Histological Analysis of Endocrine Disruptive Effects in Small Laboratory Fish

381 Pages·2009·61.584 MB·English

by  Daniel R. Dietrich, Heiko O. Krieger(auth.)

HISTOLOGICAL
ANALYSIS OF
ENDOCRINE DISRUPTIVE
EFFECTS IN SMALL
LABORATORY FISH
Testis–ovainthetestisofanadult untreatedmedaka(Oryziaslatipes).
(Courtesyof JohnW.Fournie,U.S.EPA,GulfBreeze,Florida.)
HISTOLOGICAL
ANALYSIS OF
ENDOCRINE DISRUPTIVE
EFFECTS IN SMALL
LABORATORY FISH
Daniel R. Dietrich
Heiko O. Krieger
Copyright(cid:1)2009byJohnWiley&Sons,Inc.Allrightsreserved.
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LibraryofCongressCataloging-in-PublicationData:
Histologicalanalysisofendocrinedisruptiveeffectsinsmalllaboratoryfish/DanielR.Dietrich,
HeikoO.Krieger.
p.;cm.
Includesbibliographicalreferencesandindex.
ISBN978-0-471-76358-1(cloth)
1. Endocrinedisruptingchemicals. 2. Histology,Pathological. 3. Fishes–Endocrinology.
4. Fishes–Effectofchemicalson. I. Dietrich,DanielR. II. Krieger,HeikoO.,1972-
[DNLM:1. EndocrineDisruptors–analysis.2. Fishes–anatomy&histology. 3. Animals,
Laboratory–anatomy&histology. 4. HistologicalTechniques–veterinary. 5. Xenobiotics–adverse
effects.SH171H6732009]
RA1224.2.H572009
616.4006–dc22
2008033273
PrintedintheUnitedStatesofAmerica
10987654321
CONTENTS
Preface xi
Acknowledgments xiii
Contributing Authors xv
1 Introduction 1
References 3
2 Fish Species of Interest 7
2.1 Fathead Minnow (Pimephales promelas) 8
2.2 Medaka (Oryzias latipes) 10
2.3 Zebrafish (Danio rerio) 12
2.4 Other Fish Species 13
References 15
3 Sexual Determination, Differentiation, and
Gonadal Development 19
3.1 Primordial Germ Cells in the Primordial
(Primary) Gonad 22
3.1.1 Differentiation and Number of PGCs 22
3.1.2 Molecular Markers of PGCs 25
3.2 Reproductive Strategies 26
3.3 Differentiation of the Primordial Gonad into
Ovary or Testis 27
3.4 Gonadal Duct Formation 32
3.5 Endocrinology: Influence on Gonadogenesis 40
3.6 Critical Period of Sexual Differentiation in Developing Fish 45
3.7 Bi-Potentiality of Germ Cells in Adult Fish 47
References 51
4 Female Gonad Anatomy and Morphology 66
4.1 Gonadogenesis: Ovary 66
4.1.1 Location and Gross Organization 66
4.1.2 Anatomy of the Ovary 67
v
vi CONTENTS
4.2 Hypothalamic–Pituitary–Ovarian Axis 69
4.3 Cellular Structure of the Ovary 69
4.3.1 Germ Cells (Oogenesis) 69
4.3.2 Female Somatic Supportive Tissue 78
4.3.2.1 Theca Cells 78
4.3.2.2 Granulosa Cells 81
References 81
5 Male Gonad Anatomy and Morphology 88
5.1 Gonadogenesis: Testes 88
5.1.1 Location and Gross Organization 88
5.1.2 Anatomy of the Testes 89
5.1.3 Germinal Epithelium 93
5.1.4 Male Germ Cells (Spermatogenesis) 95
5.1.5 Male Somatic Supportive Tissue 99
5.1.5.1 Sertoli Cells 99
5.1.5.2 Leydig Cells 103
5.1.5.3 Lobule Boundary Cells 104
5.1.5.4 Sex Steroid Control of Spermatogenesis 104
References 108
6 Endocrine-Disrupting Compounds 115
6.1 Individual Effects 117
6.1.1 Inhibition of Gametogenesis 117
6.1.2 Necrosis and Apoptosis (Gamete and Stromal Cells) 119
6.1.3 Atretic Follicles 121
6.1.4 Sertoli Cell Hypertrophy and Hyperplasia 123
6.1.5 Leydig Cell Hypertrophy and Hyperplasia 129
6.1.6 Fibrosis 129
6.1.7 Gonadal Duct Formation 132
6.2 Effects Associated with Exposure to Specific
Compounds or Compound Classes 133
. Table 6.1 Overt Testicular Changes in Male Fish, 134
. Table 6.2 Testis–Ova in Male Fish, 148
. Table 6.3 Testicular Fibrosis in Male Fish, 161
. Table 6.4 Germ Cell Effects in Male Fish, 165
. Table 6.5 Somatic Cell Effects in Male Fish, 181
. Table 6.6 Testicular Inflammation and Macrophage
Infiltration in Male Fish, 186
. Table 6.7 Overt Ovarian Changes in Female Fish, 187
. Table 6.8 Ovo–Testis in Female Fish, 195
CONTENTS vii
. Table 6.9 Ovarian Fibrosis in Female Fish, 196
. Table 6.10 Germ Cell Effects in Female Fish, 197
. Table 6.11 Germ Cell Atresia in Female Fish, 206
. Table 6.12 Ovarian Inflammation and Macrophage
Infiltration in Female Fish, 214
6.2.1 Effects in Male Fish Associated with Exposure
to Specific Compounds or Compound Classes 215
6.2.1.1 Testis Architecture, Integrity,
and Gross Appearance 215
6.2.1.2 Testis–Ova 215
6.2.1.3 Fibrosis 216
6.2.1.4 Germ Cell Effects 217
6.2.1.5 Somatic Cell Effects 219
6.2.1.6 Testicular Cell Inflammation
and Macrophage Infiltration 219
6.2.2 Effects in Female Fish Associated with Exposure
to Specific Compounds or Compound Classes 220
6.2.2.1 Ovary Architecture, Integrity,
and Gross Appearance 220
6.2.2.2 Ovo–Testis 221
6.2.2.3 Fibrosis 221
6.2.2.4 Germ Cell Effects 222
6.2.2.5 Germ Cell Atresia 223
6.2.2.6 Germ Cell Inflammation
and Macrophage Infiltration 225
6.3 Population Effects 225
6.3.1 Fecundity and Fertility 226
6.3.2 Breeding and Reproductive Behavior of Fishes 230
6.3.3 Transgenerational Effects 230
References 232
7 Determination of Effects of Exogenous Hormones
and Endocrine-like Active Compounds 247
7.1 Histological Processing: Microdissectionversus
Whole-Fish Sectioning 247
7.2 Optimal Tissue Preparation and Histological Techniques 250
7.3 Plane of Gonad Sectioning for Optimal Organ Representation 251
References 252
8 Evaluation of Effects in Fish Gonads 254
8.1 Qualitative (Semiquantitative) versus
Quantitative Evaluation 254
viii CONTENTS
8.2 Gonadal Staging in the Testis 255
8.2.1 Qualitative Staging in the Testis 255
8.2.2 Quantitative Staging in the Testis 257
8.3 Gonadal Staging in the Ovary 262
8.3.1 Qualitative Staging in the Ovary 262
8.3.2 Quantitative Staging in the Ovary 264
8.4 Qualitative Assessment of Histopathological Changes 265
References 267
9 Experimental Design and Statistics 270
9.1 Basic Considerations in Experimental Design 270
9.1.1 Defining the Objectives 271
9.1.2 Asking Precise Questions 271
9.1.3 The Principal Question: Is There an Effect
or Is There Not? 272
9.1.4 Basic Mechanistic Research, Risk Assessment,
and the NOEC, LOEC, and ECx Question 272
9.2 Variables to be Determined and Their Inherent Biological
and Mathematical Characteristics 274
9.2.1 Biological Characteristics 274
9.2.2 Mathematical Characteristics 278
9.3 Prerequisite Statistical Concepts 282
9.3.1 Sample Independence: The Experimental Unit 282
9.3.2 One- or Two-Sided Testing 283
9.3.3 Observable Effect Size: Minimum
Observable Effect 283
9.3.4 Power (1(cid:1)b) to Detect an Effect 284
9.3.5 Replication and Pseudoreplication 286
9.3.6 Minimum Significant Difference 287
9.4 Statistical Tests and Testing Situations
Encountered Routinely 288
9.4.1 Significance Level of Multiple Comparisons:
Variable-wise Significance 288
9.4.2 Significance Level for Many Different
Comparisons: Simultaneous Significance 288
9.4.3 Multivariate Tests 289
9.4.4 Parametric Versus Nonparametric Testing 289
References 289
10 Conclusions 295
References 297
CONTENTS ix
Appendix: Fish Preparation and Microdissection
of Organs 301
A.1 Fish Preparation 301
A.2 Microdissection of Organs 302
A.3 Tissue Fixation 303
A.3.1 Neutral Buffered Formalin 304
A.3.2 Bouin’s Fixative 304
A.3.3 Lillie’s Fixative 305
A.3.4 Davidson’s Fixative 305
A.3.5 Dietrich’s Fixative 306
A.3.6 Glutaraldehyde 306
A.4 Embedding 307
A.4.1 Paraffin 307
A.4.2 Glycolmethacrylate (GMA) 309
A.5 Tissue Sectioning 310
A.6 Sample Mounting 311
A.7 Tissue Slide Staining 312
A.7.1 Hematoxylin and Eosin 312
A.7.2 Masson’s Trichrome Stain 314
A.8 Final Processing 315
A.9 Examples 316
A.10 Summary 317
References 318
Index 319
PREFACE
Inview of the ever-expanding literature on xenobiotic [endocrine-disrupting com-
pounds(EDCs)]-inducedendocrinedisruptioninfishpopulationsoneis:
1. Easily overwhelmedand thus prone tolosing focus.
2. Oftenunsurewhethertheparametersandchangesreportedintheliteratureare
really important or evenrelevant for the task(s) definedat the outset.
3. Moreandmoreleftwiththeimpressionthatmanyofthedeleteriouspopulation
effects reportedare not asubiquitousas reported butrather,represent avery
restricted local issue.
Inviewoftheseconcernslatterandthefactthatmoststudiesemployfishgonadal
histopathologyintheassessmentofcompound-inducedendocrine-disruptiveeffects,
theEuropeanChemicalIndustryCouncil–LongRangeInitiative(CEFIC-LRI)spon-
soredalaboratoryfishgonadhistopathologyworkshop.Thisworkshopwasorganized
bytheDepartmentofHumanandEnvironmentalToxicology,UniversityofKonstanz,
Germany,andthecompanyIPMC-TMCGmbHinSwitzerland,andwascarriedoutin
Hannover, Germany in February 2004 with the participation of the leading fish
histopathologists in Europe and the United States: Helmut Segner (University of
Bern,Switzerland),LeovanderVen(RIVM,TheNetherlands),JeffWolf(EPLInc.,
Sterling,Virginia),JohnFournie(U.S.EnvironmentalProtectionAgency),andDaniel
Dietrich (University of Konstanz, Germany), as well as with statistical support by
NellyvanderHoeven,EcoStat,Leiden,Netherlands.
The workshop clearly identified several issues that appeared to hamper under-
standingofexperimentalresultswithEDCsinfish:
. Limitedunderstandingofnormalphysiologyandreproductionofthefishspecies
used
. Variant terminology indescribingeffects
. Insecurities as to the relevance of the parameters determined for evaluating
EDC-mediated effects
. Limited understandingof the natural variation of the parametersdetermined
. Flaws in the actual technical determination of parameters (variant limits of
detectionorquantification)
xi