In both models, glycerophospholipids rich in long-chain polyunsaturated fatty acids like 20:4n-6 and 22:5n-6, and sphingomyelins rich in very long chain PUFA like 28:4n-6 and 30:5n-6, disappeared as the last mature germ cells abandoned the testis and seminiferous tubules became almost exclusively populated by SC. Also in both cases, as germ cells and their endogenous phospholipids decreased approaching their minimum values, the amount per testis of cholesteryl esters and ether-linked triglycerides increased, these neutral lipids accumulating mostly PUFA and VLCPUFA among their fatty acids. The buildup of CE and ADG was considered to be a manifestation of a physiological function of SC that involves lipid processing, as these cells normally display phagocytic and lipid catabolic activities, as shown by an active acid sphingomyelinase, as well as a marked ability to oxidize fatty acids. Two weeks after in vivo experimental hyperthermia, induced by locally subjecting rat testes during 15 minutes per day to 43uC for 5 successive days, the number of germ cells was at its nadir. This not only coincided with peak levels of CE and ADG in the rat testis, but concurred with a considerable buildup of lipid droplets in SC, in agreement with the finding that these two neutral lipid classes are mostly SC products. The buildup of lipid droplets consequent to heat exposures was elegantly confirmed shortly afterwards in the mouse testis in vivo, where the expression of two lipid droplet-specific proteins, PLIN2 and PLIN3, was shown to increase after similarly short but daily repeated exposures to moderate hyperthermia. The direct effects of heat exposure on lipid and fatty acid metabolism of SC and the potential impact of these effects on spermatogenesis are still unknown. The aim of this study was to test the hypothesis that, even if SC do not die after repeated heat exposures, they may become temporarily dysfunctional enough to contribute to the reduced survival of the multitude of germ cells that they structurally and metabolically support. The described antecedents suggested that we could expect shifts in lipid metabolism. In vivo, relatively early lipid-specific changes in SC after heat exposures are difficult to ascertain in the environment of the seminiferous epithelium, where each SC is surrounded by a cohort of germ cells in different stages of their differentiation, many of which are undergoing heat-induced GDC-0879 apoptosis. For this reason, in this study we used TM4 cells in culture to investigate possibly deleterious direct effects of Y-27632 dihydrochloride 129830-38-2 hyperthermia on SC lipid and fatty acid metabolism. It was expected that lipids would be altered in a way that would correlate with evidence of SC functional and/or structural alterations. To this aim, we focused on two proteins that are markers of SC functionality and two cytoskeletal proteins. The results we present in this study demonstrate that hyperthermia induces significant alterations in SC lipid and fatty acid metabolism and that, within the same temporal frame as these lipid changes, SC-specific functions are disturbed. We propose that, although temporary and potentially reversible, most of these derangements may also occur to SC in vivo after heat exposures, this having a negative impact on the survival of the germ cells whose development SC support.