Bovine cumulus cell-oocyte gap junctional communication during in vitro maturation in response to manipulation of cell-specific cyclic adenosine 3',5'-monophosophate levels.
In the growing follicle, communication between the oocyte and its surrounding follicular cells is essential for normal oocyte and follicular development. Maturation of the fully grown oocyte in vivo is associated with the loss of cumulus cell-oocyte gap junctional communication, preventing entry of meiotic-modulating factors such as cAMP into the oocyte. We have previously shown that oocyte and cumulus cell cAMP levels can be independently regulated using inhibitors of cell-specific phosphodiesterase (PDE) isoenzymes. The objectives of this study were to examine the effects of cell type-specific PDE inhibitors on the maintenance of cumulus cell-oocyte gap junction communication (GJC) and oocyte meiotic progression. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles of abattoir-derived ovaries. Cumulus cell-oocyte GJC during oocyte maturation was quantified using the fluorescent dye, calcein-AM. COCs were cultured in the presence of specific PDE inhibitors, milrinone (an oocyte PDE3 inhibitor) or rolipram (a cumulus cell PDE4 inhibitor), and were pulsed with calcein-AM to allow dye transfer between the two cell types. Following cumulus cell removal, fluorescence in denuded oocytes was measured by microphotometry, and meiotic progression was assessed. In control COCs, dye transfer from cumulus cells to the oocyte fell progressively from 0 to 9 h, after which oocyte-cumulus cell GJC was completely lost. Loss of GJC was significantly attenuated (P < 0.05) during this time in response to treatment with milrinone and rolipram. Forskolin maintained GJC at the initial 0 h level until 3-4 h of culture, whereas treatment with milrinone and forskolin together actually increased the level of dye transfer above that in COCs treated with forskolin alone. Importantly, all treatments that prolonged GJC also delayed meiotic resumption, with meiosis generally resuming when fluorescence had fallen to approximately 40% of initial levels. These results, together with our previous studies, demonstrate that treatments that maintain or elevate cAMP levels in cumulus cells, oocytes, or both result in prolonged oocyte-cumulus cell communication and delayed meiotic resumption.
Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, 5011, Adelaide, Australia.
Rebecca E
RE
Thomas
David T
DT
Armstrong
Robert B
RB
Gilchrist
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002450:Cell Communication / Q000502:physiology*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D003584:Cytological Techniques; D005260:Female; D005452:Fluoresceins / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D017629:Gap Junctions / Q000378:metabolism*; D066298:In Vitro Techniques; D008540:Meiosis / Q000502:physiology; D020105:Milrinone / Q000494:pharmacology; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology
D005452:Fluoresceins; D005456:Fluorescent Dyes; D010726:Phosphodiesterase Inhibitors; C085925:calcein AM; D005576:Colforsin; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D020105:Milrinone