Objective: Our goal was to test the hypothesis that spermatozoal chromatin packaging changes with age and that aging affects the susceptibility of spermatozoal DNA to oxidative damage. Design: Laboratory study. Setting: Academic facility. Patient(s): Young (4 months) and old (21 months) Brown Norway rats. Intervention(s): Spermatozoa were collected from the cauda epididymidis and were incubated in saline or H 2 O 2 . Main Outcome Measurement(s): Thiols levels, chromatin condensation, DNA susceptibility to acid-induced DNA denaturation, and DNA damage were evaluated using monobromobimane, chromomycin A3 (CMA3), acridine orange, and polymerase chain reaction, respectively. Result(s): Spermatozoa from old rats had 25% fewer disulfides but similar levels of free thiols as compared with young. The CMA3 staining was decreased by 13% with age. Levels of chromatin denaturation and DNA damage were similar in control groups. After exposure to oxidant, free thiols became oxidized by about 20% irrespective of age, but CMA3 staining changed little. The acridine orange assay, however, showed a trend for greater chromatin denaturation in spermatozoa from old rats after oxidant treatment. Furthermore, the DNA from spermatozoa of old rats was significantly more susceptible to developing DNA breaks and modification after oxidative challenge.

Reactive oxygen species (ROS) play a role in male infertility, where excessive amounts impair spermatozoal motility. Epididymal antioxidant enzymes protect spermatozoa from oxidative damage in the epididymal lumen. Antioxidant secretions from the seminal vesicle protect spermatozoa after ejaculation. As it is known that with age there is increased generation of ROS, the goals of this study were to determine how aging affects the response of antioxidant enzymes in the epididymis, seminal vesicles, and liver to l-buthionine-S,R-sulfoximine (BSO) mediated glutathione (GSH) depletion, and to examine the impact of GSH depletion on motility parameters of spermatozoa from the cauda epididymidis in young (4-mo-old) and old (21-mo-old) rats. Levels of GSH and glutathione disulfide (GSSG), as well as activities of glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase, were measured in the caput, corpus and cauda epididymidis, seminal vesicles, and liver. Spermatozoal motility was assessed by computer-assisted sperm analysis. Significant age-related changes in antioxidant enzyme activities were found in the liver and cauda epididymidis. Glutathione depletion clearly affected tissues in both young and old. The compounding effect of age was most evident in the cauda epididymidis, seminal vesicles, and liver, where antioxidant enzyme activities changed significantly. Additionally, spermatozoa motility was adversely affected after BSO treatment in both age groups, but significantly more so in older animals. In summary, the male reproductive tissues and liver undergo age-related changes in antioxidant enzyme activities and in their response to GSH depletion.

Overall, we show that spermatozoa from older rats have altered chromatin packaging and integrity and that spermatozoa from the cauda epididymidis are more responsive to combined in vivo and in vitro oxidative challenge than spermatozoa from young rats.

Editorial CommentarySeligman J, Newton GL, Fahey RC, Shalgi R, Kosower NS. Nonprotein thiols and disulfides in rat epididymal spermatozoa and epididymal fluid: role of ␥-glutamyl-transpeptidase in sperm maturation. J Androl. 2005;26:629-637.