Evidence suggesting a role for sperm metalloendoprotease activity in penetration of zona‐free hamster eggs by human sperm

Abstract

It has been reported that metalloendopotease (MEP) activity is involved in somatic cell membrane fusion events and in the sea urchin sperm acrosome reaction (AR). MEP activity also has been demionstrated in human and other mammalian sperm. The present study was concerned with investigating whether a human sperm MEP is important in membrane events necessary for sperm egg fusion. Ejaculated human sperm were washed, capacitated in vitro, and preincubated with the competitive MEP inhibitors phosphoramidon (50 μM) or CBZ-L-phenylalanine (1 mM), with 100 μM diethylenetriaminepentaacetic acid (DTPA), a heavy metal chelator, or as controls, with the appropriate solvents. The AR was initiated in vitro with preovulatory human follicular fluid and the sperm washed to dilute inhibitors and then coincubated with zona-free golden hamster eggs (zonae and cumuli removed with trypsin and hyaluronidase, respectively). Eggs were washed after 0.5 h, and the number of sperm remaining bound was counted. After 2.5 h further incubation, the eggs were stained with acetolacmoid or acetoorcein and penetration was assayed by counting the number of decondensed sperm heads per egg (penetration index) and the percent of penetrated eggs. The inhibitor treatments did not decrease the percentage of penetrated eggs (range 80–90%), but a significant reduction in the penetration index was observed. Phosphoramidon reduced the penetration index by 45%, CBZ-L-phenylalanine by 57%, and DTPA by 56%. None of the inhibitors decreased the penetration index or the percentage of penetrated eggs when added directly to suspensions of acrosome-reacted sperm and zona-free eggs at the diluted levels that would have been present after washing inhibitor-treated sperm. Other studies demonstrated that the MEP inhibitors did not inhibit sperm motility, sperm binding, the occurrence of the AR, the loss of acrosomal matrix and AR vesicles, or sperm head decondensation. Phosphoramidon also significantly decreased the penetration index when the AR was initiated by the calcium ionophore ionomycin. These results suggest that during the AR, a human sperm MEP is involved (directly or indirectly) in modifying the sperm plasma membrane so as to increase its potential for fusion with the egg plasma membrane.