ESTROUS SHEEP SERUM AS A POTENT AGENT FOR OVINE IVF - EFFECT ON CHOLESTEROL EFFLUX FROM SPERMATOZOA AND THE ACROSOME REACTION

The aim of the present study was to evaluate the effect of heat-inacti vated estrous sheep serum (ESS) on sheep IVF. When the capacitation an d the fertilization media contained 20% ESS, a fertilization rate of 8 % was achieved. The beneficial effect of ESS on sheep IVF was further demonstrated since the fertilization rate was null when ESS was omitte d during sperm capacitation and fertilization. Estrous sheep serum sup ported both sperm capacitation and fertilization as shown by the resul ts of experiments in which it was omitted during one of these steps: s perm capacitation in serum-free medium resulted in delayed sperm-oocyt e penetration, while fertilization in serum-free medium significantly decreased the percentage of fertilized oocytes. To investigate the inf luence of serum on sperm ability to undergo the acrosome reaction, sal t-stored follicular sheep oocytes were inseminated, and the acrosomal status of spermatozoa attached to zonae was examined by electron micro scopy after a 4-h period of coincubation. Quantitative analysis on thi n sections demonstrated that fewer acrosome-reacted spermatozoa were o bserved when the capacitation and insemination steps were carried out in DM-H medium without serum than in DM-H-SS supplemented with 20% ESS (0.08, [0; 0.34], (median, range)/100 mu m zona vs 1.32, [0.90; 2.28] /100 mu m zona; P < 0.01). Since a higher number of spermatozoa attach ed to the zona surface in DM-H medium, the proportion of acrosome-reac ted spermatozoa was much lower (0.7%, [0%; 2.2%], (median, range) vs 5 4%, [25%; 100%]; P < 0.01) in the absence of serum. These results indi cate that in our IVF system the development of the acrosome reaction d epended on serum. Sperm cholesterol efflux during in vitro capacitatio n was measured on [H-3]cholesterol labeled spermatozoa resuspended in DM-H or DM-H-SS medium. A time-dependent cholesterol removal was obser ved in the presence of serum (60 +/- 5%, mean +/- SD, after 5 h), wher eas it was limited to 14 +/- 3 % in DM-H medium; hence addition of ser um to the capacitation medium efficiently supports cholesterol efflux, which is thought to be a key-event in the capacitation process.