P-406 Metal-phenolic network cell-membrane modification-mediated assembly strategy for re-establishing oocyte-granulosa cell communications and improving oocyte developmental potential

Abstract

Abstract Study question Re-establish the cell-cell communication of isolated oocyte and granulosa cells (GCs) to mimic the follicle microenvironment and promote the success rate of in-vitro maturation (IVM). Summary answer The Metal-phenolic network (MPN) nanoparticles-mediated cell membrane modification system was used to achieve self-assembly of GCs on the oocyte membrane and re-establishing the Oocyte-GCs communication. What is known already How to preserve the fertility of female patients of childbearing age is an important problem faced by reproductive medicine. IVM of oocytes is an important means for female fertility preservation, but the problem of oocyte maturation rate needs to be solved urgently. The Oocyte-GCs interaction determines the key events in the oocyte development and maturation. Biological materials are gradually used for IVM, but there are still significant disadvantages. MPN has been widely used for the functionalization of bacteria and cell surface due to its unique nano-network structure and good biocompatibility. Study design, size, duration The MPN nanoparticles are respectively modified on GCs and oocytes, and the GCs can be self-assembled on the surface of the oocytes to re-establish cell communication. Then explore the biosafety and cytotoxicity of MPN nanoparticles on GCs and oocytes to rule out potential impact on the secretory function of GCs, reveal the formation of gap junction and substance exchange of Oocyte-GCs, and evaluate the effects of this technology on oocyte maturation, fertilization and embryo development. Participants/materials, setting, methods The oocyte-GCs assembly could be achieved through the MPN(EGCG-Zn2+). Then MPN@GCs and MPN@Oocytes were characterized by fluorescent protein, Zeta, SEM and TEM; the cytotoxicity was assessed by Live/Dead staining, CCK-8 and flow cytometry assays. Gap junction was observed by TEM; the expression levels changes of signaling pathways for assembled oocyte and GCs were detected by single-cell RNA-seq, RT-qPCR and WB. The oocyte and embryo developmental potential were also analyzed. Main results and the role of chance We confirmed the formation of a nano-network surrounding GCs and oocytes and achieved the assembly of GCs on the surface of oocytes. Furthermore, we illustrated that MPN was non- cytotoxicity for GCs and oocytes and have no negative effect on the secretion function of GCs, indicating high biocompatibility of MPN and shows the potential application on the re-establishment of oocyte-GCs communication. Gap junctions formation and substance exchange establishment between GCs and oocytes were also proved in the study. The single-cell RNA-seq uncovered key pathways involved in oocyte-GCs interactions after the oocyte-GCs assembly. In detail, the SUMO1 ubiquitination modification of PTEN was inhibited after re-establishing cell-cell communication and decreased the localization of PTEN at the GCs membrane, which weakened its inhibition on PI3K. Higher PI3K level further up-regulated AKT level and stimulated its activation by increasing phosphorylation modification, finally improved the developmental potential and maturation rate of oocytes. The re-establishment of cell communication significantly down-regulated the P38/MAPK pathway in oocytes, including inhibited P38/MAPK expression, thereby reducing the phosphorylation of MSK1 and the activation of ATF-1, and effectively weakened the apoptosis controlled by P38/MAPK. Finally, a significantly higher fertilization and blastocyst formation rate were observed as compared to the control group. Limitations, reasons for caution Although this study has achieved promising results, there is still gap between basic research and applications, and more fundamental research should be conducted to make it a powerful tool for clinical application in the future. Wider implications of the findings This study not only provided a promising tool for establishing oocyte-GCs communication and improving oocyte maturation rate for IVM, but also showed a versatile and powerful cell-cell assembly platform for regenerative medicine including tissue engineering and organoid studies. Trial registration number not applicable