Male Reproductive Toxicology (eBook)

Front Cover 1
Male Reproductive Toxicology, Part A 4
Copyright Page 5
Table of Contents 6
Contributors 10
Chapter 1. Introduction 14
INTRODUCTION 14
STRATEGIES 15
MALES 18
FEMALES 21
REFERENCES 27
Chapter 2. Male Mouse Sexual Behavior Test 29
INTRODUCTION 29
ANIMALS 29
MATERIALS AND OTHER REQUIREMENTS 31
PROCEDURE 33
COMMENTS 37
ACKNOWLEDGMENT 38
REFERENCES 38
Chapter 3. In Vitro Techniques for Assessing Pituitary Secretory Function 39
INTRODUCTION 39
PRIMARY ANTERIOR PITUITARY CELL CULTURES 44
SHORT-TERM PITUITARY TISSUE INCUBATIONS 57
CONCLUDING REMARKS 63
REFERENCES 63
Chapter 4. Histological Methods for Evaluation of the Testis 65
INTRODUCTION 65
CHOOSING THE APPROPRIATE HISTOLOGICAL METHOD 66
FIXATIVES AND BUFFERS 70
ROUTE OF FIXATION 78
EMBEDDING MEDIUM FOR LIGHT MICROSCOPY 82
ELECTRON MICROSCOPY OF THE TESTIS 84
PHOTOMICROGRAPHY 85
EVALUATION OF HISTOLOGICAL METHODS 86
EFFECTS OF HISTOLOGICAL METHODS ON HISTOPATHOLOGICAL EVALUATIONS 94
REFERENCES 98
Chapter 5. Application of Testicular Sperm Head Counts in the Assessment of Male Reproductive Toxicity 99
INTRODUCTION 99
MATERIALS/SOLUTIONS 99
PROCEDURES 100
COMMENTS 104
ACKNOWLEDGMENT 107
REFERENCES 107
Chapter 6. Stage Synchronization in Rat Seminiferous Tubules Using Vitamin A Depletion and Repletion 108
INTRODUCTION 108
MATERIALS AND REAGENTS 109
PROCEDURES 110
COMMENTS 116
REFERENCES 118
Chapter 7. Spermatogonial Stem Cells: Assessing Their Survival and Ability to Produce Differentiated Cells 119
INTRODUCTION 119
DIRECT OBSERVATION AND COUNTS OF STEM CELLS IN WHOLE-MOUNTED TUBULES 120
CLONOGENIC ASSAY OF STEM CELL SURVIVAL 128
NONCLONOGENIC ASSAYS OF STEM CELL SURVIVAL 131
FUNCTIONAL ASSAY FOR STEM CELL SURVIVAL 134
ACKNOWLEDGMENTS 135
REFERENCES 135
Chapter 8. Dominant Lethal Tests in Male and Female Mice 137
INTRODUCTION 137
PROCEDURES IN MALES 138
PROCEDURES IN FEMALES 148
CONCLUSION 152
ACKNOWLEDGMENTS 152
REFERENCES 152
Chapter 9. Transillumination–Phase-Contrast Microscopic Techniques for Evaluation of Male Germ Cell Toxicity and Mutagenicity 155
INTRODUCTION 155
TRANSILLUMINATION METHOD 158
TRANSILLUMINATION-ASSISTED MICRODISSECTION METHOD 158
IMPROVED TRANSILLUMINATION METHOD 163
ANALYSIS OF SPECIFIC DAMAGE IN LIVING CELL SAMPLES: "GOLDEN CELL METHOD" 170
DNA FLOW CYTOMETRY OF DEFINED STAGES OF THE SEMINIFEROUS EPITHELIAL CYCLE 172
MEIOTIC MICRONUCLEUS METHOD 173
IN VIVO MEIOTIC MICRONUCLEUS TECHNIQUE 174
IN VITRO MEIOTIC MICRONUCLEUS METHOD 176
REFERENCES 178
Chapter 10. Isolation and Culture of Leydig Cells from Adult Rats 179
INTRODUCTION 179
ANIMALS, REAGENTS, AND MATERIALS 180
PROCEDURES 181
COMMENTS 189
ACKNOWLEDGMENTS 193
REFERENCES 193
Chapter 11. Isolation and Primary Culture of Leydig Cells 195
INTRODUCTION 195
ANIMALS 196
MATERIALS 196
PROCEDURES 200
COMMENTS 207
ACKNOWLEDGMENTS 209
REFERENCES 209
Chapter 12. Purification and Primary Culture of Leydig Cells 210
INTRODUCTION 210
MATERIALS/REAGENTS 210
STERILE SUPPLIES 211
SOLUTIONS 211
CONTINUOUS 11–23% METRIZAMIDE GRADIENT FOR THE SEPARATION OF MOUSE LEYDIG CELLS 212
CULTURING OF MOUSE LEYDIG CELLS 217
CONTINUOUS 5–32% METRIZAMIDE GRADIENT FOR THE PURIFICATION OF RAT LEYDIG CELLS 218
ACKNOWLEDGMENT 221
REFERENCES 221
Chapter 13. Preparation and Use of Sertoli Cell-Enriched Cultures from 18-Day-Old Rats 223
INTRODUCTION 223
MATERIALS AND REAGENTS 225
PROCEDURES 226
USE OF SERTOLI CELL-ENRICHED MONOLAYER CULTURES TO EVALUATE TOXICANT-ASSOCIATED EFFECTS 238
ACKNOWLEDGMENTS 240
REFERENCES 240
Chapter 14. Two-Compartment Cultures of Sertoli Cells—Applications in Testicular Toxicology 243
INTRODUCTION 243
POTENTIAL USES OF TWO-COMPARTMENT SERTOLI CELL CULTURES IN TESTICULAR TOXICOLOGY 244
MATERIALS AND METHODS 246
EFFECTS OF CADMIUM CHLORIDE ON SERTOLI CELLS 255
ACKNOWLEDGMENTS 257
REFERENCES 257
Chapter 15. Isolation, Separation, and Short-Term Culture of Spermatogenic Cells 259
INTRODUCTION 259
MATERIALS AND REAGENTS 260
PROCEDURES 263
CONCLUSIONS 276
ACKNOWLEDGMENTS 276
REFERENCES 276
Chapter 16. Collection of Interstitial Fluid and Seminiferous Tubule Fluid from the Rat Testis 278
INTRODUCTION 278
SOLUTIONS AND MATERIALS 279
PROCEDURES 280
DISCUSSION 284
REFERENCES 286
Chapter 17. Isolation and Culture of Epididymal Epithelial Cells from Adult Rats 287
INTRODUCTION 287
ANIMALS, REAGENTS, AND MATERIALS 288
PROCEDURES 289
COMMENTS 295
DISCLAIMER 299
REFERENCES 299
Chapter 18. Evaluation of Protein Synthesis by the Epididymis 300
PROTEIN SYNTHESIS 300
PROTOCOL 302
ANALYSIS OF PROTEIN PATTERNS 313
SUMMARY 315
REFERENCES 315
Chapter 19. Utilizing Cryo Resources CellSoft Computer-Assisted Sperm Analysis System for Rat Sperm Motility Studies 316
INTRODUCTION 316
MATERIALS 316
PROTOCOL 317
COMPUTER PROGRAM FOR CELLSOFT DATA ANALYSIS 321
COMMENTS 330
REFERENCES 331
Chapter 20. Computer-Assisted Sperm Analysis of Rodent Epididymal Sperm Motility Using the Hamilton-Thorn Motility Analyzer 332
INTRODUCTION 332
MATERIALS AND METHODS 333
SUMMARY 345
ACKNOWLEDGMENTS 346
REFERENCES 346
Chapter 21. Methods for Evaluation of Rat Epididymal Sperm Morphology 347
INTRODUCTION 347
MATERIALS AND SUPPLIES 348
PROCEDURE 348
REFERENCES 356
Chapter 22. Semen Analysis and Fertility Assessment in the Rabbit 357
INTRODUCTION 357
SEMEN COLLECTION 357
SEMEN ANALYSIS 360
FERTILITY ASSESSMENT 368
SELECTION OF MALE RABBITS AND STUDY DESIGN 372
REFERENCES 373
Chapter 23. General Techniques for Assessing Male Reproductive Potential in Human Field Studies 375
INTRODUCTION 375
PROCEDURES 376
REFERENCES 383
Index 386