[Frontiers in Bioscience E5, 755-767, January 1, 2013]

Gap junction -mediated cAMP movement between oocytes and somatic cells

Guankun Mao1, Junxia Li1, Fenghua Bian1, Yingying Han1, Meng Guo1, Baoshan Xu1, Meijia Zhang1, Guoliang Xia1

1State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing 100193, China


1. Abstract
2. Introduction
3. Materials and methods
3.1. Experimental animals
3.2 .Reagents and antibodies
3.3. Isolation and culture of ovarian follicles
3.4. Determination of cAMP and cGMP levels in oocytes
3.5. QRT-PCR analysis
3.6. Phosphodiesterase (PDE) activity assay
3.7. Western Blot Analysis
3.8. Statistical analysis
4. Results
4.1. The kinetics of CBX-induced meiotic resumption of FEOs during continuous or transient incubation.
4.2. CBX induced a short transient rise in intraoocyte cAMP level
4.3. Oocyte can produce more cAMP than each granulosa cell
4.4. Differential expression of adenylyl cyclases (ACs) in the oocyte and somatic cells of the mouse ovary
4.5. The maturation of FEOs was inhibited by GJCs blocking in the presence of forskolin but can not be inhibited in the presence of FSH
4.6. The change of cAMP level during oocyte spontaneous maturation
4.7. CBX decreases cGMP concentrations and increases the activity of cAMP-PDE3A in oocyte
4.8. The protein kinase A (PKA) I pathway takes part in the CBX-induced meiotic resumption
4.9. CBX-induced FEO maturation is also dependent on MAPK activation in oocyte
4.10. The relationship between cAMP-PKA I and MAPK signaling during CBX-induced mouse FEOs meiotic resumption
5. Discussion
6. Acknowledgment
7. References


Cyclic AMP (cAMP) plays a critical role in oocyte meiotic maturation. However, the source of cAMP surge prior to maturation and the direction of gap junction-dependent cAMP movement are unclear. In this study, inhibition of gap junctional communication (GJC) using carbenoxolone (3.5 h) induced meiotic resumption in ~90% of follicle-enclosed oocytes (FEOs). The concentration of cAMP in a single oocyte was higher than that in a single cumulus cell, suggesting that the movement of cAMP proceeds from the oocyte to cumulus cells under passive diffusion. The mRNAs of adenylyl cyclases and the corresponding proteins were mainly detected in oocytes. Persistent or transient incubation with forskolin induced meiotic resumption in FEOs. The maturation induced by persistent forskolin treatment was inhibited by carbenoxolone. However, carbenoxolone had no effect on the maturation of FEOs transiently treated with forskolin or persistently treated with follicle-stimulating hormone. Oocyte maturation was inhibited by sequential treatment with carbenoxolone followed by forskolin. The carbenoxolone-induced maturation was accompanied by a cAMP surge, increased PDE3A and MAPK activation, and decreased levels of cGMP and cAMP-dependent PKA I activation.