Expression of mutant zona proteins. (A) Immunoblot of eggs (lanes 1 to 3) or embryos (lanes 4 to 6) from normal (lanes 1 and 4), Zp2^mut transgenic (lanes 2 and 5), and Zp2^Mut (lanes 3 and 6) mice using ZP2 antibodies (3). Molecular mass is at left. (B) Eggs and embryos from normal (1 to 4) or Zp2^Mut (5 to 8) mice were stained with rhodamine-conjugated LCA to image cortical granules, which were present in ovulated (1 and 5) but not in fertilized (3 and 7) eggs. The metaphase spindle (arrows) was stained with a fluorescein-conjugated antibody to α-tubulin, and pronuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (asterisks). (C) Same as (A) but with eggs and embryos from normal (lanes 1 and 3) and Zp3^Mut (lanes 2 and 4) mice using ZP3 antibodies (19).

Sperm binding to eggs and embryos. (A) Sperm binding to Zp2^Mut eggs (1 and 2) and two-cell embryos (3 and 4) was assayed after 1 hour of incubation with normal capacitated sperm using normal two-cell embryos (5 and 6) as wash controls. The number of sperm bound to eggs and embryos was determined by confocal microscopy. (B) Same as (A) but with eggs and embryos from Zp3^Mut females.

Reversible sperm binding. (A) Normal (1), Zp2^Mut (2), and Zp3^Mut (3) eggs and Zp2^Mut embryos (4) were incubated with capacitated sperm for 1 hour and stained with Hoechst before imaging by confocal microscopy. After a brief rinse, capacitated Acr3-EGFP sperm [5 × 10⁵ ml⁻¹ of human tubal fluid (HTF)] were added and incubated for an additional 1 hour. After washing with normal two-cell embryo controls (insets) to remove nonadherent sperm, eggs and embryos were stained with Alexa 568–SBTI before imaging by confocal microscopy (5 to 8). Acrosome-reacted and -intact sperm were labeled with Alexa 568 and EGFP, respectively. Images were modified in Adobe Photoshop to remove nuclear staining from EGFP-positive sperm; thus, Hoechst-positive, EGFP-negative sperm reflect those that were not displaced by EGFP sperm. (B) Quantification of the number of sperm bound to eggs and two-cell embryos before (blue bars labeled original) and after (green bars labeled displaced) the addition of Acr3-EGFP sperm. Residual sperm (blue bars) reflect those not displaced by Acr3-EGFP after 1 hour of incubation and removal of nonadherent sperm.

Sperm binding to Zp2^Mut and Zp3^Mut eggs and embryos. (A) Normal (1 to 6), Zp2^Mut (7 to 9), and Zp3^Mut (10 to 12) eggs or Zp2^Mut two-cell embryos (13 to 15) were inseminated with capacitated Acr-EGFP sperm (1 × 10⁵ ml⁻¹ of HTF), and fertilized eggs and embryos were imaged at 1, 2, and 4 hours by differential interference contrast and confocal microscopy. (B) Capacitated Acr3-EGFP sperm were incubated in the same media as in (A) and assayed by computer-assisted sperm analysis after 1, 2, 4, 6, and 24 hours. Each data point, which is an average of three independent biological samples ± SEM, reflects the percent of sperm meeting the specific criteria. (C) Ovulated eggs in cumulus from normal, Zp2^Mut, and Zp3^Mut mice were inseminated with capacitated sperm (1 × 10⁵ sperm ml⁻¹ of HTF), and fertilization was assayed by the presence of two-cell embryos after overnight incubation. The data reflect the average of five separate experiments for each genotype (a total of 66 to 92 eggs) ± SEM.