The objectives of this study were to determine if human ejaculated sperm exhibit active caspases and if caspase-dependent apoptosis markers are identifiable. Sperm from fertile donors and infertile patients were examined after gradient separation into leukocyte-free fractions of high and low motility. Sperm were evaluated for motion parameters, morphology, caspase activation, and apoptosis markers including phosphatidylserine (PS) translocation (annexin V binding) and DNA fragmentation (TUNEL). Active caspase-3 was detected by immunofluorescent microscopy in a small proportion of sperm in situ, in fractions of high and low motility sperm of patients and donors, but low motility fractions had significantly higher numbers of positive sperm. Immunoblot analysis detected inactive procaspase-3 (32 kDa) in all fractions of low sperm motility from patients and donors, while active caspase-3 (17 kDa) was only detected by immunoblotting in a limited number of low motility fractions from patients and in even fewer fractions from donors. Caspase enzymatic activity, as measured using the fluorogenic substrate DEVD-afc, was higher in patients than in donors in both low and high motility fractions. Annexin V staining and DNA fragmentation were detected in a proportion of sperm, with a higher frequency in the low motility fractions. A significant positive correlation between in-situ active caspase-3 in the sperm midpiece and DNA fragmentation was observed in the low motility fractions of patients, suggesting that caspase-dependent apoptotic mechanisms could originate in the cytoplasmic droplet or within mitochondria and function in the nucleus. These data suggest that in some ejaculated sperm populations, caspases are present and may function to increase PS translocation and DNA fragmentation.

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In these studies, we aimed to characterize the effects of the physiological, homologous agonists of the acrosome reaction, i.e. the zona pellucida (ZP) and progesterone/follicular fluid, on human sperm. The specific aims of our studies were: (i) to examine the dependency of the solubilized ZP-induced acrosome reaction on G(i) protein activation and presence of extracellular calcium; and (ii) to determine whether progesterone/follicular fluid exert a priming or synergist effect on the solubilized ZP-induced acrosome reaction. Highly motile sperm from fertile donors were exposed to the agonists in a microassay and the acrosomal status of live sperm was determined by indirect immunofluorescence using PSA-FITC/Hoechst double-staining. Pretreatment with pertussis-toxin (100 ng/ml) and EGTA (2.5 mmol/l) significantly inhibited the ZP-induced acrosome reaction without affecting the spontaneous rate of exocytosis. Progesterone (1.25 microg/ml) and human follicular fluid (10%) exerted a priming, time-dependent effect on the ZP-induced acrosome reaction. These studies demonstrated that: (i) acrosomal exocytosis of capacitated human sperm triggered by the homologous ZP is dependent on the activation of G(i) proteins (pertussis toxin-sensitive) and the presence of extracellular calcium; and (ii) progesterone and follicular fluid exert a priming effect on the ZP-induced acrosome reaction.