Regulation of Gap Junctions in Porcine Cumulus-Oocyte Complexes: Contributions of Granulosa Cell Contact, Gonadotropins, and Lipid Rafts

Abstract

Gap-junctional communication (GJC) plays a central role in oocyte growth. However, little is known about the regulation of connexin 43 (Cx43)-based gap-junction channels in cumulus-oocyte complexes (COCs) during in vitro maturation. We show that rupture of COCs from mural granulosa cells up-regulates Cx43-mediated GJC and that gonadotropins signal GJC breakdown by recruiting Cx43 to lipid rafts when oocyte meiosis resumes. Oocyte calcein uptake through gap junctions increases during early in vitro oocyte maturation and remains high until 18 h, when it falls simultaneously with the oocyte germinal vesicle breakdown. Immunodetection of Cx43 and fluorescence recovery after photobleaching assays revealed that the increase of GJC is independent of gonadotropins but requires RNA transcription, RNA polyadenylation, and translation. GJC rupture, in contrast, is achieved by a gonadotropin-dependent mechanism involving recruitment of Cx43 to clustered lipid rafts. These results show that GJC up-regulation in COCs in in vitro culture is independent of gonadotropins and transcriptionally regulated. However, GJC breakdown is gonadotropin dependent and mediated by the clustering of Cx43 in lipid raft microdomains. In conclusion, this study supports a functional role of lipid raft clustering of Cx43 in GJC breakdown in the COCs during in vitro maturation.

Cumulus cell gene expression following the LH surge in bovine preovulatory follicles: potential early markers of oocyte competence

“…These communications played important roles in the oocyte-somatic compartment cell signaling and decreased at the GVBD (Sutovsky et al 1993, Mattioli & Barboni 2000. Breakdown of such connections was suggested to be gonadotropin-dependent in pig COCs (Sasseville et al 2009). Our data confirmed these findings in the cow at 6 h post-LH by the down-regulation of a fundamental protein of these cell-to-cell connections: GJA1.…”

Assessment of transcript and protein levels contributing to cell cycle control and gap junction connections in morphologically variable groups of porcine cumulus-oocyte complexes

“…Calder also also found a significant association between a relatively high level of Cx43 mRNA and an increased quality of bovine oocytes. Most recent findings based on the porcine model indicate that lipid raft clustering of Cx43 plays a role in the breakdown of gap junction connections during IVM of porcine COCs (Sasseville et al 2009). Based on the porcine model, there is no data demonstrating an association between the morphology of COCs and the expression of genes that are important for oocyte maturation.…”

Bidirectional communication between oocytes and follicle cells: ensuring oocyte developmental competence

“…The gap junctional connection also allows the granulosa cells to use their own ion transport mechanisms to regulate the pH of the oocyte, a capacity the oocyte itself does not possess until fully grown (FitzHarris and Baltz 2006;FitzHarris et al 2007). Importantly, signals that regulate meiotic maturation of fully-grown oocytes pass through the oocyte-granulosa cell gap junctions (reviewed by Edry et al 2006): the current view is that LH causes MAP kinase-dependent phosphorylation and disruption of the gap junctions coupling the mural with the cumulus granulosa cells, restricting the flow of meiosis-arresting cGMP into the oocyte (Norris et al 2008(Norris et al , 2009Sasseville et al 2009). Signalling in the opposite direction, the growing oocyte influences the differentiation of the surrounding granulosa cells via the gap junctional pathway: disruption of oocyte-granulosa cell gap junctional coupling in the mouse causes loss of the oocyte and, consequently, premature luteinisation of the granulosa cells such that a mature preovulatory follicle is never formed (Simon et al 1997).…”