Performing a Testicular Spermatid Head Count

Han Seung1, Gary Wolfe1, Meredith Rocca1

1 TherImmune Research Corporation, Gaithersburg, Maryland
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 16.7
DOI:  10.1002/0471140856.tx1607s16
Online Posting Date:  August, 2003
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Abstract

procedure of counting spermatid heads using hemacytometer

Keywords: testicular spermatid head count; spermatid head count

     
 
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Table of Contents

  • Basic Protocol 1: Performing a Testicular Spermatid Head Count
  • Support Protocol 1: Calculating Testicular Spermatid Head Concentration
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Performing a Testicular Spermatid Head Count

  Materials
  • Frozen testes from mouse or rat (unit 16.3; Feldman and Seeley, ), frozen individually in scintillation vials on dry ice at −70°C or below
  • DMSO/saline solution (see recipe)
  • 0.1% (w/v) trypan blue stain (see recipe)
  • 125‐ml Erlenmeyer flasks (for rats) or 12‐ml test tubes (for mice)
  • Disposable weigh boats
  • Cyclone homogenizer with 20‐mm (for rats) or 10‐mm (for mice) rotor (VirTis)
  • Scalpel or razor blades
  • Microdissecting forceps (two pairs)
  • Homogenization vessels (50‐ to 100‐ml for rats, 20‐ml for mice; Jencons Scientific)
  • Lint‐free wipes (e.g., Kimwipes)
  • 10‐, 50‐, and 500‐µl volumetric micropipettors
  • Hemacytometer with two chambers and coverslip
  • Compound microscope with 40× objective and 10× eyepiece
  • Cell counter
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Figures

Videos

Literature Cited

   Blazak, W.F., Ernst, T.L., and Stewart, B.E. 1985. Potential indicators of reproductive toxicity: Testicular sperm production and epididymal sperm number. Transit time, and motility in fischer 344 rats. Fundam. Appl. Toxicol. 5:1097‐1103.
   Centola, G.M. 1996. Comparison of manual microscopic and computer‐assisted methods for analysis of sperm count and motility. Arch. Androl. 36:1‐7.
   Feldman, D.B. and Seeley, J.C. 1988. Necropsy Guide: Rodents and the Rabbit. CRC Press, Boca Raton, Fla.
   Ku, W.W., Chapin, R.E., Wine, R.N., and Gladen, B.C. 1993. Testicular toxicity of boric acid (BA): Relationship of dose to lesion development and recovery in the F344 rat. Reprod. Toxicol. 7:305‐319.
   Linder, R.E., Klinefelter, G.R., Strader, L.F., Suarez, J.D., and Roberts, N.L. 1997. Spermatotoxicity of dichloroacetic acid. Reprod. Toxicol. 11:681‐688.
   Oakberg, E.F. 1956. Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium. Am. J. Anat. 99:507‐516.
   Robb, G.W., Amann, R.P., and Killian, G.J. 1978. Daily sperm production and epididymal sperm reserves of pubertal and adult rats. J. Reprod. Fertil. 54:103‐107.
Key Reference
   Zenick, H., Clegg, E., Perreault, D., Klinefelter, G., and Gray, L. 1994. Assessment of male reproductive toxicity: A risk assessment approach. In Principles and Methods of Toxicology, 3rd ed. (A.W. Hayes, ed.) pp. 937‐988. Raven Press, New York.
  Useful and detailed background information.
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