Sphingomyelins and ceramides with VLCPUFAs are excluded from low-density raft-like domains in differentiating spermatogenic cells

Rat spermatogenic cells contain sphingomyelins (SMs) and ceramides (Cers) with very long-chain PUFAs (VLCPUFAs) in nonhydroxylated (n-V) and 2-hydroxylated (h-V) forms. How these atypical species distribute among membrane fractions during differentiation was investigated here using a detergent-free procedure to isolate a small light raft-like low-density fraction and a large heavy fraction, mostly derived from the plasma membrane of spermatocytes, round spermatids, and late spermatids. The light fraction contained cholesterol, glycerophospholipids (GPLs), and SM with the same saturated fatty acids in all three stages. In the heavy fraction, as PUFA increased in the GPL and VLCPUFA in SM from spermatocytes to spermatids, the concentration of cholesterol was also augmented. The heavy fraction had mostly n-V SM in spermatocytes, but accumulated h-V SM and h-V Cer in spermatids. A fraction containing intracellular membranes had less SM and more Cer than the latter, but in both fractions SM and Cer species with h-V increased over species with n-V with differentiation. This accretion of h-V was consistent with the differentiation-dependent expression of fatty acid 2-hydroxylase (Fa2h), as it increased significantly from spermatocytes to spermatids. The non-raft region of the plasma membrane is thus the main target of the dynamic lipid synthesis and remodeling that is involved in germ cell differentiation.