Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa.
BACKGROUND
The formation of mature sperm involves the expression of numerous proteins during spermiogenesis and the replacement of histones with protamines to package the genome. Exposure to cyclophosphamide (CPA), an anticancer alkylating agent, during spermiogenesis may disrupt chromatin condensation with adverse consequences to the offspring.


METHODS
Adult male rats were given CPA in one of two schedules: (i) subchronic, 4 days - day 1 (100 mg kg(-1)) and days 2-4 (50 mg kg(-1) per day) or (ii) chronic - daily (6.0 mg kg(-1) per day). Animals were euthanized on days 14, 21 or 28.


RESULTS
The effects of CPA on epididymal sperm chromatin structure were germ-cell-phase specific; mid-spermiogenic spermatids were most sensitive. The acridine orange DNA denaturation assay showed significant increases in susceptibility to denaturation (P < 0.01). Chromatin packaging assessment revealed 1,4-dithiothreitol-dependent chromomycin A3 DNA binding and less condensed, protamine-deficient sperm; the total thiol (P < 0.001) and protamine contents (P < 0.01), measured using monobromobimane and the HUP1N protamine 1 antibody, respectively, were reduced. The sperm basic proteome was also altered; proteins that were identified are involved in events during spermiogenesis and fertilization.


CONCLUSIONS
Paternal exposure to CPA alters sperm chromatin structure, as well as the composition of sperm head basic proteins. We speculate that these changes underlie effects on fertilization and embryo development.
Codrington
A M
AM
Department of Pharmacology, McGill University, Montreal, Quebec, Canada.
Hales
B F
BF
Robaire
B
B
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000008:administration & dosage / Q000494:pharmacology*; D002843:Chromatin / Q000187:drug effects* / Q000648:ultrastructure*; D014128:Chromomycin A3 / Q000378:metabolism; D003520:Cyclophosphamide / Q000008:administration & dosage / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D008297:Male; D011479:Protamines / Q000378:metabolism; D020543:Proteome / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013077:Sperm Head / Q000378:metabolism; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
D018906:Antineoplastic Agents, Alkylating; D002843:Chromatin; D011479:Protamines; D020543:Proteome; D013438:Sulfhydryl Compounds; D003520:Cyclophosphamide; D014128:Chromomycin A3; D004229:Dithiothreitol