Solubilized zona pellucida proteins and progesterone induce calcium influxand the acrosome reaction in capacitated dog spermatozoa

Spermatozoa from the sperm-rich fractions of the semen of 6 beagle dogs wer e capacitated and the effect of both zona pellucida (ZP) proteins and proge sterone on calcium flux and the acrosome reaction measured. Sperm calcium f lux was determined using the dual wavelength calcium probe indo-1/AM (6 muM ) in a flow cytometric assay (one ejaculate from each dog examined; n = 6). No calcium flux was observed in the negative control treatments (RPMI medi um or DMSO). Both heat-solubilized bitch ZP proteins and progesterone cause d a similar response characterized by a gradual but marked influx of calciu m ions which was sustained over 2 min. Acrosomal status was assessed by ind irect immunofluorescence using a specific monoclonal antibody following 1 h r incubation for each treatment (four ejaculates from each dog examined; n = 24). The level of acrosomal exocytosis was very high for samples treated with ZP proteins (70.3 +/- 2.1%) and progesterone (84.6 +/- 1.5%) and was s ignificantly different from the respective controls (P < 0.001), Interestin gly the patterns of calcium flux in response to both ZP proteins and proges terone were in contrast to the situation in other species studied to date r aising the possibility that the mechanism for triggering the acrosome react ion may be different in dog spermatozoa. In addition the high degree of pro gesterone-induced acrosomal exocytosis compared to other species raises the probability that the majority of dog spermatozoa are already undergoing th e acrosome reaction before they reach the egg ZP. Mol. Reprod. Dev. 60: 491 -497, 2001, (C) 2001 Wiley-Liss, Inc.