CHARACTERIZATION OF LEYDIG CELL DEVELOPMENT IN THERAT TESTIS by Juan Zhai Submitted to the Faculty of the School of Graduate Studies of the Medical College of Georgia in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MAY, 1996 CHARACTERIZATION OF LEYDIG CELL DEVELOPMENT IN THERAT TESTIS This dissertation is submitted by Juan Zhai and has been examined and approved by an appointed committee of the faculty of the School of Graduate Studies of the Medical College of Georgia. The signatures which appear below verify the fact that all required changes have been incorporated and that the dissertation has received final approval with reference to content, fonn and accuracy of presentation. This dissertation is therefore accepted in partial fulfillment of the requirements for the degree ofDoctor ofPhilosophy. Date ' ACKNOWLEDGMENTS I would like to acknowledge and express my appreciation to my major advisor Dr. Tom Abney and my advisory committee, Drs. Virendra Mahesh, Tom Mills, William Hill and Ken Lanclos, for their support and advice. The author is appreciative of the advice and help from Drs. Darrell Brann, Roni Bollag and Lawrence Hendry. A special thanks is extended to my frie~ds Chris Reilly and Edward Pan for their support and being there to listen to all my complaints. Thanks are extended to Angela Foreman and Gene Canady for their friendship and help. lll DEDICATION This dissertation is dedicated to my father, Shouyi Zhai, whose love and wisdom had made a great contribution to my life and career. IV TABLE OF CONTENTS Page Introduction and specific aims 1 Background 5 1 Steroidogenic pathway in Leydig cells 6 Ontogenic development of Leydig cells 9 Regulation ofLeydig cell function 17 Ethylene dimethanesulphonate (EDS)-treated rats An animal model to study Leydig cell development and function 33 Materials and Methods 38 Animals and treatment 3 8 Hormones and chemicals 38 Buffers and solutions 42 Serum testosterone determination 44 Cell isolation 45 Histochemical study (313-HSD staining) 46 In vitro incubation and testosterone production 46 RNA extraction 47 Preparation of eDNA probes 48 Northern blot analysis 50 Estrogen receptor binding assay 51 Protein determination 52 Immunohistochemical study on the testicular tissue 52 Immunohistochemical study on the isolated precursor cells and Leydig cells 54 Reverse transcription-polymerase chain reaction (RT -PCR) 55 Gel electrophoresis ofRT-PCR products 57 Southern blotting and quantitative analysis 57 Flow cytometer analysis 58 Comparative study of estrogen receptor levels in the precursor cells and Leydig cells by immunofluorescent analysis 59 Statistical analysis 60 Results 62 Characterization of Leydig cell degeneration and regeneration after EDS treatment 62 Histochemical analysis of the precursor cell and Leydig cell fraction by 313-hydroxysteroid dehydrogenase (3 13-HSD) staining 64 Analysis of the precursor cell and Leydig cell fractions from the control and EDS-treated rats by flow cytometry 67 Characterization of in vitro testosterone production by Leydig cells and precursor cells during differentiation 74 LH receptor and steroidogenic enzyme (P-450.,. and P-45017a) v mRNA changes during the differentiation process 80 Estrogen receptor binding in the testis of the control and EDS-treated rats 90 Immunohistochemical studies of estrogen receptor in the testis of the control and EDS treated rats 95 Immunohistochemical detection of estrogen receptor in the isolated Leydig cells and precursor cells 95 Detection of estrogen receptor mRNA in Leydig cells and precursor cells by reverse transcription-polymerase chain reaction 99 Comparative study of estrogen receptor levels in the precursor cells and Leydig cells by immunofluorescent analysis 109 Regulation of precursor Leydig cell differentiation by LH/hCG and other possible factors Ill - Discussion 118 Characterization ofLeydig cell degeneration and regeneration after EDS treatment 118 Histochemical studies of the precursor cell and Leydig cell fraction by 3 ~-hydroxysteroid dehydrogenase (3 ~-HSD) staining 124 Analysis of the precursor cell and Leydig cell fractions from the control and EDS-treated rats by flow cytometry 127 Characterization of in vitro testosterone production by Leydig cells and precursor cells during differentiation 131 LH receptor and steroidogenic enzyme (P-450"' and P-45017a) message RNA changes during the differentiation process 135 Estrogen receptor and its mRNA levels in the testis and in the isolated precursor cells and Leydig cells 140 Regulation of precursor Leydig cell differentiation by LH!hCG and other possible factors 143 Summary 146 Literature cited 148 VI LIST OF FIGURES Page Figure I. Steroidogenic pathway for the synthesis of testosterone 7 Figure2. Amino acid sequence, orientation and proposed topology of the rat LH/hCG receptor in the plasma membrane 20 Figure 3. Genomic organization of the rat LH/hCG receptor 22 Figure 4. Gel electrophoresis of tlie LH receptor plasmid digestion 49 Figure 5. Genomic structure of the rat estrogen receptor 56 Figure 6. Temporal changes in serum testosterone levels after a single intraperitoneal injection ofEDS 63 Figure 7. The influence of a single injection ofEDS on testicular weight 65 Figure 8. The effect of a single intraperitoneal injection ofEDS on the interstitial cell number 66 Figure 9. Histochemical study of the Leydig cell and precursor cell fractions by 3f3-HSD staining 68 Figure 10. Cell size of the Leydig cell and precursor cell fractions in the control and day 20 post-EDS rats analyzed by flow cytometry 69 Figure 11. Granularity of the Leydig cell and precursor cell fractions by flow cytometry 71 Figure 12. Dio fluorescence, indicative of mitochondrial activity/number, in the Leydig cell and precursor cell populations by flow cytometry 72 Figure 13. Nile red fluorescence, indicative of lipid droplets content, in the Leydig cell and precursor cell fractions by flow cytometry 73 Figure 14. In vitro testosterone production by Leydig cells vii in the control and EDS-treated rats 75 Figure 15. In vitro testosterone production by precursor cells in the control and EDS-treated rats 76 Figure 16. Changes of the cell number in the Leydig cell fraction after EDS treatment 78 Figure 17. Changes of the cell number in the precursor cell fraction after EDS treatment 79 Figure 18. Gel electrophoresis of the total RNA extracted from Leydig cells and precursor cells 81 Figure 19. Northern blot analysis of the LH receptor in the control Leydig cells and precursor cells and in the precursor cells after EDS treatment Figure A. 83 Figure B. 84 Figure C. 85 Figure D. 86 Figure 20. Northern blot analysis ofP-450css in the control Leydig cells and precursor cells and in the precursor cells after EDS treatment 88 Figure 21. Northern blot analysis ofP-45017., in the control Leydig cells and precursor cells and in the precursor cells after EDS treatment 89 Figure22. Northern blot analysis ofLH receptor, P-450css and P-45017amRNA in the Leydig cells of the control and day 36 and 60 post-EDS rats Figure A. 91 Figure B. 92 Figure 23. Changes in the specific estrogen binding capacity of rat testicular tissue after a single intraperitoneal injection ofEDS 93 Figure 24. Representative Scatchard plot analysis of 3H-estradiol binding by control and day 16 post-EDS treated rat testicular cytosols 94 viii Figure 25. Localization of the testicular estrogen receptor by immunohistochemical analysis in the controls and day 10 EDS-treated rats Figures A and B. 96 Figures C and D. 97 Figures E and F. 98 Figure26. Detection of the estrogen receptor by immunohistochemical analysis in the Leydig cell and precursor cell fractions from the controls and day 10 EDS-treated rats 100 Figure 27. RT-PCR detection of estrogen receptor mRNA in Leydig cells and precursor cells from controls and precursor cells from EDS-treated animals 101 Figure 28. Specificity of the RT-PCR products amplified by the primer sets 103 Figure 29. Gel electrophoresis of the coamplification ofrat estrogen receptor and rabbit P -globin cDNAs 104 Figure 30. Quality control of the RT-PCR Figures A and B 106 Figure C 107 Figure 31. Southern blot and quantitative analysis of the RT-PCR products generated from precursor cells of the control and EDS-treated rats 108 Figure 32. Southern blot and quantitative analysis of the RT-PCR products generated from Leydig cells of the control and EDS-treated rats 110 Figure 33. Comparative study of the estrogen receptor levels in Leydig cells and precursor cells of the control and day 10 post-EDS rats 112 Figure 34. Average gray value, indicative of immunofluorescence of estrogen receptor, in Leydig cell and precursor cell populations 114 Figure 35. Stimulation of testosterone production by hCG in the precursor cells of the immature and day 20 post-EDS rats 116 ix Figure 36. Effects ofhCG or/and IGF-I on the in vitro testosterone production by precursor cells at day 20 post-EDS treatment 117 X LIST OF TABLES Page Table I. Comparison of estrogen receptor levels by immunofluorescent study in Leydig cells and precursor cells of the control and day 10 post-EDS rats 113 Table 2. Summary of precursor cell differentiation and Leydig cell regeneration after EDS treatment 145 XI 2 factor in Leydig cell development and function, evidence has accumulated that local factors play an important role, either in conjunction with or dependent upon LH. In recent years, the ethylene dimethanesulphonate (EDS)-treated rat has become a useful animal model for studying Leydig cell development and function. EDS, an alkylating agent, rapidly and selectively destroys mature Leydig cells in the rat testis. A single intraperitoneal injection ofEDS results in a complete destruction of Leydig cells within two days. However, Leydig cell regeneration occurs within two to three weeks after EDS treatment. The regeneration of Leydig cells in the testis is complete by five to seven weeks after EDS treatment. The regeneration of Leydig cells after EDS treatment is primarily dependent upon high levels of serum LH.