Bovine Cumulus Cell - Oocyte Gap Junctional Communication During In Vitro 1 Maturation In Response To Manipulation of Cell - Specific CAMP Levels

31 32 In the growing follicle, communication between the oocyte and its surrounding follicular 33 cells is essential for normal oocyte and follicular development. Maturation of the fully34 grown oocyte in vivo is associated with the loss of cumulus cell-oocyte gap junctional 35 communication, preventing entry of meiotic-modulating factors such as cAMP into the 36 oocyte. We have previously shown that oocyte and cumulus cell cAMP levels can be 37 independently regulated using inhibitors of cell-specific phosphodiesterase (PDE) 38 isoenzymes. The objectives of this study were to examine the effects of cell type-specific 39 PDE inhibitors on the maintenance of cumulus cell-oocyte gap junction communication 40 (GJC) and oocyte meiotic progression. Cumulus-oocyte complexes (COC) were aspirated 41 from antral follicles of abattoir-derived ovaries. Cumulus cell-oocyte gap junctional 42 communication during oocyte maturation was quantified using the fluorescent dye, calcein43 AM. COCs were cultured in the presence of specific PDE inhibitors; milrinone (MR, oocyte 44 PDE3 inhibitor) or rolipram (RP, cumulus cell PDE4 inhibitor), and were pulsed with 45 calcein-AM to allow dye transfer between the two cell types. Following cumulus cell 46 removal, fluorescence in denuded oocytes was measured by microphotometry and meiotic 47 progression assessed. In control COCs, dye transfer from cumulus cells to the oocyte fell 48 progressively from 0 to 9 h, after which oocyte-CC GJC was completely lost. Loss of gap 49 junctional communication was significantly attenuated (P<0.05) during this time in response 50 to treatment with milrinone and rolipram. Forskolin maintained gap junctional 51 communication at the initial 0 h level until 3-4 h of culture, while treatment with MR and 52 forskolin together actually increased the level of dye transfer above that in COC treated with 53 forskolin alone. Importantly, all treatments that prolonged GJC also delayed meiotic 54 resumption, with meiosis generally resuming when fluorescence had fallen to ~40% of initial 55 levels. These results, together with our previous studies, demonstrate that treatments which 56 maintain/elevate cumulus cell and/or oocyte cAMP levels result in prolonged oocyte-cumulus 57 cell communication and delayed meiotic resumption. 58 59 60