A Practical Approach, Second Edition=Ronald D. Ho.pdf

136 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONgenes was elevated significantly above control in embryos sired by cyclophosphamide-treatedmales; a peak in the expression of many of these genes was not observed until the eight-cell stagein control embryos. 111,113,114 Thus, paternal drug exposure temporally and spatially dysregulated ratzygotic gene activation, altering the developmental clock.2. Sperm Genetic IntegrityGenetic damage of the male germ cell is of major concern; such damage may be transmitted tothe offspring and lead to abnormal progeny outcome, not only in the F 1 generation but also insubsequent generations. Even fairly severely DNA-damaged sperm may be capable of fertilization.Moreover, some germ line alterations appear to cause phenotypic malformations. Measurementsof genetic damage to the male germ cell include dominant lethal and specific locus mutation testsas well as cytogenetic, fluorescent in situ hybridization (FISH), and polymerase chain reaction(PCR) based methods for the detection of DNA damage or mutations.Nonmammalian test systems have been used extensively in screening banks of drugs andchemicals for mutagenicity. These tests have been invaluable in selecting chemicals for further invivo genetic mutagenicity testing. One example of such a system involves the Japanese medaka.Use of arbitrarily primed PCR and fingerprinting in fish treated with γ-irradiation allows changesin the genomic DNA of individual progeny to be detected as bands lost or gained. 115 By takingadvantage of suitable reporter genes, one can use male germ cells of Drosophila to detect a spectrumof genetic damage, ranging from recessive lethal (or visible) mutations, deletions, reciprocaltranslocations, chromosome loss, or dominant lethal mutations to aneuploidy. 116A number of in vivo animal tests rely heavily on dominant characteristics in inbred mice.Dominant lethal and specific locus mutation tests are examples of in vivo animal tests that havebeen used extensively to identify the chemicals that are capable of mutating germ cells. 65,117–119 Inthese tests, male rodents are treated either acutely or chronically with the chemical to be investigatedand mated to control females. For the dominant lethal test, the outcome measured is embryolethality,usually postimplantation. Thus, the spermatozoa are capable of fertilizing an egg, but the conceptusfails to develop normally, either at the time of implantation or shortly thereafter. The specific locusmutation test uses mice with mutations in a number of loci coding for “visible” features, such ascoat color, and evaluates the ability of the chemical in question to cause a mutation in the malegerm cell at these loci. This approach has been very valuable in detecting a number of mammaliangerm cell mutagens. Both of these approaches use progeny outcome to measure the effects of thedrug on the male germ cell. More recently, transgenic mice have been used as a model with whichto study gene mutations during different phases of spermatogenesis. 120,121 The results of specific-locusmutation studies have suggested that exposure of spermatogonia to chemicals or radiation yields fewlarge lesions, while large lesions are common after exposure of postspermatogonial germ cells.At the chromosome level, it may be possible to detect bulky deletions, aneuploidy, or chromosomalduplications by use of cytogenetic approaches. 122 The inherent difficulty in cytogeneticanalysis of spermatozoa has been the lack of mitosis and hence of chromosomal structure. It wasonly by fertilizing denuded hamster eggs with the sperm in question that chromosomal structuresin the male pronucleus could be analyzed. This approach was used to identify the effects of age,x-irradiation, and drugs on the chromosomal banding pattern of human sperm. 123 More recently,FISH was developed for the analysis of aneuploidy in the sperm genome. 124 This method involvesthe staining of specific chromosomal regions with fluorescent-labeled complementary DNAsequences, allowing the identification of sperm with chromosomal abnormalities, such as trisomiesor aneuploidies. FISH has been used extensively to study human sperm and was adapted for therat by Wyrobek’s group to apply this powerful tool to toxicology. 125Other tests that have detected DNA damage in male germ cells include the unscheduled DNAsynthesis assay, the DNA alkaline elution assay, the single cell gel electrophoresis (SCGE or comet)assay, and the sperm chromatin structure assay (SCSA TM ). In the unscheduled DNA synthesis assay,© 2006 by Taylor & Francis Group, LLC