Fixation of Testes and Eyes Using a Modified Davidson's Fluid: Comparison with Bouin's Fluid and Conventional Davidson's Fluid

Abstract

Most recent revisions of regulatory guidelines for testing effects of chemicals on reproduction recommend Bouin's fluid (BF) or a “comparable fixative” instead of formalin to preserve the morphologic detail of testes for histopathological evaluation. However, picric acid in BF is a health and safety hazard, as well as a laboratory waste disposal problem. Furthermore, use of BF is labor intensive, requiring multiple alcohol rinses to remove picric acid for optimum preservation and immunohistochemical (IHC) detection of testicular antigens that may potentially be used to identify and quantify cells and functional proteins with critical roles in spermatogenesis. Recently a modified Davidson's fluid (mDF) has been reported as an alternative to BF to fix testes for routine histopathological examination. This study compared the overall histomorphologic clarity and the immunoand histochemical staining of testicular specimens fixed in BF and mDF. Additionally, because conventional Davidson's fixative (DF) is used routinely for optimum fixation of eyes, preservation of ocular histomorphology by DF and mDF was compared. mDF resulted in noticeably less shrinkage of the seminiferous tubules and superior overall morphologic detail compared to BF. Unlike DF, the mDF also supported excellent staining of acrosomes with periodic acid-Schiff (PAS) reagent when staging of spermatogenesis was required. IHC detection of androgen receptor and PCNA (to directly and indirectly identify Sertoli cells) as well as protein gene product 9.5 (to label spermatogonia) was superior in mDF compared to BF-fixed specimens. For histopathological examination of the eye, apposition and preservation of rods and cones, and nuclear layers of the retina were slightly inferior with mDF compared to DF. This paper has demonstrated that mDF provides comparable, and in many respects superior preservation of the testes to that of BF, both for IHC staining and for detailed histopathological examination. It also provides an acceptable fixative for eyes, although the quality of cellular preservation is inferior to that of DF.

Keywords

Immunohistochemistry histochemistry fixation histomorphology methods spermatogonia Sertoli cell antigenic markers Davidson's fixative Bouin's fixative.

References

Chapin RE , Ross MD , Lamb JC ( 1984). Immersion fixation methods for glycol methacrylate-embedded testes. Toxicol Pathol 12: 221—227.

Creasy DM (2001). Pathogenesis of male reproductive toxicity. Toxicol Pathol 29: 64—76.

Creasy DM , Jonassen H. (1999). Histological fixation of testes: Use of Davidson's fluid as an alternative to Bouin's fluid. Vet Pathol 36: 518.

Fraenkel-Conrat H. , Cooper M. , Olcott HS (1945). Reactions of formaldehyde with proteins. J Am Chem Soc 67: 950—954.

Hess RA , Moore BJ (1993). Histological methods for evaluation of the testes . In: Methods in Toxicology, Vol 3, Part A. Male Reproductive Toxicology. Chapin RE , Heindel JJ (eds). Academic Press Inc, San Diego, CA, pp 86—94.

ICH (International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use) Harmonized Tripartite Guidelines; ICH S5A: Reproductive Toxicology: Detection of Toxicity to Reproduction for Medicinal Products. Federal Register 59: 48746—48752, 1994 and ICH S5B: Reproductive Toxicology: Toxicity to Male Fertility. Federal Register 61: 15360, 1996.

Kiernan JA (1999). Fixation. In: Histological and Histochemical Methods: Theory and Practice, 3rd, ed, Butterworth-Heinemann, Oxford, pp 11—35.

McManus JFA (1948). Histological and histochemical uses of periodic acid. Stain Technol 23: 99—108.

Moore KL , Barr ML ( 1954). Nuclear morphology, according to sex, in human tissues . Acta Anat 21: 197—208.

10.

Moore KL , Graham MA , Barr ML ( 1953). The detection of chromosomal sex in hermaphrodites from a skin biopsy. Surg Gynecol Obstet 96: 641— 648.

11.

Organization for Economic Co-operation and Development (OECD) (1995). Reproduction/developmental toxicity screening test. In: OECD Guideline for Testing of Chemicals, No. 421 (Adopted July 27th 1995), OECD Paris, pp 1—10.

12.

Organization for Economic Co-operation and Development (OECD) (2001). Proposal for updating Guideline 416: Two generation reproduction toxicity study. In: OECD Guideline for Testing of Chemicals, No. 416 (Adopted January 22nd, 2001) OECD, Paris, pp 1—13.

13.

Regadera J. , Martinez-Garcia F. , Gozalez-Permato P. , Serrano A. , Nistal M. , Suarez-Quian C. (2001). Androgen receptor expression in Sertoli cells as a function of seminiferous tubule maturation in the human cryptorchid testis . J Clin Endocrinol Metab 86: 413—421.

14.

Shan LX , Bardin CW , Hardy MP (1997). Immunohistochemical analysis of androgen effects on androgen receptor expression in developing Leydig and Sertoli cells. Endocrinology 138: 1259—1266.

15.

Shan LX , Zhu LJ , Bardin CW , Hardy MP (1995). Quantitative analysis of androgen receptor messenger ribonucleic acid in developing Leydig cells and Sertoli cells by in situ hybridization . Endocrinology 136: 3856— 3862.

16.

Tokunaga Y. , Fujimiya M. , Maeda T. (1997). The presence of PGP 9.5 in the spermatogonia is associated with the spermatogenesis of the Japanese macaque (Macaca fuscata) . In: Recent Advances in Microscopy of Cells, Tissues and Organs , Motta PM (ed). Antonio Delfino Editore, Rome, pp 585—590.

17.

US Environmental Protection Agency. 1998 Health Effects Test Guidelines. OPPTS 870.3800. Reproduction and Fertility Effects. Available at 〈 http//www.epa.gov/OPPTSharmonized/870healtheffectstestguidelines/series/870_3800.pdf 〉.

18.

Wilson Pog , Barber PC , Hamid QA , Power BF , Dhillon AP , Rode J. , Day INM , Thompson RJ , Polak JM (1988). The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies . Br J Exp Pathol 69: 91—104.