Electron microscopic observations have shown a uniform physical appearance of the zona pellucida in WT oocytes in vivo, but a lack of uniformity of the zona pellucida in ZP3-KO oocytes (Fig. 2F)
Junctions were noticed between oocytes and the cumulus cells of the ovarian follicle. These junctions are known to support nutrient transfer in between the germ and somatic compartments and to allow the transfer of R112 molecules included in oocyte maturation from the granulosa/cumulus cells to the oocyte [32]. We localized junctional proteins in between oocytes and their encompassing cumulus cells. In vivo, in ZP3-KO oocytes, expression of occludin, a part of restricted junctions, was significantly less common at the interface and much more localized in the cytoplasm of oocytes (Fig. 3A). -catenin and N-cadherin, two elements of adherens junctions have been localized at the oolemma interface of the oocyte and in the zona pellucida/cumulus interface. They have been reduced in ZP3-KO mice (Fig. 3A, B). The glycogen synthase kinase-3 (GSK3), documented to mediate -catenin/Wnt signalling [33,34], also confirmed a decreased intensity of labelling in the cytoplasm of ZP3-KO oocytes (Fig. 3A). In addition, the localization of connexin37, a essential hole junction protein for oocyte/cumulus mobile conversation, was barely detectable in ZP3-KO oocytes at the zona pellucida, in contrast to wild-type oocytes (Fig. 3A, B). A comparable reduction in N-cadherin, -catenin and Connexin37 protein expression in isolated cumulus cells purified from superovulated ZP3-KO COCs was observed (Fig. 3C). Both PKA and MAPK signal transduction pathways have been observed to be involved in the junctional conversation between the cumulus/granulosa cells and the oocyte and the somatic cells of the follicle. Communication is essential for the developing oocyte and has a role in giving vitamins and minerals to the maturing oocyte [35]. Western-blot analyses have exposed a significantly lowered phosphorylation of a MAP protein, the extracellular signal-regulated kinases (ERK1/2) in ZP3-KO MII oocytes when in comparison with wild-kind oocytes (Fig. 4A, B), but not in the ZP3-KO cumulus cells (Fig. 4A, B). Concomitantly, we also observed a reduction in the protein kinase A (PKA) activity as revealed by the diminution of CREB phosphorylation (a focus on of PKA) and cAMP articles (cAMP activates PKA) in ZP3-KO MII oocytes (Fig. 4C, D). 1AMPK inactivation in oocyte. (A) Western blot investigation of the 1AMPK subunit and Cre recombinase in metaphase II (MII) oocytes, cumulus cells and liver retrieved from wild-sort and ZP3-KO mice soon after superovulation (B) Conditional invalidation of 1AMPK subunit in the oocyte.
