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Serine protease inhibitors cause egg activation defects and reduced calcium waves. A, B: Eggs fertilized in vitro in E2 medium with (B) or without (A) 5mg/ml Aprotinin. C, D: Normalized pseudo-coloured fluorescent images of eggs harvested from a Tg(actb2:GCaMP6s)lkc2 female, indicating Ca++ levels at 5 min postactivation (mpa) in E2 (C) or in 5mg/ml Aprotinin (D). E: Changes in GCaMP6s intensity over 10 min of Tg(actb2:GCaMP6s)lkc2 eggs activated with E2 (black) or 5 mg/ml Aprotinin (red). F: Corresponding Area Under Curve of GCaMP6 intensity levels presented in (E) following egg activation. n=4; Mann–Whitney test; * = p < 0.05. G–I′: Low (G, H, I) and high (G′, H, I′) magnification images of embryos fertilized in vitro in E2 (G–G′), or in E2 with 40 mg/ml Benzamidine HCl treatment and subsequently injected with 0.7 mM KCl (H–H′), or in E2 with 40 mg/ml Benzamidine HCl treatment and injected with 10 mM IP3 in KCl (I–I′). J: Proportion of embryos showing egg activation phenotypes and blastomere division. Benzamidine incubation aborts egg activation, which can be reactivated by IP3 injection. Key: CL: Chorion Lift, BD: Blastodisc, CD: Cell Division, +: Present, −: Absent. Chi-squared analyses; *** = p<0.001. Scale bars: B, I = 500 µm; D, I′ = 200 µm. See file S1 Data for underlying data.
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