Abstract

Nonylphenol (NP), a derivative of an estrogenic environmental endocrine disrupter called alkylphenol polyethoxylates (APEs), has been shown to have toxic, estrogenic, and carcinogenic effects on both aquatic and terrestrial organisms. NP is widely used in common cosmetic products and detergents. The primary objective these studies was to determine the effects of NP (dissolved in either ethanol or dimthyl sulfoxide (Me₂SO)) on rat sperm viability characteristics such as motility, acrosomal integrity, and mitochondrial membrane potential. The epididymal rat sperm were suspended (50 × 10⁶ spermatozoa mL^–1) in sodium bicarbonate-buffered Tyrode's lactate solution containing 3 mg mL^–1 bovine serum albumin (BSA) after removal of the cauda epididymis of sexually mature Sprague Dawley male rats (n = 5). Aliquots of 0.5 mL sperm suspension were added into 5-mL test tubes containing 1, 10, 100, 250, 500, and 1000 µg NP mL^–1. As negative controls, no NP–no solvent, no NP+ethanol, and no NP+Me₂SO were used. All sperm treatments were incubated for 1, 2, 3, and 4 h at 37°C with 5% CO₂ in air. Computer-aided sperm analysis system (Hamilton Thorne, Beverly, MA, USA) was used to determine sperm motility characteristics. An epifluorescent microscopy method was used to determine sperm acrosomal status after staining with 20 µg mL^–1 Alexa Fluor-488-PNA (Molecular Probes, Eugene, OR, USA) and a flow cytometric method was used to determine mitochondrial membrane potential after staining with 2 µg mL^–1 JC-1 (Molecular Probes). The data were analyzed by ANOVA by using SAS (SAS Institute, Inc., Cary, NC, USA). The results showed that, as compared to negative controls (no NP–no solvent, no NP+ethanol), exposure of rat sperm to 250, 500, or 1000 µg NP mL^–1 was highly detrimental to their motility (P < 0.05). Total loss of sperm motility was observed after exposure to 500 µg NP mL^–1. However, the time course did not seem to have a significant effect on sperm motility (P > 0.05). Nonylphenol induced dose-dependent acrosomal reaction in rat sperm (P < 0.05). The effects of NP on the mitochondrial membrane potential (MMP) of rat sperm were also significant (P < 0.05). The percentage of rat sperm with high MMP declined drastically after exposure to 100 and 250 µg NP mL^–1 as compared to negative controls (P < 0.05). However, duration of exposure to NP did not have an effect on MMP of rat sperm (P > 0.05). In conclusion, these data obtained from epididymal rat sperm provide important information about the mechanism of detrimental effects of NP on mammalian sperm, which must be seriously considered in the case of short- or long-term exposure to NP in humans as well as livestock species.