PARENT SESSION

(M394) INSULIN SIGNALING THROUGH GLYCOGEN SYNTHASE KINASE-3 REGULATES CHROMATIN CONDENSATION AND SEGREGATION IN MOUSE OOCYTES.

Smith, Gary¹, Ding, Jun¹, ¹ University of Michigan, Ann Arbor, MI

ABSTRACT- Prolonged follicle/oocyte exposure to insulin compromises embryonic developmental competence, yet the mechanism is unknown. We hypothesized that insulin acts directly on oocytes to inhibit the insulin terminal signaling enzyme glycogen synthase kinase-3 (GSK-3) and thus causes aberrant meiotic chromatin segregation. Objectives were to determine if insulin initiates signaling in oocytes and inhibits GSK-3; and if pharmacologic or physiologic GSK-3 inhibition influences meiotic chromatin behavior. Western blot analysis demonstrated that oocytes contain insulin receptors, insulin receptor substrate-2, intermediate signaling enzyme Akt, and terminal enzyme GSK-3. Receptor expression was enhanced in germinal vesicle-intact (GVI) oocytes after in vivo eCG-stimulation, and remained constant through metaphase II. In GVI oocytes Akt was phosphorylated on Ser 473 (insulin signal transduction) and GSK-3 was phosphorylated on Ser9/21 (inactivation). Conversely GSK-3 was not phosphorylated in MI or MII oocytes, indicating its activity at this transition. Pharmacological inhibition of GSK-3 with Alsterpaullone (Alster) during in vitro meiotic maturation had no effect on development to MII and oocytes were morphologically normal by light microscopy. However 10 and 20M Alster significantly increased incidence of incomplete cytokinesis of MII oocytes (23/58=40%, 40/76=53%, respectively; controls 0/55=0%; P<0.001). A significant (P<0.001) increase in chromatin condensation and segregation abnormalities occurred in Alster-treated oocytes versus controls. In vitro maturation of GVI oocytes for 17 h in 5g/ml insulin had no influence on meiotic progression to MII or incidence of abnormal karyotypes. However, culturing follicles for 10 days in 5g/ml insulin hyper-phosphorylated oocyte GSK-3, and increased (P<0.001) MI chromatin condensation problems (9/9=100%; controls:1/13=8%). In conclusion, insulin can act directly on oocytes causing GSK-3 phosphorylation and inactivation. Oocyte GSK-3 activity is important for proper meiotic chromatin condensation and segregation. While short-term insulin exposure during meiosis does not influence chromatin behavior, extended exposure during oocyte growth results in aberrant meiotic chromatin condensation.

KEY WORDS: insulin, chromatin segregation, GSK-3, oocyte