Lipid peroxides in spermatozoa; formation, r‏‏‏‏‏ôle of plasmalogen, and physiological significance

Abstract

In view of the high content of unsaturated fatty acids in the phospholipids of mammalian spermatozoa, the possibility was investigated that during aerobic incubation of spermatozoa these fatty acids, in particular arachidonic (20:4) and docosahexanoic (22:6) acid, undergo peroxidation, which in turn is responsible for the decline in viability of spermatozoa. The formation of lipid peroxides was followed by the thiobarbituric acid (TBA) reaction, iodometry and fluorimetry; it was found that during aerobic incubation of washed ram spermatozoa at 37 °C in the presence of ascorbic acid (0.1 mg/ml) and ferrous iron (2.8 μg Fe/ml), peroxides became detectable after 30 min incubation and their level reached a maximum after 60 min. Coincident with the formation of peroxides motility declined and the spermatozoa became agglutinated, but their oxygen uptake was not grossly different from that of motile spermatozoa in mixtures free of ascorbic acid or Fe. Peroxide formation could be prevented by the addition of either chelating agents, such as EDTA and sodium citrate, or antioxidants, such as tocopherol and butylated hydroxyanisole, to the incubation medium. Conclusive evidence that the substrate for the peroxidation reaction is a lipid, was obtained by extracting lipids from spermatozoa with chloroform: methanol and incubating aerobically in the presence of catalytic amounts of ascorbate and Fe, two fractions: the extracted lipid and the ‘defatted’ sperm residue; very high peroxide values were recorded in the former, but not the latter fraction. Examination by thin-layer and gas-liquid chromatography of the phospholipid and neutral lipid fractions obtained from spermatozoa, revealed that peroxidation caused a loss of approximately 50% in the content of plasmalogens, 20:4 and 22:6 fatty acids, and palmitaldehyde. Changes in the other phospholipids and their bound fatty acids were relatively small, but there was some increase in the amount of a substance which behaved chromatographically as lysolecithin. The content of cholesterol and glycerides in the neutral lipid fraction was not appreciably different in non-peroxidized and peroxidized spermatozoa, but the latter contained a higher proportion of free fatty acids. Our results indicate that under aerobic conditions the lipids in spermatozoa, in particular the plasmalogens, are susceptible to peroxidation. The peroxidation reaction apparently takes place within the lipoprotein, and is associated with a decline in the viability of spermatozoa. This decline could be due either to an increase in cellular permeability resulting from changes in membrane-bound lipids, or to a direct toxic effect of lipid peroxides on spermatozoa. Development of means for the prevention of adverse effects of the peroxidation reaction, may be of practical importance in relation to the problem of long-term storage of semen for artificial insemination.