............................................................................................................................................8 INTRODUCTION ..................................................................................................................................9 REVIEW OF THE LITERATURE.....................................................................................................10 1. GENERAL CHARACTERISTICS OF THE TRANSFORMING GROWTH FACTOR β SUPERFAMILY.................................................................................................................................10 1.1. Historical background ..............................................................................................................10 1.2. Biosynthesis and structural properties of TGFβ family members............................................11 1.3. The discovery of TGFβ receptors ............................................................................................11 1.3.1. Mammalian type II serine/threonine kinases.....................................................................12 1.3.2. Mammalian type I serine/threonine kinases......................................................................12 1.3.3. Functional characteristics of the type I and II receptors....................................................14 1.4. Additional TGFβ family member binding proteins .................................................................14 1.4.1. Type III receptors..............................................................................................................14 1.4.2. Other binding proteins ......................................................................................................15 1.5. Intracellular signaling molecules .............................................................................................15 1.5.1. The Smad family...............................................................................................................15 1.5.2. Principles for Smad activation ..........................................................................................16 1.5.3. Other signaling cascades activated by TGFβ family members .........................................18 1.6. Null mice models of ser/thr kinases and Smads.......................................................................18 1.7. Ser/thr kinase receptor and Smad involvement in human disease............................................18 1.7.1. Tumorigenesis...................................................................................................................18 1.7.2. Other disorders..................................................................................................................18 2. BONE MORPHOGENETIC PROTEINS, RECEPTORS AND SMADS IN THE MAMMALIAN OVARY ..............................................................................................................................................21 2.1. General characteristics of ovarian function..............................................................................21 2.1.1. Early stages of follicular development..............................................................................21 2.1.2. Later stages of follicular development ..............................................................................21 2.1.3. The end of the follicular lifespan ......................................................................................22 2.1.4. Ovarian steroid hormone production.................................................................................23 2.2. Expression of BMPs in the mammalian ovary.........................................................................23 2.2.1. Expression of BMP receptors in the mammalian ovary....................................................24 2.2.2. Smad expression in the mammalian ovary........................................................................24 2.3. Animal models showing involvement of BMPs for ovarian function......................................25 2.4. Biological effects of recombinant BMPs in the ovary .............................................................25 3. OVARIAN INHIBINS AND THE REGULATION OF OVARIAN INHIBIN SUBUNIT EXPRESSION ....................................................................................................................................27 3.1. Historical background of inhibins ............................................................................................27 3.2. Intraovarian effects of inhibin..................................................................................................28 3.3. Inhibin receptors ......................................................................................................................28 3.4. Assays for determination of dimeric inhibins ..........................................................................28 3.5. Inhibin subunit mRNA expression in the rat ovary..................................................................29 3.5.1. Dimeric inhibin levels during the rat estrus cycle .............................................................29 3.6. Inhibin subunit expression in the human and primate ovaries .................................................29 3.6.1. Dimeric inhibin levels during the human menstrual cycle ................................................30 3.7. In vitro regulation of inhibin subunit expression .....................................................................30 AIMS OF THE STUDY........................................................................................................................32 -Local Regulators of Inhibin Production in Ovarian Granulosa Cells 5 MATERIALS AND METHODS .........................................................................................................33 1. Human granulosa-luteal cells......................................................................................................33 2. Rat granulosa cells ......................................................................................................................33 3. MIN6 cells ..................................................................................................................................33 4. 293T cells....................................................................................................................................33 5. In vitro treatment of cells ............................................................................................................34 6. Recombinant adenoviruses .........................................................................................................34 7. Transfection of rat granulosa cells ..............................................................................................34 8. RNA extraction and preparation of filters...................................................................................34 9. Preparation of cDNA probes and hybridizations ........................................................................35 10. Western blotting........................................................................................................................35 11. Protein content ..........................................................................................................................35 12. Enzyme-linked immunosorbent assay (ELISA)........................................................................35 13. Fluorescence in situ hybridization (FISH) ................................................................................36 14. ALK7 cDNA cloning ................................................................................................................36 15. Statistical methods ....................................................................................................................36 RESULTS AND DISCUSSION ...........................................................................................................37 1. CHROMOSOMAL MAPPING OF HUMAN ACTIVIN TYPE II RECEPTORS, FOLLISTATIN AND ALK7 (I AND II) ......................................................................................................................37 1.1. Activin receptor type II (ACVRII)...........................................................................................37 1.2. Activin receptor type IIB (ACVRIIB) .....................................................................................37 1.3. Follistatin (FST).......................................................................................................................38 1.4. Activin receptor-like kinase 7 (ALK7) ....................................................................................38 2. SEARCH FOR NOVEL SERINE/THREONINE KINASE RECEPTORS (II)..............................38 2.1. Cloning and characterization of ALK7 ....................................................................................38 2.2. Ovarian ALKs..........................................................................................................................39 3. EXPRESSION OF SERINE/THREONINE KINASE RECEPTORS AND SMADS IN HUMAN GRANULOSA-LUTEAL CELLS (III AND IV)................................................................................40 3.1. Ser/thr kinases..........................................................................................................................40 3.2. Smads.......................................................................................................................................41 4. STIMULATION OF INHIBIN PRODUCTION IN HUMAN GRANULOSA-LUTEAL CELLS BY BONE MORPHOGENETIC PROTEINS (III) ............................................................................41 5. OVEREXPRESSION OF ALKS AND SMADS IN HUMAN GRANULOSA-LUTEAL CELLS STIMULATES INHIBIN B PRODUCTION (IV) ......