Cancer Chemotherapy and Sperm
Etoposide-Induced DNA Fragmentation & Mitochondrial Damage in Normal Ejaculated Sperm: A Putative In Vitro Model of Apoptosis
Wayne Kuang MD & Anthony Thomas Jr. MD
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Objective: Alterations in apoptosis (programmed cell death) may be a contributing cause of abnormal semen samples in subsets of male infertility (e.g. varicocele). A need exists for a reliable cell model system that can reveal the molecular mechanisms of apoptosis in sperm. Etoposide is a chemotherapeutic agent that can induce DNA fragmentation and mitochondrial damage in somatic cells that are undergoind apoptosis. This study investigates whether etoposide can induce similar changes in normal ejaculated sperm as a putative model to study apoptosis in mature germ cells.
Design: Prospective experimental design
Materials and Methods: Multiple ejaculated semen samples from two normal donors were collected after at least 48 hours of abstinence between samples. Resuspended sperm pellets from each sample were treated with etoposide for 24 hours. Untreated sperm served as a negative control, while Jurkat cells were a positive control. DNA fragmentation was measured with terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) flow cytometry. Motility was manually measured by phase-contrast microscopy. Mitochondrial membrane integrity was assessed with Mitosensor dye (Clontech, Palo Alto, CA) and fluorescence microscopy. Mitochondrial swelling was visualized by electron microscopy. Statistics were performed as follows: a paired non-parametric two-tailed Wilcoxon ranked-sum test for motility and TUNEL positivity and a chi-squared test for mitochondrial membrane integrity.
Results: Etoposide-induced DNA fragmentation was reproducibly seen in multiple semen samples collected from two normal donors. After a 24 hour incubation at 50 ug/ml of etoposide, TUNEL positivity in treated sperm increased on average by 986% vs. 171% in the untreated samples [P=0.016]). As expected, all treated sperm were rendered completely non-motile whereas untreated sperm demonstrated only a 27% decrease in motility [P=0.016]. All treated sperm demonstrated mitochondrial membrane damage while this was only seen in 11% of untreated sperm [P<0.0001]. Mitochondrial swelling as seen by electron microscopy was associated with treated sperm.
Conclusion: Etoposide can reliably induce DNA fragmentation in ejaculated sperm from normal donors. It is associated with a complete absence of motility and mitochondrial damage. This cell system model can potentially provide molecular insights into apoptosis-related male infertility.