Effect of protein kinase C stimulators on zona pellucida binding and the acrosome reaction of macaque sperm

Macaque sperm require treatment with dibutyryl cAMP (dbcAMP) and caffeine (termed activators; ACT) to fully capacitate in vitro. Previous studies have shown that treatment with ACT also increases sperm binding to the macaque zona pellucida and enhances the induction of acrosome reactions of the bound sperm. This study investigated whether phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-sn-glycerol (DOG), which stimulate protein kinase C (PKC), can also increase sperm binding to the zona pellucida and/or enhance induction of the acrosome reaction of bound sperm. Cyanomolgus macaque sperm were centrifuged through 60%. Percoll and then were washed with modified Biggers, Whitten and Whittingham (BWW) medium and incubated for 2.5 h at 37°C with 5% CO₂. ACT, PMA, or DOG was added to sperm during the last 15- 30 min of incubation. Sperm were evaluated immediately after incubation for motility and acrosomal status. Macaque oocytes were coincubate with stimulated sperm suspensions in BWW medium for 30 sec (pulse). Half of the oocytes were removed to sperm-free medium and further incubated for 1 h (chase). When assessed after the pulse period, sperm-zona binding increased significantly after ACT treatment compared to that in untreated controls. DOG but not PMA treatment had a similar effect on sperm binding. PMA, DOG, and ACT treatment increased the percentage of acrosome reactions in sperm bound to the zona following the 30-sec pulse compared to the percentage in controls. Inactive analogs of PMA and DOG had no effect on sperm-zona binding or the percentage of acrosome reactions (% AR) of bound sperm. The results suggest that both the production of cAMP and that of diacylglycerol second messengers are involved in mediation of the zona-induced acrosome reaction of macaque sperm. Treatment with PMA and DOG also increased the % AR in sperm suspensions incubated without oocytes. To test the hypothesis that the increase % AR of zona-bound sperm was due to preferential binding of acrosome-reacted sperm, the sperm-zona binding experiments were also performed in supplemented media with minimal Ca²⁺ (Ca²⁺-free media). The % AR of zona-bound sperm in the Ca²⁺ free media was significantly less than in media with Ca²⁺. Because Ca²⁺ is required for the zona-induced acrosome reaction, this results suggests that the increase in acrosome reactions of zona-bound sperm resulted after zona binding.