Regulatory Mechanism Of Activin-inhibin-follistatin System In The Process Of Oocyte Maturation And The Application In Human In Vitro Oocyte Maturation Technology

Up to date, mankind cannot yet reveal the regulatory mechanism ofFolliculogenesis. The crucial cause is little knowledge about the intraovarian growthfactors. Activin-inhibin-follistatin (ACT-INH-FS) system is one of the most importantregulatory system in ovary. Their auto- and paracrine Regulation mechanism in ovaryneed to be unravelled.In vitro maturation (IVM) of human oocytes, used to assist infertile couples inconceiving, is an emerging technology that has promising potential.Despitesignificant clinical improvement, pregnancy, implantation,and spontaneous abortionrates are not ideal. The possibility might underlie the limited success of human IVM:Current IVM systems induce an asynchrony in the progression of nuclear andcytoplasmic maturation. ACT-INH-FS are the important component necessary forfollicular maturation in vivo. Maybe, they are the indispensable ingredient of IVMsystems too. The present study was design to investigate The para- and autocrine roleof ACT-INH-FS in the process of oocyte maturation and To explore the value ofACT-INH-FS in human IVM.The study include three part as follow:1. The effect of ACT-INH-FS on in vitro maturation and development potentialof immature mouse oocyte.2. The developmental stage-related manner and cross-talk characteristic aboutthe ACT-INH-FS system.3. Study on the security of ACT-INH-FS utilized in IVM culture system. Part One The effect of ACT-INH-FS on in vitro maturation and developmentpotential of immature mouse oocyte【Objective】To investigate The effect of ACT-INH-FS on in vitro maturationand development potential of GV (germinal vesicle) stage mouse oocyte.【Methods】(1)oocytes isolated at GV stage were from PMSG-primed 3-4 weeks old mice.oocytes were matured for 16-18 hours in control media (HTF+10％SPS) and seventreatment media (containing 100ng/ml activin A, 100ng/ml activin A+ 100ng/mlfollistatin, 100ng/ml inhibinA, 100ng/ml inhibinA+100ng/ml follistatin,100ng/mlactivin A+100ng/ml inhibin A,100ng/ml activin A+100ng/ml inhibin A+100ng/mlfollistatin,100ng/ml follistatin respectively). MⅡstage Oocytes were fertilized andcultured using standard procedures. The germinal vesicle break down(GVBD),polarbody 1(PB1) exclusion rate, fertilizationrate rate and the frequency of blastocystformation were compared. (2) Oocytes at MⅡstage oocytes matured in either100ng/ml activin A or 100ng/ml activin A+100ng/ml inhibin A were stained withMito-Tracker Red. active mitochondria number and relocation were analyzed.【Results】(1) ACTA,ACTA+INHA,FS can improve GVBD rate obviously(P＜0.05).(2) ACTA,ACTA+INHA,INHA significantly enhanced attainment ofMⅡin oocytes (P＜0.05).(3) The fertilization rate of INHA group was lower thancontrol group (p＜0.05).Neither ACTA, nor ACTA +INHA affected the fertilizationrate.(4)In ACTA,INHA,ACTA+INHA group,the 4 cell,8cell and blastocyst formationrate were significantly higher than control group(P＜0.05). (5) FS opposed thestimulatory effect of ACTA or INHA individually (p＜0.05).But 100ng/ml FS failedto reverse the stimulatory effect of combination of the two.(6) FS could increase (notsignificant; p=0.051)the mean blastocyst yield compared with control group.(7)InMⅡstage oocyte, three mitochondrial distributions were identified: peripheral,semiperipheral, and diffused.In ACTA and ACTA+INHA group, much more oocytespresented a semiperipheral pattern or a diffused distribution of active mitochondria(P＜0.05).【Conclusion】(1) INHA and ACT A exert development-enhancing effectsduring in vitro maturation and early embryo development.(2) When ACTA is deficientor absent, follistatin may play an activin-like role.(3)ACTA and INHA could improve the synchronism in the progression of nuclear and cytoplasmic maturation.Part Two The developmental stage-related manner and cross-talkcharacteristic about ACT-INH-FS system.【Objective】(1) To determine whether ACTA and INHA exert different effecton in vitro oocyte maturation before and after LH surge of physiology cycle.(2) Toinvestigate the interreaction between ACT system and epidermal growth factor(EGF).【Methods】(1)oocytes at GV stage were isolated from PMSG-primed 3-4 weeks oldmice.oocytes were matured for 14-18 hours in the solutions with 100ng/mlACTA+100ng/ml INHA. MⅡstage Oocytes were fertilized and cultured usingstandard procedures. (2) GV stage oocytes were cultured in the different solutions for16-18 hours: 10ng/ml EGF, 10ng/ml EGF+100ng/mlFS. MⅡstage Oocytes werefertilized.The GVBD,PB1 exclusion rate, fertilizationrate rate and the frequency ofblastocyst formation were compared.【Results】(1) the percentage of COCs withfully expanded cumuli in the HCG(+) group was significantly hgher (p＜0.05) thanthe percentage in the HCG(-) group.(2) In the HCG(-) group,the mural and blastocystformation rate were significantly higher than HCG(+) group(P＜0.05).(3) FS opposedthe stimulatory effect of ACTA + INHA individually on PB1 exclusion rate (p＜0.05).But failed to reverse the stimulatory effect on embryo development(P＞0.05).【Conclusion】(1)Several hours post LH surge are the key period to improve thesynchronism in the progression of nuclear and cytoplasmic maturation.(2)Before andafter LH surge, immature oocytes showed reverse response to ACTA and INHA.(3) nuclear maturation and cytoplasmic maturation are regulated by different signalpathway.(4)Intrinsic ovarian activin system is probably a downstream mediator of Egfaction.Part Three Study on the security of IVM culture system.【Objective】(1)To explore the relationship between aneuploidy and spindleabnormalities during meiosis I by fluorescense spindle imaging. (2) To investigatethe security of ACT-INH-FS utilized in IVM culture system by karyotype analysis of in vitro matured oocytes.【Methods】(1) oocytes at GV stage were matured for 7hours in HTF medium, spindle morphology and chromosome alignment duringmetaphase I was then explored using immunocytochemistry and three-dimensionalreconstruction of confocal sections.(2) oocytes at GV stage were matured for 16-18hours in the solutions either with 100ng/ml ACTA+100ng/ml INHA or not. Followingculture, matured oocytes were isolated, spread, stained with DAPI, and the numbersof chromosomes counted.【Results】(1)There was a higher proportion of abnormalmeiotic spindle and chromosome alignment configurations among those oocytesmatured in vitro compared with oocytes matured in vivo(P＜0.05). But the percentageof aneuploid of MⅡstage oocyte matured in vitro did not increase(P＞0.05). (2)Thepercentage of aneuploid oocytes were similar between ACTA+INHA group andcontrol group (P＞0.05).【Conclusion】(1)There is a high rate of spindle abnormalityand chromosome configurations among those oocytes matured in vitro. The abnormalcytoskeleton of the oocytes might resulting in MⅠoocyte arrest and low fertilizationrate of MⅡoocytes.(2)ACTA and INHA utilized in IVM culture system have no effecton the genesis of chromosomal abnormalities.To our knowledge, this research is the first one to study the regulatorymechanism of ACT-INH-FS system on oocyte maturation in china. Most overseasreports only discuss the role of ACT-INH-FS on oocyte nuclear maturation. There arethree study investigared their effect on oocyte development potential in bovine,buttheir conclusions were controversial.In our study, for the first time,confirm thedevelopment-enhancing effects of acivin A and inhibin A on the cytoplasmicmaturation by determination of organelle in oocytes. Whereafter, we discuss thedevelopmental stage-related manner and cross-talk characteristic about theACT-INH-FS system, explore the security when they are utilized in IVM culturesystem. These contents have not been reported before.