Abstract
Very long-chain polyunsaturated fatty acids (VLCPUFA) have previously been shown to be components of sphingomyelin (SM) of mammalian testis and spermatozoa. Here we examined the fatty acids of testicular ceramide (Cer) in comparison with those of SM in some mammals with a special focus on the rat testis. In bull, cat, dog, rabbit, mouse, and rat, VLCPUFA were found in both testicular lipids, Cer having a higher percentage of VLCPUFA than SM. Rat testis had the highest percentage of VLCPUFA in both lipids, the major ones being 28:4n-6 and 30:5n-6. VLCPUFA-containing SM and Cer occurred in cells located in the seminiferous tubules, where germ cells had a higher percentage of these species than Sertoli cells. Seminiferous tubule fractionation showed that SM and Cer of mitochondria and lysosomes had mostly saturates and negligible VLCPUFA, the latter being important in the SM and Cer of microsomes and other membrane fractions. VLCPUFA were absent from the SM and Cer of rat prepuberal testis, increased with the onset of spermatogenesis to account for nearly 15 and 40% of the total fatty acids of testicular SM and Cer, respectively, remained at those levels throughout the adult life of fertile rats and tended to decrease at advanced ages. Four conditions that lead to selective death of germ cells in vivo, namely experimental cryptorchidism, post-ischemic reperfusion, focalized x-ray irradiation and treatments with the antineoplasic drug doxorubicin, caused the VLCPUFA to disappear from the testicular SM and Cer of adult fertile rats, showing that these lipids are specific traits of spermatogenic cells.
Highlights
Have been a focus of great interest in the past decade because some of their metabolites, ceramides, sphingoid bases, and phosphorylated sphingoid bases, have emerged as important messengers in cell signaling [1, 2]
Taking advantage of the knowledge that the population of germ cells in the testis is sensitive to certain cell stressors, we studied their effects on the testicular SM and Cer fatty acids
We provide qualitative and quantitative data showing that both testicular lipids have far from negligible proportions of very long chain polyunsaturated fatty acids (VLCPUFA) and present evidence that these lipids are associated to healthy cells of the spermatogenic lineage in fertile adult animals
Summary
Testis—Four-month-old Wistar rats were used in all studies involving adult animals. The testes were obtained from anesthetized animals (5 mg/kg acepromazine and 50 mg/kg ketamine, intraperitoneal) which were sacrificed immediately thereafter. One testis from an adult rat (600 – 800 g of total lipid phosphorus) was used to study Cer and its fatty acids, and 8 –10 testes per sample were pooled to analyze the Cer of 14-dayold rats or mice. Fatty Acid Analysis—The fatty acid composition of SM and Cer was determined by gas chromatography (GC) of their fatty acid methyl ester derivatives These were prepared by dissolving the lipid samples in anhydrous methanol containing 0.5 N H2SO4 [27] in Teflon-lined screw-capped tubes, closing (under N2), and warming the tubes at 45 °C overnight. Before GC, the fatty acid methyl esters containing the internal standard were routinely purified by TLC (using hexane/ether, 95:5, by volume) on silica gel G plates that had been previously washed free of contaminants with methanol/ethyl ether (75:25, by volume). The fatty acid compositions of SM and Cer were calculated on a weight percent basis
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