86 The Biochemistry of Semen 



which plasmolyse the tails (together with the middle-pieces), but not 

 the heads. In this way he obtained by centrifugation two portions, a 

 supernatant fluid representing the cytoplasm, and a deposit con- 

 sisting of sperm-heads which could be further purified by washing 

 with water. 



According to Miescher's calculations, in salmon spermatozoa the 

 heads and tails contribute 76 and 24% of fresh material, and 87 and 

 13% of the lipid-free material, respectively. Suspensions of fish 

 sperm-heads obtained by plasmolysis, centrifugation and washing, 

 are largely sperm nuclei and that is why they have been used exten- 

 sively for the study of nucleoproteins by Miescher and others who 

 followed in his footsteps. It is, however, rather uncertain what pro- 

 portion of cytoplasm defies aqueous extraction and how much 

 protein is lost from the sperm-heads in the course of washing. It is 

 quite likely that losses of varying magnitude occur, which would 

 account for the discrepancies in analytical results obtained by 

 different authors, particularly as regards the content of cytoplasmic 

 and non-basic nuclear proteins of fish spermatozoa. 



The supernatant fluid obtained by centrifugation of plasmolysed 

 salmon spermatozoa was found by Miescher to be rich in soluble 

 proteins and lipids. On addition of ethanol he obtained two frac- 

 tions, one which was ethanol-insoluble, accounted for 41-9% of dry 

 material and contained mainly protein (C 51-85, H 7-10, N 14-94, 

 S 1-37), and the other ethanol-soluble, equal to 51-8% of dry 

 material and made up of lecithin, fat and cholesterol. Salmon 

 sperm cytoplasm is known to contain phosphatases active, amongst 

 others, towards adenosine triphosphate; it is devoid of deoxynucleo- 

 proteins but contains apparently some ribonucleic acid and several 

 free amino acids, namely alanine, valine, isoleucine, tyrosine, aspar- 

 tic acid, and glutamic acid (Felix et al., 1951). 



Mammalian spermatozoa, in contrast to those of fishes, cannot be 

 plasmolysed, and their heads do not come off" in water or acid. To 

 overcome this obstacle, Zittle and O'Dell (\94la, b) exposed bovine 

 epididymal spermatozoa to ultrasonic waves and in this way dis- 

 sociated the sperm-heads from the middle-pieces and tails. On slow 

 centrifugation of the disintegrated sperm suspensions, the heads 

 settled out first; at increasing speed, the middle-pieces also formed 

 a sediment, leaving in the supernatant fluid most of the fragmented 



