Numerical Chromosomal Abnormalities in Rat Epididymal Spermatozoa following Chronic Cyclophosphamide Exposure Short Title: Sperm Aneuploidy after Cyclophosphamide Exposure Key Words: meiosis, sperm, spermatogenesis, testis, toxicology

Chronic low dose treatment of male rats with cyclophosphamide, a chemotherapeutic agent, is known to affect progeny outcome adversely in a dose and time-specific manner, resulting in increased preand post-implantation loss as well as malformations. There is concern for the genetic quality of the mature gametes exposed to cyclophosphamide during mitosis and meiosis. The goal of our study was to determine the effect of chronic cyclophosphamide treatment during spermatogenesis on the frequency of numerical chromosomal anomalies in epididymal spermatozoa. Male rats were treated with either saline or cyclophosphamide (6 mg/kg/day) for 6 or 9 weeks and cauda epididymal spermatozoa were collected. The rat sperm Y-4 FISH assay was used to assess the induction of spermatozoal disomy, nullisomy and diploidy involving chromosomes Y and 4. The overall frequency of numerically abnormal spermatozoa was elevated about 2-fold (p<0.001) after 9 weeks of cyclophosphamide treatment. Exposure for 9 weeks, but not for 6 weeks, significantly increased the frequency of spermatozoa with chromosome 4 disomy (p<0.02) and nullisomy (p<0.05), but disomy Y and diploidy were not significantly increased with treatment compared to corresponding controls. Independent of treatment, only 27% of aneuploid spermatozoa presented with morphological abnormalities, but all diploid spermatozoa were approximately twice the size of normal cells. Thus, cyclophosphamide disrupts meiotic events prior to pachynema during spermatogenesis, emphasizing the potential for adverse progeny outcomes following genotoxic damage.