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In 1957, Chang reported the presence of a substance in the seminal plasma of several mammalian species which had the ability to reverse the capacitation process (decapacitation factor or DF). Bedford & Chang (1962) were partially able to purify DF by ultra-centrifugation. Since that time, a great deal of effort has been given to the purification, isolation and identification of this substance (Chernoff, Pinsker, Dukelow 8c Williams, 1966; Dukelow, Chernoff & Williams, 1966a, b, 1967a, b; Pinsker & Williams, 1967; Williams, Abney, Chernoff, Dukelow & Pinsker, 1967; Dukelow & Chernoff, 1968). These reports have demonstrated that DF can be destroyed by crude amylase pre¬ parations but is stable to neuraminidase, pronase and glucose oxidase; that it exists in a wide variety of species but is not present in all mammalian seminal plasma; that it can be reduced in molecular size with pronase without de¬ stroying DF activity, and that it is resistant to destruction by heat and cold. Semen was collected from rabbits with an artificial vagina. These samples were centrifuged at 862 g for 15 min to remove the sperm cells, diluted with an equal volume of Krebs Ringer phosphate (KRP) and then frozen until a pooled volume of 75 ml had been accumulated. All reagents used in these extractions were analytical grade from Mallinckrodt Chemical Company, St Louis, Missouri. The seminal plasma was extracted with 200 ml of chloroform : methanol (2:1 v/v) as shown in Text-fig. 1. This extraction was repeated three times and the extracts were combined. These combined extracts were reduced to dryness and resuspended in KRP to provide a sample isotonic to spermatozoa for biological assay. This was designated the chloroform : methanol (CM) fraction. The nonlipid residue was evaporated to approximately 15 ml and called the NLR sample. In an attempt at further purification, the KRP resuspended rabbit chloro¬ form : methanol extract was extracted three times with redistilled, low boiling (40 to 60° C) petroleum ether (1:10 v/v). The petroleum ether extracts were combined and evaporated to dryness. This residue was triturated with 10 ml

DECAPACITATION FACTOR PURIFICATION WITH LIPID SOLVENTS