Clarifying much of the inconsistency currently encountered in the scientific literature, Histology and Histopathological Analysis of Endocrine Disruptive Effects on Small Laboratory Fishhelps to standardize the interpretation of results from aquatic bioassays and field observations by describing basic biological function and contrasting normal histology with histopathological conditions in a variety of fish species. The authors represent the leaders of small fish gonadal histopathology, and they incorporate views and ideas from other experts.
1. Introduction.
2. Fish species of interest.
2.1. Fathead minnow (Pimephales promelas).
2.2. Medaka (Oryzias latipes).
2.3. Zebrafish (Danio rerio).
2.4. Other fish species.
2.5. References.
3. Sexual determination, differentiation and gonadal development.
3.1. Primordial germ cells in the primordial (primary) gonad.
3.2. Reproductive strategies.
3.3. The differentiation of the primordial gonad into ovary or testis.
3.4. Gonadal duct formation.
3.5. Endocrinology: Influence on gonadogenesis.
3.6. Critical period of sexual differentiation in developing fish.
3.7. Bi-potentiality of germ cells in adult fish.
3.8. References.
4. Female gonad anatomy and morphology.
4.1. Gonadogenesis: Ovary.
4.1.1. Location and gross organization.
4.1.2. Anatomy of the ovary.
4.2. The hypothalamic-pituitary-ovarian axis.
4.3. Cellular structure of the ovary.
4.3.1. Germ cells (Oogenesis).
4.3.2. Female somatic supportive tissue.
4.3.2.1. Theca cells.
4.3.2.2. Granulosa cells.
4.4. References.
5. Male gonad anatomy and morphology.
5.1. Gonadogenesis: Testes.
5.1.1. Location and gross organization.
5.1.2. Anatomy of the testes.
5.1.3. The germinal epithelium.
5.1.4. Male germ cells (Spermatogenesis).
5.1.5. Male somatic supportive tissue.
5.1.5.1. Sertoli cells.
5.1.5.2. Leydig cells.
5.1.5.3. Lobule boundary cells.
5.1.5.4. Sex steroid control of spermatogenesis.
5.2. References.
6. Endocrine disrupting compounds: Review of effects of exogenous hormones and endocrine-like active compounds on the individual and the population.
6.1. Individual effects.
6.1.1. Inhibition of gametogenesis.
6.1.2. Necrosis and apoptosis (gamete / stromal cells).
6.1.3. Atretic follicles.
6.1.4. Sertoli cell hypertrophy / hyperplasia.
6.1.5. Leydig cells hypertrophy/hyperplasia.
6.1.6. Fibrosis.
6.1.7. Gonadal duct formation.
6.2. Effects in male and female fish associated with exposure to specific compounds or compound classes.
6.2.1. Effects in male fish associated with exposure to specific compounds or compound classes.
6.2.1.1. Testis architecture /integrity/gross appearance.
6.2.1.2. Testis-Ova.
6.2.1.3. Fibrosis.
6.2.1.4. Germ cell effects.
6.2.1.5. Somatic cell effects.
6.2.1.6. Testicular cell inflammation and macrophage infiltration.
6.2.2. Effects in female fish associated with exposure to specific compounds or compound classes.
6.2.2.1. Ovary architecture /integrity/gross appearance.
6.2.2.2. Ovo-testis.
6.2.2.3. Fibrosis.
6.2.2.4. Germ cell effects.
6.2.2.5. Germ cell atresia.
6.2.2.6. Germ cell inflammation and macrophage infiltration.
6.3. Population effects.
6.3.1. Fecundity and Fertility.
6.3.2. Breeding and reproductive behaviour of fishes.
6.3.3. Trans-generational effects.
6.4. References.
7. Determination of effects of exogenous hormones and endocrine-like active compounds: Histopathological processingand evaluation.
7.1. Histological processing.
7.1.1. Micro-dissection versus whole fish sectioning.
7.1.2. Optimal tissue preparation and histological techniques.
7.1.3. Plane of gonad sectioning for optimal organ representation.
7.2. References.
8. Evaluation of effects in fish gonads (germinal cells and supportive tissue).
8.1. Qualitative (semi-quantitative) versus quantitative evaluation.
8.2. Gonadal staging in the testis.
8.2.1. Qualitative staging in the testis.
8.2.2. Quantitative staging in the testis.
8.3. Gonadal staging in the ovary.
8.3.1. Qualitative staging in the ovary.
8.3.2. Quantitative staging in the ovary.
8.4. Qualitative assessment of histopathological changes.
8.5. References.
9. Experimental design and statistics.
9.1. Basic considerations in experimental design.
9.1.1. Defining the objectives.
9.1.2. Asking precise questions.
9.1.3. "Is there an effect, or is there not??" - the principal question.
9.1.4. Basic mechanistic research, risk assessment and the NOEC, LOEC and ECx question.
9.2. Variables to be determined and their inherent biological and mathematical characteristics.
9.2.1. Biological characteristics.
9.2.2. Mathematical characteristics.
9.3. Prerequisite statistical concepts.
9.3.1. Sample independence - the experimental unit.
9.3.2. One sided or two-sided testing.
9.3.3. The observable effect size - minimum observable effect (MOE).
9.3.4. The power (1-?) to detect an effect.
9.3.5. Replication and Pseudo-replication.
9.3.6. Minimum Significant Difference (MSD).
9.4. Statistical tests and testing situations routinely encountered.
9.4.1. Significance level of multiple comparisons (variable-wise significance).
9.4.2. Significance level in case of many different comparisons (simultaneous significance).
9.4.3. Multivariate tests.
9.4.4. Parametric versus non-parametric testing.
9.5. References.
10. Conclusions.
10.1. References.
10. Conclusions.
10.1. References.
11. Appendix.
11.1. Fish preparation and micro-dissection of organs.
11.1.1. Fish Preparation.
11.1.2. Micro-Dissection of Organs.
11.1.3. Tissue Fixation.
11.1.3.1. Neutral Buffered Formalin.
11.1.3.2. Bouin’s Fixative.
11.1.3.3. Lillie’s Fixative.
11.1.3.4. Davidson’s Fixative.
11.1.3.5. Dietrich’s Fixative.
11.1.3.6. Glutaraldehyde.
11.1.4. Embedding.
11.1.4.1. Paraffin.
11.1.4.2. Glycolmethacrylate (GMA).
11.1.5. Tissue Sectioning.
11.1.6. Sample Mounting.
11.1.7. Tissue Slide Staining.
11.1.7.1. Hematoxylin & Eosin (H&E).
11.1.7.2. Masson?s Trichrome Stain.
11.1.8. Final Processing.
11.2. Summary.
11.3. References.