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Published on: November 11, 2022
AMPK regulation of mouse oocyte meiotic resumption in vitro
Jing Chen1, Emma Hudson, Maggie M Chi
1Department of Biological Sciences, Marquette University, Milwaukee, WI 53201, USA.
Abstract:
We have previously shown that the adenosine analog 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 [Downs, S.M., Hudson, E.R., Hardie, D.G., 2002. A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev. Biol, 245, 200-212]. The present study was carried out to better define a causative role for AMPK in oocyte meiotic maturation. When microinjected with a constitutively active AMPK, about 20% of mouse oocytes maintained in meiotic arrest with dibutyryl cAMP (dbcAMP) were stimulated to undergo germinal vesicle breakdown (GVB), while there was no effect of catalytically dead kinase. Western blot analysis revealed that germinal vesicle (GV)-stage oocytes cultured in dbcAMP-containing medium plus AICAR possessed elevated levels of active AMPK, and this was confirmed by AMPK assays using a peptide substrate of AMPK to directly measure AMPK activity. AICAR-induced meiotic resumption and AMPK activation were blocked by compound C or adenine 9-beta-d-arabinofuranoside (araA, a precursor of araATP), both inhibitors of AMPK. Compound C failed to suppress adenosine uptake and phosphorylation, indicating that it did not block AICAR action by preventing its metabolism to the AMP analog, ZMP. 2'-deoxycoformycin (DCF), a potent adenosine deaminase inhibitor, reversed the inhibitory effect of adenosine on oocyte maturation by modulating intracellular AMP levels and activating AMPK. Rosiglitazone, an anti-diabetic agent, stimulated AMPK activation in oocytes and triggered meiotic resumption. In spontaneously maturing oocytes, GVB was preceded by AMPK activation and blocked by compound C. Collectively, these results support the proposition that active AMPK within mouse oocytes provides a potent meiosis-inducing signal in vitro.
Insights
AMP-activated protein kinase (AMPK) activation is crucial for inducing meiotic resumption in mouse oocytes. This study confirms AMPK
Area of Science:
- Reproductive Biology
- Molecular Endocrinology
- Cell Signaling
Background:
- AMP-activated protein kinase (AMPK) is a cellular energy sensor involved in various physiological processes.
- Previous work suggested a role for AMPK in initiating meiosis in mouse oocytes.
- The precise causative role of AMPK in oocyte meiotic maturation requires further elucidation.
Purpose of the Study:
- To definitively establish the causative role of AMPK in regulating meiotic maturation in mouse oocytes.
- To investigate the signaling pathways and molecular mechanisms by which AMPK influences oocyte meiosis.
Main Methods:
- Microinjection of constitutively active or catalytically dead AMPK into mouse oocytes arrested with dibutyryl cAMP (dbcAMP).
- Western blot analysis and direct enzyme activity assays to measure active AMPK levels in oocytes.
- Pharmacological inhibition of AMPK using compound C and adenine 9-beta-d-arabinofuranoside (araA).
- Assessment of meiotic resumption (germinal vesicle breakdown, GVB) and adenosine metabolism.
- Investigation of the effects of adenosine deaminase inhibitor (2'-deoxycoformycin) and an anti-diabetic agent (rosiglitazone).
Main Results:
- Microinjection of active AMPK induced germinal vesicle breakdown (GVB) in ~20% of dbcAMP-arrested oocytes, while inactive AMPK had no effect.
- AICAR treatment elevated active AMPK levels and promoted meiotic resumption, effects blocked by AMPK inhibitors compound C and araA.
- Compound C did not inhibit AICAR metabolism, indicating its action was downstream of AICAR phosphorylation.
- 2'-deoxycoformycin reversed adenosine's inhibitory effect on maturation by activating AMPK.
- Rosiglitazone activated AMPK and triggered meiotic resumption; spontaneous maturation was preceded by AMPK activation.
Conclusions:
- Active AMPK plays a direct and causative role in inducing meiotic resumption in mouse oocytes.
- AMPK activation provides a potent meiosis-inducing signal in vitro, independent of adenosine metabolism.
- These findings highlight AMPK as a key regulator of oocyte meiotic maturation.
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