Zygote 19 (May), pp. 97–106. C  Cambridge University Press 2010 doi:10.1017/S0967199410000195 First Published Online 23 June 2010 Activation of AMP-activated protein kinase may not be involved in AICAR- and metformin-mediated meiotic arrest in bovine denuded and cumulus-enclosed oocytes in vitro Sylvie Bilodeau-Goeseels 1 , Paul L. Panich 2 and John P. Kastelic 2 Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, Canada Date submitted: 16.11.2009. Date accepted: 22.01.2010 Summary The adenosine monophosphate-activated protein kinase (AMPK) activators, 5 ′ -aminoimidazole-4- carboxamide 1-β -D-ribofuranoside (AICAR) and metformin (MET), inhibit resumption of meiosis in bovine cumulus-enclosed oocytes (CEO) and denuded oocytes (DO). The objectives of this study were to: (1) examine the effects of AMPK inhibitors on bovine oocyte meiosis in vitro; and (2) determine if AICAR or MET activates oocyte and/or cumulus cell AMPK. The AMPK inhibitor compound C (CC; 0.5, 1, 5, and 10 μM) did not reverse the inhibitory effects of AICAR (1 mM) and MET (2 mM) on bovine oocyte meiosis. Additionally, CC (5 and 10 μM) inhibited meiosis (p < 0.05) in CEO and DO cultured for 7 h. Okadaic acid (1 μM) reversed the inhibitory effect of MET (2 mM) and CC (5 μM; p < 0.05) but not of AICAR (1 mM). Phosphorylation of the alpha subunit of AMPK on Thr172 is required for activation. Based on western blot analysis, AICAR, MET and CC did not affect Thr172 phosphorylation levels in DO and oocytes from complexes (p > 0.05). In cumulus cells, Thr172 phosphorylation decreased after 3 h of culture (p < 0.05), regardless of the presence of AMPK modulators in the culture medium. Higher concentrations of AICAR (2 mM) and MET (10 mM) did not affect Thr172 phosphorylation, but phosphorylation on Ser79 of ACC, a substrate of AMPK, was increased in response to MET (p < 0.05). In conclusion, we inferred that the inhibitory effect of AICAR and MET on bovine oocyte meiosis was probably not mediated through activation of AMPK. Moreover, these compounds probably inhibited meiosis through different pathways. Keywords: AICA riboside, AMP-activated protein kinase, Meiosis, Metformin, Oocyte Introduction In mammalian oocytes, meiosis is initiated during fetal life and is subsequently arrested at the diplotene stage of the first meiotic division. Completion of the first meiotic division is triggered by the preovulatory gonadotropin surge and meiosis progresses to the metaphase II stage where it is arrested again until fertilization. Meiotically competent oocytes spontan- 1 All correspondence to: Sylvie Bilodeau-Goeseels. Agricul- ture and Agri-Food Canada, Lethbridge Research Centre 5403 1st Avenue South, Lethbridge, Alberta, Canada T1J 4B1. Tel: +1 403 317 2290. Fax: +1 403 382 3156. e-mail: Sylvie.Bilodeau-Goeseels@AGR.GC.CA 2 Agriculture and Agri-Food Canada, Lethbridge Research Centre 5403 1st Avenue South, Lethbridge, Alberta, Canada T1J 4B1. eously resume meiotic maturation without hormonal stimulation when they are removed from follicles and cultured in suitable media. This situation suggests that the follicle provides an inhibitory factor(s) to prevent meiosis until the gonadotropin surge; however, mechanisms involved in meiotic arrest and induction are not fully understood. Cyclic adenosine monophosphate (cAMP) levels within the oocyte apparently have an important role in the control of nuclear maturation, with high levels maintaining meiotic arrest and lower levels being permissive for meiotic maturation (Downs, 1995; Shimada et al., 2002; Horner et al., 2003; Kalinowski et al., 2004; Thomas et al., 2004). Cyclic AMP is produced by the enzyme adenylate cyclase in response to hormonal stimulation and its action is mediated by cAMP-dependent protein kinase (PKA). The amount of cAMP in cells is also controlled by the rate of