Stob_1962_Nature_196_1318

A metabolite with possible anabolic and marked uterotrophic activity has been isolated from extracts of the fungus Gibberella zeae (Fusarium graminearum). Numerous reports of genital involvement in animals fed spoiled grain prompted us to investigate the possible relationship of micro-organisms to this syndrome. During 1957 and 1958 we observed seven widely separated swine herds showing vulvar hypertrophy and occasional vaginal eversion in females, preputial enlargement in castrated males and prominent mammary glands in both sexes. In each case the animals were fed moulded corn. Spoiled grain was procured from each farm, treated with sodium hypochlorite and plated on potato-dextrose agar. Predominant micro-organisms isolated from the samples were Penicillium spp., Cladosporium sp., Mucor spp. and Gibberella zeae. Cultures of these moulds were grown on sterile ground com (35 per cent moisture) in flasks stored at about 24 C for 2-3 weeks. This moulded corn was fed ad libitum to sexually immature female pigs. Within four days, vulvar and mammary enlargement were elicited only in those animals fed corn seeded with G. zeae isolates. Similarly prepared corn fed for six days to five mature, ovariectomized mice produced an average uterine weight of 943 mg compared with 226 mg for the control animals. An alcoholic extract of the Gibberella moulded corn incorporated in sesame oil and injected subcutaneously into 10 castrated female mice at a dose of 01 ml per animal daily for 3 days produced an average uterine weight of 607 mg compared with 206 mg for the controls. Nine isolates of G. zeae were obtained which produced varying degrees of uterotrophic activity. To obtain material for chemical and pharmacological tests, a study was made of the cultural requisites for the production of the active principle. substrates include wheat or barley as well as com or a synthetic medium containing mineral salts and carbohydrates ineluding com starch, high test molasses or D-glucose. An easily separable, water-soluble fraction, toxic for mice, is occasionally produced concurrently with the metabolite. This confirms the earlier work of Christiansen and Kemkamp and Mitchell and Beadles associating toxicity with the ingestion of G. zeae-infected grain. Recovery of the metabolite is effected by water extraction of the corn-mycelial mixture in a highspeed blender to remove the water-soluble fractions. The wet cake remaining after filtration is repeatedly blender-extracted with anhydrous ethanol to remove the active principle. The alcoholic extracts are combined and the solvent removed by distillation. An aqueous-organic phase partition of the resulting residue is followed by application of the concentrate to a column of synthetic magnesium-silicate. Subsequent development by gradient elution using solvents of increasing polarity separates the active component. The solvent mixture is evaporated from the active fraction resulting in a white, crystalline mass which upon recrystallization has a melting point range of 16P-163 C, (0()25/Hg= -82 (c.=24 ethanol). A methanolic solution of the metabolite exhibits a bright, greenish-blue fluorescence when exposed to ultra-violet light. Ultra-violet absorption peaks occur at about 237, 275 and 315 m[L (methanol). Additional chemical, physical and structural data will be forthcoming as pure material becomes available. Preliminary experiments indicate that partially purified concentrates of the active principle are effective in improving growth-rate and feed efficiency in sheep.