The seminal fluid of many species clots soon after ejaculation, and the vaginal plug which forms after coitus in most rodents is composed of coagulated semen. Human semen is emitted in a gelatinous form but liquefies soon afterward. Early investigations (Huggins, C, and Neal, W. J. Exper. Med., 76:527, 1942) established that the powerful proteolytic enzymes in human seminal fluid are responsible for its liquefaction. But the mechanism of semen coagulation has received little attention. L. Camus and E. Gley (Compt. rend. soc. biol., Paris, 48:787, 1896) showed that in rodents the secretions of the seminal vesicle were coagulated by an enzyme (vesiculase) of prostatic origin. G. Walker (Bull. Johns Hopkins Hosp., 21:182, 1910) demonstrated that vesiculase was secreted solely by the anterior prostate or coagulating gland. In 1955 methods were developed (22, 23) whereby the coagulable protein of the vesicular secretion of the guinea pig could be prepared in a stable form, and simple optical methods were devised to study the clotting of this material by vesiculase. The latter enzyme was partially purified and separated from another enzyme in the guinea-pig coagulating gland that hydrolyzed tosyl-L-arginine methyl ester (TAMe) and related arginine esters. This provided evidence that vesiculase was not identical with thrombin or plasmin because it is well known that the latter proteolytic enzymes readily hydrolyze TAMe. Moreover, it was found that TAMe (which inhibits the coagulation of fibrinogen by thrombin) did not affect the action of vesiculase and that thrombin (and many other proteolytic enzymes) did not clot the seminal vesicle protein. The coagulating gland of the guinea pig and the prostatic secretion of the dog were shown to contain particularly high levels of this arginine ester-hydrolyzing enzyme, which was partially purified and its properties investigated (21). Later work by others (Freund, J.; Miles, A. A.; Mill, P. J.; and Wilhelm, D. L. Nature, 182:174, 1958) showed that this enzyme is probably responsible for the extreme toxicity of the secretion of the guinea-pig coagulating gland, which induces hypotensive shock when it is injected intravenously.

Further investigations (23) showed that the rate of coagulation of seminal vesicle protein by vesiculase, and the nature of the clot which formed, were extremely sensitive to environmental factors such as pH, temperature, and ionic strength. The coagulation process was shown to involve two distinct phases. The first stage was sensitive to metal-chelating agents and did not result in the formation of insoluble protein. The second stage (leading to a coagulum) was inhibited by heavy metal ions but not by metal-chelating agents.

Glycerophosphorylcholine In Vesicular Secretion

The presence of high concentrations of L-α-glycerophosphorylcholine in the seminal vesicle secretion of rats (Lundquist, F. Nature, 172:587,1953) was confirmed (196). It was shown that the levels of this substance fell dramatically after castration and could be restored to normal by treatment with testosterone. A study was made of the biosynthesis of choline-con-taining lipids by cell-free extracts of rat seminal vesicle (196). It was established that this tissue contained active phosphorylcholine-cytidyl and phos-phorylcholine-glyceryl transferases, which catalyze the synthesis of CDP-choline and lecithin, respectively. The latter reaction was markedly stimulated, in crude extracts, by the addition of adenine nucleotides such as AMP, ATP, and DPN. It was shown that this stimulatory action resulted from a competitive inhibition by the adenine nucleotides of the cleavage of CDP-choline by a nucleotide pyrophosphatase which is particularly active in the accessory glands. No evidence for the direct incorporation of choline from CDP-choline into glycerophosphorylcholine by seminal vesicle extracts was obtained, and it seems likely that the glycerophosphorylcholine in the vesicular secretion is derived from degradation of lecithin, or possibly other choline-containing lipids, in the seminal vesicle epithelium.