Role And Mechanisms Of Brain-derived Neurotrophic Factor On Murine Immature Oocytes

Brain-derived neurotrophic factor (BDNF),a member of the family of neurotrophins,iswidely expressed not only in nervous system but also in the ovary of animal and humanbeing.It has been reported that BDNF is essential for folliculogenesis and development ofearly follicles.Moreover,Studies also reveal that the presence of BDNF promotes thepreimplantational development of mouse,bovine and porine embryos.Mechanismsunderlying ovarian BDNF actions on oocyte maturation were investigated in cellular leveland molecular level in our work.PartⅠEffect of BDNF on maturation in vitro of murineimmature oocytes and embryo developmental competenceObjective The effect of BDNF on maturation in vitro and embryo developmentalcompetence of murine immature oocytes was observed and the optimal concentration ofBDNF was selected.Methods Three experiments were performed.Firstly,Murine cumulus cell-enclosedoocytes (CEOs) were cultured in minimum essential media-α(α-MEM) supplemented with10% FBS,0.2 IU/ml FSH and different concentrations of BDNF (0,1 ng/ml,5 ng/ml,10ng/ml).After oocytes reached MⅡ,they were fertilized in vitro.The optimal concentrationof BDNF was selected according to maturation rate,fertilization rate and blastocystdevelopmental rate of murine oocytes.Secondly,to determine the relationship of BDNFand FBS,CEOs were cultured inα-MEM supplemented with different concentrations ofFBS(0,5%,10%) and BDNF (0,5ng/ml),removing FSH.Lastly,to ascertain whether therole of BDNF on immature oocytes needed cumulus cells,cumulus-free oocytes werecultured inα-MEM supplemented with 5% FBS,with and without BDNF (5ng/ml).Results Blastocyst developmental rate in the group of 5ng/ml BDNF (75.00%) washigher than those in the control oocvtes matured in vitro (56.63%),and it was similar with that in the group of oocytes matured in vivo (76.92%) in the presence of 10%FBS and FSHin the media although difference in maturation rate and fertilization rate of all groups wasnot significant.So 5ng/ml was the optimal concentration of BDNF for murine oocytematuration in vitro.Supplementation with BDNF (5ng/ml) in maturation mediasignificantly (P＜0.05) increased blastocyst development rate when the media was only withFBS (FBS5%:21.51% vs.9.09%;FBS10%:31.76% vs.18.9%).No blastocyst wasdeveloped when the media was not including FBS and FSH,but BDNF significantlyincreased fertilization rate of oocytes (29.72% vs.10.57%).Blastocyst development rate ofBDNF-treated (5ng/ml) increased by three times than that of control (18.18% vs.4.65%)when denuded oocytes were cultured although BDNF did not affect maturation andfertilization of oocytes.Conclusion The optimal concentration of BDNF for murine oocyte maturation in vitro is5ng/ml.Under different conditions of culture media,BDNF can prom ote cytoplasmicmaturation of oocytes,independent of nuclear maturation,which isessential for successfuloocyte development into preimplantation embryos.PartⅡEffect of BDNF on meiotic spindle configuration and cortical granulesdistribution of murine oocytes maturation in vitroObjective The meiotic spindle and cortical granules (CGs) of oocytes are importantorganelles.The characteristics of the spindle,including its presence and location,anddistribution of CGs during oocyte maturation could be often used as an important criterionto evaluate cytoplasmic maturation.To ascertain the effect of BDNF on ooplasm in cellularlevel,meiotic spindle morphology and location,distribution of cortical granules in cumuluscell-enclosed oocytes were investigated. Methods CEOs were cultured inα-MEM supplemented with 5%FBS,with and withoutBDNF 5ng/ml.Oocytes were collected at 2,4,8,12 and 16 h of maturation in vitro orpost-HCG.Tubulins and pericentrin were labeled with immunofluorescence andchromosomes were labeled with Hochest33258.Cortical granules were labeled withfluorescein isothiocyanate-lens culinaris agglutinin.Oocyte meiotic progression,spindleconfiguration and location,distribution of cortical granules were assessed by laser confocalmicroscopy.Results Our results revealed a fundamental distinction in meiotic progression,spindleconfiguration and location,CGs redistribution between IVM and IVO oocytes.IVOoocytes appeared more synchronously during meiotic progression when compared withIVM oocytes.Although BDNF did not affect oocyte meiotic resumption at IVM 2 h and theemission of the first polar body at IVM 16 h,kinetics of meiotic progression inBDNF-treated and control oocyteswas significantly difference at IVM 8h and 12 h.Meiotic spindles of IVO oocyteswere compact,displayed tapered poles and near theoolemma.However,most of IVM oocytes exhibited barrel-shaped spindles with flat polesand far from the oolemma.In addition,cytoplasm of IVO oocytes had more microtubuleorganizing centers (MTOCs) than that of IVM.Oocyte meiotic spindle width and areasignificantly were smaller in BDNF-treated group than those in control at 8 h of maturation(P＜0.05).65.79% at meiosisⅠand 71.93% at meiosisⅡofBDNF-treated oocytes hadspindles positioned near the oolemma,in comparison to just 34.29% at meiosisⅠand43.24% at meiosisⅡof control oocytes (P＜0.01).The tempo of cortical granulesdistribution of oocytes maturation in vivo and in vitro was different.Redistribution ofcortical granules had significantly difference at 8 h and 12 h of culture (P＜0.05) although itwas similar at 4 h and 16 h of maturation between BDNF-treated and control oocytes.AtIVM 8h,while 31.25% of oocytes in BDNF-treated showed a well-formed the first CG-freedomain (CGFD),significantly less control oocytes (12.5%) appeared the first CGFD (Stage Ⅳ).By 12 h of maturation,oocytes showed StageⅤof CGs redistribution (CGs becomeconcentrated around the cleavage furrow when the first polar body started to extrude) ofBDNF-treated are more than those of control,whereas oocytes entered StageⅣ(a stage ofa second CGFD of CG redistribution) of BDNF-treated were less than those of control.Conclusions BDNF can affect meiotic progression,spindle configuration and location,redistribution of CGs.BDNF improves spindle configuration and location,which is onereason that BDNF promotes cytoplasmic maturation.PartⅢSignal transduction pathways of BDNF action on oocytesObjective To ascertain signal transduction pathways of BDNF,the effect of BDNF onthe activity of protein kinase B (PKB) and mitogen-activated protein kinase (MAPK) inoocytes and cumulus cells was investigated.Methods CEOs were divided into three groups:α-MEM supplemented with 5%FBS(control);α-MEM supplemented with 5%FBS and BDNF 5ng/ml (BDNF-treated);α-MEMsupplemented with 5%FBS,BDNF 5ng/ml and 100nMK252a (a pan-specific Trk inhibitor,K252a-treated).Oocytes and cumulus cells were collected at 0,1,2,3,6 and 16 h ofmaturation in vitro,respectively.The phosphorylation of PKB and MAPK within oocytesand cumulus cells were detected by western blot.Results The presence of BDNF in maturation media enhanced the activity of PKB andelongated activation time in oocytes.K252a inhibited completely PKB activation in oocytes.So activation of PKB resulted from activation of the high-affinity TrkB receptor by BDNF.BDNF increased slightly the activity of MAPK in oocytes,which did not cause byactivation of TrkB receptor because K252a could not inhibited MAPK activation in oocytes. In cumulus cells,BDNF elongated activation time of PKB and MAPK,and increased totalprotein of PKB and MAPK at IVM 16h.K252a inhibited temporarily PKB activation,butdid not affect the activity of MAPK in cumulus cells.Conclusions BDNF enhances the activity of PKB and MAPK within oocytes andelongates activation time within cumulus cells,which may be relevant to the promotion ofBDNF on ooplasmic maturation.PKB pathway is one signaling cascade activated byBDNF combination of the TrkB receptor,whereas MAPK pathway is not.