P-836: Oxidative stress induced caspase activation and associated toxicity in human sperm

Abstract

ObjectiveOxidative stress is a result of an imbalance caused by: 1] over production of reactive oxygen species (ROS), and/or 2] reduced scavenger capacity. Oxidative stress can occur when cellular antioxidant mechanisms are overwhelmed by pathologies of the male reproductive system such as infection/inflammation, varicocele and/or continuous exposure to toxic agents. High levels of ROS are believed to adversely affect biomolecules, particularly cellular lipids, proteins and DNA. These damaged biomolecules can result in defective sperm function that may ultimately result in male infertility. We have previously reported that when sperm from a fertile male was treated in vitro with ROS (50 and 100 μM H2O2), lipid peroxidation levels were significantly increased compared to untreated controls. Further, ROS damaged sperm showed defective DNA decondensation and recondensation when analyzed by the human sperm activation assay. The aims of this study were to determine if ROS concentrations 1) are correlated with lipid peroxidation and apoptosis, and 2) induce detrimental effects on sperm function as defined by World Health Organization criteria.DesignIn vitro dose response study.Materials and methodsSperm were isolated from a semen sample of a fertile male using Percoll gradients. Sperm from the 80% pellet were divided into 5 equivalent aliquots and treated with 0, 10, 50, 100 or 500 μM H2O2 in PBS. Following an incubation period, sperm were evaluated for: (A) lipid peroxidation; (B) sperm function [viability, motility, acrosomal integrity, hypo-osmotic swelling test (HOST)]; and (C) apoptosis.ResultsLinear regression analyses indicated that 1) lipid peroxidation (r= 0.8628, p<0.01) and 2) the number of sperm positive for apoptosis via Caspase 3 activation (r= 1.0, p< 0.0109) were H2O2-dose dependent. To determine the effects of increasing H2O2 concentration on sperm function, ANOVA was calculated for: a) viability, b) rapid progressive motility, c) acrosomal integrity and c) HOST positive response. The results indicate that the means for each sperm function parameter in sperm exposed to 10, 50 100 and 500 μM H2O2 was statistically significant when compared with the the untreated control (0 μM H2O2) means (p< 0.0107).ConclusionLipid peroxidation was H2O2-dose dependent. ROS, at physiological concentrations (∼10 μM), are required for proper sperm function. However, human sperm exposed to high concentrations of ROS (50, 100 and 500 μM H2O2) are susceptible to oxidative damage, which is mediated by lipid peroxidation that leads to apoptosis via Caspase 3 activation. It therefore appears that oxidative stress is a causative mediator of defective sperm function, which is associated with male infertility. ObjectiveOxidative stress is a result of an imbalance caused by: 1] over production of reactive oxygen species (ROS), and/or 2] reduced scavenger capacity. Oxidative stress can occur when cellular antioxidant mechanisms are overwhelmed by pathologies of the male reproductive system such as infection/inflammation, varicocele and/or continuous exposure to toxic agents. High levels of ROS are believed to adversely affect biomolecules, particularly cellular lipids, proteins and DNA. These damaged biomolecules can result in defective sperm function that may ultimately result in male infertility. We have previously reported that when sperm from a fertile male was treated in vitro with ROS (50 and 100 μM H2O2), lipid peroxidation levels were significantly increased compared to untreated controls. Further, ROS damaged sperm showed defective DNA decondensation and recondensation when analyzed by the human sperm activation assay. The aims of this study were to determine if ROS concentrations 1) are correlated with lipid peroxidation and apoptosis, and 2) induce detrimental effects on sperm function as defined by World Health Organization criteria. Oxidative stress is a result of an imbalance caused by: 1] over production of reactive oxygen species (ROS), and/or 2] reduced scavenger capacity. Oxidative stress can occur when cellular antioxidant mechanisms are overwhelmed by pathologies of the male reproductive system such as infection/inflammation, varicocele and/or continuous exposure to toxic agents. High levels of ROS are believed to adversely affect biomolecules, particularly cellular lipids, proteins and DNA. These damaged biomolecules can result in defective sperm function that may ultimately result in male infertility. We have previously reported that when sperm from a fertile male was treated in vitro with ROS (50 and 100 μM H2O2), lipid peroxidation levels were significantly increased compared to untreated controls. Further, ROS damaged sperm showed defective DNA decondensation and recondensation when analyzed by the human sperm activation assay. The aims of this study were to determine if ROS concentrations 1) are correlated with lipid peroxidation and apoptosis, and 2) induce detrimental effects on sperm function as defined by World Health Organization criteria. DesignIn vitro dose response study. In vitro dose response study. Materials and methodsSperm were isolated from a semen sample of a fertile male using Percoll gradients. Sperm from the 80% pellet were divided into 5 equivalent aliquots and treated with 0, 10, 50, 100 or 500 μM H2O2 in PBS. Following an incubation period, sperm were evaluated for: (A) lipid peroxidation; (B) sperm function [viability, motility, acrosomal integrity, hypo-osmotic swelling test (HOST)]; and (C) apoptosis. Sperm were isolated from a semen sample of a fertile male using Percoll gradients. Sperm from the 80% pellet were divided into 5 equivalent aliquots and treated with 0, 10, 50, 100 or 500 μM H2O2 in PBS. Following an incubation period, sperm were evaluated for: (A) lipid peroxidation; (B) sperm function [viability, motility, acrosomal integrity, hypo-osmotic swelling test (HOST)]; and (C) apoptosis. ResultsLinear regression analyses indicated that 1) lipid peroxidation (r= 0.8628, p<0.01) and 2) the number of sperm positive for apoptosis via Caspase 3 activation (r= 1.0, p< 0.0109) were H2O2-dose dependent. To determine the effects of increasing H2O2 concentration on sperm function, ANOVA was calculated for: a) viability, b) rapid progressive motility, c) acrosomal integrity and c) HOST positive response. The results indicate that the means for each sperm function parameter in sperm exposed to 10, 50 100 and 500 μM H2O2 was statistically significant when compared with the the untreated control (0 μM H2O2) means (p< 0.0107). Linear regression analyses indicated that 1) lipid peroxidation (r= 0.8628, p<0.01) and 2) the number of sperm positive for apoptosis via Caspase 3 activation (r= 1.0, p< 0.0109) were H2O2-dose dependent. To determine the effects of increasing H2O2 concentration on sperm function, ANOVA was calculated for: a) viability, b) rapid progressive motility, c) acrosomal integrity and c) HOST positive response. The results indicate that the means for each sperm function parameter in sperm exposed to 10, 50 100 and 500 μM H2O2 was statistically significant when compared with the the untreated control (0 μM H2O2) means (p< 0.0107). ConclusionLipid peroxidation was H2O2-dose dependent. ROS, at physiological concentrations (∼10 μM), are required for proper sperm function. However, human sperm exposed to high concentrations of ROS (50, 100 and 500 μM H2O2) are susceptible to oxidative damage, which is mediated by lipid peroxidation that leads to apoptosis via Caspase 3 activation. It therefore appears that oxidative stress is a causative mediator of defective sperm function, which is associated with male infertility. Lipid peroxidation was H2O2-dose dependent. ROS, at physiological concentrations (∼10 μM), are required for proper sperm function. However, human sperm exposed to high concentrations of ROS (50, 100 and 500 μM H2O2) are susceptible to oxidative damage, which is mediated by lipid peroxidation that leads to apoptosis via Caspase 3 activation. It therefore appears that oxidative stress is a causative mediator of defective sperm function, which is associated with male infertility.