Fig. 3. Dynein and dynactin are required for pronuclear migration and apposition. (A) Pronuclear apposition is normal when control IgG antibodies are transfected into bovine zygotes, but female pronuclear migration is inhibited following transfection of antibodies against either dynein intermediate chain (70.1) or dynactin p150^Glued. Dynein shows normal distribution around the female (F), but not the male (M), pronucleus in controls (left) and localizes to the female pronuclear surface proximal to the sperm aster after anti-dynein antibody transfection (center). Dynactin concentration around both pronuclei is reduced following anti-dynactin antibody transfection (right). Microtubule (MT) organization is unperturbed in zygotes transfected with anti-dynein and anti-dynactin antibodies, with sperm asters clearly visible. Scale bars, 10 µm. (B) Quantification of the effects of antibody transfection on pronuclear apposition. Zygotes were transfected 12 hours post-insemination (HPI) and developed until 24 HPI, when they were then fixed and analysed by immunocytochemistry. Pronuclear apposition was scored by measuring the distance between pronuclei, with 10 µm representing the average diameter of a pronucleus. Pre-absorbed antibodies refer to anti-dynein and anti-dynactin antibodies incubated with their antigens or cell lysates prior to transfection.

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