317 RESISTANCE AGAINST DIFFERENT REACTIVE OXYGEN SPECIES IN BOVINE EPIDIDYMAL SPERM UNDER DISTINCT MATURATION STATUS

Abstract

Oxidative stress is caused by reactive oxygen species (ROS) that may cause structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components. Evidence indicates that oxidation products are also deleterious to biological systems. Spermatozoa are particularly susceptible the oxidative stress, mainly due to the reduced cytoplasm and the high content of polyunsaturated fatty acids in its membrane. The mechanisms by which sperm acquire antioxidant capacity are still not completely elucidated. The aim was to study the resistance of sperm derived from different epididymal compartments (caudae and head) to the different ROS and to the lipid peroxidation product malondialdehyde (MDA). Epididymal sperm samples from 4 testicles were collected from the head and caudae epididymides. Sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate and ferrous sulfate (4 mM; produces hydroxyl radical), and MDA. Samples were analyzed for 3-3′ diaminobenzidine stain, as an index of mitochondrial activity; the eosin nigrosin stain, as an index of membrane integrity; the simple stain (fast green/Bengal rose), as an index of acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Pearson correlation). Results showed that immature sperm (head epididymides) were significantly more susceptible to the MDA and to the hydroxyl radical in all studied variables, especially acrosomes, membranes, and mitochondrial potential. Semen derived from the caudae epididymides was more susceptible to the hydrogen peroxide and to the MDA, especially regarding mitochondrial potential. In semen from the epididymal head, a positive correlation was found between TBARS and sperm showing no mitochondrial potential (r = 0.66, P = 0.01). On the other hand, negative correlations were found between TBARS and sperm with damaged acrosome and membrane (r = -0.63, P = 0.01 and r = -0.58, P = 0.02, respectively) in samples collected from the caudae epididymides. The present results suggest that sperm susceptibility to the attack of ROS is different throughout maturation. Although immature sperm are more susceptible to the hydroxyl radical, mature sperm are more susceptible to the hydrogen peroxide. Furthermore, MDA, a product of lipid peroxidation, is also deleterious to the sperm, indicating that once oxidative stress starts, further damage may be caused by their products.

The authors thankNutricell for the media used in the experiment andFAPESP for financial support (process #06/05736-1).