Identification and regulation of glycogen synthase kinase-3 during bovine embryo development

Abstract

The aim of this study was to examine the presence and regulation of glycogen synthase kinase-3a (GSK3A) and GSK-3b (GSK3B) in bovine embryos and their possible roles in embryo development. Our results show that GSK3A and GSK3B are present in bovine embryos at the two-cell stage to the hatched blastocyst stage. Bovine embryo development was associated with an increase in the phosphorylation of both isoforms, being statistically significant at blastocyst and hatched blastocyst stages, compared with earlier stages. Inhibition of GSK3 with CT99021 (3 mM) resulted in a significant increase in the percentage and quality of blastocysts, while inhibition of GSK3 with lithium chloride (LiCl; 20 mM) significantly reduced at the proportion of eight-cell embryos on day 3 and inhibited blastocyst formation. The use of LY294002 (10 mM), a specific inhibitor of phosphatidylinositol-3 kinase, also produced a significant decrease in embryo development. In addition, treatment with LiCl and LY294002 produced a significant decrease in the serine phosphorylation of both isoforms of GSK3. Finally, CT99021 and LiCl reduced the phosphorylation of b-catenin on Ser45 in two-cell embryos, while LY294002 increased it. Despite the fact that LiCl inhibited GSK3 activity, as demonstrated by b-catenin phosphorylation, its effects on the bovine embryo could be mediated through other signaling pathways leading finally to a decrease in the phosphorylation of GSK3 and a reduction in embryo development. Therefore, in conclusion, GSK3A/B serine phosphorylation was positively correlated with embryo development, indicating the importance of an accurate regulation of GSK3 activity during developmental stages to achieve normal bovine embryo development.

Insulin Increases Epiblast Cell Number of In Vitro Cultured Mouse Embryos via the PI3K/GSK3/p53 Pathway

“…Furthermore, a culture of ESCs in which GSK3 a and b had been deleted with CT99021 did not produce the effects seen when wild-type ESCs were cultured with CT99021 [48]. This decrease in b-catnin phosphorylation was also observed in embryos (bovine 2-cell) cultured with CT99021 at 3 mM, an effect that was the same as LiCl, another recognized inhibitor of GSK3 [49]. CT99021 at 1 mM was shown to reduce GSK3b activity to 1% in an in vitro assay in which the inhibitor was shown to be highly specific [32].…”

GSK3 Inhibitors Regulate MYCN mRNA Levels and Reduce Neuroblastoma Cell Viability through Multiple Mechanisms, Including p53 and Wnt Signaling

“…Different GSK3 inhibitors resulted in divergent cellular responses and transcriptional changes. At the organismal level, different GSK3 inhibitors can also produce different cellular outcomes and phenotypes (17)(18)(19)(20)(21). Such differences are unlikely to be due to varying inhibitor affinity to GSK3-a or GSK3-b, as they occur even in basal metazoans that possess only a single GSK3 gene.…”

GSK3β, But Not GSK3α, Inhibits the Neuronal Differentiation of Neural Progenitor Cells As a Downstream Target of Mammalian Target of Rapamycin Complex1

“…Although the reason for the discrepancy between these two studies is not clear, the germline deletion of GSK3a might have caused indirect effects, as GSK3 is an important regulator during embryogenesis [27]. Both GSK3a and GSK3b are known to be expressed from the two-cell stage embryo, and their activities are tightly controlled as the embryo develops [28]. Therefore, germ-line manipulation of either form of GSK3 may have produced unexpected outcomes in the later stages of development, especially when the other form, which could have compensated for the loss, was deleted.…”