Biology, Division of Medical Cell Biology Director/Chairperson: Prof. Dr. Eveline Baumgart-Vogt Faculty of Medicine of Justus Liebig University Giessen First Supervisor: Prof. Dr. Eveline Baumgart-Vogt Second Supervisor: Prof. Dr. Christiane Herden Declaration “I declare that I have completed this dissertation single-handedly without the unauthorized help of a second party and only with the assistance acknowledged therein. I have appropriately acknowledged and referenced all text passages that are derived literally from or are based on the content of published or unpublished work of others, and all information that relates to verbal communications. I have abided by the principles of good scientific conduct laid down in the charter of the Justus Liebig University of Giessen in carrying out the investigations described in the dissertation.” Shan, Wang November 27th 2017 Giessen,Germany Table of Contents 1 Introduction ...................................................................................................................... 3 1.1 Peroxisomal morphology, biogenesis, and metabolic function ...................................... 3 1.1.1 Morphology of peroxisomes ................................................................................... 3 1.1.2 Biogenesis of peroxisomes .................................................................................... 4 1.1.3 Peroxisomal metabolic functions ........................................................................... 7 1.2 Peroxisome biogenesis disorder .................................................................................12 1.2.1 Peroxisome related human disease ......................................................................12 1.2.2 Mouse model with peroxisomal deficiency ............................................................14 1.2.3 Peroxisomal disorders and human fertility ............................................................15 1.3 Mouse ovary ...............................................................................................................16 1.3.1 Follicular development of mouse ovary .................................................................17 1.3.2 Estrous cycle in the mouse ovary .........................................................................18 1.3.3 Steroid biosynthesis in the ovary ..........................................................................19 1.4 Peroxisomes in the ovary ............................................................................................24 1.5 Up to date evidences for the involvement of peroxisomes in steroid hormone metabolism .......................................................................................................................25 2 Aims of the study ............................................................................................................27 3 Materials and methods ....................................................................................................28 3.1 Materials .....................................................................................................................28 3.1.1 Chemicals for the general experiments .................................................................28 3.1.2 General Instruments used in the laboratory ..........................................................28 3.1.3 General materials used for cell culture ..................................................................29 3.2 Methods ......................................................................................................................30 3.2.1 Morphological experiments ...................................................................................30 3.2.2 Cell culture experiments .......................................................................................35 3.2.3 Biochemical experiments ......................................................................................38 3.2.4 Molecular biological experiments ..........................................................................41 3.3 Statistical Analysis ......................................................................................................48 4 Results .............................................................................................................................49 4.1 Part 1: Investigation of peroxisomal functions during mouse ovarian folliculogenesis .49 4.1.1 Regulation of peroxisomal enzymes during follicular maturation ...........................49 4.1.2 SOD2 and glutathione reductase regulation during follicular maturation ...............55 4.2 Part 2 Investigation of peroxisomal functions on steroidogenesis in mouse granulosa tumor cell lines— KK-1 cells .............................................................................................56 4.2.1 Peroxisome abundance in KK-1 granulosa cells ...................................................56 4.2.2 KK-1 granulosa cells respond to hCG and FSH treatment and produce higher amount of steroids .........................................................................................................57 4.2.3 Regulation of the peroxisomal compartment involved in fatty acid transport and β- oxidation under hCG treatment ......................................................................................58 1 4.2.4 Regulation of peroxisomal metabolism involved in ROS degradation under hCG treatment .......................................................................................................................59 4.2.5 Establishment of a siRNA-mediated Pex13 knock-down in KK-1 cells to induce peroxisome deficiency ...................................................................................................60 4.2.6 Impact of Pex13 KD on steroid synthesis under hCG treatment ...........................62 4.2.7 AKAP1 overexpression in KK-1 cells ....................................................................67 4.2.8 The mitochondrial steroidogenesis defect was rescued by AKAP1-myc overexpression in peroxisome deficient KK-1 cells ........................................................69 4.2.9 Peroxisomal enzyme regulation after Akap1-5B overexpression in KK-1 cells ......72 4.2.10 ROS production was increased in KK-1 cells with Pex13 knock-down ................72 4.2.11 Effects of H2O2 on steroid biogenesis and steroidogenic enzymes .....................75 4.2.12 Effects of H2O2 on peroxisomal and mitochondrial compartments.......................78 4.2.13 The reduced mitochondrial steroidogenesis observed in KK-1 cells after Pex13 knock-down was rescued by the addition of the antioxidant α-tocopherol ......................79 5 Discussion .......................................................................................................................82 5.1 Part 1 Peroxisomal functions on mouse ovarian folliculogenesis .................................82 5.1.1 The history of peroxisomes in the mouse ovary ....................................................82 5.1.2 Peroxisomes detected with PEX14p as optimal marker protein in mouse ovarian follicular maturation .......................................................................................................83 5.1.3 Antioxidative enzymes catalase, SOD2 and glutathione reductase (GR) in mouse ovarian follicular maturation ...........................................................................................83 5.1.4 Lipid metabolism in mouse ovarian follicular maturation .......................................86 5.1.5 Peroxisomes in the corpus luteum (CL) ................................................................88 5.2 Part 2 Peroxisomal functions on steroidogenesis in mouse granulosa tumor cell lines— KK-1 cells .........................................................................................................................88 5.2.1 Peroxisomal involvement in steroidogenesis ........................................................88 5.2.2 The link between peroxisomal β-oxidation and steroid synthesis ..........................89 5.2.3 ROS production and anti-oxidants regulation during steroid synthesis ..................90 5.2.4 Pex13 knock-down in vitro and its influence on steroidogenesis ...........................91 5.2.5 Underlying mechanisms for StAR inhibition under Pex13 knock-down conditions.94 5.2.6 Protective role of tocopherol on steroidogenic enzymes and steroid biosynthesis 95 6 Summary ..........................................................................................................................97 7 Zusammenfassung ..........................................................................................................99 8 References ..................................................................................................................... 101 9 Index of abbreviation .................................................................................................... 116 10 Acknowledgements ..................................................................................................... 119 11 Curriculum Vitae