Inhibition of phosphorylated Ser473-Akt from translocating into the nucleus contributes to 2-cell arrest and defective zygotic genome activation in mouse preimplantation embryogenesis.

Phosphorylated Ser473-Akt (p-Ser473-Akt) is extensively studied as a correlate for the activity of Akt, which plays an important role in mouse oogenesis and preimplantation embryogenesis. However, little progress has been made about its effect on the mouse zygotic genome activation (ZGA) of 2-cell stage in mouse preimplantation embryos. In this study, we confirmed its localization in the pronuclei of 1-cell embryos and found that p-Ser473-Akt acquired prominent nucleus localization in 2-cell embryos physiologically. Akt specific inhibitors API-2 and MK2206 could inhibit the development of mouse preimplantation embryos in vitro, and induce 2-cell arrest at certain concentrations. 2-cell embryos exposed to 2.0 µmol/L API-2 or 30 µmol/L MK2206 displayed attenuated immunofluorescence intensity of p-Ser473-Akt in the nucleus. Simultaneously, qRT-PCR results revealed that 2.0 µmol/L API-2 treatment significantly downregulated the mRNA pattern of MuERV-L and eIF-1A, two marker genes of ZGA, suggesting a defect in ZGA compared with that of control group. Collectively, our work demonstrated the nuclear localization of p-Ser473-Akt during major ZGA, and Akt specific inhibitors API-2 and MK2206 which led to 2-cell arrest inhibited p-Ser473-Akt from translocating into the nucleus of 2-cell embryos with defective ZGA as well, implying p-Ser473-Akt may be a potential player in the major ZGA of 2-cell mouse embryos.

Roles of ERα during mouse trophectoderm lineage differentiation: revealed by antagonist and agonist of ERα.

During mouse early embryogenesis, blastomeres increase in number by the morula stage. Among them, the outer cells are polarized and differentiated into trophectoderm (TE), while the inner cells remain unpolarized and give rise to inner cell mass (ICM). TE provides an important liquid environment for ICM development. In spite of extensive research, the molecular mechanisms underlying TE formation are still obscure. In order to investigate the roles of estrogen receptor α (ERα) in this course, mouse 8-cell embryos were collected and cultured in media containing ERα specific antagonist MPP and/or agonist PPT. The results indicated that MPP treatment inhibits blastocyst formation in a dose-dependent manner, while PPT, at proper concentration, promotes the cavitation ratio of mouse embryos. Immunofluorescence staining results showed that MPP significantly decreased the nuclear expression of CDX2 in morula, but no significant changes of OCT4 were observed. Moreover, after MPP treatment, the expression levels of the genes related to TE specification, Tead4, Gata3 and Cdx2, were significantly reduced. Overall, these results indicated that ERα might affect mouse embryo cavitation by regulating TE lineage differentiation.

Effects of ERα-specific antagonist on mouse preimplantation embryo development and zygotic genome activation.

Zygotic genome activation (ZGA) is essential for normal development of mammalian preimplantation embryos. Estrogen receptor alpha (ERα) has been implicated in early embryogenesis, and controls the expression of genes associated with proliferation, differentiation and development of cell and target organs via a genomic effect. The objective of this study was to determine whether ERα plays a role in early embryo development and affects ZGA gene expression. Toward this objective, 1-cell embryos from B6C3F1 mouse were cultured with the antiestrogen ICI182780, ERα-specific antagonist MPP, ERα-specific antibody and ERß-specific antagonist PHTPP. Development of 2-cell to 4-cell in vitro was significantly blocked by ICI182780, MPP and ERα-antibody treatment in a dose-dependent manner but not affected by PHTPP exposure. MPP decreased nuclear ERα protein levels and reduced mRNA expression levels of MuERV-L, one of the ZGA related genes. The results indicate that ERα has a functional role in early embryo development by regulation of ZGA-related genes.

Role of CD4+ CD25+ regulatory T cells in melatonin-mediated inhibition of murine gastric cancer cell growth in vivo and in vitro.

Melatonin is an important immune modulator with antitumor functions, and increased CD4(+) CD25(+) regulatory T cells (Tregs) have been observed in tumor tissues of patients and animal models with gastric cancer. However, the relationship between melatonin and Tregs remains unclear. To explore this potential connection, we performed an in vivo study by inoculating the murine foregastric carcinoma (MFC) cell line in mice and then treated them with different doses of melatonin (0, 25, 50, and 100 mg/kg, i.p.) for 1 week. The results showed that melatonin could reduce the tumor tissue and decrease Tregs numbers and Forkhead box p3 (Foxp3) expression in the tumor tissue. An in vitro study was also performed to test the effects of purified Tregs on melatonin-mediated inhibition of MFC cells. The cell cultures were divided into three groups: 1) MFC+ Tregs; 2) MFC only; and 3) MFC+CD4(+) CD25(-) T cells. After treatment with different concentrations of melatonin (0, 2, 4, 6, 8, and 10 mM) for 24 h, a dose-dependent apoptosis and cell cycle arrest at the G2/M phase was detected in melatonin-treated MFC at melatonin concentration higher than 4 mM. There were no significant differences in the rates of apoptosis and cell cycle distributions of MFC among the three groups. In conclusion, the antigastric cancer effect of melatonin is associated with downregulation of CD4(+) CD25(+) Tregs and its Foxp3 expression in the tumor tissue.