OXIDATIVE AND BIOSYNTHETIC REACTIONS 95 



£ul glycolytic system, which is lacking in fish sperm. From the evo- 

 lutionary point of view, glycolysis is believed to be the more primi- 

 tive mechanism. But there is no reason why this should be so in the 

 case of spermatozoa which are produced at an evolutionary stage 

 at which respiration is already firmly established. It is possible that 

 the maintenance of motility of spermatozoa of certain lower forms 

 depends on oxidative metabolism and that the development of sper- 

 matozoa capable of deriving energy from glycolytic processes is a 

 later development in mammals. 



Now to return to studies of biosynthetic reactions in bull sperma- 

 tozoa: These were washed free from seminal plasma and incubated 

 with C 14 -labeled glucose, glycerol, or acetate on as large a scale as 

 possible. It took several months to accumulate enough material for 

 each run. The cells were extracted with lipid solvents, and the ex- 

 tracts were fractionated by silicic acid column and paper chroma- 

 tography. Thus far there have been isolated a diglyceride fraction 

 of high specific activity, less strongly labeled triglyceride and phos- 

 phatide fractions, four weakly radioactive zones which appear to be 

 ketosteroids, and cholesterol, which contained very little, if any, C 14 

 label. The diglycerides, cholesterol, and ketosteroids were eluted 

 from the silicic acid column in one strongly radioactive fraction (25% 

 ether/75% hexane). They were further fractionated by paper chro- 

 matography in the Zaffaroni system. This treatment left several zones 

 on the paper which gave a positive Zimmerman reaction. The fastest 

 moving zone was allowed to run off, and it contained cholesterol 

 and glycerides. The cholesterol was removed by treatment with 

 digitonin and the digitonin-soluble supernatant was again purified 

 on a silicic acid column. This yielded a relatively homogeneous frac- 

 tion which was identified as consisting of diglycerides by its running 

 properties on the column and on paper (Schlenk et al., 1957), and by 

 infrared spectrophotometry which showed the progression curve 

 characteristic of long-chain fatty acids. The diglyceride fraction 

 which was strongly C 14 -labeled was transesterified, and the fatty acid 

 methyl esters were analyzed by gas chromatography. The fatty acids 

 found were mainly myristic, palmitic, and stearic acids, but in some 

 samples oleic acid and lauric acid were also present. The C 14 label 

 was not in the fatty acids but in the glycerol moiety, when the incu- 

 bation substrate had been C 14 -glucose or C 14 -glycerol. The glycerides 



