100 The Biochemistry of Semen 



Unfortunately however, the extraction with M-NaCl is not a 

 universal means for the separation of sperm nucleoproteins. In the 

 key-hole limpet or freshwater clam, the sperm nucleoprotein 

 resists extraction with M-NaCl, but can be brought into solution 

 with a 2m salt solution, whereas no nucleoprotein can be extracted 

 with NaCl of either concentration from the sperm of man, bull, 

 boar or ram. Moreover, dialysis against M-NaCl or extraction with 

 dilute mineral acids both prove inadequate for the removal of 

 nuclear proteins from mammalian spermatozoa. In such cases, the 

 separation of protein can be brought about with a chloroform- 

 octanol mixture (Sevag, Lackman and Smolens, 1938), but before 

 this is applied it is necessary to separate the sperm nucleus from the 

 remainder of the sperm cell by ultrasonic or mechanical treatment. 



Deoxyribonucleic acid 



This when freed from nuclear protein, is composed of mono- 

 nucleotides, each consisting of one molecule of phosphoric acid, 

 one molecule of the sugar D(-)2-deoxyribose, and one molecule of 

 a purine or pyrimidine base: adenine, guanine, cytosine or thymine. 

 A small amount of yet another base, 5-methylcytosine (Wyatt, 1950, 

 1951), has been found so far in the sperm deoxyribonucleic acid of 

 man, bull, ram, herring and sea-urchin {Echinus esculentus), but 

 probably it occurs also in other species. 



In all species, deoxyribonucleic acid is confined entirely to the 

 sperm nucleus as can be demonstrated by various staining methods, 

 and particularly by the 'Feulgen nucleal reaction'. This reaction was 

 described by Feulgen (1914, 1917) at first as a colour test for thymo- 

 nucleic acid, but later it was adapted for the staining of cell nuclei 

 (Feulgen and Rosenbeck, 1924). 



With thymonucleic acid itself, the test is carried out best on a 

 solution of sodium nucleinate (01 g./l ml.) prepared in a boiling 

 tube on the water-bath. The solution is treated with 1 ml. 2N-H2SO4, 

 and left at 100° for 3 minutes, then cooled and neutralized. When a 

 drop of the hydrolysate is mixed with a few ml. of Schiff^'s fuchsin- 

 sulphurous acid reagent (a 0-5% solution of fuchsin decolorized 

 with SO2 and the excess of SO2 removed by suction), an intense 

 purple colour develops. The chemistry of the Feulgen colour re- 

 action is as yet only partly understood but is believed to involve the 



