Figure 3. Effect of PYK2 inhibition on sperm binding, fertilization, and oocyte activation.

The effectiveness of PF04594755 during a 30minute pre-treatment incubation was tested by incubating zona free oocytes with concentrations of PF04594755 between 10 and 75μM, then assessing total oocyte PYK2 PY⁵⁷⁹ content (Panel A (relative fluorescence intensity)) as described in “Materials and Methods”. The effect of different concentrations of PF04594755 or SKI606 on sperm binding (Panel B), fertilization (Panel C), and oocyte activation (Panel D), was tested as described in “Materials and Methods”. After 2 hrs incubation with capacitated sperm, oocytes were fixed and prepared for confocal fluorescence to assess sperm binding, sperm penetration, and anaphase resumption (egg activation). In some experiments, oocytes recovered from flox-Fak-CAT-eGFP homozygous females carrying the Zp3-cre transgene (Fak-null oocytes) were used for fertilization. The number of bound sperm per oocyte (Panel B), number of fertilized eggs containing incorporated sperm heads in the ooplasm (Panel C), or number of oocytes showing evidence of anaphase resumption (activated oocytes) (Panel D). Values represent the mean of at least 5 experiments +/− SEM. (*) indicates a value significantly different from that of unfertilized oocytes (P<0.05).