Peroxynitrite-mediated nitrosative stress decreases motility and mitochondrial membrane potential in human spermatozoa

Abstract

Nitrosative stress is produced by high levels of reactive nitrogen species (RNS). The RNS include peroxynitrite, a highly reactive free radical produced from a diffusion-controlled reaction between nitric oxide and superoxide anion. Peroxynitrite causes nitration and oxidation of lipids, proteins and DNA, and is thus considered an important pathogenic mechanism in various diseases. Although high levels of peroxynitrite are associated with astenozoospermia, few reports exist regarding the in vitro effect of high levels of this RNS on human sperm. The aim of this study was to evaluate the in vitro effect of nitrosative stress caused by peroxynitrite on the viability, motility and mitochondrial membrane potential of human spermatozoa. To do this, human spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a molecule that generates peroxynitrite. Incubations were done at 378C for up to 4 h with SIN-1 concentrations between 0.2 and 1.0 mmol/l. Generation of peroxynitrite was confirmed using dihydrorhodamine 123 (DHR) by spectrophotometry and flow cytometry. Sperm viability was assessed by propidium iodide staining; sperm motility was analyzed by CASA, and the state of mitochondrial membrane potential (DCm) by JC-1 staining. Viability and DCm were measured by flow cytometry. The results showed an increase in DHR oxidation, demonstrating the generation of peroxynitrite through SIN-1. Peroxynitrite decreased progressive and total motility, as well as some sperm kinetic parameters. Mitochondrial membrane potential also decreased. These alterations occurred with no decrease in sperm viability. In conclusion, peroxynitriteinduced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility.

Raffia palm (Raphia hookeri) wine extenuates redox imbalance and modulates activities of glycolytic and cholinergic enzymes in hyperglycemia-induced testicular injury in type 2 diabetic rats

“…This also correlates with the increased NO level on the onset of T2D (Figure a), as NO reacts with O 2 ▪ − to produce peroxynitrite (ONOO − ). This triggers nitrosative stress and proinflammation leading to testicular dysfunction, impaired sperm motility, decreased gonadotropin secretion, and finally infertility (Doshi, Khullar, Sharma, & Agarwal, ; Uribe, Boguen, Treulen, Sánchez, & Villegas, ). The elevated SOD activities in the treated groups (Figure b), therefore indicates a reduced testicular level of O 2 ▪ − and its dismutation to H 2 O 2 and O 2.…”

From Nitric Oxide Toward S-Nitrosylation: Expanding Roles in Gametes and Embryos

“…NO is, on the other hand, considered a major free radical involved in sperm damage at sperm motility level. Nitrosative stress produced by high levels of reactive nitrogen species decreases progressive and total motility, as well as several sperm kinetic parameters, meanwhile, sperm viability is not affected [60,61].…”

Damage to Sperm DNA Mediated by Reactive Oxygen Species: Its Impact on Human Reproduction and the Health Trajectory of Offspring

“…If fertilization does not occur, it has been proposed that the continued generation of ONOO − by capacitating spermatozoa leads to a state of "over-capacitation" whereby the ROS generation that drives capacitation eventually overwhelms the limited antioxidant capacity of these cells, leading to a state of oxidative stress (Aitken 2011 ). The appearance of ONOO − -mediated oxidative stress leads to a loss of sperm function (Uribe et al 2015 ) and the generation of lipid aldehydes that via the mechanisms described above, stimulate yet more ROS generation, ultimately precipitating a state of apoptosis characterized by rapid motility loss, mitochondrial ROS generation, caspase activation in the cytosol, annexin V binding to the cell surface, cytoplasmic vacuolization, and oxidative DNA damage (Koppers et al 2011 ). During this intrinsic apoptotic cascade, it is only after the spermatozoa have become immobilized, and therefore prone to phagocytosis, that markers of apoptosis such as phosphatidylserine externalization start to appear (Koppers et al 2011 ).…”