Invetigation Of MKLN1 Regulating Mouse Oocytes Maturation

Cumulus cells are a kind of specialized granulosa cells which surround the oocytes.They play an important role in the maturation process of oocytes.Cumulus cells can through gap connection or cell protuberance provide the oocytes with a variety of nutrients,such as pyruvic acid,amino acids and growth factors,and also regulate oocyte meiosis by transmission of cyclic adenosine(c AMP).The close association between cumulus cells and oocytes suggests that cumulus cells may serve as an important noninvasive biomarker for oocyte developmental competence.In vitro maturation(IVM)is a technique that culture the unmature oocytes in vitro.But IVM still faces many challenges,such as the low maturation rate and poor developmental competence.How to improve the quality of oocytes after IVM and select top-quality oocytes is an urgent problem in clinical practice.Due to the special enviroment in vitro,oocytes are often facing oxidative stress.By transmission of glutathione.In our previous experiments,we compared the transcriptional differences between oocytes with difference developmental competence and the transcriptional difference between in vivo and in vitro matruation cumulus cells.We found a number of potential biological and prospective genes,one of which was MKLN1.Preliminary studies showed that if cumulus cells expressed MKLN1 highly,the oocytes showed better develpomental competence.MKLN1 is a scaffold protein in cytoplasm,it has been found playing an important role in regulating cytoskeletopn,binding anf transporting GABA receptors.The biological role of MKLN1 in cumulus cells and its potential role in regulating oocytes maturation are not reported.In this paper,we uesd the technique of small RNA interference to regulate the expression of MKLN1 in the cumulus cells,and to study the biological function of MKLN1 in cumulus cells and its mechanism.The research will help us to understand the mechanism of how cumulus cells improves oocytes maturation in vitro,and whether MKLN1 cound be a target forimproving IVM outcome.Materials and methods: 1.In vitro culture mouse follicles and COCs,spearated th cumulus cells and oocytes before and after IVM,regulate MKLN1 expression in cumulus cells by small RNA interference.QRT-PCR and Western blot were used to verify the effect.2.Find the effect of knock-down MKLN1 on the maturation and developmental competence of oocytes:The IVM group and the control group were used to compare the maturation rate of oocytes.And then observe the blastocyst formation in two groups.3.Whether the knock-down MKLN1 can affect oocyte maturation by increasing themselves apoptosis?The apoptosis of cumulus cells in the interference group and the control group was detected by Annexin-V/PI.The expression levels of Bcl-2 and Bax m RNA and protein in cumulus cells of two groups were detected.And discuss the potential mechanism of apoptosis.4.Find the intracellular mechanism of MKLN1 regulating the apoptosis of cumulus cells:We searched the database and the published literature to find the interaction protein of MKLN1;Used the immunoprecipitation method to verify it,and determine the location of MKLN1 and its interaction protein by immunofluorescence cell localization.5.Statistical method:We used SPSS 19.0 statistical software for data analysis,counting data statistics using chi square test,measurement data inthe form of mean number of standard deviation,the independent sample was analysised by t test.P<0.05 was set as cut point and defined to be statistical significance.Result: 1.By q RT-PCR and Western blot validation,the expression of m RNA and the protein expression of MKLN1 in the interference group were lower than the control group.2.The maturation rate in interference group was lower than the control.And the blastocyst formation rate also was lower than the control group.3.The cumulus cells had higher apoptosis level in the interference group,and the level of Bcl-2 decreased in the interference group,and higher expression of Bax.4.By searching the database and literatures,we found MKLN1 may interact with RANBP9,and verified by co-immunoprecipitation.5.By immunofluorescence we found MKLN1 mainly expressed in cytoplasm,and RANBP9 expressed in nucleus and cytoplasm.They had obvious co-location in cytoplasm.Conclusion: 1.Low expression of MKLN1 in cumulus cells reduces the developmental competence of oocytes.2.Low expression of MKLN1 in cumulus cells may increase the apoptosis of cumulus cells,which may be one of the reasons for low maturation rate of IVM oocytes.3.MKLN1 may regulate the disturbution of RANBP9 in nucleus and cytoplasm by binding to RANBP9,and then participate in the regulation of cumulus cells apoptosis.4.MKLN1 and its binding protein RANBP9 in cumulus cells can be uesd as potential targets to enhance the maturation of IVM oocytes.