Oral Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

Cyclic AMP modulated-IVM differentially alters oocyte and cumulus cell adenine nucleotides and AMP-activated protein kinase (#57)

Dulama Richani 1 , Sonia Bustamante 2 , Cathy F. Lavea 1 , David J. Agapiou 1 , Michael J. Bertoldo 1 , Robert B. Gilchrist 1
  1. School of Women’s & Children’s Health, University Of New South Wales, Sydney, NSW, Australia
  2. Bioanalytical Mass Spectrometry Facility, UNSW Australia, Sydney, NSW, Australia

Cyclic adenosine monophosphate (cAMP) is a key regulator of oocyte maturation. Contrary to in vivo maturation, cumulus-oocyte complexes (COCs) matured via in vitro maturation (IVM) are deficient in cAMP. Pharmacological elevation of cAMP in COCs during IVM decidedly improves oocyte developmental competence. In a model of cAMP-modulated IVM, COCs are loaded with cAMP prior to maturation in a short “pre-IVM” phase by exposure to IBMX, an inhibitor of cAMP breakdown, and forskolin, a potent promoter of adenylate cyclase conversion of ATP to cAMP. The cAMP is then hydrolysed to AMP to resume oocyte maturation. This study investigated the effect of pre-IVM on oocyte and cumulus cell adenine nucleotide homeostasis and the AMP-activated protein kinase (AMPK), a cellular energy sensor activated by alterations in relative adenine nucleotide levels. Mouse COCs were subjected to +/-2 hours pre-IVM with forskolin+IBMX, followed by standard IVM with FSH. Following COC culture, adenine nucleotides (ATP, ADP and AMP) were measured in whole COCs or cumulus-denuded oocytes (CDOs) using an in-house LC-MS/MS method developed for use on oocytes. Phosphorylated (pAMPK) and total (tAMPK) AMPK were measured using Western blotting. CDOs exhibited decreased ATP and increases in ADP and the ADP:ATP ratio throughout IVM (0-16h, P≤0.02) when exposed to pre-IVM. This was supported by an increase in CDO pAMPK and tAMPK at 16h (P≤0.05). Contrary to CDOs, in COCs pre-IVM led to an increase in ATP (P=0.02), however COC AMP, AMPK activity, and the cellular energy charge (a metabolic regulatory parameter) were not significantly different to control. In conclusion, pre-IVM decreases oocyte ATP yet increases cumulus cell ATP, indicating a differential metabolic response to cAMP modulation between the two compartments. Moreover, pre-IVM increases AMPK activity in the oocyte, which may be a mechanism contributing to cAMP-mediated increased developmental competence as it is a master regulator of ovarian function.