AMP-ACTIVATED PROTEIN KINASE (AMPK) IS INVOLVED IN FOLLICLE-STIMULATING HORMONE (FSH)-INDUCED MOUSE OOCYTE MEIOTIC MATURATION

Abstract

We have previously shown that AMPK activation can induce mouse oocyte meiotic resumption in vitro. The present study was carried out to determine whether AMPK activity is involved in follicle-stimulating hormone (FSH)-induced maturation. The upstream kinase of AMPK specifically phosphorylates AMPK on Thr172 of the catalytic subunit (PT172-AMPK) to activate the enzyme. Acetyl-CoA carboxylase (ACC) is a major substrate of AMPK, and its phosphorylation state is a common indicator of AMPK activity. By western analysis, a time course experiment was carried out to test phospho-ACC levels in FSH-treated oocytes. Cumulus cell-enclosed oocytes were denuded and extracted for western analysis after 3, 6, or 9 h culture with or without FSH in 300 μM dbcAMP. Within 3 h of FSH treatment, phospho-ACC levels were increased in germinal vesicle (GV)-stage oocytes when compared to nonstimulated controls and remained elevated in these oocytes throughout 9 h of culture. Interestingly, phospho-ACC returned to the basal level in oocytes undergoing GV breakdown by 9 h of FSH treatment. Using PT172 antibody (binds only to the activated kinase), western analysis showed that PT172-AMPK was elevated in FSH-treated GV-stage oocytes within 6 h. These results indicate that AMPK is activated in FSH-stimulated oocytes before meiotic resumption. Compound C, a potent small-molecule AMPK inhibitor, or adenine 9-beta-d-arabinofuranoside, a precursor of araATP (a competitive inhibitor of AMPK), dose-dependently prevented FSH-induced meiotic resumption and ACC phosphorylation in GV-stage oocytes, further supporting a role for AMPK in FSH-induced maturation. To directly observe active AMPK, oocytes were fixed, permeabilized and processed for immunofluorescent staining using anti-PT172 antiserum. Fluorescence was generally increased throughout the entire GV-stage oocyte after 6 h of FSH treatment compared to controls. A shift of active AMPK localization was observed after GVB, with accumulation around the condensed chromosomes. Compound C downregulated this FSHinduced increase in fluorescence. Taken together, these results support the idea that AMPK activation within the mouse oocyte plays a mediating role in FSH-stimulated meiotic resumption. Supported by funds from the NIH (HD040392). (platform)