AMP-activated protein kinase is involved in hormone-induced mouse oocyte meiotic maturation in vitro

Abstract

We have previously shown that AMP-activated protein kinase (AMPK) can induce the resumption of meiosis in mouse oocytes maintained in meiotic arrest in vitro. The present study was carried out to determine whether AMPK activation is involved in hormone-induced maturation. Follicle-stimulating hormone (FSH) and the EGF-like peptide, amphiregulin (AR), are potent inducers of maturation in cumulus cell-enclosed oocytes (CEO). Within 3 h of FSH treatment, phospho-acetyl CoA carboxylase (ACC) levels were increased in germinal vesicle (GV)-stage oocytes when compared to non-stimulated controls and remained elevated throughout 9 h of culture, indicating AMPK activation. A similar response to AR was observed after 6 h of culture. Using anti-PT172 antibody (binds only to activated AMPK), Western analysis demonstrated active AMPK in both FSH- or AR-treated GV-stage oocytes within 6 h. The AMPK inhibitors, compound C and adenine 9-beta-d-arabinofuranoside (araA), blocked FSH- or AR-induced meiotic resumption and ACC phosphorylation, further supporting a causal role for AMPK in hormone-induced meiotic resumption. Immunocytochemistry using anti-PT172-AMPK antibody showed an increased diffuse cytoplasmic staining and more intense punctate staining in the germinal vesicles of oocytes following treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or with FSH or AR, and this staining was eliminated by compound C or a blocking peptide for the anti-PT172 antibody. Staining of oocytes from hCG-stimulated mice with the anti-PT172 antibody also showed pronounced label in the germinal vesicles within 1-2 h. Furthermore, in oocytes from all groups, active AMPK was always observed in association with the condensed chromosomes of maturing oocytes. Taken together, these results support a role for AMPK in FSH and AR-induced maturation in vitro and hCG-induced maturation in vivo.

Figure 1

AMPK activation in FSH-or AR-treated GV-stage oocytes. (A) Western analysis of phospho-ACC in FSH-stimulated oocytes. CEO were denuded and extracted after culture with or without FSH in dbcAMP-supplemented medium for the indicated times. All oocytes were at the GV-stage, except a group of GVB-stage oocytes were collected after 9 h FSH treatment. Western analysis of PT172-AMPK in GV-stage oocytes after 6 h culture in the absence (Con) or presence of 0.1 μl/ml FSH (B) or 50 ng/ml AR (C) in dbcAMP-containing medium.

Figure 2

Effects of compound C and araA on FSH-induced maturation and ACC phosphorylation. CEO were precultured for 0.5 h with increasing doses of compound C (A) or araA (B) in 300 μM dbcAMP. FSH (0.1 μg/ml) was then added and cultures were continued for 17–18 h before assessment of GVB percentage. Groups with no common letters are significantly different. (C) Western analysis of phospho-ACC in compound C or araA-treated GV-stage oocytes. CEO were preincubated 0.5 h with 5 μM compound C or 1 mM araA in 300 μM dbcAMP before addition of FSH. After 6 h of culture, GV-stage oocytes were prepared for the Western analysis.

Figure 3

Effects of compound C and araA on AR-induced maturation and ACC phosphorylation. CEO were pretreated with increasing concentrations of compound C (A) or araA (B) in 300 μM dbcAMP for 0.5 h. AR (50ng/ml) was then added and cultures were continued for 17–18 h before assessment of GVB. Groups with no common letters are significantly different. (C) Western analysis of phospho-ACC in compound C or araA-treated oocytes. CEO were preincubated 0.5 h with 5 μM compound C or 2 mM araA in 300 μM dbcAMP before addition of AR. CEO were denuded after 6 h of culture and GV-stage oocytes were collected and processed for Western analysis.

Figure 4

Localization of active AMPK in AICAR-stimulated CEO. After 6 h of culture, cumulus cells were removed and DO were fixed and stained with rabbit anti-PT172 AMPK antiserum followed by FITC-labeled sheep anti-rabbit antiserum. (A) dbcAMP alone (control), GV-stage; (B) AICAR, GV-stage; (C) AICAR, GVB-stage; (D) AICAR, GV-stage; primary antibody was neutralized with a specific blocking peptide; (E) AICAR plus compound C, GV-stage; CEO were preincubated with 5 μM compound C in 300 μM dbcAMP before the addition of 1 mM AICAR. Oocytes were stained with propidium iodide (PI) for detection of chromatin. The bar represents 50 μm.

Figure 5

Effect of compound C on the percentage of GV-stage oocytes that contained nuclear PT172 staining after 6 h of culture in 1 mM AICAR.

Figure 6

Localization of active AMPK in FSH-stimulated CEO. After 6 h of culture, cumulus cells were removed and DO were fixed and stained with rabbit anti-PT172 AMPK antiserum followed by FITC-labeled sheep anti-rabbit antiserum. (A) dbcAMP alone (control), GV-stage; (B) FSH (0.1μg/ml), GV-stage; (C) FSH (0.1μg/ml), GVB-stage; (D) FSH (0.5 μg/ml) plus compound C, GV-stage; CEO were preincubated with 5 μM compound C in 300 μM dbcAMP before the addition of FSH. Oocytes were stained with propidium iodide (PI) for detection of chromatin. The bar represents 50 μm.

Figure 7

Effect of compound C on the percentage of GV-stage oocytes that contained nuclear PT172-AMPK staining after 6 h of culture in FSH.

Figure 8

Localization of active AMPK in AR-stimulated CEO. After 6 h of culture, cumulus cells were removed and DO were fixed and stained with rabbit anti-PT172 AMPK antiserum followed by FITC-labeled sheep anti-rabbit antiserum. (A) dbcAMP alone (control), GV-stage; (B) AR, GV-stage; (C) AR, GVB-stage; (D) AR plus compound C, GV-stage; CEO were preincubated with 5 μM compound C in 300 μM dbcAMP before the addition of AR. Oocytes were stained with propidium iodide (PI) for detection of chromatin. The bar represents 50 μm.

Figure 9

Effects of AR and compound C on nuclear PT172-AMPK staining. (A) Effect of AR dose on oocyte maturation and nuclear staining. CEO were cultured 6 h in medium containing 300 μM dbcAMP plus increasing dose of AR and the frequencies of both GVB and nuclear staining in GV-stage oocytes were determined. (B) Inhibitory effect of compound C on the percentage of GV-stage oocytes that contained nuclear PT172-AMPK staining after 6 h of culture in AR.

Figure 10

Localization of active AMPK in oocytes from hCG-stimualted mice. eCG-primed mice were injected with 5 IU hCG and 0, 1, 2 or 3.5 h later oocytes were isolated, fixed and stained with rabbit anti-PT172 AMPK antiserum followed by FITC-labeled sheep anti-rabbit antiserum. Oocytes were stained with propidium iodide (PI) for detection of chromatin. Note that two cumulus cell nuclei stain intensely with propidium iodide on the bottom portion of the micrograph of the 1 h group. The bar represents 50 μm.