The Effects Of Maternal Protein Elmod2 On Mouse Oocyte Maturation And Fertilization

ELMOD2 is one of the ELMO protein family members.The human ELMO protein family has six members,known as ELMOD1,ELMOD2,ELMOD3,ELMO1,ELM02 and ELM03.These six proteins have been further divided into two subgroups:ELMODs and ELMOs by homology comparison.ELMOD1-3 has a highly conserved "catalytic arginine residue" which is critical for its GTPase-activating protein(GAP)activity.While except a conserved ELMO domain,ELMO1-3 also has an unknown function 3361 domain and a PH domain.But they don't have the conserved "catalytic arginine residue".Recent research has compared the GAP activity of human ELMOD1-3 against six Arf protein members,and then found that ELMOD2 is the most acticity protein among ELMO families.Though has been identified as a maternal protein in mature mouse oocyte,there is no report on the effects and mechanism of Elmod2 in oocyte maturation and fertilization process.Thus,in this research we did a deeply detailed study on the function of Elmod2 in mouse oocyte and made a preliminary discussion on its mechanism.First,we analyzed the expression of Elmod2 in mouse ovary and oocyte,found that compared with other Elmo family members,Elmod2 has the highest mRNA abundance in mouse oocytes.Moreover,the expression of Elmod2 is more preferential in oocytes than in granular cells(GCs).The immunofluorescent result of day-21 mouse ovary sections showed that Elmod2 is riched in oocytes cortex and germinal vesicle region,while the immunofluorescent result of mouse oocytes showed that Elmod2 locates in the nucleus in germinal vesicle(GV)phase and distributes in the region of the spindle in other meiotic phases.The result of western blot indicated that Elmod2 has a higher expression in day-3(phase of primordial follicle assembly)and day-21(phase of first batch of follicle maturation)mouse ovary and its expression is relatively stable during different periods of oocyte development.To study the function of Elmod2 in mouse oocytes,we microinjected specific Elmod2 siRNA into the cytoplasm of mouse GV oocytes,both RT-PCR and western blot displayed a high efficiency of interference.The immunofluorescent result showed that Elmod2 knockdown cause a significant delay in meiosis progress after 8h(MI phase)and 16h(M? phase)of oocytes culture with severely disrupted spindles and misaligned chromosomes.Consistent with this observation,incidence of aneuploidy is also markedly increased in Elmod2-interfered oocytes.Further more,the rate of fertilization and 2-pronuclear zygote(2PN)is obversely decreased in vitro fertilization experiment(IVF)following Elmod2 knockdown.Altogether,our data reveal a novel function of Elmod2 during oocytes meiosis and fertilization process.Finally,we did some preliminary explorations on the potential mechanisms by which Elmod2 control oocytes development.On the basis of available data,Elmod2 is an effective GAP for Arl2 and Arl2 required for mitochondrial morphology,motility,and maintenance of ATP levels.So after the same treatment,we marked the mitochondria and measured ATP levels of mouse oocytes,found that knockdown of Elmod2 showed an obvious defect on mitochondria distribution and ATP levels;at the same time,the immunofluorescent result showed the co-localization of them.Immunoprecipitation-Mass Spectroscopy analysis with mouse oocytes found that Elmod2 can interact with Arf5.The immunofluorescent result also showed the co-localization of Elmod2 and Arf5.Knockdown of Elmod2 can cause the fluorescent signal enhancement of Arf5.In summary,we suggest that Elmod2,as a maternal factor with specific expression in mouse oocytes,regulates mitochondria distribution and ATP levels to influence spindle assembly and chromosome alignment by interacting with Arl2 and Arf5 during oocyte meiotic maturation and fertilization process.