Cumulus Cells Gene Expression As A Biomarker Of Early Embryo Development

Background and ObjectivesFolliculogenesis is a consective process,mainly consist of primordial follicles,primary follicles,secondary follicles and preovulatory follicles,primary and secondary follicles formed growing follicles.During the early process,granulosa cells surrounding the oocyte become cuboidal.With the oocyte cytoplasm increase,then granulosa cells proliferate and form multiple layers of somatic cells that surround the oocyte,resulting in the formation of a secondary follicle.This is followed by the formation of small,fluid-filled cavities within the follicle that coalesce to form the antral follicle.Under the influence of follicle stimulating hormone(FSH),the antrum continues to enlarge,resulting in the formation of a preovulatory follicle.As antrum formed,granulosa cells differentiate into two separated antrum cell subtypes:the mural granulosa cells(MGCs),thoses locating the follicle wall promote follicle ruptPre,and the cumulus cells(CCs)5 which surrounding the zona pellucida maintain the development of oocyte.CCs together with oocyte formed cumulus-oocyte complex(COCs).Follicular development and oocyte maturation rely on carefully orchestrated cross talk between the oocyte and CCs.On one hand,CCs by gap junctions transfer micromolecules such as cAMP to oocyte to maintain meiotic arrest;On the other hand,oocytes secrete factors promote CCs proliferation and mucification.Because oocyte themselves poorly metabolize energy production.CCs offer nutrition,such as pyruvate,gluthatione for oocyte;inversely,ooctye also stimuli the expressions of relevant metabolic enzymes in CCs,which are indespensible for oocyte and embryo development.CCs facilitate spern trap,select,capacitation and prevent harening of zona pellucida.It suggests that gene expressions,molecules and signal signatures of CCs could serve as a biomarker for oocyte developmental potential and embryo implantation potential.In previous study we analyzed mouse CC's genomic expression profiles before and after in vitro maturation,suggest that genes upregulated in CCs were primarily involved in matrix metalloprotease and epidermal growth factor receptor,whereas genes that were downregulated were mainly linked to endonuclear-related functions.Further study revealed that four function genes(EFEMPl,FDXI,CCND2,GJA1)in CCs for the prediction of oocyte quality.Besides,the research about AMH have an effect on IVM medium,the results indicate that addition of AMH to IVM medium not only improves oocytes quality by upregulating oocyte secrete factors expression but also increase blastocysts formation rate.The study using mouse early embryo development model,get differentiate gene expressions in CCs derived from in vitro matured oocytes with and without blastocyst formation by RNA sequencing,and further explore the selected biomarkers from the differentiate gene expressions.At the same time,these targeted genes were identified in CCs derived from human oocytes with and without blastocyst formation.Besides,we detect the expressions of AIFM2 and FDX1 mRNA in CCs wrapped around the COCs undergoing IVM or in vivo maturation,and investigate their potential functions in regulating oocyte maturation.Material and Methods(1)Establishment of the mouse early embryo development model,zona pellucida become thinner and the presence of antrum cavity consider blastocyst formation.Collection of CCs derived from IVM with or without blastocyst formation and in vivo maturation with blastocyst.Total RNA was extracted and used to hybridize to the RNA sequencing to get the differential gene expression.(2)Bioinformatic analysis of RNA sequencing results:Identifying differentially expressed transcripts between the IVM blastocyst and IVM non-blastocystAssessing the function of the differentially expressed transcripts.(3)Targeted genes selection standard:First,the genes obtained from differential gene expression.Second,based on the biological functions,those genes ranked in the top three were selected.Third,genes are participated in embryo development reported in the literature.Fourth,genes are designed specific primers for RT-qPCR study.Fifth,genes can easily get unique antibody.The RNA derived from the same samples was used to validate the results of differential gene expression by RT-qPCR.(4)Detected the targeted gene mRNA levels in human CCs:After human oocyte in vitro fertilization,obtainment of CCs derived from with and without blastocyst formation.Compare the mRNA expression in two groups's CCs.(5)Analyze AIFM2 and FDX1 mRNA expression and subcellular location:In vivo maturation COCs(IVV-COCs)and Germinal vesicle COCs(GV-COCs)were isolated from 3 weeks old mice,and then cultivated the latter to metaphase ?stage(IVM-COCs)by in vitro matiration.CCs were digested from GV-COCs,IVM-COCs and IVV-COCs respectively and defined as GV-CCs group,IVM-CCs group and IVV-CCs group.The expression of AIFM2 and FDX1 mRNA were tested by RT-qRCR.The subcellular location of AIFM2 protein and FDX1 protein in GV-COCs,IVM-COCs and IVV-COCs were observed by laser scanning confocal microscope,investigate their potential functions in regulating oocyte maturation.(6)Statistical methods:Raw real-time PCR data were statistically analyzed by SPSS version 20.0.The significance of differences in target gene RNA expression in independent samples was evaluated by a two-tailed Student's t-test and data were presented as mean ± SD.P<0.05 was set as cut point and defined to be statistical significance.Results(1)Mouse COCs were in vitro cultured,the maturated oocytes was those with the first polar body.In vivo matured COCs served as the positive control group.?we detected 57,801 transcripts in the IVM groups.48,506 transcripts overlapped in these two groups(83.92%);6,219 transcripts expressed in only the IVM blastocyst group(10.76%)and 3076 transcripts expressed in only the IVM non-blastocyst group(5.32%).?Setting of FC cut-off=2.0,higher RPKM cut-ofi?1 and P eut-off?0.05.Excluded 9 transcripts that were differentially expressed between the positive control and IVM blastocyst groups.Thus,we obtained 97 up-regulated and 103 down-regulated transcripts in the comparison of the IVM non-blastocyst and blastocyst groups.Many differentially expressed genes were involved in biological processes relevant to skeletal system morphogenesis,cell adhesion and nucleus.(2)Wnt signaling related genes(ARRB1,LGR4,SLX2,SMC2),cell adhesion related genes(CDH5,CNTNAP1),skeletal system related genes(MKLN1,RHOBTB1)and related gene transports calcium ions(ATP2C1).The expression patterms of these 9 genes in the IVM blastocyst and non-blastocyst groups were similar in the real-time PCR data and the RNA.sequencing data.ARRB1,ATP2C1,CDH5,LGR4,MKLN1,RHOBTB1and SMC2 were significantly up-regulated in CCs from the IVM blastocyst group compared to the non-blastocyst group.The expression of CNTNAP1 and SIX2 showed an increasing trend in the IVM blastocyst group;the differences were not significant in any of the replicate trials,but the same trends were observed.(3)The expressions of the target genes in human CCs derived from the oocytes that developed to the blastocyst stage(B+)and the oocytes that did not develop to the blastocyst stage(B-)were compared.The mRNA levels of ARRB1(p=0.016),LGR4(p=0,025)and SMC2(p=0.013)were significantly lower in the B+ group than in the B-group.However,there were no significant differences in the mRNA levels of ATP2C1,CNTNAP1,CDH5S MKLN1,RHOBTB1 or SIX2 between the two groups.(4)The expression of AIFM2 and FDX1 mRNA in the IVV-CCs group and IVM-CCs group were significantly higher than thoes in GV-CCs group(p<0.01),howeyer,they are no difference beteeen IVV-CCs group and IVM-CCs group(p>0.05).By immunofluorescence,both AIFM2 and FDX1 were maialy expressed in the cytoplasm,with little expression in the nucleus.After confocal microscope,they were localised to the outer as well as inner layers of GV-COCs,whereas the proportion of positively stained CCs and oocytes were higher after in vitro or vivo maturation.Conclusions(1)With oocyte maturation and blastocyst information,lots of genetic changes occurred in CCs during early embryo development.Oocyte through IVMJVF and embryo cultivation in vitro.Genes involved in skeletal system morphogenesis,cell adhesion or cell junction were up regulated.ARRB1,ATP2C1,CDH5,LGR4,MKLN1,RHOBTB1 and SMC2 were significantly upregulated in CCs from the mouse IVM blastocyst group compared to the non-blastocyst group.(2)The expressions of ARRB1,LGR4 and SMC2 mRNA levels in the human CCs were significantly lower in the blastocyst group than in the non-blastocyst group.(3)After mouse oocyte reached ruaturated,the expressions of AIFM2 and FDX1 were highered,shows that thse genes participate in oocyte maturation and may be as potential biomarkers for predicted oocyte development competence.Addition FDX1 and AIFM2 products to IVM culture medium could facilitate oocyte maturation,which will be investigated further in our future study.