The Roles And Mechanisms Of Maternal Proteins Gm364 And Elmod2 In Meiosis Progression Of Mouse Oocyte

Meiotic failure in oocytes is the major determinant of human zygote-originated reproductive diseases,the successful accomplishment of meiosis largely relay on the normal functions of many maternal proteins.These proteins were synthesized during the development of oogonia to oocyte.Some of them participate in the formation of primordial follicles.The others,however,are stored in the oocyte and will function after meiosis resumption,mainly work on meosis progression,fertilization and development of early embryo.Defects of maternal proteins have been found to result in reproduction-related diseases,e.g.infertility and miscarriage.Thus research of the function and underlying mechanism of maternal proteins is of great importance.The focus of our study—Gm364 and Elmod2,were both screened as maternal proteins highly expressed in mouse oocytes as well as fertilized eggs,thus were identified as maternal proteins.But their actual physiologic roles in mammalian oocytes remain unknown.The current study is mainly about the expression,role(s)and mechanism(s)of the two maternal proteins.Part 1.Gm364 plays important roles in meiosis progression through Deltex2/Notch2/Aurka?Akt signalingBackground:The maternal protein Gm364,which contains 9 transmebrane domains,is one of the transmembrane 9 superfamily.Gm364 was upregulated in a mouse model of which follicles undergo synchronous activation,thus was identified as one of "female fertility factors",but till now there were no any functional research of it.Methods and Results:1.RT-PCR,immunofluorescence and western blot showed that Gm364 is highly expressed in mouse ovaries,and is much more predominant in oocytes than in granulosa cells,mainly concentrates on the membrane.2.Through comprehensive phenotype analysis after knocking down Gm364 with specific siRNA,we found loss of Gm364 causes significant meiosis delay mainly characterized by chromosome congression problem.Not surprisingly,in-vitro fertilization results showed significantly lower fertilization and 2-pronuclei(PN)rates.3.Through IP-Maldi several interactive proteins associated with Deltex2/Notch2 signaling were characterized.The evidences of the interaction of Gm364 and Deltex2/Notch2 signaling are as follows:(a)Notch2 intracellular domain(NICD2)and Gm364 are co-immunoprecipitated well and co-localized at cell membrane;(b)Inhibition of Notch2 caused similar phenotype as knockdown of Gm364,including the decrease of chromosome congression as well as the failure of fertilization;(c)Protein level of NICD2 and the downstream kinases Akt and Aurka was down-regulated after Gm3 64 knockdown;(d)The intracellular Deltex2 increased while extracelluar Deltex2 decreased after deletion of Gm364,suggesting excytosis of Deltex2 was disrupted.4 Gm364 activity was regulated by Gramd 1 a-mediated phosphorylation and phosphorylated Gm364(p-Gm364)concentrated in cytoplasm.5.P-Gm364 interacted well with Psma3,which was involved in the ubiquitin-proteasome system,indicating p-Gm364 might be the inactive form and could be ubiquitinated at unique sites for degradation.Conclusion:In mouse oocytes,multi-transmembrane protein Gm364 triggers excytosis of the ligand of Notch2—Deltex2,then activates Notch2 and its downstream kinases,Akt and Aurka,to directly affect meiosis progression and fertilization.Activity of Gm364 might be regulated by Gramd la-mediated phosphorylation,and phosphorylated Gm364 might be the inactive form for further ubiquitin-proteasome degradation system.Part 2.GTPase-activating protein Elmod2 is involved in meiosis progression of mouse oocytes through activating Arl2 Background;Elmod2(ELMO/CED-12 domain containing 2)is one of Elmod GTPase activating protein(GAP)family,and could activate GTPase Arl2(ADP ribosylation factor-like 2),which is involved in the mitochondrial morphology,motility and maintenance of ATP level in somatic cells.We have found inhibition of Arl2 in mouse oocytes caused delay of meiosis progression.The aim of the current study is to investigate the interaction of Elmod2 and Arl2 in mouse oocyte,and to find their role in meiosis progression.Methods and Results:1.Elmod2 was the most abundant among Elmod family members in mouse oocytes.2.Deletion of Elmod2 by specific siRNA caused severe meiotic delay as well as significant increase of aneuploidy M?oocytes.3.After Elmod2 knockdown,the distribution of mitochondria changed largely,as great aggregation appeared in the cytopalsm.Accordingly,ATP level decreased dramatically.4.Similar defects occurs after inhibition of Arl2,including the delay of meiosis progression and the change of mitochondria activity.5 Immunoflourescence showed Elmod2 and Arl2 co-localized in the region of spindle,while co-IP revealed they were interated well with each other.Conclusion:In mouse oocytes,Elmod2 might function as the GAP for GTPase Arl2,and the interaction of them is involved in meiotic progression through regulating the distribution of mitochondria and maintenance of proper ATP level.