Investigations into Metabolism, Transport and Function of Sulfonated Steroids in the Porcine Testicular-Epididymal Compartment Yaser Dezhkam INAUGURAL DISSERTATION Submitted to the Faculty of Veterinary 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 Supported by the German Research Foundation (DFG) DFG Research Group "Sulfated Steroids in Reproduction" (FOR1369) 1 Investigations into Metabolism, Transport and Function of Sulfonated Steroids in the Porcine Testicular-Epididymal Compartment Inaugural Dissertation Submitted to the Faculty of Veterinary 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 Yaser Dezhkam from Urmia, Iran Giessen 2015 2 From the Clinic for Obstetrics, Gynecology and Andrology for Large and Small Animals with Ambulatory Service Faculty of Veterinary Medicine, Justus-Liebig-University Giessen First Supervisor: Prof. Dr. Gerhard Schuler Second Supervisor: Prof. Dr. Klaus Steger Committee Members: Prof. Dr. Gerhard Schuler (Internal Thesis Examiner) Committee Member: Prof. Dr. Marion Piechotta (External Thesis Examiner) Other committee Members: Prof. Dr. Klaus-Dieter Schlüter (Chairman of Oral Panel) Committee Member: Prof. Dr. Klaus Steger (Chairman of Oral Panel) Date of Doctoral Defense: 23. April 2015 3 DECLARATIONS “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and referenced all text passages that are derived literally from or are based on the content of published or unpublished work of others, and all information that relates to verbal communications.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.” Yaser Dezhkam 4 TABLE OF CONTENTS 1. Introduction ............................................................................................... 19 2. Literature review ....................................................................................... 22 2.1 Anatomy, histology and functions of the testis ....................................... 22 2.1.1 General information and gross morphology of the testis ...................... 22 2.1.2 Seminiferous tubules, spermatogenic epithelium and spermatogenesis ..................................................................................................................... 23 2.1.2.1 Seminiferous tubules ................................................................................. 23 2.1.2.2 Sertoli cells ................................................................................................. 23 2.1.2.3 Germ cells and spermatogenesis .............................................................. 25 2.1.2.3.1 Spermatogonia ........................................................................................... 26 2.1.2.3.2 Primary spermatocytes ............................................................................. 26 2.1.2.3.3 Secondary spermatocytes .......................................................................... 26 2.1.2.3.4 Spermatids, spermiogenesis and sperm release ...................................... 26 2.1.2.4 Peritubular cells ......................................................................................... 27 2.1.3 Tubuli seminiferi recti, rete testis and ductuli efferentes ...................... 28 2.1.4. Leydig cells ................................................................................................. 29 2.2 Anatomy, histology and function of the epididymis ............................... 29 2.2.1 Anatomy and histomorphology ................................................................ 29 2.2.1.1 Epithelial cell types of the epididymis and epididymosomes................. 30 2.2.1.1.1 Principal cells ............................................................................................. 30 2.2.1.1.2 Epididymosomes ........................................................................................ 30 2.2.1.1.3 Basal cells ................................................................................................... 31 2.2.1.1.4 Other cell types of the epididymal epithelium ........................................ 32 2.2.2 Functions of the epididymis ...................................................................... 32 2.2.2.1 The blood-epididymis barrier .................................................................. 32 2.2.2.2 Reabsorption of tubular fluid ................................................................... 33 2.2.2.3 Microenvironment for maturation .......................................................... 33 2.2.2.4 Storage of sperm cells in the epididymis ................................................. 35 2.2.2.5 Control of epididymal differentiation and functions by androgens and estrogens ..................................................................................................... 35 5 2.3 Vas deferens ............................................................................................... 37 2.4 The vascular and lymph system of the testis and epididymis ............... 38 2.4.1 The vascular and lymph system of the testis ........................................... 38 2.4.2 The vascular and lymph system of the epididymis ................................. 39 2.4.3 Architecture of the vascular cone of the spermatic cord ....................... 40 2.5 Steroid hormones and steroidogenesis .................................................... 41 2.5.1 Initiation of steroidogenesis and steroid acute regulatory protein (StAR) ..................................................................................................................... 43 2.5.2 Cytochrome P450 side chain cleavage (CYP11A1) ................................ 45 2.5.3 3β-Hydroxysteroid dehydrogenase Δ5/4 isomerase (3β–HSD, HSD3B) ..................................................................................................................... 45 2.5.4 17α-Hydroxylase-C17/20-lyase (CYP17, P450c17) ................................. 46 2.5.5 17β-Hydroxysteroid dehydrogenases (17β-HSD, HSD17B) .................. 47 2.5.6 5α-Reductase (steroid-5-alpha-reductase, SRD5A) ............................... 48 2.5.7 Cytochrome P450 aromatase (CYP19) .................................................... 48 2.6 Sulfonated steroids .................................................................................... 50 2.6.1 Enzymatic reaction of sulfonation ........................................................... 50 2.6.2 Effects of steroid sulfonation and possible roles of sulfonated steroids 51 2.7 Cytosolic sulfotransferases (SULTs) ........................................................ 53 2.7.1 SULTs relevant to the sulfonation of steroids ........................................ 55 2.7.1.1 SULT1E1 .................................................................................................... 55 2.7.1.2 SULT2A1 .................................................................................................... 57 2.7.1.3 SULT2B1 .................................................................................................... 59 2.8 Steroid sulfatase and sulfatase pathway .................................................. 61 2.8.1 Steroid sulfatases ....................................................................................... 61 2.8.2 Physico-chemical and structural properties ........................................... 61 2.8.3 Sulfatase pathway ...................................................................................... 63 2.9 Testicular steroidogenesis in boars .......................................................... 63 2.9.1 Steroidal pheromones ................................................................................ 64 2.9.2 Sulfonated steroids in boars ..................................................................... 65 2.10 Hypotheses and aims of this study ........................................................... 68 3. Material and methods ............................................................................... 70 6 3.1 Animal experiments and sample collection ............................................. 70 3.1.1 Collection of blood samples ...................................................................... 70 3.1.1.1 Long-term profiles in unstimulated boars .............................................. 70 3.1.1.2 Long-term profiles in hCG-stimulated boars ......................................... 71 3.1.1.3 Sample collection from local blood vessels of the testicular-epididymal compartment .............................................................................................. 72 3.1.2. Collection and processing of tissue samples ............................................ 73 3.2 Steroid measurements ............................................................................... 74 3.2.1 Radioimmunological measurements of steroid concentrations ............ 75 3.2.1.1 Radioimmunological measurement of testosterone ............................... 77 3.2.1.2 Radioimmunological measurement of estradiol-17ß ............................. 77 3.2.1.3 Radioimmunological measurement