PARENT SESSION
GAMETE BIOLOGY AND GAMETOGENESIS - C

Wednesday, August 4, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(700) ALSTERPAULLONE INHIBITION OF OOCYTE GLYCOGEN SYNTHASE KINASE-3 IMPAIRS SPINDLE FORMATION, CENTROSOME LOCALIZATION, AND POLAR BODY EXTRUSION.

Acevedo, Nicole ^{1, 3}, Ding, Jun², Smith, Gary ^{1, 2, 3}, ¹ Department of Molecular and Integrative Physiology, Ann Arbor, MI³ Reproductive Sciences Program, Ann Arbor, MI² Department of OB/GYN, Ann Arbor, MI

ABSTRACT- Pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) significantly increases incidence of abnormal homologue segregation and spindle formation during meiosis. A competent spindle is comprised of microtubules (MT) nucleated by a functional centrosome. Impaired centrosome positioning and/or nucleation ability during mitosis can lead to aberrant MT organization, resulting in chromosome segregation abnormalities and defective cytokinesis. Objective of this study was to elucidate causes of aberrant chromatin segregation during oocyte meiosis resulting from pharmacological inhibition of GSK-3, focusing on the spatial relationship between chromatin, spindle, and centrosomal components. Germinal vesicle intact (GV-intact) mouse oocytes obtained from eCG-primed d19-21 CF-1 mice were cultured for 19h in HTF media containing either 20M Alsterpaullone (Alster; selective inhibitor of GSK-3 activity) dissolved in DMSO or DMSO alone (Control) until meiotic arrest at metaphase II (MII). Basal levels of GSK-3 activity in GV-intact oocytes were inhibited by 20M Alster. Inhibition of GSK-3 significantly (P<0.001) delayed timing of germinal vesicle breakdown (2h: Control=85%; Alster= 16%) and development to MII (19h: Control= 88%; Alster= 57%). Immunocytochemical evaluation of GSK-3 inhibited oocytes indicated abnormal chromatin condensation and/or chromatid misalignment on the MII plate. Alsterpaullone treatment prevented proper microtubule polymerization and/or meiotic spindle formation (Control=100%; Alster= 0%) and compromised proper localization of centrosomes at spindle poles (Control=100%; Alster= 25%). Karyotyping confirmed that GSK-3 inhibition significantly compromised correct homologue segregation, separation, and condensation of MII chromatin (Control=100%; Alster=17%). Inhibition of GSK-3 also significantly decreased incidence of complete cytokinesis during extrusion of the first polar body (Control=100%; Alster= 43%). In conclusion, inhibition of GSK-3 compromised oocyte meiotic progression through incorrect centrosomal localization, impaired spindle formation, aberrant chromatin remodeling, and incomplete cytokinesis during polar body formation.

KEY WORDS: glycogen synthase kinase-3, meiotic spindle, alsterpaullone, mouse oocyte