Etoposide Model to Study Apoptosis in Normal Ejaculated Sperm

Abstract

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 the mitochondrial apoptotic pathway by disrupting mitochondrial membranes. Such mitochondrial damage should cause mitochondrial swelling and render normal spermatozoa non-motile. This study investigates whether ejaculated spermatozoa from normal donors can be treated with etoposide to create a reproducible apoptotic sperm cell model that provides insight into the mitochondrial apoptotic pathway. Prospective experimental design 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 fragmentationwas used as a marker of apoptosis and 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 swellingwas visualized by electron microscopy. Statistics were performed as follows: a paired non-parametric two-tailed Wilcoxon ranked-sum test for motility and apoptosis and a chi-squared test for mitochondrial membrane integrity. Etoposide-induced apoptosis was reproducibly seen in multiple semen samples collected from two normal donors.After a 24 hour incubation at 50 ug/ml of etoposide, apoptosis 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. Etoposide can reliably induce apoptosis in ejaculated sperm from normal donors. It is associated with a complete absence of motility and damage to mitochondria. Such a cell system model can provide molecular insights that can help us understand apoptosis-related changes in semen that is associated with male infertility.