PARENT SESSION
GAMETE BIOLOGY AND GAMETOGENESIS - B

Tuesday, August 3, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(405) REGULATION OF MEIOTIC RESUMPTION IN MOUSE OOCYTES BY AMP-ACTIVATED PROTEIN KINASE (AMPK).

Chen, Jing¹, Downs, Stephen¹, ¹ Department of Biological Sciences, Milwaukee, WI

ABSTRACT- We have previously shown that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK), stimulates an increase in AMPK activity and induces meiotic resumption in mouse oocytes (Dev Biol 245: 200, 2002). The present study was carried out to better define a causative role for AMPK in meiotic maturation. Oocytes from eCG-primed, immature mice were maintained in meiotic arrest with dibutyryl cyclic AMP (dbcAMP) and microinjected with either a constitutively active AMPK or a catalytically dead AMPK. About 20% of the oocytes receiving active kinase were stimulated to undergo germinal vesicle breakdown (GVB), while the dead kinase had no effect. Acetyl-CoA carboxylase (ACC) is a substrate of AMPK, and its phosphorylation state is commonly used to indirectly show AMPK activity. When oocytes were cultured in medium containing dbcAMP plus AICAR, western analysis revealed phosphorylation of ACC in GV-stage oocytes well before GVB. Compound C, a presumptive inhibitor of AMPK, blocked both AICAR-stimulated maturation and phosphorylation of ACC. The inhibitor had no effect on the nucleoside transporter as measured by adenosine uptake, nor did it appear to block adenosine kinase, the enzyme that converts AICAR to the AMP analog, ZMP. These results suggest that compound C does not act by preventing the uptake and metabolism of AICAR in the oocyte. The AMPK upstream kinase was recently identified as LKB1, which we have shown to be present in mouse oocytes by western analysis. LKB1 is stabilized by association with heat shock protein and other protein chaperones. Geldanamycin and radicicol interfere with such association and promote LKB1 degradation. Treatment with these compounds completely suppressed meiotic induction by AICAR. These latter results suggest that reducing LKB1 activity prevents AMPK activation and meiotic induction by AICAR, a possibility we are currently testing. Other reported stimulators of AMPK such as leptin, metformin and rosiglitazone were unable to consistently trigger GVB, but it is not yet known if they can stimulate AMPK activity within mouse oocytes. Taken together, these data support the proposition that an increase in AMPK activity within mouse oocytes provides a potent meiosis-inducing signal. Supported by funds from the NIH.

KEY WORDS: meiotic resumption , AMP-activated protein kinase, mouse oocytes