Submission Number: GAR-4-19-9

Abstract Number: 425

LOCALIZATION OF PHOSPHORYLATED-MAPK AND PP2A DURING MOUSE OOCYTE MEIOSIS AND THEIR ROLES IN MICROTUBULE POLYMERIZATION.

Qing Lu, Rowena Angeles and Gary D Smith*

Depts of OB/GYN, Physiology, and Urology, Reproductive Sciences Program, University of Michigan, Ann Arbor, MI. ¹

Abstract:
Phosphorylated-MAPK (phospho-MAPK) is believed to be required for meiotic spindle formation. PP2A also has been suggested to be important for spindle formation. However, little is known about their subcellular distributions, interactions or mechanism(s) by which they operate in spindle formation. In the present study, we investigated the intracellular localization of phospho-MAPK and PP2A during mouse oocyte meiosis and their relationship with microtubules (MTs). Using immunocytochemistry, confocal microscopy and Western blot analysis, we showed that phospho-MAPK was absent in germinal vesicle-intact (GVI) oocytes and those undergoing germinal vesicle breakdown (GVBD). Pronounced phospho-MAPK staining was observed at MT arrays of the first meiotic spindle. Within metaphase II (MII) oocytes, phospho-MAPK staining was predominantly congregated at spindle plates. PP2A was dispersed throughout the cytoplasm in GVI oocytes. At GVBD, PP2A displayed a clustering within the center of oocytes. Throughout the MI/MII transition an intense staining of PP2A was present at MT arrays of meiotic spindles. These results indicate that phospho-MAPK and PP2A co-localize to MT arrays of meiotic spindles, suggesting that both proteins are important in regulating spindle assembly. To address whether MAPK and PP2A physically associate with MT during polymerization, we utilized in vitro polymerized MTs obtained from Cos-7 and HeLa cells. Both phospho-MAPK and PP2A co-purify with polymerized MTs. We then asked whether MAPK activation and PP2A are involved in MT polymerization in oocytes. Treating MII oocytes with Taxol (MT polymerization agonist) increased the level of phospho-MAPK, while the level of phospho-MAPK was reduced following nocodazole treatment (MT polymerization antagonist). Treatment of GVI and MII oocytes with okadaic acid, a PP1/PP2A inhibitor, increased the level of phospho-MAPK, yet blocked normal MT polymerization. These data suggest that both MAPK activation and PP2A are required for MT polymerization and normal spindle formation. (Supported by NIH Grant HD35125).

Keywords: oocyte, microtubules, MAPK, PP2A

This abstract is being presented at: 3:00 PM in session:
SESSION 15: OOCYTE DEVELOPMENT