The Role of N-WASP in ORC4 Mediated Polar Body Extrusion

Abstract

Oocyte meiosis is completed though two asymmetric cellular divisions, where the oocyte extrudes half of its DNA two times, into two smaller daughter cells called polar bodies. N-WASP is an important factor in the actin polarization pathway. We investigated whether N-WASP is required for the extrusion of both polar bodies during mouse female meiosis. Previous work in our laboratory demonstrated that the DNA replication protein origin recognition complex 4 (ORC4) forms a cage around the chromosomes that will be extruded during polar body extrusion (PBE), but not the chromosomes that remain in the oocyte. My hypothesis is that N-WASP’s involvement in action polarization may be important for PBE, and related to ORC4 cage formation. We first localized N-WASP during oocyte progression through meiosis and found that it co-localizes with ORC4, except that N-WASP was not present in the initial stage, GV oocytes, while ORC4 was. This finding suggested the possibility that N-WASP was synthesized during meiosis. We tested this by injecting siRNA into GV oocytes upon collection, then allowing them to mature. We found that only 23.2% + 2.5% (MEAN+SD) of oocytes injected with siRNA directed towards N-WASP progressed to MII (the normal end point for in vitro GV maturation), while 92.9% + 10.1% of oocytes injected with control siRNA progressed to MII. These data suggest that (1) N-WASP may function in the same process that regulates PBE that ORC4 does, (2) that N-WASP is synthesized during meiosis, and (3) that N-WASP is required for progression to MII.