The Role And Mechanism Of Maternal Protein KBTBD8 In Meiosis Progression Of Mouse Oocyte

During oogen esis,oocytes synthesize and accumulate a number of maternal proteins.Some of maternal function in the formation of follicles and/or the growth of the oocytes,including GDF9,and BMP 15 and so on.Besides many maternal proteins also play significant roles in later stages,namely fertilization and early embryogenesis including MATER.We have found that defects of maternal proteins lead to reproduction-related diseases,e.g.infertility and miscarriage.It is very important to research the function and underlying mechanism of maternal proteins.The focus of our study—KBTBD8,was screened as maternal protein that highly expressed in mouse oocytes as well as fertilizated eggs.KBTBD8 was the member of the BTB-kelch family proteins which had highly conserved BTB/POZ domain,BACK domain and three kelch repeats.The BTB/POZ domain was mainly involved in facilitating self oligomerization or mediating protein-protein interactions with other proteins;Kelch β-propellers primarily functioned as scaffolds for protein-protein interactions.Proteins of the BTB-kelch family were known to be involved in multiple biological processes such as migration,cytoskeleton arrangement,regulation of cell morphology,protein ubiquitination and gene expression.Most importantly,many kelch proteins functioned as substrate-specific adaptors for Cullin E3 ubiquitin ligase(Cul3),a core component of the ubiquitin-proteasome system to regulate the protein turnover.It was reported that Kbtbd8 was located in the Golgi apparatus and translocated to the spindle apparatus during mitosis.And Achim Werner et al found that CUL3KBTBD8 was an essential regulator of neural crest specification.CUL3KBTBD8 monoubiquitylated NOLC1 and its paralog TCOFI,whose mutation underlied the neurocristopathy Treacher Collins Syndrome.Ubiquitylation drived formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodeled the translational program of differentiating cells in favor of neural crest specification.But its actual physiologic roles in mammalian oocytes meiosis progression remained unknown.The current study was mainly about the expression,role(s)and mechanism(s)of the maternal proteins.Methods and Results:1.Immunofluorescence and Western Blot showed that Kbtbd8 was highly expressed in mouse ovaries,and was much more predominant in oocytes than in granulosa cells,mainly concentrates on the spindle apparatus.2.Through comprehensive phenotype analysis after knocking down Kbtbd8 with microinjecting specific siRNA,we found loss of Kbtbd8 caused significant meiosis delay mainly characterized by chromosome disarrangement and spindle apparatus disorder problem.In-vitro fertilization results showed significantly lower fertilization and 2-pronuclei(PN)rates.3.Detailed molecular mechanisms were as follows:a)Protein level of p-Cdkl was up-regulated and Cyclin B1 was down-regulated after Kbtbd8 knockdowned that failed to active the MPF and meiotic resumption.b)After Kbtbd8 knockdowned,the distribution of mitochondria changed largely,as great aggregation appeared in the cytopalsm.Accordingly,ATP level decreased dramatically.c)Kinases p-Akt,p-Erkl/2,ace-α-Tubulin was down-regulated after Kbtbd8 knockdowned,which illustrated the phenotype of spindle apparatus disorder and chromosome disarrangement.Conclusion:In mouse oocytes,KBTBD8 which located in the spindle apparatus affected mouse oocyte meiosis progression and fertilization by regulating the Akt,Erkl/2,MPF and mitochondria signal pathway.