Protein Constituents of Spermatozoa 91 



but even here, the demonstration of the absorption bands of cyto- 

 chrome became possible (Mann, \95\d) by the application of the 

 technique of Keilin and Hartree (1949, 1950), whereby manifold 

 intensification of absorption bands is brought about by means of 

 liquid air. 



Our evidence for the occurrence and active participation of the 

 cytochrome system in the oxidative metabolism of mammalian 

 spermatozoa can be briefly summarized as follows. Whole fresh 

 semen examined a little while after ejaculation shows the diffuse 

 spectrum of oxidized cytochrome as well as, weakly, the absorption 

 bands of reduced cytochrome a, b, and c. However, after the addi- 

 tion of a reducing agent or on anaerobic incubation of the semen, 

 the bands of the reduced cytochromes become much more pro- 

 nounced, the cytochrome a band being more distinct than c, and 

 the latter stronger than cytochrome b. The picture is similar with 

 washed sperm suspensions in fructose-Ringer-phosphate solution; 

 freshly prepared sperm suspensions show mainly the spectrum of 

 oxidized cytochrome, which becomes reduced in the course of 

 anaerobic incubation; on aeration of the incubated suspension 

 cytochrome reverts to the oxidized form. The band of cytochrome a 

 can be shown to undergo a typical change under the influence of 

 carbon monoxide; the reaction product thus formed in the sperm 

 resembling closely the carbon monoxide compound of cytochrome 

 oxidase or cytochrome a^, originally described by Keilin and 

 Hartree (1939) in heart muscle preparations. Carbon monoxide, 

 cyanide, azide, hydroxylamine, and other typical inhibitors of the 

 cytochrome system in respiring tissues, all inhibit also sperm 

 respiration. 



An elegant experiment on the behaviour of cytochrome in sperma- 

 tozoa was performed by Rothschild (1948a, d) who demonstrated 

 that the oxygen uptake of sea-urchin sperm is inhibited by carbon 

 monoxide and that the inhibition can be completely reversed by 

 white light but not by light of the 548 m^^ wavelength; the non- 

 reversal at that particular wavelength being due to lack of absorp- 

 tion by cytochrome oxidase in this region of the spectrum. By 

 interposing between the source of light and the microscope a colour 

 filter transmitting light of the 548 m/n wavelength, Rothschild was 

 able to observe spermatozoa microscopically, in the presence of 



