Control of Spontaneous Activation of Rat Oocytes by Regulating Plasma Membrane Na+/Ca2+ Exchanger Activities

Abstract

Inhibiting oocyte spontaneous activation (SA) is essential for successful rat cloning by nuclear transfer (NT). This study tested the hypothesis that activities of the Na(+)/Ca(2+) exchanger (NCX) would decrease with oocyte aging and that SA of rat oocytes could be inhibited if the intraoocyte Ca(2+) rises were prevented by activating the NCX through increasing Na(+) concentrations in the culture medium. Elevating Na(+) levels in culture medium by supplementing NaCl inhibited SA of rat oocytes, while maintaining a constant level of maturation-promoting factor and mitogen-activated protein kinase activities. Experiments using the NCX inhibitor bepridil, the Na(+)/K(+)-ATPase inhibitor ouabain, and an assay for intraoocyte Ca(2+) concentrations showed that extracellular Na(+) inhibited rat oocyte SA by enhancing NCX activity and preventing intracellular Ca(2+) rises. Immunohistochemical quantification indicated that the density of NCX1 decreased significantly in aged oocytes that were prone to SA compared with that in freshly ovulated oocytes whose SA rates were low during in vitro culture. Cumulus cell NT showed that sham enucleation caused marked SA in freshly ovulated rat oocytes and that Na(+) supplementation prevented the manipulation-induced SA and improved the in vitro and in vivo development of rat somatic cell NT embryos. Taken together, the results have confirmed our hypothesis that the NCX is active in rat oocytes and its activity decreases with oocyte aging and that activating the NCX by increasing extracellular Na(+) inhibits SA of rat oocytes and improves the development of rat somatic cell NT embryos. These data are also important for understanding the mechanisms of oocyte aging.

Effects of embryo-derived exosomes on the development of bovine cloned embryos

“…Interestingly, although exosome supplementation enhanced only ratio of ICM/TE to a similar level as that in nonrenewal group (Table 3), no overt difference in pregnancy or calving rate was detected between these two groups, suggesting ratio of ICM/TE during cell lineage specification might be a more critical marker than others for the following in vivo development to term. This suggestion actually also supports previous findings that satisfactory blastocyst rate of SCNT embryos did not guarantee the following in vivo development to term [49, 52]. …”

Role of Na+/Ca2+ Exchanger (NCX) in Modulating Postovulatory Aging of Mouse and Rat Oocytes

“…Both the present study and our previous studies [31], [37] demonstrated that in the Sprague-Dawley rats, whereas many oocytes recovered 19 h post hCG injection underwent spontaneous activation during in vitro culture, spontaneous activation was less frequently observed in oocytes collected 13 h after hCG. To explore the mechanisms causing the difference in activation susceptibility between newly-ovulated and aged rat oocytes, the present results indicated that in the 13-h oocytes, an unknown activating stimulus (maybe the manipulation for their release from the oviduct) induced mild Ca 2+ rises, which activated NCX under a low ROS level (Fig.…”

“…The Na + /Ca 2+ exchange has been observed in immature and mature mouse [34], [35], porcine [36] and rat [37] oocytes. However, although our recent study indicated a role for Na + /Ca 2+ exchange in the spontaneous activation of rat oocytes [37], systematic studies are lacking on roles of the Na + /Ca 2+ exchanger (NCX) activity in controlling postovulatory oocyte aging of different species. Furthermore, in previous studies the NCX activity was usually determined by functioning assays (by measuring Ca 2+ flux, for example), with little attention paid to the dynamic changes in NCX contents.…”

“…There are two major mechanisms responsible for Ca 2+ extrusion across the plasma membrane: the Ca 2+ –ATPase and Na + /Ca 2+ exchange [32], [33]. The Na + /Ca 2+ exchange has been observed in immature and mature mouse [34], [35], porcine [36] and rat [37] oocytes. However, although our recent study indicated a role for Na + /Ca 2+ exchange in the spontaneous activation of rat oocytes [37], systematic studies are lacking on roles of the Na + /Ca 2+ exchanger (NCX) activity in controlling postovulatory oocyte aging of different species.…”

Towards Functional Annotation of the Preimplantation Transcriptome: An RNAi Screen in Mammalian Embryos

“…In summary, GO term analysis offered no striking outliers in functional categories within our screen results and suggests that preimplantation development equally requires all aspects of cellular and molecular function for success. Supporting this notion, previous studies have found essential roles during preimplantation for genes within each of these functional categories (including DNA/RNA/protein binding4546, catalytic activity4748, various transporter activity495051). …”