Involvement Of Calmodulin(CaM)and Protein Kinase Cδ(PKCδ)in The Release Of Diplotene-arrested Mouse Oocytes

Objective:Congenital Anorectal Malformation（ARM）is the most common digestive tract malformation caused by embryonic intestinal development disorders.The development of oocytes,embryos and fetuses are all affected by the maturity and quality of oocytes.The process for oocytes from Germinal Vesicle（GV）stage to Germinal Vesicle Breakdown（GVBD）is called as the release of diplotene-arrested oocytes.GVBD is the marker of oocyte maturation and a key process to ensure the completion of meiosis and the normal development of embryo.Previous studies have demonstrated that Protein Kinase Bα（PKBα/Akt1）promotes the release of diplotene-arrested mouse oocytes,and Calmodulin（CaM）and Protein Kinase Cδ（PKCδ）regulate PKBα/Akt1.Therefore,during the release of diplotene-arrested mouse oocytes,CaM and PKCδmay play an important role in the regulation of normal embryonic development and the formation of congenital malformations.However,the mechanism of their regulations during the release of diplotene-arrested mouse oocytes is not very clear.Mouse oocytes are the most simple and natural cell cycle models in vertebrates that are closest to human embryos,however,the use of mouse oocytes in the study of embryonic regulatory mechanisms and birth defects was limited.In this study we used mouse as model animal to set up the model of oocyte maturation in vitro（from GV to GVBD period）.We firstly examined the effects of commonly used medium M2 or M16 in mouse embryos on the survival rate and cell morphology of mouse oocytes during the release of diplotene-arrested oocytes.In this study,dimethyl sulfoxide（DMSO）was used as the dissolution reagent for CaM and PKCδinhibitors,so we also investigated the effects of different concentrations of DMSO on the oocytes during the release of diplotene-arrested oocytes.Finally,under the M2 covered mineral oil training environment and reasonable concentration of DMSO,we used CaM and PKCδinhibitors,Calmidazolium chloride and Sotrastaurin,to test the CaM,PKCδ（Thr505）and pCaMKII（Thr286）expression and the change of localization and analyzed the effects of CaM and PKCδon the release of diplotene-arrested oocytes and the interaction of them.Our study has important theoretical and clinical significance for understanding the regulatory mechanism of normal embryonic development and the congenital anorectal malformation derived from embryonic dysplasia.Methods:1.Detect the effects of M16 and M2 medium on the development of GVBD oocytes:GV stage oocytes were collected and randomly divided into 60groups（30 oocytes in each group）.Under the microscope,60 groups of oocytes were randomly placed into mineral oil covered M2 droplet culture,mineral oil covered M16 droplet culture and M16 droplet culture respectively（20 groups in each solution）,then we placed them in an incubator at 37℃and 5%CO₂ for 4 hours.The survival rate and morphology of oocytes in each group were observed under microscope.2.Detect the effects of different concentrations of DMSO on the development of GVBD oocytes:Oocytes were randomly divided into 30 groups（50 oocytes in each group）and cultured in the 0,1%and 2%DMSO,then we also placed them in an incubator at37℃and 5%CO₂ for 4 hours.The survival rate of each group of oocytes and the number of oocytes in GV and GVBD were observed under the microscope.3.Detect the effects of CaM and PKCδon pCaMKII（Thr286）:The percentage of GVBD oocytes was compared between the treatment group and the control group,and the expression and localization changes of pCaMKII（Thr286）were detected.4.Detect the interaction between CaM and pPKC（Thr505）during the release of diplotene-arrested oocytes:Oocytes were treated with the Calmidazolium chloride to detect the level and localization of pPKCδ（Thr505）,then oocytes were treated with the Sotrastaurin to detect the level of CaM.Results:1.During the release of mouse oocytes from diplotene arrest,oocytes in M2covered with mineral oil had much better morphology and higher survival rate than those in M16.2.The effect of DMSO on the survival rate of oocyte was not statistically significant in the 0,1%and 2%DMSO,but the 2%DMSO could promote the release of mouse oocytes from diplotene arrest.3.After a treatment with Calmidazolium chloride,GVBD percentage and pCaMKII（Thr286）levels were decreased,and immunofluorescence showed changes in the localizations of pCaMKII（Thr286）compared with control group.After a treatment with Sotrastaurin,the level of pCaMKII（Thr286）was also decreased.And immunofluorescence showed changes in the localizations of pCaMKII（Thr286）compared with control group.4.The level of pPKCδ（Thr505）was decreased with the treatment of Calmidazolium chloride.And the immunofluorescence results showed that the localizations of pPKCδ（Thr505）was changed compared with the control group.However,after a treatment with Sotrastaurin,the expression level of CaM remained unchanged.Conclusion:1.This study showed that oocytes in M2 covered with mineral oil had much better morphology and higher survival rate than those in M16 during the release of mouse oocytes from diplotene arrest.2.1%DMSO and 2%DMSO had no effects on the survival rate of oocyte,however,2%DMSO could promote the release of mouse oocytes from diplotene arrest.3.Down-regulation of CaM inhibited the release of mouse oocytes from diplotene arrest and the expression of pCaMKII（Thr286）;Down-regulation of pPKCδ（Thr505）,the expression of pCaMKII（Thr286）is decreased,suggesting that CaM/pPKCδ（Thr505）might be involved in the regulation of pCaMKII（Thr286）.4.When CaM was inhibited,the level of pPKCδ（Thr505）is decreased.However,When pPKCδ（Thr505）was inhibited,the expression of CaM remained unchanged,suggesting that CaM might be an upstream kinase of pPKCδ（Thr505）during the release of mouse oocytes from diplotene arrest.The research on the culture technique and protein regulation of oocytes during the release of diplotene-arrested oocytes has an important theoretical and clinical significance for understanding the regulation mechanism of normal embryo development and the congenital anorectal malformation caused by embryonic dysplasia.