PARENT SESSION
PLATFORM SESSION 19: SPERM-EGG BINDING AND EGG ACTIVATION
Chair: Miller, David¹, ¹
Co-chair: Rivera, Rocio¹, ¹
Harborside E
2:00 PM-4:00 PM

(439) CHARACTERIZATION OF CYTOPLASMIC DYNEIN AND DYNACTIN AND THE PRONUCLEAR MOVEMENTS DURING MAMMALIAN FERTILIZATION.

Payne, Christopher^1,3, Takahashi, Diana¹, Simerly, Calvin^1,2, Schatten, Gerald^1,2, ¹ Pittsburgh Development Center, Pittsburgh, PA³ Department of Cell and Developmental Biology, Portland, OR² Departments of OB/GYN-Repro. Sci. and Cell Biol.-Physiology, Pittsburgh, PA

ABSTRACT- Mammalian fertilization is a complex and remarkable process, during which the diploid genome is restored through the union of the haploid oocyte and sperm. This restoration is achieved in most mammalian zygotes through the migration of the female pronucleus (FPN) towards the male pronucleus (MPN) along sperm aster microtubules. While dependence of pronuclear movements upon microtubules has been known for some time, the characterization of molecular motors on the pronuclear migration events is just now underway. Here, using immunocytochemistry, SDS-PAGE, and Western blotting, we examined the microtubule-based motor protein cytoplasmic dynein, along with its cofactor dynactin, in pronucleate-stage bovine zygotes. We found that both cytoplasmic dynein and dynactin associate with the surface of the FPN, while only dynactin associates with the surface of the MPN. Treatment of zygotes with 20 uM nocodazole, a microtubule-disrupting drug, significantly decreased the association of cytoplasmic dynein with the FPN (7±2.9%, vs. 81±6.4% control; n=489; p<0.001). Nocodazole treatment did not, however, significantly inhibit the association of dynactin with both pronuclei (62±9.5%, vs. 80±4.9% control; n=479). This result suggests that dynactin may be interacting with a component of the pronuclear envelope in the absence of microtubules. Co-immunoprecipitation and co-localization experiments revealed that the p150^Glued subunit of dynactin interacts with p62 nucleoporin, a member of the Nuclear Pore Complex (NPC). Microinjection of p150^Glued antibodies into oocytes, followed by in vitro fertilization, both inhibited the association of dynactin with the surfaces of the pronuclei, as well as inhibited pronuclear migration, with 84% (95/113) of zygotes showing pronuclei at or > 10 um apart. Thus, the association of dynactin with the NPCs of the male and female pronuclei is required for pronuclear migration.

KEY WORDS: cytoplasmic dynein, dynactin, nuclear pore complex, pronuclear migration