Studies On The Correlation Between Oocyte Chromatin Configuration Classification And Developmental Competence In Mammals

In mammalian oocytes,chromatin in the germinal vesicle（GV）is initially decondensed,but condenses into different configurations as oocytes grow.The GV chromatin configuration is closely related to the developmental ability of oocytes.Reported classifications of germinal vesicle（GV）chromatin configurations in oocytes were not done by uniform standards and they were not well correlated with oocyte competence.Therefore,there is an urgent necessary to establish a standard for classification of GV chromatin configuration so as to clarify its relationship with oocytes developmental ability.Since zearalenone（ZEN）can enter food chain and cause health problems,studies on its toxic effects on oocytes are of great importance for healthy reproduction in both humans and animals.However,although both in vivo and in vitro ZEN exposure were found to impair oocyte competence,the mechanisms by which ZEA damage the oocyte are largely unclear.The purpose of this study was to categorize the oocyte chromatin configuration,explore the effect of ZEN exposure on GV chromatin configuration.And we explored the potential mechanism of ZEN influence on oocyte quality from the perspective of chromatin configuration to further study the correlation between oocyte chromatin configuration and development competence.In this study,a new classification of GV chromatin configurations in porcine and mouse oocytes was classified into 8 types by using an improved method that allows a clearer visualization of both nucleolus and chromatin after Hoechst staining.Then we compared the chromatin configuration under different conditions,such as different follicle size,the health or atresia,ovary handling to correlate the oocyte development competence with various chromatin configurations.Finally,we performed study in vivo to explore the effect of ZEN on GV chromatin configurations and gene expression by immunofluorescence staining and one step RT-q PCR and other methods,so as to futher understand the mechanism of ZEN impairs oocyte competence.The main findings are as follows:（1）The GV chromatin was classified into eight configurations including nonsurrounded nucleolus（NSN）,surrounded nucleolus（SN）,partly NSN（p NSN）and SN（p SN）,prematurely condensed NSN（c NSN）,p NSN（cp NSN）and p SN（cp SN）,early diakinesis（ED）and re-decondensed（RDC）patterns.（2）Under favorable conditions such as in vitro maturation in complex 199 medium, NSN oocytes from 1 to 2 mm follicles went consecutively through p NSN,p SN,cp SN,and ED before undergoing GVBD,and chromatin in some SN oocytes from 3 to 6 mm follicles re-decondensed into a re-decondensation（RDC）configuration.Compared to those in oocytes cultured in 199 medium without a prior GVBD inhibition the most prominent change in chromatin configurations after the GVBD inhibition was a significant increase in the proportion of oocytes with an RDC configuration,while the percentage of SN oocytes decreased.（3）Under unfavorable conditions such as follicle atresia,ovary handling or maturation in simple MEM medium,however,premature chromatin condensation occurred,forming c NSN,cp NSN,and cp SN patterns.（4）While all the NSN and p NSN oocytes and some p SN,RDC oocytes actively transcribing,no c NSN,cp NSN or cp SN oocytes show any transcriptional activity.（5）Maturation and embryo culture suggested that SN and p SN oocytes were more competent than NSN and p NSN oocytes;cp SN oocytes were more competent than c NSN/cp NSN oocytes;and only RDC oocytes could develop into blastocysts.（6）Porcine oocytes exposure to ZEN treatment can cause a significant premature chromatin condensation（PMC）in NSN oocytes and affect the maturation rate of pig oocytes during in vitro culture,suggesting that ZEN disturbed oocytes developmental competence via impairing chromatin configuration.The result shows that ZEN exposure can also decreased the4-cell and blastocyst developmental potential in mouse oocytes.（7）The in vivo ZEN exposure can do some harm to the spindle assembly in mouse MI oocytes and lead to the increased levels of MII aneuploidy,which suggested that ZEN exposure significantly disturbed oocyte chromosome segregation via impairing spindle assembly.（8）While the H₂O₂ level was elevated by ZEN treatment,both MMP and the GSH/GSSG ratio were lower in the oocytes of ZEN treated mice.（9）The in vivo ZEN exposure caused a significant premature chromatin condensation（PMC）in NSN,p NSN and p SN oocytes,resulting in the formation of c NSN,cp NSN and cp SN configurations.（10）Our analysis on global gene transcription indicated that in vivo ZEA exposure significantly not only decreased the percentages of transcribing oocytes,but also reduced the transcription intensity in these oocytes.Our further experiment analysis verified the significantly down-regulated the expression of the oocyte competence-,spindle assembly-,antioxidant-and apoptosis-related genes,such as Bcl2、Nfe212、Mater、Plk1、Aurka and Cat m RNA level in ZEN exposure GV oocytes.While ZEN exposure up-regulated the expression of pre-apoptosis and redox potential-related genes such as Bax、Gpx、Gsr and Sod2.It is concluded that the newly classified chromatin configurations are more closely correlated with oocyte competence than those reported previously.What’s more,our results indicated that ZEN exposure compromised competence of mouse oocytes by impairing chromatin configuration and gene transcription.