Sphingomyelins and ceramides with very-long-chain PUFA are excluded from lowdensity, raft-like domains in differentiating spermatogenic cells

Abstract

Rat spermatogenic cells contain sphingomyelins (SM) and ceramides (Cer) with very long chain (VLC) PUFA, 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 (L) fraction and a large heavy (H) fraction, mostly derived from the plasma membrane, of spermatocytes, round, and late spermatids. The L fraction contained cholesterol, glycerophospholipids and SM with the same saturated fatty acids in all three stages. In the H fraction, as PUFA increased in the glycerophospholipid and VLCPUFA in SM from spermatocytes to spermatids, the concentration of cholesterol also augmented. The H 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 H, 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.