Bovine Cumulus Cell-Oocyte Gap Junctional Communication During In Vitro Maturation in Response to Manipulation of Cell-Specific Cyclic Adenosine 3′,5′-Monophosophate Levels1

Abstract

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.

Role of gap junction-mediated communications in regulating large-scale chromatin configuration remodeling and embryonic developmental competence acquisition in fully grown bovine oocyte

“…cAMP is generated also by cumulus cells and then transported into the oocyte through gap junctions [37,38]. The manipulation of intracellular cAMP concentration has been demonstrated to influence functional coupling in bovine COCs [14,39]. In our culture system, the maintenance of open GJ-mediated communications in cumulus oocyte complexes isolated from mid-antral follicles was achieved with the combined administration of IBMX and of rhFSH at a concentration lower than what is usually adopted during IVM.…”

Regulation of sheep oocyte maturation using cAMP modulators

“…In parallel, increases in CC cAMP induce expression of EGF-like peptides and subsequent activation of the EGF receptor and MAPK pathway, thereby stimulating cumulus expansion and oocyte maturation [40][41][42]. It is widely reported that cAMP modulators in cattle IVM extended CC-oocyte GJC and increased oocyte developmental competence [13,19,20,32,43,44].…”

“…Oocyte developmental competence is compromised in spontaneous IVM due to many factors, including presumably an absence of the full ovulatory molecular EGF cascade in CC and premature breakdown of oocyte-cumulus cell gap junctions; together this reduced exchange of positive regulatory molecules and metabolites from the follicular compartment [32]. Furthermore, meiotic resumption occurs in a rapid and uncontrolled manner [25].…”

“…Furthermore, meiotic resumption occurs in a rapid and uncontrolled manner [25]. However, the SPOM and related IVM models using elevated cAMP concentrations attenuated spontaneous meiosis, thereby resulting in a more gradual cessation of oocyte transcription and transition into the M-phase and promoting and prolonging oocyte-cumulus cell gap junction communication (GJC) [13,[32][33][34][35]. Part of the rationale for these approaches were substantial increases in cAMP occurred in vivo following LH-induction of the ovulatory cascade in porcine [36] and murine COCs [13,37].…”

“…The pre-IVM phase in the SPOM system primarily targets CC to increase cAMP concentrations in CC and also serves to maintain CC-oocyte GJC [13,32]. These two effects combine to load the oocyte with cAMP from the CC [39].…”