The process of capacitation is poorly understood for ejaculated sperm. We have shown that ejaculated sperm undergo the acrosome reaction in the ampulla of the oviduct. A lysophosphatidylcholine (LC) sensitivity test was developed to separate capacitation from the acrosome reaction. A sperm capacitating factor possessing characteristics of heparin or heparan sulfate was found in oviduct fluid. Capacitation induced by heparin was Ca++ dependent and blocked by glucose with the block reversed by 8-bromo cAMP. The presence of seminal plasma alters sperm capacitation characteristics by inhibiting spontaneous capacitation of epididymal sperm. We hypothesize that the ejaculated sperm capacitating factor of oviduct fluid is heparan sulfate and that it is either masked or modulated during the estrous cycle such that only estrual oviduct fluid capacitates ejaculated sperm. We further hypothesize that capacitating agents capacitate sperm by first binding to sperm followed by sterol depletion and modification of the plasma membrane resulting in Ca++ uptake. We propose to test hypotheses supporting the following aims: A) to identify naturally occurring sperm capacitating agents in the bovine oviduct; by isolation of the factor using HPLC and affinity chromatography and by use of the LC sensitivity and in vitro fertilization assays we have developed to detect capacitation of bovine sperm. The factor will be identified from its structural characteristics. B) To determine the mechanism whereby capacitating agents cause capacitation of bovine sperm; by determining if binding of the agent to sperm is required, if cholesterol efflux occurs, if cAMP levels are modulated and if capacitation is dependent on Ca++ and blocked by agents blocking Ca++ uptake. C) To identify and isolate by HPLC, seminal plasma components which inhibit capacitation in vitro and to determine using the techniques of Aim B, the mechanisms whereby they prevent capacitation of bovine sperm. These experiments will for the first time isolate and identify capacitating agents from the mammalian oviduct. Results of these experiments will enhance our understanding of how capacitation occurs and thus our ability to manipulate the fertilization process.
