Ligands & Signaling Components of the Transforming Growth Factor β Family : Local Regulators of Inhibin Production in Ovarian Granulosa Cells

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) .