PROGESTERONE IN MARE FOLLICULAR-FLUID INDUCES THE ACROSOME REACTION IN STALLION SPERMATOZOA AND ENHANCES IN-VITRO BINDING TO THE ZONA-PELLUCIDA

The aim of this study was to investigate whether mare follicular fluid (FF) induces the acrosome reaction (AR) in stallion spermatozoa and, if so, to identify the component in FF responsible for it. Furthermore , the effect of this component on sperm-zona binding and dir subsequen t AR was studied. Pooled FF, aspirated from the preovulatory follicles of mal-rs in oestrous, was used and aliquots of the fluid were treate d with charcoal to remove steroids (CFF). Charcoal treatment reduced t he progesterone concentration in FF from 153 to <2 ng/mL. Spermatozoa from fertile stallions collected by a swim-up procedure were preincuba ted in modified Tyrode's medium for 5 h and then incubated for 30 min at 37 degrees C with either (1) 50% FF + 50% CFF, (2) 50% FF + 50% CFF + 150 ng/mL progesterone, (3) 50% CFF + 150 ng/mL progesterone, ( 1) 150 ng/mL progesterone or (5) modified Tyrode's medium alone. The sper m-hemizona assay was applied: (a) to compare the number of spermatozoa bound to a hemizona in the presence and absence of 1.5, 15 or 150 ng/ mL progesterone after 1 h co-incubation of spermatozoa and hemizonae, (b) to compare the incidence of the Ali in spermhemizona complexes inc ubated for 1 h in the presence and absence of 1 mu g/mL progsterone. B oth spermatozoa in suspension and bound to a hemizona were treated wit h the supravital dye Ethidium homodimer and fixed. Their plasma membra nes were permeabilized, and the outer acrosomal membranes were labelle d with FITC-PNA. Viable spermatozoa without the outer acrosomal membra ne were considered as physiologically acrosome-reacted. Results showed that (1) FF induced a higher percentage of AR than did CFF or modifie d Tyrode's medium, (2) addition of 150 ng/mL progesterone to CFF resto red 77% of the AR-inducing activity and (3) CFF and modified Tyrode's medium both induced the AR to a similar extent when supplemented with 150 ng/mL progesterone. Neither FF nor progesterone treatment affected sperm viability severely. The number of spermatozoa bound to a hemizo na in the presence of 15 and 150 ng/mL progesterone was significantly higher (p < 0.05) than the number of spermatozoa bound in the absence of progesterone. A higher incidence of the AR was found in sperm-hemiz ona complexes incubated in the presence of progesterone (55.6 +/- 3.3% vs. 27.1 +/- 4.3%, in the presence and absence of progesterone, respe ctively) (n = 15, p < 0.05). It is concluded that mare FF can induce t he AR in stallion spermatozoa. Progesterone is the physiological compo nent responsible for this AR-inducing capacity. Progesterone enhances sperm-zona binding activity and exerts an additive effect on the zona- induced AR.