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Protocol no. 21

BOVINE SPERMATOZOA CYTOTOXICITY TEST

The cytotoxic effect of test compounds on bovine spermatozoa is determined by the measurement of spermatozoa motility and velocity using videomicrography and automatic computer analysis, and ATP contents.

CONTACT

Dr Hasso Seibert Department of Toxicology Christian-Albrecht-Universitat Brunswiker Strasse 10 2300 Kiel 1 Germany Tel: Germany - 0431 597 4931 Fax: Germany - 0431 597 3558

RATIONALE

The use of ejaculated mammalian spermatozoa for studying the cytotoxic effect of chemicals may provide several advantages compared to other widely used cell types and in vitro systems. This is especially true of their swimming activity, which is dependent upon intact cellular structures and functions, and which consequently offers an endpoint for quantitative evaluation of the cytotoxic potential of chemical substances.

BASIC PROCEDURE

2ml aliquot of freshly collected bovine spermatozoa (30 x 106 cells/ml medium) is exposed to test compound for 1 hour. 4ml of this suspension is aliquoted into a prewarmed haemocytometer-like chamber (37°C) and transferred to a similarly heated microscope stage. Five independent microscopic fields are examined and videotaped for 10 seconds each. This procedure is repeated so that 10 fields are examined in all. Videoimage analysis equipment is then used to analyse the videotape produced, from which the percentage of motile spermatozoa and the mean velocity of the moving spermatozoa can be determined.

CRITICAL ASSESSMENT

An important prerequisite for the use of spermatozoa is a method for fast, objective and reproducible measurements of motion parameters. Existing methods are either subjective and inaccurate (direct microscopic observation) or too time consuming, such as cinemicrographic and photomicrographic techniques, non-automatic videomicrographic approaches, and combination of cinematography and computer image analysis. This test system, however, employs a method based on computer assisted image analysis of videomicrographic recordings of sperm samples. Videomicroscopy Videomicroscopy enables cells to be studied while still in the intact living state and before the occurrence of irreversible damage. The development of toxic events may be followed through the continuous observation of one cell sample, and potentially allows the determination of concentrations of test substances at which impairment of certain cellular parameters, such as motility, is still reversible. This may prove to be a more valid set of endpoints than those based on cell death. While videomicroscopy may seem at first sight to be a complicated procedure, it should be viewed not as one difficult technique, but as a bundle of many relatively simple techniques. If a good research microscope is available, the cost of setting up videomicroscopy need not be greater than, for example, that of obtaining a good spectrophotometer for the neutral red assay. Use of automatic computer analysis Automatic computer analysis of videomicrographic recording enables reproducible measurements of the concentration dependent effects of the test chemicals on spermatozoa motility and mean velocity. The main advantage of computer videomicrography compared to other existing objective and precise methods for the assessment of spermatozoa motion, such as photomicrographic techniques, lies in its rapidity. Use of bovine spermatozoa Sperm samples from breeding bulls offer the advantage of a very stable biological material with a high degree of motility (usually 80-95%), and viability. The use of human spermatozoa has also been studied in this test system; however, for the purposes of use in routine cytotoxicity testing the use of human spermatozoa has many disadvantages compared to bovine spermatozoa e.g. low ejaculate volume, low sperm density, low proportion of motile cells, high percentage of morphologically anomalous sperm, etc. Also, considerable problems may be encountered when trying to find human volunteers who can provide ejaculates fulfilling the criteria for "normal values" according to WHO guidelines (WHO, 1987). Thus, the authors recommend that human spermatozoa be used with regards to more specific questions concerning the possible reproductive toxic effects of chemicals. Comparison to other in vitro methods Hong et al. (1981) developed a transmembrane migration method specifically as a pharmacological test for comparing the influence of various pharmacological agents on spermatozoa motility. This test measures the proportion of spermatozoa in an aliquot of semen to move across 5mm pores of a Nucleopore membrane into buffer during a 2 hour incubation at 37°C. Though this method appears to be simple, quantitative, and reproducible, it suffers (besides possible problems concerning exposure conditions), from the disadvantage that only overall changes in spermatozoa swimming activity can be recorded and not changes in the moving pattern of individual spermatozoa. Computer videomicrography, however, offers the possibility to detect and analyse subtle effects of chemical compounds on the swimming pattern of spermatozoa. For instance, when a series of chlorophenol compounds were tested in this system, it was found that the velocity of spermatozoa started to decrease at concentrations where motility was still unaffected. The computer analysis yields additional results on the straightness of swimming trajectories or linearity (percentage of straightforward swimming spermatozoa), respectively. The results indicate that after exposure to those chlorophenols tested this motion parameter was impaired at considerably lower concentrations than velocity. It can be surmised that the application of improved image analysis systems will yield detailed and valuable data on the action of chemicals on spermatozoa motion. One possible way to judge the sensitivity of this short-term motion assay is by comparison of the test results with those from cytotoxicity tests with cultured cells. When the assay is compared to other in vitro systems such as the MIT-24 test (HeLa cells) or the neutral red assay using bluegill sunfish, BF-2 cells, it appears that the short-term bovine spermatozoa motion assay is at least as sensitive as the cited cell culture assays using an exposure time of 24 hours. However, it is obvious that detailed comparisons of the reported midpoint toxicities are very difficult because of the different cell types, endpoints, and incubation conditions. The measurement of spermatozoa motion inhibition alone gives no information about the mode of action of a chemical substance. However, it is the author's opinion that in vitro cytotoxicity tests should not only yield quantitative results about the toxic potential of chemicals but additionally at least preliminary information about cellular targets. Therefore, they recommend that other parameters e.g. cellular adenylate pool, membrane integrity and respiration rates, should also be examined.

TEST STATUS

Currently being evaluated, in the "Multicentre Evaluation of In Vitro Cytotoxicity" (MEIC) project, organised by the Scandinavian Society for Cell Toxicology, as a potential replacement alternative for acute toxicity tests. The system is also being evaluated by Dr Seibert and co-workers for inclusion in a battery of in vitro tests, which include the following: a) Primary cultured hepatocytes (Aschmann et al., 1989) b) Primary cultured skeletal muscle cells (Tesseraux et al., 1987; Gülden & Burghoff, 1990) c) Balb 3T3-cells d) Co-culture of microcarrier-attached rat hepatocytes with different target cells (Voss & Seibert, submitted).

OTHER ORGANISATIONS USING THE TEST

None at present.

CHEMICALS TESTED

Antimycin A 2,4-Dinitrophenol Ethanol Hexachlorophene Methylmercury Triton-X-100 Chlorophenols: Pentachlorophenol (PCP, purity > 99%) 2,3,4,5-Tetrachlorophenol (2,3,4,5-TCP) 2,4,5-Trichlorophenol (2,4,5-TCP) 2,4-Dichlorophenol (2,4-DCP) 4-Monochlorophenol (4-MCP)

REFERENCES

  1. Aschmann, C., Stork, T. & Wassermann, O. (1989) Short-term effects of chlorophenols on the function and viability of primary cultured rat hepatocytes. Arch. Toxicol., 63, 121-126. Babich, H. & Borenfreund, E. (1987) in vitro cytotoxicity of organic pollutants to bluegill sunfish (BF-2) cells. Environ. Res., 42, 229-237.
  2. Ekwall, B., Selling, J. & Johnels, D. (1987) Toxicity of chlorophenols to HeLA cells as measured in the MIT-24 system. ATLA, 14, 178-181
  3. Gülden, M. & Burghoff, C. (1990) Effects of membrane directed neurotoxicants on the contractile activity of cultured skeletal muscle cells. ATLA, 17, 215-217.
  4. Hong, C.Y., Chaput de Saintonge, D.M. & Turner, P. (1981) A simple method to measure drug effects on human spermatozoa motility. Br. J. Clin. Pharmacol., 11, 385-387. Kolossa, M. & Seibert, H. (1990) A chemically "defined" diluent for cryopreservation of bovine spermatozoa. Andrologia, 22, 445-454.
  5. Seibert, H. (1988) Messung der Bewegungsaktivität der Spermatozen von Mensch und Rind mit Hilfe von Videomikrographie und Computerbildanalyse. Fertilität, 4, 215-218.
  6. Seibert, H., Kolossa, M. & Wasserman, O. (1989) Bovine spermatozoa as an in vitro model for studies on the cytotoxicity of chemicals: Effects of chlorophenols. Cell Biology and Toxicology, 5, 315-330. Seibert, H. & Gosch, U. (1990) A short-term bovine sperm cell assay for the evaluation of the in vitro cytotoxicity of chemicals. ATLA, 17, 228-232.
  7. Tessereaux, I., Gülden, M., & Wassermann, O. (1987) Cultured myotubes from skeletal muscle of adult rats. Characterization and action of Anemonia sulcata toxin II. Naunyn Schmiedeberg's Arch. Pharmacol., 336, 232-239.
  8. Voss, J.-U. & Seibert, H. (1991) Microcarrier-attached rat hepatocytes as a xenobiotic-metabolizing system in cocultures. Cell. Biol. Toxicol. (Submitted) WHO (1987) WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. Cambridge University Press, Cambridge.
  9. Videomicrographic techniques Weiss, D.G. (1989) Videomicroscopic measurements in living cells: dynamic determination of multiple endpoints for in vitro toxicology. Journal of Molecular Toxicology, 1, 465-488.

IP-21© 1991