Background Dynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently, lactylation has been reported as a novel epigenetic modification that regulates various cellular processes, but its role during early embryogenesis has not been elucidated. Results We examined nuclear accumulation of H3K23la, H3K18la and pan histone lactylation in mouse oocytes and pre-implantation embryos by immunofluorescence with specific antibodies. All of the three modifications were abundant in GV stage oocytes, and both H3K23la and pan histone lactylation could be detected on the condensed chromosomes of the MII oocytes, while H3K18la were not detected. After fertilization, the nuclear staining of H3K23la, H3K18la and pan histone lactylation was faint in zygotes but homogeneously stained both of the parental pronuclei. The signal remained weak in the early cleavage stage embryos and increased remarkably in the blastocyst stage embryos. Comparison of the embryos cultured in four different conditions with varying concentrations of oxygen found that H3K23la, H3K18la and pan histone lactylation showed similar and comparable staining pattern in embryos cultured in atmospheric oxygen concentration (20% O2), gradient oxygen concentration (5% O2 to 2% O2) and embryos obtained from in vivo, but the modifications were greatly reduced in embryos cultured in hypoxic condition (2% O2). In contrast, nuclear accumulation of H3K18ac or H3K23ac was not significantly affected under hypoxic condition. Moreover, the developmental rate of in vitro cultured embryo was significantly reduced by low oxygen concentration and small molecule inhibition of LDHA activity led to decreased lactate production, as well as reduced histone lactylation and compromised developmental rate. Conclusions We provided for the first time the dynamic landscape of H3K23la, H3K18la and pan histone lactylation in oocytes and pre-implantation embryos in mice. Our data suggested that histone lactylation is subjected to oxygen concentration in the culture environment and hypoxic in vitro culture reduces histone lactylation, which in turn compromises developmental potential of pre-implantation embryos in mice.
In addition to serving as the building blocks for protein synthesis, amino acids serve as critical signaling molecules in cells. However, the mechanism through which amino acid signals are sensed in cells is not yet fully understood. This study examined differences in the phosphorylation levels of proteins in response to amino acid signals in Cashmere goat fetal fibroblasts (GFb). Amino acid deficiency was found to induce autophagy and attenuate mammalian/mechanistic target of rapamycin complex (mTORC1)/Unc-51-like autophagy activating kinase 1 (ULK1) signaling in GFb cells. A total of 144 phosphosites on 102 proteins positively associated with amino acid signaling were screened using phosphorylation-based proteomics analysis. The mitogen-activated protein kinase (MAPK) signaling pathway was found to play a potentially important role in the interaction network involved in the response to amino acid signals, according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and MAPK1/3 may serve as a central hub for the entire network. Motif analysis identified three master motifs, xxx_S_Pxx, xxx_S_xxE, and xxx_S_xDx, which were centered on those phosphosites at which phosphorylation was positively regulated by amino acid signaling. Additionally, the phosphorylation levels of three membrane proteins, the zinc transporter SLC39A7, the sodium-dependent neutral amino acid transporters SLC1A5 and SLC38A7, and three translation initiation factors, eukaryotic initiation factor (eIF)5B, eIF4G, and eIF3C, were positively regulated by amino acid signals. These pivotal proteins were added to currently known signaling pathways to generate a novel model of the network pathways associated with amino acid signals. Finally, the phosphorylation levels of threonine 203 and tyrosine 205 on MAPK3 in response to amino acid signals were examined by western blot analysis, and the results were consistent with the data from the phosphoproteomics analysis. The findings of this study provide new evidence and insights into the precise mechanism through which amino acid signals are sensed and conducted in Cashmere goat fetal fibroblasts.
Background: Sexual reproduction is the basic way for higher eukaryotes to reproduce. Fusion of sperm with oocyte is the key process for success of sexual reproduction. Multiple proteins have been identified to be implicated in mammalian sperm-egg fusion, in which only a few, such as IZUMO1 of sperm and JUNO of egg, have been verified by knockout mice to be indispensable to gamete fusion. However, interaction between IZUMO1 and its receptor JUNO mediates sperm-egg recognition and binding, but is unqualified for membrane fusion. Thus, there might be other accessorial proteins interacting with IZUMO1 or/and JUNO for the sperm-egg binding and succeeding membrane fusion. Results: Here, we reported that treatment of rat sperm and egg with anti-Galectin-1 antibody significantly inhibits IVF rate. Galectin-1 was detected on the head and the front half flagellum of rat sperms and in cytoplasm of spermatocytes and the Sertoli cells. It was also found on cell membrane of rat MII eggs as well as in cytoplasm of cumulus cells. Galectin-1 interacts with JUNO and co-localizes with JUNO on the cell membrane and in cytoplasm of rat MII eggs. Conclusions: Our results suggested that Galectin-1 be involved in the process of sperm-egg fusion through interaction with JUNO.
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