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. 2010 Mar 18;285(20):15627–15636. doi: 10.1074/jbc.M109.079483

FIGURE 4.

FIGURE 4.

Inhibitory effect of dicalcin on sperm binding and sperm penetration through the VE protein layer. A, representative micrographs of unfertilized (Unfertilized) and fertilized (Fertilized) eggs after sperm treatment. Scale bar: 20 μm. B, effect of pretreatment with anti-dicalcin antibody on sperm binding to egg. Ovulated eggs were pretreated with preimmune antibody (Pre) or anti-dicalcin antibody (Anti-dica) followed by insemination. After rinse, bound sperm at an equatorial plane were counted (n = 9; *, p = 0.027). Almost no sperm remained attached to fertilized eggs (Fertilized). C, effect of pretreatment with dicalcin on sperm binding to egg. Ovulated eggs were pretreated with BSA or dicalcin at the indicated concentrations, and bound sperm were counted (n = 9; *, p = 0.003). D, a scheme that represents our in vitro penetration assay. Suspension of viscous VE proteins (VE) was placed on the mesh of the upper chamber. Some sperm (S) successfully penetrated through the VE. E, representative confocal image of Rhodamine-RCAI-treated VE proteins on the mesh of the upper chamber (red square in D). VE proteins stained with Rhodamine-RCAI (red) were observed. A penetrating spermatozoon stained with Hoechst 33342 was recognized in XZ and YZ planes (arrows). The potential trait of the spermatozoon through the VE is indicated (arrowheads). A direction from the upper chamber to the lower one is indicated (arrows at upper right). m, mesh. Scale bar: 50 μm. F, time course of sperm penetration. Sperm in the lower chamber were collected at the indicated times, and the percentage of the number of the penetrated sperm was calculated. The extent of penetration reached maximum at around 10 min. G, effect of pretreatment with dicalcin on sperm penetration. VE proteins on the upper chamber were pretreated either with dicalcin (4 μm) or BSA (4 μm) for 15 min, followed by sperm placement. After 5 min, sperm in the lower chamber were collected and the number of the penetrated sperm was calculated. The extent of penetration pretreated with BSA was set to ∼100% (n = 6; *, p = 0.039). BSA, pretreated with BSA; dicalcin, pretreated with dicalcin.