EFFECTS OF CHRONIC LOW-DOSE CYCLOPHOSPHAMIDE EXPOSURE ON THE NUCLEI OF RAT SPERMATOZOA

Previous studies from our laboratory have shown that chronic exposure of the father to low doses of cyclophosphamide results in a significant increase in early embryonic death with little effect on the male reproductive system in rats. Such effects on progeny outcome are hypothesized to be mediated by an action of the drug on the nucleus of spermatozoa. The purpose of the present studies was to investigate the effects of cyclophosphamide treatment for 1 or 6 wk on the pattern of decondensation of sperm nuclei and on the sulfhydryl content of sperm nuclear proteins. Adult male rats were treated with cyclophosphamide (6.1 mg/kg/day) daily for 1 or 6 wk. Cauda epididymal spermatozoa were collected, demembranated, and then incubated with dithiothreitol (DTT) and proteinase K. The in vitro decondensation pattern of the nuclei of spermatozoa was divided into two phases: nuclear swelling and nuclear elongation. Spermatozoa from animals treated for 1 wk with cyclophosphamide showed the same decondensation pattern as those treated with vehicle (saline) alone. However, spermatozoa from animals treated for 6 wk with cyclophosphamide showed normal initial nuclear swelling but had a markedly affected nuclear elongation pattern. The changes with time in the decondensation pattern of these spermatozoa were quantitated by morphometric analysis of the head region of the spermatozoa. The nuclear area, curvature, and length of spermatozoa obtained from chronically drug-treated males were all significantly smaller than for those obtained from controls, while cell diameter was not affected. The effect of treatment with cyclophosphamide on the disulfide cross-linking of sperm nuclear proteins was determined by assessing C-14-iodoacetamide incorporation into spermatozoa from the testis and caput and cauda epididymides. Spermatozoal heads were isolated, purified, demembranated, and then incubated with C-14-iodoacetamide, with or without a DTT preincubation. One week of cyclophosphamide treatment had no effect on the C-14-iodoacetamide incorporation. After 6 wk of treatment with cyclophosphamide, a significant decrease in C-14-iodoacetamide binding to spermatozoal nuclei was seen; this effect occurred in testicular as well as in epididymal spermatozoa. Together these results demonstrate that long-term cyclophosphamide treatment affects the decondensation potential of spermatozoa. These changes in decondensation pattern may be due to the alkylation of nuclear proteins or DNA or to the cross-linking of these macromolecules by metabolites of cyclophosphamide.