Supplemental Figure 1
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Fig. S1. Acr3-EGFP sperm bind, acrosome exocytose and fertilize mouse eggs. (A) Composite confocal photomicrographs of Acr3-EGFP sperm after staining with Alexa 568-conjugated soy bean trypsin inhibitor (SBTI) and Hoechst. Intact acrosomes are indicated by green on the anterior surface of sperm heads and nuclei are imaged as blue. The upper portion is a confocal image of the heads and midpieces of sperm imaged by brightfield in the lower portion. Scale bar: 5 μm. (B) Same as in A except after induction of the sperm acrosome reaction with the calcium ionophore A23187 (3 μM). Acrosome-reacted sperm lack EGFP and bind Alexa 568-SBTI (red). (C) Bar graph (mean±s.e.m.) of Acr3-EGFP sperm binding to cumulus-free ovulated eggs. Acr3-EGFP and normal sperm (5×105) were mixed in different ratios (1, 100% Acr3-EGFP sperm; 2, 75% Acr3-EGFP and 25% normal sperm; 3, 50% Acr3EGFP and 50% normal sperm; 4, 25% Acr3-EGFP and 75% normal sperm; 5, 100% normal sperm) and incubated with eggs for 30 minutes. Using sperm binding to two-cell embryo as negative wash controls, Acr3-EGFP sperm binding to eggs was determined as a percent of total sperm binding. (D) Eggs or one-cell embryos were recovered 0, 15, 30 or 60 minutes after insemination with capacitated Acr3-EGFP sperm. Following permeabilization, eggs and embryos were stained with Rhodamine-conjugated Lens culinaris agglutinin (LCA) lectin to detect cortical granules (red), Hoechst to identify nuclei (blue) and imaged by confocal microscopy to identify intact acrosomes (green) on the anterior head of sperm.
