The Study On The Regulatory Mechanism Of GPER In Mouse Oocyte Meiosis And Maternal MRNA Translation

The quality of oocyte maturation and the development competence after fertilization are key factors supporting the reproductive capacity of female animals.Different from symmetrical mitosis,oocytes undergo cytoplasmic extremely asymmetric meiosis while achieving chromosome halving in order to preserve the maternal material to the greatest extent.Because the fully grown oocyte is in a state of transcriptional silencing before initiating meiosis,the substances required for the meiotic process and early embryonic development after fertilization depend on the continuous accumulation of maternal factors(m RNA,protein and metabolites,etc.)during the follicular growth stage.Studies have shown that heterodimeric G proteins can be asymmetrically activated in a receptor-independent manner to participate in a variety of asymmetric cell division processes.G protein-coupled estrogen receptor(GPER)were expressed in mouse oocytes,and GPER showed asymmetric distribution during the maturation of mouse oocytes,with higher expression on the side of the cortex near the spindle.Based on these results,this study mainly explored the molecular mechanism of GPER regulation of spindle migration,localization,asymmetric division and translation of maternal m RNA during oocyte maturation.Blocked maternal m RNA translation,abnormal spindle assembly,and faulty chromosome segregation during oocyte meiosis lead to abnormal embryonic development,miscarriage,or severe genetic disease.After germinal vesicle breakdown(GVBD),maternal m RNA translation,assembly of the meiotic spindle,establishment of spindle-chromosome interactions,and oocyte polarity formation are key processes to complete asymmetric division,revealing the regulation of these important processes.The molecular targets of oocytes and their regulatory mechanisms on oocyte meiosis are the main goals of embryo engineering research in reproductive biology.The research work has important reference value for revealing the reproductive mechanism of female animals,improving the reproductive ability of domestic animals and treating female infertility.The main research contents and research results obtained are as follows:(1)GPER is involved in asymmetric division of oocytesThe GPER plays an important role in the regulation of oocyte meiosis.Asymmetric oocyte division and first polar body ejection depend primarily on cortical spindle positioning,cortical remodeling,oocyte polarization and subsequent actin-dependent spindle migration into the cortex.This study mainly explores the mechanism of GPER in asymmetric division of oocytes.Mouse oocytes were isolated,and the expression and localization of GPER in different stages of oocytes were detected by western blotting and immunofluorescence staining.GPER is located in the cytoplasm and is enriched on the oocyte membrane during the period of GV);after GVBD,GPER is mainly distributed around chromosomes;from the first meiotic metaphase(MI),with the microtubules form bipolar spindles around chromosomes,while GPER is mainly concentrated in the bipolar spindle region.From anaphase,GPER is mainly located on the side of the chromosome near the poles of the spindle.Using small interfering RNA(si RNA)microinjection technology to knock down the expression of endogenous GPER in oocytes,it was found that GPER knockdown did not affect the occurrence of GVBD in oocytes,but led to a decrease in the rate of polar body excretion,and the MI spindle could not be transferred to the oocyte.Cortical areas migrate,expelling larger polar bodies.In addition,the assembly level of cytoplasmic filaments and the formation rate of filament caps were significantly reduced after GPER knockdown,and chromosome segregation errors in abnormally assembled spindles were significantly increased.Western blotting results showed that knockdown of endogenous GPER protein expression in mouse oocytes resulted in decreased activity of PI3K-AKT signaling pathway in MI stage oocytes,and decreased protein expression levels of Rho family small molecule GTPase members RAC1,Rho A,and CDC42,inhibiting the phosphorylation of Cofilin,a key protein in actin depolymerization.By injecting in vitro transcribed Gper m RNA into GPER knockdown oocytes,the expression of GPER protein in oocytes can be rescued.It is found that the expression of exogenous GPER can rescue the failure of polar body expulsion,abnormal polar body size and caused by GPER knockdown.(2)GPER is involved in the regulation of maternal m RNA translation during oocyte meiosisGrowing oocytes are transcriptionally silent,and the bulk of the proteins required to drive meiosis come from the continuous translation of maternal m RNAs stored in the oocyte’s growth phase.The translation process of these maternal m RNAs mainly depends on the regulation of cytoplasmic polyadenylation and translation activation,however,the upstream regulatory pathways involved in the regulation of cytoplasmic polyadenylation and maternal m RNA translation and its role in oocyte meiosis is not very clear.This study aimed to explore the regulatory mechanism of GPER on maternal m RNA translation during mouse oocyte meiosis.The Cre/Loxp system was used to construct oocyte Gper-specific knockout mice(Gper-CKO).HPG protein translation assay found that the overall translation activity of GPER-deficient oocytes during meiosis was significantly decreased;PAT polyadenylation tail length analysis showed that GPER-deficient oocytes were involved in the regulation of meiosis genes(Mos,Tpx2,Dazl,Ccnb1,Btg4,Cnot7,etc.)m RNA polyadenylation was blocked;Oocyte transcriptome sequencing results showed that 161 genes were significantly up-regulated in GPER-deficient oocytes,and 1212 genes were significantly up-regulated down.KEGG analysis showed that the down-regulated genes of Gper-CKO oocytes were mainly enriched in EGFR signaling pathway,MAPK signaling pathway,actin cytoskeleton and endocytosis related pathway genes.Gene Ontology(GO)analysis revealed that genes related to RNA binding,m RNA 3’UTR binding and translation initiation factor activity were significantly downregulated in Gper-CKO oocytes.In addition,genes related to PKB binding and actin binding were significantly down-regulated in Gper-CKO oocytes.These signaling pathways or biological processes are all related to the regulation of m RNA translation and the actin assembly.The activities of ERK1/2 and PI3K-AKT signaling pathways in oocytes were further analyzed,and the results showed that the phosphorylation levels of key proteins of ERK1/2 and PI3K-AKT signaling pathways in GPER-deficient oocytes were significantly down-regulated.These results suggest that GPER regulates oocyte-derived m RNA polyadenylation and translational activation by regulating ERK1/2 and PI3K-AKT signaling pathway activity.(3)GPER is involved in mediating the effect of EGF on the regulation of maternal m RNA translation during oocyte meiosisThe ability of oocytes to mature and develop after fertilization is largely dependent on complex interactions with the cumulus/granulosa cells that surround it during development.In addition to providing critical metabolite support to the oocyte,cumulus cells are also involved in oocyte meiosis resume,cytoskeleton assembly,and maternal m RNA translation regulation.EGF can regulate maternal m RNA translation in a PI3K-AKT signaling pathwaydependent manner during oocyte meiosis by activating EGFR in cumulus cells.But it was not clear how EGFR,which is only expressed on cumulus cells,transmits the signal across the cell to the oocyte when activated.Therefore,this study aimed to investigate the mechanism that GPER is involved in mediating the translation regulation of oocyte maternal m RNA by EGF.The HPG protein translation assay found that EGF treatment of cumulus-oocyte complexes(COCs)could significantly increase the overall translation level of oocytes,but had no effect on cumulus-depleted oocytes(DO),indicating that EGF improve maternal m RNA dependent on the cumulus cells;Western blot results showed that EGF increased GPER protein level and the cortex enrichment of GPER in the oocyte from COCs;Immunofluorescence staining and immunoelectron microscopy analysis showed that GPER positive signal existed in oocytes zona pellucida,and the GPER positive signal in EGF-treated COCs-derived oocytes was higher than that in the control group.Further studies showed that GPER co-localized with vesicle marker proteins,and EGF promoted the formation of GPER-positive vesicles in cumulus cells and the transition from cumulus cells to oocytes.EGF inhibited the localization of GPER in lysosomes and enhanced the localization of GPER on the cell membrane by promoting the expression of RAB11 A.These results suggest that EGF regulates maternal m RNA translation in oocytes by promoting the delivery of GPER-positive vesicles from cumulus cells to the oocyte.In conclusion,GPER is expressed in all stages of mouse oocyte meiosis,and GPER is involved in regulating oocyte maternal m RNA polyadenylation and translation activation through ERK1/2 and PI3K-AKT signaling pathways,which is important Rho GTPasemediated dynamic assembly of microfilaments,spindle migration,oocyte maturation and embryonic development.