Daniel R. Dietrich, PhD, is a Professor and Head of the Human and Environmental Toxicology Group, Faculty of Sciences of the University of Konstanz, Konstanz, Germany. Heiko O. Krieger, MSc, is a scientific research assistant and PhD student in the Human and Environmental Toxicology Group, Faculty of Sciences of the University of Konstanz, Konstanz, Germany.

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• Preface xiAcknowledgments xiiiContributing Authors xv1 Introduction 1References 32 Fish Species of Interest 72.1 Fathead Minnow (Pimephales promelas) 82.2 Medaka (Oryzias latipes) 102.3 Zebrafish (Danio rerio) 122.4 Other Fish Species 13References 153 Sexual Determination, Differentiation, and Gonadal Development 193.1 Primordial Germ Cells in the Primordial (Primary) Gonad 223.1.1 Differentiation and Number of PGCs 223.1.2 Molecular Markers of PGCs 253.2 Reproductive Strategies 263.3 Differentiation of the Primordial Gonad into Ovary or Testis 273.4 Gonadal Duct Formation 323.5 Endocrinology: Influence on Gonadogenesis 403.6 Critical Period of Sexual Differentiation in Developing Fish 453.7 Bi-Potentiality of Germ Cells in Adult Fish 47References 514 Female Gonad Anatomy and Morphology 664.1 Gonadogenesis: Ovary 664.1.1 Location and Gross Organization 664.1.2 Anatomy of the Ovary 674.2 Hypothalamic–Pituitary–Ovarian Axis 694.3 Cellular Structure of the Ovary 694.3.1 Germ Cells (Oogenesis) 694.3.2 Female Somatic Supportive Tissue 784.3.2.1 Theca Cells 784.3.2.2 Granulosa Cells 81References 815 Male Gonad Anatomy and Morphology 885.1 Gonadogenesis: Testes 885.1.1 Location and Gross Organization 885.1.2 Anatomy of the Testes 895.1.3 Germinal Epithelium 935.1.4 Male Germ Cells (Spermatogenesis) 955.1.5 Male Somatic Supportive Tissue 995.1.5.1 Sertoli Cells 995.1.5.2 Leydig Cells 1035.1.5.3 Lobule Boundary Cells 1045.1.5.4 Sex Steroid Control of Spermatogenesis 104References 1086 Endocrine-Disrupting Compounds 1156.1 Individual Effects 1176.1.1 Inhibition of Gametogenesis 1176.1.2 Necrosis and Apoptosis (Gamete and Stromal Cells) 1196.1.3 Atretic Follicles 1216.1.4 Sertoli Cell Hypertrophy and Hyperplasia 1236.1.5 Leydig Cell Hypertrophy and Hyperplasia 1296.1.6 Fibrosis 1296.1.7 Gonadal Duct Formation 1326.2 Effects Associated with Exposure to SpecificCompounds or Compound Classes 133Table 6.1 Overt Testicular Changes in Male Fish, 134Table 6.2 Testis–Ova in Male Fish, 148Table 6.3 Testicular Fibrosis in Male Fish, 161Table 6.4 Germ Cell Effects in Male Fish, 165Table 6.5 Somatic Cell Effects in Male Fish, 181Table 6.6 Testicular Inflammation and MacrophageInfiltration in Male Fish, 186Table 6.7 Overt Ovarian Changes in Female Fish, 187Table 6.8 Ovo–Testis in Female Fish, 195Table 6.9 Ovarian Fibrosis in Female Fish, 196Table 6.10 Germ Cell Effects in Female Fish, 197Table 6.11 Germ Cell Atresia in Female Fish, 206Table 6.12 Ovarian Inflammation and Macrophage Infiltration in Female Fish, 2146.2.1 Effects in Male Fish Associated with Exposure to Specific Compounds or Compound Classes 2156.2.1.1 Testis Architecture, Integrity, and Gross Appearance 2156.2.1.2 Testis–Ova 2156.2.1.3 Fibrosis 2166.2.1.4 Germ Cell Effects 2176.2.1.5 Somatic Cell Effects 2196.2.1.6 Testicular Cell Inflammation and Macrophage Infiltration 2196.2.2 Effects in Female Fish Associated with Exposure to Specific Compounds or Compound Classes 2206.2.2.1 Ovary Architecture, Integrity, and Gross Appearance 2206.2.2.2 Ovo–Testis 2216.2.2.3 Fibrosis 2216.2.2.4 Germ Cell Effects 2226.2.2.5 Germ Cell Atresia 2236.2.2.6 Germ Cell Inflammation and Macrophage Infiltration 2256.3 Population Effects 2256.3.1 Fecundity and Fertility 2266.3.2 Breeding and Reproductive Behavior of Fishes 2306.3.3 Transgenerational Effects 230References 2327 Determination of Effects of Exogenous Hormones and Endocrine-like Active Compounds 2477.1 Histological Processing: Microdissection versus Whole-Fish Sectioning 2477.2 Optimal Tissue Preparation and Histological Techniques 2507.3 Plane of Gonad Sectioning for Optimal Organ Representation 251References 2528 Evaluation of Effects in Fish Gonads 2548.1 Qualitative (Semiquantitative) versus Quantitative Evaluation 2548.2 Gonadal Staging in the Testis 2558.2.1 Qualitative Staging in the Testis 2558.2.2 Quantitative Staging in the Testis 2578.3 Gonadal Staging in the Ovary 2628.3.1 Qualitative Staging in the Ovary 2628.3.2 Quantitative Staging in the Ovary 2648.4 Qualitative Assessment of Histopathological Changes 265References 2679 Experimental Design and Statistics 2709.1 Basic Considerations in Experimental Design 2709.1.1 Defining the Objectives 2719.1.2 Asking Precise Questions 2719.1.3 The Principal Question: Is There an Effect or Is There Not? 2729.1.4 Basic Mechanistic Research, Risk Assessment, and the NOEC, LOEC, and ECx Question 2729.2 Variables to be Determined and Their Inherent Biological and Mathematical Characteristics 2749.2.1 Biological Characteristics 2749.2.2 Mathematical Characteristics 2789.3 Prerequisite Statistical Concepts 2829.3.1 Sample Independence: The Experimental Unit 2829.3.2 One- or Two-Sided Testing 2839.3.3 Observable Effect Size: Minimum Observable Effect 2839.3.4 Power (1  b) to Detect an Effect 2849.3.5 Replication and Pseudoreplication 2869.3.6 Minimum Significant Difference 2879.4 Statistical Tests and Testing Situations Encountered Routinely 2889.4.1 Significance Level of Multiple Comparisons: Variable-wise Significance 2889.4.2 Significance Level for Many Different Comparisons: Simultaneous Significance 2889.4.3 Multivariate Tests 2899.4.4 Parametric Versus Nonparametric Testing 289References 28910 Conclusions 295References 297Appendix: Fish Preparation and Microdissection of Organs 301A.1 Fish Preparation 301A.2 Microdissection of Organs 302A.3 Tissue Fixation 303A.3.1 Neutral Buffered Formalin 304A.3.2 Bouin’s Fixative 304A.3.3 Lillie’s Fixative 305A.3.4 Davidson’s Fixative 305A.3.5 Dietrich’s Fixative 306A.3.6 Glutaraldehyde 306A.4 Embedding 307A.4.1 Paraffin 307A.4.2 Glycolmethacrylate (GMA) 309A.5 Tissue Sectioning 310A.6 Sample Mounting 311A.7 Tissue Slide Staining 312A.7.1 Hematoxylin and Eosin 312A.7.2 Masson’s Trichrome Stain 314A.8 Final Processing 315A.9 Examples 316A.10 Summary 317References 318Index 319

Daniel R. Dietrich, PhD, is a Professor and Head of the Human and Environmental Toxicology Group, Faculty of Sciences of the University of Konstanz, Konstanz, Germany. Heiko O. Krieger, MSc, is a scientific research assistant and PhD student in the Human and Environmental Toxicology Group, Faculty of Sciences of the University of Konstanz, Konstanz, Germany.

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• Preface xiAcknowledgments xiiiContributing Authors xv1 Introduction 1References 32 Fish Species of Interest 72.1 Fathead Minnow (Pimephales promelas) 82.2 Medaka (Oryzias latipes) 102.3 Zebrafish (Danio rerio) 122.4 Other Fish Species 13References 153 Sexual Determination, Differentiation, and Gonadal Development 193.1 Primordial Germ Cells in the Primordial (Primary) Gonad 223.1.1 Differentiation and Number of PGCs 223.1.2 Molecular Markers of PGCs 253.2 Reproductive Strategies 263.3 Differentiation of the Primordial Gonad into Ovary or Testis 273.4 Gonadal Duct Formation 323.5 Endocrinology: Influence on Gonadogenesis 403.6 Critical Period of Sexual Differentiation in Developing Fish 453.7 Bi-Potentiality of Germ Cells in Adult Fish 47References 514 Female Gonad Anatomy and Morphology 664.1 Gonadogenesis: Ovary 664.1.1 Location and Gross Organization 664.1.2 Anatomy of the Ovary 674.2 Hypothalamic–Pituitary–Ovarian Axis 694.3 Cellular Structure of the Ovary 694.3.1 Germ Cells (Oogenesis) 694.3.2 Female Somatic Supportive Tissue 784.3.2.1 Theca Cells 784.3.2.2 Granulosa Cells 81References 815 Male Gonad Anatomy and Morphology 885.1 Gonadogenesis: Testes 885.1.1 Location and Gross Organization 885.1.2 Anatomy of the Testes 895.1.3 Germinal Epithelium 935.1.4 Male Germ Cells (Spermatogenesis) 955.1.5 Male Somatic Supportive Tissue 995.1.5.1 Sertoli Cells 995.1.5.2 Leydig Cells 1035.1.5.3 Lobule Boundary Cells 1045.1.5.4 Sex Steroid Control of Spermatogenesis 104References 1086 Endocrine-Disrupting Compounds 1156.1 Individual Effects 1176.1.1 Inhibition of Gametogenesis 1176.1.2 Necrosis and Apoptosis (Gamete and Stromal Cells) 1196.1.3 Atretic Follicles 1216.1.4 Sertoli Cell Hypertrophy and Hyperplasia 1236.1.5 Leydig Cell Hypertrophy and Hyperplasia 1296.1.6 Fibrosis 1296.1.7 Gonadal Duct Formation 1326.2 Effects Associated with Exposure to SpecificCompounds or Compound Classes 133Table 6.1 Overt Testicular Changes in Male Fish, 134Table 6.2 Testis–Ova in Male Fish, 148Table 6.3 Testicular Fibrosis in Male Fish, 161Table 6.4 Germ Cell Effects in Male Fish, 165Table 6.5 Somatic Cell Effects in Male Fish, 181Table 6.6 Testicular Inflammation and MacrophageInfiltration in Male Fish, 186Table 6.7 Overt Ovarian Changes in Female Fish, 187Table 6.8 Ovo–Testis in Female Fish, 195Table 6.9 Ovarian Fibrosis in Female Fish, 196Table 6.10 Germ Cell Effects in Female Fish, 197Table 6.11 Germ Cell Atresia in Female Fish, 206Table 6.12 Ovarian Inflammation and Macrophage Infiltration in Female Fish, 2146.2.1 Effects in Male Fish Associated with Exposure to Specific Compounds or Compound Classes 2156.2.1.1 Testis Architecture, Integrity, and Gross Appearance 2156.2.1.2 Testis–Ova 2156.2.1.3 Fibrosis 2166.2.1.4 Germ Cell Effects 2176.2.1.5 Somatic Cell Effects 2196.2.1.6 Testicular Cell Inflammation and Macrophage Infiltration 2196.2.2 Effects in Female Fish Associated with Exposure to Specific Compounds or Compound Classes 2206.2.2.1 Ovary Architecture, Integrity, and Gross Appearance 2206.2.2.2 Ovo–Testis 2216.2.2.3 Fibrosis 2216.2.2.4 Germ Cell Effects 2226.2.2.5 Germ Cell Atresia 2236.2.2.6 Germ Cell Inflammation and Macrophage Infiltration 2256.3 Population Effects 2256.3.1 Fecundity and Fertility 2266.3.2 Breeding and Reproductive Behavior of Fishes 2306.3.3 Transgenerational Effects 230References 2327 Determination of Effects of Exogenous Hormones and Endocrine-like Active Compounds 2477.1 Histological Processing: Microdissection versus Whole-Fish Sectioning 2477.2 Optimal Tissue Preparation and Histological Techniques 2507.3 Plane of Gonad Sectioning for Optimal Organ Representation 251References 2528 Evaluation of Effects in Fish Gonads 2548.1 Qualitative (Semiquantitative) versus Quantitative Evaluation 2548.2 Gonadal Staging in the Testis 2558.2.1 Qualitative Staging in the Testis 2558.2.2 Quantitative Staging in the Testis 2578.3 Gonadal Staging in the Ovary 2628.3.1 Qualitative Staging in the Ovary 2628.3.2 Quantitative Staging in the Ovary 2648.4 Qualitative Assessment of Histopathological Changes 265References 2679 Experimental Design and Statistics 2709.1 Basic Considerations in Experimental Design 2709.1.1 Defining the Objectives 2719.1.2 Asking Precise Questions 2719.1.3 The Principal Question: Is There an Effect or Is There Not? 2729.1.4 Basic Mechanistic Research, Risk Assessment, and the NOEC, LOEC, and ECx Question 2729.2 Variables to be Determined and Their Inherent Biological and Mathematical Characteristics 2749.2.1 Biological Characteristics 2749.2.2 Mathematical Characteristics 2789.3 Prerequisite Statistical Concepts 2829.3.1 Sample Independence: The Experimental Unit 2829.3.2 One- or Two-Sided Testing 2839.3.3 Observable Effect Size: Minimum Observable Effect 2839.3.4 Power (1  b) to Detect an Effect 2849.3.5 Replication and Pseudoreplication 2869.3.6 Minimum Significant Difference 2879.4 Statistical Tests and Testing Situations Encountered Routinely 2889.4.1 Significance Level of Multiple Comparisons: Variable-wise Significance 2889.4.2 Significance Level for Many Different Comparisons: Simultaneous Significance 2889.4.3 Multivariate Tests 2899.4.4 Parametric Versus Nonparametric Testing 289References 28910 Conclusions 295References 297Appendix: Fish Preparation and Microdissection of Organs 301A.1 Fish Preparation 301A.2 Microdissection of Organs 302A.3 Tissue Fixation 303A.3.1 Neutral Buffered Formalin 304A.3.2 Bouin’s Fixative 304A.3.3 Lillie’s Fixative 305A.3.4 Davidson’s Fixative 305A.3.5 Dietrich’s Fixative 306A.3.6 Glutaraldehyde 306A.4 Embedding 307A.4.1 Paraffin 307A.4.2 Glycolmethacrylate (GMA) 309A.5 Tissue Sectioning 310A.6 Sample Mounting 311A.7 Tissue Slide Staining 312A.7.1 Hematoxylin and Eosin 312A.7.2 Masson’s Trichrome Stain 314A.8 Final Processing 315A.9 Examples 316A.10 Summary 317References 318Index 319

Daniel R. Dietrich, PhD, is a Professor and Head of the Human and Environmental Toxicology Group, Faculty of Sciences of the University of Konstanz, Konstanz, Germany. Heiko O. Krieger, MSc, is a scientific research assistant and PhD student in the Human and Environmental Toxicology Group, Faculty of Sciences of the University of Konstanz, Konstanz, Germany.

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• Preface xiAcknowledgments xiiiContributing Authors xv1 Introduction 1References 32 Fish Species of Interest 72.1 Fathead Minnow (Pimephales promelas) 82.2 Medaka (Oryzias latipes) 102.3 Zebrafish (Danio rerio) 122.4 Other Fish Species 13References 153 Sexual Determination, Differentiation, and Gonadal Development 193.1 Primordial Germ Cells in the Primordial (Primary) Gonad 223.1.1 Differentiation and Number of PGCs 223.1.2 Molecular Markers of PGCs 253.2 Reproductive Strategies 263.3 Differentiation of the Primordial Gonad into Ovary or Testis 273.4 Gonadal Duct Formation 323.5 Endocrinology: Influence on Gonadogenesis 403.6 Critical Period of Sexual Differentiation in Developing Fish 453.7 Bi-Potentiality of Germ Cells in Adult Fish 47References 514 Female Gonad Anatomy and Morphology 664.1 Gonadogenesis: Ovary 664.1.1 Location and Gross Organization 664.1.2 Anatomy of the Ovary 674.2 Hypothalamic–Pituitary–Ovarian Axis 694.3 Cellular Structure of the Ovary 694.3.1 Germ Cells (Oogenesis) 694.3.2 Female Somatic Supportive Tissue 784.3.2.1 Theca Cells 784.3.2.2 Granulosa Cells 81References 815 Male Gonad Anatomy and Morphology 885.1 Gonadogenesis: Testes 885.1.1 Location and Gross Organization 885.1.2 Anatomy of the Testes 895.1.3 Germinal Epithelium 935.1.4 Male Germ Cells (Spermatogenesis) 955.1.5 Male Somatic Supportive Tissue 995.1.5.1 Sertoli Cells 995.1.5.2 Leydig Cells 1035.1.5.3 Lobule Boundary Cells 1045.1.5.4 Sex Steroid Control of Spermatogenesis 104References 1086 Endocrine-Disrupting Compounds 1156.1 Individual Effects 1176.1.1 Inhibition of Gametogenesis 1176.1.2 Necrosis and Apoptosis (Gamete and Stromal Cells) 1196.1.3 Atretic Follicles 1216.1.4 Sertoli Cell Hypertrophy and Hyperplasia 1236.1.5 Leydig Cell Hypertrophy and Hyperplasia 1296.1.6 Fibrosis 1296.1.7 Gonadal Duct Formation 1326.2 Effects Associated with Exposure to SpecificCompounds or Compound Classes 133Table 6.1 Overt Testicular Changes in Male Fish, 134Table 6.2 Testis–Ova in Male Fish, 148Table 6.3 Testicular Fibrosis in Male Fish, 161Table 6.4 Germ Cell Effects in Male Fish, 165Table 6.5 Somatic Cell Effects in Male Fish, 181Table 6.6 Testicular Inflammation and MacrophageInfiltration in Male Fish, 186Table 6.7 Overt Ovarian Changes in Female Fish, 187Table 6.8 Ovo–Testis in Female Fish, 195Table 6.9 Ovarian Fibrosis in Female Fish, 196Table 6.10 Germ Cell Effects in Female Fish, 197Table 6.11 Germ Cell Atresia in Female Fish, 206Table 6.12 Ovarian Inflammation and Macrophage Infiltration in Female Fish, 2146.2.1 Effects in Male Fish Associated with Exposure to Specific Compounds or Compound Classes 2156.2.1.1 Testis Architecture, Integrity, and Gross Appearance 2156.2.1.2 Testis–Ova 2156.2.1.3 Fibrosis 2166.2.1.4 Germ Cell Effects 2176.2.1.5 Somatic Cell Effects 2196.2.1.6 Testicular Cell Inflammation and Macrophage Infiltration 2196.2.2 Effects in Female Fish Associated with Exposure to Specific Compounds or Compound Classes 2206.2.2.1 Ovary Architecture, Integrity, and Gross Appearance 2206.2.2.2 Ovo–Testis 2216.2.2.3 Fibrosis 2216.2.2.4 Germ Cell Effects 2226.2.2.5 Germ Cell Atresia 2236.2.2.6 Germ Cell Inflammation and Macrophage Infiltration 2256.3 Population Effects 2256.3.1 Fecundity and Fertility 2266.3.2 Breeding and Reproductive Behavior of Fishes 2306.3.3 Transgenerational Effects 230References 2327 Determination of Effects of Exogenous Hormones and Endocrine-like Active Compounds 2477.1 Histological Processing: Microdissection versus Whole-Fish Sectioning 2477.2 Optimal Tissue Preparation and Histological Techniques 2507.3 Plane of Gonad Sectioning for Optimal Organ Representation 251References 2528 Evaluation of Effects in Fish Gonads 2548.1 Qualitative (Semiquantitative) versus Quantitative Evaluation 2548.2 Gonadal Staging in the Testis 2558.2.1 Qualitative Staging in the Testis 2558.2.2 Quantitative Staging in the Testis 2578.3 Gonadal Staging in the Ovary 2628.3.1 Qualitative Staging in the Ovary 2628.3.2 Quantitative Staging in the Ovary 2648.4 Qualitative Assessment of Histopathological Changes 265References 2679 Experimental Design and Statistics 2709.1 Basic Considerations in Experimental Design 2709.1.1 Defining the Objectives 2719.1.2 Asking Precise Questions 2719.1.3 The Principal Question: Is There an Effect or Is There Not? 2729.1.4 Basic Mechanistic Research, Risk Assessment, and the NOEC, LOEC, and ECx Question 2729.2 Variables to be Determined and Their Inherent Biological and Mathematical Characteristics 2749.2.1 Biological Characteristics 2749.2.2 Mathematical Characteristics 2789.3 Prerequisite Statistical Concepts 2829.3.1 Sample Independence: The Experimental Unit 2829.3.2 One- or Two-Sided Testing 2839.3.3 Observable Effect Size: Minimum Observable Effect 2839.3.4 Power (1  b) to Detect an Effect 2849.3.5 Replication and Pseudoreplication 2869.3.6 Minimum Significant Difference 2879.4 Statistical Tests and Testing Situations Encountered Routinely 2889.4.1 Significance Level of Multiple Comparisons: Variable-wise Significance 2889.4.2 Significance Level for Many Different Comparisons: Simultaneous Significance 2889.4.3 Multivariate Tests 2899.4.4 Parametric Versus Nonparametric Testing 289References 28910 Conclusions 295References 297Appendix: Fish Preparation and Microdissection of Organs 301A.1 Fish Preparation 301A.2 Microdissection of Organs 302A.3 Tissue Fixation 303A.3.1 Neutral Buffered Formalin 304A.3.2 Bouin’s Fixative 304A.3.3 Lillie’s Fixative 305A.3.4 Davidson’s Fixative 305A.3.5 Dietrich’s Fixative 306A.3.6 Glutaraldehyde 306A.4 Embedding 307A.4.1 Paraffin 307A.4.2 Glycolmethacrylate (GMA) 309A.5 Tissue Sectioning 310A.6 Sample Mounting 311A.7 Tissue Slide Staining 312A.7.1 Hematoxylin and Eosin 312A.7.2 Masson’s Trichrome Stain 314A.8 Final Processing 315A.9 Examples 316A.10 Summary 317References 318Index 319