Structural and numerical aberrations of germ cell chromosomes are major causes of human disease, fetal death, and sterility and may be inducible by chemicals and environmental agents. Experiments proposed in this application are part of a long-term objective to develop an inexpensive in vitro assay system to measure the ability of chemicals to induce chromosomal abnormalities in vertebrate spermatogenic cells. The first specific aim is to evaluate the sensitivity, reproducibility, and validity of in vitro cytogenetic assays for chemically induced meiotic nondisjunction and structural aberrations in cultured spermatocytes from the frog Xenopus laevis. Spermatocytes from premeiotic S to metaphase I will be isolted, exposed to a variety of chemical concentrations, and cultured 26 days. During the culture period, samples will be removed and analyzed cytogenetically for chromatid exchanges, breaks, and fragments (diakinesis and metaphase II) or aneuploid frequency (metaphase II). Sixteen chemicals will be tested, including direct action S-independent and S-dependent clastogens, non-clastogens and chemicals which induce mitotic nondisjunction. The same chemicals will also be tested in the second specific aim by a rapid spermatid micronucleus assay for meiotic chromosome malsegregation resulting from fragmentation and nondisjunction induced by chemicals in cultured spermatocytes. The third specific aim is to develop and evaluate in vitro unscheduled DNA synthesis (UDS) assays for chemical induction of DNA repair in pachytene spermatocytes and acrosomal spermatids. Purified spermatocytes and spermatids will be exposed in culture to various chemical concentrations and 3H-labelled thymidine for 4 hrs and then assayed for DNA repair synthesis by liquid scintillation counting and autoradiography. In the fourth specific aim, the cytogenetics, micronuclear, and UDS assays will be conducted using 4 genotoxic chemicals which require metabolic activation to mutagenic form. The assays will be conducted with and without rat liver S9 microsomal activating systems. The final goal is to develop co-cultures between Sertoli cells and spermatogonia or late spermatids which will promote the in vitro differentiation of the germ cells and ultimately allow in vitro chemical genotoxicity testing at all spermatogenic stages.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Toxicology Study Section (TOX)
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Fordham University
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Risley, M S; Morse-Gaudio, M (1992) Comparative aspects of spermatogenic cell metabolism and Sertoli cell function in Xenopus laevis and mammals. J Exp Zool 261:185-93
Risley, M S; Tan, I P; Roy, C et al. (1992) Cell-, age- and stage-dependent distribution of connexin43 gap junctions in testes. J Cell Sci 103 ( Pt 1):81-96
Risley, M S; Pohorenec, G M (1991) Micronuclei and chromosome aberrations in Xenopus laevis spermatocytes and spermatids exposed to adriamycin and colcemid. Mutat Res 247:29-38
Risley, M S (1990) Support of Xenopus laevis spermatogenesis in vitro by different energy substrates. Biol Reprod 42:511-22
Risley, M S; Saccomanno, C F; Pohorenec, G M (1990) An improved method for cytogenetic analysis of meiotic aneuploidy in rodent and frog spermatocytes. Mutat Res 234:361-8
Li, L Y; Seddon, A P; Meister, A et al. (1989) Spermatogenic cell-somatic cell interactions are required for maintenance of spermatogenic cell glutathione. Biol Reprod 40:317-31
Risley, M S; Miller, A; Bumcrot, D A (1988) In vitro analysis of germ cell genotoxicity in testis explant cultures: spermatid micronucleus assays. Mutat Res 203:125-33
Risley, M S; Miller, A; Bumcrot, D A (1987) In vitro maintenance of spermatogenesis in Xenopus laevis testis explants cultured in serum-free media. Biol Reprod 36:985-97
Risley, M S; Einheber, S; Bumcrot, D A (1986) Changes in DNA topology during spermatogenesis. Chromosoma 94:217-27