Histopathology of the Male Reproductive System I: Techniques

Dianne M. Creasy1

1 Huntingdon Life Sciences, East Millstone, New Jersey
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 16.3
DOI:  10.1002/0471140856.tx1603s12
Online Posting Date:  August, 2002
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Abstract

Histopathology of the Male Reproductive System: Techniques (Diane Creasy, Huntingdon Life Sciences, East Millstone, New Jersey). Fixation and sampling of the male reproductive system for routine histopathological examination requires special procedures including the use of nonroutine fixatives and nonroutine stains. High‐resolution light microscopy and electron microscopy require additional specialized techniques including perfusion fixation and plastic embedding. Aspects of comparative anatomy are also important for ensuring that specific structures within the reproductive tract are sampled adequately in various species. This unit provides methodology and guidance on what structures to sample and how to fix and prepare tissues from the male reproductive tract for routine and special investigations.

     
 
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Table of Contents

  • Basic Protocol 1: Dissection, Immersion Fixation, and Paraffin Embedding of Male Reproductive‐Tract Tissues
  • Basic Protocol 2: Perfusion Fixation of the Male Rodent Reproductive Tract by Cardiac Perfusion and Resin Embedding of Tissues
  • Support Protocol 1: Removal, Trimming, and Orientation of Male Testes, Epididymides, and Accessory Sex Glands
  • Support Protocol 2: Processing and Embedding of Tissues in Epoxy Resin
  • Support Protocol 3: Processing and Embedding of Tissues in Glycol Methacrylate
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Dissection, Immersion Fixation, and Paraffin Embedding of Male Reproductive‐Tract Tissues

  Materials
  • Male rodent, dog, or primate
  • Modified Davidson's fixative (see recipe)
  • 10% neutral buffered formalin (∼4% [w/v] formaldehyde; Sigma or standard recipe, see Bancroft et al., )
  • Surgical instruments for dissection
  • Additional reagents and equipment for euthanization; dissecting the testes, epididymides, and accessory sex glands (see protocol 3); dehydration, clearing, and infiltration with paraffin wax (Bancroft et al., ); and paraffin sectioning and staining (Bancroft et al., )

Basic Protocol 2: Perfusion Fixation of the Male Rodent Reproductive Tract by Cardiac Perfusion and Resin Embedding of Tissues

  Materials
  • Krebs Ringer's solution (see recipe)
  • Karnovsky's fixative (see recipe)
  • Male rodent
  • 65 mg/ml sodium pentobarbital solution (e.g., Sleepaway; Fort Dodge Animal Health) or equivalent for anesthesia
  • 0.1 M sodium cacodylate buffer (see recipe)
  • Osmium tetroxide/potassium ferrocyanide fixative (see recipe)
  • Perfusion setup (Fig. ), including:
  •  Two 1‐liter stoppered bottles, each connected to a length of silastic tubing (∼3.0 mm internal diameter) and each vented to air
  •  Silastic tubing, ∼3.0 mm internal diameter (e.g., Masterflex L/S 16 pump tubing; Cole‐Parmer Instrument)
  •  Three‐way stopcock
  •  Variable‐speed peristaltic pump (e.g., Masterflex; Cole‐Parmer Instrument)
  •  Manometer
  •  Dosing needle: stainless steel intubation needle with bulbous end, 16 to 18G (depending on size of animal), attached to a cut‐off 1‐ml tuberculin syringe
  •  Dissection board suspended in a tray
  •  Vacuum pump connected to 1‐liter bottle trap
  • Rubber bands
  • Dissecting tools, including:
  •  Scalpel
  •  Hemostats
  •  Forceps
  • 5‐cm (2‐in) Dieffenbach serrefine (bulldog) clamp
  • Single‐edged blades
  • Additional reagents and equipment for dehydrating, infiltrating, and embedding in epoxy resin (see protocol 4); sectioning and staining for electron microscopy or dehydrating, infiltrating, and embedding in glycol methacrylate (see protocol 5); sectioning and staining for high‐resolution light microscopy

Support Protocol 1: Removal, Trimming, and Orientation of Male Testes, Epididymides, and Accessory Sex Glands

  Materials
  • Fixed tissue sample postfixed in 1% osmium tetroxide/1.25% potassium ferrocyanide (see protocol 2)
  • 30%, 50%, 70%, 85%, 95%, and 100% (v/v) ethanol
  • Propylene oxide (e.g., Electron Microscopy Sciences)
  • 1:1 propylene oxide/epoxy resin
  • Epoxy resin (e.g., Araldite 502 or 506 lufts; Electron Microscopy Sciences)
  • Mold suitable for electron microscopy blocks (e.g., Electron Microscopy Sciences)
NOTE: During processing, all steps should be carried out with agitation.

Support Protocol 2: Processing and Embedding of Tissues in Epoxy Resin

  Materials
  • Fixed tissue sample, 3 to 5 mm thick (see protocol 2)
  • 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences or see recipe), optional
  • Osmium tetroxide/potassium ferrocyanide fixative (see recipe), optional
  • 50%, 70%, 80%, 95%, and 100% (v/v) ethanol
  • JB4 glycol methacrylate embedding kit (e.g., Polysciences), including:
  •  Solution A (acrylic monomer)
  •  Catalyst (organic peroxide)
  •  Solution B (accelerator)
  • Ice bath
  • Glycol methocrylate embedding molds, available as block‐mold system that allows polymerization directly onto a metal or plastic microtome chuck (Polysciences)
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Figures

Videos

Literature Cited

Literature Cited
   Bancroft, J.D., Stevens, A., and Turner, D.R. 1990. Theory and Practice of Histological Techniques, 3rd ed. Churchill Livingston, New York.
   Chapin, R.E., Ross, M.D., and Lamb, J.C. 1984. Immersion fixation methods for glycol methacrylate‐embedded testes. Toxicol. Pathol. 12:221‐227.
   Environmental Protection Agency (EPA). 1998. Health Effects Test Guidelines, Report No. OPPTS 870.3800, Reproduction and Fertility Effects. pp. 1‐12. EPA 712‐C‐ Office of Prevention, Pesticides and Toxic Substances (OPPTS), U.S. EPA, Washington, D.C.
   Evans, H.E. 2000. Guide to the Dissection of the Dog, 5th ed. W.B. Saunders, Philadelphia.
   Feldman, D.B. and Seeley, J.C. 1988. Necropsy Guide: Rodents and the Rabbit. CRC Press, Boca Raton, Fla.
   Frederick, P.M. and Doorn, L.G. 1973. A technique for perfusion of rat testis in situ through internal spermatic arteries. J. Reprod. Fertil. 35:117‐121.
   Harleman, J.H. and Nolte, T. 1997. Testicular toxicity: Regulatory guidelines—the end of formalin fixation? Toxicol. Pathol. 25:414‐417.
   Hayat, M.A. 1989. Principles and techniques of electron microscopy: Biological applications, 3rd ed. CRC Press, Boca Raton, Fla.
   Hess, R.A. 1998. Effects of environmental toxicants on the efferent ducts, epididymis and fertility. J. Reprod. Fertil. Suppl. 53:247‐259.
   Hess, R.A. and Moore, B.J. 1993. Histological methods for evaluation of the testes. In Methods in Toxicology, Vol. 3, Part A. Male Reproductive Toxicology (R.E. Chapin and J.J. Heindel, eds.) pp. 86‐94. Academic Press, San Diego.
   International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). 1994. Tripartite Harmonized ICH Guideline S5A: Reproductive Toxicology: Detection of toxicity to reproduction for medicinal products/CPMP/ICH/386/95. Fed. Regist. 59:48746‐48752.
   International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). 1996. Tripartite Harmonized ICH Guideline S5B: Reproductive Toxicology: Male fertility studies/CPMP/ICH/136/95. Fed. Regist. 61:15360.
   Lee, C. and Holland, J.M. 1987. Anatomy, histology and ultrastructure, prostate, rat. In Monographs on Pathology of Laboratory Animals: Genital System (T.C. Jones, U. Mohr, and R.D. Hint, eds.) pp. 239‐251. Springer‐Verlag, New York.
   Organization for Economic Cooperation and Development (OECD). 1995. Reproduction/developmental toxicity screening test. In Guideline for Testing of Chemicals, No. 421 (adopted July 27, 1995) pp. 1‐10. OECD, Paris.
   Organization for Economic Cooperation and Development (OECD). 2001. Proposal for updating Guideline 416: Two generation reproduction toxicity study. In Guideline for Testing of Chemicals, No. 416 (adopted January 22, 2001) pp. 1‐13. OECD, Paris
   Russell, L. and Burguet, S. 1977. Ultrastructure of Leydig cells as revealed by secondary tissue treatment with a ferrocyanide‐osmium mixture. Tissue Cell 9:751‐766.
   Russell, L.D., Ettlin, R., Sinha Hikim, A.P., and Clegg, E.D. 1990. Tissue Preparation for Evaluation of the Testis. In Histological and Histopathological Evaluation of the Testis (L.D. Russell, R. Ettlin, A.P. Sinha Hikim, and E.D. Clegg, eds.) pp. 195‐209. Cache River Press, Clearwater, Fla.
   Sprando, R.L. 1990. Perfusion of the rat testis through the heart using heparin. In Histological and Histopathological Evaluation of the Testis (L.D. Russell, R. Ettlin, A.P. Sinha Hikim, and E.D. Clegg, eds.) pp. 277‐280. Cache River Press, Clearwater, Fla.
   Suwa, R., Nyska, A., Peckham, J.C., Hanley, J.R., Mahler, J.F., Haseman, J.K., and Maronpot, R.R. 2001. A retrospective analysis of background lesions and tissue accountability for male accessory sex organs in Fisher‐344 rats. Toxicol. Pathol. 29:467‐478.
   Yuan, Y.D., Ulrich, R.G., and Carlson, R.G. 1987. Histology and ultrastructure, glands of the ductus deferens (ampullary gland), rat. In Monographs on Pathology of Laboratory Animals: Genital System. (T.C. Jones, U. Mohr, and R.D. Hint, eds.) pp. 229‐234. Springer‐Verlag, New York.
Key References
   Hess, R.A. and Moore, B.J. 1993. See above.
  Provides a detailed comparison of the results achieved using different combinations of fixatives and embedding procedures to examine the testes. Also provides technical details for perfusion fixation and subsequent processing and embedding in resin for light and electron microscopy.
   Russell et al., 1990. See above.
  Provides various protocols for fixation, tissue processing, and staining of the testis and discusses the advantages and disadvantages of various methods.
   Sprando, R.L. 1990. See above.
  Provides detailed technical guidance on the art of perfusion fixation for the testes.
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