Evidence for diplasmalogen as the major component of rabbit sperm phosphatidylethanolamine

The question of whether diplasmalogens [1,2-di(O-1'-alkenyl) phosphatidyl derivatives] make up part of the plasmalogen component of cell phospholipids was examined using rabbit epididymal spermatozoa. These cells are readily obtained as a highly homogeneous suspension and long have been known to have high plasmalogen content. Phospholipids were determined by thin layer chromatography (TLC) with CuSO4 staining. Plasmalogens were determined by hydrolysis of the phospholipids with TCA/HCl, followed by TLC and CuSO4 staining. Ethanolamine derivatives were determined by ninhydrin. The phosphatidylethanolamine (PE) content of these cells was 29 +/- 2 micrograms/10(8) cells, 90% of which was assayed as diplasmalogen and 10% as diacyl PE. No monoplasmalogen could be detected. The presence of diplasmalogen as the major component of PE was given further support from infrared and proton nuclear magnetic resonance (1H-NMR) spectroscopy, which showed the presence of O-1'-alkenyl substituents but near absence of O-acyl substituents. The phosphatidylcholine (PC) content of the cells was 104 +/- 5 mu/10(8) cells, of which 50% was monoplasmalogen with the 1'-alkenyl group on the 2 position of the glycerol moiety. No diplasmalogen was found in PC. The other phospholipids in rabbit sperm were phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SP) and lysophosphatidylcholine (LPC). Phosphatidylserine (PS) and phosphatidylinositol (PI) were present at the limits of detectability of the TLC method. None of these phospholipids contained plasmalogen. The PE component of rabbit sperm phospholipids appears to differ from that of the other cells in having the previously unreported diplasmalogen as its major constituent.