The Function And Mechanism Of LncRNA Uc.308 In Oocyte Maturation

Objective:In recent years,the incidence of infertility has increased year by year.Oocyte maturation in vitro(IVM)technology as a treatment mean plays an important role in the treatment of poor ovarian response and the fertility of some cancer patients.Although the application of IVM technology in animal has matured,there are still problems in human application.Therefore,it is important to clarify the molecular mechanism of oocyte maturation and obtain high quality eggs.The regulation mechanism of oocyte maturation is complex,involving the ubiquitin proteasome pathway,phosphatidyl inositol pathway and adenosine cyclic adenosine pathway.The post genome era,long noncoding RNA(lncRNA)function is revealed by participating in chromosome recombination,genomic imprinting,endogenous siRNA formation and regulation of protein function,influence the gametogenesis and embryogenesis,cell differentiation and growth and other biological processes.Previous studies have found that lncRNA uc.308 is highly expressed in immature oocytes and complemented with the Btrc gene sequence.The purpose of this study is to analyze the role of lncRNA uc.308 in the process of mammalian oocyte maturation,take Btrc as the breakthrough point of the mechanism research,further analyze the molecular mechanism of lncRNA uc.308 affecting oocyte maturation.Methods:(1)Detect the expression level of lncRNA uc.308 in oocyte maturation by real time fluorescence quantitative PCR.(2)Detect the subcellular localization of lncRNA uc.308 by the technique of fluorescence in situ hybridization.(3)Explore the function of lncRNA uc.308 in oocyte meiosis by microinjecting lncRNA uc.308 in GV oocyte making it overexpression,observe the structure of the oocyte cytoskeleton through immunofluorescence staining of F-actin and b-tubulin.(4)Detect the expression level of Btrc gene after microinjecting lncRNA uc.308 in GV oocyte by real time fluorescence quantitative PCR.Detect the expression levels of Btrc gene and the rate of the first polar body after microinjecting lncRNA uc.308+Btrc siRNA,studying the possible mechanism of lncRNA uc.308 in oocyte maturation.Results:(1)LncRNA uc.308 was expressed in all stages of oocyte development,the expression level in GV and GVBD phase was higher,MI stage decreased obviously,and slightly increased in MII phase.(2)During the GV and GVBD phases,the signal of lncRNA uc.308 dispersed in the cytoplasm,MI phase,mainly located around the chromosome,and a small amount aggregate in the cell membrane.(3)After microinjection of lncRNA uc.308 in GV phase,the MI formation rate of oocytes was slightly higher than that of the control group,while the MII formation rate was significantly lower than that of the control group,and the morphology of spindle was not significantly abnormal.(4)Microinjection of lncRNA uc.308 in GV phase,the expression of Btrc gene in GV phase decreased compared with the control group,increased in MII phase compared with the control group.In the rescue experiment,the level of overexpression of Btrc gene was lower than that in the lncRNA uc.308 overexpression group,and the level of the decrease in the discharge rate of the first polar body of oocyte was relieved.Conclusions:LncRNA uc.308 was expressed in all stages of oocyte development,and the expression level was higher in GV and GVBD phase,dispersed in the cytoplasm,and MI phase decreased significantly,aggregated around chromosome and a small amount in cell membrane.After overexpression of lncRNA uc.308,the meiotic maturation process of oocytes was abnormal,and the formation rate of the first polar body decreased and the probability of abnormal division increased.And after overexpression of lncRNA uc.308,the expression level of related gene Btrc decreased at GV stage,MII phase increased significantly.When decreased the expression of Btrc gene by coinjection of Btrc siRNA,Btrc expression level decreased at MII stage,and the first polar body excretion rate decreasing get relief.It is suggested that the effect of lncRNA uc.308 in the maturation of oocyte is closely related to the Btrc gene.