Low-density membrane fractions from male germ cells lack sphingolipids with very long chain PUFA

Abstract

Sphingomyelins (SM) and ceramides (Cer) with very long-chain PUFA (VLCPUFA), in nonhydroxy (n-V) and 2-hydroxy (h-V) forms, are specific components of rat spermatogenic cells. Here we evaluated how differentiation affects their distribution among membrane fractions from such cells. Using a detergent-free procedure, a small light, raft-like low-density (L) fraction and a large heavier (H) fraction, both showing markers typical of cell plasma membranes, were separated from pachytene spermatocytes, round, and late spermatids. MALDI-TOF spectra showed that the L fraction had mostly SM species with saturated fatty acids regardless of the cell stage, while the H fraction was rich in stage-varying SM and Cer species with VLCPUFA. In this fraction spermatocytes accumulated mostly n-V SM and spermatids h-V SM and h-V Cer species. A third fraction made of intracellular membranes had less SM and more Cer than the H fraction, differentiation also increasing the h-V/n-V ratio in both lipids. The buildup of 2-hydroxy fatty acids correlated with the expression (mRNA) of fatty acid 2-hydroxylase (Fa2h), higher in spermatids than in spermatocytes. The differentiation-dependent rise in h-V Cer in the germ cell H fraction during spermatogenesis is consistent with the eventually uneven distribution that n-V and h-V species of SM and Cer display between the head and the tail of mature spermatozoa.