BACKGROUND: Metabolic homeostasis is closely related to early impairment of cell fate determination and embryo development. The protein kinase mechanistic target of rapamycin (mTOR) is a key regulator of cellular metabolism in the body. Inhibition of mTOR signaling in early embryo causes postimplantation development failure, yet the mechanisms are still poorly understood.
METHODS: Pregnancy mice and preimplantation mouse embryo were treated with mTOR inhibitor in vivo and in vitro respectively, and subsequently examined the blastocyst formation, implantation, and post-implantation development. We used immunofluorescence staining, RNA-Seq smart2, and genome-wide bisulfite sequencing technologies to investigate the impact of mTOR inhibitors on the quality, cell fate determination, and molecular alterations in developing embryos.
RESULTS: We showed mTOR suppression during preimplantation decreases the rate of blastocyst formation and the competency of implantation, impairs the post implantation embryonic development. We discovered that blocking mTOR signaling negatively affected the transformation of 8-cell embryos into blastocysts and caused various deficiencies in blastocyst quality. These included problems with compromised trophectoderm cell differentiation, as well as disruptions in cell fate specification. mTOR suppression significantly affected the transcription and DNA methylation of embryos. Treatment with mTOR inhibitors increase lysosomal activation and disrupts the organization and dynamics of the actin cytoskeleton in blastocysts.
CONCLUSIONS: These results demonstrate that mTOR plays a crucial role in 8-cell to blastocyst transition and safeguards embryo quality during early embryo development.

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Stroke is considered a leading cause of mortality and neurological disability, which puts a huge burden on individuals and the community. To date, effective therapy for stroke has been limited by its complex pathological mechanisms. Autophagy refers to an intracellular degrading process with the involvement of lysosomes. Autophagy plays a critical role in maintaining the homeostasis and survival of cells by eliminating damaged or non-essential cellular constituents. Increasing evidence support that autophagy protects neuronal cells from ischemic injury. However, under certain circumstances, autophagy activation induces cell death and aggravates ischemic brain injury. Diverse naturally derived compounds have been found to modulate autophagy and exert neuroprotection against stroke. In the present work, we have reviewed recent advances in naturally derived compounds that regulate autophagy and discussed their potential application in stroke treatment.

Cerebral ischemia is a severe neurological disorder with limited therapy. Autophagy refers to the intracellular degradation process via an autophagosome-lysosome pathway. Emerging studies indicated the neuroprotective effects of autophagy against ischemic neuronal injury, suggesting the potential neuroprotection of autophagy-inducing compounds. Tomatidine is a gut microbiota-derived metabolite from unripe tomatoes. Tomatidine activates autophagy either in mammal cells or C elegans. However, potential neuroprotection of tomatidine against ischemic neuronal injury has not been determined. In the present investigation, N2a cells and primary cultured mice cortical neurons were subjected to oxygen-glucose deprivation followed by reperfusion (OGD/R). Cell injury was determined by MTT and lactate dehydrogenase release. Autophagosomes and autolysosomes were visualized by transfecting mCherry-GFP-tandem fluorescent LC3. The protein levels of LC3, Cathepsin D, Cathepsin B, and transcription factor EB (TFEB) were detected by Western blot. Lysosomes were stained with LysoTracker Red and dequenched-bovine serum albumin (DQ-BSA red). Tomatidine alleviated OGD/R-induced injury in N2a cells and neurons. Interestingly, tomatidine treatment attenuated, rather than reinforced, the OGD/R-elevated LC3-II, which can be reversed by lysosome inhibitor. These results indicated enhanced lysosomal activity rather than autophagosome generation with tomatidine treatment in our models. Indeed, tomatidine increased the lysosome number, proteolytic activities, as well as the expression of Cathepsin D and Cathepsin B. In addition, tomatidine increased the expression and nucleus translocation of (TFEB). Besides, lysosomal inhibitors chloroquine and bafilomycin, but not wortmannin, abolished the protection of tomatidine. In conclusion, the present study revealed the neuroprotection of tomatidine against ischemic injury by promoting lysosomal activity, possibly with the involvement of TFEB-related mechanisms.

This study shows that Cd induces autophagy in the human\'s embryonic normal liver cell line (WRL-68). The expression of LC3B-II and the mature cathepsin L were analyzed by Western blotting. The autophagosomes and lysosomes were directly visualized by electron microscopy and confocal microscopy analysis in Cd-exposed WRL-68 cells. In this study, we first found that autophagy induced the activation of lysosomal function in WRL-68 cells. The lysosomal activation was markedly decreased when the cells were co-treated with 3-MA (an inhibitor of autophagy). Secondly, we provided the evidence that the activation of lysosomal function depended on autophagosome-lysosome fusion. The colocalization of lysosome-associated membrane protein-2 (LAMP2) and GFP-LC3 was significantly reduced, when they were treated with thapsigargin (an inhibitor of autophagosome-lysosome fusion). We demonstrated that deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, which suggests that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Thirdly, we provided evidence that the activation of lysosomal function was associated with lysosomal acid. We investigated the relationship between autophagosome-lysosome fusion and pH in acidic compartments by visualizing fusion process in WRL-68 cells. This suggests that increasing pH in acidic compartments in WRL-68 cells inhibits the autophagosome-lysosome fusion. Finally, we found that the activation of lysosomal function was associated with Ca(2+) stores and the intracellular Ca(2+) channels or pumps were possibly pH-dependent.