Régulation des jonctions communicantes chez les complexes ovocyte-cumulus de porc au cours de la maturation in vitro
|Advisor:||Richard, François; Robert, Claude|
|Abstract:||Intercellular gap-junctional communication (GJC) plays an important role in ovarian cell physiology. Closure of GJC has been proposed to be involved in oocyte maturation, particularly in the resumption of meiosis, both in vivo and in vitro, by controlling the flow of meiosis inhibitors, such as cAMP and cGMP. In the present study, we have first developed an assay based on recovery of calcein fluorescence in photobleached cumulus cells (FRAP: Fluorescent Recovery After Photobleaching) in the three-dimensional cumulus-oocyte complex (COC), during the first hours of porcine in vitro maturation (IVM). Results obtained using FRAP technology provide evidence that the composition of the IVM medium in terms of hormones and follicular fluid clearly affects cumulus-cumulus cell GJC. We then turned our attention to the effect of gonadotropins on connexins regulation. Indeed, GJC are composed of connexins proteins. We indentified Cx43, Cx45 and Cx60 as the main connexins expressed in swine COC. We show that gonadotropins regulate Cx43 protein level, degradation and localisation in the COC during the first hours of IVM. We finally turned our attention on the effect of natriuretic peptide C (CNP) on GJC regulation and meiotic resumption during porcine IVM. Indeed, CNP has been proposed to bind to receptor natriuretic peptide receptor B (NPR-B) where it triggers the production of cyclic guanosine monophosphate (cGMP). The cGMP produced is then transferred to the oocyte through gap junctions where it promotes meiotic arrest. Here we show that CNP and a cGMP analogue have different effects on GJC and Cx43 regulation suggesting that cGMP signaling may not be the only signaling pathway involving CNP. Moreover, we found that CNP is likely to bind to receptor natriuretic peptide receptor C (NPR-C) to play a role in maintaining meiotic arrest during porcine IVM, when activated in addition to NPR-B. In conclusion, we describe new mechanisms involved in the complex regulation of dynamic changes in GJCs and meiotic resumption. A better understanding of GJC regulation during IVM may in turn provide a powerful tool to improve assisted reproduction technologies and their efficacy in mammals.|
|Document Type:||Thèse de doctorat|
|Open Access Date:||19 April 2018|
|Collection:||Thèses et mémoires|
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