The Role Of Endoplasmic Reticulum Stress Induced By Brefeldin A In Mouse Oocyte Division And Maturation

The division of mother cells into two daughter cells with different fate is called asymmetric division of cells,which is essential for the development of diversity.Mammal female meiosis produces a haploid egg and two micropolar bodies through two consecutive rounds of asymmetric cell division.This asymmetric cell division is based on asymmetric spindle positioning.Polarity is established by this asymmetric division of oocytes,which is essential for the determination of polarity in early embryos.This process requires co-regulation of tubulin,actin,and a variety of polar relative proteins.As the largest membrane organelle in the cell,the endoplasmic reticulum is involved in the synthesis and processing of various proteins.When the endoplasmic reticulum stresses,the synthesis and processing of various proteins are blocked,which leads to the obstruction of oocyte development.Brefeldin A(BFA)inhibits protein transport from the endoplasmic reticulum to the Golgi,is an inducer of endoplasmic reticulum stress,and it can disrupt the asymmetric division of oocytes.In this experiment,we use mouse oocytes as a material,with BFA treatment,the asymmetric division of mouse oocytes was disrupted,and the up-regulation of the stress marker molecule was detected by real-time PCR.Therefore,we think that the failure of asymmetric division of oocytes may be related to endoplasmic reticulum stress.Subsequently,we cloned the calreticulin(CRT)gene,which has a remission effect on endoplasmic reticulum stress,and constructed a recombinant plasmid pCRT-Venus.Then overexpressed it in mouse oocytes by in vitro transcription and micromanipulation techniques.By counting its effect on oocyte maturation rate,it was investigated whether it had a salvage effect on BFA-induced divisional damage.This study exploresd the relationship between endoplasmic reticulum stress and female meiosis during oocyte culture in vitro,and the role of calreticulin in endoplasmic reticulum stress in mouse oocytes.Studies have shown that:1.BFA inhibits polar body excretion: BFA treatment at different concentrations(0.5μM,1μM,2.5μM,5μM)does not affect the GVBD rate of mouse oocytes.Low concentration of BFA treatment resulted in symmetric division of some oocytes,and high concentration of BFA treatment significantly inhibited PB1.After removing BFA at different time points,the ratio of PB1 decreased with the extension of the action time.2.BFA inhibits spindle migration and disrupts the localization of polar-associated proteins: Immunofluorescence assay showed that BFA could inhibit the migration of spindle,affect the distribution of p-MAPK,destroy the formation of actin cap,It also disturbs the distribution of the polar related proteins Par6 a,Dlg1,and Crb3.3.BFA affects the expression of stress factors: the expression of stress factors was detected by real-time fluorescent quantitative PCR.Compared with the normal group,the stress marker factor was significantly up-regulated in the treatment group.4.Construction of the pCRT-Venus recombinant plasmid: The mouse CRT target band was cloned using mouse testis tissue cDNA as a template,and ligated with p-Venus vector to construct pCRT-Venus recombinant plasmid.5.Overexpression of CRT does not promote mouse oocyte maturation: CRT mRNA is injected into mouse oocytes by microinjection techniques and cultured in M2,and the rate of oocyte GVBD and PB1 is counted in the oocyte.There was no significant difference in the rate of PB1 between cells overexpressing CRT and the control group.