An improved mouse sperm-oocyte plasmalemma binding assay: studies on characteristics of sperm binding in medium with...

Sperm binding to the oocyte plasmalemma is crucial to subsequent steps in fertilization. However, the usual in vitro assay for sperm-oocyte binding does not distinguish between nonspecific attachment and specific binding leading to fusion and penetration. Since zona pellucida-free pronuclear zygotes should not bind sperm (because of the block to polyspermy at the level of the oocyte membrane), a procedure has been developed to remove virtually all sperm from zona-free pronuclear zygotes (2PNZ). After six washes with a 90-microm-bore pipette, there were 0.5 +/- 0.2 sperm/2PNZ (n = 83). Therefore, these washing conditions were used to define sperm-oocyte binding. The relationship of binding to oocyte penetration was determined for outbred mouse oocytes coincubated in complete medium with (B6 x 129)F1 hybrid sperm (10(7)/ml). Binding was maximal (29 +/- 5 sperm per oocyte) during the first 30 minutes but decreased significantly to 4.6 +/- 1.4 by 60 minutes of coincubation (over 10 trials). Oocyte penetration was 99 +/- 1% by 30 minutes, while the number of decondensed sperm nuclei per oocyte increased significantly to 7.5 +/- 0.6 at 60 minutes. These data suggest that the block to polyspermy involves detachment of bound sperm. Similar coincubations were carried out in medium without glucose (NoG), as this medium has been reported to inhibit fusion without affecting binding. However, binding was only 11 +/- 2 at 30 minutes but increased to 25 +/- 4 at 60 minutes, suggesting that binding was retarded in NoG. When gametes were coincubated in NoG for 5 hours, about half of the oocytes were penetrated, suggesting that the lack of glucose did not inhibit fusion but instead delayed it. These data suggest that if sperm binding is to be determined in complete medium, the time of the block to polyspermy should be determined prior to selecting the appropriate time to assay binding. Furthermore, using the same coincubation period for the binding assay in control and treated sperm may not be appropriate if the treatment alters the time of maximal binding.