Studies on the metabolism of semen. 8. Ergothioneine as a normal constituent of boar seminal plasma. Purification and crystallization. Site of formation and function

Abstract

Ergothioneine was found in boar semen plasma but not in the spermatozoa. Produced only in the seminal vesicles, it accounts for the major part of the total reducing material which reacts with 2,6-dichlorophenolindophenol in the cold, and which yields the blue color with Folin''s phosphotungstic acid reagent for uric acid. The concn. of ergothioneine in the vesicular secretion as detd. in samples from 20 boars, 29-256 mg. /100 ml., avg. 79 mg./100 ml. From 1300 ml. of boar vesicular secretion 0.48 g. pure crystalline ergothioneine was isolated and identified by elementary analysis, detn. of the bromine-oxidizable S, diazo reaction, reducing properties, and by prepn. of certain derivatives including the crystalline Hg compound. For detection of small amts. of ergothioneine in tissues, urine and other body fluids, a chromatographic method was developed, which depends on localization of.u.-v.-absorbing areas on paper chromatograms followed by spraying with diazo reagent or certain other color-yielding reagents. In this manner it was possible to detect less than 5 [mu]g. ergothioneine, and to distinguish it from thiolhistidine, uric acid and ascorbic acid. Thiolhistidine, either free or bound, does not accompany ergothioneine in the boar seminal vesicles or in the vesicular secretion. The content of ascorbic acid in the boar vesicular secretion was low. In the bull, there is little ergothioneine, and the high reducing value of the vesicular secretion is largely due to ascorbic acid. Ram and human semen showed the presence of some reducing diazo-reactive substances which, though not identical with, may possibly be related to ergothioneine. Boar urine, unlike boar semen, contained only a negligible amt. of ergothioneine. The claim that human urine contains ergothioneine was not confirmed. None, or only a trace of it, was found in pig fetal fluid, vitreous fluid of the eye, corpora lutea, adrenal glands and thyroid gland. Ergothioneine, in a concn. corresponding to that found in the boar vesicular secretion, protected spermatozoa against the action of sulfhydryl inhibitors. Both motility and fructolysis, under anaerobic as well as aerobic conditions, were abolished in sperm suspensions by 10-3M iodosobenzoic acid, but could be maintained at a normal level if 2 x 10-3M ergothioneine was also added. Ergothioneine also counteracted the inhibition caused by cupric ions and H2O2. The protective effect of ergothioneine against inhibitors of SH-groups was not limited to sperm but could be demonstrated in muscle glycogenolysis and yeast fermentation.